KR101469325B1 - Composition comprising an extract of combined crude drug including Xanthium strumarium L. for preventing and treating inflammatory disease or allergic disease - Google Patents

Abstract

The present invention relates to a composition for the prophylaxis and treatment of inflammatory diseases or allergic diseases comprising as an active ingredient a complex herbal medicine extract comprising a mixture of Lactobacillus casei, Lactobacillus, Bacillus, and white mite. The extract of the present invention secretes IL-5, and IL-6 secreted from splenocytes in a rhinitis animal model induced by ovalbumin, and inhibits the production of NO and cytokines (TNF-alpha, IL- 13 by inhibiting the production of an anti-inflammatory, anti-allergic and anti-inflammatory effect, thereby being useful as a pharmaceutical composition for the prevention and treatment of an inflammatory disease or an allergic disease.

Description

[0001] The present invention relates to a composition for treating and preventing an inflammatory disease or an allergic disease,

The present invention relates to a composition for the prophylaxis and treatment of an inflammatory disease or an allergic disease containing a complex herbal medicine extract.

[Document 1] Busse WW and Lemanske RF, Asthma, N Engl Med 2001 , 344 (5), 350-362.

[Literature 2] Lemanske RF and Busse WW, Asthma, J Allergy Clin Immunol 2003 , 110 (5), 703-705.

[Literature 3] Woodfolk JA, Cytokines as a therapeutic target for a allergic disease: a complex picture, Curr Pharm Des 2006 , 12 (19), 2349-2396.

[4] Harizi H, et al., Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology, Trends Mol Med 2008 , 14 (10), 461-469.

[Literature 5] Cicardo VH, Mechanism of the hypotensive effect of prostaglandins A2 and E2, Acta Physiol Lat Am 1981 , 85-91.

[Literature 6] Liu F, et al., Prostaglandin B2-induced pulmonary hypertension is mediated by TxA2 / PGH2 receptor stimulation, Am . J. Physiol 1994 , 267, 602-608).

[Literature 7] Gollman HM et al., Comparative pyrogenic potency of endogenous prostanoids and of prostanoid-mimetics injected into the anterior hypothalamic / preoptic region of the cat, Brain Res 1988 , 449, 281-293.

[8] Collins, et al., Cutaneous sympathetic motor rhythms during a fever-like response induced by prostaglandin E (1), Neuroscience , 2002 , 110, 351-360.

[0019] Shiraishi, et al., Cycloxygenase-2 / prostaglandin D2 / CRTH2 pathway mediates double-stranded RNA-induced enhancement of allergic airway inflammation, J Immunol 2008 , 180, 541-549.

[10] Dahlen SE, Treatment of asthma with antileukotrienes: first line or last resort therapy, Eur J Pharmacol 2006 , 533, 40-56.

[Document 11] Kitamura S, Leukotriene (B4, C4, D4, E4), Nippon Rinsho 2005 , Suppl 8, 28-33.

[Literature 12] Metabolism and Shin Min-gyo-ji, Metaphorical view of herbal medicine , Young Lim, pp. 1080-1082, 1998.

[Literature 13] Metabolism of medicinal plants and medicinal plants , Young Lim, pp. 960-963, 1998.

[Literature 14] Metabolism of medicinal plants and medicinal plants , Young Lim, pp. 409-411, 1998.

[Literature 15] Metabolism and Shin Min-gyo, The metabolism of medicinal herbs ( medicinal herb) - Medicinal plants , Young Lim, pp. 851-852, 1998).

[Literature 16] Choi JH et al., Flowers of Inula japonica attenuate inflammatory responses, Immune Network 2010 , 10 (5), 145-152.

[17] Yang JH et al., Anti-inflammatory activity of ethylacetate fraction of Cliona celata, Immunopharmacol Immunotoxicol 2011 , 33 (2), 373-379.

[18] Jeon EJ et al., The effects of peroxisome proliferator-activated receptor-agonist on a murine model of experimental allergic rhinitis, Otolaryngology - Head and Neck Surgery 2008 , 139, 124-130.

Inflammation is the defense of biological tissues against injury to the body. That is, in response to various harmful stressors, the biological defense reaction to recover the original state by removing the injury caused by stimulation is an inflammatory reaction. Irritation of inflammation includes infection, chemical, and physical stimulation. Biocomponents related to the inflammatory reaction include free radicals, proteins of low molecular weight and polymers such as carbohydrates and lipids, plasma, blood cells, blood vessels and connective tissues. The process of inflammation is usually divided into two, acute and chronic inflammation can be divided into. Acute inflammation is a short-term reaction within a few days. Plasma components and blood cells are involved in dephosphoremia through micro-circulatory system. Chronic inflammation has a long duration and tissue proliferation.

Most allergic diseases are mediators (mainly histamine, leukotrienes, tumor necrosis factor α (TNFα), leukocyte and eosinophil-releasing agents in tissues of mast cells and blood activated by antigen-antibody reaction, , Cytokines, etc.). The use of currently used allergy treatment drugs is in relief of symptoms, so development of more fundamental therapeutic drugs is urgently required.

In addition, key mediators that induce allergic diseases such as inflammation and asthma, prostaglandins, acids (prostaglandines, PGs), leukotrienes acids (leukotriens, LTs), platelet activating factor (PAF) Phospholipase A ₂ (phospholipase A _2), such as Arachidonic acid-derived eicosanoids are produced from arachidonic acid, a precursor by cyclooxygenase (COX) and lipoxygenase (Harizi H, et al., Roles in biology and immunopathology , Trends Mol Med 2008 , 14 (10), 461-469).

Prostaglandins bind to specific cell surface receptors and act to increase intracellular concentrations of cAMP (and possibly cGMP). The effect of increasing cAMP depends on the cell type, and PGA ₂ and PGB _{2 lower} blood pressure (Cicardo VH, Mechanism of the hypotensive effect of prostaglandins A2 and E2, Acta Physiol Lat Am 1981 , 85-91; Liu F, et al., Prostaglandin B2-induced pulmonary hypertension is mediated by TxA2 / PGH2 receptor stimulation, Am . J. Physiol 1994 , 267, 602-608), PGD ₂ , and PGE ₁ are known to be involved in inflammation such as pain and fever (Gollman HM et al., Comparative pyrogenic potency of endogenous prostanoids and of prostanoid-mimetics injected into the anterior hypothalamic / preoptic region of the cat, Brain Res 1988 , 449, 281-293; Collins, et al., Cutaneous sympathetic motor rhythms during a fever-like response induced by prostaglandin E (1), Neuroscience , 2002 , 110, 351-360). In addition, PGD ₂ is known to be a major contributor to asthma exacerbation by contraction of smooth muscle in bronchial asthma patients (Shiraishi et al., Cyclooxygenase-2 / prostaglandin D2 / CRTH2 pathway mediates double-stranded RNA-induced enhancement of allergic airway inflammation, J Immunol 2008 , 180, 541-549).

Leukotriene constitutes a local functional hormone group produced in vivo from arachidonic acid, and important leukotrienes include leukotriene B ₄ (LTB ₄ ), leukotriene C ₄ (LTC ₄ ), leukotriene D ₄ (LTD ₄ ) and leukotriene E ₄ LTE ₄ ). The biosynthesis of these leukotrienes is initiated by the enzyme 5-lipoxygenase acting on arachidonic acid to produce an epoxide known as leukotriene A ₄ , which can be converted to other leukotrienes (LTB ₄ , LTC ₄ , LTD ₄ , LTE ₄ ). Leukotriene is involved in allergic and allergic reactions such as pulmonary artery disease such as asthma, chronic bronchitis and related obstructive airways disease, allergic rhinitis, contact dermatitis, allergic conjunctivitis, inflammation such as arthritis or inflammatory bowel disease, pain . Recently, drugs that are attracting attention as an allergic asthma medication are those that simultaneously inhibit histamine release, inhibit leukotriene C ₄ production, and inhibit platelet activating factor production (Dahlen SE, Treatment of asthma with antileukotrienes: first line or last resort therapy , Eur J Pharmacol 2006 , 533, 40-56; Kitamura S, Leukotriene (B4, C4, D4, E4), Nippon Rinsho 2005 , Suppl 8, 28-33).

Xanthium , a year-old from the Asteraceae family, strumarium L. (= X. sibiricum PATR ex WIDD.)) is the fruit of Xanthium strumarium, medicine in the leaf, root, flower, and use, negligence glass column toast Marine (xanthostrumarin) 1.2%, 3.3% resins, fatty oil, Alkaloids, vitamin C (vitamin C) and pigments. Dried fruits contain 9.2% of fatty oil and the fatty acids are 64.20% linoleic acid, 26.8% oleic acid, 5.32% palmitic acid, 3.36% stearic acid, Respectively. In the unsaturated fat, ceryl alcohol, β-, γ-, ε-sitosterol (β-, γ-, ε-sitosterol) and acetone insoluble fat were found in 33.2% of lecithin, and cephalin (66.8%). Species, moisture 6-7%, fatty oil containing 40%, and seed coat, the pen is known to be acid (pentosan) contains the component of 15.86% (jeongboseop and sinmingyo low, illustrated hyangyak (herbal) Dictionary-medicinal plant pieces, Younglim Pp. 1080-1082, 1998 ).

The perennial ginseng ( Trichosanthes kirilowii Maxim.) Belonging to the bacillus subtilis is a seed of the horntart, which is called gwaluja and perilla. The medicinal materials are fruits, roots, foliage, perilla and seeds. The fruit of the hanontari contains triterpenoid saponins, organic acids, resins, sugars and coloring matters, and the root contains trichosanthin and saponin. Seed gangbuin contains glycosides, saponins, organic acids, salts, plant gums, resins, pigments and fatty acids such as trichosanic acid, which is an unsaturated fatty acid, and arginine, (Lysine), alanine (alanine), valine, leucine, isoleucine, glycine and alkaloid are known to be contained ( Medicinal herbs) metabolism - medicinal plants , Yeonglim, pp. 960-963, 1998 ).

A perennial white paper belonging to the dragonfly family ( Angelicae Dahurica (FISCH.) BENTH. et Hook. f.) is a herbal medicine obtained from the roots of pebbles and related plants, and contains the essential oil in the outpouring of griffins, and byak-angelicin, byak-angelicol, But are not limited to, oxypeucedanin, imperatorin, torin, isoimperatorin, phellopterin, xanthotoxin, marmesin, scopoletin, - contains isobyak-angelicol, neobyak-angelicol, alloisoimperatorin, 5-methoxy-8-hydroxypsoralen, it is known that the (jeongboseop and sinmingyo low, illustrating direction of about (herbal) Dictionary, Younglim four, pp 409-411, 1998).

Mentha , a perennial plant belonging to the family Lamiaceae arvensis L. var. piperascens MALINV.) is the outpost or leaf of peppermint ㆍ peppermint, fresh leaf contains 0.8 ~ 1% of essential oil and dried leaf contains 1.3 ~ 2%. The main ingredient of the essential oil is menthol, which contains about 77-78% of menthone and 8-12% of menthone. It contains menthol menthol, camphene, limonene, isomenthone, pinene Menthenone, resin and a small amount of tannin and rosmarinic acid are known to be contained in the medicinal herb ( medicinal herbs and medicinal herbs) , medicinal plants , pp 851-852, 1998 ).

However, none of the above references discloses or discloses any effect of the herbal composition extract of the present invention consisting of ginseng, ginseng, white ginseng and mint on inflammatory or allergic diseases.

Accordingly, the present inventors found that the herbal extracts inhibit the production of NO and cytokines (TNF-α, IL-1) secreted from macrophages after LPS stimulation, and decrease serum IgE concentration and spleen Anti-inflammatory, anti-allergic and anti-inflammatory effects that inhibit the production of IL-4, IL-5, and IL-13 secreted from the cells.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of an inflammatory disease or an allergic disease, which comprises an extract of a complex herbal medicine consisting of a Chinese medicine, a Chinese medicine, a white lotus,

Specifically, the conjugated herbs defined herein have a relative weight ratio (w / w) of from 1: 20: 1 to 10: 1 to 20: 1 to 20, preferably 1: Is mixed at a blending ratio of 10: 1 to 8: 1 to 10: 1 to 10, more preferably 1: 3: 1 to 3: 1 to 3: 1 to 3.

The extracts defined herein may be extracted with water, methanol, ethanol, butanol or a solvent mixture thereof, preferably water, methanol, butanol or a mixture thereof, more preferably water and ethanol Solvent, more preferably an extract which is soluble in 50 to 90% ethanol mixed solvent.

The inflammatory diseases as defined herein include, but are not limited to, acute or chronic inflammatory diseases, chronic bronchitis and related obstructive airways diseases, rhinitis and rhinitis, arthritis or inflammatory bowel disease, pain, arteriosclerosis, heart disease, multiple sclerosis, Parkinson ' Colon cancer and the like, preferably acute or chronic inflammatory diseases, arthritis or inflammatory bowel disease.

The allergic diseases defined herein include allergic and allergic reactions such as rhinitis, asthma, contact dermatitis, allergic conjunctivitis, allergic diseases such as atopic dermatitis, preferably rhinitis, asthma, contact dermatitis or rhinitis or atopic dermatitis .

Hereinafter, the method for obtaining the extract of the present invention will be described in detail.

For example, the extract can be used as a raw material in a dry state as a raw material, a gum, a white paper, and a mint as a raw material, preferably in a ratio of 1: 20: 1 to 10: 1 to 20: 1 to 3: 1 to 3: 1 to 3, preferably 1: 1 to 10: 1 to 8: 1 to 10: 1 to 10, more preferably 1: ; Water, a lower alcohol of C ₁ to C ₄ or a mixed solvent thereof, preferably water and an ethanol mixed solvent, is added by about 1 to 5 times, preferably 2 to 4 times the weight of the dried raw material, More preferably at room temperature or at 80 占 폚 for 10 to 30 hours, preferably for about 18 to 25 hours, at a temperature of 5 to 100 占 폚, preferably 20 to 90 占 폚, Or a heat extraction method, preferably a cold extraction method, followed by filtration and concentration under reduced pressure to obtain the extract of the present invention.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease or an allergic disease, which comprises a complex herbal medicine extract obtained by the above method and comprising a herbal extract, a gingiva, a white gingiva, and a peppermint as an active ingredient.

The AR5 herbal composition extract prepared above inhibited the production of NO and cytokine (TNF-α, IL-1) secreted from macrophages after LPS stimulation, and decreased serum IgE concentration in ovalbumin-induced rhinitis animal models Anti-inflammatory, anti-allergic and anti-asthmatic effects that inhibit the production of IL-4, IL-5, and IL-13 secreted from splenocytes.

The pharmaceutical composition of the present invention comprises 0.1 to 50% by weight of the above extract, based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Since the extract of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for the purpose of prevention.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preparation containing the extract of the present invention is preferably administered by parenteral administration, for example, in the form of a spray in the form of an inhalation, non-forcible, and the like.

Accordingly, the present invention provides a spray formulation, a non-formulation formulation or a dentifrice formulation for the treatment and prevention of an inflammatory disease or an allergic disease containing the extract as an active ingredient by the above production method.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.5 g / kg to 5 g / kg per day, preferably 1 g / kg to 3 g / kg per day. The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Further, the present invention provides a health functional food for preventing or ameliorating an inflammatory disease or an allergic disease, which comprises a complex herbal medicine extract comprising an active ingredient, consisting of ginseng, ginseng, white paper and mint.

&Quot; Health functional food "as defined herein means food prepared and processed using raw materials or ingredients having functionality useful to the human body in accordance with Law No. 6727 on Health Functional Foods." Functional " Structure and function of the nutrient to control or physiological effects, such as to obtain a beneficial effect for health is intended to eat.

The health functional food for preventing the inflammatory disease or allergic disease of the present invention contains 0.01 to 95% by weight, preferably 1 to 80% by weight of the extract, based on the total weight of the composition.

In addition, for the purpose of preventing inflammatory diseases or allergic diseases, it can be manufactured and processed into health functional foods in the form of tablets, capsules, powders, granules, liquids, and circles.

The present invention provides a health supplement containing a herbal extract comprising the above ingredients, gingiva, gingiva, white papules and peppermint which are effective for the prevention and treatment of inflammatory diseases or allergic diseases.

Examples of foods to which the extract can be added include various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

The composition comprising the extract of the present invention can be used variously for medicines, foods and beverages for the prevention and improvement of inflammatory diseases or allergic diseases. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used as powders, granules, tablets, capsules or beverages have.

Further, the present invention provides a food or food additive containing as its main component a complex herbal medicine extract having a prophylactic and / or ameliorating effect of an inflammatory disease or an allergic disease, comprising:

The extract of the present invention can be added to foods or beverages for the purpose of prevention and improvement of inflammatory diseases or allergic diseases. At this time, the amount of the extract in the food or beverage is generally from 1 to 5% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is preferably 0.02 to 10 g based on 100 ml, Can be added at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient, as long as it contains the extract as an essential ingredient at the indicated ratio, and there is no particular limitation to the liquid ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate 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-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The AR5 complex herbal extract of the present invention inhibits the production of NO and cytokines (TNF-α, IL-1) secreted by macrophages after LPS stimulation, and decreases serum IgE concentration and spleen IL-4, IL-5, and IL-13 secreted by the cells, and thus can be usefully used in compositions for the prevention and treatment of various inflammatory diseases and allergic diseases.

FIG. 1 is a graph showing the cell survival rate by the MTS assay method for the herbal medicine extract,
Figures 2-A, 2-B, and 2-C show the inhibitory effects of NO on the herbal extract; 1b in the supernatant of cells treated with LPS, and FIG.
FIG. 3 is a graph showing the amount of serum IgE in the group of mice induced with orbumin of the herbal extract,
Figures 4-A, 4B and 4C show the results on the inhibition of IL-4, IL-5 and IL-13 production, which are cytokines secreted in splenocytes of the non-saline model induced by ovalbumin.

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

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

Example  1. Preparation of complex herbal medicine extract

(150 g), gulluin (150 g), white paper (150 g), and peppermint (60 g) purchased from Omni Hub Co., Ltd. (http://www.omniherb.com/) The combined extracts (510 g) were immersed in 7,650 ml of 70% ethanol for 24 hours at 60 DEG C, and then an extract was obtained at room temperature. Further, 7,650 ml of 70% ethanol was added thereto for further extraction twice. The filtered filtrate was concentrated under reduced pressure to evaporate the solvent with Vaccum rotary evaporator (Nihon Seiko, VR-205c, Japan) and dried to obtain 59.6 g (yield: 11.7%, hereinafter referred to as Compound Called crude drug).

Reference Example  1. Experimental preparation

Experimental material

Cell culture reagents such as RPMI-1640, Modifed Eagle Medium (MEM), non-essential amino acids solution, fetal bovine serum (FBS), streptomycin and penicillin were purchased from Hyclone (Logan, UT, USA). The IgE measurement products were obtained from BD Biosciences (San Jose, CA, USA), IL-4, IL-6, -5 and IL-13 were purchased from e-Bioscience (Vienna, Austria).

Cell culture

Mouse macrophage RAW 264.7 cells were cultured in DMEM (Hyclone) medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37 ° C and 5% CO ₂ .

Sample Preparation

The herbal extracts were diluted to a concentration of 100, 50, 25, 12.5, 6.25μg / ㎖ in DMSO solvent and diluted with 25, 12.5, 6.25μg / ㎖ of soluble extract Respectively. In asthmatic animal models induced by ovalbumin (OVA), the herbal extracts were administered at a dose of 200 mg / kg.

Experimental Example  1. Cells Survival number  Measure( MTS assay )

In order to examine the effect of the MTS assay method on the cell viability of the samples obtained in the above examples, the following experiment was carried out using the method described in the existing literature (Choi JH et al., Flowers of Inula japonica attenuate inflammatory responses, Immune Network 2010 , 10 (5), 145-152).

MTS solution (Promega, USA) was added to RAW 264.7 cells at a concentration of 2 × 10 ⁵ cells / ml, and the cells were incubated for 24 hours with 100, 50, 25, and 12.5 μg / And the absorbance was measured at 490 nm after 1 hour.

As shown in FIG. 1, the results of this experiment did not show toxicity at the concentrations of the herbal extracts treated.

Experimental Example  2. Nitric oxide ( NO ) And Proinflammatory  Measurement of cytokine production

Experiments were carried out as described below using the method described in the existing literature (Yang JH et al., Proc. Natl. Acad. Sci. Anti-inflammatory activity of ethylacetate fraction of Cliona celata, Immunopharmacol Immunotoxicol 2011 , 33 (2), 373-379).

RAW 264.7 cells (2 × 10 ⁵ cells / well) were pre-cultured on a 24-well plate, and the complex herbal extracts were pretreated for 1 hour at concentrations of 6.25, 12.5, 25, 50 and 100 μg / ml 100 ng / ml of LPS was treated in each well and cultured at 37 ° C and 5% CO ₂ . After incubation for 24 hours, the supernatant was collected and reacted with 100 μl of the same amount of Griess reagent (G4410, Sigma). The absorbance was measured at 540 nm using an ELISA reader (Sunrise-Basic Tecan, TECAN) The amount of NO produced is shown graphically using a standard curve. TNF-α and IL-1b were measured by ELISA kit (R & D System, USA).

As shown in FIG. 2A, in order to examine the inhibitory effect on NO production of the herb extracts, pretreatment of the extracts showed that the concentrations of 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml and 50 μg / ml , Nitrite production inhibited 92%, 79%, 57% and 32%, respectively. As shown in FIGS. 2B and 2C, cytokine levels were significantly increased in the supernatant of LPS-treated cells (27 ng / ml for TNF-α and 160 pg / ml for IL-1b) 25, 23, 20 and 18 ng / ml for TNF-α and 95, 68, 26 and 15 pg / ml for IL-1b in the cell supernatants pretreated with Dependent inhibitory activity against cine production.

Experimental Example  3. With ovalbumin  Induced rhinitis animal model

In order to examine the inhibitory effect on rhinitis using the ovalbumin (OVA) -induced rhinitis animal model of the sample obtained in the above-mentioned example, the experiment described in the existing literature was applied as follows (Jeon EJ et al., The effects of peroxisome proliferator-activated receptor-g agonist on a murine model of experimental allergic rhinitis, Otolaryngology - Head and Neck Surgery 2008 , 139, 124-130).

OVA (A5503, Sigma) was dissolved in saline at a concentration of 200 μg / ml using 6-week-old female BALB / C mice (17 to 18 g, Coatec) and the mixture was mixed with alum (77161, Thermo) 200 [mu] l per mouse (OVA 20 [mu] g) was sensitized peritoneally on days 0 and 14. For induction of antigen, OVA (100 ug) was injected into the nasal passages of mice for 6 days after the last intraperitoneal injection for 5 days. Dexamethasone (Dexa, D4702, Sigma) was used as a reference drug at a dose of 1 mg / kg, and the herbal extract was administered at a dose of 200 mg / kg twice a day for 12 days orally for 12 days. When OVA was administered to the nasal cavity, The drug was administered orally before the hour.

Experimental Example  4. With ovalbumin  In the induced rhinitis animal model IgE  Measurements

In order to examine the inhibitory effect on rhinitis using the ovalbumin (OVA) -induced rhinitis animal model of the sample obtained in the above-mentioned example, the experiment described in the existing literature was applied as follows (Jeon EJ et al., The effects of peroxisome proliferator-activated receptor-g agonist on a murine model of experimental allergic rhinitis, Otolaryngology - Head and Neck Surgery 2008 , 139, 124-130).

In order to measure the amount of rhinitis-related protein produced, blood was collected from the heart of the rhinitis model and serum was separated and the amount of IgE produced was measured by ELISA method. The product was measured using an ELISA reader (Sunrise-Basic Tecan, TECAN) using a Mouse IgE ELISA Set (BD Bioscience).

As shown in FIG. 3, the amount of serum IgE (PC, 13419 ng / ml) in the group of mice induced to orbumin significantly increased when compared with the negative control (NC, 1136 ng / ml) In the mouse group treated with the extract, the amount of IgE decreased (10638 ng / ml), indicating that the IgE level was regulated.

Experimental Example  5. With ovalbumin  In the induced rhinitis animal model IL  Measure

In order to examine the inhibitory effect on rhinitis using the ovalbumin (OVA) -induced rhinitis animal model of the sample obtained in the above-mentioned example, the experiment described in the existing literature was applied as follows (Jeon EJ et al., The effects of peroxisome proliferator-activated receptor-g agonist on a murine model of experimental allergic rhinitis, Otolaryngology - Head and Neck Surgery 2008 , 139, 124-130).

In order to measure the amount of rhinitis-associated protein, IL-4, IL-5, and IL-13 were incubated for 72 hours in OVA (50 μg / ml) Was determined by ELISA method. The measurement kit was measured with an ELISA reader (Sunrise-Basic Tecan, TECAN) using a Platinum ELISA kit (Vienna, Austria) of e-Bioscience.

As shown in FIGS. 4A, 4B, and 4C, cytokines secreted from splenocytes of the non-saline model induced by ovalbumin were quantitatively determined to be 83 pg for IL-4 ml, 104 pg / ml, and 2865 pg / ml, respectively, in the mice treated with the herbal extracts, Lt; RTI ID = 0.0 > cytokine < / RTI > production.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically explained.

Formulation example  One. Sanje  Produce

AR5 extract -------------------------- 20 mg

Lactose ------------------------------- 100 mg

Talc -------------------------------- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

AR5 extract -------------------------- 10 mg

Corn starch ------------------------- 100 mg

Lactose ------------------------------- 100 mg

Magnesium stearate ------------------ 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

AR5 extract -------------------------- 10 mg

Crystalline cellulose -------------------- 3 mg

Lactose -------------------------- 14.8 mg

Magnesium stearate -------------- 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

AR5 extract -------------------------- 10 mg

Mannitol ----------------------------- 180 mg

Sterile sterile distilled water for injection ---------------- 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O ------------------------ 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

AR5 extract -------------------------- 20 mg

Isomerization Party ----------------------------- 10 g

Mannitol -------------------------------- 5 g

Purified water -------------------------------

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of spray formulations

Ingredients and Content (in 100 ml) (specific gravity: 0.812 g / ml)

AR5 extract: 0.9 g

Glycerin: 6.30 g

Stevioside (100%): 0.020 g

Anhydrous ethanol: 74.8 g

6.30 g of glycerin, 70.0 g of anhydrous ethanol and 0.9 g of AR5 extract were dissolved and dissolved in 0.1 g of purified water separately, and 0.02 g of stevioside (100% purity) was dissolved in purified water in accordance with a conventional spray preparation method After adding 4.8 g of anhydrous ethanol to the above solution, the total volume is made to 100 ml. It is put into a spray bottle and capped. When using, the separately sprayed spraying device is replaced with a cap and assembled.

Claims (11)

The present invention relates to a pharmaceutical composition comprising a complex herbal medicine having a relative ratio (w / w) ratio of 1: 1 to 20: 1 to 10: 1 to 20: 1 to 20 as an active ingredient For the prevention and treatment of rhinitis, arthritis, inflammatory bowel disease, contact dermatitis or atopic dermatitis. delete delete delete delete delete The present invention relates to a pharmaceutical composition comprising a complex herbal medicine having a relative ratio (w / w) ratio of 1: 1 to 20: 1 to 10: 1 to 20: 1 to 20 as an active ingredient A health functional food for preventing and improving rhinitis, arthritis, inflammatory bowel disease, contact dermatitis or atopic dermatitis. delete delete delete delete

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