KR100912735B1 - Composition for the anti-inflammatory and analgesia comprising rutin-contained Sophora japonica extract - Google Patents

Abstract

The invention goehwa containing routines (Rutin) of 10.0 ~ 25.0% by weight in terms of active material (Sophora japonica ) method for producing an extract and an anti-inflammatory analgesic composition comprising the same as an active ingredient, and more particularly, a lumped extract prepared to contain a certain amount of rutin using a method such as filtration and concentration under reduced pressure with an aqueous alcohol solution. An anti-inflammatory analgesic composition comprising as an active ingredient.

The lumped extract containing a certain amount of rutin according to the present invention is a substance derived from natural products and has no toxicity and no side effects. As it has an excellent anti-inflammatory effect such as inhibitory action, anti-inflammatory analgesic action, acute edema suppression effect, and acute arthritis suppression action, the composition containing it is an effective and safe medicine for the treatment and prevention of inflammatory diseases such as arthritis, edema, pain, etc. It can be usefully used.

Description

Composition for the anti-inflammatory and analgesia comprising rutin-contained Sophora japonica extract

The present invention relates to a method for preparing Sophora japonica extract containing rutin (Rutin) of 10.0 to 25.0% by weight as an active substance, and to an anti-inflammatory analgesic composition using the same as an active ingredient, more specifically, an aqueous alcohol solution extract solvent. It relates to an anti-inflammatory analgesic composition comprising as an active ingredient a lump extract which is prepared to contain a certain amount of rutin by using methods such as filtration and vacuum concentration.

Inflammation refers to the pathological state of an abscess formed by the invasion of external bacteria, and the inflammatory response is a biological defense reaction process for repairing and repairing damage caused by an invasion that causes any organic change in cells or tissues of a living body. When infected with (cell) damage or external infectious agents (bacteria, fungi, viruses, various types of allergens), various inflammatory mediators and immune cells in local blood vessels and body fluids are involved, resulting in enzyme activation, secretion of inflammatory mediators, and body fluids. It exhibits a series of complex physiological reactions such as infiltration, cell migration and tissue destruction, as well as external symptoms such as erythema and edema fever pain. In normal cases, the inflammatory response removes external infectious agents and regenerates damaged tissues to restore the function of life.However, if the inflammatory response occurs excessively or continuously due to the absence of antigens or internal substances, the major pathology of the disease (sensitizing disease, Chronic inflammation) and is also a barrier to the treatment of blood transfusions, drug administration, and organ transplantation.

Inflammatory factor NO (nitric oxide), which is involved in this inflammatory reaction, is passed through the intermediate metabolite hydroxyarginine (L-arginine) from L-arginine by NOS (nitric oxide synthase (NOS)). And citrulline are made. NO is biologically active and has a very small molecular weight. It is a mediator involved in vasodilation, platelet adhesion and aggregation, neurotransmission, and digestive system movement by acting on the vascular system in vivo. It has been shown to be responsible for the action (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, and thus the stabilized end products NO ₃ (nitrate) and NO ₂ (nitrite) It is known to convert quickly to (Snyder, SH et al., Scientific American, May, pp 28-35, 1992).

On the other hand, NOS participating in NO production is classified as a calcium (Ca ^{2 +)} or calmodulin (camodulin), there are two main i.e., cNOS (constitutive NOS) and iNOS (inducible NOS) in accordance with the dependence whether, cNOS is calcium, 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).

Goehwa, also known as paintings in the herbaceous wood, kills roundworms and governs fever, chiru, and long winds. It is a deciduous arborescent tree of legumes, reaching 10 m in height, and the leaf is composed of 6 to 8 pairs of leaflets of egg-shaped leaves. Aggregation has been prescribed in traditional Chinese medicines and related literature such as Dongbobom, Hyanggyeobbang and light-dose saliva as a single-sided herbal medicine. There is no description of active active ingredients expressing drug efficacy among the lumped ingredients.

On the other hand, recently, in Korea, it has been disclosed for the treatment of inflammatory periodontal disease of lump extract extracted with 100% methanol (Eui Eun et al., Journal of Korean Periodontology, 35 (1): 9-17, 2005), and also registered Korean Patent No. 509682 Is for the composition for the prevention and treatment of menopausal diseases containing the extract as an active ingredient, Korean Patent No. 539457 is registered in Korea patent for the composition for the prevention and treatment of hyperlipidemia, arteriosclerosis and fatty liver containing a gangrene extract No. 10-0539456 relates to a composition for the prevention and treatment of cancer comprising a lump extract, in Korean Patent Application No. 10-2004-0082006, various herbal medicines including lumps are used for the treatment of hemorrhoids or gynecological diseases And it discloses about a bath bag containing the same. Absorption extracts in rat model experiments that caused cerebral infarction overseas have the effect of reducing interleukin-1 production, microglial cell activity, and brain cell death like Ginkgo biloba, which leads to brain diseases such as human cerebral infarction. A paper has been published that suggests it will be effective in treatment (Lao CJ et al., Am J Chin Med. 33 (3): 425-438, 2005).

However, it has not been disclosed to date which of the lumping components inhibits mediators and cells related to inflammation.

Accordingly, the present inventors conducted a thorough study to determine the anti-inflammatory effect of the specific active ingredient and the extract according to the content of the active ingredient having a significant anti-inflammatory effect in the lump extract, lump extract containing 10.0 ~ 25.0% by weight of rutin as the active substance This excellent anti-inflammatory effect was confirmed to complete the present invention.

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

Still another object of the present invention is to provide a method for preparing a lump extract containing 10.0-25.0% by weight of rutin as an active substance having excellent anti-inflammatory effect.

In order to achieve the above object, the present invention is Sophora containing 5.0 to 30.0% by weight of rutin as the active material japonica ) provides an anti-inflammatory analgesic composition comprising the extract as an active ingredient.

In the present invention, the routine may be characterized in that 10.0 ~ 25.0% by weight, characterized in that excellent anti-inflammatory analgesic effect against arthritis, otitis media, edema, pain or degenerative diseases caused by inflammation and inflammatory reactions can do.

In addition, the present invention comprises the steps of: a) heating the solidified solution with 30% aqueous ethanol (v / v) of 5 to 8 times the amount of the monohydrate at 80 ° C. for 3 hours, followed by extraction, cooling and filtration at room temperature to obtain a filtrate; b) The residue remaining after the filtrate was obtained in step a) was heated with 80% aqueous solution of 30% ethanol (v / v) at 5 to 8 times the amount of residue for 3 hours at 80 ° C. and then reextracted, cooled and filtered at room temperature. To obtain a filtrate; And c) combining the filtrates obtained in steps a) and b) and concentrating under reduced pressure at 60 to 80 ° C., and obtaining a lump extract through thermal vacuum drying and pulverization. It provides a method of manufacturing.

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

In addition, in the present invention, the lumped extract may be characterized in that the powder.

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

The present invention relates to a method for preparing Sophora japonica extract containing a certain amount of Rutin as an active substance and an anti-inflammatory analgesic composition comprising the same as an active ingredient.

The anti-inflammatory analgesic composition of the present invention contains a lump extract as an active ingredient, and the lump extract contains 5.0 to 30.0 wt% Rutin as an indicator and an active substance, more preferably 10.0 to 25.0 wt 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, rutin was selected as an indicator and an active substance to obtain a lump extract to maximize the expression of the desired anti-inflammatory effect.

The lumped extract used in the treatment of inflammatory diseases of the present invention can be used by extracting the specific active substance routine from the extract itself (medicinal extract) extracted from the lump or the lumped extract. The blending ratio at this time is determined by the content of rutin, which is an indicator substance and an active substance, irrespective of the original content of the block, preferably contains 5.0 to 30.0% by weight of rutin in the block, and more preferably 10.0 to 25.0. It should contain a weight percent routine.

In the present invention, when the content of the rutin is less than 10.0% by weight, the result is relatively poor in the treatment of arthritis and the like. Therefore, the content of the rutin 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 routine content, but if it contains more than a certain level, the effect does not increase any more, and is not preferable in terms of manufacturing, technical and economical 30.0% by weight. It is good to make it contain below about 25.0 weight% more preferably.

In the present invention, when the indicator substance and the active substance, rutin, which 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 lump extract, but the lump obtained in the present invention It is not possible to rule out the action of other ingredients than the above routines in the extract.

The preparation method of the lump extract containing 10.0-25.0% by weight of rutin having a significant anti-inflammatory effect according to the present invention comprises the following steps.

a) heating the solidified solution with a 30% aqueous ethanol solution (v / v) of 5 to 8 times the amount of the monohydrate at 80 ° C. for 3 hours, followed by extraction, cooling and filtration at room temperature to obtain a filtrate;

b) The residue remaining after the filtrate was obtained in step a) was heated with 80% aqueous solution of 30% ethanol (v / v) at 5 to 8 times the amount of residue for 3 hours at 80 ° C. and then reextracted, cooled and filtered at room temperature. To obtain a filtrate; And

c) combining the filtrates obtained in steps a) and b) and concentrating under reduced pressure at 60-80 ° C. to obtain a lump extract through thermal vacuum drying and grinding.

To explain the above production method in more detail, 30% ethanol aqueous solution (v / v) was added to the crude probiotic, repeated extraction for 2 to 3 hours, 2 to 5 times, and then cooled slowly at room temperature, followed by centrifugation and the like. Filtration with water separates the residue. 30% ethanol aqueous solution (v / v) of 5 to 8 times the amount of the residue was added to the residue, and the mixture was filtered again after heating, re-extracting and filtering the filtrate. In the present invention, 30% ethanol aqueous solution (v / v) is added to the residue as described above, and the extraction efficiency can be improved by re-extracting and filtering, wherein the amount of 30% ethanol aqueous solution (v / v) used for extraction is 5 times. If less than the solubility of the extract is poor extraction efficiency, if the amount is more than 8 times the use of the decompression concentration may take a long time, such as economical or handling problems may occur.

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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.

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The concentrate was thermally 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 solidified extract containing 10.0 to 25.0 wt% of rutin as the active substance. have.

Lipopolysaccharide was examined for contamination by the residual amount of lipopolysaccharide (hereinafter abbreviated as "LPS"), which is endotoxin of bacteria, to the lump extracts containing a certain amount of rutin obtained as described above. The residual amount of was found to be 17 endotoxin units (EU) / ml. This is compared with 350 EU / mL (Scheer, Arzenimittelforschung, 43 (7): 975 to 800, July 1993), which is the standard permissible range of LPS residues in European countries. It shows that it is 20 or less.

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

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

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

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

Therefore, the lump extract containing 10.0 to 25.0% by weight of rutin as the active substance of the present invention can 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 formulated into tablets according to the present invention in the form of tablets, it is preferable to combine lactose, microcrystalline cellulose, magnesium stearate, and the like, which are used as bases, and to use the monohydrate extract in a ratio of 1: 1.

In addition, the lump 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, patient age, sex, health status, diet, administration time, administration method , The rate of excretion, the severity of the disease, and the like. However, for the desired effect, the lumped 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.

The lump extract of the present invention can be administered to mammals such as mice, mice, livestock, etc. by various routes. 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: containing rutin Lump  Preparation of Extract

Sophora japonica , washed with water and dried well, 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 to 80 mesh to obtain a lump extract. It was.

Rutin content was 10.0 ~ 25.0% by weight in the lump extract extracted and purified by the above method. In addition, the rutin-containing monohydrate 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: Inflammatory mediated precursor from macrophages iNOS  Screening of Inhibitory Effects of Rutin-Contained Monohydrate Extracts in Production

In order to investigate the extent to which the rutin-containing lump 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 the administration of the mice. 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). Full 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 rutin-containing lump 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 the incubation, the culture medium was removed and macrophages were stimulated for 2 hours by treatment with the concentration of monoclonal extract containing rutin under fresh incomplete RPMI-1640 medium. 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. Inhibition of iNOS Production by Routine-Contained Monohydrate Extract Using Immunoblotting

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 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 a concentration of 1 μg / ml of rutin-containing lump extract, and the maximum iNOS production by LPS was inhibited at 100 μg / ml (see FIG. 2; a is The result of immunoblotting, b represents the light density measurement of the data a).

As described above, the rutin-containing lump extract was found to have an effect of significantly inhibiting iNOS production in a concentration-dependent manner from the macrophage stimulated LPS.

Experimental Example  2: nitric oxide, an inflammation mediator ( NO Contains rutin to suppress production Lump  Examine the Effect of Extracts

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

2-1. Pretreated macrophages with rutin-containing lump extracts and stimulated with LPS

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 period, the culture medium was removed and the macrophages were stimulated for 2 hours by treatment with the concentration-containing lumped extracts under the condition of fresh incomplete RPMI-1640 medium. 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 (1,000 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 NOx

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, at 1 ㎍ / ㎖ concentration of lumps containing rutin began to show the effect of inhibiting NO production by LPS, an inflammation-causing substance, showed a maximum NO production inhibitory effect at a concentration of 100 ㎍ / ㎖ (see Figure 3).

As described above, the rutin-containing lump extract was found to have an effect of significantly inhibiting the production of inflammatory markers NO in a concentration-dependent manner.

Experimental Example 3: Expression inhibitory effect test of rutin-containing lump extract in the expression of TNF-α gene (TNF-alpha mRNA)

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

3-1. Pretreatment of macrophages with rutin-containing lump extract and stimulation with IPS-inducing LPS and isolation of total 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 inoculated with incomplete RPMI-1640 medium (manufactured by Gibco, USA) containing only 100 U / ml of penicillin-streptomycin, and the concentrations of rutin-containing monohydrate extract under appropriate medium conditions Treatment was stimulated for 2 hours. Then, macrophages were further stimulated for 6 hours with lipopolysaccharide (10 [mu] g / ml; Sigma, USA).

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, forward primer (5'-GGCAGGTCTTTGGAGTCATTGC-3 ') and reverse primer (5'-CATTCGAGGCTCCAGTGAATTCCAG-3') of TNF-α, and beta-actin forward primer (β-actin forward primer; 5'-CAAAGAAAATGGACGCCGCCGAACTTGG-3 ') and β-actin reverse primer (5'-CCTGCTTGCTTGCTGATCCACATCTGCTGG-3') were used.

To synthesize cDNA by polymerase chain reaction, 5 ㅧ reverse transcriptase buffer, 2.5 mM dNTP, 500 ng primer / μl, 200 U / μl MMLV reverse transcriptase (from 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. The difference in mRNA expression of the TNF-α gene was confirmed.

As a result, the rutin-containing monoclonal extract of the present invention began to suppress the expression of the TNF-α gene at a concentration of 1 μg / ml, but showed the inhibition of expression of the TNF-α gene at the highest concentration of 100 μg / ml (see FIG. 4). a is the result of reverse transcriptase-polymerase chain reaction, and b is the light density measurement of the a data.

As described above, it was confirmed that rutin containing rutin significantly inhibited TNF-α gene expression as an index of inflammation.

Experimental Example 4: Test of the inhibitory effect of rutin-containing lump extract to inhibit the secretion of inflammatory mediator TNF-α in macrophages

As shown in Experiment 3, the rutin-containing lump extracts have the ability to inhibit TNF-α gene expression, which is an index of inflammation, in macrophages mainly involved in the inflammatory response, but such 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. Pretreatment of macrophages with rutin-containing lump extracts stimulated with LPS

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 with rutin-containing monoclonal extracts at different concentrations with appropriate incomplete RPMI-1640 medium. 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. Quantitative Analysis of TNF-α Proteins Using Immunoblotting

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 charge direction and the gel in the 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 for about 3 minutes in the develop solution), the X-ray film was again washed with water for 2 minutes and then reacted for 3 minutes in a fixing solution and washed with water for 2 minutes to analyze the bands.

As a result, the concentration of TNF-α protein began to be suppressed at the concentration of 1 μg / ml of rutin-containing monoclonal extract of the present invention, and the highest concentration of TNF-α was inhibited at 100 μg / ml (see FIG. 5; Results of immunoblotting, 5b represents light concentration measurement analysis of 5a data).

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

Experimental Example 5: Inhibition efficacy test of rutin-containing lump extract that inhibits NF-κB phosphorylation (p-NF-κB) in macrophages

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. Pretreatment of macrophages with rutin-containing lump extracts stimulated with LPS

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 stimulated for 2 hours by treatment with the concentration of monoclonal extract containing rutin under fresh incomplete RPMI-1640 medium. 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 Rutin Containing Extracts Using Immunoblotting

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 electrophoretic 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. 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 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 about 200 ml of TBS-T buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) in a blocking buffer (5% nonfat dry milk). Locked with Red-Rocker (manufactured by Hoefer, USA) for 1 hour to block the non-specific binding of the antibody, and then once again for 15 minutes with TBS-T buffer, twice for 10 minutes. Thereafter, 3 ml of TBS-T buffer was dispensed into a siliconized bottle, to which a rabbit anti-murine phospho-polyclonal primary antibody; 500 30 µl (1: 300 seats) was added to the PBS buffer solution at a concentration of ㎍ / 1.5 ml, and the NC paper was placed in a silicone container at 10 rpm in a 4 ° C hybridization incubator (Robins, USA). After incubation, the cells were incubated for 20 minutes with 70 ml TBS-T buffer. Washed at 12 rpm in a hybridization incubator three times each, and then subjected to affinity separation using a secondary antibody, ie goat anti-rabbit IgG horseradish peroxidase binding. -rabbit IgG horseradish peroxidase conjugated affinity purified antibody; stock solution dissolved in PBS buffer at a concentration of 0.02 mg / 4ml; add 30 µl (1: 20,000 dilution) to 3 ml TBS-T buffer, After incubation for 3 hours in the incubation incubator for 4 minutes, each wash for 20 minutes with 70 ml TBS-T buffer After washing, the NC paper is placed in a thin transparent plastic and the reaction solution 1 and 2 of ECL (peroxidase substrate) reaction 50% each 3 ml of the mixture was 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 rutin-containing lump extract was inhibited and the phosphorylation of NF-κB by LPS was inhibited to the maximum at 100 μg / ml (FIG. 6). 6a is the result of immunoblotting and 6b is the light density measurement of the 6a data.

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

Experimental Example 6: 12-O-tetradecanoylphorbol 13-acetate-induced ear edema suppression test by rutin-containing lump extract

Rutin-containing lump 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 in 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 lump extracts 20, 100, 200, 400 mg / kg. Induced. At this time, the existing anti-inflammatory drugs for comparison were 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 Rutin-induced Monohydrate 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 Massification (400 mg / kg-rutin content 20.0 wt%) 0.41 ± 0.02 39 Agglomeration (200 mg / kg-rutin content 20.0 wt%) 0.46 ± 0.03 31 Agglomeration (100 mg / kg-rutin content 20.0 wt%) 0.59 ± 0.01 12 Agglomeration (20 mg / kg-rutin content 20.0 wt%) 0.61 ± 0.02 9 Data represent the mean of difference in ear thickness (mm) ± S.E.M (n = 12) 2. Control group: Experimental group that did not cause edema. Negative Control: No Drug Treatments After Edema Induction

As shown in Table 1, when the concentration of 400 mg / kg containing rutin-containing extract was the highest inhibition rate of 39%, which did not show a significant difference compared to the existing anti-inflammatory analgesic SK-JOINS, the present invention Rutin-containing monoclonal extracts were found to have a concentration-dependent effect of inhibiting TPA-induced ear edema.

Experimental Example 7: A test for inhibiting arachidonic acid induced ear edema with rutin-containing lump extract

In order to confirm the arachidonic acid-induced ear edema inhibitory effect of the rutin-containing lump extract of the present invention, the experiment was carried out as follows.

Seven-week-old ICR mice were isolated for each experimental group, and then SK-JOINS was dissolved in acetone after 1 hour after oral administration of 400 mg / kg of SK-JOINS (as a comparative drug) and 20, 100, 200, 400 mg / kg of lump 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 rutin-containing monohydrate 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 Massification (400 mg / kg-rutin content 20.0 wt%) 0.27 ± 0.02 51 Agglomeration (200 mg / kg-rutin content 20.0 wt%) 0.33 ± 0.05 40 Agglomeration (100 mg / kg-rutin content 20.0 wt%) 0.45 ± 0.03 18 Agglomeration (20 mg / kg-rutin content 20.0 wt%) 0.52 ± 0.03 5 Data represent the mean of difference in ear thickness (mm) ± S.E.M (n = 12) 2. Control group: Experimental group that did not cause edema. Negative Control: No Drug Treatment After Edema

As shown in Table 2, it was found that the concentration of arachidonic acid-induced ear edema inflammation was highest when the concentration of rutin-containing lump extract 400 mg / kg, and the routine-containing lumped extract of the present invention was concentration-dependent on arachidonic acid-induced ear edema. It was confirmed that remarkably suppressed.

Experimental Example  8: containing rutin Lump  Acetic acid-induced stretching inhibition test by extract

In order to confirm whether rutin-containing lump extract of the present invention is effective in relieving and suppressing pain as well as ear edema, an experimental method of a 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 SK-JOINS (comparative drug) and 1 hour after oral administration of lump extract 20, 100, 200, 400 mg / kg ) Intraperitoneal administration (0.1 mL / 10 g) caused organ inflammation. Stretching of the mouse was observed for 10 minutes after 10 minutes, because it causes itching in edema, which can be used as a pain index.

Inhibition of Acetic Acid-Induced Stretching by Rutin-Containing Monohydrate Extract process Writhing number Pain inhibition rate (%) Control 30.60 ± 1.6 - SK-JOINS (400 mg / kg) 19.5 ± 1.9 36 Massification (400 mg / kg-rutin content 20.0 wt%) 20.5 ± 1.8 33 Agglomeration (200 mg / kg-rutin content 20.0 wt%) 25.5 ± 1.3 17 Agglomeration (100 mg / kg-rutin content 20.0 wt%) 25 ± 1.6 18 Agglomeration (20 mg / kg-rutin content 20.0 wt%) 28 ± 0.8 8 1. Control group: Drug-free experimental group after stretching induction

As shown in Table 3, it was found that the concentration of 400 mg / kg of rutin-containing monohydrate extract showed the highest acetic acid-induced stretching inhibitory effect, which showed a similar analgesic inhibition rate as that of SK-JOINS, a comparative anti-inflammatory analgesic. Rutin-containing monoclonal extract of the present invention was confirmed to have a concentration-dependent acetic acid-induced stretching inhibitory effect.

Experimental Example  9: containing rutin Lump  Treatment Effect of Acute Arthritis by Extracts

In order to assay the acute arthritis therapeutic effect of rutin-containing lump 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), oral administration of 1,2 mg of carbohydrate extract 20, 100, 200, 400 mg / kg 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 Rutin Extract process Inflammation-induced degree (%) Control 0 Positive control 84 SK-JOINS (400 mg / kg) 51 Agglomeration (400 mg / kg-rutin content 20.0 wt%) 46 Agglomeration (200 mg / kg-rutin content 20.0 wt%) 49 Agglomeration (100 mg / kg-rutin content 20.0 wt%) 70 Agglomeration (20 mg / kg-rutin content 20.0 wt%) 79 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 acute arthritis treatment effect is the highest when the concentration of glucoside extract 400 mg / kg containing the routine, which showed a better effect than the SK-JOINS comparative anti-inflammatory drugs, of the present invention Rutin-containing lump extract was found to have a significant acute arthritis treatment effect in a concentration-dependent manner.

Experimental Example  10: containing rutin Lump  Cytotoxicity test of extract

In order to investigate the stability of the rutin-containing rutin-containing lump extract of the present invention to the cytotoxicity, the experiment was carried out as follows.

10-1. Splenocyte 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, the well-containing monohydrate extract was added to each well by concentration and incubated for 2 days to perform MTT analysis.

10-2. MTT assay for chemical cytotoxicity testing

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 lump extract extract routine of the present invention on the spleen cells of the experimental animal, the concentration of lump extract extract routine is 1 ~ 100 ㎍ / ㎖ and the stimulation time is 2 days, NS (non -stimulation was stimulated with saline instead of lump extract.

Degree As shown in Fig. 7, the rutin-containing lump extract of the present invention did not have a toxic effect on splenocytes even at a high concentration of 100 µg / ml, but showed a slightly higher survival rate than the control group.

As described above, the rutin-containing lump extract of the present invention was found to be cytotoxic and safe as an anti-inflammatory analgesic.

Experimental Example 11: Long-term toxicity test of rutin-containing lump extract

In order to examine the effect of the rutin-containing lump 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 performed. Renal 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) Blood levels of both enzymes of pyruvic transaminase were measured and tested.

Using 7-week-old mice (Balb / c; Daejeon Chemical Research Institute, Korea), 10 ㎍ and 100 ㎍ of rutin-containing lump extracts 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 rutin-containing lump extract, respectively, and 30 ㎍ and 300 ㎍ of rutin-containing lump extract of the present invention were daily administered to mice, and blood was collected at 1, 5 and 10 days, respectively. Toxicity on liver and kidney tissue was examined (GOT, GPT and CRE, BUN), and control group (NS) was stimulated with saline instead of lump extract.

As shown in FIGS. 8 and 9, in the experimental group administered 10 ㎍ of rutin-containing lump extract, blood levels of GOT, GPT, CRE, and BUN were almost the same as the normal group, but slightly higher than the normal group in the experimental group administered 100 μg. There was no difference (FIG. 8 and FIG. 9).

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

Experimental Example 12: Weight Change Test of Experimental Animals by Rutin-Contained Soybean Extract

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

Rutin-containing lump extracts were dissolved in 0.01 M PBS buffer to concentrations of 10 μg / ml and 100 μg / ml and subcutaneously injected daily into immature mice at day 1 and intraperitoneally injected into mature mice at 7 weeks of age. (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 rutin-containing lump extracts in both immature and mature mice did not show a significant difference compared to the control.

As described above, the rutin-containing lump 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.

Production Example  1: Preparation of Tablet

Tablets for oral administration are prepared using the wet granulation method and the dry granulation method with the following composition using the rutin-containing monohydrate extract of the present invention.

[Furtherance]

Rutin-containing lump 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

An ointment is prepared using the rutin-containing monohydrate extract of the present invention with the following composition.

[Furtherance]

Rutin-containing lump 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 rutin-containing lump extract of the present invention with the following composition.

[Furtherance]

Rutin-containing lump 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

A transdermal agent is prepared using the rutin-containing lump extract of the present invention with the following composition.

[Furtherance]

Rutin-containing lump 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 lumped extract containing a certain amount of rutin 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. Since the substance 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, edema 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 pain, or degenerative diseases.

1 shows an HPLC chromatogram of rutin-containing lump 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 rutin-containing lump extract of the present invention (a: immunoblotting results, b: light concentration measurement results of a data).

Figure 3 shows the effect of inhibiting the concentration dependent production of nitric oxide (NO) of the rutin-containing lump extract of the present invention.

Figure 4 shows the concentration-dependent effect on the inhibition of gene mRNA expression of TNF-α (tumor necrosis factor-alpha) of rutin-containing lump extract of the present invention (a: results of reverse transcriptase-polymerase chain reaction (RT-PCR) , b: results of light density measurement of the data).

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

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

Figure 7 shows the toxicity of the lumped extract of the present invention on the splenocytes of the experimental animals in a graph of survival rate according to the concentration.

8 is a graph showing the toxicity (GOT, GPT) of the rutin-containing lump extract of the present invention on liver tissue of the experimental animal.

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

Figure 10 shows the weight change of the rutin-containing lump extract of the present invention on the growth of immature experimental animals.

Figure 11 shows the weight change of the rutin-containing lump extract of the present invention on the growth of mature experimental animals.

<110> Konkuk University Industrial Cooperation Corp. <120> Composition for the anti-inflammatory and analgesia comprising rutin-contained Sophora japonica extract <130> P07-E203 <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 Sophora japonica extract containing 5.0-30.0% by weight of rutin as an active ingredient. 2. The anti-inflammatory analgesic composition of claim 1 wherein the rutin is 10.0-25.0 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. delete delete delete delete