WO2010036065A2 - Pharmaceutical composition for preventing and treating inflammatory diseases, containing an ethyl acetate fraction of dried extract of trachelospermi caulis as an active ingredient, and method for producing the fraction - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment and prevention of inflammatory diseases, containing an ethyl acetate fraction of dried extract of trachelospermi caulis as an active ingredient, and to a method for producing same. More particularly, the present invention relates to a composition for the prevention and treatment of inflammatory diseases, containing an ethyl acetate fraction (partition) of dried extract of trachelospermi caulis as an active ingredient, wherein the extract of trachelospermi caulis is refined and concentrated to contain 0.05 to 12 wt % of arctigenin as an index material.

Description

Pharmaceutical composition for the prevention and treatment of inflammatory diseases containing ethyl acetate fraction of dry extract such as rockfall, etc.

The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing an ethyl acetate fraction of a dry extract of rockfall, etc. as an active ingredient and a method for producing the fraction.

In general, arthritis is a medical name collectively referred to as an inflammatory change in a joint caused by some cause such as bacteria or trauma. Arthritis is divided into acute and chronic depending on the duration of inflammation.

At this time, acute arthritis is classified as follows (Doosan Encyclopedia).

Serous arthritis is usually caused by trauma, with some unknown causes, usually occurring in only one joint.

Serous fibrotic arthritis occurs during acute joint rheumatism, which is characterized by turbid effusions in the joint cavity, and in general, fibrous gastric membranes that cause severe movement disorders even when inflammation subsides. .

Purulent arthritis is common in open windows of joints or infectious diseases such as gonorrhea, typhoid fever, sun red fever, and sepsis. Infants who are 1 to 2 months old are severely injured and cannot be treated. Caused by periosteal osteomyelitis in adults, the pus burst into the joints and often go into the joints, this is called secondary arthritis.

Chronic arthritis is classified as follows:

Specific inflammation refers to gouty arthritis caused by tuberculous syphilis toxicity or metabolic disorders of many uric acid in middle-aged men. Multiple arthritis is caused by chronic arthritis. (移行) or in the course of tuberculosis, syphilis, gonorrhea may appear multiple, and one of the sepsis.

In addition, it also includes arthritis called Still's disease.

Deformed osteoarthritis is caused by aging or trauma to bones or joints, and hemophilia (관절 友 病 性) arthritis is caused by bleeding in the joints when hemophilia.

Many drugs have been developed for the purpose of treating diseases caused by inflammatory changes typified by arthritis as described above.

The rockfall lamp (Trachelospermi caulis), also known as the bark line, is a evergreen vine of the oleander family. It grows up to 5 meters long. The petals are deeply divided into five, with a pinwheel-shaped white flower from May to June, and the fragrance is very good. The fruit is held from September to November. The two long fruits often represent human beings, and sometimes they make a round circle like a bracelet and admire them. In fact, rockfall grows on the southern coast of Korea, in the foothills and fields, and in the wasteland, and usually grows on rocks, fences, and other trees and plants. In places where there are many rockfalls, there is a place where no other grass grows and all sides are covered with rockfalls. In North Korea's Dictionary of Synonyms, Rockfall, etc., is dried stems and leaves. Taste is bitter and a little cold. It acts on the heart, liver and nerves. Eliminate customs and make meridians work well. It is used for nausea, limb spasms, back pain, joint pain, tonsillitis, swelling, etc. 5 ~ 10g per day with water and eat it '.

Falling rocks are used for adding paraplegia, dew, etc. to limb paralysis pain, muscle cramps, and inability to flex. Since the blood cools, sore throat, boil, water, etc., take the month to take. The fruit of the rockfall is called the rockfall family (絡 石 果), and ripe mature fruits are collected around July and used. Leaves and stems are mainly used, and they can be harvested at any time regardless of the season and dried in the sun to be chopped.

China's 'Honam Pharmaceuticals' records 37-74g of 'falling stone' in musculoskeletal pain, and it is recorded.'Gangseo Herbal Medicine 'has an' arthritis line, organ skin root shell 37g, and knee grass root 18.5g. It is sweetened with water and taken as a liquor. ' You can take dried rocks, etc., once a month with 8-12 grams of water, until you lose about two-thirds of the water. Or soak it in alcohol or use it as a powder. For external use, powder it, open it, apply it, or apply fresh juice to the affected area or wash it. Rockfall is a vine that is good for our body to treat back pain, strengthen the musculature, and smooth the joints. Among the known rockfalls, such as tracheloside (Tracheloside), actin (Arctiin), materesinoside (Matairesinoside), arctigenin (notracheloside) and the like are the most widely known.

Traditionally, traditional rock medicines such as Dongbobom, Hyanggyeolbang, and light-dose are prescribed as rocky herbal medicines.However, such prescriptions are used to determine the shape of rockfalls and to prepare herbal medicines and liquids. It's just a brief comment about it. In other words, opinions regarding the active ingredients expressing the efficacy of the components extracted through the above-described method could not be obtained.

Thus, the present inventors have been filed under the name of the drug for the prevention and treatment of inflammatory diseases containing rock extracts, etc., and has been registered as patent No. 0847439.

However, in general, natural extracts have a high effective dose for showing efficacy and contain various ingredients, and therefore, there are various inconveniences for applying them to pharmaceuticals.

Accordingly, an object of the present invention is to provide a method for preparing an ethyl acetate fraction of a dried rock extract, which has been purified and concentrated to have the best anti-inflammatory activity in the rock extract.

In addition, another object is to provide a pharmaceutical composition for preventing and treating inflammatory diseases containing ethyl acetate fraction of the dry extract such as rockfall as an active ingredient.

In order to solve the above problems, the present invention is an ethyl acetate fraction of the dry extract of rockfall, etc., containing an extract of purified and concentrated so as to contain 0.05 to 12% by weight of actinogen, an indicator, as an active ingredient. It is characterized by a pharmaceutical composition for preventing and treating inflammatory diseases.

In addition, in order to achieve another object, the present invention

(a) dissolving a dry extract such as rockfall in water, adding ethyl acetate to the lysate and mixing the mixture to separate the water layer and the ethyl acetate layer;

(b) separating and recovering the ethyl acetate layer; And

(c) removing the solvent from the recovered ethyl acetate layer and concentrating to contain 0.05 to 12% by weight of actinogen, an indicator, to obtain an anti-inflammatory active fraction;

It characterized by another method for producing an ethyl acetate fraction of dry extract such as rockfall.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

The present invention has developed an ethyl acetate fraction (hereinafter referred to as ethyl acetate fraction of rockfall) and the preparation method of the dried rockfall extract which significantly improved the anti-inflammatory effect of the conventional rock extract such as rockfall, and applied to the pharmaceutical composition It is to be done.

The ethyl acetate fraction of rockfall, etc., in the present invention refers to a material obtained by adding a solvent to a rockfall, etc. dry extract and then partitioning and concentrating. At this time, the dry extract of rockfall lamp refers to the dry rock extract extracted with water or an organic solvent before fractionation.

In the present invention, it is preferable to use water or alcohols selected from methanol, ethanol and butanol as the solvent used for extraction and fractionation of rockfall. This is because water and alcohol meet the pharmacopeia regulations.

In the present invention, when the ethyl acetate fractions such as rockfall are purified, chromatography is preferably performed. Specifically, it is preferable to use a method of separating the polar mobile phase and the nonpolar stationary phase or the nonpolar mobile phase and the polar stationary phase. More specifically, it is preferable to perform normal phase and reverse phase chromatography repeatedly to further increase the purity.

In the present invention, the normal phase chromatography refers to a silica gel base stationary phase, and the reverse phase chromatography refers to an ODS (Octar-desil-silica) -based stationary phase, specifically, C-18 ODS, and the like. Solvents used in the art will be apparent to those skilled in the art, and detailed descriptions thereof will be omitted.

Actigenin in the present invention is a substance consisting of a molecular weight of 372 of the molecular formula C21H24O6, Dibenzylbutyroactone ligand (Dibenzylbutyroactone ligand), one of the substances that are actively studied on the biological activity in various plants, particularly Asteraceae, Actinin ) Is a substance consisting of molecular weight 534 of the molecular formula C27H34O11 and refers to a substance found in various plants and having a glycoside-bound form of actinogen.

The present invention is an ethyl acetate fraction of the dry extract of rockfall, etc., which should contain 0.05 to 12% by weight of the actinogen, indicator material, which is less than 0.05% by weight, the synergistic effect of the drug is reduced and exceeds 12% by weight. Since the lower surface process is added and the production cost is increased while the drug efficacy is not increased, it is preferable to apply the above-described content range of the indicator substance to the manufacturing process. In general, it is more preferable to standardize the content of actinizenin, which is an indicator, at 0.4 to 3.0% by weight in order to simplify the manufacturing cost and the process. In addition, it is more preferable that 6-20 wt% of actin is contained as another indicator of the fraction of the present invention.

Ethyl acetate fractions such as rockfall extracted and purified according to the present invention can be used as such, but are mixed with pharmaceutically acceptable carriers, excipients, diluents, powders, granules, capsules, Ointments, transdermal or injectables can be prepared. In addition, fractions such as rockfall according to the present invention has been used for food and medicinal use since ancient times, and there is no particular restriction on the dosage, body absorption, weight, age, sex, health condition, diet, administration time, administration of the body. It may vary depending on the method, the rate of excretion, the severity of the disease and the like. However, in terms of efficacy, ethyl acetate fractions such as rockfall are preferably administered in an amount of 100 to 1000 mg / day for the adult (60 kg basis). Since the ethyl acetate fraction of rockfall according to the present invention shows an excellent effect of about 20 ~ 300% compared to the efficacy of the ordinary rockfall extract, it can be administered by setting a dose applied thereto.

Therefore, the pharmaceutical composition comprising the active ingredient of the present invention is to be prepared in consideration of the effective amount range, the unit dosage form formulated in this way is the needs of the individual and the judgment of the expert to monitor or observe the administration of the drug as needed Depending on the specific dosage regimen, it may be administered several times at regular time intervals.

Inflammatory diseases in the present invention include acute, chronic inflammatory diseases, rheumatic inflammatory diseases caused by autoimmune reactions, degenerative inflammatory diseases and the like.

Hereinafter, the method of purifying rock extracts or fractions according to the present invention by process.

[First Step] Step of Obtaining Ethyl Acetate Fraction

In this step, a dry extract of rockfall or the like is dissolved in water, and ethyl acetate (ethyl acetate) is added to the dissolved solution and mixed to separate the water layer and the ethyl acetate layer.

Then, the solvent is removed from the separated ethyl acetate layer and concentrated to contain 0.05 to 12% by weight of the actinogen, which is an indicator, to obtain an ethyl acetate fraction having excellent anti-inflammatory effect.

This is because the extraction efficiency is improved by adding ethyl acetate having a relatively non-polar characteristic to the extract such as rockfall.

In addition, in the present invention, in consideration of extraction efficiency and safety in connection with ethyl acetate, it is preferable to extract rockfall and the like with alcohols, and in particular, extract with ethanol, methanol or butanol, and preferably dry one. This is because when extracted with alcohols, the solubility in the reaction with water is good and the efficiency of separating the active ingredient from water when fractionating from ethyl acetate is excellent. More specifically, 20 to 99.9% (v / v) ethanol is used, and more preferably 30 to 95% (v / v) ethanol is used. This is because using less than 20% of ethanol lowers the extraction efficiency of the active ingredient, and using ethanol of 99.9% or higher purity increases the production cost and does not improve the extraction efficiency and efficacy. However, in the present invention, rock extracts, such as rockfall, are not limited to alcohols, and may be extracted using any solvent as long as it is a solvent that can obtain a lot of active ingredients and is contained in the pharmacopoeia or is safe for human body.

Hereinafter, the second process and the third process may be selectively performed according to the efficacy or effective concentration of the drug, the physical properties of the extract and the like.

[Step 2] Refining Process

In this step, the ethyl acetate layer separated in the first step is chromatographed to obtain an active fraction. At this time, in the present invention, it is particularly preferable to perform normal phase, reverse phase or reverse phase chromatography with a normal phase to obtain a more purified active fraction. Here, various mobile phase or fixed phase solvents used in the normal phase or reverse phase chromatography will be apparent to those of ordinary skill in the art and will not be described in detail.

[Step 3] Extraction Purification Process

This process is carried out in the second process to obtain an active fraction with a higher degree of purification, and then dried or concentrated to obtain an extract purified.

As described above, the ethyl acetate fraction of rockfall according to the present invention exhibits a markedly improved anti-inflammatory effect compared to rockfall light extract obtained by the conventional method, and can be developed as a pharmaceutical composition or functional food for preventing and treating inflammatory diseases. It was made possible.

1 is a photograph showing the results of TLC development pattern of rock extract and fractions according to the present invention [a: Example 2, b: Example 3, c: Example 1, d: Example 4],

Figure 2a and Figure 2b is a photograph showing the chromatogram of actin and actinigen of the rock extract and fractions (Examples 1 and 4) according to the present invention,

FIG. 3 is a photograph showing column fractions of ethyl acetate fractions according to the present invention separated by normal phase chromatography, separated by TLC, and

4 is a graph showing the IL-1β, TNF-α generation inhibitory effect of the column fractions of the ethyl acetate fraction such as rockfall according to the present invention,

5 is a photograph showing the expression of MMP-1 and MMP-3 stimulated with TNF-α in arthritis synovial tissue cells (FLS) of Example 3 of rockfall, etc., according to the present invention.

Figure 6 is a photograph showing the arthritis inhibitory effect of the arthritis inflammation edema evaluation test using the arthritis-induced mouse model (Example 3),

Figure 7 is a photograph showing the results of H & E staining of the CIA mouse joint of the rockfall fraction (Example 3) according to the present invention,

Figure 8 is a photograph showing the inhibitory effect of IL-1β, IL-6, RANTES, MMP-3 mRNA expression of the rockfall fraction (Example 3) in arthritis induced mouse joints,

9 is a photograph showing the inhibitory effect of MMP-1, 3 protein expression in arthritis model animal joint tissue of the rockfall fraction (Example 3) according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples, and it will be apparent to those skilled in the art that various changes or modifications can be made within the spirit and scope of the present invention.

Example 1 Preparation of 95% Ethanol Extracts, such as Rockfall

After washing with clean purified water and drying well, 20 kg of rockfall, etc. was added at about 5-10 times the weight of the crude drug in 95% ethanol aqueous solution, and the mixture was extracted at about 80 ° C. for 5 hours while stirring well. The extract was slowly cooled to room temperature, filtered to remove debris, the filtrates were combined, and the ethanol solvent was concentrated by evaporation under reduced pressure to obtain 1 kg of ethanol extract such as rockfall (yield about 5%).

Example 2: Preparation of 30% Ethanol Extract, such as Rockfall

After washing with clean purified water and drying well, 40 kg of rockfall and the like were added with 30% aqueous ethanol solution 5 to 8 times the weight of the crude drug, followed by hot boiling extraction every two hours with stirring. The extract was slowly cooled to room temperature, centrifugally filtered to remove debris, and the filtrates were combined and concentrated under reduced pressure at 60 to 80 ° C.

After the ethanol recovered through the ethanol fraction was added to the concentrate, the concentrate was sufficiently suspended, and the filtrate was centrifugally filtered at 1000 rpm. The filtrate was concentrated under reduced pressure, and then vacuum dried at 60 ° C. and 0.08 pa. Sterilization during the grinding process yielded 1 kg of ethanol extracts such as powdered rockfall (yield 2.5%).

Example 3: Preparation of Ethyl Acetate Fraction, such as Rockfall

100 g of the 30% ethanol extract prepared in Example 2 was dissolved in about 0.8 L of water, dissolved, and then mixed with the same amount of ethyl acetate (330 mL X 5), followed by fractionation, to obtain an ethyl acetate soluble fraction. An appropriate amount (about 125 g) of sodium thiosulfate was added to the ethyl acetate soluble fraction, the water molecules of the fraction were absorbed and filtered to purify the ethyl acetate soluble fraction. The purified fractions were concentrated using a vacuum filter to obtain about 11 g of ethyl acetate fractions such as rockfall.

Example 4 Preparation of Ethyl Acetate Fraction, such as Rockfall

100 g of the 95% ethanol extract prepared in Example 1 was dissolved in about 0.8 to 2 L of water, dissolved, and the same amount to 2 times of ethyl acetate were mixed and fractionated to obtain an ethyl acetate soluble fraction. An appropriate amount (about 125 g) of sodium thiosulfate was added to the ethyl acetate soluble fraction, the water molecules of the fraction were absorbed and filtered to purify the ethyl acetate soluble fraction. The purified fractions were concentrated using a vacuum filter to obtain about 16.7 g of ethyl acetate fractions such as rockfall.

Example 5 TLC Identification of Ethyl Acetate Fractions, such as Rockfall

Prepared for TLC development by melting a small amount (about 1 mg per 5 mL methanol) of each of the rockfall extract and fractions prepared in Examples 1 to 4, and then capturing the ethylacetate fraction of rockfall dissolved in methanol in Silica TLC (normal phase). After loading by using a dot and completely dried, it was developed in a closed TLC tank using different mobile phase solvent, it was shown in Figure 1 using a color developing reagent. At this time, the mobile phase was (A) ethyl acetate 66%, ethanol 19%, water 15%, (B) chloroform 80%, methanol 15%, water 5%.

According to the results, comparing the ethanol extracts (a, c) of rockfall and ethyl acetate fractions (b, d) of rockfall was different from the starting point of TLC development. In other words, it could be clearly observed that the ethyl acetate fraction was further purified, and the difference of the components developed from the starting point was significantly reduced by fractionation with ethyl acetate, and only certain portions were purified with the ethyl acetate fraction.

Example 6 Rock Formation Indicator Component High Performance Liquid Chromatography (HPLC) Analysis

The content of the indicator component Actin of the rockfall lamp extract of Example 2 and the rockfall lamp fraction of Example 3 was analyzed using a high performance liquid chromatography apparatus.

[Actin analysis condition]

1) Equipment used: Waters HPLC system

2) Mobile phase: 50% methanol

3) Analysis time: 30 minutes

3) Flow Rate: 1ml / min

4) Column: YMC-pack Pro C18 RS (250X4.6mm ID. 5particles)

5) Detector: UV 280nm

6) Standard Actin: Provided by Hunan Guohua Pharmaceutical Co., Ltd, China

In Example 2, the actin content was found to contain about 3% by weight, and the actin content in Example 3 was changed in content every fraction. That is, the largest amount of ethyl acetate fraction was detected in about 19% at one time, and the actin content was gradually decreased from 2 to 5 times, indicating the content of about 6%. The ethyl acetate fraction fractionated five times was analyzed for an average content of about 12-15%.

In addition, the contents of actin and actinigenin of the rockfall lamp extract of Example 1 and the rockfall lamp fraction of Example # 4 were analyzed by high performance liquid chromatography (HPLC). The method of analyzing actin is the same as that of Examples 2 and 3, and the method of analyzing actigenin is as follows.

Actinogen analysis conditions

1) Equipment used: Waters HPLC system

2) Mobile phase: 40% acetonitrile (CH3CN)

3) Analysis time: 30 minutes

3) Flow Rate: 1ml / min

4) Column: YMC-pack Pro C18 RS (250 X 4.6 mm ID. 5 particles)

5) Detector: UV 280 nm

6) Standard Product: Sigma-Aldrich Product No. A1854. (Lot. 074K4701, Germany)

In Example 1, the actin content was found to contain about 2% by weight and 0.5% by weight of actinigen, and the actin content in Example 4 was about 6% by weight, and about 1.2% by weight of actigenin when fractionated three times. This was measured.

Chromatograms of actin and actinigen are shown in FIG. 2.

Example 7 Normal Phase Column Fraction from Ethyl Acetate Fraction, such as Rockfall

Ethyl acetate fractions such as rockfall obtained in Example 3 were prepared using silica gel column chromatography using a step gradient solvent system composed of methylene chloride: methanol (1000: 5 to 95: 5). Separated.

That is, the rock acetate ethyl acetate fraction is suspended about 12 times with silica gel in the ratio of methylene chloride 1000: methanol 5, which is a mobile phase, and finely stacked on a glass column.Then, the rock acetate ethyl acetate fraction is about 0.5 to 1 times. Suspended in silica gel of and adsorbed by concentrated under reduced pressure was injected into a column formed. The concentration gradient was changed stepwise to the ratio of methylene chloride 95: methanol 5 at about 5 times the capacity of the silica gel to receive the fractions in the capacity of the silica gel column (about 100 mL units), and each of the obtained fractions was obtained in Example 5 above. Separation was confirmed through silica gel TLC as in the method (see FIG. 3). In this case, 95% of methylene chloride, 5% of methanol or 80% of chloroform, 15% of methanol, and 5% of water were used.

Test Example

In this test example, in order to confirm the anti-inflammatory effect of the fractions according to the present invention, the effects of intracellular nitric oxide and cytokine (Cytokine) on extracts and fractions were analyzed. In addition, the swelling and inflammation inhibition of extracts were analyzed using arthritis-induced mice through animal experiments, and histologic staining, inhibitory effects of mRNA and protein of arthritis factors were analyzed through articular biological samples of animal models.

1. Cell Culture and Extract Purification Treatment

RAW 264.7 (ATCC, # TIB-71) cells, mouse-derived macrophages, were treated with Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), penicillin (100 units / ml) and streptophore at 5% CO2 and 37 ° C. Cultures were made using mycin sulfate (100 g / ml). For each assay, RAW 264.7 cells were seeded at 96 × well plates at 2 × 10 4 cells / well and treated with concentrations of 300 ng / ml LPS and fractions after 24 hours.

2. Measurement of Cell cytotoxicity

Cytotoxicity was incubated for 4 hours using TACS XTT Cell Proliferation / Viability Assay kit (R & D systems, Cat. 4891-025-K) and measured at OD490nm.

3. Statistical Processing

Statistical significance test for each experimental group was performed as follows. Levene's test is performed on the data obtained from each experimental group to confirm the variance homogeneity, and when the variance is homogeneous, one-way ANOVA is performed to confirm the significance at the level of p = 0.05. Dunnett's t-test was used to compare the differences between the experimental groups.

Test Example 1: Nitric Oxide (NO assay)

40 mg / ml grease reagent (Sigma, G4410) was added to each culture supernatant (100 μl), mixed, and reacted at room temperature for 30 minutes, and then absorbance was measured at OD540 nm. As a result of analysis, the percentage of LPS stimulation-treated control group (control) was expressed as a relative percentage (% of LPS treated control) or the unit IC50 at a concentration showing a 50% inhibitory effect or the inhibition rate is shown in Table 1 and Table 2 below. It was.

Table 1

Table 1 is a result of converting the NO production inhibitory effect of the 30% ethanol extract (Example 2), such as rockfall and ethyl acetate extract (Example 3), converted to IC50. It was found that the effect was improved to 379 μg / ml while the range of concentration inhibiting 50% was purified from Example 1-3 at 1180 μg / ml in Example 2.

In addition, the NO production inhibition rate of the extracts and fractions (Examples 1 and 4) of rockfall, etc. was analyzed in the same manner as described above. At the highest concentration of 320 μg / ml showing no cytotoxicity of Examples 1 and 4, the NO production inhibition rate of the ethanol extract of Example 1 showed a relatively high effect of 68%. In addition, it can be seen that Example 4, which is an ethyl acetate extract, showed a higher effect by showing a higher inhibition rate of 80% [Table 2].

[Table 2]

Test Example 2: Cytokine Analysis

The levels of IL-1β and TNF-α in the culture were measured by ELISA using an IL-1β assay kit (R & D systems, Cat. MLB00B) and TNF-α assay kit (R & D systems, Cat. MTA00). Absorbance was measured using a microplate reader at OD450nm UV. The results of the analysis were obtained in relative% based on the LPS-stimulated treatment group.

At this time, the treatment concentration of the sample in the cytokine analysis was performed based on a concentration range that does not exhibit cytotoxicity as a result of cytotoxicity experiments.

Comparative analysis of the 30% ethanol rockfall extract (Example 2) and the rock acetate ethyl acetate fraction (Example 3) is shown in Table 3 below. In the case of IL-1β and TNF-α, the inhibition rate of 38% and 42% was shown in Example 3, respectively, which showed higher inhibitory effect than that of Example 2 [Table 3]. This is the value analyzed in the highest toxicity range (Example 2: 640 [mu] g / ml, Example 3: 160 [mu] g / ml,) showing no effect on cytotoxicity.

Table 3

As a result, when comparing ethanol extracts such as rockfall and ethyl acetate fractions such as rockfall, the ethyl acetate fractions showed relatively superior inhibitory effects of TNF-α and IL-β, which are classified as inflammatory cytokines. Compared to the ethanol extract, the active ingredients are indirectly more friendly to nonpolar solvents, and it can be seen that the ethyl acetate solubles of the active substances were purified.

In addition to this, the purification result of the cytokine analysis of fractions 2, 5, and 6 fractions purified in Example 7 for further purification test is shown in FIG. 4. IL-1β, TNF-α inhibitory effect was shown to be a more purified form showing a high inhibitory effect at a lower concentration.

Therefore, the extract purified according to the present invention is considered to have a very good anti-inflammatory effect in comparison with the conventional extract.

Test Example 3 Isolation and Culture of Synovial Cells (FLS: Fibroblast Like Synoviocyte) from Synovial Tissues

Synovial tissues from arthritis patients were cut with scissors to about 1 × 1 × 1 mm and incubated at 37 ° C. for 2 hours in HBSS buffer in which 1 mg / ml collagenase was dissolved. Only the supernatant was separated using a 70 ㎛ nylon cell strainer (BD, NJ, USA). The isolated cells were washed by centrifugation at 1500 rpm for 2 minutes at 10 minutes. After removing the supernatant, add α-MEM medium (10% FBS, 1% L-Glutamine, 1% penicillin-streptomycin) to the pellet, transfer to T75-flask, 37 ℃, 2 ~ 3 hours in CO2 incubator After incubation, washed twice and incubated. More than 95% FLS was obtained after 3-6 passages (> 95% surface markers for fibroblasts (CD90 +), <1% CD3 +, <1% CD11b, <1% Fcγ Receptor II positive). It was.

Test Example 4: Immunoblotting Analysis

The FLS obtained in Test Example 3 was dispensed into 6 well plates at 2 × 10 5 / well. After incubation for 3 days, the culture medium was replaced with incomplete α-MEM (1% L-glutamin, 1% antibiotics), and after treatment of fractions such as rockfall (Example 3) at 10, 25, 50 μg / ml concentration, 30 Minutes later, TNF-α was treated with 20 ng / ml, and after 24 hours, only the culture medium was separated and extracted. The separated extracted culture was subjected to immunoblotting analysis using a 10% SDS-PAGE gel. Antibodies MMP-1 and MMP-3 were used to confirm the effects on MMPs secreted from FLS and are shown in FIG. 5. As shown in FIG. 5, significant decreases of MMP-1 and MMP-3 were confirmed depending on the concentrations of 10, 25, and 50 μg / ml of the rockfall fractions of Example 3.

Test Example 5: Collagen-induced arthritis (CIA) mouse model

Experimental animals were purchased 5 weeks old male DBA / 1J mice, purified for 2 weeks, and then tested at 7 weeks of age. 100 μg bovine Type II collagen (in 0.05M acetic acid) was mixed with the same amount of Freund's complete adjuvant (Chondrex Inc., Redmond, Washington) and injected into the tail muscles (1st Immunization). After 21 days, 50 μl (2 mg / ml) bovine type II collagen was injected again by intraperitoneal injection (i.p) (2nd Immunization). As in the 2nd immunoassay, distilled water (vehicle), IND (indomethacin), and rockfall fractions (Example 3) were injected orally every day as a suspension in 5% Arabian gum.

Arthritis score was visually evaluated every 3 days, and each evaluator (6 persons) was blinded.

0 = normal, 1 = mild, apparent swelling limited to individual digits, 2 = moderate, redness and swelling of the ankle 3 = redness and swelling of the paw including digits, and 4 = maximally inflamed limb with involvement The arthritis score was evaluated based on the evaluation criteria of multiple joints. Evaluation was measured up to 18 days after 2nd immunization. 6 shows the results of visual evaluation of the CIA test.

Figure 7 shows the evaluation results of the tissue evaluation picture (H & E staining). CIA animal model hind paws sacrificed 38 days after 1st immunization were extracted, immobilized CIA in fixative solution (4% paraform-alehyde in PBS), and then in demineralized solution (10% EDTA, 4% paraform-aldehyde in PBS). Weekly demineralization process was performed. Next, paraffin was treated and sectioned to 5 μm in thickness, followed by H & E (hematoxylin and eosin) staining for cartilage destruction, bone erosion, infiltration of immune cells, and degree of pannus formation. Measured.

The measurement criteria were 0 = normal, 1 = immune cell infiltration, 2 = synovial hyperplasia, pannus formation, 3 = bone erosion, and collapse.

Test Example 6: mRNA Extraction from Experimental Animal Tissue

The paw and hind leg tissues collected from CIA experimental animals (Test Example 5) were frozen in liquid nitrogen, crushed first using a mortar and then crushed using RNA isolation kit (Intron, Korea). RNA was extracted and separated. After the RNA isolation lysis buffer was added, vortex was added for 30 minutes, and 200 µl of chloroform was added. After centrifugation at 13,000 rpm, 4 ° C. for 30 minutes, the supernatant was obtained, and total RNA was extracted using an RNA separation filter. The total extracted RNA was quantified using a spectrophotometer (Eppendorf, German).

Test Example 7: Reverse transcription-polymerase chain reaction (RT-PCR)

1 μg of total RNA extracted in Test Example 6 was carried out using a Superscript first strand synthesis system (Invitrogen, CA, USA) to perform RT-PCR. After 5 min reaction at 72 ° C. with OligodT 18mer (invitrogen, CA, USA) at 1 ug of total RNA, reaction buffer Mix (3 mM MgCl 2, 0.5 mM dNTP, 5x reaction buffer, 20U ribonuclase inhibitor, 1 μl reverse transcriptase ) Was added, followed by reaction at 25 ° C-5 minutes, 42 ° C-1 hour, and 72 ° C-5 minutes. The template thus produced was used for the PCR reaction. In the PCR reaction, IL-1β was 33 cycles (IL-6: 35 cycles) at 94 ° C-2 minutes, 94 ° C-20 seconds, 60 ° C-10 seconds (RANTES, MMP-3, GAPDH: 50 ° C) and 72 ° C-40 seconds. , RANTES, MMP-3: 35 cycles), and finally the experiment was carried out by reacting for 5 minutes at 72 ℃. The product thus obtained was electrophoresed on a 2% agarose gel, stained with ethidium bromide and confirmed in a UV-system as shown in FIG. 8. Each PCR primer sequence is shown in Table 4 below.

Table 4

RT-PCR primer sequence

* Molecular Pharmacology. 1997, 52: 30-37.

Test Example 8: Protein Extraction from Experimental Animal Tissue

The paw and hind leg tissues collected from CIA experimental animals (Test Example 5) were frozen in liquid nitrogen, and first crushed using a mortar and then lysis buffer (20 mM HEPES, pH 7.5, 150 mM). NaCl, 1% Nonidet p-40, 10% glycerol, 60 mM octyl β-glucoside, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM nitrophenylphosphate, 0.7 μg / ml pepstatin, and a protease-inhibitor cocktail tablet ) Was added to extract the total protein. The separated and extracted proteins were quantified by Bradford assay, and immunoblot analysis was performed.

Expression of MMP-1 and MMP-3 in the forefoot and hind paw tissues collected from CIA experimental animals at the protein level was confirmed that the inhibitory effect in the fraction (Example 3), such as rockfall, is shown in FIG.

Preparation Example 1 Preparation of Tablet

Tablets for oral administration were prepared using the wet granules method and the dry granules method using the ethyl acetate fraction of the present invention.

[Furtherance]

Rockfall ethyl acetate fraction 200 mg, light silicic anhydride 10 mg, magnesium stearate 2 mg, microcrystalline cellulose 50 mg, starch glycolate 25 mg, lactose 101 mg, povidone 12 mg, ethanol anhydride.

Preparation Example 2 Preparation of Ointment

The ointment was prepared using the ethyl acetate fraction of the rockfall of the present invention with the following composition.

[Furtherance]

Rockfall ethyl acetate fraction 5 g, cetyl palmitate 20 g, cetanol 40 g, stearyl alcohol 40 g, myristan isopropyl 80 g, monostearic acid sorbitan 20 g, polysorbate 60 g, paraoxybenzoic acid propyl 1 g, 1 g of methyl paraoxybenzoate, phosphoric acid and purified water.

Preparation Example 3 Preparation of Injection

Injectables were prepared using the ethyl acetate fraction of the present invention, such as rockfall, in the following composition.

[Furtherance]

Ethyl acetate fraction 100 mg, mannitol 180 mg, sodium dihydrogen phosphate 25 mg, purified water 2974 mg

Preparation Example 4 Preparation of Transdermal Agent

Using the ethyl acetate fraction of rockfall of the present invention, a transdermal agent was prepared in the following composition.

[Furtherance]

0.4 g of ethyl acetate fractions such as rockfall, 1.3 g of sodium polyacrylate, 3.6 g of glycerin, 0.004 g of aluminum oxide, 0.2 g of methyl paraben, and 14 ml of acrylic adhesive solution.

App file attached to the electronic application PCT-SAFE-FM.

Claims (6)

Ethyl acetate fraction of the dry extract of rockfall, etc., A composition for the prevention and treatment of inflammatory diseases containing an extract of purified and concentrated so as to contain 0.05 ~ 12% by weight of actinogen, an indicator. The method of claim 1, The rock-fall extract dried composition for preventing and treating inflammatory diseases, characterized in that the dried extract extracted from methanol or ethanol. The method of claim 1, The extract, such as rockfall extract is dried using 20 ~ 99.9% (v / v) ethanol extract for the prevention and treatment of inflammatory diseases, characterized in that the composition. The method of claim 1, Ethyl acetate fraction of the dry extract, such as rockfall, the composition for preventing and treating inflammatory diseases, characterized in that it contains 6 to 20% by weight of actin. (a) dissolving a dry extract such as rockfall in water and separating the water layer and the ethyl acetate layer by mixing ethyl acetate with the lysate; (b) separating and recovering the ethyl acetate layer; And (c) removing the solvent from the recovered ethyl acetate layer and concentrating to contain 0.05 to 12% by weight of actinogen, an indicator, to obtain an anti-inflammatory active fraction; Method for producing an ethyl acetate fraction of the dry extract such as rockfall, characterized in that comprises a. The method of claim 5, Mixing the ethyl acetate in the water layer separated in step (a) to separate the water layer and the ethyl acetate layer; After separating and recovering the ethyl acetate layer, the separation and recovery steps are repeated 2 to 5 times, the solvent is removed from the recovered ethyl acetate layer, and the content of actinizenin, which is an indicator, is contained in an amount of 0.05 to 12% by weight. Concentrating so as to obtain an anti-inflammatory active fraction.

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