WO2003047599A1

WO2003047599A1 - An antiinflammatory composition containing catalposide isolated from stem bark of catalpa ovata

Info

Publication number: WO2003047599A1
Application number: PCT/KR2002/001013
Authority: WO
Inventors: Hun-Taeg Chung; Ho-Sub Lee; Seon-Il Jang; Hyun-Cheol Oh; Tae-Oh Kwon; Hyun-Ock Pae
Original assignee: Hun-Taeg Chung; Ho-Sub Lee; Seon-Il Jang; Hyun-Cheol Oh; Tae-Oh Kwon; Hyun-Ock Pae
Priority date: 2001-12-06
Filing date: 2002-05-29
Publication date: 2003-06-12
Also published as: KR100421625B1; KR20030046796A; AU2002304195A1

Abstract

There is provided an anti-inflammatory composition containing iridoid glycoside compound, catalposide isolated from stem bark of Catalpa ovata as an active ingredient, for treating inflammatory response by inhibiting inducible nitric oxygen synthase (iNOS), NF-κB, TNF-α, IL-1β and IL-6.

Description

AN ANTIINFLAMMATORY COMPOSITION CONTAINING CATALPOSIDE ISOLATED FROM STEM BARK OF

CATALPA OVATA

FIELD OF THE INVENTION

The present invention relates generally to an anti-inflammatory composition containing catalposide, iridoid glycoside compound, isolated from stem bark of Catalpa ovata as an active ingredient. More particularly, the present invention relates to an anti-inflammatory composition for treating inflammatory response by inhibiting inducible nitric oxygen synthase (iNOS), NF-KB, TNF-α, IL-lβ and IL-6.

BACKGROUND OF THE INVENTION

Inflammation is caused by a complex biochemical reaction and various mediators, such as prostaglandins, leukotrienes and platelet activating factor, have been reported to be involved in the development of inflammatory diseases. Thus, many efforts have been made to find anti-inflammatory agents by screening substances which can inhibit synthesis of mediators or lessen the activities thereof. And, various in vivo and in vitro experimental models have been set up for the assessment of anti-inflammatory activity. Some macrophage-derived mediators which are called pro-inflammatory cytokines, such as TNF-α, IL-lβ and IL-6, are also considered to play a key role in inflammatory and immune responses. Particularly, since these pro- inflammatory cytokines are detected at high levels in afflicted joint fluids, these are considered to have a role in the pathogenesis of rheumatoid arthritis caused by excessive inflammatory response. That is, rheumatoid arthritis is known as an autoimmune disease wherein immune-inducible T-cells(CD4+T cells) penetrate excessively into lymphocytes of joint to release various pro- inflammatory cytokines, such as TNF-α, IL-lβ and IL-6, and interferon, inducing excessive immune response and destructing bone and cartilage. For example, it has been reported in this art, that detection of high level of pro- inflammatory cytokines, such as TNF-α, IL-lβ and IL-6 around the joint is estimated to be diagnosed as rheumatoid arthritis. According to recent reports, pretreatment of animal with anti-TNF-α prevented the development of arthritis. And, injection of IL-lβ into the knee joint of rabbits results in the degradation of cartilage, whereas the injection of antibodies against IL-lβ ameliorates collagen- induced arthritis and decreases the damages to cartilage. Further, it was reported that antibody against the receptor for IL-6 has shown remarkable efficacy in mice with collagen- induced arthritis. Based on these results, screening of substances for inhibiting productions of and activities of pro-inflammatory cytokines is advantageously employed for development of novel anti-inflammatory and anti-rheumatic agents and evaluation the activities thereof.

In addition to said pro-inflammatory cytokines, inflammatory response is mediated by NF-κB which is known as a transcription factor. NF- B is a transcription factor ubiquitous in various cells which are associated with the development of rheumatoid arthritis and asthma. Under normal conditions, NF- KB is retained in the cytoplasm as a complex protein consisting of p50, p65 and IκBα subunits. Once activated by outer environment, IκBα is then phosphorylated, ubiquitinated, and degraded; this causes the p50 and p65 heterodimers to be translocated to the nucleus. After NF-κB reaches the nucleus, it induces the expressions of genes encoding pro-inflammatory cytokines such as TNF-α, IL-l β and IL-6, and inflammatory mediators. These inflammatory mediators activate inflammatory cells or cause inflammatory response directly. Therefore, NF-κB-inhibiting substances can be used as new anti-inflammatory agents. For example, it was reported in this art, administration of selective inhibitors of NF- B pathway abrogated the production of IL-l β and TNF-α in animal models with chronic inflammation. And, non-steroidal anti-inflammatory drugs such as aspirin and indomethacin also exerted their anti-inflammatory effects by inhibiting translocation of NF- B into the nucleus (Hackstein EL, et al., J. Immunol 2001 Jun 15;166(12):7053-62; Weber C. et al, Circulation 1995 Apr 1;91(7): 1914-7).

While, various inflammatory agents including the endotoxin lipopolysaccharide (LPS) induce the activation of NF-κB to cause inflammatory response.

Apart from mediators involved in said inflammation, nitric oxide(NO) is also implicated in inflammation. This reactive species synthesized by NO synthase, particularly inducible nitric oxide synthase (iNOS) is involved in pathological aspect of many inflammatory diseases. iNOS can be over- expressed in response to LPS, leading to cause autoimmune disease (Jang, D. and Murrell, G. A. C, Nitric oxide in arthritis, Free Radical Biology & Medicine, 1998, 24: 1511-1519; Miyasaka, N. and Hirata, Y., Nitric oxide and inflammatory arthritides, 1997, 61 : 2073-2081; Lafaille, J. L, The role of helper T cell subsets in autoimmune disease, 1998, 9: 139-151). In case that over-expression of iNOS is induced by other stimuli, such as pro-inflammatory cytokines, it leads to some inflammatory and autoimmune diseases. In summary, once NF-κB in the cytoplasm is activated by outer stimuli, it induces the expressions of genes encoding pro-inflammatory cytokines such as TNF-α, IL-lβ and IL-6. The formed pro-inflammatory cytokines stimulate the gene encoding iNOS, to produce reactive species such as NO implicated in inflammatory response. In addition to this chain of mechanism, NF-κB activates directly the expressions of iNOS gene or other inflammatory genes, and pro-inflammatory cytokines, such as TNF-α, IL-lβ and IL-6, activates other biological responses involved in inflammation. Thus, NF-κB transcription factor implicated in inflammatory response; pro-inflammatory cytokines such as TNF-α, IL-lβ and IL-6; and iNOS gene can be used as target for screening new anti-inflammatory agents for treatment of diseases caused by excessive inflammatory response, such as septic shock and rheumatoid arthritis.

SUMMARY OF THE INVENTION

In order to develop new anti-inflammatory agents, the present inventors have conducted extensive researches for screening substances which can inhibit NF-κB transcription factor, pro-inflammatory cytokines such as TNF-α, IL-lβ and IL-6; and iNOS gene. As a result thereof, we found that iridoid glycoside compound, catalposide isolated from stem bark of Catalpa ovata inhibited the activation of NF- B; suppressed the expressions of genes encoding pro- inflammatory cytokines such as TNF-α, IL-lβ and IL-6, so to inhibit the productions thereof; and suppresses the expression of iNOS gene. Thus, composition containing said catalposide as an active ingredient can be advantageously used as an anti-inflammatory medicament and we completed the present invention.

Therefore, a major object of the present invention is to provide an anti- inflammatory composition.

In order to achieve said object, the present composition contains catalposide as an active ingredient for treating diseases caused by excessive inflammatory response, such as rheumatoid arthritis.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the present invention.

The object of the present invention is to provide an anti-inflammatory composition containing catalposide as an active ingredient, based on finding that catalposide can 1) inhibit the production of pro-inflammatory cytokines implicated in inflammatory response and the gene expression thereof in macrophages; 2) suppress the activation of gene transcription by inhibiting activation and nuclear translocation of NF- B; and 3) reduce the production of NO in macrophases by suppressing expression of iNOS gene.

Catalpa ovata G. Don. has been cultivated as an ornamental tree. The stem thereof contains mainly iridoid type of secondary metabolites, for example 0.1% of catalposide and other compounds such as catapol deshydroxybenzoyl catalposide, catalpalactone, para-hydroxy benzoic acid and KN0₃(25%). Various parts of Catalpa ovata, i.e. fruit, pericarp and stem have been used as a crude drug in Korea for chronic nephritis and edema.

Iridoids are cyclopentanoid monoterpene derivatives that fall into four distinct groups : iridoid glycosides, non-glycosidic iridoids, secoiridoids and bisridoids. The most numerous group of these is the iridoid glycosides. Iridoid glycosides exist broadly in plants of many families and commonly possess a bitter taste and, in addition, a wide variety of biological activities. According to the literature, the therapeutic effects of many plants are attributable to the iridoid compounds. Indeed, certain iridoid-producing plants have often been used as anti-inflammatory remedies.

Catalpa ovata G. Don. (Bignoniaceae) is one of iridoid-producing plants, and exhibits anti-inflammatory activity. In Korea folk medicine, it has been used for the treatment of rheumatoid inflammation. However, numerous kinds of iridoid compounds are contained in Catalpa ovata and it has not been known which component of these compounds exhibits anti-inflammatory and anti- rheumatoid activities.

Among these iridoid compounds contained in Catalpa ovata, the present invention demonstrated that catalposide exhibited anti-inflammatory activity. Moreover, we clarified the mechanism of catalposide in macrophages involved in inflammatory response. Thus, it is suggested that catalposide may be advantageously used for the treatment of autoimmune disease such as rheumatoid inflammation.

Catalposide can be extracted from Catalpa ovata or catalposide-producing plant or biochemically synthesized by conventional method in this art. For example, extraction of catalposide may be performed with methanol, which will be described in the following examples.

Further, as an inflammatory disease in the present invention, it may be conventionally known to this art and obtain therapeutical effect by administrating the present composition therefor. Said disease caused by excessive inflammatory response and to be treated by the present composition may include, but not limited thereto, autoimmune disease selected from the group consisting of rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), psoriasis, inflammatory bowel disease and autoimmune hepatitis; or septic shock. Also, inflammatory diseases known to this art may be included to the scope of the present invention. According to the present invention, catalposide isolated from the stem of

Catalpa ovata can significantly inhibit the production of cytokines including TNF-α, IL-lβ and IL-6 in RAW 264.7 macrophages in a concentration- dependent manner. This inhibition is because catalposide suppresses the expressions of genes encoding TNF-α, IL-lβ and IL-6. Furthermore, catalposide can significantly inhibit the nuclear translocation of p65 subunit of NF- B. Thus, catalposide can block the excessive productions of TNF-α, IL- lβ and IL-6 by suppressing the expressions of TNF-α, IL-lβ and IL-6 genes, and can inhibit the transcriptions of inflammatory genes due to activation of NF-κB. Therefore, catalposide can be used as anti-inflammatory and anti-rheumatic drugs.

On the other hand, the present invention isolated two iridoid glycoside compounds having similar structure to that of catalposide and conducted the same experiments as conducted as to catalposide. As results, they did not show any inhibitory effects on LPS-induced NO production, iNOS gene expression and NF- B activation. This result demonstrates that C-6 substituent moiety of catalposide may have anti-inflammatory activities. In the anti-inflammatory composition according to the present invention, an therapeutically-effective dose of catalposide as an active ingredient may be controlled depending on administration route, formulation and patient's condition such as age, weight, sensibility and disease symptoms. In detail, it may be, but not limited thereto, formulated for catalposide to be administrated in a unit dose of 0.001~500mg/kg of patient's weight. Of course, the dose of the active ingredient may not be limited within said range.

In order to administrate catalposide in said dose, the present composition may be provided in the form of tablets, capsules, drinks, medicines or drugs. It may be orally or parenterally administrated for the treatment of inflammatory diseases. And, it may contain pharmaceutically acceptable excipient, carrier or diluent, which is known to this art, depending on the type of formulation. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of catalposide (la); of C-6 substituent with activity thereof (lb); and of specioside (lc) and picroside (Id), which are iridoid compounds.

FIG. 2 shows the inhibitory effect of catalposide on the LPS-induced productions of pro-inflammatory cytokines such as TNF-α (FIG.2a), IL-lβ (FIG.2b) and IL-6 (FIG.2c); and shows cell viability (FIG. 2d).

FIG. 3 shows the inhibitory effect of catalposide on the LPS-induced m- RNA transcriptions of TNF-α, IL- 1 β and IL-6 genes.

FIG. 4 is a result of western blot analysis of p65 subunit, showing that catalposide inhibits the translocation of p65 subunit of NF-κB from cytoplasm into the nucleus.

FIG. 5 shows the inhibitory effects of catalposide on the LPS-induced NO production and iNOS(inducible NO synthase) gene expression.

FIG. 6 shows the inhibitory effect of catalposide on the LPS-induced activation of NF-κB analyzed by electrophoretic mobility shift assay (EMS A).

PREFERRED EMBODIMENT OF THE INVENTION

The present invention will be described in more detail by way of the following examples, which should not be considered to limit the scope of the present invention. All experiments were repeated at least three times and Student's t-test was used to assess the statistical significance of differences using Microcal Origin version 5.0 software (Microcal Software, USA). A confidence level (p<0.05) was considered significant. <Reference Example 1> Isolation of catalposide

Catalpa ovata G. Don. (Bignoniaceae) employed in this experiment was collected in Iksan, Chonbuk province, Korea, in July 2000. A voucher specimen (No. KTO 51) was deposited at the Herbarium of the Medicinal Resources Research Center, Wonkwang University.

Catalposide was isolated from the stem bark of Catalpa ovata G. Don. (Bignoniaceae) (No. NOSEA-4-5-4) with high purity of 95% or more by the method of Naggar SF (Naggar SF, Doskotch RW. Specioside: a new iridoid glycoside from Catalpa speciosa. J. Nat. Prod. 1980; 43: 524-526). In detail, 500g of the dried stems of C. ovata was crushed and extracted with 51 of MeOH for 24 hrs. The MeOH extract was concentrated, suspended in distilled water, and sequentially partitioned with n-hexane, EtOAc and BuOH.

12.6G (dried weight) of EtOAc-soluble fraction showed potent inhibitory effect on the productions of TNF-α, IL-lβ, IL-6 and NO at the concentration of lOO g/m . Thus, said EtOAc-soluble fraction was subjected to silica gel (Merck Kieselgel 60; 0.063~0.2mm particle size) column chromatography (column : 2.5cm diameter; 40cm height). The column was eluted with a gradient elution using mixtures of MeOH in CH₂C1₂, i.e. 10% with 600ml, 20% with 300ml and 30% with 150mA, followed by 300ml of 100% MeOH, yielding 8 fractions, 150m per each fraction. Fraction 4(5.3g) eluted between 450~600ml retained the activity, thus fractionated further Sephadex LH-20 column chromatography (3cm diameter; 50cm height; stepwise gradient of 150ml of MeOH-H₂O (4: 1), followed by 150ml of MeOH; collecting 50ml fractions).

Each fraction was tested by the assay. A portion (1.15g) eluted between 200~250ml was detected to have activity. This active fraction was subjected to semi-preparative reversed-phase HPLC using a gradient from 30~40% CH₃CN in H₂0 over 40 min to yield catalposide [tr 24.7 min; 7.8mg; calculated 0.022% w/w; [α]²³ _D: -116.8°(c 0.13, MeOH)]; specioside [tr 27.3 min; 16.3mg; calculated 0.047% w/w; [α]²³ _D: -155.6°(c 0.27, MeOH)]; and picroside [tr 29.0 min; 19.0mg; calculated 0.055% w/w; [α]²³ _D: -115.6°(c 0.32, MeOH)].

Each compound was identified by the comparison of [α]_D, MS, ¹H-NMR and C-NMR data with those in said literature of Naggar et al. The structure of catalposide is shown in FIG. 1.

<Reference Example 2> Reagents

Lipopolysaccharide (LPS), RPMI-1640 medium and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazoleum (MTT) were obtained from Sigma Chemical

Company.

Fetal bovine serum (FBS), oligo (dT) 18 primers, Moloney leukemia virus

(M-MLV) reverse transcripase, dNTP solution, RNA inhibitor and antibiotics were purchased from Gibco/RBL. AmpliTag™ DNA polymerase was obtained from Takara Technologies.

Polymerase chain reaction oligonucleotide primer pairs were purchased from

Bioneer Co. (Korea).

All reagents and media for tissue culture experiments were tested for endotoxin content with use of a colorimetric Limulus amoebocyte lysate assay (detection limit, 10 pg/ml; Whittaker Bioproducts). None of these reagents contained endotoxin. Tissue culture plates and 100mm diameter petri dishes were obtained from Nunc, Inc. (USA).

<Reference Example 3> RAW264.7 macrophage cell line and culture The murine macrophage cell line, RAW264.7 was obtained from the

American Tissue Culture Collection (ATCC).

The macrophage cell line was maintained in complete RPMI 1640 medium supplemented with, as antibiotic/antimycotic, lOOU/ml of penicillin G and lOOU/ml of streptomycin and 10% heat inactivated FBS, at 37^°C in a humidified 5% C0₂ atmosphere.

<Reference Example 4> Cell viability

The cell viability was measured by the following method. That is, 50 zg/ml of MTT was added to cell suspension of Reference Example 3 (1x10 cells/ml) and then reacted for 4 hrs. The formed formazan was dissolved in acidic 2-propanol (Sigma, USA) and absorbance was measured at 590nm. The absorbance measured in control (test sample-untreated) cells was taken as 100% of viability and relative cell viability was calculated by the comparison of absorbance in test sample-treated cells. <Example 1> Measurement of cvtokine production

Because interference with the biosynthesis or action of macrophage- mediated pro-inflammatory cytokines, such as TNF-α, IL-lβ and LL-6, has become an important strategy for pharmacological intervention in a variety of inflammatory and rheumatic diseases, said pro-inflammatory cytokines can be employed as a criteria for the evaluation of pharmacological efficacy of anti- inflammatory agents. This example examined whether catalposide could inhibit the productions of pro- inflammatory cytokines such as TNF-α, IL-lβ and IL-6 in activated RAW264.7 macrophages.

The gram-negative bacterial endotoxin LPS has been well known to this art as an inducer of the productions of pro-inflammatory cytokines, such as TNF-α, IL-lβ and IL-6, in macrophages. Thus, in this example, 1/zg/ml of LPS was used to induce the productions of pro-inflammatory cytokines in macrophages.

In detail, the productions of cytokines including TNF-α, IL-lβ and IL-6 were measured by immunoassay. Ixl0⁶/ml of RAW264.7 cells were pretreated with 10, 50 or lOOng/m of catalposide for 1 hr and further treated with 1/tg/m of LPS, for 6 hrs to induce TNF-α and IL-6 productions or for 12 hrs to induce IL-1 β production. Then, the medium was centrifuged to remove the cells and to collect supernatant. Within the supernatant, TNF-α, IL-lβ and IL-6 productions were quantified by means of kits supplied by Quantikine™(USA) and R&D Systems(USA), according to the manufacture's instructions, respectively.

The results are shown in FIG. 2. The resulting values were obtained by repeating the experiments three times and represented by average± standard deviation.

As shown in FIG. 2, the pretreatment of RAW264.7 macrophages with catalposide remarkably inhibited the LPS-induced productions of pro- inflammatory cytokines in a concentration-dependent manner. It was confirmed that this effect was not due to the cytotoxicity of catalposide.

<Example 2> RNA extraction

RNA was extracted from cells treated with LPS plus catalposide; from cells treated with LPS alone as positive control; and from untreated cells as negative control (each lxlO⁷ cells/ml) by TRIZOL™ reagent (GIBCO/BRL (USA) according to the manufacture's instructions. Before use, the integrity and purity of the RNA samples were checked by electrophoresis.

<Example 3> RT-PCR

As described in Example 1, catalposide inhibited macrophage-mediated productions of pro-inflammatory cytokines without cytotoxicity. In order to confirm whether the inhibitory action of catalposide may also suppress the transcriptions of TNF-α, IL-lβ and IL-6 genes, RT-PCR assay was conducted. RAW264.7 macrophage cells were treated with LPS and catalposide by the same method as described in Example 1. After collecting the cells from the medium, RNA was extracted by the method of Example 2. For the RNA samples, RT-PCR assay was performed as follows. In detail, the reverse transcription reaction mixture contained ImM dNTPs,

1.75units//zl of RNAse inhibitor, 2.5units/ l of M-MLV reverse transcriptase, 25units/ l of oligo(dT) primers, 25ng of said extracted RNA, 5mM MgCl₂ and PCR buffer(5mM KC1; lOmM Tris-HCl pH8.3) to final volume 20 t The prepared mixture was incubated at room temperature for 10 min and then reverse transcription protocol was performed in a thermal cycler : 42 ^°C for 60 min; 94 ^°C for 3 min. After the generation of the first strand cDNA, the tubes were placed on ice for 5 min prior to either to being stored at -20 °C or used for the following PCR reaction.

The PCR reaction solution contained 2.5units of AmpliTag™ DNA polymerase, 5μg/ l of sense primer and antisense primer pair, 20 μi of said reverse transcription solution, 2mM MgCl₂ and PCR buffer to final volume 100μβ. The sense and antisense primer sequences used in this example were shown in Table 1. [Table 1]

Each gene was amplified using said primer sequences, to yield PCR products of 692bp for TNF-α, 563bp for IL-lβ, 638bp for IL-6 and 153bp for β- actin. PCR was performed using the GeneAmp PCR system (Perkin Elmer).

The pretreatment was performed at 94 ^°C for 5 min, at 60 ^°C for 5 min and at 70 °C for 90 sec. PCR with temperature profile : denaturation at 94 °C for 45 sec, annealing at 60 °C for 45 sec and extension at 75 °C for 90 sec was performed 35 cycles for cytokine cDNA and 25 cycles for β-actin cDNA, and followed with a final cycle of 94 °C for 45 sec, 60 ^°C for 45 sec and 72 °C for 10 min.

Depending upon the expected amount of product, about 0.05-0.2 volumes of the RT-PCT reaction mixture were electrophoresed through 2.5% agarose gel and amplification was confirmed by visualizing with ethidium bromide staining and UV irradiation. The results are shown in FIG. 3

As shown in FIG. 3, catalposide inhibited the mRNA transcriptions of cytokines, TNF-α, IL-lβ and IL-6 genes in a concentration-dependent manner. While, control β-actin gene was expressed and was unaffected by catalposide.

<Example 4> Western blot analysis of p65

As described above, catalposide inhibited the expressions of pro- inflammatory cytokine genes. In order to confirm whether the inhibition of gene expression may be caused by suppressing the activation of NF- B transcription factor, this example examined whether catalposide may inhibit the activation of NF-κB. Once NF- B is activated, the subunits p50 and p65 heterodimers are formed to be translocated into the nucleus and activate various genes. Thus, the activation of NF-κB is confirmed by observing the nuclear translocation of p65 subunit. This example measured the levels of p65 in the cytoplasm and nucleus by western blot analysis, to confirm the inhibitory effect of catalposide on the activation of NF-κB.

RAW264.7 macrophage cells were pretreated with lOOng/ml of catalposide for 1 hr or untreated as control, and further treated with Iμg/mi of LPS, for o min, 30 min or 60 min. Then, from each cell, nucleus and cytoplasm were extracted by the following method:

The extract of cytoplasm was performed by the method of Wang, Y, et al. (Wang, Y., et al., Mitochondrial Katp Channal and End Effector of Cardioprotection During Late Preconditioning: Triggering Role of Nitric Oxide., J. Mol. Cell Cardiol, 33 : 2037-2046).

In detail, the cells were crushed with the buffer containing 25mM Tris-HCl, 5mM EGTA, 2mM EDTA, lOOmM NaF, 0.02mM leupeptin, O.OlmM E64, 0.12mM peppstatin, 0.2mM PMSF and 5mM DTT, and then centrifuged at 14,000g for 15 min, to collect supernatant for western blot analysis. The extract of nucleus was performed by the method of Schreiber, E., et al.

(Schreiber, E., et al, Rapid detection of octamer binding proteins with 'mini- extracts' prepared from a small number of cells., Nucleic Acids Res. 1989: 17: 6419-).

In detail, the cells were injected with 1ml of cold buffer containing lOmM HEPES(pH 7.9), lOmM KCl, O.lmM EDTA, O.lmM EGTA, 1.5mM MgCl₂,

ImM DTT, 10 g/ml of leupeptin, 4mM Pefabloc SC, 50mM NaF and Na₃VO₄

(Said reagents were purchased from Sigma (USA) and Sata Cruz Biotech. INC.), and then, injected with 62.5 μJl of 10% Nonidet P-40. After, homo-mixing for 10 sec, the mixture was placed on the ice for 30 min and then centrifuged at 4 ^°C, 5,000rpm for 10 min, to collect nuclear precipitate. To the nuclear precipitate was added 150 β of cold buffer containing 20mM HEPES(pH 7.9), 0.4M NaCl, ImM EDTA, ImM EGTA, 1.5mM MgCl₂, 20% (w/v) glycerol, ImM DTT, 10 g/ml of leupeptin, 10 zg/ml of aprotinin, 4mM Pefabloc SC, 50 mM NaF and Na₃V0₄, to be floated. After standing at 4^°C for 30 min, the mixture was centrifuged at 15,000rpm for 10 min, to collect supernatant for western blot analysis. In order to determine the p65 levels in nuclear and cytoplasmic extracts, electrophoresis was performed with 8.5% SDS-polyacrylamide gels. Then, the proteins were electrotransferred to nitrocellulose filters, probed with a rabbit polyclonal antibody to p65 (Santa Cruz Biotech. INC.), and detected by chemiluminescence using ECL kit (Amersham, USA). The results are shown in FIG. 4.

As shown in FIG. 4, in case of LPS alone treatment, the level of p65 declined in the cytoplasm and increased in the nucleus depending on treatment times. In contrast, in case of LPS plus catalposide treatment, p65 was scarcely observed in the nucleus and the level of p65 in the cytoplasm was not changed. These results show that catalposide inhibits the translocation of p65 to the nucleus, for NF-κB not to be activated.

<Example 5> Analysis of iNOS mRNA expression

RAW264.7 macrophage cells were treated with 0, 10, 50 or lOOng/ml of catalposide in DMSO or with 0 or lOOng/ml of specioside for 1 hr. Thereto were injected 5U/m! of IFN-γ and lOng/ml of LPS, and then cultured for 18 hr.

The final concentration of DMSO in the medium was 0.1%. For the product, the NO production and the iNOS mRNA expression were examined as follows.

Because, the produced NO is transformed to nitrite which is stable end- product in the medium, the production of NO was confirmed by measuring the concentration of nitrite in the medium by the method of Griess. In detail, the culture supernatant of macrophage cells was collected and thereto was injected the same amount of Griess reagent (0.1% of N-(l-naphthyl)- ethylenediamine dihydrochloride in 1% sulfanylamide and 2.5% phosphoric acid). The mixture was placed at room temperature for 10 min. The concentration of nitrite was determined by measuring absorbance at 540nm using ELISA plate reader. The concentration of nitrite in the medium without cells was 5~8M which was used as a reference value for measurement of nitrite in test samples.

Further, iNOS gene expression was evaluated as follows. Firstly, total RNA was extracted by the acid-guanidinium isothiocyanate phenol chloroform (AGPC) method (Chomezynski P, et al, Analytical Biochemistry, 1987, 162, 156-9) and then for iNOS, RT-PCR assay was performed by the method of Choi CY, et al. (Choi CY, et al, International Immunopharmacology, 2001, 1, 1141- 51).

In detail, performance profile of RT-PCR and other reagents and their contents of the reaction solution, except primers and RNA strand, were same as described in Example 3. The primer sequences for RT-PCR of iNOS gene were 5-.TTTGGAGCAGAAGTGCAAAGTCTC-3 and 5'-

GATCAGGAGGGATTTCAAAGACCT-3'. The primer sequences for RT-PCR of β-actin, as control gene were 5'-CCTCTATGCCAACACAGT-3' and 5'- AGCCACCAATCCACACAG-3'. The amplified products of RT-PCR were electrophoresed through 2.5% agarose gel and confirmed by visualizing with ethidium bromide (EtBr) staining and UV irradiation. Further, IX 10⁶ cells/ml of RAW 264.7 macrophage cells were pretreated with lOOng/ml of specioside, other iridoid glycoside compound, instead of catalposide and further treated with l g/ml of LPS. The NO production and iNOS gene expression were confirmed by the same method as described above. In this example, all the resulting values were obtained by repeating the experiments three times and represented by average + standard deviation. The results are shown in FIG. 5.

As shown in FIG. 5, RAW 264.7 microphage cells were treated with 1 zg/mlof LPS alone, nitrite as stable end-product was accumulated by 15.90+0.22μM. But, catalposide reduced nitrite accumulation in a concentration-dependent manner (IC₅₀ = 46±0.12ng/ml). On the other hand, other iridoid glycoside compound, specioside did not show any inhibitory effect on nitrite accumulation.

Further, the result for iNOS gene expression was the same as that for said nitrite production. That is, catalposide inhibited the LPS-induced iNOS gene expression, while specioside did not inhibit at all.

Although the result was not shown in this example, another iridoid compound, picroside did not show any inhibitory effects on iNOS mRNA expresstion and NO production, alike specioside.

<Example 6> NF- B DNA binding assay

RAW 264.7 macrophage cells were treated with LPS, catalposide and/or specioside by the same method as described in Example 5. After the culture for 1 hr, the nucleus was extracted from the cells by the method of Dignam JD. et al. (Dignam JD. et al, Nucleic Acids Research, 1983, 11, 1475-89). For the nuclear extract, NF-κB DNA binding activity was analyzed by electrophoretic mobility shift assay (EMSA) (Oh G-S, et al, Cancer Letters, 2001, 174, 17-24). The results are shown in FIG. 6.

As shown in FIG. 6, catalposide reduced gel shift by NF-κB binding to the nucleus in a concentration-dependent manner. On the contrary, specioside accelerated gel shift. These results indicate that catalposide can inhibit the activation of NF-κB which is a major transcription factor involved in iNOS gene expression. Further, it indicate that catalposide is related to the upstream signaling pathway of NF- B activation.

On the other hand, iridoid glycoside compounds, specioside and picroside did not show any inhibitory effects on iNOS gene expression and NF- B activation. These results show that C-6 substituent of catalposide may play an important role in exerting anti-inflammatory activity. The chemical structures of C-6 substituent of catalposide and of specioside and picroside, which have similar structure to that of catalposide, are shown in FIG. 1.

INDUSTRIAL USE OF THE INVENTION

As above described, catalposide can inhibit the productions of various mediators, i.e. pro-inflammatory cytokines including TNF-α, IL-l β and IL-6, involved in inflammatory response; iNOS gene expression; and NF-κB activation. Thus, the present composition containing catalposide can be advantageously used for the treatment of diseases caused by excessive inflammatory response, such as rheumatoid arthritis and septic shock. In particular, because the present composition contains catalposide isolated from natural sources, it may not cause any serious side effects, even if it may be administrated for long terms. <Reference>

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Claims

1. An anti-inflammatory composition for treating the diseases caused by excessive inflammatory response, containing catalposide as an active ingredient.

2. The anti-inflammatory composition according to Claim 1, wherein said catalposide is isolated from stem bark of Catalpa ovata.

3. The anti-inflammatory composition according to Claim 1, which inhibits productions of pro-inflammatory cytokines including TNF-α, IL-1 β and IL-6.

4. The anti-inflammatory composition according to Claim 3, which suppresses expressions of genes encoding TNF-α, IL-lβ and IL-6.

5. The anti-inflammatory composition according to Claim 1, which inhibits activation of NF-κB.

6. The anti-inflammatory composition according to Claim 5, which inhibits nuclear translocation of p65 subunit of NF-κB.

7. The anti- inflammatory composition according to Claim 1, which inhibits production of nitric oxide (NO).

8. The anti-inflammatory composition according to Claim 7, which suppresses expression of iNOS (inducible NO synthase) gene.

9. The anti-inflammatory composition according to Claim 1, wherein said catalposide is contained in an amount of 0.001~500mg/kg of patient's weight to be administrated in a unit dose.

10. The anti-inflammatory composition according to any one of Claims 1 to 9, wherein said disease caused by excessive inflammatory response is autoimmune disease selected from the group consisting of rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), psoriasis, inflammatory bowel disease and autoimmune hepatitis; or septic shock.