KR101403999B1 - A method for preparing a purified extract and the composition comprising the same for treating and preventing asthma and allergic disease - Google Patents

Abstract

The present invention relates to a method for producing a Safflower leaf blade purified water and fraction from which a toxic substance is removed, and a composition for preventing or treating asthma or allergic diseases containing the Safflower leaf blade purified product and fraction prepared by the above production method as an effective ingredient.

Description

TECHNICAL FIELD The present invention relates to a method for preparing purified water and fractions obtained by removing toxic substances, and a composition for treating and preventing asthma and allergic diseases containing the purified product as an active ingredient. and preventing asthma and allergic disease.

TECHNICAL FIELD The present invention relates to a method for preparing purified saury and its fractions by removing toxic substances, and a composition for preventing or treating asthma or allergic diseases containing the saururus purified product and fractions prepared by the method as an active ingredient.

[Literature 1] Bae Ki-Hwan, Korean Medicinal Plants, Kyung-Soo, p204, 2000

[Document 2] Korean Patent Registration No. 10-0675618

[Literature 3] Chang HW et al .. Planta. Medica., 70 (5), pp. 474-476, 2004

[Literature 4] Lionel A. Samayawardhena and Catherine J. Pallen J. Immunol 185, 5993-6002, 2010

The present invention relates to a method for producing a Safflower leaf blade purified water and fraction from which a toxic substance is removed, and a composition for preventing or treating asthma or allergic diseases containing the Safflower leaf blade purified product and fraction prepared by the above production method as an effective ingredient.

Most allergic diseases are caused by mast cells in the tissues of the tissues activated by the antigen-antibody reaction and mediators (mainly histamine, leukotriene, prostaglandin, TNFα, cytokine etc.) liberated in the eosinophils and basophilic cells of the blood. The use of currently used allergic medicines remains a symptomatic relief, so the development of more fundamental therapeutic drugs is urgently required.

Prostaglandins, leukotriens, and platelet activating factor (PAF) are the major mediators that induce allergic diseases, including phospholipase A2 and cyclooxygenase, And arachidonic acid, which is a precursor by lipoxygenase.

On the other hand, recently, drugs that are attracting attention as an allergic asthma treatment drug have drugs that simultaneously inhibit histamine release, inhibit leukotriene C ₄ production, and inhibit platelet activation factor production.

On the other hand, Houttuynia cordata (S aururus chinensis ) is a perennial plant belonging to the genus Peppermint Saury, with a height of 0.5 ~ 1m. The undergrowth is long and sideways, the leaves are alternate and oval, and the base is heart-shaped. Two to three leaves under the inflorescence are white. In late June to July, the water inflorescence is opposite to the leaf and hangs down. From the donation to the end, the white flowers slowly bloom, and the inflorescence is straight and long. The flower stipule is not on the axis of the inflorescence, but attaches to the end of a small peduncle of 2 ~ 3㎜ in length. Flowers are bisexual and have no perianum. Surgery is in six, facing the epidermis, and the donation is attached to the epidermis. The pistil is made of 4 pieces of cardamom, and the donations are attached to each other. There are two seeds per seedling in the bottom seed, and one seedling in one seedling. Leaves, flowers, and roots are white, and two or three leaves on the upper part are white. It grows in wetlands and is distributed in Korea, Japan, and Southeast Asia. There are two species of Saururus species, of which S. cernuus is distributed in the eastern part of North America.

It is also called red horse hemp or red horse horse ridge which is taken out and dried out. It has the efficacy of improving blood circulation, releasing blood, releasing fever, releasing poison, stopping cramping, stopping pain, overwork, muscular and bony symptoms, bruises , Arthralgia, post-operative pain, and snakebite poisoning (Bae Ki-hwan, Korean Medicinal Plants, Kyongsoo, p204, 2000).

On the other hand, Korean Patent Registration No. 10-0675618 has developed a prior art "therapeutic agent for asthma or allergic disease of purified water of Saururus chinensis" invented by the inventor of the present invention. However, the saururic acid ethanol refined product disclosed in the above- Toxic substances such as manassatin A and manassatin B are contained in large quantities, which have hindered development as a product.

Accordingly, the inventors of the present invention have conducted research and development on extraction and fractionation methods for removing the above-mentioned toxic substances on the Saururus chinensis leaf, and as a result, the present inventors have completed a preparation method for producing Saururus chinensis leaf material with the toxic substances removed and developed Inhibited the degranulation reaction liberated after stimulation with antigen-antibody to mouse bone marrow mast cells, inhibited the production of COX-2-dependent PGD2, inhibited 5-lipoxygenase-dependent LTC4 production and inhibited the inflammatory cytokine Tumor Necrosis In addition to suppression of Factor-α (TNF-α) and IL-6 production, passive systemic anaphylaxis (PSA), which is an in vivo animal model of systemic allergy model induced by antigen-antibody, Lt; RTI ID = 0.0 > LTC4 < / RTI > production in a dose-dependent manner.

In order to accomplish the above object, the present invention provides a method for preparing Safflower lantern refined water and fractions by removing toxic substances.

In one embodiment of the present invention, the present invention relates to a method for the production of a saururus chinensis leaf having a weight of about 0.5 to 200 times (w / v), preferably 1 to 100 times (w / v) Preferably 5 to 20 times by weight (w / v) of a non-polar solvent selected from hexane, heptane, methylene chloride, dichloromethane, chloroform or ethyl acetate or a mixed solvent thereof, preferably hexane, heptane, Adding a solvent, more preferably a hexane, or a heptane solvent; The solution is allowed to stand for about 30 minutes to about 1 week, preferably for about 1 hour to about 72 hours, more preferably for about 3 hours to about 12 hours, at about 10 to 150 degrees Celsius, preferably 20 to 100 degrees Celsius, A second step of extracting at a temperature of from -80 to < RTI ID = 0.0 > 80 C < / RTI > by an extraction method such as cold extraction, hot water extraction, ultrasonic extraction or reflux cooling extraction, A third step of repeating the extraction process from one time to ten times, preferably from two times to five times; And a fourth step of removing the extractant from the extraction solvent and drying the extract, wherein the toxic material is removed.

(W / w), preferably 10 to 100 times (w / w) of the weight of the sample, to the dried Saururus chinensis leaf, Water, a water, or a mixed solvent of 50-99% water and ethanol, preferably water or a mixture of water and ethanol, more preferably water or a mixture of 50-99% water and ethanol Extraction method such as cold extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction at 10 to 150 ° C, preferably 20 to 100 ° C for 12 hours to 1 week, preferably 48 hours to 72 hours, A second step of extracting the crude extract by a reflux extraction method; Adding the crude extract to water in an amount of about 0.05 to 50 times, preferably 0.5 to 5 times by volume (v / w%) of the weight of the crude extract to produce a suspension; A non-polar solvent selected from n-hexane, heptane, methylene chloride, chloroform or ethyl acetate in an amount of about 0.5 to 20 times, preferably 1 to 5 times (v / w) A fourth step of obtaining a non-polar solvent-soluble fraction by fractionation with a hexane or ethyl acetate solvent; And a fifth step of recovering the non-polar solvent-soluble fraction and evaporating and drying it. The present invention also provides a method for producing the Saururus chinensis leaf fraction from which a toxic substance is removed.

It was confirmed that the purified product and fractions of Saururus chinensis leaf prepared by the above method contain almost no toxic substances such as manassatin A and manassatin B, which are contained in a large amount of conventionally known ethanol refined products.

The present inventors also found that fractions developed by the above-described method inhibit the degranulation reaction liberated after stimulation with an antigen-antibody to mouse bone marrow mast cells, suppress the production of COX-2-dependent PGD2, inhibit 5-lipoxygenase-dependent LTC4 (TNF-α) and IL-6 production, as well as passive systemic anaphylaxis (PSA), an in vivo animal model that is a systemic allergy model induced by antigen-antibody, Was used to confirm that the production of PGD2 and LTC4 produced in the blood was strongly inhibited dose-dependently.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating an asthma or allergic disease, which comprises the Saururus chinensis leaf purified product or fraction prepared by the above-described method as an active ingredient.

Asthma or allergic diseases as defined herein include bronchial asthma, allergic rhinitis, allergic asthma or allergic dermatitis.

The composition of the present invention preferably contains 0.01 to 99.9%, preferably 0.1 to 90%, of Saururus chinense leaf purified water. However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

The composition comprising the Saururus chinensis leaf bladder of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The composition containing the purified product according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions Examples of the carrier, excipient and diluent which can be contained in the composition including the purified product 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, sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the tablet of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the purified product of the present invention is preferably administered at 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections. The pharmaceutical dosage forms of the tablets of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

The present invention provides a health functional food for preventing or ameliorating an asthma or allergic disease, which comprises the purified Saururus chinensis leaf or fraction prepared by the above-described method as an active ingredient. Examples of foods to which this can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, tea vitamins, and health functional foods.

The purified product or fraction itself of the present invention has little toxicity and side effects, and therefore can be safely used for prolonged use for preventive purposes.

The present invention provides a health supplement comprising a Saururus chinensis leaf refined product or fraction and a pharmaceutically acceptable food supplementary additive produced by the above method showing the effect of preventing asthma or allergic diseases.

The composition containing the purified product of the present invention can be used variously for medicines, foods and beverages for the prevention of asthma or allergic diseases. Examples of the food to which the present Saururus chinense leaf refined product can be added include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and may be used in the form of powders, granules, tablets, capsules or beverages .

Since the sama grass leaf blend itself of the present invention has little toxicity and side effects, it can be safely used for long-term use for preventive purposes.

The purified product of the present invention may be added to foods or beverages for the purpose of preventing allergic diseases or inflammatory diseases. In this case, the amount of the purified product in the food or beverage may generally be from 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is preferably 0.02 to 30 g based on 100 ml, Can be added at a ratio of 0.3 to 10 g.

The health beverage composition of the present invention contains the above-mentioned purified product as an essential ingredient at the indicated ratio, and there is no particular limitation on the liquid ingredient, and it 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 disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavorings other than those mentioned above, natural flavors (such as tau martin, stevia tablets (e.g., rebaudioside A, glycyrrhizin) and synthetic flavors (saccharin, aspartame, etc.) The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As described above, the purified saururus of the present invention exhibits an anti-asthmatic and anti-allergic effect and is a composition for preventing or treating asthma or allergic diseases. It can be used as medicines and health supplements composed of natural medicines without toxicity have.

1 is a view showing a calibration curve of standard materials,
Figure 2 is an HPLC chromatogram of a reference material,
Figure 3 is an HPLC chromatogram of Saururus chinensis ethanol (A), nucleic acid fraction (B), ethyl acetate (EtoAc) fraction (C), butanol fraction (D) and water fraction (E) of a reference material;
Figure 4 is a diagram showing the HPLC form of nucleic acid (hexane) purified and haptane purified,
FIG. 5 is a graph showing the effect of inhibiting degranulation of the nucleic acid fraction,
6 is a graph showing the effect of the nucleic acid fraction on PGD2 production inhibition,
FIG. 7 is a graph showing a 5-lipoxygenase (5-LO) -dependent LTC4 production inhibitory effect produced after stimulation with an antigen-antibody to mouse bone marrow mast cells of a nucleic acid fraction,
FIG. 8 shows the effect of nucleic acid fractions on the production of TNF-.alpha. And IL-6 produced after stimulation with antigen-antibody in mouse bone marrow mast cells.
FIG. 9 shows the effect of nucleic acid fractions on passive systemic anaphylaxis (PSA), a systemic allergy model induced by an antigen-antibody, on the production of PGD2 and LTC4 in serum,
10 is a graph showing the degranulation inhibitory effect of purified nucleic acid,
11 is a graph showing the effect of purifying nucleic acid on PGD2 production inhibition,
FIG. 12 is a graph showing 5-lipoxygenase (5-LO) -dependent LTC4 production inhibitory activity produced after stimulation with antigen-antibody to mouse bone marrow mast cells of nucleic acid purified product,
FIG. 13 is a graph showing the effect of purified nucleic acid on the production of TNF-.alpha. And IL-6 produced after stimulation with an antigen-antibody to mouse bone marrow mast cells,
Figure 14 shows the effect of nucleic acid purification on passive systemic anaphylaxis (PSA), a systemic allergy model induced by an antigen-antibody, on the production of PGD2 and LTC4 in serum.

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

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

EXAMPLES Example 1 Preparation of Saururus chinensis refinement for removing toxic substances

1-1. Saururus chinensis Hexane  Extraction method

The Saururus used in this experiment chinensis ) was purchased from Baekdu Pharmaceutical Company located in the Yakju market of Daegu, Republic of Korea. Twenty grams of naturally-dried Saururus ground powder was taken and 150 ml of hexane was added. When the solution was a hexane solvent, the solution was refluxed at 40 ° C for 5 hours. The procedure was repeated three times. The hexane solution Was evaporated to dryness to obtain 212.86 mg of purified hexane (hereinafter referred to as SCEHX)

1-2. Saururus chinensis Heptane  Extraction method

The Saururus used in this experiment chinensis ) was purchased from Baekdu Pharmaceutical Company located in the Yakju market of Daegu, Republic of Korea. 20 g of the ground powder of naturally-dried saururus was taken and 150 ml of heptane was added. When the heptane solvent was used, the solution was refluxed for 5 hours at 70 ° C. The procedure was repeated three times. The heptane solution Evaporated to dryness to obtain 222.20 mg of a heptane purified product (hereinafter referred to as SCEHP).

Example 2. Preparation of Saururus chinensis Fractions

2-1. Saururus chinensis EtOH Purified water  Produce

The Saururus used in this experiment chinensis ) was purchased from Baekdu Pharmaceutical Company located in the Yakju market of Daegu, Republic of Korea. (10 Kg) of naturally-dried Saururus chinensis were subjected to reflux extraction at 80 ° C for 12 hours with 70%, 90% and 100% ethanol respectively of 25 L, 25 L and 25 L, respectively, and the extracts were transferred to a Vaccum rotary evaporator ; Nihon Seiko Co., Ltd., VR-205c, Japan) to yield 1.2 kg, 1.1 kg and 1.3 kg of 70% ethanol, 90% ethanol and 100% ethanol soluble tablets, respectively.

2-2. Saururus chinensis Solvent fraction  Produce

The Sacheberry leaf ethanol solutions were evaporated to dryness and suspended in 3.0 L of water. The suspension was fractionated with 2-3 times the volume of hexane, ethyl acetate and butanol solvents, and evaporated to dryness. 51.3 g of hexane fraction, 248.4 g of ethyl acetate fraction , 189.4 g of a butanol fraction and 346.7 g of a water fraction (hereinafter, referred to as SCHE, SCEA, SCBU and SCWA, respectively).

Example 3. Component analysis

In order to analyze the components of the samples obtained in the above examples, the components were analyzed by HPLC analysis as follows.

3-1. Instruments and reagents

HPLC system for content analysis LC-20AD pump (Shimadzu, Japan ), SPD-M20A (Shimadzu) diode array detector, SIL-20A (Shimadzu) auto-sampler injector, DGU-20A 5 (Shimadzu) solvent degasser and CTO- 20A (Shimadzu) column oven.

The standard substance, manassantin B, was isolated from Saururus chinensis, and the purity of each standard was over 95%. The HPLC system for the analysis of methanol and acetonitrile for HPLC analysis was purchased from LC-20AD pump (Shimadzu, Japan) and SPD-M20A (Burdick & Jackson (USA)).

3-2. Preparation of standard solution

Sauchinone, manassantin A and manassantin B standard solutions were weighed accurately and dissolved in methanol, adjusted to a concentration of 1 mg / ml, stored at 4 ° C and diluted prior to use.

3-3. HPLC  Analysis condition

The content of Sauchinone (S), manassantin A (MA) and manassantin B (MB) in the Saururus chinensis was determined using Shimadzu LC-20A system. The column used for the analysis was an Agilent Eclipse XDB-C18 (5 μm, 4.6 × 150 mm) column and the column temperature was maintained at 40 ° C. The flow rate was 1.0 mL / min and the injection volume was 10 μL. The mobile phase was eluted with 50% acetonitrile (v / v) and the detection wavelength was detected at 241 nm.

3-4. HPLC  Analysis ( Fraction )

The calibration curves for the peak areas of sauchinone, manassantin A and manassantin B were in the range of 25200.00 μg / mL, y = 26132x + 51114 (R ² = 0.9998), y = 13439x + 32736 (R ² = 0.9998) + 30421 (R ² = 0.9998) (FIG. 1).

The three components were detected at 13.517 (sauchinone), 26.025 (manassantin A) and 39.612 min (manassantin B) (Fig. 2).

The chromatogram of the water fraction of Saururus chinensis leaf refinement is shown in FIG. The content of Sauchinone, manassantin A and manassantin B in Saururus chinensis is shown in Table 1.

Comparison of the content of sauchinone (S), manassantin A (MA) and manassantin B (MB) in each fraction S (mg / g) MA (mg / g) MB (mg / g) EtOH Extraction 1.09 0.53 0.98 Hexane fraction 0.21 0.00 0.00 The EtOAc fraction 0.92 0.35 0.69 BuOH fraction 0.00 0.00 0.00 H2O fraction 0.00 0.00 0.00

As shown in Table 1, it was confirmed that the nucleic acid components hardly contain manassantin A (MA) and manassantin B (MB), which are toxic.

1. An ethanolic refined product of Saururus chinensis was obtained and the ethanol purified product was subjected to solvent fractionation to obtain a hexane fraction, an ethyl acetate fraction, a butanol fraction and a water fraction, and the content of sauchinone contained in each fraction was quantified by the above-described HPLC analysis .

As a result of this experiment, it was found that the above-mentioned three compounds contained 1.09 mg, 0.53 mg and 0.98 mg per gram of the crude drug sample, respectively. In the purified nucleic acid fractions fractionated from the ethanol purified product, sauchinone 0.21 mg per gram, and it was confirmed that manassatin A and manassatin B, which are toxic substances contained in the conventional ethanol refined product, are not contained (FIG. 3 and Table 1).

3-5. HPLC  Analysis result (toxic substance removal Purified water )

The calibration curves for the peak areas of sauchinone, manassantin A and manassantin B were in the range of 25200.00 μg / mL, y = 26132x + 51114 (R ² = 0.9998), y = 13439x + 32736 (R ² = 0.9998) + 30421 (R ² = 0.9998) (FIG. 1).

The three components were detected at 13.517 (sauchinone), 26.025 (manassantin A) and 39.612 min (manassantin B) (Fig. 2). The chromatogram of the purified product of the Saururus chinensis leaf obtained from the above example using hexane and heptane extraction solvent is shown in FIG. The contents of Sauchinone, manassantin A and manassantin B in Saururus chinensis are shown in Table 2.

Comparison of sauchinone (S), manassantin A (MA) and manassantin B (MB) contents in nucleic acid and heptane tablets S (mg / g) MA (mg / g) MB (mg / g) Hexane extraction 0.33 0.00 0.08 Heptane extraction 0.30 0.00 0.13

In order to selectively extract sauchinone from Saururus chinensis, sauchinone and manassatin B contained 0.33 mg and 0.08 mg per gram of crude drug sample, and manassatin A , And heptane tablets contained 0.30 mg and 0.13 mg of sauchinone and manassatin B per gram of the crude drug sample and no manassatin A showing toxicity (Fig. 4 and Table 2).

Reference example  1. Preparation of experimental material

Cell culture reagents such as RPMI-1640, modified Eagle Medium (MEM), non-essential amino acids solution, fetal bovine serum (FBS), streptomycin, penicillin, (Grand Island, USA). Among the reagents used in the experiments, IgE and DNP-HSA (antigen) were purchased from Sigma (St. Louis, USA). EIA kits for prostaglandin D2 and leukotriene C4 were purchased from Cayman (Ann Arbor, USA).

Reference Example 2. Experimental cell culture

(BMMC, mouse bone marrow-derived mast cells) were isolated from bone marrow from BALB / C mice and cultured in RPMI-1640 medium containing 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin and IL -3 (culture medium in which rat spleen cells were stimulated with pokeweed mitogen to give a final concentration of 20%) for about 3 weeks to obtain 95% or more homogeneous BMMC.

Experimental Example 1. Measurement of degranulation inhibition in mast cells

After 3 weeks from the culture of rat bone marrow-derived mast cells of Reference Example 2, the cells were treated with 100 ng / ml of IgE at a cell concentration of 2 × 10 ⁵ cells for 2 hours or longer (IgE-sensitized mast cells) Then 25 ng / ml DNP-HSA (antigen) was stimulated for 15 minutes and centrifuged at 3000 rpm, 4 캜 for 5 minutes. The supernatant was mixed with a β-hex substrate [100 mM citrate buffer (0.955% citric acid, 1.478% sodium citrate dihydrate, pH 4.5), 1.3 mg / ml p-nitrophenyl-N-acetyl-bD-glucosaminide] The reaction was stopped with 0.2 M glycine (pH 10.7), and the absorbance at 405 nm wavelength was measured using an ELISA (BIO-TECK INSTRUMENTS, INC, ELx800) Was calculated according to Equation (1).

As a result, as shown in FIG. 5 and FIG. 10, the deagglutination reaction liberated after antigen-antibody stimulation of mouse bone marrow mast cells with sample-nucleic acid purified extracts of hexane fraction and Saururus chinensis leaf, Respectively.

Experimental Example  2. Cyclooxygenase -2-dependent prostaglandin D2  Production inhibition measurement

The amount of prostaglandin D2 (PGD2) produced using a prostaglandin D2 EIA kit (Cayman, PGD2-MOX EIA kit, product no. 512011) was measured in order to confirm the degree of inhibition of the purified products and fractions obtained in the above- Were measured.

After 3 weeks of the culture of rat bone marrow-derived mast cells (BMMC) in Reference Example 2, the purified product and fractions (final concentration 125 μg / ml) obtained in the above Example were added to an IgE-sensitized mast cell concentration of 2 × 10 ⁵ cells / After treatment with DNP-HSA at 25 ng / ml for 1 hour, 8 hours after cell stimulation, the supernatant prostaglandin D2 was measured by EIA (enzyme linked immunoassay) using a prostaglandin D2 assay kit (Cayman) Respectively. At this time, in order to inhibit the production of prostaglandin D2 produced by cyclooxygenase-1, 10 쨉 g / ml of aspirin was pre-treated for 1 hour before use in mouse bone marrow-derived mast cells. After washing with a washing buffer 4 times, the substrate solution was treated with 200 μl each for 5-20 minutes, treated with 50 μl of stop solution, and absorbance at 450 nm Were measured by ELx800 (BIO-TEK, Instrument, Inc, USA). As a positive control, licofelon (Toronto Research Chemicals Inc., North York, ON, Canada), a COX-2-2 / 5-LO dual inhibitor, was used.

As a result of the experiment, as shown in FIG. 6 and FIG. 11, the sample-nucleic acid refinement obtained by directly extracting the hexane fraction and Saururus chinensis leaf of the Saururus chinensis leaf with the nucleic acid is COX-2 dependent , And inhibited the expression of COX-2 protein.

Experimental Example  3. Leukotriene  Analysis of the effects on production

In order to confirm the effect of the purified product and the fraction obtained in the above Example on the production of leukotriene C4, an experiment was carried out by applying the method disclosed in the literature (Chang HW et al .. Planta. Medica., 70 (5), pp.474-476, 2004).

After 3 weeks from the culture of the bone marrow-derived mast cells from Reference Example 2, the purified product and fractions obtained in the above Example were added to IgE-sensitized mast cells (2 x 10 ⁵ cells / well) at a constant concentration (1, 5, ml) and treated with 25 ng / ml of DNP-HSA (Sigma, S9915) for 15 minutes. Quantification of leukotriene C4 in the supernatant after cell stimulation was measured using a leukotriene C4 EIA kit (Enzyme linked immuno assay, Cayman, product no. 520211).

As a result of the experiment, as shown in FIG. 7 and FIG. 12, the hexane fraction of Saururus chinensis leaf and the sample-nucleic acid purified product obtained by directly extracting the Saururus chinensis leaf with the nucleic acid were subjected to 5-lipoxygenase-dependent Lt; RTI ID = 0.0 > LTC4 < / RTI >

Experimental Example  4. Proinflammatory ( proinflammatory ) Effect on cytokine production

In order to confirm the effect on the proinflammatory cytokine production of the purified products obtained in the above examples, IgE-sensitized mast cells (2 × 10 ⁵ cells / well) were added with the purified product and fraction obtained in the above example Was pre-treated for 1 hour at a constant concentration (5, 10 μg / ml) and treated with 25 ng / ml of DNP-HSA (Sigma, S9915) for 15 minutes. TNF-α and IL-6 were measured in the supernatants after cell stimulation using an enzyme-linked immunoassay (TNF-α and IL-6 EIA kit, R & D Systems Inc., Minneapolis, MN, USA)

As a result of this experiment, as shown in Figs. 8 and 13, the hexane fraction of Saururus chinensis leaf and the specimen-nucleic acid purified extract of the Saururus chinensis leaf were directly detected by Tumor, which is an inflammatory cytokine generated after stimulation with anti- Ncrosis Factor-α (TNF-α) and IL-6 production in a dose-dependent manner.

Experimental Example  5. In animal models allergy  Inhibitory effect

In order to confirm the anti-allergic effect of the tablets obtained in the above examples in vivo, the passive systemic anaphylaxis method disclosed in the literature was applied and the experiment was conducted as follows Lionel A. Samayawardhena and Catherine J. Pallen J. Immunol 185, 5993-6002, 2010).

ICR mice are injected with 2 μg IgE administered in 100 μl of saline solution. After 24 hours, the purified product and fractions obtained in the Examples are orally administered at a predetermined amount (50, 100 mg / kg, dissolved in CMC) 1 hour before the antigen administration in advance. The antigen is intravenously injected with 4 mg of DNP-HSA dissolved in 200 μl of physiological saline. As a control group, 200 μl of CMC (Carboxymethylcellulose) alone was orally administered. At this time, fexofenadin, an H1 receptor antagonist, was used as a positive control.

The experimental results, as shown in Figs. 9 and 14, the sample is extracted with a bar of the hexane fraction and three hundred seconds leaves of three hundred seconds leaves the nucleic acid-nucleic acid purification waters antigens - of systemic allergy model induced by antibody passive systemic anaphylaxis (PSA) Was used to strongly inhibit PGD2 and LTC4 production in the blood in a dose-dependent manner,

Hereinafter, formulation examples of the pharmaceutical composition containing the purified product and the fractions of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  1. Preparation of injection preparation

SCEHX fractions ........................................ 10 mg

Sodium Metabisulfite .............................. 3.0㎎

Methylparaben ......................................... 0.8 mg

Propylparaben ....................................... 0.1mg

Sterilized distilled water for injection ....................................

The above ingredients are mixed and made into 2 ml by a conventional method, filled in a 2 ml ampule and sterilized to prepare an injection.

Formulation Example 2. Preparation of tablets

SCEHP fractions ........................................ 200 mg

Lactose ................................................ 100 Mg

Starch ................................................ 100 Mg

Magnesium stearate .................................. Amount

The above components are mixed and tablets are prepared by tableting according to a conventional method for producing tablets.

Formulation Example 3. Preparation of capsules

SCHE fractions ......................................... 10 mg

Lactose ................................................ 50 Mg

Starch ................................................ 50 Mg

Talc ................................................. 2 mg

Magnesium stearate .................................. Amount

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

Formulation Example 4. Preparation of liquid preparation

SCEA fraction ........................................ 1000㎎

Sugar................................................. .20g

Isomeric sugar .............................................. 20g

Lemon flavor ................................................ Amount

Purified water was added to the total volume to 1000 ml. The above components are mixed according to a usual method for producing a liquid agent, and then filled in a brown bottle and sterilized to prepare a liquid agent.

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

Formulation Example 5. Preparation of Healthy Foods

SCEHX fractions ......................................... 1000 mg

Vitamin mixture ............................................

Vitamin A Acetate .................................... 70 ㎍

Vitamin E .............................................. 1.0 mg

Vitamin B1 ............................................ 0.13 mg

Vitamin B2 ............................................ 0.15 mg

Vitamin B6 .............................................. 0.5 mg

Vitamin B12 ............................................ 0.2 ㎍

Vitamin C ............................................... 10 Mg

Biotin ................................................. 10 [mu] g

Nicotinic acid amide ....................................... 1.7 mg

Folic acid ................................................. ..50 [mu] g

Calcium pantothenate .......................................... 0.5 mg

Mineral mixture ............................................

Ferrous sulfate ............................................ 1.75 mg

Zinc oxide .............................................. 0.82 mg

Magnesium carbonate ......................................... 25.3 mg

Potassium Phosphate ............................................... 15 mg

Secondary Calcium Phosphate ............................................ 55 mg

Potassium citrate ............................................. 90 mg

Calcium carbonate .............................................. 100 mg

Magnesium chloride ............................................. 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation Example 6. Preparation of Health Drink

SCHE fractions ......................................... 1000 mg

Citric acid .............................................. 1000 mg

Oligosaccharides .............................................. 100 g

Plum concentrate ............................................. 2 g

Taurine ................................................. .1 g

Purified water was added to the whole ... 900 ml

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

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

Claims (9)

A first step of adding 5 to 20 times (w / v) weight of hexane, heptane, or a mixed solvent thereof to the dried saururus leaves; A second step of extracting the solution by a reflux cooling extraction method at 20 ° C to 100 ° C for 1 to 72 hours; A third step of repeating the extraction process two to five times; And a fourth step of removing the extract from the extraction solvent and drying the extracted extract, wherein the toxic material is removed. delete Water or a water and ethanol mixed solvent of 10 to 100 times (w / w) the weight of the sample is added to the dried Saururus chinensis leaf and extracted by a reflux cooling extraction method at 20 to 100 ° C for 48 to 72 hours, A first step of obtaining an extract; A second step of adding the crude extract to water in an amount of 0.5 to 5 times the volume of the crude extract (v / w) to prepare a suspension; A third step of obtaining a non-polar solvent-soluble fraction by fractionating the suspension with hexane, ethyl acetate or a mixed solvent thereof in an amount of 1 to 5 times the volume of the suspension (v / w); And a fourth step of recovering and evaporating the non-polar solvent-soluble fraction, and removing the toxic substance. delete A pharmaceutical composition for preventing or treating bronchial asthma, allergic rhinitis, or allergic asthma comprising the Saururus chinensis leaf purified water or fraction of claim 1 or 3 as an active ingredient. delete A health functional food for preventing or ameliorating bronchial asthma, allergic rhinitis, or allergic asthma comprising the Saururus chinensis leaf purified product or fraction of claim 1 or 3 as an active ingredient. delete delete

Images