KR100361090B1 - A novel prenylated flavonoid-type compound showing antiinflammatory activity purified from Broussonetia papyrifera and extracts containing the said compound as a major component, a method for preparing them and pharmaceutical compositions containing them - Google Patents

Abstract

The present invention relates to a novel prenylated flavonoid compound and a coconut oil extract containing the same as a main component thereof, a process for producing the same, and a pharmaceutical composition containing the same. More particularly, the present invention relates to a method for producing a prenylated flavonoid compound of Broussonetia papyrifera, A novel plenylated flavonoid compound represented by the following formula (1), which is excellent in antiinflammatory effect, which is separated from muscle, by methanol and acetate extraction and chromatography, and a cocoa tree extract containing it as a main component; A salt thereof, a process for producing the compound and an extract thereof, and a pharmaceutical composition for treating an inflammatory disease comprising the same as an active ingredient. The novel plenylated flavonoid compound of the present invention and the extract of Cucurbitaceae containing it as a main component inhibits or inhibits the activity of enzymes involved in the production of inflammatory mediators and thus exerts excellent antiinflammatory effects in vitro and in vivo Inflammatory diseases such as acute inflammation, intraarticular diseases such as rheumatoid arthritis, skin diseases such as psoriasis, allergic inflammatory diseases such as bronchial asthma, and the like.

Description

TECHNICAL FIELD The present invention relates to a novel plenylated flavonoid compound exhibiting an anti-inflammatory activity isolated from a cedar tree, and a method for producing the same, and a pharmaceutical composition containing the same, wherein the novel prenylated flavonoid-type compound has antiinflammatory activity The present invention relates to a method for preparing a compound of formula (I)

The present invention relates to a novel prenylated flavonoid compound and a coconut oil extract containing the same as a main component thereof, a process for producing the same, and a pharmaceutical composition containing the same. More particularly, the present invention relates to a method for producing a prenylated flavonoid compound of Broussonetia papyrifera, A novel plenylated flavonoid compound represented by the above formula (1) which is excellent in anti-inflammatory effect, which is separated from muscle, by methanol and acetate extraction and chromatography, and a cocoa tree extract containing the same as a main component thereof; A salt thereof, a process for producing the compound and an extract thereof, and a pharmaceutical composition for treating an inflammatory disease comprising the same as an active ingredient.

Broussonetia spp. Is a deciduous broad-leaved shrub classified as mulberry (Broussonetia kazinoki Sieb.) And cucumber (Broussonetia papyrifera Vent.) And distributed in most parts of Korea, China, Taiwan and Japan. Grows at the foot of the mountain or in the den.

The bark fiber of cedar wood is used as a raw material of paper, and various ingredients extracted from cedar wood have been reported to have various effects. Specifically, the cedarwood contains ingredients that are effective for tannage, acne, acne, species, waxing, diuretic, etc., ingredients that can remove the wind, ingredients that have the effect of clearing blood, Have been reported.

Inflammation, on the other hand, refers to the pathological condition of an abscess formed by the invasion of external bacteria. Specifically, when external bacteria invade a specific tissue and proliferate, the leukocyte of the living body recognizes it and actively attacks the proliferated foreign germs. In this process, the dead cells of the leukocyte are accumulated in the invaded tissue At the same time, the cell debris of invading microorganisms killed by leukocytes melts into the invading tissues and abscess forms. The treatment of abscess due to inflammation can be promoted through the anti-inflammatory action. The anti-inflammatory action is to inhibit the proliferation of invading microorganisms by using an antibacterial agent or to activate macrophages that digest foreign substances accumulated in the abscess, It is an inflammation treatment promoting action such as exerting the function of excreted macrophages.

In general, an inflammatory reaction is a biological defense reaction process for repairing and regenerating damage caused by an invasion that causes a change in a biological substance in cells or tissues. In this reaction process, localized blood vessels, various tissue cells of body fluids and immune cells Lt; / RTI > As described above, normally, the inflammatory reaction induced by the foreign invading bacteria is a defense system for protecting the living body. However, when abnormally excessive inflammatory reaction is induced, various diseases appear. Such diseases are collectively referred to as inflammatory diseases. The inflammatory disease is a disease in which various inflammatory mediators secreted from target cells activated by external stimuli amplify and sustain inflammation and cause life-threatening diseases of the human body. These diseases include acute inflammation, diseases in joints such as rheumatoid arthritis, And allergic inflammatory diseases such as bronchial asthma and the like.

The key inflammatory mediators that induce inflammatory diseases are prostaglandins, hydroxy fatty acids, hydroxyeicosatetraenoic acid (hereinafter abbreviated as " HETE ") and leukotrienes leukotrienes, which are cyclooxygenase and lipoxygenase, which are known as eicosanoids, such as cyclooxygenase and lipoxygenase,

genase) from the precursor arachidonic acid.

Various antiinflammatory substances have been developed for the treatment of the above inflammatory diseases. The antiinflammatory substances reported to date are physicochemicals from acute inflammation to damaged tissue cells, leukocyte cells immobilized by inflammation-related cells or coinage factors (Moyazawa, K and Mikami, T., Japan J. Pharmacol. , 38, 199, 1985), which inhibit the biosynthesis of prostaglandins activated by physiological stimulation. However, these substances have a therapeutic effect on acute inflammation, but only chronic inflammation such as rheumatoid arthritis is mitigated only by primary inflammation phenomenon, and it is reported that an immunological therapeutic effect can not be expected.

On the other hand, active studies on HETE and leukotrienes produced from arachidonic acid by cyclooxygenase and lipoxygenase in addition to prostaglandins have shown that substances that inhibit the activity of the two enzymes are expected to have an effect on chronic inflammation and (Huskisson, ED et al., Inflammation, Mechanism and Treatment, 55, 1980) there is recently a cyclooxygenase isozyme (isozyme) in cyclooctane dioxygenase inhibitor of claim -2 found in the spotlight as an anti-inflammatory cycloalkyl There is a need to develop a novel anti-inflammatory agent having an inhibitory effect on oxigenase and lipoxygenase activity.

Accordingly, the present inventors have made efforts to develop an inhibitor of enzymes mediating the production of eicosanoids, which is a key substance that induces inflammation, to prepare an extract containing a novel phenylalanide compound as a main component , A novel plenililide compound was isolated and purified from the extract, and the structure was determined to obtain 5,7,3 ', 4'-tetrahydroxy-6,5'-diphenyl levovolol (5,7, 3 ', 4'-tetrahydroxy-6,5'-diprenylflavonol). The inventors of the present invention have shown that the novel plenililide compound and the extract of Cucurbitaceae containing the main component thereof exhibit an excellent anti-inflammatory effect as well as an anti-allergic effect in vitro and in vivo The present invention has been completed upon confirming that it can be effectively used as a therapeutic agent for inflammatory diseases.

It is an object of the present invention to provide a novel plenylated flavonoid compound exhibiting excellent anti-inflammatory effects as a therapeutic agent for an inflammatory disease and a pharmaceutically acceptable salt thereof.

It is also an object of the present invention to provide a preparation method for separating and purifying novel plinilated flavonoid compounds from corn.

In addition, the object of the present invention is to provide a coconut extract, a pharmaceutically acceptable salt thereof, and a process for producing the same, wherein the extract contains the compound as a main component.

Finally, it is an object of the present invention to provide a pharmaceutical composition for the treatment of an inflammatory disease containing as an active ingredient, a novel plenylated flavonoid compound and a cocoa tree extract containing the same as a main ingredient.

Figure 1 shows the correlation of ¹ H- ¹³ C observed in the HMBC spectrum,

2The The results obtained by Western blotting show the effect of the novel plenilyl flavonoid series of the present invention to inhibit the expression of cyclooxygenase-2,

1: Cells not inducing the expression of cyclooxygenase-2

2: Cells inducing expression of cyclooxygenase-2

3: induction of the expression of cyclooxygenase-2 and simultaneously adding 10 [mu] M of the plenylated flavonoid compound of the present invention

4: induction of the expression of cyclooxygenase-2 and simultaneously adding 100 μM of the pleuronylated flavonoid compound of the present invention

FIG. 3 shows the results of investigation of the in vivo antiallergic activity of the plenylated flavonoid compound of the present invention using PCA (passive cutaneous anaphylaxis) method.

Control group: Negative control without addition of test compound

Blouse-K1 (A): An experimental group administered with the plenylated flavonoid compound of the present invention at a dose of 20 mg / kg

Blouse-K1 (B): Experimental group in which the plenylated flavonoid compound of the present invention was administered at a dose of 40 mg / kg

In order to accomplish the above object, the present invention provides a novel plenylated flavonoid compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof.

Formula 1

Analysis a melting temperature with respect to the compound represented by the formula (1) and EIMS (Electron Impact Mass Spectrum) and then using the method of measuring the mass of a hydrogen nuclear magnetic resonance spectrum ⁽¹ H-NMR spectrum) and carbon nuclear magnetic resonance spectra ⁽¹³ C -NMR spectrum) was performed to confirm that the compound of the present invention was a plenylated flavonoid compound. Spectral data obtained by performing ¹ H- ¹ H COZY (Correlation spectroscopy), HMQC (Heteronuclear multi-quantum correlation) and HMBC (Heteronuclear multiple bond connectivity) NMR experiments on the plenylated flavonoid compound of formula Compared with the model compounds based on literature data, the compound of formula (I) is a compound of 5,7,3 ', 4'-tetrahydroxy (γ, γ-dimethylallyl group) -6,5 ' -difrenyl flavanol. ≪ / RTI >

Specifically, the results of the ¹ H- ¹ H COSY, HMQC and HMBC NMR with respect to the player carbonyl federated flavonoid compound of general formula (I) of the present invention, a cake of the 4-carbon in the HMBC spectrum combined earthenware (keto group) and hydrogen In addition to the correlation between the absorption spectrum (δ 12.72) of the hydroxyl group (δ 12.72) and the absorption spectrum of the 6th carbon (δ 110.1) The correlation of the absorption spectrum of the methylene hydrogen (CH ₂ -9, δ 3.22) of the dimethylaryl group was observed, and it was found that the prenyl group was bonded to the carbon 6 and the carbon 5 ' (See Fig. 1 ).

In view of the above-described spectral data results, the plenylated flavonoid compound of the present invention has a structural formula in which two γ, γ-dimethylaryl groups are attached to the 6-carbon and 5'-carbon, And was identified as a novel 5,7,3 ', 4'-tetrahydroxy-6,5'-diphenylflavone compound represented by the formula (1).

The plenylated flavonoid compound of the present invention represented by the formula (1) can be used in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful . The compounds of formula (I) may form acid addition salts which are pharmaceutically acceptable according to methods conventional in the art. Examples of the free acid include free acid and inorganic acid. Examples of the inorganic acid include hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid. Examples of the organic acid include citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid or aspartic acid Can be used.

In addition, the present invention provides a production method for separating and purifying a novel plasticized flavonoid compound from cedarwood.

The process for preparing the plenylated flavonoid compounds of the present invention comprises

1) adding methanol to the root of the cucumber tree, refluxing it and then distilling it under reduced pressure to obtain a methanol extract (step 1);

2) a step of dissolving the methanol extract in distilled water and then extracting the ethyl acetate layer with a solvent of ethylacetate to obtain an ethyl acetate layer (step 2);

3) distilling off the ethyl acetate layer to obtain an ethyl acetate extract (step 3); And

4) Chromatography of the ethyl acetate extract to obtain an anti-inflammatory active material (step 4).

In addition, the present invention provides an extract of Kjellmani japonica, which exhibits an anti-inflammatory effect, a pharmaceutically acceptable salt thereof and a process for producing the same.

The extract of the present invention contains a novel plasticized flavonoid compound as a main component and exhibits excellent anti-inflammatory effect both in vitro and in vivo.

The extract of the present invention can be used in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The compounds of formula (I) may form acid addition salts which are pharmaceutically acceptable according to methods conventional in the art. As the free acid, a free acid and an inorganic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid and the like can be used. Examples of the organic acid include citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, It is possible to use fluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid or aspartic acid.

In addition, the process for preparing the extract of the present invention

1) adding methanol to the root of the cucumber tree, refluxing it and then distilling it under reduced pressure to obtain a methanol extract (step 1);

2) dissolving the methanol extract in distilled water and then extracting with a solvent of ethylacetate to obtain an ethyl acetate layer (step 2); And

3) distilling the ethyl acetate layer under reduced pressure to obtain an ethyl acetate extract (step 3).

Finally, the present invention relates to a pharmaceutical composition for the treatment of inflammatory diseases containing, as an active ingredient, a novel plenylated flavonoid compound represented by the general formula (1) or a coconut extract and / or a pharmaceutically acceptable salt thereof as a main component thereof .

The plenylated flavonoid compound represented by Formula 1 of the present invention and the extract of Cucurbitaceae including the main ingredient thereof inhibit the activity of enzymes involved in the production of eosinoid mediating inflammation or inhibit the expression of some enzymes, Inflammatory effect.

The compound of formula (I) and the extract of Cucurbitaceae including the main ingredient thereof can be administered orally or parenterally at the time of clinical administration, such as intravenous, subcutaneous, intraperitoneal or skin application, .

The pharmaceutical compositions of the present invention may be formulated for oral administration, for example as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs (elixirs). Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation in tablets, capsules and the like; Excipients such as dicalcium phosphate; Disintegrating agents such as corn starch or sweet potato starch; Lubricating oil such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the pharmaceutical composition of the present invention can be administered parenterally, and parenteral administration is by skin application, subcutaneous injection, intravenous injection, intramuscular injection, or intramuscular injection. For formulation into parenteral administration formulations, the compound of formula (I) is mixed with a stabilizer or buffer in water to prepare a solution or suspension, which is then formulated into unit dosage forms of ampoules or vials.

The effective dose of the compound of the formula (1) is generally from 1 to 300 mg / kg, preferably from 10 to 100 mg / kg for an adult, and is determined by a doctor or pharmacist several times a day, It may be administered once to three times in divided doses. In addition, the above compounds were found to be very stable compounds with an MLD value of 300 mg / kg or more as a result of the acute toxicity test.

The effective dose of the extract of Cedarwood containing the compound of formula (1) as a main ingredient is generally 10 to 1000 mg / kg, preferably 100 to 300 mg / kg for an adult, Several times a day, preferably once a day or three times a day. In addition, the above compounds were found to be very stable compounds with an MLD value of 300 mg / kg or more as a result of the acute toxicity test.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

The present inventors carried out the following experiment to prepare an extract of Fusarium oxysporum (Fusarium oxysporum) containing as a main component a plenylate flavonoid compound exhibiting an anti-inflammatory effect from the root of Cucumber mildew.

Methanol was added to 1 kg of cocoa beans, refluxed for 5 hours, and then distilled under reduced pressure to obtain 140 g of a dried methanol extract. The extract was dissolved in distilled water, and extracted with ethyl acetate. The extract was vacuum distilled to obtain 30 g of ethyl Acetate extracts were prepared to produce extracts of Cedarwood extract containing a plonylate flavonoid compound as an active ingredient.

Example 2: Preparation of novel plenylated flavonoid compound showing anti-inflammatory effect

The present inventors carried out the following experiment to isolate and purify the plenilyl flavonoid compound showing anti-inflammatory effect from the root of Cucumber spp.

Methanol was added to 1 kg of cocoa beans and refluxed for 5 hours, followed by distillation under reduced pressure to obtain 140 g of a dried methanol extract. The extract was fractionated with distilled water and ethyl acetate and distilled under reduced pressure to obtain 30 g of ethyl acetate extract The fraction eluted with hexane-ethyl acetate (8: 5) in a fraction obtained by performing silica gel column chromatography using hexane-ethylacetate as a gradient elution solvent was used as a new To obtain a phenylated flavonoid compound (Rf: 0.16, hexane-ethyl acetate = 8: 5).

The melting point of the compound was analyzed and the mass was measured using the EIMS method. The hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum were carried out to confirm that the compound was a plasticized flavonoid compound. As a result of ¹ H- ¹ H COZY, HMQC and HMBC NMR spectroscopy on the phenylated flavonoid compound, it was found that the hydrogen bond with the keto group of the 4th carbon in the HMBC spectrum yielded a hydroxyl group (CH ₂ -9, 隆 3.22) of the γ, γ-dimethylaryl group, as well as the correlation between the absorption spectrum of the 6th carbon and the absorption spectrum of the 6th carbon (δ 110.1), as well as the correlation between the absorption spectrum (δ 12.72) ( Fig. 1 ). The results are shown in Fig. 1 and Fig .

In view of the above-described spectral data results, it has been found that a compound having a structural formula in which two γ and γ-dimethylaryl groups are attached to carbon number 6 and carbon number 5 ' The plenylated flavonoid compound of the present invention was identified as a novel 5,7,3 ', 4'-tetrahydroxy-6,5'-diphenyl flavanol.

mp 202-204 ^o C

EIMS, m / z 438 [M +] 395 (MC 3 H 7) +, 383 (MC 4 H 7) +, 365 (MC 4 H 7 -H 2 O) +, 327 (MC 4 H 7 -C 4 H ₈ ) ⁺

^{1 H-NMR (500 MHz,} DMSO-d 6) 1.62 (3H, s, CH 3 -17), 1,69 (6H, s, CH 3 -18, 13), 1.72 (3H, s, CH 3 - 12), 3.22 (2H, d , J = 7.0 Hz, CH 2 -9), 3.27 (2H, d, J = 7.2 Hz, H-14), 5.18 (1H, m, H-10), 5.28 (1H , m, H-15), 6.45 (1H, s, H-8), 7.44 (1H, d, J = 2.1 Hz, H-6 '), 7.59 (1H, d, J = 2.1 Hz, H-6 '), 8.88, 9.24, 9.63 (OH), 12.72 (5-OH); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) 144.5 (C-2), 135.6 (C-3), 175.7 C-6), 161.6 (C-7), 92.5 (C-8), 153.9 (C-8a), 121.2 (C-4 '), 128.0 (C-5'), 120.1 (C-6 '), 128.2 (C-9), 122.3 ), 17.7 (C-13), 21.0 (C-14), 122.7 (C-15), 131.5 (C-16), 25.5 (C-17), 25.5 (C-18).

<Experimental Example 1> Measurement of the inhibitory effect on the prostaglandin E2 production of the plenylated flavonoid compound of Example 2 and the extract of Kjellmani japonica extract of Example 1 containing it as a main component

The plastilized flavonoid compound obtained in Example 2 and proximal gladin E ₂ (hereinafter referred to as " PGE ₂ &quot;), which plays a key role in mediating inflammation, The abbreviated term &quot; abbreviated &quot;), the present inventors conducted the following experiments.

First, RAW 264.7 cells (ATCC) were cultured in a 96-well cell culture plate using a method of Kim et al., Biochem. Pharmacol. 58, 759-765, 2 x 10 &lt; ⁵ &gt; cells / well) were cultured and then lipopolysaccharide (Lipopolysa-

The amount of PGE ₂ produced in the medium was determined by the enzyme-linked immunoabsorbant assay (EIA) method after 24 hours of induction of the inflammatory reaction at a concentration of 1 μg / ml, Are shown in Table 1 below. At this time, the plenylated flavonoid compound of Example 2 and the cucumber extract of Example 1 containing it as a main component were treated simultaneously with LPS.

Table 1 Inhibitory Effect of Plenilized Flavonoid Compound (Compound of Example 2) on PGE ₂ Formation of the Extract of Curcuma bean (containing the compound of Example 1) as a main ingredient

compound density PGE ₂ production rate (%) Control group - 0 LPS 100 The extract of cedarwood of Example 1 10 [mu] g / ml 88 50 [mu] g / ml 70 100 [mu] g / ml 52 200 [mu] g / ml 42 The compound of Example 2 1 [mu] M 95 10 μM 81 50 μM 62 100 [mu] M 26

As shown in the above Table 1, the PGE ₂ production rates of the cells treated with the 50 μM and 100 μM concentrations of the plenylated flavonoid compound of Example 2 shown in the formula (1) were 62% and 26%, respectively, And the PGE ₂ production rate of the cells treated with the extracts of the extracts of Example 1, which contained the above-described pleinylated flavonoid compound as a main component, at a concentration of 100 μg / ml and 200 μg / ml, 52% and 42%, respectively, compared with cells induced by LPS. Therefore, the plenylated flavonoid compound of the present invention represented by the formula (I) and the extract of Cucurbitaceae containing the main component thereof inhibited the production of PGE ₂ in RAW 264.7 cells. In particular, the pleuronylated flavonoid compound of the present invention, isolated and purified from the extract of Fusarium oxysora, strongly inhibited the production of PGE ₂ .

From the above results, it can be seen that the plenylated flavonoid compound of the present invention and the extract of Corydalis koreanum extract containing it as a main ingredient strongly inhibit the production of PGE ₂ , which is a key mediator of inflammation in vitro, Respectively.

<Experimental Example 2> Measurement of the inhibitory effect on the enzyme activity of cyclohexenase-2 of the extract of the extract of Chenopodium species of Example 1 containing the plenylated flavonoid compound of Example 2 and its main component

The activity of the cyclooxygenase-2 involved in the production of eicosanoids, which is known as the major inflammatory mediator, in the plenylated flavonoid compound of the present invention obtained in Example 2, in order to to ensure that represents the effect that reduces the PGE ₂ inhibitory generated, the present inventors have performed an experiment as described below by using the enzyme stock solution from cyclooctane claim -2 dioxygenase extracted from the cell, the old cycle with the standard comparison substance NS-398, an oxigenase-2 inhibitor, was used.

First, aspirin was added to RAW cells at a concentration of 10 μg / ml and pretreated for 3 hours, followed by the addition of LPS at a concentration of 1 μg / ml to induce the expression of cyclooxygenase-2. After induction of expression for 24 hours, the medium was discarded and only the cells were collected and disrupted with a teflon homogenizer, and centrifuged at 12,000 rpm for 15 minutes to obtain supernatant, which was used as the enzyme solution of cyclooxygenase-2. The method such as You the enzyme stock solution (You, et al., Arch . Pharm. Res. 22, 18-24, 1999) of the ¹⁴ C-PGE is ¹⁴ [C- arachidonic acid and the precursor of Example 2 with ₂ The experimental group to which the plasticized flavonoid compound was added and the group containing the ^14C -arachidonic acid and the plasticylated flavonoid compound as the main components were added to the test group for 20 minutes, and then chloroform: methanol ^14C -PGE ₂ , which is a product of cyclooxygenase- _2, was extracted with a 1: 2 (2: 1) solution.

The extract was developed in Thin layer chromatography (hereinafter abbreviated as "TLC") and the amount of PGE ₂ produced was measured by quantifying ¹⁴ C as a labeling substance of PGE ₂ using autoradiography From this, the activity of cyclooxygenase-2 was determined. The inhibition rate of enzyme activity against cyclooxygenase-2 was calculated based on the inhibitory effect of enzyme activity on cyclooxygenase-2 of the control group to which the test compound was not added, and the results are shown in Table 2 below.

Table 2 Effect of Plenylated Flavonoid Compound (Example 2) and Cucumber tree extract containing it as a main ingredient (enzyme extract of Example 1) on inhibition of enzyme activity on cyclooxygenase-2

compound density PGE ₂ production rate (%) Control group - 100 NS-398 0.1 μM 104 1 [mu] M 95 10 μM 38 100 [mu] M 5 The extract of cedarwood of Example 1 10 [mu] g / ml 108 50 [mu] g / ml 97 100 [mu] g / ml 99 200 [mu] g / ml 101 The compound of Example 2 1 [mu] M 96 10 μM 99 50 μM 98 100 [mu] M 100

As shown in Table 2, when the enzyme solution inducing inflammation reaction with LPS was treated with 100 μM of NS-398, the amount of PGE ₂ was drastically decreased, whereas the amount of the pleuronylated flavonoid compound of Example 2 was 100 mu] M, the amount of PGE ₂ produced was not reduced. Even when the extract of Example 1 containing the above-described pleinylated flavonoid compound as the main component was treated to a concentration of 200 [mu] g / ml, PGE ₂ Was not reduced.

To the player carbonyl federated flavonoid compounds of the invention From the above results, directly inhibiting the enzyme activity of cyclooxygenase -2 seen that inhibit the production of PGE ₂ to the other indirect way as to reduce the production of PGE ₂ I could.

<Experimental Example 3> Measurement of the inhibitory effect on the expression of cyclohexenase-2 in the extract of the extracts of Example 1 containing the plenylated flavonoid compound of Example 2 and its main component

Whether or not the plenylated flavonoid compound of the present invention obtained in Example 2 and the extract of the cucumber extract of Example 1 containing the same as the main component inhibited the expression of cyclooxygenase-2 and showed the effect of reducing PGE ₂ production To do this, we performed western blotting as follows.

First, RAW cells were treated with LPS at a concentration of 1 μg / ml, and the expression of cyclooxygenase-2 was induced for 24 hours. Then, the cells were collected and disrupted using a Teflon crusher and centrifuged at 12,000 rpm for 15 minutes to obtain a supernatant Afterwards, polyacrylamide gel electrophoresis (PAGE) was performed to separate the proteins according to their sizes. The separated proteins were blotted on a polyvinylidene fluoride membrane and treated with an anti-cyclooxygenase-2 antibody (Cayman Co.) to remove the expressed cyclooxygenase-2 The amount was measured.

As a result, the amount of the cyclooxygenase-2 expression was measured at a concentration of 100 μM and 200 μg / ml, respectively, in the plenylated flavonoid compound of the present invention obtained in Example 2 and the extract of Example 1 containing the same as the main component, . However, only the results using the plenylated flavonoid compound of Example 2 were shown in this specification ( Fig. 2 ). The results indicate that kkuji tree extract containing the player carbonyl federated flavonoid compound, and this main component of the present invention cyclooctane reduces the PGE ₂ production by inhibiting the expression instead of directly inhibiting the enzymatic activity of the dioxygenase -2 Respectively.

EXPERIMENTAL EXAMPLE 4 A mixture of the pleuronylated flavonoid compound of Example 2 and the cyclooxygenase-1, 5-lipoxygenase and 12-lipoxygenase of the extract of Example 1, Measurement of inhibitory effect on enzyme activity

The plenylated flavonoid compound of the present invention obtained in Example 2 and the extract of the extract of the present invention containing the extract of Example 1 as thromboxane B ₂ which acts as a key substance mediating inflammation and cyclohexanone Lipoxygenase and 12-lipoxygenase, the present inventors carried out the following in vitro experiments as follows.

The present inventors used the disruption of platelets isolated from human blood with a stock solution of cyclooxygenase-1, and the enzyme activity of cyclooxygenase-1 was measured using the method of Experimental Example 2 . At this time, the amount of thromboxane B ₂ boksan was measured by quantification of the ¹⁴ C- ¹⁴ C labeling of thromboxane B ₂ boksan material.

In addition, the present inventors quantitatively determined the amount of ¹⁴ C-12-HETE produced in the same manner as in Experimental Example 2 by using platelet disruption liquid isolated from the blood of a ruminant using an enzyme solution of 12-lipoxygenase, Enzyme activity was measured and the amount of ¹⁴ C-5-HETE produced was determined by the same method as Experimental Example 2 using a disruption solution of neutrophil isolated from the blood of a female with an enzyme solution of 5-lipoxygenase to obtain 5-lipoxygenase And the results are shown in Tables 3 and 4 below.

<Example 3> Inhibitory effect of 5-lipoxygenase on the activity of the phenylated flavonoid compound (compound of Example 2) and the extract of Coconut tree (containing the extract of Cedarwood of Example 1)

compound density 5-HETE Production Rate (%) Control group - 100 NDGA 1 [mu] M 33 10 μM 22 100 [mu] M 22 The extract of cedarwood of Example 1 10 [mu] g / ml 92 50 [mu] g / ml 80 100 [mu] g / ml 61 200 [mu] g / ml 49 The compound of Example 2 1 [mu] M 83 10 μM 46 50 μM 25 100 [mu] M 18

Table 4 Effect of the Plenylated Flavonoid Compound (Compound of Example 2) and the extract of Cucurbitaceae containing the same as the main ingredient (enzyme extract of Example 1) on inhibition of enzyme activity on 12-lipoxygenase

compound density 12-HETE Production Rate (%) Control group - 100 NDGA 1 [mu] M 62 10 μM 26 100 [mu] M 18 The extract of cedarwood of Example 1 10 [mu] g / ml 103 50 [mu] g / ml 90 100 [mu] g / ml 72 200 [mu] g / ml 57 The compound of Example 2 1 [mu] M 92 10 μM 73 50 μM 71 100 [mu] M 29

As shown in the results of Tables 3 and 4, the enzyme activity inhibitory concentration (IC) for 5-lipoxygenase of the compound of Example 2 and the extract of the extract of the present invention,₅₀, Concentration for 50% active inhibition) were measured as 7 [mu] M and 193 [mu] g / ml, respectively The inhibitory concentration of the enzyme activity against 12-lipoxygenase was measured to be 69 μM or more and 200 μg / ml or more, respectively. Thus, the plenylated flavonoid compound of the present invention and the extract of Cucurbitaceae containing it as a main component were analyzed by 5-lipoxygenase It was confirmed that the enzyme activity of 12-lipoxygenase was strongly inhibited. However, the inhibitory effect on cyclooxygenase-1 was not shown.

From the above results, it can be seen that the plenylated flavonoid compound of the present invention and the extract of Cucurbitaceae containing it as a main component do not inhibit the enzyme activity of cyclooxygenase-1, and the enzyme of 5-lipoxygenase and 12-lipoxygenase Inhibited the production of eicosanoids, which is a key inflammatory mediator.

<Experimental Example 5> Measurement of antiinflammatory effect in vivo of the pleuronylated flavonoid compound of Example 2 and the extract of Curcuma longa extract of Example 1 containing it as a main component

In order to confirm that the plenylated flavonoid compound of the present invention obtained in Example 2 and the extract of Curcuma japonica extract of Example 1 containing it as a main component exhibit antiinflammatory effects in vivo, The same experiment was carried out.

SD male mice were subjected to the carageenan-induced paw edema method of Winter et al. (Winter et al., Proc. Soc. Exp Biol Med 111, 544-547, 1962) After the oral administration of the plenylated flavonoid compound of Example 2 and the extract of Curcuma japonica extract of Example 1 containing it as a main component, the anti-inflammatory effect in vivo was measured and the results are shown in Table 5 below.

Antiinflammatory effect of the plenylated flavonoid compound (compound of Example 2) in vivo and the extract of cedarwood (the extract of cedarwood of Example 1) containing the compound as its main ingredient

compound amount Inhibition rate (%) Indomethacin 1 mg / kg 52 The extract of cedarwood of Example 1 50 mg / kg 18 100 mg / kg 32 200 mg / kg 43 The compound of Example 2 10 mg / kg 38 50 mg / kg 54 100 mg / kg 69

As shown in Table 5, when the flavonoid compound of Example 2 was orally administered at a dose of 10 mg / kg, oral administration at a dose of 38% and 50 mg / kg resulted in 54% and 100 mg / The inhibition rate of inflammation was 69%. In the case of oral administration of the extract of the extract of Example 1 at a dose of 50 mg / kg, oral administration at a dose of 18%, 100 mg / kg, orally administered at a dose of 32% and 200 mg / kg, 43% of the inhibition rate of inflammation. Thus, it was confirmed that the plenylated flavonoid compound of the present invention prepared in Example 2, and the extract of Koojiba japonica of Example 1 containing the same as the main component, exhibited excellent antiinflammatory effects not only ex vivo but also in vivo.

Experimental Example 6 Measurement of anti-allergic activity in vivo of the pleuronylated flavonoid compound of Example 2 and the extract of Coconut Tree of Example 1 containing it as a main component

In order to confirm that the plenylated flavonoid compound of the present invention obtained in Example 2 and the extract of Curcuma japonica extract of Example 1 containing it as an active ingredient exhibit anti-allergic activity in vivo, the inventors of the present invention found that the passive skin anaphylactic reaction (Passive cutaneous anaphylaxis test).

DNP-BSA was used as an antigen, and IgE monoclonal antibody purchased from Miles Co. was used as an antibody. SD rats weighing 250-300 g were first removed with hair clippers and 100 μl of the antibody DNP-BSA was injected intradermally. After 48 hours from the administration, 20 and 40 mg / kg of the plenylated flavonoid compound of the present invention obtained in Example 2 and the extracts of Example 1, which contained 50 and 100 mg / Lt; / RTI &gt; 30 minutes after the administration, 200 μl of 0.5% Evans blue solution containing the antigen was injected into the tail vein. After 30 minutes, 1 ml of 1 N potassium hydroxide solution (KOH) was added to the pigment released to the skin Lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; The skin pigment was added with 0.6 N phosphate acetone solution, and the absorbance was measured at 620 nm after shaking.

As a result, when the plenylated flavonoid compound of the present invention obtained in Example 2 was administered at a dose of 20 mg / kg, it showed about 40% in vivo antiallergic activity as compared with the control, and a dose of 40 mg / kg Showed about 72% in vivo anti-allergic activity compared to the control group ( Fig. 6 ). In addition, when the extract of the present invention was administered at a dose of 50 mg / kg, the extract of Kjellmani japonica extract of Example 1 containing the plenylated flavonoid compound showed about 28% in vivo antiallergic activity as compared with the control, / Kg, the anti - allergic activity in vivo was about 52% as compared with the control group. Therefore, it was confirmed that the plenylated flavonoid compound of the present invention and the extract of Coconut tree containing the main component thereof exhibit anti-allergic activity in vivo.

<Experimental Example 7> Oral administration of acute toxicity test to mouse and rat

The inventors of the present invention conducted the following toxicity tests in order to confirm whether or not the plenylated flavonoid compound of the present invention and the extract of Cucurbitaceae containing the main component thereof exhibit toxicity in vivo.

In accordance with the announcement of the Food and Drug Administration, the plenylated flavonoid compound of the present invention and the extract of Cucurbitaceae containing the main component thereof were administered to ICR mice and SD rats in male and female at a dose of 0.1 to 300 mg / kg. After 2 weeks from the administration of the test substance, animal death, clinical symptoms and weight change were observed, hematologic test and blood biochemical test were performed, and autopsy was performed to observe the abnormality of abdominal organs and thoracic organs. As a result of the test, there were no clinically symptomatic or dead animals in all the animals to which the test substance was administered, and no toxic change was observed in weight change, blood test, blood biochemical test, and autopsy findings. As a result, the tested compounds showed no toxic effects up to 300 ㎎ / kg in experimental animals, and the minimum LDL (oral dose) of LD ₅₀ was more than 300 ㎎ / kg.

As described above, the plenylated flavonoid compound of the present invention and the extract of Cucurbitaceae containing the main component thereof exhibit excellent anti-inflammatory effects and can be formulated into a pharmaceutical composition for the treatment of inflammatory diseases.

&Lt; Formulation Example 1 >

A syrup containing the plenylated flavonoid compound of the present invention or a cocoa tree extract containing it as a main component and a pharmaceutically acceptable salt thereof in an amount of 2% (weight / volume) of the active ingredient is prepared by the following method.

The acid addition salt, saccharin and saccharide of the plenylated flavonoid compound of the present invention or the extract of Cucurbitaceae containing it as a main component were dissolved in 80 g of hot water. After the solution was cooled, a solution composed of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed. Water was added to the mixture to make 100 ml. The addition salt may be replaced with another salt according to the embodiment.

The components of the syrup are as follows.

Plenilized flavonoid compound or its main ingredient Coconut extract, hydrochloric acid ······················· 2 g

Saccharin ························· 0.8 g

Per day ····························· 25.4 g

Glycerin · · · · · · · · 8.0 g

Spices · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Ethanol ······················· 4.0 g

Sorbic acid ······························· 0.4 g

Distilled water · · · · · · · · · · ·

&Lt; Formulation Example 2 >

Tablets containing 15 mg of active ingredient are prepared as follows.

250 g of the plasticized flavonoid compound of the present invention or the extract of Curcuma bean extract containing it as a main component was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

The components of the tablet are as follows.

Plenilized flavonoid compounds or extracts of Cucurbitaceae containing hydrochloric acid as its main component · Hydrochloric acid ················· 250 g

Lactose · · · · · · · · · · · · · 175.9 g

Potato starter ····························· 180 g

Colloidal silicic acid · · · · · · · · · · · · · · · 32 g

10% gelatin solution

Potato starter · · · · · · · · · · · · 160 g

Talc ··········································· 50 g

Magnesium stearate ························· 5 g

&Lt; Formulation Example 3 >

An injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of hydrochloric acid salt, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml of the plasticized flavonoid compound of the present invention or a main component thereof. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

The components of the injection solution are as follows.

Plenilized flavonoid compound or its main ingredient Cranberry extract Extract · Hydrochloric acid ············· 1 g

Sodium chloride ····················· 0.6 g

Ascorbic acid ·················· 0.1 g

Distilled water ·························································

As described above, the plenylated flavonoid compound of the present invention represented by the formula (I) and the extract of Cucurbitaceae containing it as a main component directly inhibits the enzyme activity of 5-lipoxygenase and 12-lipoxygenase Inhibits the expression of cyclooxygenase-2 and inhibits the production of eicosanoids, an inflammatory mediator, thereby exhibiting an excellent anti-inflammatory effect in vitro and in vivo. In addition, it exhibits no toxicity, and thus can prevent acute inflammation, joints such as rheumatoid arthritis Inflammatory diseases such as asthma, bronchial asthma, and the like, and the like. In addition, the plenylated flavonoid compound of the present invention and the extract of Cucurbitaceae containing it as a main component exhibit excellent anti-allergic activity in vivo and can be usefully used as an antiallergic agent.

Claims (7)

A novel novel prenylated flavonoid compound represented by the following formula (1). Formula 1 1) adding methanol to the root of the cucumber tree, refluxing it and then distilling it under reduced pressure to obtain a methanol extract (step 1); 2) a step of dissolving the methanol extract in distilled water and then extracting the ethyl acetate layer with a solvent of ethylacetate to obtain an ethyl acetate layer (step 2); 3) distilling off the ethyl acetate layer to obtain an ethyl acetate extract (step 3); And 4) obtaining an anti-inflammatory active substance by performing chromatography on the ethyl acetate extract (step 4). 1) adding methanol to the root of the cucumber tree, refluxing it and then distilling it under reduced pressure to obtain a methanol extract (step 1); 2) dissolving the methanol extract in distilled water and then extracting with a solvent of ethylacetate to obtain an ethyl acetate layer (step 2); And 3) a step of extracting the ethyl acetate layer by vacuum distillation to obtain an ethyl acetate extract (step 3). A pharmaceutical composition for the treatment of inflammatory diseases containing the novel plenylated flavonoid compound of claim 1 and / or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for an anti-allergic agent containing the novel plenylated flavonoid compound of claim 1 and / or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for the treatment of an inflammatory disease, comprising as an active ingredient an extract of Curcuma japonica as claimed in claim 2 and / or a pharmaceutically acceptable salt thereof, which contains the novel plenylated flavonoid compound of claim 3 as a main ingredient. A pharmaceutical composition for use as an anti-allergic agent containing, as an active ingredient, the extract of Curcuma japonica as claimed in claim 2 and / or a pharmaceutically acceptable salt thereof, which contains the novel plenylated flavonoid compound of claim 3 as an active ingredient.