KR20080101344A - Composition for the anti-inflammatory and analgesia comprising tannin-contained kalopanacis cortex extract - Google Patents

Abstract

An anti-inflammatory analgesic agent composition is provided to cause no toxicity and side effect using a tannin containing Kalopanacis Cortex extracts as an active ingredient, to remarkably suppress a production of TNF-alpha and NO which are an inflammatory-mediating material, to have an NF-kappaB phosphorylation inhibiting property, an antiphlogistic alleviation effect, an acute form edema inhibiting property and an acute arthritis inhibiting property and to cure and prevent inflammation diseases including arthritis, tympanitis, edema, pain or degenerative disease. An anti-inflammatory analgesic agent composition includes a tannin containing Kalopanacis Cortex extracts as an active ingredient which is Actinidia polygama extracts containing a Tannin of 5.0 ~ 30.0 weight%. A manufacturing method of the Kalopanacis Cortex extracts containing the tannin comprises steps of: 1) extracting the Kalopanacis Cortex extracts using water, alcohol or alcohol water solution and cooling and filtering the Kalopanacis Cortex; 2) decompression-concentrating a filtrate at 60 ~ 80°C after the filtrate is layer-separated; 3) obtaining Kalopanacis Cortex extracts by decompression-concentrating, vacuum-drying, pulverizing and sterilizing the filtrate after adding an alcohol to the condensed products and centrifuging at 500 ~ 1,000 rpm.

Description

Composition for the anti-inflammatory and analgesia comprising tannin-contained Kalopanacis Cortex extract}

Figure 1 shows the HPLC chromatogram of the tannin-containing thawing extract according to the present invention.

Figure 2 shows the inhibitory effect of the concentration-dependent production of inducible nitrogen oxide synthase (iNOS) of the tannin-containing thawed skin extract of the present invention (a: immunoblotting results, b: light concentration measurement analysis results of a data).

Figure 3 shows the inhibitory effect of concentration-dependent production of nitrogen oxide (nitric oxide; NO) of the tannin-containing thawed skin extract of the present invention.

Figure 4 shows the concentration-dependent effect on the inhibition of gene mRNA expression of tumor necrosis factor-alpha (TNF-α) of the tannin-containing thaw extracts of the present invention (a: reverse transcription-reverse transcription-polymerase chain reaction (RT-PCR) B, a light density measurement of the data.

Figure 5 shows the concentration-dependent effect on the inhibition of the production of TNF-α of the tannin-containing thawed skin extract of the present invention (a: results of immunoblotting, b: light concentration measurement results of a data).

Figure 6 shows the concentration-dependent effect on the inhibition of NF-κB phosphorylation of tannin-containing thawed skin extract of the present invention (a: results of immunoblotting, b: light concentration measurement analysis of a data).

Figure 7 shows the toxicity of the tannin-containing thawed skin extract of the present invention on the spleen cells of the experimental animal in a graph of survival rate according to the concentration.

FIG. 8 is a graph showing the toxicity (GOT, GPT) of the tannin-containing thaw extracts of the present invention on liver tissue of experimental animals.

9 is a graph showing the toxicity (CRE, BUN) of the tannin-containing thawing extract of the present invention on the kidney tissue of the experimental animal.

Figure 10 shows the effect of the tannin-containing thawed skin extract of the present invention on the growth of immature experimental animals by weight change.

Figure 11 shows the effect of the tannin-containing thawed skin extract of the present invention on the growth of mature experimental animals by weight change.

The present invention is thawed skin containing 10.0 ~ 25.0% by weight of tannin (Tannin) as an active substance ( Kalopianacis Cortex ) The present invention relates to a method for preparing an extract and an anti-inflammatory analgesic composition using the same as an active ingredient, and more particularly, to a method for extracting thaw skin, which is prepared to contain a predetermined amount of tannin by filtration and concentration under reduced pressure. An anti-inflammatory analgesic composition comprising the component.

Inflammation refers to the pathological state of an abscess formed by the invasion of external bacteria, and the inflammatory response is a biological method for restoring and repairing damage caused by an invasion that causes any organic change in cells or tissues. When infected with external infectious agents such as damage to (cells), bacteria, fungi, viruses, and various kinds of allergens, various inflammatory mediators and immune cells are involved in local blood vessels and contractions, resulting in enzyme activation, secretion of inflammatory mediators, and body fluids. It exhibits a series of complex physiological reactions including infiltration, cell migration, tissue destruction, and external symptoms such as erythema and edema pain.

In normal cases, the inflammatory response is to restore the function of life by removing external sources of infection and regenerating damaged tissues.However, if the inflammatory response is excessive or persistent due to the absence of antigens or internal substances, the major pathology of the disease (sensitizing disease) , Chronic inflammation) and also become a disorder in the treatment process, such as blood transfusion, drug administration, organ transplantation.

Nitric oxide (NO), an inflammatory factor involved in this inflammatory response, is an intermediate metabolite of hydroxyarginine from L-arginine by nitric oxide synthase (NOS). NO and citrulline are produced. NO is biologically active and has a very small molecular weight. It is a mediator that acts on the vascular system in vivo and is involved in vasodilation, platelet adhesion and aggregation, neurotransmission, and digestive system movement. It has been shown to be responsible for the dragon (neural transmission, blood coagulation, blood pressure control, immunity to cancer cells, etc.), and is produced not only in inflammatory cells but also in non-immune cells to protect against microbial infection. In general, NO exists in the form of gas in the atmosphere, and because it is a free radical structure, it is very toxic and its half-life is very unstable with 6 to 10 seconds, so that NO ₃ (nitrate) and NO ₂ (nitrite) are stabilized final products. It is known to change rapidly (Snyder, SH et al., Scientific American, May, pp 28-35, 1992).

On the other hand, NOS involved in NO production is largely classified into two types, namely, cNOS (constitutive NOS) and iNOS (inducible NOS), depending on whether calcium (Ca ²⁺ ) or calmodulin is dependent. Calmodulin-dependent, mainly in brain, epithelial, neutrophil, and gastrointestinal mucosa cells. INOS is independent of calcium or calmodulin. INOS is present in macrophages, hepatocytes, and cancer cells. IL-1β (interlukin-1β) , Cytokines such as IFN-γ (tumor necrosis factor-α) and bacterial endogenous bacterial LPS (lipopolysaccharide) (Dinerman, JL, et al., Circ.Res., 73, pp 217-222, 1993). This stimulation also activates COX-2 (cyclooxygenase-2), which produces inflammatory mediators PGs (prostaglandines), which correlate closely with iNOS expression and COX-2 expression. It also affects expression (Robert C., et al., J. Immunol. 165, pp1582-1587, 2000; Daniela S., et al., Proc. Natl. Acad. Sci. USA, 90, pp7240-7244 , 1993).

Therefore, various efforts have been made to secure a substance that inhibits the production of NO by iNOS and develop it as a medicine.

Inflammatory diseases caused by these inflammatory reactions include arthritis, gastritis, colitis, arthritis, nephritis, hepatitis, cancer or degenerative diseases (Gobert AP et al., J. Immunol. 168 (12), pp6002-6006, 2002; Dijkstra G. et al., Scand. J. Gastroenterol. 37 (5), pp546-554, 2002; Sakac V. and Sakac M. Med. Pregl. 53, pp463-474, 2000; Albrecht EW et al., Am. J. Transplant, 29 (5), 448-453, 2002; Ramachandran A. et al., Free Radical Biol. Med. 33 (11), pp1465-1474, 2002; Sartor L. et al., Biochemical Pharmacol. 64 , pp 229-237, 2002; Sadowska-Krowicka H. et al., Proc. Soc.Exp. biol.Med. 217 (3), pp351-357, 1998; Lo AH. et al., Carcinogenesis, 23 (6) , pp983-991, 2002).

Inflammatory inhibitory effect test as described above, in the cellular aspect to determine whether the production of inflammatory mediators (Nitric oxide; NO), tumor necrosis factor-alpha (TNF-α), IL-1β, etc. inhibits the animal, In the experimental aspect, capillary permeability inhibitory effect and ear edema and foot edema inhibitory effect are examined. In animal experiments for analgesic effects, the analgesic effect is generally examined using acetic acid-induced stretching inhibition test (Eun et al., 26 (2): 122-129, 1995; Choi J et al., J Ethnopharmacol 79 (2): 199-204, February 2002; Kim YK et al., Arch Pharm Res. 26 (12): 1061-1066, December 2003; Kim IT et al., J Ethnopharmacol. 94 ( 1): 165-173, September 2004; Lee Ji-eun et al., The Korean Periodontological Society, 35 (1): 9-17, 2005; Lao CJ et al., Am J Chin Med. 33 (3): 425-438 , 2005; Lee YC et al., Int Immunopharmacol. 6 (4): 703-713, April 2006).

Haedongpi is the bark of an oak tree ( kalopanax pictus Nakai ), and its name is KALOPANACIS CORTEX. The oak is a deciduous arboreous tree, growing up to 25 m in height, with thorns on its branches. The leaves are palm-shaped, a common feature of the Galapaceae, and are usually divided into 5-9 branches. Fruit is nuclear, spherical, ripened in black in October, with 1-2 seeds in it. The taste is bitter, flat and grows well in deep mountain forests throughout Korea, Japan, China, Manchuria Ussuri and Sakhalin.

It is said that the drug is effective in the wind, dehumidification, blood circulation, and insecticide.

Haedongpi is traditionally prescribed as a medicinal herb in traditional Chinese medicines and related literature such as Dongbobogam, Hyangjegbang, and light-dose.However, such prescription is given to the simple form of the method of differentiating the appearance of Haedongpi and the preparation of herbal medicine and liquid solution. There is no description of active active ingredients expressing medicinal effects among thawed skin components.

On the other hand, in Korea, edema inhibitory effect and analgesic effect of thawed skin extract Liriodendrin extracted with methanol have been disclosed (Eun et al., Journal of Pharmacognosy, 26 (2): 122-129, 1995). Analgesic activity has been published (Kim IT et al., J Ethnopharmacol. 94 (1): 165-173, September 2004).

In addition, Korean Patent Registration No. 10-0547554 discloses a composition containing anti-inflammatory activity, thaw skin, browning, and lacquer extract, and Korean Patent Registration No. 10-0547560 discloses thawing skin, anti-inflammatory activity having anti-inflammatory activity, The pharmaceutical composition containing the lacquer extract is disclosed, and Korean Patent Application No. 10-2001-0011226 (Registration Abandoned) discloses the extract of Umber, its preparation method and uses, and in Korean Patent No. 10-0555660 Disclosed is a composition for preventing and treating arthritis, including a carlopanax saponin A derivative isolated from ethyl acetate soluble extract of thawed skin.

It is also known that Haedongpi extract has a toxic effect on malaria (Park H et al., Biol Pharm Bull. 26 (11): 1623-1624, November 2003). Also published (Li DW, Biol Pharm Bull. 26 (4): 429-433, April 2003).

In particular, thawing methanol extract has been reported to reduce arthritis in certain diseases (Choi J et al., J Ethnopharmacol. 79 (2): 199-204, February 2002).

However, according to what is known to date, except for thawed blood saponin, it is not disclosed which components of thawed blood are inhibited by mediators and cells related to inflammation.

Accordingly, the present inventors have made intensive efforts to identify the anti-inflammatory effects of the extracts of thaw skin according to the specific active ingredient and its content, except for saponins, among the thaw skin extracts, and tannin (Tannin) is 10.0-25.0 weight. It was confirmed that the thawed extract containing% has an excellent anti-inflammatory effect and completed the present invention.

After all, the main object of the present invention is to provide an anti-inflammatory analgesic composition comprising the thaw skin extract containing 10.0 ~ 25.0% by weight of tannin as the active ingredient.

Still another object of the present invention is to provide a method for preparing thaw extract, which contains 10.0 to 25.0% by weight of tannin as an active substance having excellent anti-inflammatory effect.

In order to achieve the above object, the present invention provides an anti-inflammatory analgesic composition comprising the extract of Kalopanacis Cortex containing 5.0 to 30.0% by weight of tannin (Tannin) as an active ingredient.

In the present invention, the tannin may be characterized in that 10.0 ~ 25.0% by weight, characterized in that the anti-inflammatory analgesic effect to be turbid for arthritis, otitis media, pain, edema or degenerative diseases caused by inflammation and inflammatory reactions You can do

In addition, the present invention comprises the steps of a) extracting, cooling and filtering the thawed blood with an aqueous solution of 5 to 8 times water, alcohol or alcohol to obtain a filtrate; b) separating the filtrate with an aqueous solution of alcohol and then concentrating under reduced pressure at 60-80 ° C. to obtain a concentrate; And c) adding alcohol to the concentrate, followed by centrifugation at 500 to 1,000 rpm, and then concentrating the filtrate under reduced pressure, vacuum drying, pulverizing and sterilizing to obtain a thawed skin extract. Provide a method.

In the present invention, the tannin may be characterized in that 10.0 ~ 25.0% by weight.

In addition, in the present invention, the thawing extract may be characterized in that the powder, the alcohol may be characterized in that the lower alcohol having 1 to 6 carbon atoms, such as methanol, ethanol, propanol, butanol and the like.

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

The present invention relates to a method for preparing a extract of Kalopanacis Cortex containing a certain amount of tannin (Tannin) as an active substance, and to an anti-inflammatory analgesic composition comprising the same as an active ingredient.

The anti-inflammatory analgesic composition of the present invention contains the thawed extract as an active ingredient, the thawed extract contains 5.0 to 30.0% by weight of tannin (Tannin) as an indicator and active substance, more preferably 10.0 to 25.0 weight It may contain%.

Since the amount of the active bioactive substance contained in the original herbal medicine can vary greatly depending on the place of origin, the harvesting time, the storage period and the storage condition, the content of the specific active substance is more limited than the limitation of the weight ratio of the ingredients used in the composition of each herbal composition. It is more preferable to limit.

Therefore, in the present invention, tannin was selected as an indicator and an active substance in order to obtain a thawing extract that maximizes the expression of the desired anti-inflammatory effect.

Haedongpi extract used in the treatment of inflammatory diseases of the present invention can be used by extracting tannin which is a specific active substance from the extract itself (medicinal extract) extracted from the thawed blood or the said thawed extract. In this case, the blending ratio is determined by the content of tannin, which is an indicator substance and an active substance, irrespective of the original content of thawed bark, and preferably contains 5.0 to 30.0% by weight of tannin in the extract of thaw bark, more preferably 10.0 to 25.0 It should contain tannins by weight.

In the present invention, when the content of the tannin is less than 10.0% by weight, the result is relatively poor in the treatment of arthritis, etc., so that the content of the tannin is preferably 10.0% by weight or more. In addition, in the present invention, there is no particular limitation on the upper limit of the tannin content, but if it is contained in excess of a certain level, the effect does not increase any more, and is not preferable in terms of manufacturing, technical and economical 30.0 weight Or less, more preferably about 25.0% by weight.

In the present invention, when the indicator substance and the active substance tannin is contained in 10.0 ~ 25.0% by weight, the synergistic effect of the desired drug can be expressed to show an excellent anti-inflammatory effect of the thawed extract, but in the present invention the thawed extract It is not possible to exclude the action of other components other than the tannin.

Method for producing a thaw skin extract containing 10.0 ~ 25.0% by weight of tannin having a significant anti-inflammatory effect according to the present invention,

a) extracting, cooling and filtering thawed skin with 5 to 8 times the amount of water, alcohol or aqueous alcohol solution to obtain a filtrate;

b) separating the filtrate with an aqueous alcohol solution, and then concentrating under reduced pressure at 60-80 ° C. to obtain a concentrate; And

c) adding alcohol to the concentrate, centrifuging at 500 to 1,000 rpm, and then concentrating the filtrate under reduced pressure, vacuum drying, pulverizing and sterilizing to obtain a thawed skin extract.

In more detail, the method of adding the water, alcohol or aqueous alcohol solution to the thawed medicinal herb extract, repeated extraction for 2 to 3 hours, 2 to 5 times, then slowly cooled to room temperature and filtered by centrifugation, etc. Separate from. To the residue is added 5-8 times the amount of water, alcohol, or aqueous alcohol solution of the weight of the mixed herbal medicines, heated and re-extracted, filtered and then mixed with the previous filtrate and filtered.

In the present invention, the extraction efficiency can be increased by adding water or an aqueous alcohol solution to the residue as described above, and re-extracting and filtering. When the amount of the water, alcohol or alcohol aqueous solution used for extraction is less than 5 times, the solubility of the extract is poor. If the extraction efficiency is lowered, and the amount of use exceeds 8 times, the amount of the aqueous alcohol solution used for the separation of the layers increases, and the economical or handling problems may occur such as taking a long time under reduced pressure concentration.

In the present invention, the alcohol may be used both aliphatic and aromatic alcohol generally used in the art, preferably aliphatic alcohol, more preferably of 1 to 6 carbon atoms, such as methanol, ethanol, propanol, butanol, etc. Preference is given to using lower alcohols.

In the present invention, the method of re-extracting after the first extraction is adopted. Even in the case of mass production of the herbal extracts, even though the filtration is effective, the loss occurs because the water content of the herbal medicine itself is high, so the extraction efficiency is reduced only by the first extraction. This is to prevent this. In addition, as a result of verifying the extraction efficiency of each step, it was found that about 80 to 90% of the total extraction amount is extracted by the second extraction, and the third or more multistage extraction is considered to be economical.

As described above, the extract obtained by extraction with water, alcohol or an aqueous alcohol solution over the first and second stages is filtered and concentrated, and then purified impurities, such as unnecessary proteins, polysaccharides and fatty acids contained in the filtrate, in the present invention the same amount as the filtrate The impurities may be purified by performing a layer separation of the aqueous alcohol solution of 2 to 5 times with a solvent to obtain a solvent fraction.

The alcohol used for the layer separation is preferably a lower alcohol having 1 to 6 carbon atoms such as methanol, ethanol, propanol, butanol, and the like, and more preferably a 30% ethanol aqueous solution. In addition, in the layer separation, when the amount of the lower alcohol aqueous solution is less than the filtrate, fine particles are formed by unnecessary components such as fatty acids, so that the layer separation is not smooth and the extraction content of the active active material is lowered. Not efficient

The layered extract was concentrated under reduced pressure at 60 to 80 ° C. to remove residual solvent. The alcohol was recovered by adding the recovered alcohol during concentration under reduced pressure, and then the filtrate was centrifuged at 500 to 1,000 rpm. It may be cloudy and then concentrated under reduced pressure again.

In the present invention, when the reduced pressure concentration temperature is less than 60 ℃ it is difficult to remove the solvent 100%, if it exceeds 80 ℃ may cause a problem in the stability of the concentrate, when the rpm is less than 500 and The separation of alcohol is difficult, and if it is more than 1,000 may cause problems in the stability of the concentrate.

The concentrate is vacuum dried at 0.08 to 0.3 pa at 60 to 80 ° C., and then pulverized and sterilized to 30 to 80 mesh to obtain a powdery thawing extract containing 10.0 to 25.0 wt% of tannin as an active substance. .

Residual amount of lipopolysaccharide was examined as a result of examining the presence or absence of contamination with the residual amount of endotoxin lipopolysaccharide (hereinafter abbreviated as "LPS") with respect to the thawed skin extract containing a certain amount of tannin obtained as described above. Has been found to be 17 endotoxin units (EU) / ml. This is comparable to 350 EU / ml, the standard acceptable range of LPS residues for these test reagents in European countries (Scheer, Arzenimittelforschung, 43 (7): 975-800, July 1993). It was confirmed that it was 1/20 or less of this residual flow allowable range.

The present invention investigated the inhibitory effect of thaw extracts using macrophages, primary immune response cells and representative inflammatory response cells, and used ICR mice, male SD rats, and Balb / c mice (provided by Daejeon Chemical Research Institute). The edema and analgesic inhibition test, the arthritis treatment effect test, and the toxicity on splenocytes, liver, kidney and growth were examined.

As a result, the thawing extract of the present invention significantly inhibits the production of NO and TNF-α, which are inflammatory mediators, inhibits NF-κB phosphorylation, anti-inflammatory analgesic, acute edema, and acute arthritis inhibitory activity. It was confirmed that it has an excellent anti-inflammatory effect.

In addition, the thawing extract of the present invention did not show harmful toxicity in both splenocytes, liver and kidney, it was confirmed that the harmful toxicity to the development of both immature rats and mature mice.

Therefore, it can be seen that the thawing extract of the present invention is a safe substance having excellent anti-inflammatory effect and no toxicity.

Therefore, the thawing extract containing 10.0 to 25.0% by weight of tannin as the active substance of the present invention may be usefully used for medicines, health supplements, etc. for the treatment and prevention of inflammatory diseases, but is not limited thereto.

When the composition of the present invention is used as a medicine, it may include only the herbal extract itself, but may further include appropriate carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, and according to conventional methods Formulations may be made in the form of an acceptable form, for example powders, tablets, granules, capsules or injections.

The carrier or excipient includes water, dextrin, calcium carbonate, lactose, propylene glycol, liquid, paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals, which may be used one or more, but are not limited thereto. Both carriers and excipients can be used. In addition, when formulating the antioxidant composition, it may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

When the thawed skin extract according to the present invention is formulated into tablets by a conventional method, it is preferable to use a combination of lactose, microcrystalline cellulose, magnesium stearate, and the like, and the thawed skin extract in a ratio of 1: 1.

In addition, Haedongpi extract according to the present invention has been used for edible and medicinal since ancient times, there is no particular restriction on the dosage, body absorption, weight, age, sex, health status, diet, time of administration, method of administration , The rate of excretion, the severity of the disease, and the like. However, for the desired effect, the thawing extract of the present invention is preferably administered at 0.001-50 mg, preferably 0.1-10 mg per kg of body weight per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

Haedongpi extract of the present invention can be administered to a variety of routes to mammals, such as mice, mice, livestock. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Tannin-Containing Thawed Extract

Thawed skin washed with water and dried well ( Actinidia polygama ) was added to a 30% ethanol aqueous solution, followed by stirring with hot water twice every two hours with stirring. The hot water extract was slowly cooled to room temperature and then centrifugally filtered to remove the residue. The filtrates were combined and concentrated under reduced pressure at 60 to 80 ° C. The ethanol recovered through the ethanol fraction was added to the concentrate, suspended, centrifugally filtered at 1,000 rpm, concentrated under reduced pressure, dried under vacuum at 60 ° C and 0.08 pa, and then pulverized and sterilized at 80 mesh to obtain a thawed extract. It was.

Tannin (Tannin) content in the thawed extract extracted and purified by the above method was 10.0 ~ 25.0% by weight. In addition, the tannin-containing thaw extract obtained by the above method was analyzed by HPLC under the following conditions, and the results are shown in FIG. 1 (see FIG. 1).

1) Solvent: 50% methanol

2) Column: C-18 COSMOSIL PACKED, 10 ㎛, 4.6 * 250 ㎜

3) flow rate: 0.8 ml / min

4) Column temperature: 20 ℃

5) Detector: UV 280 nm

Experimental Example 1: Examination of the inhibitory effect of tannin-containing thawing skin extract on iNOS, an inflammation mediator from macrophages

In order to investigate the extent to which the tannin-containing thawed skin extract of the present invention inhibits the production of iNOS, a precursor of NO, an inflammatory mediator, it was tested as follows.

1-1. Isolation of Macrophages from Mouse Abdominal cavity

To extract macrophages from the abdominal cavity of mice, 6- to 10-week-old Balb / c mice were inoculated with a thioglycollate medium (produced by Gibco, USA) for at least 2 months, and then 4 days after administration. Was sacrificed by cervical dislocation. Incomplete RPMI-1640 medium (produced by Gibco, USA) with 100 U / ml penicillin-streptomycin added to the abdominal cavity of the sacrificed mice was extracted and centrifuged into macrophages in a 6-well plate. Aliquots were made at 10 ⁶ cells / well concentration. This is about 1 hour CO ₂ After incubation in the incubator, cells and other suspensions that did not adhere to the plate were washed twice with incomplete RPMI-1640 medium followed by 100 mL / well penicillin-streptomycin and 10% fetal bovine serum (1.5 mL / well). Complete RPMI-1640 medium with fetal bovine serum) was injected and incubated in a CO ₂ thermostat (100% humidity, 37 ° C., 5% CO ₂ ) for 3 days.

Purity of macrophages was measured by separating adherent cells, attaching them to slide glass using Cytospin-III (500 rpm, 5 minutes), and using light and Giemsa staining. It was measured by staining, and the purity was 95% or more (data not shown).

1-2. Pretreatment of macrophages with tannin-containing thawing extracts stimulated with LPS

The macrophages obtained in the mouse macrophage isolation process of 1-1 were plated at 3 × 10 ⁶ cells / well in a 6 well plate (product of Falcon, USA), and 100 U / ml penicillin-streptomycin and 10% fetal calf serum were added. Cultured for 3 days using complete RPMI-1640 medium (manufactured by Gibco, USA). After incubation, the culture medium was removed and macronin-containing thawing extracts were stimulated for 2 hours at different concentrations under fresh incomplete RPMI-1640 medium conditions. Then, macrophages were further stimulated for 6 hours with lipopolysaccharide (10 [mu] g / ml; Sigma, USA).

Immediately after completion of the culture for stimulation, the cells were washed three times with cold PBS buffer, and the cells were lysed using a lysis buffer containing various protease inhibitors. After cell lysis, the lysis samples were examined for the amount of iNOS present in the cells using the immunoblotting technique as follows.

1-3. Analysis of Inhibitory Effect of iNOS Production by Tannin-Containing Thawing Extract Using Immunoblotting Technique

Separation gel and top gel of 0.1% SDS polyacrylamide electrophoresis phase used 16% and 3%, respectively, and the electrophoresis current was 55 mA (Powersupply; Pharmacia, Sweden) and temperature cooling system (Sweden) LKB company) was run for about 4 to 5 hours while maintaining 4 ℃. The electrophoresis system used here was Hoefer SE 600 (manufactured by Hoefer-Pharmacia, USA), and each sample was electrophoresed by loading the same total protein amount into the gel.

The electrophoresis gel was pre-wet a nitrocellulose membrane in transfer buffer (39 mM glycine, 2.9 g, 48 mM tris-base, 5.8 g, 0.037% SDS, 20% methanol; pH 8.3). , NC) put on the fiber pad (fiber pad) and assembled. The immunoblotting transfer cassette was then placed in the transfer chamber with the NC membrane in the positive charge direction and the gel in the negative (negative charge) direction. The current was supplied at 1 A, and the temperature was transferred for 1 hour and 30 minutes while maintaining the temperature of 4 DEG C using a cooling system. The NC paper is a TBS-T buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) dissolved in a blocking buffer (5% nonfat dry milk) about 200 Lock 1 ml of Red-Rocker (manufactured by Hoefer, USA) in 1 ml to block the non-specific binding of the antibody, and then again with TBS-T buffer once for 15 minutes, 2 minutes for 10 minutes. Washed twice. Subsequently, 3 ml TBS-T buffer solution is dispensed into a siliconized bottle, into which a rabbit anti-murine iNOS polyclonal primary antibody; PBS buffer at a concentration of 500 μg / 1.5 ml. After dissolving in the solution) 30 μl (1: 300 dilution) was added, the NC paper was placed in a silicone container and reacted at 10 rpm in a 4 ° C hybridization incubator (Robins, USA) for 12 hours. After incubation, washing at 12 rpm in a hybridization incubator three times with 70 ml TBS-T buffer for 20 minutes each, followed by secondary antibody, ie goat anti-rabbit IgG horseradish fur 30 μl (goat anti-rabbit IgG horseradish peroxidase conjugated affinity purified antibody; stock solution dissolved in PBS buffer at 0.02 mg / 4ml) using oxidase binding in 3 ml TBS-T buffer 1: 20,000 dilution) was added and incubated for 3 hours in a hybridization incubator at 4 ° C., and washed 4 times with 70 ml TBS-T buffer for 4 minutes each. After washing, the NC paper was placed in thin transparent vinyl, and 3 ml of 50% of ECL (peroxidase substrate) reaction solution 1 and 2 were dispensed into NC paper and incubated for 5 minutes. Subsequently, in a dark room (10 W safety lamp, manufactured by Kodak, USA), the X-ray film is placed in a cassette containing NC, and the photosensitive film is exposed for 5 or 10 minutes or more, and the X-ray film is developed in a developing box. solution) and reacted for about 3 minutes. After the above process, the X-ray film was washed again in water for 2 minutes, and then reacted for 3 minutes in a fixing solution (fixing solution) and washed again with water for 2 minutes to analyze the band (band).

As a result, the production of iNOS protein by LPS began to be suppressed at the concentration of 1 μg / ml of tannin-containing thawed extract of the present invention, and it was shown that iNOS production by LPS was inhibited to the maximum at 100 μg / ml (see FIG. 2). (a) is the result of immunoblotting, and (b) shows the light concentration measurement analysis of the data (a).

As described above, it was confirmed that the tannin-containing thawed skin extract significantly inhibited iNOS production in a concentration-dependent manner from LPS-stimulated macrophages.

Experimental Example 2: Examination of the effects of tannin-containing thawed skin extracts that inhibit the production of nitrogen oxides (NO)

In order to confirm the inhibitory effect of NO production, which is an inflammation mediator of tannin-containing thawed skin extract of the present invention, it was tested as follows.

2-1. Thawed blood  Extract With tannins  Inflammatory Substances After Pretreatment of Macrophages LPS Stimulated by

The macrophages of Experimental Example 1-1 were plated at 3 × 10 ⁶ cells / well concentration in a 6 well plate (Falcon Co., Ltd.) to complete RPMI to which 100 U / ml penicillin-streptomycin and 10% fetal bovine serum were added. Incubated for 3 days using -1640 medium (available from Gibco, USA). After the incubation, the culture medium was removed and the macrophages-containing thawing extracts were treated at different concentrations and stimulated for 2 hours under fresh incomplete RPMI-1640 medium conditions. Then, macrophages were further stimulated for 6 hours with lipopolysaccharide (10 [mu] g / ml; manufactured by Sigma, USA) as an inducer of inflammation.

Immediately after the cultivation for stimulation, each cell culture was collected and centrifuged (1000 g, 4 ° C., 5 minutes) to collect the culture supernatant, and the amount of NO produced was measured as follows.

2-2. Quantitative Analysis of Nitric Oxide (NO)

Nitrogen oxide NO produced from activated macrophages reacts with oxygen and is converted into nitrite directly, so NO production was examined by measuring nitrite in culture. Nitrite was measured as follows using a grease reaction solution (Griess reagent, Green LC et al., 1982, Anal Biochem., 126 (1), 131-138). The reaction was mixed with 100 μl culture supernatant and 100 μl grease reaction solution and incubated at constant temperature for 10 minutes. After the incubation, the absorbance was measured at 540 nm using an ELISA reader. Reagent for was used sodium nitrite (Sigma, USA).

As a result, the tannin-containing thawed skin extract of the present invention started showing the inhibitory effect of NO production by LPS, an inflammatory-inducing substance, at a concentration of 1 ㎍ / ml, and showed the highest NO production inhibitory effect at the concentration of 100 ㎍ / mL (see FIG. 3). .

As described above, it was confirmed that the tannin-containing thawed skin extract significantly inhibited the production of NO, an inflammatory marker, in a concentration-dependent manner.

Experimental Example 3: Expression inhibitory effect test of tannin-containing thawing skin extract in TNF-α gene (TNF-αmRNA) expression

In order to examine the gene expression inhibitory effect of TNF-α (tumor necrosis factor-α) of the tannin-containing thawed skin extract of the present invention, it was tested as follows.

3-1. Pretreatment of macrophages with tannin-containing thaw extracts stimulated with LPS and isolated RNA

The macrophage cells of Experimental Example 1-1 were plated at a concentration of 3 × 10 ⁶ cells / well in 6-well plates (Falcon, USA) and 100 U / ml of penicillin-streptomycin and 10% fetal calf serum were added thereto. Incubated for 3 days using complete RPMI-1640 medium (available from Gibco, USA). After the cell culture was finished, the culture medium was washed, and then injected with incomplete RPMI-1640 medium (produced by Gibco, USA) containing only 100 U / ml of penicillin-streptomycin. Treatment was stimulated for 2 hours. Then, macrophages were further stimulated for 6 hours with lipopolysaccharide (10 [mu] g / ml; Sigma, USA) as an inducer of inflammation.

After cell stimulation, the cultured cells were washed twice with cold PBS buffer and then incubated at room temperature for 5 minutes by administering 1 ml of Trizol to each well from which the PBS buffer was removed. After the incubation, the solution of the cell dissolved in Trizol was pipetted three times, placed in an E-tube, added 0.2 ml of chloroform, vortexed for 10 seconds to mix the cells well, and centrifuged again (speed 12,000 rpm). , 60 minutes, 4 ° C). Then only the supernatant, the entire RNA layer, was transferred to the E-tubes and precipitated for at least one day with cold 100% isopropanol. Again centrifuged for 20 minutes under the same conditions to discard the supernatant and repeated this process to remove pure total RNA. Then, 75% ethanol was added to the mixture, and the mixture was stirred well by hand and centrifuged. After centrifugation, the supernatant was discarded and 0.1% DEPC-H ₂ O was added and heated at 65 ° C for 10 minutes, and then used at -20 ° C. Saved until.

3-2. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

In order to perform reverse transcription-polymerase chain reaction, all the primers described below were ordered by Bioneer Co., Ltd. (Cheongwon, Chungbuk). Specifically, TNF-α forward primers (5'-GGCAGGTCTTTGGAGTCATTGC-3 ') and reverse primers (5'-CATTCGAGGCTCCAGTGAATTCCAG-3'), and beta-actin forward primers (β-actin forward primers; 5'-CAAAGAAAATGGACGCCGCCGAACTTGG-3 ') and β-actin reverse primer (5'-CCTGCTTGCTTGCTGATCCACATCTGCTGG-3') were used.

To synthesize cDNA by polymerase chain reaction, 5 × reverse transcriptase buffer, 2.5 mM dNTP, 500 ng primer / μl, 200 U / μl MMLV reverse transcriptase (manufactured by Takara, Japan), total RNA, 0.1% The reaction mixture was prepared by adding DEPC-treated secondary distilled water (DDW). The primer used at this time was a reverse primer. Then incubated for 30 minutes at 42 ℃, 30 minutes at 75 ℃ to obtain a cDNA. Then RT mixture containing cDNA obtained during the reverse transcription reaction, 10 × Taq polymerase buffer, 2.5 mM dNTP, forward primer (25 pmol / μl), reverse primer (25 pmol / μl), Taq polymerase (2.5 units / μl; manufactured by Takara Co., Ltd.), and distilled water were mixed well to amplify the cDNA by repeating the reaction conditions for 30 seconds at 95 ° C., 1 minute at 55 ° C., and 30 seconds at 72 ° C., followed by electrophoresis of the cDNA And the difference in mRNA expression of the TNF-α gene was confirmed.

As a result, the tannin-containing thawed extract of the present invention began to suppress the expression of the TNF-α gene at a concentration of 30 ㎍ / ㎖ and showed the suppression of expression of the TNF-α gene at the peak of 300 ㎍ / ㎖ (see FIG. 4). (a) is the result of reverse transcriptase-polymerase chain reaction (RT-PCR), and (b) shows the result of densitometric analysis of (a).

As described above, it was confirmed that tannin-containing thawed skin extract (Tannin) significantly inhibited TNF-α gene expression, which is an index of inflammation.

Experimental Example  4: in macrophages TNF inhibits secretion of -α Tannins  contain Thawed blood  Inhibitory Effect of Extracts

As described in Experiment 3, the tannin-containing thawed skin extract has the ability to inhibit TNF-α gene expression, a marker of inflammation, in macrophages mainly involved in the inflammatory response, but the TNF-α gene expression inhibition is In order to investigate whether it leads to production and secretion inhibition, the following experiments were carried out.

4-1. Tannins  contain Thawed blood  Inflammatory Substances after Pretreatment of Macrophages with Extracts LPS Stimulated by

Macrophages of Experimental Example 1-1 were inoculated in 6-well plates (product of Falcon, USA) at a concentration of 3 × 10 ⁶ cells / well and completely added with 100 U / ml penicillin-streptomycin and 10% fetal calf serum. Cultured for 3 days using RPMI-1640 medium (manufactured by Gibco, USA). After the cell culture was finished, the culture medium was washed and stimulated for 2 hours by treating the macrophages-containing thawing extracts with concentrations with the new incomplete RPMI-1640 medium under the proper medium conditions. Then, macrophages were further stimulated for 6 hours with lipopolysaccharide (10 [mu] g / ml; Sigma, USA).

Immediately after the end of the culture for cell stimulation, each culture was collected, centrifuged (1000 g, 4 ° C., 5 minutes), the culture supernatant was collected, and the amount of TNF-α protein present in the culture supernatant was measured as follows. .

4-2. immune Blotting  Used TNF Quantitative Analysis of -α Proteins

Separation gel and top gel of 0.1% SDS polyacrylamide electrophoresis phase used 16% and 3%, respectively. During electrophoresis, current was supplied at 55 mA (Powersupply; Pharmacia, Sweden) and temperature was cooled. system (manufactured by LKB, Sweden) was run for 4 to 5 hours at 4 ℃. In addition, the electrophoresis system was used Hoefer SE 600 (product of Hoefer-Pharmacia, USA). After the electrophoresis, the obtained gel is a nitrocellulose membrane pre-soaked in transfer buffer (39 mM glycine 2.9 g, 48 mM Tris base 5.8 g, 0.037% SDS, 20% methanol, pH 8.3); NC) was put on and assembled in a fiber pad. The immunoblotting transfer cassette was then placed in the transfer chamber with the nitrocellulose membrane in the positive direction and the gel in the negative (negative charge) direction. The current was supplied at 1 A and the temperature was transferred for 1 hour and 30 minutes while maintaining 4 ° C using a cooling system. The nitrocellulose membrane is a blocking buffer (TBS-T buffer solution dissolved in 5% nonfat dry milk (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20)) Lock at 200 ml using Red-Rocker (Hoefer; CA, USA) for 1 hour and block the non-specific binding of the antibody, then again with TBS-T buffer for 15 minutes. Times, washed twice for 10 minutes.

3 ml of TBS-T buffer solution was then dispensed into a siliconeized bottle, into which a rabbit anti-murine TNF-α polyclonal primary antibody; 500 μg in PBS buffer 30 mL (1: 300 dilution ratio) were added to each of the nitrocellulose membranes in a siliconized bottle, followed by 10 ° C in a hybridization incubator (Robins, USA). The reaction was carried out for 12 hours at rpm speed. Then, using 70 ml of TBS-T buffer, wash at 12 rpm for 3 minutes each in a hybridization reactor for 20 minutes, and use goat anti-rabbit IgG horseradish peroxidase as a secondary antibody. Conjugated affinity purified antibody; stock solution was affinity-separated 0.02 mg / 4 ml in PBS buffer, added 30 µl (1: 20,000 dilution ratio) to 3 ml TBS-T buffer, and then adjusted to 4 ° C. The cells were incubated for 3 hours in the reactivation reactor and washed four times for 20 minutes each using 70 ml TBS-T buffer. Then, the nitrocellulose membrane was placed in thin transparent vinyl, and 3 ml of a reaction solution in which 50% of the peroxidase substrate (ECL) reaction solution 1 and the reaction solution 2 were mixed was dispensed thereon and incubated for 5 minutes. After the reaction, insert the X-ray film into the cassette containing the NC in a dark room (10 W safety lamp; manufactured by Kodak, USA), and after 5 minutes or 10 minutes or more, the developer solution in the box for developing the X-ray film ( After reacting in a develop solution for about 3 minutes, the X-ray film was again washed with water for 2 minutes, then reacted for 3 minutes in a fixing solution, and then washed with water for 2 minutes to analyze the bands. .

As a result, TNF-α protein secretion was suppressed at the concentration of 1 μg / ml of the tannin-containing thawing extract of the present invention, and at the 100 μg / mL concentration, the highest TNF-α secretion was inhibited (see FIG. 5; (a ) Is the result of immunoblotting, and (b) shows (a) light density measurement analysis of the data).

As described above, it was confirmed that the tannin-containing thaw extracts inhibited TNF-α protein production and secretion, which are inflammatory markers, in a concentration-dependent manner.

Experimental Example  5: in macrophages NF -κB phosphorylation (p- NF -κB) Tannins  contain Thawed blood  Inhibitory efficacy test of extract

The production of inflammatory markers NO and TNF-α is produced through the transcription factor NF-κB. Therefore, whether the inflammatory markers NO and TNF-α production were inhibited by NF-κB activation or phosphorylation was inhibited. In order to investigate, it experimented as follows.

5-1. Tannins  contain Thawed blood  Inflammatory Substances after Pretreatment of Macrophages with Extracts LPS Stimulated by

The macrophages of Experimental Example 1-1 were plated at 3 × 10 ⁶ cells / well in 6-well plates (manufactured by Falcon, USA) and completely RPMI- was added with 100 U / ml penicillin-streptomycin and 10% fetal bovine serum. The cells were incubated for 3 days using 1640 medium (available from Gibco, USA). After the incubation, the culture medium was removed and macrophages were treated with tannin-containing thawing extracts by concentration under fresh incomplete RPMI-1640 medium conditions for 2 hours. Then, macrophages were further stimulated for 6 hours with lipopolysaccharide (10 [mu] g / ml; Sigma, USA).

Immediately after completion of the culture for stimulation, the cells were washed three times with cold PBS buffer, and the cells were lysed using a lysis buffer containing various protease inhibitors. After cell lysis, the lysis samples were examined for the amount of NF-κB phosphorylation (p-NF-κB) present in cells using immunoblotting.

5-2. Analysis of NF-κB Phosphorylation (p-NF-κB) Inhibition by Tannin-Containing Thawed Extracts Using Immunoblotting Technique

Separation gel and top gel of 0.1% SDS polyacrylamide electrophoresis phase used 16% and 3%, respectively, and the electrophoresis current was 55 mA (Powersupply; Pharmacia, Sweden) and temperature cooling system (Sweden) LKB company) was run for about 4 to 5 hours while maintaining 4 ℃. The electrophoresis system used here was Hoefer SE 600 (manufactured by Hoefer-Pharmacia, USA). Each sample was electrophoresed by loading the same total protein amount into the gel.

The electrophoresis gel was pre-wet a nitrocellulose membrane in transfer buffer (39 mM glycine, 2.9 g, 48 mM tris-base, 5.8 g, 0.037% SDS, 20% methanol; pH 8.3). , NC) put on the fiber pad (fiber pad) and assembled. An immunoblotting transfer cassette was then placed in the transfer chamber with the NC membrane in the positive direction and the gel in the negative (negative charge) direction. The current was supplied at 1 A, and the temperature was transferred for 1 hour and 30 minutes while maintaining the temperature of 4 DEG C using a cooling system. The NC paper is a TBS-T buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) dissolved in a blocking buffer (5% nonfat dry milk) about 200 Lock 1 ml of Red-Rocker (manufactured by Hoefer, USA) in 1 ml to block the non-specific binding of the antibody, and then again with TBS-T buffer once for 15 minutes, 2 minutes for 10 minutes. Washed twice. Subsequently, 3 ml TBS-T buffer solution was dispensed into a siliconeized bottle, to which a rabbit anti-murine phospho-polyclonal primary antibody; 500 μg / 1.5 30 ml (1: 300 seats) was added to the PBS buffer solution at a concentration of ㎖, and the NC paper was placed in a silicon container at a temperature of 4 ° C in a hybridization incubator (Robins, USA) at 10 rpm for 12 hours. Reacted for a while. After incubation, the cells were washed with 70 ml TBS-T buffer at 20 rpm for 3 minutes each at a hybridization incubator at 12 rpm, followed by a secondary antibody, ie goat anti-rabbit IgG horseradish. 30 μl of affinity isolated antibody (goat anti-rabbit IgG horseradish peroxidase conjugated affinity purified antibody; stock solution dissolved in PBS buffer at 0.02 mg / 4 ml) in 3 ml TBS-T buffer (1: 20,000 dilution) was added and incubated for 3 hours at 4 DEG C, a hybridization incubator, and washed 4 times with 70 ml TBS-T buffer for 4 minutes each. After washing, the NC paper was placed in thin transparent vinyl, and 3 ml of 50% of ECL (peroxidase substrate) reaction solution 1 and 2 were dispensed into NC paper and incubated for 5 minutes. Subsequently, in a dark room (10 W safety lamp, manufactured by Kodak, USA), the X-ray film is placed in a cassette containing NC, and the photosensitive film is exposed for 5 or 10 minutes or more, and the X-ray film is developed in a developing box. solution for 3 minutes, and then the X-ray film was washed again in water for 2 minutes, and then reacted for 3 minutes in a fixing solution and then washed with water for 2 minutes to analyze the band. It was.

As a result, it was shown that the phosphorylation of NF-κB by LPS at the concentration of 1 μg / ml of tannin-containing thawing bark extract was inhibited and the phosphorylation of NF-κB by LPS was inhibited to the maximum at 100 μg / ml (FIG. 6). (A) is the result of immunoblotting, and (b) is the light concentration measurement analysis of the data (a) above).

As described above, it was confirmed that the tannin-containing thawed skin extract of the present invention significantly inhibited the phosphorylation of NF-κB by LPS from macrophages in a concentration-dependent manner.

Experimental Example  6: Tannins  contain Thawed blood  By extract Flame retardant  12-O-tetradecanoylphorbol 13- acetate  ( TPA ) Judo Edema  Suppression test

Tannin-containing thawed skin extract of the present invention was tested as follows to determine whether the anti-inflammatory effect is applied in animal experiments.

Seven-week-old ICR mice were isolated for each experimental group and edema in the ears of rats using TPA (2.5 ㎍ / 20 μl) dissolved in acetone 1 hour after oral administration of 20, 100, 200, 400 mg / kg of thawed blood extract. Induced. At this time, the existing anti-inflammatory agent for comparison was used SK-JOINS product (400 mg / kg). Ear edema was measured in each experimental group 4 hours after the induction of edema, and when the measurement was performed, the mouse was culled using the cervical dislocation method and accurate measurement was induced. The results are shown in Table 1 below.

Inhibitory Effect of Tannin Containing Thapa Extract on TPA-induced Ear Edema process Thickness of the ear Inhibition rate of inflammation (%) Control 0.67 ± 0.02 - Negative control 0.23 ± 0.06 65 Control reagent (SK-JOINS 400 mg / kg) 0.44 ± 0.07 34 Thawed blood (400 mg / kg-tannin content 15.0 wt%) 0.43 ± 0.03 36 Thawing blood (200 mg / kg-tannin content 15.0 wt%) 0.50 ± 0.07 31 Thawed blood (100 mg / kg-tannin content 15.0 wt%) 0.59 ± 0.01 25 Thawing blood (20 mg / kg-tannin content 15.0 wt%) 0.61 ± 0.02 9 1. Data represent the mean of difference in ear thickness (mm) ± S.E.M (n = 12) 2. Control group: no experimental group causing edema 3. Negative control group: no drug treatment group after edema induction

As shown in Table 1, when the tannin-containing thawed skin extract 400 mg / kg concentration was the highest inhibition rate of 39%, which did not show a significant difference compared to the existing anti-inflammatory agent SK-JOINS, of the present invention It was confirmed that tannin-containing thawed skin extracts had a concentration-dependent effect of inhibiting TPA induced ear edema.

Experimental Example 7: A test for suppressing arachidonic acid induced ear edema by tannin-containing thawing extract

In order to confirm the arachidonic acid-induced ear edema inhibitory effect of the tannin-containing thaw skin extract of the present invention, it was tested as follows.

Seven-week-old ICR mice were isolated in each experimental group, and then SK-JOINS was dissolved in acetone after 1 hour after oral administration of 400 mg / kg SK-JOINS (comparative drug) and 20, 100, 200, 400 mg / kg of Haedongpi extract. Arachidonic acid (2 μg / 20 μl) was used to cause edema in the ear. Ear edema was measured in each experimental group one hour after the induction of edema, and when the measurement was performed, the mouse was selected using the cervical dislocation method and accurate measurement was induced. The results are shown in Table 2 below.

Inhibition of Arachidonic Acid-Induced Ear Edema by Tannin-Containing Thawing Extract process Thickness of the ear Inhibition rate of inflammation (%) Control 0.55 ± 0.01 - Negative control 0.21 ± 0.021 62 SK-JOINS 400 mg / kg 0.30 ± 0.02 45 Thawed blood (400 mg / kg-tannin content 15.0 wt%) 0.25 ± 0.05 55 Thawing blood (200 mg / kg-tannin content 15.0 wt%) 0.31 ± 0.05 44 Thawed blood (100 mg / kg-tannin content 15.0 wt%) 0.47 ± 0.08 15 Thawing blood (20 mg / kg-tannin content 15.0 wt%) 0.53 ± 0.01 4 1. Data represent the mean of difference in ear thickness (mm) ± S.E.M (n = 12) 2. Control group: no experimental group causing edema 3. Negative control group: no drug treatment group after edema induction

As shown in Table 2, when the tannin-containing thawed skin extract 400 mg / kg concentration it was found that the highest inhibition of arachidonic acid-induced ear edema inflammation, the tannin-containing thawed skin extract of the present invention concentration-dependent arachidonic acid-induced ear edema It was confirmed that remarkably suppressed.

Experimental Example 8: Acetic acid-induced stretching inhibition test by tannin-containing thawing extract

In order to confirm whether the tannin-containing thawed skin extract of the present invention is effective in relieving and suppressing pain as well as ear edema, the experimental method of the conventional acetic acid induced stretching inhibition test (Hermendez-Perez M, Rabanal RM, J. Ethnopharmacol. 81 : 43-47, 2002).

Seven-week-old ICR mice were isolated for each herbal candidate, followed by 400 mg / kg of SK-JOINS (as a comparative drug), and 1 hour after oral administration of 20, 100, 200, 400 mg / kg of Haedongpi extract (0.7%). ) Intraperitoneal administration (0.1 mL / 10 g) caused organ inflammation. After 10 minutes, we observed stretching of the mouse for 10 minutes because it causes itching during edema, which can be used as a pain index.

Inhibition of Acetic Acid-Induced Stretch by Tannin-Containing Thawing Extract process Writhing number % Pain inhibition Control 30.60 ± 1.6 - SK-JOINS (400 mg / kg) 19.5 ± 1.9 36 Thawed blood (400 mg / kg-tannin content 20.0 wt%) 15.2 ± 1.4 50 Thawing blood (200 mg / kg-tannin content 20.0 wt%) 20.1 ± 1.1 34 Thawed blood (100 mg / kg-tannin content 20.0 wt%) 24.3 ± 1.3 21 Thawed skin (20 mg / kg-tannin content 20.0 wt%) 26.2 ± 0.8 13 1. Control group: Drug-free experimental group after stretching induction

As shown in Table 3, it was found that the tannin-containing thaw extracts had a high acetic acid-induced stretching inhibitory effect at a concentration of 400 mg / kg, which showed more analgesic inhibition rate than SK-JOINS, a comparative anti-inflammatory agent, containing tannins of the present invention. Haedipi extract was found to have a concentration-dependent acetic acid-induced stretching inhibitory effect.

Experimental Example  9: Tannins  contain Thawed blood  Treatment Effect of Acute Arthritis by Extracts

In order to test the acute arthritis therapeutic effect of the tannin-containing thawing extract of the present invention, the experiment was carried out as follows.

Pre-prepared SK-JOINS in male SD rats (about 200 g) (400 mg / kg) (used as a comparative drug), and 1 ml of 20%, 100, 200 and 400 mg / kg of thawing extracts were orally administered and 1% carrageenan Acute foot edema was induced by injection of 100 μl of saline into the left foot. Edema was measured after 3 hours using a plethysmometer (LE 7500) and the results are shown in Table 4 below.

Treatment of Acute Arthritis by Tannin-Containing Thawing Extract process Inflammation-induced degree (%) Control 0 Positive control 84 SK-JOINS (400 mg / kg) 51 Thawed blood (400 mg / kg-tannin content 15.0 wt%) 53 Thawing blood (200 mg / kg-tannin content 15.0 wt%) 60 Thawed blood (100 mg / kg-tannin content 15.0 wt%) 72 Thawing blood (20 mg / kg-tannin content 15.0 wt%) 79 1. Control group: Experimental group that did not cause edema 2. Positive control group: Drug-free experimental group after edema induction

As shown in Table 4, it can be seen that the tannin-containing thaw extract extract has the highest acute arthritis treatment effect at a concentration of 400 mg / kg, which shows a better effect than the SK-JOINS anti-inflammatory agent, tannin of the present invention. It was found that the extracts of the thawed skin had a significant acute arthritis treatment effect in a concentration-dependent manner.

Experimental Example 10: Cytotoxicity Test of Tannin-Containing Thawed Skin Extract

In order to investigate the cytotoxic stability of the tannin-containing tannin-containing thaw extract of the present invention, the following experiments were carried out.

10-1. Splenocytes  Extraction and culture

Seven-week-old Balb / c mice (Daejeon Chemistry Institute, Korea) were sacrificed by cervical dislocation and the spleen was recovered aseptically, followed by cold incomplete RPMI-1640 medium supplemented with 100 U / ml penicillin-streptomycin alone (Gibco, USA). After washing twice with), the cold incomplete RPMI-1640 medium was injected again using a Daunce cell homogenizer (manufactured by Wheaton, USA) to make each cell and incubated for 10 minutes in ice water. After incubation, only the cell supernatant was collected, centrifuged (1,000 g, 5 min, 4 ° C.), and the supernatant was again poured out to obtain cells. Splenocytes were dispensed in 96-well plates at a concentration of 1.5 × 10 ⁵ cells / well and cultured in complete RPMI-1640 medium (produced by Gibco, USA) with 100 U / ml penicillin-streptomycin and 10% fetal calf serum. It was. Then, tannin-containing thawed skin extract was added to each well by concentration and incubated for 2 days to perform MTT analysis.

10-2. For chemical cytotoxicity testing MTT  analysis

MTT analysis was performed according to the method of Francois D and Lita L., J. Immunological methods, 89, 271-277, 1986. When the culturing of splenocytes was completed, 50 μl of MTT reaction solution (concentration 1 mg / ml in HBSS buffer solution) was added to each well, and the cells were cultured again at 37 ° C. for 4 hours. Subsequently, the culture supernatant was removed, and 100 µl of dimethylsulfoxide (DMSO) was added to each well to dissolve the precipitated insoluble formazan, followed by stirring for 15 minutes, followed by ELISA reader (ELISA reader; US Pharmacia Co., Ltd.) was used to measure the absorbance (OD) at the wavelength of 540 nm to measure% cytotoxicity according to the following formula, and the results are shown in FIG. 7.

Figure 7 shows the toxicity of the tannin-containing thawed skin extract of the present invention on the spleen cells of the experimental animal as a graph of survival rate, wherein the concentration of tannin, the thawed skin extract ranges from 1 to 100 ㎍ / ㎖ and the stimulation time is 2 days, NS (non-stimulation) was stimulated with saline instead of thawing extract.

Degree As shown in Figure 7, the tannin-containing thawed skin extract of the present invention did not have a toxic effect on splenocytes even at a high concentration of 100 ㎍ / ㎖, but showed a slightly higher survival rate compared to the control.

As described above, it was confirmed that the tannin-containing thawed skin extract of the present invention had no cytotoxicity and was safe as an anti-inflammatory agent.

Experimental Example 11: Long-term toxicity test of tannin-containing thawing extract

In order to examine the effects of the tannin-containing thawed skin extract of the present invention on liver and kidney, which are the main organs sensitive to metabolism and toxicity of living organisms, the following experiments were carried out. Kidney function is examined by measuring blood levels of blood creatin (CRE) and blood urea nitrogen (BUN), and liver function is measured by GOT (glutamic-oxalacetate transaminase) and GPT (glutamic pyruvic) transaminase) was tested by measuring blood levels of both enzymes.

Using 7-week-old mice (Balb / c; Daejeon Chemical Research Institute, Korea), 10 ㎍ and 100 ㎍ of tannin-containing thawing bark extract were dissolved in 2 ml of 0.01 M PBS buffer solution and administered 2 ml once daily to the vein. In the normal group, only 2 ml of PBS buffer solution was administered once daily, and the same procedure was performed. After 1, 5 and 10 days after intravenous injection, 10 μl of serum obtained from the eye of the mouse was placed on a multilayer film, and the Fuji Dry Chem System (Fugi Photo Film, Japan) was used. CRE, BUN, GOT and GPT values were measured, respectively, and the results are shown in FIGS. 8 and 9.

8 and 9 are graphs showing the liver and kidney toxicity effects of tannin-containing thawed skin extract, respectively, 30 and 300 μg of tannin-containing thawed skin extracts of the present invention were administered daily to mice, and blood was collected at 1, 5 and 10 days. Toxic effects on liver and kidney tissues were examined (GOT, GPT, CRE, BUN), and control group (NS) was stimulated with saline instead of thawed extract.

As shown in FIGS. 8 and 9, in the experimental group administered 30 ㎍ of tannin-containing thawed skin extract, blood levels of GOT, GPT, CRE, and BUN were almost the same as those of the normal group. There was no difference (FIG. 8 and FIG. 9).

As described above, it was confirmed that the tannin-containing thawed skin extract of the present invention did not have harmful toxicity in both liver and kidney.

Experimental Example 12: Weight Change Test of Experiment Animals Using Tannin-Containing Thawing Extract

In order to confirm the safety of the tannin-containing thawed extract of the present invention, the effect on body development of immature or mature rats was investigated by weight change.

Tannin-containing thawed bark extracts were dissolved in 0.01 M PBS buffer solution at a concentration of 10 μg / ml and 100 μg / ml, and subcutaneously injected daily in immature mice at 1 day of age, and injected intraperitoneally daily in 7-week-old mature mice. (NS) was injected subcutaneously or intraperitoneally with 0.01 M PBS buffer solution. The results are shown in FIGS. 10 (for immature rats) and 11 (for immature rats).

As shown in FIGS. 10 and 11, it was found that the concentrations of 10 μg / ml and 100 μg / ml of the tannin-containing thaw extracts of both immature and mature mice did not show a significant difference compared to the control.

As described above, the tannin-containing thawed skin extract of the present invention not only had a strong anti-inflammatory and analgesic effect, but also showed no toxicity to cells, tissues and body development in animal experiments using mice.

Hereinafter, the preparation examples of the pharmaceutical composition will be described, but the present invention is not intended to limit the present invention, but is only intended to be described in detail.

Preparation Example 1 Preparation of Tablet

Using the tannin-containing thawed skin extract of the present invention, tablets for oral administration with the following compositions are prepared using wet granulation and dry granulation.

[Furtherance]

Tannin-Containing Thawed Extract ........... 200 mg

Hard silicic anhydride ......................................... 10 mg

Magnesium Stearate ......................................... 2 mg

Microcrystalline cellulose ............... 50 mg

Sodium starch glycolate ........................ 25 mg

Lactose ............... 101 mg

Povidone ...................................... 12 mg

Ethanol anhydrous .........................

Preparation Example 2 Preparation of Ointment

Using the tannin-containing thawed skin extract of the present invention to prepare an ointment with the following composition.

[Furtherance]

Tannin-containing thawed skin extract ..................... 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 profile ....................... 1 g

Methyl paraoxybenzoate ......................... 1 g

Phosphoric Acid and Purified Water ...................

Preparation Example 3 Preparation of Injection

An injection is prepared using the tannin-containing thaw extract of the present invention with the following composition.

[Furtherance]

Tannin-Containing Thawed Extract ...... 100 mg

Mannitol ............... 180 mg

Sodium dihydrogen phosphate ............. 25 mg

Purified water for injection ............. 2,974 mg

Preparation Example 4 Preparation of Transdermal Agent

Using the tannin-containing thawed extract of the present invention to prepare a transdermal agent with the following composition.

[Furtherance]

Tannin-containing thawed skin extract ............ 0.4 g

Sodium polyacrylate ............... 1.3 g

Glycerin ......................... 3.6 g

Aluminum hydroxide ........................ 0.004 g

Methyl Paraben ......................... 0.2 g

Acrylic adhesive solution ................................. 14 ml

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described above, the thawing extract containing a certain amount of tannins according to the present invention is a substance derived from natural products, has no toxicity and side effects, significantly inhibits the production of inflammatory mediators NO and TNF-α, and mediates inflammation. As it has excellent anti-inflammatory effects such as NF-κB phosphorylation inhibitory action, anti-inflammatory analgesic action, acute edema suppression effect, and acute arthritis inhibitory action, the composition containing it is arthritis, otitis media, pain caused by inflammation and inflammatory reaction It can be usefully used as an effective and safe medicine for the treatment and prevention of inflammatory diseases such as edema or degenerative diseases.

<110> Konkuk University Industrial Coopertaion Corp. <120> Composition for the anti-inflammatory and alagesia comprising          tannin-contained Kalopanacis Cortex extract <130> P07-E208 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggcaggtctt tggagtcatt gc 22 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cattcgaggc tccagtgaat tccag 25 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 caaagaaaat ggacgccgcc gaacttgg 28 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cctgcttgct tgctgatcca catctgctgg 30  

Claims (7)

An anti-inflammatory analgesic composition comprising as an active ingredient an extract of Actinidia polygama containing 5.0 to 30.0% by weight of tannin. The anti-inflammatory analgesic composition according to claim 1, wherein the tannin is 10.0 to 25.0 wt%. The anti-inflammatory analgesic composition according to claim 1, which has an anti-inflammatory analgesic effect against arthritis, otitis media, edema, pain or degenerative diseases caused by inflammation and inflammatory reactions. 1) extracting, cooling and filtering thawed blood with 5 to 8 times the amount of water, alcohol or aqueous alcohol solution; 2) separating the filtrate with an aqueous alcohol solution and then concentrating under reduced pressure at 60 to 80 ° C; And 3) adding alcohol to the concentrate, centrifuging at 500 to 1,000 rpm, and then concentrating the filtrate under reduced pressure, vacuum drying, pulverizing and sterilizing to obtain a thawed blood extract; thawed blood containing tannins, comprising the Method of Preparation of Extracts. 5. The method of claim 4, wherein the alcohol is 30% ethanol aqueous solution. 5. The method of claim 4, wherein the thawing extract is a powder. 5. The method of claim 4, wherein the tannin is 10.0-25.0 wt%.

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