KR101150485B1 - A pharmaceutical composition comprising extract of Alchornea triplinervia for prevention and treatment of asthma or inflammatory diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of asthma or inflammatory diseases, which contains Alchornea triplinervia (Spreng.) Muell. Arg. (Euphorbiaceae) extract. Suppressant extract inhibits the rapidly increased NO production due to inflammation, inhibits the secretion of immunoglobulin E (IgE) and cytokines from bronchoalveolar lavage fluid in ovalbumin-induced asthma models, and increases the inflammation caused by eosinophils. By inhibiting the Alkonia triple nubia The composition containing the extract as an active ingredient may be usefully used in medicines or health functional foods for the prevention and treatment of asthma or inflammatory diseases.

Description

A pharmaceutical composition comprising extract of Alchornea triplinervia for prevention and treatment of asthma or inflammatory diseases

The present invention relates to a composition for the prevention and treatment of asthma or inflammatory diseases containing Alchornea triplinervia extract.

Inflammation is a continuous and complex reaction that destroys the cells that make up the body. Inflammatory reactions are pathological conditions formed by the invasion of external infectious agents (bacteria, fungi, viruses, various allergens), and their damage when invasion results in any organic change in cells or tissues. It is a defensive reaction process of a living body trying to repair and repair a site. These defense mechanisms are cytokines, plasma enzyme systems (complementary system, coagulation factor, kinin, etc.), lipid transporters secreted from other cells (prostagladins, nucotrienes), mast cells, leukocytes. basophils, regulated by the action of modulators in the blood vessels that augment from platelets (Ross A. et al., 2002). Macrophages are involved in innate immunity and adaptive immunity, and macrophages are activated against bacteria, fungi, viruses, and various types of allergens.

In general, the inflammatory response is a biological defense mechanism for regenerating damage caused by invasion that causes any organic change in cells or tissues of the body, and local blood vessels, various tissue cells of body fluids and immune cells work in this process. do. Normally, the inflammatory response induced by external invading bacteria is a protective action to protect the living body, while abnormally excessive inflammatory response is induced various diseases. These are life-threatening diseases such as acute inflammation, diseases in the joints such as rheumatoid arthritis, skin diseases appearing in the form of psoriasis, and allergic inflammatory diseases such as bronchial asthma.

Asthma, a chronic inflammatory disease, is caused by airway hyperresponsiveness, deposition of inflammatory cells into the lung tissue, and overproduction of mucus (Kon and Kay, 1999), interleukin-4 (IL-4), and interleukin- 5, cytokines, including interleukin-13, and chemokines such as eotexin and lentis are overexpressed, leading to allergic asthma (Berkman et al., 1996). In activated B cells, mast cells and eosinophils cells produce a variety of inflammatory mediators (Kay AB.2001). Eosinophilic cells increase the levels of particulate proteins and generate reactive oxygen species as the primary mediators of asthma development (Elsner J et al., 1999).

Asthma is the most allergic disease. Clinical symptoms such as wheezing, dyspnea and cough caused by extensive narrowing of the airways can be reversibly improved naturally or by treatment (Minoguchi K and Adachi M. 1999). The number of deaths from asthma is increasing worldwide, with 3-10% of people suffering from asthma (Martin BL et al., 1997). Some medicines for the treatment of asthma exist but have side effects.

Treatments for allergic diseases include dexamethasone and cortisone using corticosteroids. In the treatment of asthma, corticosteroids inhibit erosion of eosinophils, inhibit the migration of eosinophils and lymphocytes to the bronchial mucosa and inhibit the expression of TH2 cytokines (Trigg CJ et al., 1994 and Caramori G et. al., 2005). However, they act as inflammatory drugs, but they are highly toxic and can cause edema-like symptoms as a side effect. In addition, there is a case that a problem that causes a severe immunosuppression occurs due to the selective action on the cause of inflammation (Check WA, Kaliner MA, 1990).

The anti-inflammatory drugs presently present are a steroid treatment using corticosteroids and thus have side effects. Therefore, there is a need for a nonsteroidal therapeutic agent having no side effects. Therefore, in consideration of the side effects and stability in vivo, the interest in the herbal medicine, using the ingredients extracted from the plant is conducting a study on the development of therapeutic agents for inflammation and asthma.

Alconia triplenervia is euphorbiaceae, 5-20 m high, mainly distributed in low land and lowland forests (Berruy et al., 1999). Three kinds of bones exist as basics, and are distributed in southern Brazil and southern United States. In general, the ring is almost faint or absent, the tree is brown and without strings (Mainieri and Peres Chimelo, 1989).

The leaves and thin parts of alconia triplenervia have been widely used by Brazilians as a tea-drinking drug for treating digestive disorders in general (Silva EM et al., 2000). Alchornea cordifolia, belonging to the same genus as Alkonia Triplenervia, has anti-inflammatory effects and has antibacterial effects against Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli. Have Alchornea castaneaefolia and Alchornea glandulosa are used to treat ulcers (Manga MH et al., 2004, Hiruma-Lima CA et al., 2006, Calvo TR et al., 2007). Alconia triplenervia leaves were separated from amentoflavone, isocorilagin, gallic acid and methyl gallate (Brace A et al., 2002). It is known to have an effect of protecting the stomach and has an antibacterial effect (ZP Lima et al., 2008).

Accordingly, the present inventors conducted continuous research to develop a substance capable of preventing, treating or improving asthma or inflammation as described above, and as a result, the alconia triplenervia extract suppresses NO production which is rapidly increased by inflammation, Anti-asthmatic or anti-inflammatory drugs, processed foods, and functional foods by inhibiting the secretion of immunoglobulin E (IgE) and cytokines from bronchoalveolar lavage fluid and suppressing the increased inflammation caused by eosinophils in ovalbumin-induced asthma models The present invention was completed by confirming that it can be usefully used in compositions such as food additives, functional drinks or beverage additives.

An object of the present invention to provide a composition, health food or cosmetic composition for the prevention and treatment of inflammatory diseases using Alchornea triplinervia L. extract.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing Alchonia triplenervia ( Alchornea triplinervia ) extract as an active ingredient.

In addition, the present invention provides an external preparation for skin for the prevention and improvement of inflammatory diseases containing an alconia triplenervia extract as an active ingredient.

In another aspect, the present invention provides a cosmetic composition for the prevention and improvement of inflammatory diseases containing alkania triple nubia extract as an active ingredient.

In addition, the present invention provides a health food or food additives for the prevention and improvement of inflammatory diseases containing alkania triple nubia extract as an active ingredient.

Alchonia triplenervia ( Alchornea triplinervia ) extract of the present invention has excellent anti-inflammatory and anti-asthmatic activity, almost no cytotoxicity, pharmaceuticals, processed food, functional for the prevention and treatment of inflammation-related diseases, allergies and asthma It can be usefully used as an active ingredient of a composition such as food, food additives, functional beverages or beverage additives.

1 is a graph showing the cytotoxicity of the Alconia triplenervia extract in RAW264.7 cells:
-Negative control group administered only DMSO;
30: experimental group treated with 30 μg / ml of alconia triplenervia methanol extract; And
50: Experimental group treated with 50 μg / ml of Alkonia triplenervia methanol extract.
Figure 2 is a graph showing the inhibitory effect of the Alconia triplenervia extract on the production of NO induced by LPS (P value (*) is 0.05, (**) is 0.005 or less):
-Negative control group administered only DMSO;
+: Positive control group induced by LPS;
30: LPS-induced experimental group after treatment with 30 μg / ml of the alconia triplenervia methanol extract; And
50: Experimental group induced by LPS after treatment with 50 μg / ml of Alkonia triplenervia methanol extract.
FIG. 3 is a graph showing the effect of Alkonia triplenervia extract on the total inflammatory cell number and eosinophil count of bronchoalveolar lavage fluid after induction of airway inflammation:
NC: non-inflammatory voice control;
OVA: positive control group airway sensitized with egg white albumin; And
AT; Experimental group treated with 30 mg / kg of alconia triplenervia methanol extract.
4 is a graph showing the results of measuring the amount of immunoglobulin-E (IgE) that specifically binds egg white albumin in serum after induction of asthma with egg white albumin.
Figure 5 is a graph showing the results of measuring the content of the total immunoglobulin-E and immunoglobulin-E specifically binding to egg white albumin after induction of airway inflammation, BALF.
6 is a graph showing the results of measuring the cytokine content of bronchoalveolar lavage fluid after inducing airway inflammation:
A: interleukin-4;
B: interleukin-13; And
C: Eothexin.
Figure 7 is a graph showing the result of measuring the cytokine through the polymerase chain reaction to make DNA by using RNA isolated from the lung tissue induced airway inflammation, reverse transcriptase.
8 is a graph showing the results of measuring the antioxidant effect of bronchial alveolar lavage fluid after inducing airway inflammation.
9 is a diagram showing the results of staining bronchial lung tissue after inducing airway inflammation.

Hereinafter, terms used in the present invention will be described.

As used herein, the term "inflammation" refers to a pathological condition of abscesses formed by the invasion of external infectious agents (bacteria, fungi, viruses, various types of allergens).

As used herein, the term "prevention" means any action that inhibits or delays the progression of an inflammatory disease by administration of a composition of the present invention.

As used herein, the terms "treatment" and "improvement" refer to any action in which the symptoms of an inflammatory disease improve or benefit from administration of a composition of the present invention.

As used herein, the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner.

The term "individual" as used herein refers to all animals, including humans, monkeys, dogs, goats, pigs, or rats, having a disease in which the symptoms of an inflammatory disease can be improved by administering the composition of the present invention.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which means the type of disease, the severity, the activity of the drug, the drug Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including drug used concurrently, and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing Alchornea triplinervia extract as an active ingredient.

The inflammatory diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia (fibromyalgia) ), Psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases Any one is preferred, but is not limited thereto.

The alconia triplenervia extract is preferably prepared by a manufacturing method comprising the following steps, but not always limited thereto:

1) extracting by adding an extraction solvent to Alchornea triplinervia ;

2) filtering the extracted extract of step 1); And

3) drying the filtered extract of step 2) under reduced pressure.

In the above method, Alchornea triplinervia of step 1) may be used without limitation, such as being grown or commercially available.

The extraction solvent is preferably water, alcohol or a mixture thereof. As the alcohol is preferred to use a C ₁ to C ₄ lower alcohol, it is preferable to use the lower alcohol is methanol or. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but is not limited thereto. The extraction solvent is preferably extracted by adding 1 to 10 times the dried alconia triplenervia amount. The extraction temperature is preferably 30 to 100 ° C., but is not limited thereto. In addition, the extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, but is not limited thereto. In addition, the number of times the extraction is preferably 3 to 5 times, more preferably three times repeated extraction is not limited thereto.

In the above method, the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

In order to determine the cytotoxicity of the alkalonia triple nubbia extract, the present inventors performed MTT test by treating alkalonia triple nubbia extract to macrophages by concentration, and the alconia triple nubbia extract was 50 ug / ml. It was confirmed that there is no cytotoxicity at the concentration (see Table 1 and FIG. 1).

In addition, the present inventors analyzed the degree of generation of NO in cells treated with an inflammation-inducing substance in order to determine the NO production inhibitory effect of the alconia triplenervia extract, the alconia triplenervia extract was NO compared to the control group. The production was significantly inhibited (see Table 2 and FIG. 2).

In addition, the present inventors analyzed the number of inflammatory cells and eosinophils by using a mouse model inducing airway sensitization with egg white albumin to determine the effect of inflammatory cells and eosinophils of the alconia triple nubia extract. Poach extract significantly reduced the number of inflammatory cells and eosinophils compared to the control (see Table 3 and Figure 3).

In addition, the present inventors analyzed the IgE levels in serum and bronchial alveolar lavage fluid using a mouse model that induced airway sensitization with egg white albumin to determine the effect of IgE reduction of alkoonia triple nubia extract. Poach extract significantly reduced the production of IgE compared to the control (see FIGS. 4 and 5).

In addition, the present inventors measured the contents of IL-4 and IL-13 using a mouse model that induced airway sensitization with egg white albumin, in order to determine the cytokine production inhibitory effect of alconia triple nubia extract Konya triplenervia extract significantly reduced the production of cytokines compared to the control (see Table 5 and FIG. 6).

In addition, the present inventors analyzed the amount of cytokine mRNA using RT-PCR in a mouse model inducing airway sensitization with egg white albumin to determine the inhibitory effect of cytokine mRNA production of alconia triplenervia extract. As a result, the alkania triplenervia extract significantly reduced the amount of cytokine mRNA compared to the control (see FIG. 7).

In addition, the present inventors analyzed the amount of reactive oxygen species in a mouse model inducing airway sensitization with egg white albumin, in order to determine the inhibitory effect of active oxygen species (ROS) production of the alconia triple nubia extract, The extract of Lipoblastia significantly reduced the production of reactive oxygen species compared to the control group (see FIG. 8). Therefore, it was found that the alkania triplenervia extract has an antioxidant effect.

In addition, the present inventors observed the change of bronchial inflammatory cells and epithelial cells in a mouse model that induced airway sensitization with egg white albumin, in order to determine the anti-asthma effect of the alconia triple nubia extract, The extract significantly reduced the number of inflammatory cells, including eosinophils, compared to the control group, and reduced the damage of the epithelial cells to an almost invisible level (see FIG. 9). Therefore, the alconia triplenervia extract can prevent infiltration of inflammatory cells, including eosinophils, thereby inhibiting asthma and preventing damage to epithelial cells, thereby preventing or treating allergic diseases caused by infiltration of inflammatory cells. It can be seen.

Taken together, the alconia triplenervia extract of the present invention inhibits NO production, which is rapidly increased by inflammation, and significantly inhibits the secretion of IgE and cytokines in bronchoalveolar lavage fluid in egg white albumin-induced asthma models. In addition, it can be seen that it can be usefully used as an active ingredient in preventing and treating asthma or inflammatory diseases by inhibiting increased inflammation caused by eosinophils.

The composition of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the alconia triplenervia extract.

The composition of the present invention is preferably 0.1 to 99 parts by weight of the alkania triplenervia extract based on the total weight, but is not limited thereto.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose , Mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opiodry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate , Sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition of 0.1 to 90 parts by weight, but is not limited thereto.

That is, the composition of the present invention can be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used It can be prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, It may be prepared by mixing sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc may also be 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, water and liquid paraffin. . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, 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 composition of the present invention may be administered orally or parenterally according to a desired method, and when administered parenterally, external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method It is preferable to select. Dosage ranges depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and the severity of the disease.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease of the patient, the daily dosage is Alkonia Triplenervia 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, based on the amount of extract, and may be administered 1 to 6 times per day.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of inflammatory diseases.

In addition, the present invention provides a method for preventing inflammatory disease, comprising administering to a subject a composition comprising a pharmaceutically effective amount of an Alconia triplenervia extract as an active ingredient.

The present invention also provides a method for treating an inflammatory disease comprising administering to a subject suffering from an inflammatory disease a composition comprising an pharmaceutically effective amount of an alkonia triplenervia extract as an active ingredient.

The inflammatory diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia (fibromyalgia) ), Psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases Any one is preferred, but is not limited thereto.

In addition, the present invention provides an external preparation for skin for the prevention and improvement of inflammatory diseases containing an alconia triplenervia extract as an active ingredient.

The inflammatory diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia (fibromyalgia) ), Psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases Any one is preferred, but is not limited thereto.

Suitable formulations for use as external skin preparations include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase, It may be provided in the form of a nonionic vesicle dispersant, in the form of a cream, skin, lotion, powder, ointment or spray. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The external skin preparations include fatty acid, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water in addition to the alconia triplenervia extract. Commonly used in ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or external preparations for skin And any other ingredients that may be used, such as adjuvants conventionally used in the field of dermatology. In addition, the ingredients may be introduced in amounts generally used in the field of dermatology.

In another aspect, the present invention provides a cosmetic composition for the prevention and improvement of inflammatory diseases containing alkania triple nubia extract as an active ingredient.

The inflammatory diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia (fibromyalgia) ), Psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases Any one is preferred, but is not limited thereto.

The cosmetic composition of the present invention includes, but is not limited to, a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids, and seaweed extracts in addition to the alconia triplenervia extract.

The cosmetic composition of the present invention may be blended with the above essential components, if necessary, with other ingredients normally formulated into cosmetics. The other components include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings, blood circulation accelerators, Cooling agents, limiting agents or purified water are preferred, but not limited thereto.

In addition, the compounding component which may be added other than this is not limited to this, Although it can mix | blend any of the above components in the range which does not impair the objective and effect of this invention, It is preferable to mix | blend in 0.001-10% weight percentage with respect to gross weight, It is not limited to this.

The cosmetic composition of the present invention may take the form of a solution, an emulsion or a viscous mixture, but is not limited thereto. Ingredients included in the cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the alkania triplenervia extract of the present invention as an active ingredient, such as stabilizers, solubilizers, vitamins, pigments and fragrances Conventional auxiliaries and carriers may be included but are not limited to these.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, but may be prepared in an emulsion, cream, lotion, pack, foundation, lotion, essence or hair cosmetic, but is not limited thereto. Specifically, the cosmetic composition of the present invention skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, Formulations of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions or body cleansers.

In addition, the present invention provides a health functional food or food additives for the prevention and improvement of inflammatory diseases containing alconia triple nubia extract as an active ingredient.

The inflammatory diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia (fibromyalgia) ), Psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases Any one is preferred, but is not limited thereto.

The alkania triplenervia extract of the present invention may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.

There is no particular limitation on the kind of the food. Examples of foods to which the Alkonia triple nubia extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages , Tea, drink, alcoholic beverages and vitamin complexes, etc., and includes all of the health food in the usual sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the alkania triple nubbia extract of the present invention has various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, and preservatives. , Glycerin, alcohol, carbonation agent used in carbonated beverages, and the like. In addition, the alconia triplenervia extract of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by Examples and Preparation Examples.

However, the following Examples and Production Examples specifically illustrate the present invention, and the content of the present invention is not limited to the Examples and Production Examples.

Example 1 Alkonia triple nubia ( Alchornea triplinervia A) extract manufacturer

<1-1> Alkonia Triplenervia Methanol Extract

The inventors dried and pulverized 10 kg of leaves of Alchornea triplinervia (Iquitos, Loreto, Peru) and pulverized, followed by adding 20 liters of methanol to the samples of alconia triplenervia and circulating at room temperature. Extraction over time recovered the filtrate supernatant. This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure to obtain 2.9 kg of alkania triplenervia methanol extract.

<1-2> Alkonia Triplenervia Ethanol Extract

In the manufacturing method of Example <1-1>, 1.0 kg of alkania triplenervia ethanol extract was obtained using ethanol instead of methanol.

<1-3> Alkonia Triplenervia Water Extract

1.0 kg of the alconia triplenervia water extract was obtained using water instead of methanol in the preparation method of Example <1-1>.

Example 2 Cytotoxicity Test of Alkonia Triplenervia Extract

The present inventors suspended 10026ul of raw macrophage cells of mice in DMEM (Dulbecco's Modified Eagle Medium, Gibco) medium with 10% Fetal Bovine Serum, and then suspended them at a concentration of 5 × 10 ⁴ / ml. Each well was placed in a 96 well plate (96 well plate) and incubated in an incubator until the cells were attached. After approximately 4 hours, each alcoonia triplenervia extract was treated at a concentration of 50 ug / ml and 30 ug / ml. After incubation for 24 hours, 10 mg of 5 mg / ml MTT solution was added to each well, followed by 4 hours of incubation. After the incubation was completed, the supernatant was removed, and 100 ul of DMSO was added. Absorbance was measured at 570 nm. Cell viability was calculated according to Equation 1 below with 100% of the negative control treated with 0.1% DMSO.

As a result, as shown in Table 1 and FIG. 1, it was confirmed that the alconia triplenervia extract had no cytotoxicity at concentrations of 30 and 50 ug / ml (Table 1 and FIG. 1).

sample Cell survival rate (%) Negative Control 100.0 ± 1.2 Example <1-1> Extract 30 ug / ml 99.2 ± 1.7 Example <1-1> Extract 50 ug / ml 97.6 ± 2.4 Example <1-2> 30 ug / ml Extract 99.0 ± 1.5 Example <1-2> 50 ug / ml Extract 97.2 ± 2.2 Example <1-3> 30 ug / ml extract 99.2 ± 1.5 Example <1-3> Extract 50 ug / ml 97.5 ± 1.8

Example 3 NO Production Inhibitory Effect of Alkonia Triplenervia Extract

The present inventors treated LPS with Raw264.7 cells to randomly induce inflammation, and then measured the amount of inducible nitric oxide (NO) in order to determine the inhibitory effect on inflammation. 96-well plate with 10% Fetal Bovine Serum added to DMEM (Dulbecco's Modified Eagle Medium, Gibco) medium containing no phenol-Red and suspended 5 × 10 ⁴ / ml cells (96 well plate) was incubated uniformly. After attaching the cells for 4 hours, the alconia triplenervia methanol extract was treated at a concentration of 30 and 50 ug / ml and incubated for 1 hour, followed by 1 ug / ml lipopolysaccharide (LPS, lipopolysaccharide, Sigma). ) Was incubated for 24 hours. Thereafter, 100 µl of the supernatant was collected, placed in a new 96-well plate, and the same amount of grease reagent (Griess reagent, Sigma) was added and reacted at room temperature for 10 minutes, followed by a microplate reader (Bio-Rad). Absorbance was measured at 550 nm. The NO production amount of the samples was quantified using various concentrations of sodium nitrite standard curves dissolved in the same medium, and the inhibition rate of each sample was expressed as a percentage by 100% of the NO production amount of the LPS-treated group. Statistical analysis was performed by Student's t-test, and significance was expressed by p value (p <0.05).

As a result, as shown in Table 2 and FIG. 2, the amount of NO produced in the LPS treatment group was significantly increased compared to the negative control group (2.76 μM) treated with the excipient. However, when treated with LPS-treated macrophages triple nubbia extract significantly inhibited the production of nitrite than the LPS-only group (Table 2 and Figure 2).

sample Concentration (ug / ml) NO (uM) Inhibition Rate (%) Negative Control 2.76 ± 0.21 LPS treatment group One 13.82 ± 0.25 Example <1-1> Extract 30 ug / ml 30 9.77 ± 0.58 29.3 Example <1-1> Extract 50 ug / ml 50 7.24 ± 0.30 47.6

Example 4 Analysis of Effects of Alkonia Triplenervia Extract on Inflammatory Cells and Eosinophils

We purchased 8-week-old uninfected pathogen females (Balb / c) (weight: about 20 g) from Orient (Seoul, Korea), followed by 2 mg aluminum hydroxide (Sigma A8222) at two-week intervals. 200 µl of phosphate buffer solution (pH 7.4) suspended in 20 µg egg white albumin (Sigma A5503) was injected into the abdominal cavity twice. After 28 days, 29 days and 30 days, the phosphate buffer solution containing 1% egg white albumin was sprayed in a closed container containing mice for 20 minutes using an ultrasonic nebulizer, and the negative control group did not cause airway sensitization. 6 mice), positive control group airway sensitized with egg white albumin (6 mice), control group administered with anti-asthma medicine Montecaster 30 mg / kg (6 mice), Alkonia triple width as experimental group The methanol extract of 30 mg / kg was suspended in a phosphate buffer solution and then experimented with a group of mice (6 mice) orally administered 1 hour prior to antigen administration. 48 hours after the last antigen administration, an excess of pentobarbital (Sigma P3761) was administered and lethal before bronchial incision was performed. Bronchoalveolar lavage fluid (BALF) was obtained by inhalation of 0.6 ml three times by cannula insertion into the trachea. Total inflammatory cells and eosinophil counts were measured as follows. 100 μl of the bronchial alveolar fluid of each experimental group was placed on the slide and centrifuged using a cytospin device (Hanil, Korea) to fix the cells on the slide. Total cell number except dead cells stained with Trypan blue was calculated using a hemocytometer and repeated three times (Daigle I, et al., 2001). Eosinophil counts were determined after staining with Diff-Quick reagent (Sysmex, Cat No. 38721, Switzerland) and calculated in the same manner as total cell numbers. Statistical analysis was performed by Student's t-test, and significance was expressed by p value.

As a result, as shown in Table 3 and Figure 3, in the case of normal mice without airway sensitization as a negative control, the total number of inflammatory cells (bronchoalveolar alveoli) of the bronchial alveolar (inflammatory cells) was 109 ± 31 × 10 ⁴ cells / ㎖, Eosinophils were almost 0%. As a positive control group, the total inflammatory cell count was increased more than 10 times to 2302 ± 345 × 10 ⁴ cells / ml when sensitized with egg white albumin and phosphate buffer solution. The total number of inflammatory cells in the case of Alkonia triplenervia extract 854 ± 220 × 10 ⁴ cells / ml decreased 37% and eosinophil count also decreased 41.3%. In addition, when the administration of the anti-asthma drug Montecaster as a comparison group, the total number of inflammatory cells was 548 ± 141 × 10 ⁴ cells / ㎖ (Table 3 and Figure 3).

Kinds Per mouse
Total number of inflammatory cells (in thousands) Per mouse
Number of eosinophils (in thousands) Normal mouse 100 or less Not observed Phosphate buffer solution treatment after sensitization of egg white albumin 2302 ± 345 725 ± 108 Example <1-1> Egg white albumin sensitization after extract treatment 854 ± 220 304 ± 72 Egg white albumin sensitization after montecaster treatment 548 ± 141 335 ± 143

Example 5 Analysis of the Effect of Alkonia Triplenervia Extract on Immunoglobulin E (IgE)

<5-1> IgE Analysis of Specific Binding to Egg White Albumin in Serum

In order to measure the IgE present in the serum of the animal, the present inventors dilute the serum of each experimental group obtained in Example 4 in a diluted solution 40 times, and then use 100 µl of egg white albumin used to induce asthma. The antigen-antibody reactions were induced at room temperature for 2 hours in μg-attached 96-well plates.

As a result, as shown in Figure 4, the rats sensitized with egg white albumin, while the content of IgE was sharply increased, whereas the production of IgE was significantly reduced in the Alkonia triple nubbia methanol extract treatment compared to egg white albumin treatment (Fig. 4).

<5-2> IgE analysis in bronchial alveolar lavage fluid

Sandwich-type enzyme-linked immunosorbent assay (ELISA) was used to measure IgE present in bronchial alveoli. 100 μl of the bronchial alveolar fluid of each experimental group obtained in Example 4 was placed in a 96 well plate with capturing IgE antibody to induce antigen-antibody reaction at room temperature for 1 hour. Plates were previously blocked with 5% bovine serum albumin (BSA) for 1 hour and then washed three times with washing solution (PBS + 0.1% tween 20). After the antigen-antibody reaction was finished, three washings were performed, followed by 100 μl of a detection IgE antibody (mouse, PharMingen, San Diego, USA) bound with horseradish peroxidase (HRP). Was added to the wells and allowed to react for 1 hour. After the reaction, the solution was washed five times with ortho-phenylene diamine (OPD) and hydrogen peroxide (H ₂ O ₂ ) as a substrate to induce a color reaction, and then measured the absorbance at 405 nm. The amount of immunoglobulin E in bronchial alveolar fluid was measured.

As a result, in the mice sensitized with egg white albumin, as shown in Table 4 and FIG. 5, the content of IgE was rapidly increased, whereas the production of IgE was higher in the Alkonia triplenervia methanol extract treatment than in the egg white albumin treatment. This was significantly reduced by more than 50% (Table 4 and FIG. 5).

Kinds Immunoglobulin E Content (ng / ml) Immunoglobulin E Content Combined with Egg White Albumin Normal mouse 0 or less 0 or less Egg white albumin sensitization 11.32 ± 4.99 7.04 ± 1.56 Example <1-1> Egg white albumin sensitization after extract treatment 4.16 ± 4.82 6.20 ± 1.06 Egg white albumin sensitization after montecaster treatment 3.24 ± 1.50 5.36 ± 0.93

Example 6 Cytokine Production Inhibitory Effect of Alkonia Triplenervia Extract

Sandwich-type enzyme-linked immunosorbent assay (ELISA) was used to measure cytokines present in bronchial alveoli. 100 μl of the bronchial alveolar solution of each experimental group obtained in Example 4 was placed in a 96-well plate to which a cytokine antibody was attached to induce an antigen-antibody reaction at room temperature for 2 hours. In order to measure the contents of interleukin-4 (IL-4) and interleukin-13 (IL-13), an ELISA kit (BioSource International, Camarillo, CA) that specifically reacts with each cytokine was used. The content of each cytokine was measured.

As a result, as shown in Table 5 and FIG. 6, in the rat sensitized with egg white albumin, the content of interleukin-4 was increased, and the cytokine production was higher in the alkonia triplenervia methanol extract treatment than the egg white albumin treatment. It was significantly reduced (Table 5 and FIG. 6).

Kinds Interleukin-4 content Interleukin-13 content Eotexin Content Normal mouse   66.3 ± 11.3  17.3 ± 2.96  16.7 ± 3.3 Egg white albumin sensitization 107.7 ± 7.1  28.1 ± 1.88  27.7 ± 2.0 Egg white sensitization after extract treatment  88.7 ± 5.6  24.1 ± 2.46  24.1 ± 2.5 Egg white albumin sensitization after montecaster treatment   60.1 ± 12.0  29.3 ± 7.76  23.9 ± 3.4

Example 7 Cytokine mRNA (messenger RNA) Inhibition Effect of Alkonia Triplenervia Extract

The present inventors performed reverse transcription polymerase chain reaction (quantitative RT-PCR) (Promega, Madison, Wis.) In order to measure the amount of cytokine RNA present in the lung tissue of the experimental group. After extracting the whole Rn from the tissue, the complementary DNA (cDNA) was made through reverse transcriptase polymerase chain reaction, and then polymerase chain reaction was performed using primers of cytokines.

As a result, As shown in FIG. 7, in rats sensitized with egg white albumin, the amount of RNA of interleukin-4 was increased, and the production of cytokine R & A was significantly higher in the alconia triplenervia methanol extract treatment than the egg white albumin treatment. Was reduced (FIG. 7).

Example 8 Inhibitory Effect of Alkonia Triplenervia Extract on Reactive Oxygen Species (ROS) Production

In order to investigate the association between asthma-induced and antioxidant activity in this experimental animal, free radical species production experiments were performed. Oxygen species, which are essential for organic respiratory organisms, are incompletely reduced or generated by peptide growth factors, cytokines, and various actions during the enzymatic and cellular electron transfer or energy metabolism processes. It refers to an unstable state with fee radicals. Excessive production results in toxicity, or oxidative stress, to the living body. By oxidizing huge molecules (proteins, lipids, etc.) in the cell, it destroys the homeostasis of the cell, kills the cell, and causes fatal damage in the tissue.

In order to measure the reactive oxygen species present in bronchial alveolar, the amount was measured using DCFDA (Molecular Probes, OR), and as shown in FIG. 8, the result of the rat sensitized with egg white albumin, The amount was increased, and the production of active oxygen species was significantly decreased in the Alkonia triple nubbia methanol extract treatment compared to egg white albumin treatment. Therefore, it was found that the alkania triplenervia extract is also effective in antioxidant activity.

Example 9 Anti-asthma Effect of Alkonia Triplenervia Extract

The present inventors performed the following experiment to determine the effect of the alconia triplenervia extract on bronchial inflammatory cells and epithelial cells.

The lung tissue of each experimental group of <Example 4> was soaked in 10% neutral buffer-formalin for 24 hours, and paraffin embedding was then performed. Embedding tissue was cut into 4 mm thick sections to make sections, stained with hematoxylin and Eosin Y (ThermoShandon, Pittsburgh, Pa.) And then encapsulated with Dako-cytosumation (Dakocytomation, Denmark). The stained and sealed slides were examined under an optical microscope.

As a result, as shown in Figure 9, when sensitized with egg white albumin than the normal state without airway sensitization, asthma was induced in the alveolar and bronchioles of the lung tissue, epithelial cells were damaged, and many eosinophils, including around the bronchioles Inflammatory cells were infiltrated. In addition, when the alkania triplenervia methanol extract was administered, inflammatory cells, including eosinophils, were markedly reduced, and epithelial cell damage was hardly seen. This coincided with a decrease in the number of cells causing inflammation and the number of eosinophils when the Alkonia triplenervia methanol extract of <Example 4> was administered.

Therefore, the alkania triplenervia extract can prevent the treatment of allergic diseases caused by the infiltration of inflammatory cells by reducing the infiltration of inflammatory cells, including eosinophils, inhibiting the induction of asthma and preventing the damage of epithelial cells. It can be seen.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Preparation of powder

2 g of extract of Example <1-1>

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of Tablet

100 mg of extract of Example <1-1>

Corn starch 100 mg

Lactose 100 mg

Stearic Acid Magnesium 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of extract of Example <1-1>

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

&Lt; 1-4 >

1 g of extract of Example <1-1>

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

<1-5> Preparation of granules

150 mg of extract of Example <1-1>

Soy extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added, dried at 60 ° C. to form granules, and then filled into fabrics.

Production Example 2 Preparation of Food

<2-1> Production of flour food

0.5-5.0 parts by weight of the extract of Example <1-2> of the present invention was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<2-2> Preparation of Soups and Gravys

0.1 ~ 5.0 parts by weight of the extract of Example <1-2> of the present invention was added to soups and broths to prepare meat products for health promotion, soups of noodles, and broths.

<2-3> Preparation of Ground Beef

10 parts by weight of the extract of Example <1-2> of the present invention was added to ground beef to prepare a ground beef for health promotion.

<2-4> Production of Dairy Products

5 to 10 parts by weight of the extract of Example <1-2> of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Preparation of Wire

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The extract of Example <1-3> of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds, and extracts of Example <1-3> prepared above were prepared by combining the following ratios.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Extract (3 parts by weight) of Example <1-3>,

(0.5 part by weight),

(0.5 parts by weight)

Preparation Example 3 Preparation of Beverage

<3-1> Preparation of health drink

Substances such as liquid fructose (0.5 parts by weight), oligosaccharides (2 parts by weight), sugar (2 parts by weight), salt (0.5 parts by weight), water (75 parts by weight) and the examples of the present invention <1-3> 5 g of the extract was homogeneously mixed and sterilized immediately, and then packaged in a small packaging container such as a glass bottle or a plastic bottle.

<3-2> Preparation of Vegetable Juice

5 g of the extract of Example <1-3> of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<3-3> Preparation of Fruit Juice

1 g of the extract of Example <1-3> of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

As can be seen from the above, since the alconia triplenervia extract of the present invention has an excellent effect on anti-inflammatory, the development and prevention and treatment of inflammatory diseases, functional foods for improving inflammation, and functional feed additives for improving inflammation, and It can be usefully used for the study of treatment methods.

Claims (10)

A pharmaceutical composition for the prevention and treatment of asthma, which contains Alchornea triplinervia L. extract as an active ingredient.
The pharmaceutical composition for preventing and treating asthma according to claim 1, wherein the alkania triplenervia extract is extracted using water, a lower alcohol of C ₁ to C ₄ , or a mixture thereof as a solvent.
3. The pharmaceutical composition for preventing and treating asthma according to claim 2, wherein the lower alcohol is ethanol or methanol.
delete delete delete delete delete Health food for the prevention and improvement of asthma containing Alchornea triplinervia L. extract as an active ingredient. delete

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