KR101010741B1 - A composition comprising the extract of complex herbs as an active ingredient for preventing and treating inflammatory disease - Google Patents

Abstract

The present invention ( Artemisiae Capillaris Herba ), Gardenia ( Gardeniae) Fructus ) and rhubarb ( Rhei Radix et Rhizoma ) relates to a composition containing the complex herbal extract of the present invention as an active ingredient, specifically, the complex herbal extract of the present invention LPS-induced NO, PGE ₂ , iNOS expression, COX- in RAW 264.7 cells, which are mouse macrophages 2 Significantly reduced the expression and content of inflammatory cytokines (proinflammatory cytokines), the composition can be usefully used as a pharmaceutical composition or health functional food for the prevention and treatment of inflammatory diseases.

Human, Gardenia, Rhubarb, Inflammatory diseases

Description

A composition comprising the extract of complex herbs as an active ingredient for preventing and treating inflammatory disease}

The present invention relates to a pharmaceutical composition or health functional food for the prevention and treatment of inflammatory diseases containing a complex herbal extract of phosphorus, gardenia and rhubarb as an active ingredient.

[1] Lee TH et al., Mol. Cells. , 23 (3), pp 398-404, 2007

2 Nishida T et al., Dig. Dis. Sci. , 52 (8), pp 1890-1896, 2007

[Reference 3] Kim Ho-cheol. Herbal Pharmacology, Seoul, Moon Mundang, 2004

[Document 4] Park, Young-Soon, Pharmacological Commentary on Oriental Medicine, Academy Books, pp153-154, 2002

[5] Lee IA et al., Biol . Pharm . Bull . , 28 (11), pp 2106-2110, 2005

6 Peng CH et al., Curr . Cancer Drug Targets , 5 (4) pp 299-305, 2005

[Reference 7] 曾 野 維 喜,, 東西 醫學 臨 床 漢 方 處方 學, 南山 堂, 1995

[Reference 8] Kim Young-myung et al. Korean Journal of Food Science and Nutrition, 35 (7), pp841-846, 2006

[Reference 9] Kim B.H et al., The Journal of Korean Oriental Medicine, 23 (3), pp74-84, 2002

10 Desai A et al., J. Pharm . Sci . [Epub ahead of print], 2007

[11] Wang S et al., J. Ethnopharmacol . , 114 (3), pp 458-462, 2007

Heo et al., J. Immunol ., 179 (9), pp6305-6310, 2007

Heo et al., J. Leukoc Biol ., 79 (2), pp 330-338, 2006

Inflammation is one of the defenses of biological tissues against external stimuli. When inflammatory reactions occur, various inflammatory factors (pro-inflammatory mediators) are produced, which causes clinical symptoms such as redness, fever, swelling, pain and dysfunction. Inflammatory factors include nitric oxide (NO), which is produced by inducible nitric oxide synthase (iNOS), and prostaglandins, which are produced by cyclooxygenase-2 (COX-2). E ₂ (prostagrandinE ₂ , PGE ₂ ). These inflammatory factors activate nuclear transcription factor-κB (NF-κB), a transcription factor of the inflammatory response, resulting in excess NO and PGE ₂ , resulting in inflammation (Lee TH et al., Mol . Cells . , 23 (3), pp 398-404, 2007; Nishida T et al., Dig . Dis. Sci . , 52 (8), pp 1890-1896, 2007). In the case of nitric oxide synthase (NOS) in mammalian cells, there are three similar forms: neuronal nitric oxide synthase (neuronal NOS, nNOS) and endothelial nitric oxide synthase (eNOS). And iNOS. In particular, iNOS is involved in the inflammatory response. nNOS and eNOS are always expressed and iNOS is expressed in the presence of stimulation of interferon-γ, lipopolysaccharide (LPS), and various inflammatory cytokines. Normally eNOS is a potent vasodilator that plays an important role in maintaining vascular homeostasis. NO also modulates acute and chronic inflammatory responses. PGE ₂ is produced from arachidonic acid by COX. COX was mainly studied in the early 1990s, but there are also two similar forms. COX-1 is expressed in almost all tissues and produces prostaglandins that regulate physiological functions such as regulating blood flow in the kidneys or protecting the cells of the stomach. In contrast, COX-2 is expressed in macrophage in response to infection or damage caused by microorganisms or stress of various factors. In other words, iNOS and COX-2 expression, NO and PGE ₂ production are representative inflammatory factors of immune cells.

Other inflammatory factors include tumor necrosis factor-α (TNF-α), interleuken-1β, IL-6, monocyte chemotactic protein-1, which are inflammatory cytokines. (monocyte chemoattractant protein-1, MCP-1), and the like.

Recently, natural resources have been developed as leading substances in many therapeutic agents and have been used as valuable resources for the pharmaceutical industry. Therefore, it is possible to reduce the side effects than artificial chemicals by developing anti-inflammatory disease prevention and treatment materials from natural resources, and it is also competitive in price.

Injin ( Artemisiae Capillaris Herba ) is known to have edema, hepatic function protection, antibacterial and antiviral effects, and also has an antihypertensive effect on blood pressure and arteriosclerosis (Kim, Ho-Cheol. Among the components of the phosphorus, esculetin promotes the secretion of bile, while chlorogenic acid and caffetannic acid release cholesterol and β-lipoprotein in the blood. Lowering prevents the deposition of fat on the walls of blood vessels, blood lipid lowering, cholesterol lowering action. In addition, eugenol and eugenic acid are known to have anti-thrombotic action, bile secretion promoting action and antioxidant action (Park, Young-Soon, Pharmacological Commentary of Oriental Medicine, Academy Books, pp153-154, 2002).

Gardenia ( Gardeniae Fructus ) has the effect of inhibiting skin fungi, dipharymia and pancreatic secretion, protecting liver function and inhibiting myocardial contraction (Kim Ho-cheol. Herbal Pharmacology, Seoul, Munmundang. Pp125-127, 2004). Gardenia and its components, crocin and crocetin, have been reported to improve hyperlipidemia by inhibiting pancreatic lipase activity (Lee IA et al., Biol . Pharm . Bull . 28 (11), pp 2106-2110, 2005). Recently, gardenia Jenny Forsythe (geniposide) the liver, heart, and was reported to be a stand Anti yanginja do harm to the kidneys (Peng CH et al., Curr . Cancer Drug Targets , 5 (4) pp 299-305, 2005). Gardenia jasminoides, geniposide, genipin, galdenoside, shanzhiside, galdenin, mannitol, β-sitosterol, Nonnacosane, pectin, tannin, etc. are known to promote bile secretion, antihypercholesterolemia, antacid, antiacetylcholine, and antihistamine. (曾 野 維 喜, 續 東西 醫學臨 床 漢 方 處方 學, 南山 堂, p98, 1995).

Rhubarb (Rhei Radix et Rhizoma ) contains anthracene glycosides, which are a lower component, and has antibacterial activity against various bacteria. In addition, there is edema, hemostasis, diuretic effect and immunomodulatory activity (Ho Chul Kim. Pharmacology of Korean Medicine, Moon Mundang, pp174-177, 2004). After extracting laminaran from rhubarb and administering it to high-fat diet rats, it improves serum lipid composition (Kim Young-Myung et al., The Korean Journal of Food Science and Nutrition, 35 (7), pp841-846, 2006). Inhibition of Bax / Bcl-2 spontaneous cell death was also reported by neuroprotective effect of Mogolian gerbil in reversible whole cerebral ischemia model (Kim BH et al., The Korean Journal of Oriental Medicine, 23 (3), pp74-). 84, 2002). Rhubarb consists of anthraquinone derivatives rhein, emodin, chrysophanol, aloe-emodin, physcion, and glycosides thereof. And various pharmacological actions are revealed. Sennoside A, in particular, has been proved to have potent action in the digestive system, increasing plasma invasive pressure by action on blood, moving tissue moisture into blood vessels, and replenishing blood volume by bleeding blood. It is known to improve microcirculation disorder by reducing blood viscosity. In addition, antihyperlipidemia, anti-infection, anti-tumor, diuresis, and the effects of female hormones have been identified and used (曾 野 維 喜, 續 東西 醫學 臨 床 漢 方 學, 南山 堂. Pp207-208, 1995).

In this regard, the present inventors treated a complex herbal extract of human, gardenia and rhubarb on RAW 264.7 cells, which are mouse macrophages, by MTS assay, NO production, PGE ₂ production, iNOS, COX-2 and inflammatory cytokines TNF-α, IL-1β By measuring the amount of IL-6 and MCP-1 produced, the anti-inflammatory effect was confirmed to complete the present invention.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing a composite herbal extract of phosphorus, gardenia and rhubarb as an active ingredient.

In another aspect, the present invention provides a health functional food for the prevention and improvement of inflammatory diseases containing a composite herbal extract of phosphorus, gardenia and rhubarb as an active ingredient.

Extracts as defined herein include solvents selected from water, including purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably methanol extracts.

The complex herbal extract as defined herein is characterized in that the dry weight ratio (w / w) of phosphorus, gardenia and rhubarb comprises from 5 to 15: 1 to 10: 1 to 6, preferably 10: 5: 3.

Inflammatory diseases as defined herein include atherosclerosis, conjunctivitis, atopic, arthritis, rhinitis, ulcerative colitis, hypertension, diabetes, cancer or asthma, preferably arteriosclerosis, arthritis, hypertension, diabetes or cancer, more preferably arteriosclerosis Or hypertension.

Hereinafter, the present invention will be described in detail.

The herbal extract of the present invention can be prepared as follows. The dried phosphorus, gardenia and rhubarb are each selected from water containing 1 to 5 times, preferably 3 times purified water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably 15 to 100 with methanol. A first step of extraction at 24 ° C., preferably at 20 ° C. to 30 ° C. for 24 hours to 60 hours, preferably for 48 hours; A second step of repeating the first step twice; Extracts of the present invention can be obtained through a manufacturing method including a third step of concentrating the filtration and freeze-drying.

In addition, the herbal extract of the present invention can be prepared as follows. A first step of mixing the dry weight ratio (w / w) of phosphorus, gardenia and rhubarb in 5 to 15: 1 to 10: 1 to 6, preferably 10: 5; A solvent selected from water containing 1 to 5 times, preferably 3 times purified water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably 15 to 100 ℃, preferably 20 to A second step of extracting at 30 ° C. for 24 to 60 hours, preferably 48 hours; A third step of repeating the second step twice; Through the manufacturing method comprising the fourth step of filtration concentrated and freeze-dried can be obtained the complex herbal extract of the present invention.

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the extract of the complex herbal medicine obtained by the above method as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of inflammatory diseases containing a composite herbal extract obtained by the above production method as an active ingredient.

The pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the complex herbal extract of the present invention comprises 0.1 to 50% by weight of the complex herbal extract based on the total weight of the composition.

The pharmaceutical composition containing the complex herbal extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Pharmaceutical compositions containing the complex herbal extract of the present invention may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents which may be contained in the composition containing the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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, which form at least one excipient such as starch, calcium carbonate, sucrose, etc. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate 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 combined herbal extract of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered from 0.1 to 100 mg / kg once or several times daily. The dosage may also be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

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

The present invention provides a dietary supplement for the prevention and improvement of inflammatory diseases comprising a composite herbal extract as an active ingredient. Foods to which it may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to food or beverages for the purpose of preventing inflammatory diseases. In this case, the amount of the composite herbal extract in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, and the health functional beverage composition may be added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extract) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extract of the present invention may contain a pulp for the production of natural fruit juices and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

As described above, the complex herbal extract of the present invention is the production of NO, PGE ₂ , iNOS expression, COX-2 expression, inflammatory cytokine TNF-α in RAW 264.7 cells It can be used as a composition for the prevention and treatment of inflammatory diseases by showing an anti-inflammatory effect that significantly reduces the production of IL-1β, IL-6 and MCP-1.

Hereinafter, the present invention will be described in detail. However, the following Examples, Reference Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Example 1 Preparation of Herbal Extracts

1-1. Preparation of Methanol Soluble Extract of Injin

Injin ( Artemisiae Capillaris Herba ) was selected from the Department of Herbal Medicine, College of Oriental Medicine, Dongguk University, added 3 times 100% methanol to 300 g, extracted at 25 ° C for 48 hours, and filtered through this process twice. Concentrated and lyophilized to give 16.08 g (yield 5.36%) of the sample of the present invention (hereinafter referred to as "AC").

1-2. Preparation of Methanol Soluble Extract of Gardenia Gardenia

Gardenia ( Gardeniae Fructus ) was selected from the Department of Herbal Medicine, Dongguk University, College of Oriental Medicine, added 3 times 100% methanol to 300 g, extracted at 25 ℃ for 48 hours, this process was filtered twice, concentrated and frozen Drying gave 76.5 g (yield 25.5%) of the sample of the present invention (hereinafter referred to as "GF").

1-3. Preparation of Methanol Soluble Extract of Rhubarb

Rhubarb ( Rhei Radix et Rhizoma ) was selected from the Department of Herbal Medicine, College of Oriental Medicine, Dongguk University, added 3 times 100% methanol to 300 g, extracted at 25 ° C for 48 hours, and filtered through this process twice. After concentrating and freeze-drying, 57.9 g (yield 19.3%) of the sample of the present invention (hereinafter referred to as “RR”) were obtained.

1-4. Preparation of Complex Herbal Extracts

Injin, Gardenia and Rhubarb (provided with the Department of Herbal Medicine, Dongguk University) were mixed with 150 g, 75 g and 45 g, respectively, and 3 times of 100% methanol was added to a total of 270 g, followed by extraction at 25 ° C. for 48 hours. This process was repeated twice, filtered, concentrated and lyophilized to give a sample of the present invention (hereinafter referred to as “IJHT”) to obtain 48.06 g (yield 17.8%) of the following experiment.

Reference Example 1. Preparation of Experimental Materials

Cell culture reagents such as Dulbecco's Modifide Eagle Medium (DMEM), fetal bovine serum (FBS), and streptomycin-penicillin were purchased from Gibco BRL (Grand Island, USA), Sodium Dodesyl Sulfate (SDS), Acrylamide, Bis, Protein Assay reagents were purchased from Bio-Rad (Hercules, USA), NP-40, CAPS, tween20, protease inhibitors, etc. were purchased from Sigma (St. Louis, USA). The primary antibody iNOS monoclonal antibody (mAb) and the secondary antibody anti-rabbit IgG horseradish peroxidase (HRP) -conjugated antibody were produced by SantaCruz Biotechnology (Santa Cruz, CA), and COX-2 polyclonal antibody and β. -actin mAb was purchased from Cell Signaling Technology (Beverly, MA). Aqueous One Solution Cell Priliferation Assay (MTS) kit and Griess Reagent System were purchased from Promega (Madison, USA) and ELISA kits for various cytokine measurements were purchased from Pierce Biotechnology (Rockford, USA) and PGE ₂ analysis The kit was purchased from R & D (Minneapolis, USA). All reagents used in the experiments were used above the analytical grade.

Reference Example 2. Cell Culture

Macrophage lines of mice, RAW 264.7 cells (KCLB), were treated with DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS and 1% penicillin-streptomycin (37%, 5% CO). Cultured under the conditions of ₂ was used in the following experiment. In addition, all experimental results are expressed as mean and standard deviation, and significance test is performed by using student's t- test (p <0.001) using sigma plot (Sigma Plot, version 7.0 for Window). Significance between LPS test groups (*) is indicated.

Experimental Example 1. MTS Analysis

5- (3-caroboxymeth-oxyphenyl) described in Desai A et al., J. Pharm . Sci. [Epub ahead of print], 2007 for measuring the toxicity of the extracts obtained in Example 1 above . The experiment was performed using the -2H-tetra-zolium inner salt (MTS) analysis method as follows.

RAW 264.7 cells cultured by the method of Reference Example 2 were divided into 1 × 10 ⁴ / well in 96 well plates, and the extracts obtained in Example 1 were prepared by concentration (0, 50, 100, 300, 500, 700, respectively). μg / ml) for 18 hours. After 20 μl MTS solution was added per well for 4 hours in a 37 ° C., 5% CO ₂ incubator, the change in absorbance at 450 nm was measured using a microplate reader (DYNEX, Opsys MR, USA). By expressing the cell viability of the control group without the extract was expressed as a percentage. In order to correct the absorbance of the drug by each concentration, the culture medium without the cells were cultured together to compare and correct the absorbance of the control group and the experimental group to express the cell viability as a percentage (mean value ± standard deviation) (see Equation 1 below).

% Cell viability = {(absorbance of test group-absorbance of sample only) / absorbance of control group} X 100

sample Concentration (㎍ / ml) Cell survival rate (%) AC 0 100 ± 0 50 112 ± 7 100 105 ± 8 300 99 ± 4 500 38 ± 2 700 32 ± 3 GF 0 100 ± 0 50 123 ± 15 100 122 ± 14 300 115 ± 13 500 112 ± 8 700 102 ± 5 RR 0 100 ± 0 50 118 ± 11 100 108 ± 9 300 100 ± 9 500 79 ± 2 700 58 ± 4 IJHT 0 100 ± 0 50 119 ± 14 100 122 ± 15 300 123 ± 12 500 119 ± 11 700 106 ± 15

As shown in Table 1, IJHT and GF had little effect on the cell viability of RAW 264.7 at all concentrations, whereas AC and RR decreased cell viability at high concentrations. Therefore, the following experiment was carried out at a concentration of 300 μg / ㎖ of IJHT does not affect the cell viability of RAW 264.7.

Experimental Example 2 Measurement of NO Production

In order to determine the amount of NO produced, experiments were performed using the method described in Wang S et al., J. Ethnopharmacol. , 114 (3), pp458-462, 2007.

RAW 264.7 cells cultured by the method of Reference Example 2 were pretreated with the IJHT obtained in Example 1-4 and incubated for 18 hours by treatment with 100 ng / ml LPS after 1 hour. Add 50 g of culture medium with the same amount of Greases Reagent and allow to react at room temperature for 10 minutes, using Automatic ELISA reader (precision microplate reader, Molecular Devices, BIO-TEX EL800uv-PC, USA) at 540 nm. Absorbance was measured. NO concentration in the culture was determined using a standard curve of concentration of sodium nitrite.

As a result, as shown in FIG. 1A, when the LPS was treated at a concentration of 100 ng / ml, it was confirmed that the amount of NO produced was increased by about 10 times compared to the control which did not have any treatment. In addition, when the concentration of IJHT 300 μg / ㎖ pretreated for 1 hour, it was confirmed that the amount of LPS-induced NO production reduced by about 74%.

Experimental Example 3. PGE ₂ Measurement of production

In order to measure the amount of PGE ₂ , experiments were performed using a commercial competitive enzyme immunoassay kit (R & D systems, Minneapolis) as follows.

RAW 264.7 cells cultured by the method of Reference Example 2 were pretreated with 300 μg / ml of IJHT obtained in Example 1-4 for 1 hour and then treated with 100 ng / ml of LPS. After 18 hours, 100 µl of each culture was loaded into a 96-well plate coated with goat anti-mouse. 50 μl of primary antibody solution and 50 μl of PGE ₂ conjugate were added thereto, and the plate was left at 4 ° C. overnight. After washing 4 times with washing buffer and treating with 200 μl of substrate solution for 5-20 minutes, the solution was treated with 50 μl of stop solution and absorbance at 450 nm. Was measured.

As a result, as shown in Figure 1B, it was confirmed that the amount of PGE ₂ production is increased by about 20 times compared to the control without any treatment at a concentration of 100 ng / ml LPS. In addition, it was confirmed that the experimental group treated with IJHT 300 ㎍ / ml reduced the amount of LPS-induced PGE ₂ by about 35%.

Experimental Example 4. Western blot analysis

Western blot analysis was performed using Hu et al. (Heo et al., J. Immunol., 179 (9), pp6305-6310, 2007) to measure iNOS and COX-2 protein expression.

300 μg / ml of IJHT obtained in Example 1-4 was three times with ice-cold Tris buffered saline (TBS: 20 mM Tris-HCl, pH 8.0, 137 mM NaCl) in cells cultured by the method of Reference Example 2. After washing, add lysis buffer (lysis buffer: TBS, 1% NP-40, 1 mM sodium orthovanadata, 10 μg / ml aprotinin, 10 μg / ml leupeptin and 1 mM PMSF) and react at 30 ° C. for 30 minutes. The supernatant was collected by centrifugation at g for 10 minutes. The same amount of protein was separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the protein was transferred to a nitrocellulose membrane (Whatman, Germany). The membrane was reacted for 1 hour in a blocking buffer (blocking buffer: TBS solution containing 5% non-fat milk and 0.1% Tween20) to block nonspecific binding of the antibody, followed by antibody to each protein (anti-iNOS). And anti-COX2) were added and reacted for 1 to 2 hours. It was then washed with a TBST solution containing 0.1% Tween20 for 40 minutes and then reacted with a secondary antibody (anti-rabbit IgG horseradish peroxidase-conjugated antibody). Subsequently, the protein was confirmed on the X-ray film (AGFA, Belgium) after the reaction with the ECL system. Protein quantification of each sample was performed by measuring the absorbance at 595 nm using a Bradford protein assay kit.

As a result, as shown in Figure 2, when treated with LPS 100 ng / ml, it was confirmed that the expression of iNOS and COX-2 significantly increased compared to the control. In addition, when the concentration of IJHT 300 μg / ㎖ pretreated for 1 hour, it was confirmed that the expression of iNOS and COX-2 induced by LPS 100 ng / ml 52% and 79%, respectively.

Experimental Example 5. Measurement of cytokines in cell culture

As an indicator of inflammation, Hu et al. (Heo et al., J. Leukoc Biol., 79 (2), pp330-338, 2006) were used to measure the amount of inflammatory cytokines in cell culture. ELISA assay (Enzyme-Linked Immunosorbent Assay) was performed as follows.

RAW 264.7 cells cultured by the method of Reference Example 2 were treated with IJHT obtained in Example 1-4 at a concentration of 300 μg / ml for 1 hour, and then treated with LPS 100 ng / ml for 18 hours. After dilution to concentration, 50 μl each was added to a cytokine-coated 96 well plate and overnight at 4 ° C. Wash three times with washing buffer and 100 μl of biotinylated antibody reagent to each well, react at room temperature for 1 hour, wash three times, and then 100 μl of strep. Tavidin-HRP solution (streptavidine-HRP solution) was treated in each well and allowed to react at room temperature for 1 hour, and then washed three times with washing buffer again. 100 μl of di (2-ethylhexyl) -2,4,5-trimethoxybenzalmalonate (TMB) substrate was treated for 5-30 minutes, followed by 100 μl reaction stop solution at 450 nm. Absorbance was measured.

As a result, as shown in Figure 3 when treated with a concentration of LPS 100 ng / ml in RAW 264.7 cells, TNF-α is 4.3 times, IL-1β 6.2 times, IL-6 57.3 times, MCP-1 is 5.2 times increased. After pretreatment with IJHT concentration of 300 μg / ml for 1 hour, 100 ng / ml LPS was treated for 18 hours to produce 44% of TNF-α induced by LPS, 67% of IL-1β, and IL. The production amount of -6 was found to be effectively reduced by 34%, MCP-1 production by 69%.

Although the formulation example of the composition comprising the complex herbal extract of the present invention will be described, the present invention is not intended to limit it, it is intended to explain in detail only.

Formulation example  One. Powder  pharmacy

IJHT 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

IJHT 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are usually prepared by tableting according to the method for preparing tablets.

Formulation example  3. Manufacture of capsule

IJHT 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

Formulation example  4. Preparation of injections

IJHT 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 mg

It is usually prepared in the above ingredient content per ampoules (2 ml) according to the preparation method of the injection.

Formulation example  5. Liquid  Produce

IJHT 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified Water Proper Amount

Normally, each component is added to the purified water to dissolve according to the preparation method of the liquid solution, lemon flavor is added, the above ingredients are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle and sterilized. To prepare a liquid solution.

Formulation example  6. Preparation of dietary supplements

IJHT 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

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

Vitamin C 10 mg

10 μg biotin

Nicotinamide 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is a composition that is relatively suitable for the health functional food, the composition is mixed in a preferred embodiment, but the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method. Then, the granules may be prepared and used for preparing the nutraceutical composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

IJHT 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

900 ml of purified water whole

After mixing the above components according to the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

1 is a diagram measuring the effect on the LPS-induced NO (A) and PGE ₂ production (B) of the complex herbal extracts,

Figure 2 is a measure of the effect on the expression of LPS-induced iNOS and COX-2 of the complex herbal extracts,

Figure 3 is a measure of the effect on the production (D) of LPS-induced TNF-α (A), IL-1β (B), IL-6 (C) and MCP-1 of the complex herbal extracts.

Claims (8)

A pharmaceutical composition for the prevention and treatment of atherosclerosis, which contains a complex herbal methanol extract having a dry weight ratio (w / w) of phosphorus, gardenia and rhubarb (w / w) of 10: 5: 3 as an active ingredient. delete delete delete delete delete Health functional food for the prevention and improvement of atherosclerosis, which contains a compound herbal methanol extract having a dry weight ratio (w / w) of phosphorus, gardenia and rhubarb as 10: 5: 3 as an active ingredient. delete

Images