KR20100118667A - The pharmaceutical composition for arthritis therapy having anti-inflammation activity - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for preventing and treating arthritis is provided to suppress NO and TNF-alpha generation and to ensure anti-inflammation effect. CONSTITUTION: A pharmaceutical composition for anti-inflammation contains extract of Cynanchum atratum, Euphorbia ebracteolata, Inula helianthus-aquatilis, or Stephania delavayi as an active ingredient; and pharmaceutically acceptable carrier or excipient. The extract is water or methanol solution. A health food for anti-inflammation contains the extract as an active ingredient.

Description

The pharmaceutical composition for arthritis therapy having anti-inflammation activity}

The present invention relates to a method for extracting and purifying an active ingredient from natural products as a composition for treating arthritis, a pharmaceutical composition containing the extract, and an anti-inflammatory method using the same. More specifically, Cynanchum atratum, folk drama (Euphorbia ebracteolata), Inura helianthus-aquatilis (Inula helianthus-aquatilis), Stefania delavayi extracts and concentrates the active ingredients of arthritis protective effect and method for the preparation thereof by extracting and concentrating will be.

Inflammation refers to a pathological condition 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. In the reaction process, local blood vessels, various tissue cells and immune cells of the body fluids and the like. In general, the inflammatory response is induced by stimulation such as infection, ischemia, wound, etc. and shows clinical symptoms such as erythema, edema, and pain. Substances involved in this inflammatory response include nitric oxide, prostaglandin E ₂ and thromboxane. Prostaglandins and thromboxanes are synthesized by cyclooxygenase (COX), and cyclooxykenase-2 (COX-2), whose expression is induced by external stimulation, is used for degenerative diseases including cancer and cancer. It is known to affect the onset and progression (Shen et al., Eur, J. Pharmacol., 446, pp 187-194, 2002). Nitric oxide (NO) is synthesized by inducible nitrixc oxide (iNOS) and binds to superoxide anions, which are produced in macrophages, to form powerful peroxynitrite and hydroxyperoxide. ) And damage tissues (Yen and Lai, food and Chemical Toxicology, 40, pp1433-1440, 2002). Expression of these molecular substances is regulated by signaling of nuclear factor-κB (NF-κB), which is an inducible transcription factor. NF-κB is a nuclear protein of the Rel gene family and is present in an inactive form by binding to IκB in the cytoplasm, but cytokines and liposes such as reactive oxygen, TNF-α and IL-1β It is known to promote gene expression that is activated by various stimuli such as polysaccharides (lipopolysaccharides (LPS)) and then migrates to the nucleus to induce inflammatory responses (Oh GT., Et al., Atherosclerosis, 159 (1), pp 17-26, 2001; Epstein, FH., et al., The New England J. of Medicine, 336 (15), pp 1066-1071, 1997; Zhang WJ., et al., FASEB J., 15 (13) , pp 2423-2432, 2001; Denk a., et al., J. of Biol. Chem., 276 (30), pp28451-28458, 2001; Sahnoun Z., et al., Therapie, 53 (4), pp315 -339, 1998; Lindner V., et al., Pathobiology, 66 (6), pp 311-320, 1998; Landry DB., Et al., Am J. of Pathol., 151 (4), pp 1085-1095, 1997; Gerritsen ME., Et al., Am J. of Pathol., 147 (2), pp278-292, 1995).

Inflammatory diseases caused by this inflammatory response include gastritis, colitis, arthritis, nephritis, hepatitis, arteriosclerosis, 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), pp 448-453, 2002; Ramachandran A. et al., Free Radical Biol. Med. 33 (11), pp 1465-1474, 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).

It is already known that immune function protects living organisms from various pathogens, and this immune function is used as a method of preventing and treating diseases, and its representative methods include vaccination and antitoxin use. have.

Recently, as the mechanism of action of the immune function is known, attempts have been made to directly use immunomodulators capable of regulating immune function.

The immunomodulatory substance non-specifically stimulates immune cells to enhance the immune function of the living body, thereby enhancing the defense of the living body from disease factors. As such immunomodulatory substances, chemical synthetic substances, microbial compositions, biologics, and the like are utilized.

However, most of the above immunomodulators have a limit to be applied to a living body due to side effects or toxicity.

In an effort to solve these problems, recently, studies on immunomodulators have been carried out through the use and validation of traditional herbal medicines and active ingredients extracted from non-toxic food ingredients or natural products. It is proven to be highly effective in degenerative diseases. In addition, research on nutritional physiology and pharmacological mechanisms for such efficacy is being actively conducted.

As the active search for a bioactive substance that promotes bioregulation and biological defense system from such natural products is actively progressing, it is entering a practical stage of use as a therapeutic agent or health supplement of alternative medicine.

Currently, natural products for activating immune function include ginseng, thorny ginseng, herbal medicines containing ginseng or herbal medicines selected from conventional herbal medicines.

Therefore, the present inventors have verified the efficacy of non-toxic food ingredients, natural products or conventional herbal medicines in the process of searching for substances that may exhibit anti-inflammatory effects by enhancing immune function, White Rice (Cynanchum atratum), Euphorbia ebracteolata (Euphorbia ebracteolata) , Inula helianthus-aquatilis (Inula helianthus-aquatilis), was prepared an extract of a mixture containing Stefania delavayi, water extract of the mixture shows a high immune cell activation effect, in vivo The present invention was completed by revealing that it is excellent in the ability to destroy inflammatory cells and effectively inhibit the growth of inflammatory cells to prolong its survival.

An object of the present invention is to provide a mixture of each of the extracts consisting of white rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, Stephania delavayi and the mixture It provides a pharmaceutical composition useful for the treatment and prevention of immune function enhancement and inflammatory diseases containing the extract.

In order to achieve the above object, the present invention has a white rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, Stefania Delabay, which exhibits immune activity enhancement and anti-inflammatory effects. (Stephania delavayi) Anti-inflammatory pharmaceutical composition containing each extract or mixed extract as an active ingredient and pharmaceutical compositions for the treatment and prevention of inflammatory diseases.

Each extract of the White Rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, Stefania delavayi is a lower alcohol such as water, ethanol, methanol And preferably extracts soluble in methanol.

According to a preferred embodiment of the present invention for solving the above problems, White rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis (Stephania delavayi) It provides an anti-inflammatory pharmaceutical composition containing one or two or more extracts selected from the group consisting of) as an active ingredient, and containing a pharmaceutically acceptable carrier or excipient.

According to a preferred embodiment of the present invention, the extract is preferably a solvent selected from the group consisting of water or methanol.

According to a preferred embodiment of the present invention, it is selected from the group consisting of Cinnanchum atratum, Euphorbia ebracteolata, Inula helianthus-aquatilis or Stefania delavayi It provides a health functional food containing one or two or more extracts as an active ingredient.

The extracts and mixed extracts of Cinnanchum atratum, Euphorbia ebracteolata, Inula helianthus-aquatilis, and Stephenia delavayi of the present invention are inflamed in the above experiment. by inhibiting Nitric Oxide and inflammatory mediator cytokine is Tumor necrosis factor-alpha, Interleukin- 1 beta, inflammation mediator of prostaglandin E ₂ (prostaglandin E ₂₎ activity in macrophages which participate in the reaction anti-inflammatory agent containing the extract It provides a basis for the development of anti-inflammatory pharmaceutical composition and inflammatory diseases because it can be helpful for the prevention of degenerative diseases such as gastritis, colitis, hepatitis, arthritis, arteriosclerosis and cancer caused by inflammatory reactions. And as a medicament for prevention.

Hereinafter, the present invention will be described in detail.

Each extract of Cinnanchum atratum, Euphorbia ebracteolata, Inula helianthus-aquatilis, and Stephenia delavayi of the present invention is dried and pulverized, and then dried. About 5 to 25 times the weight, preferably about 20 times the volume of water, methanol, lower alcohols such as ethanol or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably methanol Furnace at an extraction temperature of 20 to 100 ° C., preferably 20 to 50 ° C. for about 0.5 hour to 2 days, preferably 1 hour to 1 day, by extraction method such as hot water extraction, cold extraction and reflux cooling extraction. To 5 times, preferably 2 to 3 times of continuous extraction to obtain, filtered under reduced pressure and the filtrate was concentrated under reduced pressure at 20 to 100 ℃, preferably 50 to 70 ℃ with a vacuum rotary concentrator and the extracted residue was vacuum Gun with lyophilizer Each extract of white rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, and Stephania delavayi available in water, lower alcohols can be obtained. In addition, a mixed extract thereof was also obtained in the same manner as above. Each extract obtained was used for the experiment while stored at -20 ℃.

The present invention is a mixed extract of white rice (Cynanchum atratum) obtained by the above method, Euphorbia ebracteolata, Inula helianthus-aquatilis, Stefania delavai (Stephania delavayi) It provides an anti-inflammatory pharmaceutical composition containing as an active ingredient.

In addition, the present invention is mixed with each extract of White rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, Stephania delavayi obtained by the above method It provides a pharmaceutical composition for the treatment and prevention of inflammatory diseases containing the extract as an active ingredient.

As an anti-inflammatory agent of the respective extracts and mixed extracts of White Rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, and Stephania delavayi obtained above In order to investigate the efficacy of lipopolysaccharide (LPS) -stimulated RAW 264.7 mouse macrophages and C57BL / 6 mice, the extracts were added at various concentrations and their effects on inflammatory response factors, along with biochemical analysis. As a result of molecular biology experiments, the extract according to the present invention is a nitric oxide (Nitric Oxide) production in macrophages and inflammatory cytokines TNF-a (Tumor necrosis factor-alpha), IL-1β (Interleukin-1 beta) and pro-tags Prostaglandin E _{2 which} is an inflammation-inducing factor.

Anti-inflammatory containing each extract and mixed extract of White rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, Stephania delavayi of the present invention The pharmaceutical composition for the treatment and prevention of red and inflammatory diseases, comprises 0.1 to 50% by weight of the extract relative to the total weight of the composition.

In addition, the composition comprising a white rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, Stefania delavayi (Stephania delavayi) of the present invention Silver may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Carriers, excipients and diluents which may be included in the composition comprising the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The powder or extract of the composition comprising the extract according to the present invention can be used in various forms, respectively, formulated according to conventional methods. It may be formulated as a conventional agent in the pharmaceutical field according to the therapeutic purpose, and various forms suitable for oral, parenteral, and topical administration are possible. For example, extracts, elixirs, capsules, granules, pills, suspensions, granules, powders, tablets, syrups, solutions, suspensions, emulsions, warnings, ointments, sprays, Oils, gels, spirits, tinctures, baths, linings, lotions, patches, pads, creams, suppositories, and sterile injectable solutions may be used. Topical agents are generally used for the purpose of topical application directly to the affected area. Extracts, granules, powders, tablets, capsules, lotions, sprays, oils, ointments, gels and the like can also be preferably used.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations may contain at least one excipient, for example, starch, calcium carbonate, sucrose ( sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of the herbal composition of the present invention may vary depending on the age, sex, and weight of the patient, but in general, an amount of 0.02 to 10 g, preferably 1 to 5 g, may be administered once to three times daily. . In addition, the dosage of the herbal composition may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention is described in more detail based on the following examples, although the present invention is not limited thereto.

Example 1 Preparation of Polar Solvent Soluble Extract by Hot Water Extraction Method

1-1. Hot water extraction method using water solvent

The dried and pulverized medicinal herbs Cynanchum atratum, Euphorbia ebracteolata, Inula helianthus-aquatilis, and Stefania delavayi purchased from Junghan Intercorp, respectively. 500 g of each was pulverized until it was completely powdered with a grinder. 10 L of methanol, which is about 20 times of the dry weight, is added and extracted twice at 25 ° C. at regular intervals (12h, 24h) for 2 hours under reflux cooling. After filtration under reduced pressure, the filtrates are collected and collected at 60 ° C in a vacuum rotary concentrator. The residue was dried under reduced pressure at 80 ° C. and dried in a vacuum freeze dryer. 49.2 g of dried extract of Cynanchum atratum, 55.1 g of dried extract of Euphorbia ebracteolata, and Inula helianthus- Inura herlianthus- aquatilis) dry extract was obtained 38.4g, Stefania delavayi dry extract 44.7g, their mixed extract was obtained 53.6g. The extracts were used for the experiments while stored at -20 ° C. (See Figure 1).

1-2. Hot water extraction method using water-alcohol mixed solvent

10L of 25% alcohol was added to the pulverized mixed herbal medicine of Example 1-1, stirred well, and heated to reflux for 3 hours at an extraction temperature of 90 to 95 ° C., and then the filtrate was separated. The filtrate was collected three times, and the crude drug extract was concentrated under reduced pressure at 55 to 65 ° C., and then 60 g of the herbal extract powder extract was used as a sample (see FIG. 1).

Reference Example Experimental Materials.

DMEM culture, Fetal bovine serum (FBS), penicillin and streptomycin were purchased from Life Technologies Inc. (Grand Island, NY). 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt: MTS (a)]

[3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS (a)], dimethyl sulfoxide (DMSO), and E. Coli Lipopolysaccharide (Lipopolysaccharide, LPS) were purchased from Sigma Chemical Co., MO, U.S.A. Griries reagent system kit for nitric oxide was purchased from Promega (Promega, WI, USA), and Elisa kit for measuring TNF-α and IL-1β cytokine levels. ELISA kit) was purchased from ebioscience, CA, USA.

Experimental Example 1. Cell Culture

RAW 264.7 mouse macrophage (Macrophage) contains fetal bovine serum (FBS) (10%) and antibiotics (antibiotics-antimycotics, 100U / ml penicillin G sodium, 100ug / ml streptomycin sulfate and 0.25ug / ml amphotericin B) It was passaged every other day at 37 ℃, 5% CO ₂ conditions in DMEM medium containing.

Experimental Example 2. Cytotoxicity Test (MTS assay)

RAW 264.7 mouse macrophage (Macrophage) was dispensed in 96-well plate at 5 x 10 ³ per well and incubated for 24 hours at 37 ℃, 5% CO ₂ conditions to test material Administration by concentration. After administration again incubated for 48 to 72 hours at 37 ℃, 5% CO ₂ conditions. After incubation, [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS (a)] solution is added to 20 μl of each well and allowed to react while incubating at 37 ° C., 5% CO ₂ for 1 to 4 hours. After the reaction was stopped, absorbance was measured using an absorbance spectrometer at 490 nm. The survival rate in the experimental group for the test substance was calculated by converting the positive control group without the test substance into 100% survival rate.

Experimental Example 3. Measurement of Nitric Oxide (NO) Formation

RAW 264.7 cells were suspended in DMEM medium containing 10% FBS without phenol red and placed in 5 × 10 ⁵ cells for each well of a 24-well plate. The cells were incubated for 24 hours at% CO ₂ incubated for 20 hours with LPS (1ug / ml) and test or DMSO in RAW 264.7 cells, and 100 μl of the supernatant was added to Griries reagent (0.1% Naph. Absorbance was measured at 540 nm by reacting with 180 μl of a solution of naphthylethylenediamine and 1% sulfanilamide in 5% H ₃ PO ₄ solution.The calibration curve was measured using a sodium nitrite solution. The absorbance average was converted to the amount of nitrite by using the non-linear recovery analysis (NO-) in the NO treatment inhibitory activity of the test substance treated group based on the amount of nitrite in the LPS-treated group. each specimen using linear regression analysis The IC ₅₀ compared the effect between the test material as a way to determine the (a concentration to inhibit NO production, 50%).

Experimental Example 4 Measurement of TNF-α and IL-1β Cytokine Levels

Incubate the RAW 264.7 cells by dispensing 5 × 10 ⁵ cells per well into a 24-well plate, treating the test material by concentration, and then again applying 37 ℃, 5% CO for 24 hours. ₂ was cultured by taking the cell culture medium was measured using TNF-α, IL-1β content of the enzyme immunoassay kit (enzyme immuno assay, EIA) kit (kit). Cell culture solution was added to a 96-well plate attached with capture antibodies for each cytokine, and after 24 hours reaction, diluted TNF-α, IL-1β antibody and horseradish peroxy Secondary antibodies bound to horseradish peroxidase were each reacted at room temperature for 2 hours. After the reaction, the mixture was washed 5 times with 1 × phosphate buffered saline tween (1 × PBST), and then reacted with an enzyme substrate for 30 minutes. Then 2N-sulfuric acid was added to stop the reaction. Absorbance developed after the reaction was stopped at 450 nm wavelength using an absorbance spectrophotometer. The contents of TNF-α and IL-1β were converted using a standard curve obtained from the reaction of the standard.

Experimental Example 5. prostaglandin _{E 2 (Prostaglandin E 2 (PGE} 2)) measured produce

RAW 264.7 cells were suspended in DMEM medium containing 10% FBS and 1 × 10 ⁵ cells were added to each well of a 96-well plate for 24 hours at 37 ° C. and 5% CO ₂ culture conditions. Incubated for RAW 264.7 cells were washed once with phosphate buffered saline (PBS), replaced with fresh DMEM medium, and treated simultaneously with LPS (1ug / ml) and solvent dissolved in test or test material (DMSO) for 20 hours. Incubated for Dilute the supernatant was added to the PGE ₂ antibody plates (PGE ₂ antibody plate) and cultured for 18 hours or more at room temperature by treating the PGE ₂ -AchE teuraenseo (tracer). After incubation, the PGE ₂ antibody plate (PGE ₂ antibody plate) was washed 5 times with 0.05% Tween in PBS for 1 minute, and then incubated at room temperature for about 7 hours by treatment with Ellman's reagent. After absorbance was measured at 405 nm, the numerical value was substituted into the calibration curve prepared with PGE ₂ standard solution to convert the amount of PGE ₂ produced. PGE ₂ production inhibitory activity in the LPS-treated group was determined by non-linear regression analysis to determine IC ₅₀ (concentration that inhibits PGE ₂ production by 50%) of each test substance. The potency of the livers was compared.

2. Experimental results

In vitro experiments in the present invention using a RAW 264.7 cell line, a mouse macrophage line (Cynanchum atratum), Euphorbia ebracteolata, Inura helianthus-aquatilis (Stepula), Stefania The anti-inflammatory effects of each extract of Delabay (Stephania delavayi) and their extracts were confirmed.

As confirmed in FIG. 2, White rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, Stefania della in RAW 264.7 mouse macrophage cells The cytotoxic effects of each extract and the mixed extracts of the bay (Stephania delavayi) showed almost the same results compared to the positive control group without any test substance and showed no toxicity to cells at all. .

Figure 3 shows the RAW for each extract of White rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, Stephanie delabay (Stephania delavayi) and mixed extracts thereof 264.7 relates to the expression of nitrite concentrations on anti-inflammatory effects in mouse macrophage cells. Compared to the negative control group treated with LPS (lipopolysaccharide) that caused inflammation in RAW 264.7 cells and the positive control group treated with no treatment, the experimental group treated with the natural mixed extract showed almost similar nitrite concentrations. Expression was found to be effective in anti-inflammatory.

FIG. 4A shows RAW for each extract of Cynanchum atratum, Euphorbia ebracteolata, Inula helianthus-aquatilis, Stephanie delabay and mixed extracts thereof. Inflammation was induced in 264.7 mouse macrophage cells and the expression levels of the inflammatory cytokine, TNF-α (Tumor necrosis factor-α), were determined. The negative control group added with LPS (lipopolysaccharide), which causes inflammation, showed a concentration of about 410 ng / ml, and the positive control group without adding LPS (lipopolysaccharide) was about 21 The concentration was about ng / ml. In the experimental group to which the extract of Cynanchum atratum was added, the concentration was about 37 ng / ml. In the experimental group to which the extract of Euphorbia ebracteolata was added, the concentration was about 41 ng / ml. (Inula helianthus-aquatilis) in the experimental group was added to the concentration of about 44 ng / ml, while the experimental group added to the stephania delavayi extract showed a concentration of about 23 ng / ml. In the experimental group added, the concentration was about 26 ng / ml, and all of the extracts showed significantly lower inhibitory levels of inflammatory cytokines than the negatively-induced negative controls.

In FIG. 4b, the expression level of the inflammatory cytokine IL-1β (Interleukin-1β) was confirmed after inducing inflammation in RAW 264.7 mouse macrophage cells with respect to the natural mixed extract. The negative control group added with LPS (lipopolysaccharide), which causes inflammation, showed a concentration of about 330 ng / ml, while the positive control group without adding LPS (lipopolysaccharide) was found. The concentration was about 15 ng / ml. In the experimental group to which the extract of Cynanchum atratum was added, the concentration was about 47 ng / ml. In the experimental group to which the Euphorbia ebracteolata extract was added, the concentration was about 21 ng / ml, and the Inula helianthus-aquatilis extract was added. In the experimental group, the concentration was about 38 ng / ml, and in the experimental group to which the Stefania delavayi extract was added, the concentration was about 11 ng / ml, and in the experimental group to which the mixed extracts were added, the concentration was about 20 ng / ml. Concentration was shown in all the extracts showed significantly lower inhibition of inflammatory cytokines than inflammation-induced negative controls.

In Figure 5 induces inflammation in RAW 264.7 mouse macrophage cells (natural macromolecular extract) against the natural mixed extract through the quantification of prostaglandin (Prostaglandin E ₂ ) which is one of the causes of inflammation to the natural mixed extract The anti-inflammatory effect was confirmed. The negative control group added with LPS (lipopolysaccharide), which causes inflammation, showed a concentration of about 237 pg / ml, and the experimental group added with Cinnanchum atratum extract showed a concentration of about 21 ng / ml. The concentration of 37 ng / ml in the experimental group to which Euphorbia ebracteolata extract was added, and the concentration of 18 ng / ml to the experimental group in which Inula helianthus-aquatilis extract was added. In the experimental group to which the Stephanieia delavayi extract was added, the concentration was about 25 ng / ml. In the experimental group to which the mixed extracts were added, the concentration was about 31 ng / ml. The concentration of PGE _{2 was} significantly lower than that of the negative control.

Examples of the formulation of the pharmaceutical composition will be described below, which is intended to explain in detail only and not intended to limit the present invention.

Preparation Example 1 Preparation of Capsule

200 mg of the composite extract of Example 1

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate proper amount

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Preparation Example 2 Preparation of Tablet

200 mg of the composite extract of Example 1

Lactose 100 mg

Starch 100 mg

Talc 2 mg

Magnesium stearate proper amount

Tablets are prepared by mixing and tableting the components according to conventional tablet production methods.

Preparation Example 3 Preparation of Liquid

1000 mg of composite extract of Example 1

2000 mg of sugar

Isomerized sugar 2000 mg

Lemon flavor

Purified water is added to adjust the total amount to 1000 ml, and then the above ingredients are mixed according to a conventional liquid preparation method, and then filled into a brown bottle and sterilized to prepare a liquid preparation.

Preparation Example 4 Preparation of Health Functional Food

1000 mg of composite extract of Example 1

Vitamin mixture proper amount

Vitamin A Acetate 70 ㎍

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Nicotinamide 1.8 mg

Folic acid 50 μg μgtotenate calcium 0.5 mg

Inorganic Mixtures

Zinc Oxide 0.82 mg

Magnesium carbonate 25 mg

Potassium Citrate 90 mg

Calcium carbonate

The above ingredients may be mixed and granules prepared in accordance with a conventional health food preparation method, and may be used for preparing a health food composition according to a conventional method. The composition ratio of the vitamin and mineral mixtures described above may be modified arbitrarily.

Preparation Example 5 Preparation of Health Functional Drink

1000 mg of composite extract of Example 1

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

900 mg total, including purified water

After mixing the ingredients according to a conventional healthy beverage production method, and stirred and heated at 80 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 ml container, sealed sterilization and refrigerated. Used to prepare healthy beverage compositions.

Preparation Example 6 Preparation of Capsule

200 mg of the composite extract of Example 1

50 mg of conventional arthritis medications (NSAIDs)

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate proper amount

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules. The content of NSAIDs can be adjusted appropriately.

1 is a composite herbal composition of the present invention (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis (Inula helianthus-aquatilis), Stephanie Delabay (Stephania delavayi) to prepare an extract Schematic diagram showing the process.

Figure 2 shows the cytotoxicity of Cinnanchum atratum, Euphorbia ebracteolata, Inula helianthus-aquatilis, and Stephenia delavayi extracts on RAW264.7 cells. Is observed.

Figure 3 shows the effect of white rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, Stephania delavayi extract on the NO production ability of RAW264.7 cells This is a measure of the impact.

4A and 4B show inflammatory activity of RAW264.7 cells with white rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, and Stephenia delavayi extract. As a result of measuring cytokine factors, FIG. 4A shows TNF-α, and FIG. 4B shows inflammatory inhibition of natural extracts related to IL-1β production.

FIG. 5 shows PGE 2 (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis, and Stefania delavayi extract of PGE ₂ (RAW264.7 cells). Prostglandin E ₂ ) is a measure of the effect on the production capacity.

Claims (3)

One or two or more extracts selected from the group consisting of white rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis or Stefania delavayi An anti-inflammatory pharmaceutical composition containing as a component and containing a pharmaceutically acceptable carrier or excipient. According to claim 1, wherein the extract is an anti-inflammatory pharmaceutical composition, characterized in that the solvent selected from the group consisting of water or methanol. One or more extracts selected from the group consisting of white rice (Cynanchum atratum), Euphorbia ebracteolata, Inula helianthus-aquatilis or Stefania delavayi Health functional foods containing.

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