KR20170054315A - Composition comprising extract of Nelumbo nucifera Gaertn. stamen and ovary for prevention and treatment of allergic diseases or inflammatory diseases - Google Patents

Abstract

The present invention relates to a composition for preventing or treating allergic diseases or inflammatory diseases containing an extract of ovary and stamen of lotus as an active ingredient. More specifically, the present invention relates to a composition which is effective in treatment of allergic diseases or inflammatory diseases by preventing the release of beta-hexosaminidase and inhibiting inflammatory cytokines and immunoglobulin E (IgE) as well as lipoxygenases.

Description

Technical Field [0001] The present invention relates to a composition for preventing and treating allergic diseases or inflammatory diseases, which comprises an extract of soft noodles and an extract as an active ingredient. stamen and ovary for prevention and treatment of allergic or inflammatory diseases.

The present invention relates to a composition for preventing and treating an allergic disease or an inflammatory disease, which comprises an extract of soft and dry leaves as an active ingredient.

When foreign substances come into the body from the outside of the body, an immune reaction that protects our body from these bodies occurs, which is called a normal immune response. However, when the immune response is excessive, an allergic reaction is caused by hypersensitivity reaction that causes physical abnormality, and when a specific mechanism of hypersensitivity causes an abnormality in our body.

The cause of the allergic reaction is caused by the immune system of the human body reacting to external substances acting as allergen, allergen. Allergens are presented on the surface of cells by antigen presenting cells, which are recognized by B lymphocytes and converted to plasma cells, releasing antibodies (IgE) that specifically recognize the antigen.

Allergic diseases are caused by repeated immune reactions to allergens in memory cells, and IgE binding to antigen binds to receptors of mast cells (FcεRI) of tissues, leading to degranulation of mast cells . The degranulation of mast cells secretes histamine, heparin, leukotrienes, prostaglandine and other anaphylatic agents. These secreted immune activation factors activate the immune system and cause inflammation and allergic reactions.

Hypersensitivity reactions can be classified into four types. Type 1 hypersensitivity reactions, commonly referred to as allergic reactions, are reactions that occur to specific antigens and are induced by binding IgE antibodies specific for that antigen to mast cells or basophils.

When allergen-specific IgE antibodies are produced, these antibodies bind to FcεRI receptors on the surface of mast cells or basophils, and allergens bind to these antibodies to activate these cells, secrete various physiologically active substances, Expansion, increased penetration of blood vessels, smooth muscle contraction, and inflammatory response.

These reactions may occur locally or systemically depending on the type and amount of mediators produced by these cells. Specific allergic diseases include atopic dermatitis, rhinitis, and asthma.

Mast cells are very important in various allergic diseases such as atopic dermatitis, asthma, allergic rhinitis and rheumatoid arthritis. In contrast to other cells, mast cells contain large and coarsely stained granules. When IgE is stimulated by an antigen in the state of being bound to FcεRI (high-affinity receptor for IgE) of mast cells, It is involved in the inflammatory reaction.

Thus, mast cells may be involved in a variety of physiological, immunological, and pathological processes that occur during allergic reactions, such as wound healing, angiogenesis, host response to parasites and neoplasms, and chronic inflammatory and IgE mediated immediate allergic responses .

The mediators secreted during mast cell activation in allergic reactions include histamine and 5-HT (serotonin), biogenic amines that cause vasodilatation and pain, and IL-1, 4, 6, 10 , 13, MIF, and TNF to induce inflammation and leukocyte migration and proliferation.

In addition, by secretion of the phospholipid metabolites leukotrien B4, LTB4, leukotrien C4, LTC4, platelet activating factor (PAF), prostaglandin D2 (prostaglandin D2, PGD2) Leukocyte chemotaxis, airway constriction and pain.

In addition, there is an increased risk of tissue damage, pain, enzymes such as chymase and tryptase that induce angiotensin synthesis, corticotropin-releasing hormone (CRH), endolphins, , Endothelin, kinins, somatostatin substance P, SP, vasoactive intestinal peptide (VIP), urocortin, vascular endothelial growth factor growth factor, VEGF) to induce a series of allergic reactions such as inflammation, inflammation, pain, vasodilation, and airway constriction.

The allergic disease drugs developed so far are predominantly antagonistic to histamine and leukotriene receptors secreted by allergens in mast cells. However, since these drugs show tolerance within a short period of time after administration, most of the patients are symptomatic to improve their symptoms, not only to prevent the cause of the disease but also to have side effects due to medicines (Rabe KF, et al. , Eur Respir J Suppl., 34: 34s-40s, 2001).

Meanwhile, the lotus flower ( Nelumbo nucifera ) is a perennial aquatic plant with yellow flowers. It is widely used as a food material and is used as a variety of medicines in eastern Asia, especially in China. Specifically, seeds of lotus have been applied to various uses such as tissue inflammation, cancer and leprosy treatment, and poisoning. In addition, rhizome rootstocks have been reported to have antioxidant efficacy (Jin-Tuarn L., Ying-Shu L., Chien-Jung L., Ann-Ru W., Food Chem. Tox., 45, 486-493 2007); Dongmei et al., 2007). In addition, lotus leaves are used as traditional Chinese medicines with a hemostatic effect and are known to alleviate hyperlipemia in experimental animals (la Cour et al., 1995).

In addition, the operation of lotus flower can be made into Chinese tea leaves with good fragrance when dried, and it has been proved that it has antioxidative effect in kidney cells (Jung et al ., 2003). However, it has not been reported so far that lotus leaf extract and mint extract have therapeutic effect for allergy or inflammation disease.

The present invention relates to a pharmaceutical composition for preventing or treating an allergic disease or an inflammatory disease, which comprises an extract of soft and dry leaves as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention relates to a process for the production of Nelumbo nucifera Gaertn., stamen, and Nelumbo nucifera Gaertn., ovary) as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating an allergic disease or an inflammatory disease.

Wherein the extract is selected from the group consisting of water, C ₁ -C ₄ alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid, ether, chloroform, . ≪ / RTI >

After the first extraction, the extract may be secondarily extracted with any one solvent selected from the group consisting of butanol, dichloromethane and hexane.

The extract may be contained in the composition at a concentration of 0.1 to 100 占 퐂 / ml.

The extracts of Nelumbo nucifera Gaertn., Stamen and Nelumbo nucifera Gaertn ovary were prepared by using 1-eicosanol, cycloartenol, petadecanoic acid, trans- Squalene, 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl, hexadecanoic acid methyl ester, beta- Sitosterol, and steroidal lipid. The term " pharmaceutically acceptable carrier "

The allergic disease may be selected from the group consisting of allergic rhinitis, allergic conjunctivitis, allergic asthma, anaphylactic shock, food allergy, hay fever, drug allergy, plant allergies, urticaria, eczema and allergic dermatitis.

The inflammatory disease may be a skin inflammatory disease or atopic dermatitis.

The soft tissue and soft tissue extracts may inhibit the secretion of beta-hexoxaminidase, inhibit inflammatory cytokines and immunoglobulin E, and inhibit lipoxygenase.

The inflammatory cytokine may be IL-4 (Interleukin 4).

In one embodiment of the present invention, Nelumbo nucifera Gaertn., stamen) and open surgery ( Nelumbo nucifera Gaertn., Ovary) extract as an active ingredient for preventing or ameliorating an allergic disease or an inflammatory disease.

The soft and dry extracts according to the present invention are effective for the treatment of allergic diseases or inflammatory diseases by inhibiting beta-hexosaminidase release, inhibiting the secretion of inflammatory cytokines and IgE, and inhibiting lipoxygenase . In addition, when applied to human body, high cytotoxicity can be administered because of low cytotoxicity.

FIG. 1 shows data obtained by MTT analysis of the cytotoxicity of the compounds isolated from the extracts of soft-tissue and soft-tissue extracts.
Fig. 2 is data obtained by measuring the beta-hexosaminidase inhibitory activity of the fractions obtained by extracting the solvent from the soft tissue and the soft tissue.
FIG. 3 is a graph showing the inhibitory effect of beta-hexosaminidase on the treatment concentration of the extracts of soft tissue and soft tissue, soft tissue extract and soft tissue extract.
FIG. 4 is a graph showing IL-4 production inhibition ratios by treatment concentration of soft tissue and soft tissue extract, soft tissue extract, and soft tissue extract.
FIG. 5 shows data on the content of IgE in plasma of an atopy-induced mouse.
FIG. 6 is a graph showing the effect of improving the atopy of the soft and dry extracts, the soft and soft extracts, and the soft and soft extracts.

The present inventors have confirmed the atopy treatment effect, the skin inflammation inhibitory effect and the antiallergic effect of the soft and dry extracts, which are natural substances with little side effects, and completed the present invention.

In the present specification, the term " soft-boiled rice "is also referred to as a soft rice flour, a federation, or a saliva, and refers to a region surrounding the seed of the lotus flower. The soft rice flour preferably includes immature pistil or mature pistil Do not.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for the production of Nelumbo nucifera Gaertn., stamen, and Nelumbo nucifera Gaertn., ovary) as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating an allergic disease or an inflammatory disease.

The soft and dry extracts of the present invention inhibit beta-hexosaminidase release, inhibit inflammatory cytokines and IgE, inhibit lipoxygenase and are effective in the treatment of inflammatory diseases or allergic diseases, Which is effective because of low cytotoxicity. Specifically, as shown in FIG. 3 and FIG. 4, it was confirmed that the extracts of soft tissue and soft tissue were superior to those of soft tissue extract or soft tissue extract to inhibit the release of beta-hexonimidase and inhibit IL-4 release As a result, as shown in FIG. 6, it was confirmed that the number of scratch operations was reduced by 30% to 50% or more in atopic-induced mice. Thus, the soft and dry extract of the present invention may be effective for inflammatory or allergic diseases.

Wherein the extract is selected from the group consisting of water, C ₁ -C ₄ alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid, ether, chloroform, . ≪ / RTI >

As the extraction method, extraction methods such as hot water extraction, reflux cooling extraction, ultrasonic extraction, and room temperature extraction may be used, preferably room temperature extraction method, and most preferably 60% to 100% methanol at 40 to 60 And then extracted secondarily with any one solvent selected from the group consisting of butanol, dichloromethane and hexane, but the present invention is not limited thereto.

The concentration of methanol for preparing the soft and dry extracts may be 60% to 100%, preferably 70% to 90%, but is not limited thereto. At this time, when the concentration of methanol is less than the above range, there is a problem that an effective compound in the soft and dry extract is not extracted sufficiently.

The extracting temperature of the soft-tissue extract and the soft-tissue extract may be 40 캜 to 60 캜, preferably 45 캜 to 55 캜, but is not limited thereto. In this case, when the extraction temperature is lower than the above range, there is a problem that a compound effective for the treatment of an immune disease or an inflammatory disease is not extracted sufficiently. When the extraction temperature exceeds the above range, .

The extract may be contained in the composition at a concentration of 0.1 μg / ml to 100 μg / ml, and is preferably contained at a concentration of 2 μg / ml to 50 μg / ml, but is not limited thereto. At this time, when the concentration of the extract is less than the above range, there is a problem that cell proliferation is not effective, and when the concentration of the extract exceeds the above range, there is a problem of cytotoxicity. Specifically, the cells showed a cell proliferating ability of 94% or more at a concentration of 100 μg / ml. Thus, the soft-tissue and soft-tissue extracts of the present invention had no toxicity or side effects.

The Nerumbo nucifera Gaertn., stamen) and sternal surgery ( Nelumbo nucifera Gaertn., ovary) extracts were prepared from 1-eicosanol, cycloartenol, petadecanoic acid, trans-squalene, 2,6,10,14, 18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl), hexadecanoic acid methyl ester, beta-sitosterol and steroidal lipid. Or a combination thereof.

Specifically, 1-eicosanol, cycloartenol, petadecanoic acid, beta-sitosterol, and steroidal lipid in the above-mentioned compounds are excellent IL -4 release inhibitory effect, and is effective for the treatment of various diseases such as pentadecanoic acid, trans-squalene (2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23- hexamethyl), and beta-sitosterol have excellent? -hexosaminidase inhibitory effect.

The allergic disease may be selected from the group consisting of allergic rhinitis, allergic conjunctivitis, allergic asthma, anaphylactic shock, food allergy, hay fever, drug allergy, plant allergies, urticaria, eczema and allergic dermatitis.

In addition, the inflammatory disease of the present invention may be a skin inflammatory disease and is preferably atopic dermatitis, but the present invention is not limited thereto.

The soft and dry extracts of the present invention inhibit the secretion of beta-hexoxaminidase, inhibit inflammatory cytokines and immunoglobulin E, and inhibit lipoxygenase , And the inflammatory cytokine is preferably IL-4 (Interleukin 4), but is not limited thereto. Specifically, as shown in Fig. 3, it was confirmed that the extract of soft tissue and soft tissue was significantly superior to that of soft tissue extract or soft tissue extract in terms of beta-hexosaminidase inhibition. In addition, as shown in FIG. 4, it was confirmed that the extracts of soft tissue and soft tissue were significantly superior to those of soft tissue extract or soft tissue extract in inhibiting IL-4 secretion.

Therefore, the compound may be effective for inflammatory diseases or allergic diseases, and the soft and dry extracts having both of these compounds are effective for the inflammatory diseases or allergic diseases.

The pharmaceutical composition containing the extract according to the present invention can be administered orally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And can be used as formulations. 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 and 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 preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route 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 a daily dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The extract of the present invention can 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.

The present invention is open and open surgical ovary (Nelumbo nucifera Gaertn., stamen & ovary) extract as an active ingredient for preventing or ameliorating allergic diseases or inflammatory diseases. The detailed contents of the soft tissue and soft tissue extracts are as described above.

Examples of foods that can be supplemented with soft and dry surgical extracts include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, tea, vitamin complexes and health functional foods. It can also be added to foods or beverages for the purpose of preventing hypertension.

In this case, the amount of the extract in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, .

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. 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 polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above 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, the extract of the present invention can also be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (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 extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. 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 extract of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1.

1-1. Preparation of soft and dry surgical extracts

The soft tissue and soft tissue were collected from the voice on July 17, 2008, dried and pulverized. The soft tissues contained immature or mature pistil in the navel. Then, 34 g was taken and extracted with 50% to 80% methanol using an accelerated solvent extraction apparatus, and then the solvent was removed with a reduced pressure concentrator. This gave 10.44 g crude extract. The yield of soft tissue and soft tissue was 30.7%. The other samples used in the experiment are shown in Table 1 below and the extraction method is the same. The extracts prepared from the soft and dry extracts were used for in vitro and in vivo activities.

Botanical name Academic name Extraction site mountainous district Collection / Availability Amount of sample (g) used for extraction yield(%) year Nelumbo nucifera Gaertn. leaf Ginseng Herbal Research Institute July 17, 2008 11 24.8 year Nelumbo nucifera Gaertn. Surgery and Nesting Ginseng Herbal Research Institute July 17, 2008 34 30.7 year Nelumbo nucifera Gaertn. stem Ginseng Herbal Research Institute July 17, 2008 43 22.7 year Nelumbo nucifera Gaertn. leaf Ginseng Herbal Research Institute July 17, 2008 23 15.7 year Nelumbo nucifera Gaertn. Home Ginseng Herbal Research Institute July 17, 2008 59.57 32.2 year Nelumbo nucifera Gaertn. Operation Ginseng Herbal Research Institute July 17, 2008 84.42 27.3

1-2. Fraction preparation and component separation

After extracting 80% methanol from Yeoncheon and Yangyang in Seocheon and Yangyang in 2014, the extract was filtered and the solvent was removed under reduced pressure of 40 or less. 200 ml of distilled water was added to the crude extract, and the fractions of each solvent were successively extracted with n-hexane, dichloromethane, ethyl acetate and butanol (200 ml each) to obtain various fractions.

Among the fractions thus prepared, 5 g of the n-hexane layer was applied to column chromatography using silica gel or the like, and the compound was separated using a concentration gradient of n-hexane: ethyl acetate and dichloromethane: methanol. The isolated compounds were identified by MS instrumental analysis and the six compounds were identified as shown in Table 2 below. The fractionated compounds were used for in vitro activity experiments.

No. Sample The Molecular Weight Compound name One 9W C ₂ O H ₄₂ O 298.32 1-Eicosanol 2 9_3 C ₃ O H ₅₀ O 426.72 Cycloartenol 3 10 C ₁₅ H ₃₀ O ₂ 242.39 Petadecanoic acid 4 9_11 C ₃ O H ₅₀ 410.71 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl 5 16_31 - - Steroidal lipid 6 45_70 C ₁₇ H ₃₄ O ₂ 270.25 Hexadecanoic acid, methyl ester 7 7W C ₂₉ H ₅₀ O 414.71 β-Sitosterol

1-3. Cell culture

The RBL-2H3 cell line used in the experiment was purchased from Korean Cell Line Bank (KCLB), and the cells were cultured in RPMI-1640 supplemented with 15% fetal bovine serum (FBS), 100 mM sodium pyruvate, well culture plate in 2.5 ⁵ cells / well were seeded in culture in 5% CO _2, incubator of 37.

Example 2

2-1. Assessment of cytotoxicity of extracts from soft tissues

After the mast cells prepared in Example 1-3 were cultured, the soft and dry extract prepared in Example 1-1 was treated for 24 hours in mast cells and dimethyl sulfoxide (DMSO) as a negative control was added thereto. The cells were cultured in a 37, 5% CO 2 incubator for one day. To measure cytotoxicity, 200 μg / ml of MTT (USB, Cleveland, Ohio, USA) solution was added and cultured for 3 hours in a 37.5% CO 2 incubator for 4 hours . After removing the MTT solution, 100 μl of DMSO was added and the absorbance was measured at 550 nm using a Biotek Synergy HT plate reader (Biotek, Winooski, VT, USA).

As a result, as shown in Table 3, the kite extract showed a very high cell proliferation effect at a concentration of 100 μg / ml. The cell proliferation effect of the soft petiole extract was the highest at 96%, followed by the cell proliferation effect of the soft and dry extract.

Name of sample Usage site Cell viability (% of control), final concentration 100 [mu] g / ml Nelumbo nucifera Gaertn. petal 96.7 ± 1.2 Nelumbo nucifera Gaertn. Navel and Surgery 94.0 ± 1.1 Nelumbo nucifera Gaertn. leaf 61.2 ± 1.2 Nelumbo nucifera Gaertn. stem 89.9 ± 1.6

2-1. Evaluation of cytotoxicity of compounds derived from extracts of soft and dry leaves

After the mast cells prepared in Example 1-3 were cultured, the compounds derived from the soft and dry extract prepared in Example 1-2 were treated for 24 hours in mast cells. Each treatment concentration was from 0.32 to 200 μM. As a negative control, dimethyl sulfoxide (DMSO) as a solvent was added and R406 was treated as a positive control. The cells were cultured in a 37, 5% CO 2 incubator for one day. To measure cytotoxicity, 200 μg / ml of MTT (USB, Cleveland, Ohio, USA) solution was added and cultured for 3 hours in a 37.5% CO 2 incubator for 4 hours . After removing the MTT solution, 100 μl of DMSO was added and the absorbance was measured at 550 nm using a Biotek Synergy HT plate reader (Biotek, Winooski, VT, USA).

As a result, the cell proliferation effect on various solvent fractions is as shown in Fig. Compounds isolated from soft tissue and soft tissue were not found to significantly inhibit cell proliferation and generally showed lower cytotoxicity than R406, a positive control. Thus, most of the compounds were found to be cytotoxic.

Example 3

3-1. Determination of beta-hexosaminidase release of the extracts of each soft spot

Allergic reactions are a type of inflammatory disease that occurs when mast cells are degranulated, releasing histamine. It is known that histamine is synthesized and stored in mast cells and has a great influence on acute inflammatory reaction. Beta-hexosaminidase is an enzyme which coexists in histamine and mast cell and is known to be proportional to the amount of histamine released by degranulation And is used as an index of the anti-allergy effect confirmation. Therefore, in order to confirm the antiallergic response of the extracts of soft tissue and soft tissue, the release of beta - hexosaminidase was measured.

The mast cells prepared in Example 1-3 were cultured and then replaced with Tyrode's buffer and 200 ng / ml of DNP-IgE (Sigma-Aldrich, St. Louis, Mo., USA) Respectively. Then, 50 ng / ml of DNP-BSA (Sigma-Aldrich, St. Louis, Mo., USA) was added to the extract prepared in Example 1-1 for 1 hour and then cultured at 37 for 2 hours. Then the degranulation reaction was stopped on ice. 25 μl of the supernatant and 100 μl of 2.4 mM 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich, St. Louis, Mo., USA) were mixed on a 96-well plate and incubated for 1 hour at 37 ° C. The reaction was stopped by adding 175 μl of 0.1 M glycine-carbonate buffer (pH 10.0) and the fluorescence was exited (ex: 360 nm em: 450 nm) using a Biotek Synergy HT plate reader (Biotek, Winooski, Were measured.

As a result, as shown in Table 4, in the experimental group treated with 20 ㎍ / ㎖ of soft and dry extracts, the inhibitory effect of beta-hexosaminidase release was 38.3%, and the relatively high beta-hexosaminidase Day.

Name of sample Usage site Beta-hexosaminidase release inhibition rate (%), final concentration 20 [mu] g / ml Nelumbo nucifera Gaertn. petal 13.1 ± 2.7 Nelumbo nucifera Gaertn. Surgery and Nesting 38.3 ± 7.7 Nelumbo nucifera Gaertn. leaf 56.3 ± 1.5 Nelumbo nucifera Gaertn. stem 32.7 ± 5.2

3-2. Solvent star Home  And beta - Hexosa Mini-Days  Emission measurement

Beta-hexosaminidase release of the extracts of the soft-tissue and soft-tissue extracts by solvent was measured in the same manner as in Example 3-1.

As a result, as shown in FIG. 2, the extract of dichloromethane or hexane extracted from the soft tissue and the soft tissue showed a high beta-hexosaminidase release ability, and then the extract of soft tissue and soft tissue extracted with butanol Respectively. On the other hand, the soft and dry extracts extracted with ethyl acetate solvent showed an average inhibition of beta - hexosaminidase release by 15%.

3-3. year Home  And Yan Surgery Derived from extract  The beta- Hexosa Mini-Days  Emission measurement

The ability to inhibit beta-hexosaminidization release of compounds 3, 4 and 7 derived from soft and dry extracts was confirmed in the same manner as in Example 3-1.

As a result, as shown in Table 5, the IC ₅₀ of the compounds 3, 4, and 7 was 0.1 to 3.2 μM, which was superior to the 6.8 μM of the positive control substance R406 in inhibiting beta-hexosaminidase release.

Sample IC ₅₀ (? M) compound 3 1.1 compound 4 3.2 compound 7 0.1 R406 * 6.8 * Positive control: selleck, S2194

3-4. year Home  And stomach, stomach, and stomach Home  By concentration of extract Measure the release of beta-hexosaminidays

Extract 2 / / ml, 10 / / ㎖ and 50 / / ㎖, respectively.

As a result, as shown in FIG. 3, it was confirmed that the inhibition of beta-hexosaminidase release of the soft and dry extracts from all the concentration ranges was superior to that of the kernal extract or soft extract.

Example 4

4-1. Inhibition of the production of IL-4 cytokine by extracts from soft tissues

Overproduction of cytokine IL-4 is associated with allergic disease. Thus, in Example 4, the amount of IL-4 cytokine production was measured according to the extract treatment of the soft tissue.

Blank control, each standard substance (positive control group R406) and 20 μg / ml of each extract sample were added to the wells of the mast cells prepared in Example 1-2, and 1 × anti-IL -4 and incubated for 4 hours at room temperature (18 ~ 25). After removing the cover and washing the plates several times, 100 μl of 1 × solution was added, re-covered at room temperature for 30 min, and incubated and washed. 100 [mu] l of chromogen TMB substrate solution was added to each well, and the plate in the aluminum foil was wrapped and incubated at room temperature for 10 to 20 minutes in a dark place. 100 μl of stop reagent was added to each well and the absorbance was immediately measured at 450 nm using a Biotek Synergy HT plate reader (Biotek, Winooski, VT, USA).

As a result, as shown in Table 6, the extracts of soft tissue and soft tissue were superior to the other sites in inhibiting IL-4 production, and the cell proliferation effect was also excellent at 94% or more.

Name of sample Usage site IL-4 release Nelumbo nucifera Gaertn. petal 59.2 ± 3.6 Nelumbo nucifera Gaertn. Navel and Surgery 59.2 ± 1.2 Nelumbo nucifera Gaertn. leaf 46.5 ± 0.4 Nelumbo nucifera Gaertn. stem 36.8 ± 2.5

4-2. IL-4 release of compounds derived from soft and dry surgical extracts

IL-4 release inhibitory effects of the compounds 1, 3, 5, and 7 derived from the soft and dry extracts were confirmed in the same manner as in Example 4-1.

As a result, as shown in Table 7, compounds 1, 3 and 7 showed an IC ₅₀ of 1.8 to 5.7 μM which was superior to that of R406 as a positive control, and Compound 5 had an IC ₅₀ of 3.0 μg / ml, which was superior to R406, which showed a value of 3.6 μg / Release inhibition effect.

Sample IC ₅₀ ([mu] M) IC ₅₀ ([mu] g / ml) compound 1 1.8 - compound 3 2.7 - compound 7 5.7 - R406 * 31.3 - compound 5 - 3.0 R406 * - 3.6 * Positive control: selleck, S2194

4-3. year Home  And stomach, stomach, and stomach Home  Inhibition of IL-4 cytokine production by extract concentration

Except that the extract samples were treated at 2 / / ㎖, 10 / / ㎖ and 50 / / ㎖, respectively.

As a result, as shown in FIG. 4, IL-4 cytokine production inhibitory activity of the soft tissue and soft tissue extracts of IL-4 produced at the concentrations of 2 μg / ml and 10 μg / Inhibitory activity.

Example 5.

Determination of lipoxygenase inhibitory activity of extracts of soft and dry leaves

Lipoxygenase (5-LO, 5 ') is widely distributed in plant and plant cells and exhibits various biological activities such as inflammation reaction mediator and signal transduction mediator. It is a kind of enzyme such as linoleic acid, linolenic acid, arachidonic acid, 1,4- It is an enzyme that produces 1,3-cis, trans-diene 5-hydroperoxide by adding molecular oxygen to unsaturated fatty acid having cis-pentadiene (1,4-pentadiene) structure from arachidonic acid to various inflammation and allergic diseases Therefore, we tried to evaluate the anti-allergic effect by measuring lipoxygenase inhibitory activity using structurally and mechanically SLO (soybean lipoxygenase) similar to 5-LO in the first reaction of leucotriene biosynthesis involved.

As a test method, 20 μl of each sample was added to 2 ml of 0.1 M Tris buffer (pH 8.5), 20 μl of linolenic acid (final concentration 110 μM) was mixed and reacted at room temperature for 5 minutes. 20 μl of soybean lipoxygenase (type, 500 UNIT / final concentration) was added to the reaction solution, followed by reaction for 5 minutes. The absorbance at 234 nm was measured and the IC ₅₀ value was determined.

As a result, as shown in the following Table 8, the inhibitory effect of the soft tissue and the surgical extract on the lipoxygenase was lower than that of the positive control substance NDGA 4.53 占 퐂 / ml as the IC _{50 of} 8.43 占 퐂 / ml, The genase inhibitory effect was excellent.

Sample IC ₅₀ ([mu] g / ml) Nelumbo nucifera Gaertn. (Stomatitis, necropsy and surgery) 8.43 NDGA * 4.53 * Positive control: NDGA (Nordihydroguaiaretic acid)

Example 6.

6-1. Preparation of experimental animals

NC / Nga mouse was used as an experimental animal for atopic experiment, and it was adapted to the experimental environment for more than one week prior to the experiment. The incubation room maintained a 12 hour light cycle and room temperature of 23, the feed was fed sterilized solid feed purchased from Korea BioLink, and water sterilized by ultraviolet light was supplied.

Atopic induction was induced by barrier disruption of the skin with 4% SDS (sodium dodecyl sulfate) 24 hours later and 3 hours after application of house dust mite twice a week for 14 days in total of 50 ㎎ / Lt; / RTI >

6-2. Two-week experimental animal treated with soft-tissue and soft-tissue extracts

The soft and dry extracts were prepared by dissolving in 50% ethanol at a concentration of 5%, and then applied to the skin every morning for 13 days six times a week in the atopy-induced mice prepared by the method of Example 6-1.

6-3. year Home  And atopy in the mouse serum IgE  Reduction effect

The serum of the mouse prepared by the method of Example 6-1 was taken from the abdominal aorta and stored for 15 minutes in a refrigerator, followed by centrifugation and shaking at room temperature for 2.5 hours. After discarding the solution, 100 μl of 1X Biotinylated IgE Detection Antibody was added, incubated at room temperature for 1 hour with shaking, and again the solution was removed and washed. 100 μl of 1X HRP-Streptavidin solution was added, incubated at room temperature for 45 min with shaking, and solution removal and washing were repeated. Then, 100 μl of TMB One-Step Substrate Reagent was added to each well and incubated at room temperature for 30 minutes. 50 μl of Stop Solution was added to each well and immediately measured at 450 nm.

As a result, as shown in FIG. 5, the amount of IgE was increased in the negative control group (NC) than in the atopy induced normal group, but the content thereof was decreased in the mouse treated with the soft extract. In addition, the extracts of soft tissue and soft tissue were more effective in reducing IgE than lotus leaf extract, which was superior to positive control material (PC, tacrolimus ointment).

6-4. year Home  And open surgery, Home  Effects of extracts and keratocyte extracts on atopy

Atopy was induced in mouse skin in the same manner as in Example 6-1 in order to confirm the effect of improving the atopy of the soft and dry extracts. Atopy-induced mice were prepared by dissolving 50% ethanol in 5% concentration of each of soft tissue and soft tissue extracts, soft tissue extracts, and soft tissue extracts, and applied on the morning of 6 times / week for 6 weeks.

On the 39th day after the application of the atopy-induced sample, the scratch behavior of each animal was measured using an animal behavior measuring device (LABORAS, Netherland).

As a result, as shown in FIG. 6, it was confirmed that the scratch copper acceptance of the mice treated with the soft tissue and soft tissue extracts was significantly lower than that of the mice treated with the soft tissue extract or soft tissue extract.

The preparation examples of the pharmaceutical 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 1] Preparation of powders

20 mg of the extract of Smooth and Soft Surgery of Example 1

Lactose 100 mg

Talc 10 mg

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

[Formulation Example 2] Preparation of tablet

10 mg of the extract from the soft tissue and soft tissue of Example 1

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

10 mg of the extract from the soft tissue and soft tissue of Example 1

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

10 mg of the extract from the soft tissue and soft tissue of Example 1

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

[Formulation Example 5] Preparation of liquid agent

20 mg of the extract of Smooth and Soft Surgery of Example 1

10 g per isomer

5 g mannitol

Purified water quantity

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] Preparation of health drink

100 mg of the extract from the soft tissue and soft tissue of Example 1

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3 g

water    dose

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (10)

Nelumbo nucifera Gaertn., stamen) and sternal surgery ( Nelumbo nucifera A pharmaceutical composition for preventing or treating an allergic disease or an inflammatory disease, which comprises an extract of Gaertn., Ovary as an active ingredient.
The method according to claim 1,
Wherein the extract is selected from the group consisting of water, C ₁ -C ₄ alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid, ether, chloroform, . ≪ / RTI >
A pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases.
The method according to claim 1,
The extract is firstly extracted and then extracted with a solvent selected from the group consisting of butanol, dichloromethane and hexane
A pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases.
The method according to claim 1,
Wherein the extract is contained in the composition at a concentration of 0.1 to 100 占 퐂 / ml
A pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases.
The method according to claim 1,
The extracts of Nelumbo nucifera Gaertn., Stamen and Nelumbo nucifera Gaertn ovary were prepared by using 1-eicosanol, cycloartenol, petadecanoic acid, trans- Squalene, 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl, hexadecanoic acid methyl ester, beta- Wherein the composition comprises at least one compound selected from the group consisting of sitosterol (beta-sitosterol) and steroidal lipid (steroidal lipid)
A pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases.
The method according to claim 1,
Wherein said allergic disease is selected from the group consisting of allergic rhinitis, allergic conjunctivitis, allergic asthma, anaphylactic shock, food allergy, hay fever, drug allergy, plant allergies, urticaria, eczema and allergic dermatitis
A pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases.
The method according to claim 1,
Wherein the inflammatory disease is skin inflammatory disease or atopic dermatitis
A pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases.
The method according to claim 1,
The soft tissue and soft tissue extracts are characterized by inhibiting the secretion of beta -hexoxaminidase, inhibiting inflammatory cytokines and immunoglobulin E, and inhibiting lipoxygenase
A pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases.
9. The method of claim 8,
Wherein the inflammatory cytokine is IL-4 (Interleukin 4)
A pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases.
Nelumbo nucifera Gaertn., stamen) and sternal surgery ( Nelumbo nucifera Gaertn., Ovary) extract as an active ingredient for preventing or ameliorating allergic diseases or inflammatory diseases.

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