KR100416482B1 - Anti-inflammatory extract of Ligularia fischeri Turcz. and their fractions - Google Patents

Abstract

The present invention relates to a bear odor extract and its fractions, which have an anti-inflammatory action, and more specifically, a bear odor extract using a water-soluble alcohol from Ligularia fischeri Turcz. Which is an edible plant, and ethyl acetate from the odor extract. The present invention relates to a fraction and an anti-inflammatory pharmaceutical composition comprising the same as an active ingredient, wherein the extracts of these odors and their fractions inhibit the activity and expression of cyclooxygenase-2, which is associated with the expression of inflammation, and thus the inflammatory eicosanoids ( As it exhibits anti-inflammatory action by preventing the production of eicosanoids, it can be usefully used as an anti-inflammatory agent for chronic inflammatory diseases such as acute inflammation and rheumatoid arthritis.

Description

Anti-inflammatory extract and its fractions having anti-inflammatory action {Anti-inflammatory extract of Ligularia fischeri Turcz. and their fractions}

The present invention relates to a bear odor extract and its fractions, which have an anti-inflammatory action, and more specifically, a bear odor extract using a water-soluble alcohol from Ligularia fischeri Turcz. Which is an edible plant, and ethyl acetate from the odor extract. The present invention relates to a fraction and an anti-inflammatory pharmaceutical composition comprising the same as an active ingredient, wherein the extracts of these odors and their fractions inhibit the activity and expression of cyclooxygenase-2, which is associated with the expression of inflammation, and thus the inflammatory eicosanoids ( As it exhibits anti-inflammatory action by preventing the production of eicosanoids, it can be usefully used as an anti-inflammatory agent for chronic inflammatory diseases such as acute inflammation and rheumatoid arthritis.

Ligularia fischeri Turcz. Is a dicotyledonous plant that is a perennial plant of the Asteraceae family, grows in wetlands of highlands or deep mountains, and is distributed in Korea, Japan, China, Sakhalin Island and East Siberia. In oriental medicine, the roots are cut and dried in the autumn, called Horochil, which is used as a therapeutic agent for Jinhae, expectorant, analgesic, and bleeding. However, the mechanism of anti-inflammatory action and anti-inflammatory activity are not known.

Inflammation refers to a pathological condition of the abscess formed by the invasion of external bacteria. Specifically, when the external bacteria invade specific tissues and proliferate, the white blood cells of the living body recognize the same and actively attack the multiplying external bacteria. The dead sea of white blood cells generated during this process accumulates in the tissues invaded by the bacteria. At the same time, cell destruction of invading bacteria killed by leukocytes is fused into the invaded tissue to form an abscess. Inflammation of the abscess may be promoted through anti-inflammatory action. The anti-inflammatory action is an antimicrobial agent that inhibits the proliferation of invading bacteria or activates macrophage that feeds on the foreign matter accumulated in the abscess. It promotes inflammation by increasing the function of excreted macrophages.

In general, an inflammatory reaction is a biological defense reaction process for repairing and repairing damage caused by an invasion that causes some organic changes in cells or tissues of the body, and this reaction process includes local blood vessels, various tissue cells of body fluids and immune cells. This works. Normally, the inflammatory response induced by external invading bacteria is a defense system to protect the living body, while abnormally excessive inflammatory response is induced, and various diseases appear. These diseases are collectively called inflammatory diseases. The inflammatory disease is a disease that threatens human life by amplifying and sustaining inflammation of various inflammatory mediators secreted from target cells activated by external stimuli, such as diseases in the joints such as acute inflammation and rheumatoid arthritis, psoriasis, etc. Allergic inflammatory diseases such as skin diseases and bronchial asthma, and the like.

Key inflammatory mediators that induce inflammatory diseases are eicosanoids such as prostaglandins, hydroxy fatty acids, hydroxyeicosatetraenoic acid and leukotrienes. ) And are produced from arachidonic acid, a precursor, by cyclooxygenase and lipoxygenase.

In order to treat the inflammatory diseases, various anti-inflammatory substances have been developed. The anti-inflammatory substances reported to date are physicochemical from the cell membranes of leukocytes injured by tissue cells damaged by acute inflammation, cells involved in inflammation, or chemotactic factors. And drugs that inhibit the biosynthesis of prostaglandins that are activated by physiological stimulation. However, these substances have a therapeutic effect on acute inflammation but only alleviate the primary inflammation for chronic inflammation such as rheumatoid arthritis, and immunological therapeutic effects are not expected [Moyazawa, K and Mikami, T. , Japan J. Pharmacol ., 38 , 199, 1985 ].

Recent reports, on the other hand, refer to cyclooxygenase-2 (hereinafter referred to as "COX-2"), an isoform of cyclooxygenase (hereinafter referred to as "COX") to these diseases. ) Is known to be involved. The inventors of the present invention found that the bear extract shows a strong inhibitory effect while studying a plant extract that inhibits COX-2.

The present inventors have found that the water-soluble alcohol extract and ethyl acetate fraction of bear odor are prostaglandin E ₂ (prostaglandin E ₂ , PGE ₂ ) in a RAW 264.7 cell line, which is a mouse macrophage cell line induced with lipopolysaccharide (LPS). The present invention was completed by finding that they strongly inhibit the production of c) and have a mechanism of inhibiting the activity of COX-2 and suppressing expression.

It is an object of the present invention to provide a bear odor extract and its fractions which exhibit an anti-inflammatory effect.

The present invention also provides an anti-inflammatory pharmaceutical composition containing the bear extract and its fractions as an active ingredient.

Figure 1 shows the results of measurement of the effect of inhibiting the expression of cyclooxygenase-2 protein bear extract of the present invention and fractions thereof by Western blot method.

1: Cell showing a negative control (control (-))

2: Cell showing a positive control (control (+))

3: cells added with NS-398 10 uM

4: Cell added 1 μg / ml of ethyl acetate fraction

5: Cells to which 10 µg / ml of ethyl acetate fraction was added

6: cells added with 100 g / ml of ethyl acetate fraction

7: Cell added 5 ㎍ / ㎖ of odor extract extracted with methanol

8: Cells added with 25 ㎍ / ml of bear extract extracted with methanol

In order to achieve the above object, an object of the present invention provides an extract and bear fractions that bears an anti-inflammatory effect.

In addition, the present invention provides an anti-inflammatory pharmaceutical composition containing the bear extract and its fractions as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present invention includes a bear extract and fractions thereof prepared by the following method.

Bear extract of the present invention is a single or two from the group consisting of water, C _{1 ~} C ₄ lower alcohol, glycerol, propylene glycol, 1,3-butylene glycol, ethyl acetate, benzene, hexane, diethyl ether and dichloromethane It is prepared by mixing and immersing more than one mixed solvent and then extracting and filtering and concentrating the extraction stock solution. Preferably, the bear extract is prepared using methanol or a mixed solvent of methanol and water.

The fraction of the present invention is prepared by suspending the extract of Goji odor with distilled water, extracting with ethyl acetate, and then filtration and concentration.

In addition, the present invention includes the pharmaceutical composition for anti-inflammatory containing the bear extract and fractions thereof as an active ingredient.

In order to investigate the efficacy of the above-mentioned bear extract and its fractions as anti-inflammatory agents, that is, to investigate the effect on COX-2, the bear extract of the present invention and its fractions on cells stimulated with lipopolysaccharide (LPS) After the addition of each concentration, the production amount of prostaglandin E ₂ , which is an inflammation-inducing substance, was measured by ELISA. As a result, bear extract and its fractions significantly inhibited the production of prostaglandin E ₂ . In addition, the results of treatment of the bear odor extract and fractions thereof with COX-2 preexpressed cells were also found to inhibit the production of prostaglandin E ₂ . Through this result, it can be seen that the bear extract of the present invention and its fraction directly inhibits COX-2.

In addition, after the addition of the substance of the present invention to the cells stimulated with lipopolysaccharide, the amount of COX-2 expressed by Western blotting was assayed, and the extracts and fractions of the present invention inhibit COX-2 expression. And it was found. Therefore, these substances not only directly inhibited COX-2, but also suppressed the expression of prostaglandin production.

Bear extract and fractions thereof may be applied orally or parenterally during clinical administration, for example topical application such as intravenous, subcutaneous, intraperitoneal or dermal application and may be used in the form of general pharmaceutical formulations.

The pharmaceutical compositions of the present invention may be used in oral formulations such as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs ( formulated into elixirs). Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

In addition, the pharmaceutical composition of the present invention may be administered parenterally, and parenteral administration may be by skin application, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection. In order to formulate into a parenteral formulation, bear extract and its extracts of the present invention are mixed in water with stabilizers or buffers to prepare solutions or suspensions, which are formulated in unit dosage forms of ampoules or vials.

The effective dose of bear odor extract and its fractions is generally 1 to 800 mg / kg in adults, preferably 50 to 500 mg / kg, several times a day at preferred time intervals, preferably at the discretion of the physician or pharmacist. It may be administered in divided doses once to three times a day. In addition, the bear extract of the present invention and its fractions were found to be very stable as a result of acute toxicity test LD ₅₀ value of 2 g / kg or more.

Hereinafter, the present invention will be described with reference to Examples.

However, the present invention is not limited by the examples.

Example 1 Bear Extract and Fraction Extraction

(1) Bear Extract

Methanol was added to 180 g of bear odor, extracted once in a cold dark, and concentrated to prepare about 30 g of bear odor extract. 1 g of this was taken as an active detection sample.

(2) fraction extraction

The extract obtained from the above was taken in a fraction filter, and distilled water was added thereto, followed by fractionation twice with the same amount of hexane. The aqueous layer was shaken again with ethyl acetate or butanol and left to prepare about 6 g of each fraction. About 1 g of this was taken and used as an active screening sample.

Experimental Example 1 Prostaglandin E ₂ (PGE ₂ ) Inhibitory effect

The following experiment was performed to know the effect on the activity of COX-2 in the extracts and fractions prepared in Example 1.

To the amount as is water arachidonic acid metabolism ELISA the amount of prostaglandin E ₂ method (Cayman Co.) which is generated by the RAW 264.7 cells in a culture by methods such as Chi and the addition of lipopolysaccharide, and then COX-2 after 24 hours Table 1 is shown. In addition, in order to measure the direct COX-2 inhibitory ability, after washing for 24 hours after lipopolysaccharide treatment, it was measured 30 minutes after addition of the achidonic acid and the extract and fraction prepared in Example 1. The results are shown in Table 1 by three repeated experiments [ Biochem. Pharmacol. 61 , 1195-1203 ( 2001 ).

As can be seen in Table 1 , it can be seen that the bear extract and ethyl acetate fraction of the present invention inhibits the production of prostaglandin E ₂ at a concentration of 1 to 25 μg / ml without cytotoxicity. In particular, it was found that COX-2 was also directly inhibited as shown in the results of the 30-minute experiment.

Inhibitory Effect of Prostaglandin E ₂ (PGE ₂ ) Production in RAW 264.7 Cells sample Dose (μg / ml) Inhibition (%) 24 hours Inhibition ability (%) 30 minutes Negative Control - - - Positive control group (+ LPS) - - - Bear smell methanol extract 5 24 ^* - 25 78 ^* 62 ^* Hexane fraction One - - 10 - - Ethyl acetate fraction One - - 10 56 ^* 40 ^* Butanol fraction One - - 10 - -

*: P <0.005, Student-t test (n = 3)

Experimental Example 2 Effect of Inhibiting Cyclooxygenase-2 (COX-2) Expression

The following experiment was conducted to know the effect on the inhibition of expression of COX-2 in the extracts and fractions prepared in Example 1.

RAW 264.7 cells were treated with 1 [mu] g / ml lipopolysaccharide and at the same time the methanol extract of Goji and its ethyl acetate fraction were treated at respective concentrations and incubated at 37 ° C., 5% CO ₂ for 24 hours. Total cell lysates were prepared from the cultured cells, and the amount of expressed COX-2 was analyzed by Western blot.

As shown in FIG. 1 , COX-2 protein induced and produced by lipopolysaccharide was detected, and it was found that the treated substances significantly inhibited the expression of COX-2.

As a result, the extract of bear odor and its ethyl acetate fraction not only inhibit the expression of COX-2, an enzyme involved in inflammatory expression, but also directly inhibit the activity to prevent the production of inflammatory eicosanoids. Inflammatory action was confirmed.

Experimental Example 3 Inhibitory Effect of Inflammation in Animal Model 1

An experiment was conducted to determine the in vivo anti-inflammatory activity of the bear odor extract of Example 1 and its ethyl acetate fraction.

Six rats per experimental group were tested by rat carageenan-induced paw edema for rats using Sprague-Dawley rats [Winter et al., Proc. Soc. Exp. Biol. Med. 111 , 544 ( 1962 ).

As shown in Table 2 below, the substances of the present invention showed significant anti-inflammatory activity in vivo. From this, the possibility as an anti-inflammatory agent was confirmed.

Carrageenan-induced plantar edema suppression effect sample Dose (mg / kg) % Inhibition Indomethacin One 42 ^* 10 78 ^* Bear smell methanol extract 10 5 100 22 ^* 250 29 ^* 500 38 ^* Ethyl acetate fraction 10 8 ^* 100 28 ^* 250 33 ^* 500 42 ^*

*: P <0.05, Student-t test (n = 5)

Experimental Example 4 Inhibition of Inflammation in Animal Model 2

An experiment was conducted to determine the in vivo anti-inflammatory activity of the bear odor extract of Example 1 and its ethyl acetate fraction.

Six rats per experimental group were tested by rat adjuvant-induced arthritis method using Splague-Dawley rats. Inhibition of inflammation is shown in Table 3 below in comparison with arthritic inflammation edema of the untreated foot on day 16 after adjuvant injection [Kim et al. , Planta Med. 65 , 465 ( 1999 ).

As can be seen in Table 3 , the substances of the present invention showed a significant anti-inflammatory action in the chronic inflammation model in vivo.

Arthritis Inhibitory Effect on Rats sample Dose (mg / kg / day) % Inhibition ^a Indomethacin One 78 ^* Bear smell methanol extract 100 27 500 36 ^* Ethyl acetate fraction 100 33 ^* 500 44 ^*

*: P <0.05, Student-t test (n = 6)

Experimental Example 5 In vivo Acute Toxicity Test

Experiments were conducted to determine the toxicity of the bear extract of the present invention and its ethyl acetate fraction.

According to the notification of the Korea Food and Drug Administration, the odor extract and ethyl acetate fraction of the present invention were administered orally in five-fold dose increments from 0.1 mg / kg to 2 g / kg in male and female ICR mice and SD rats. The mortality, clinical symptoms, and body weight were examined for 2 weeks after the administration of the test substance. Hematological and hematological examinations were performed.

As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. The results determined that the bear extract of the present invention and its ethyl acetate fraction were nontoxic at normal doses because they all showed no acute toxicity up to 2 g / kg.

In the clinic, they are effective at 50 to 500 mg / kg, and can be used not only orally, but also as injections, skin ointments, and the like.

Preparation Example 1 Preparation of Tablet

Inflammatory agent containing the bear extract of the present invention and the ethyl acetate fraction thereof as an active ingredient can be administered parenterally and orally, and prepared as a tablet in an oral dosage form below.

A tablet containing 100 mg of active ingredient is prepared by the following method.

250 g of the extract of Example 1 were mixed with 175.9 g lactose, 180 g potato starch and 32 g colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. After drying, the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

Extract of Example 1 250 g

Lactose ········ 175.9 g

Potato starch ·············· 340 g

Colloidal silicic acid 32 g

10% gelatin solution

Talc · 50 g

Magnesium stearate 5 g

As described above, bear odor extract of the present invention, its fractions and anti-inflammatory pharmaceutical composition comprising them as an active ingredient not only inhibits the expression of COX-2, but also directly inhibits the activity of the inflammation-inducing eicosanoids (eicosanoid) Inhibition of the production of prostaglandin E ₂ has an anti-inflammatory effect, and also has a significant anti-inflammatory effect in vivo can be used significantly in chronic inflammatory diseases such as acute inflammation and rheumatoid arthritis.

Claims (7)

Mix a single or two or more mixed solvents from the group consisting of water, C _{1 to} C ₄ lower alcohols, glycerol, propylene glycol, 1,3-butylene glycol, ethyl acetate, benzene, hexane, diethyl ether and dichloromethane The anti-inflammatory bear odor extract obtained by immersing and extracting and then extracting and filtering the extract. The anti-inflammatory bear extract of claim 1, wherein the extraction solvent is methanol or a mixed solvent of methanol and water. The anti-inflammatory bear extract of claim 1, wherein the anti-inflammatory bear extract inhibits cyclooxygenase-2. The anti-inflammatory bear extract of claim 1, wherein the anti-inflammatory bear extract inhibits the production of eicosanoids. Anti-inflammatory ethyl acetate fraction obtained by suspending the bear extract of claim 1, suspended with distilled water and then extracted with ethyl acetate. An anti-inflammatory pharmaceutical composition comprising the bear extract of claim 1 as an active ingredient. An anti-inflammatory pharmaceutical composition comprising the ethyl acetate fraction of claim 5 as an active ingredient.