KR20080028630A - Antagonistic pharmaceutical composition for neurokinin-1 receptor - Google Patents

Abstract

A composition comprising a water fraction of Bupleurum falcatum L. is provided to show excellent SP binding inhibitory activity, thereby being usefully used as a natural drug for preventing and treating various diseases requiring an NK-1 receptor antagonist such as inflammatory liver diseases, asthma, arthritis, diabetes, depression, vasodilation, vomiting, pain, and anxiety. An antagonistic pharmaceutical composition for neurokinin-1(NK-1) receptor comprises a water fraction of Bupleurum falcatum L. with excellent SP binding inhibitory activity. A method for preparing the water fraction of Bupleurum falcatum L. comprises the steps of: (a) after adding 50% ethanol to Bupleurum falcatum L. and subjecting it to ultrasonic extraction, filtering the extract and concentrating the filtrate under reduced pressure to obtain an ethanol extract; (b) after suspending the ethanol extract in distilled water, fractionating the suspension with n-hexane to obtain an n-hexane fraction and a water fraction; (c) fractionating the water fraction with CHCl3 to obtain a chloroform fraction and a water fraction; and (d) fractionating the water fraction isolated from the step(c) with ethyl acetate to obtain an ethyl acetate fraction and a water fraction, thereby obtaining the water fraction as a final water fraction of Bupleurum falcatum L..

Description

A method for preparing Shiho water extract exhibiting neurokinin-1 receptor antagonistic effect and a neurokinin-1 receptor antagonistic pharmaceutical composition containing Shiho water extract {ANTAGONISTIC PHARMACEUTICAL COMPOSITION FOR NEUROKININ-1 RECEPTOR}

1 is a graph showing the SP binding inhibitory activity (%) by concentration of the NK-1 receptor antagonist (Notice: L-733,060).

Figure 2 is a graph comparing the effect of the NK-1 receptor antagonist of the total extract and each solvent fraction of Shiho according to the method of the present invention.

Figure 3 is a graph showing the effect of NK-1 receptor antagonism represented by five small fractions of the final water fraction of Shiho according to the present invention.

The present invention provides an NK containing a water fraction of Bupleurum falcatum L. that has similar or superior NK-1 receptor antagonistic efficacy to that of known non-peptide synthetic antagonists. -1 receptor antagonist compositions and methods for their preparation.

Neurokinin receptor (NK) binds to the intracellular ligand substance P (substance P; SP) belonging to the tachykinins system in neuropeptides that act on the central and peripheral nervous systems. Substance P. Int J Biochem Cell Biol 33: 555-576 (2001).

SP was first discovered in the brain and intestine of horses in 1931, and has 11 amino acids (HOOC-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH ₂ ) As a neurotransmitter consisting of NK-1, NK-2, NK-3 and three receptors, but binds mainly to the NK-1 receptor (affinity 0.05-0.5 nM) performs a physiological function [ Substance P. Int J Biochem Cell Biol 33: 555-576 (2001).

화학식 1

Formula 1

Currently, the NK-1 receptor is known to be evenly distributed in the central nervous system such as the amygdala, hippocampus, hypothalamus, striatum, and spinal cord of the brain and peripheral nervous system such as skin, inflammatory cells, digestive system, respiratory system, and cardiovascular system. The NK-1 receptor is a G-coupled receptor that is activated by the binding of the ligand SP and plays an important physiological function to play the role of a neuromodulator or neurotransmitter. P. Int J Biochem Cell Biol 33: 555-576 (2001) and Expression of functional NK-1 receptors in murine microglia. Glia 37: 258-267 (2002)].

NK-1 receptor activation by SP in the central nervous system causes symptoms such as depression, pain, vomiting and anxiety [Distinct mechanism for antidepressant activity by blockade of central substance P receptors. Science 281: 1640-1645 (1998). In the peripheral nervous system, changes in epithelial cell penetration, vasoconstriction, increased sputum secretion, airway contraction, and proinflmmatory mediators are known to cause migraine, arthritis, asthma, inflammatory bowel syndrome (IBS), and hyperalgesia. Targeting tachykinins for the treatment of obstructive airways disease. Treat Respir Med 3: 201-216 (2004); Role of substance P in inflammatory arthritis. Ann Rheum Dis 51: 1014-1018 (1992); The role of substance P in inflammatory disease. J Cell Physiol 201: 167-180 (2004)].

These phenomena in the body, which are mediated by NK-1 receptors, are the main causes of neurological disorders such as depression, migraine, and major adult diseases such as asthma, hypertension, diabetes, arthritis, arteriosclerosis, and hepatitis. As such, the NK-1 receptor may be a key point of action for the development of therapeutics for various diseases known to have high mortality, particularly adult diseases. Therefore, there is an urgent need for the discovery of substances that control them.

Recent studies have reported that NK-1 receptor antagonists are effective in suppressing inflammatory liver disease, asthma, arthritis, diabetes, depression, vasodilation and vomiting. This suggests that targeting the development of NK-1 receptor antagonists increases the likelihood of development of prophylactic and therapeutic agents for various diseases [Potential therapeutic targets for neurokinin-1 receptor antagonists. Expert Opinion on Emerging Drugs 9: 9-21 (2004).

From the 1960s to the present, the world's leading pharmaceutical companies (eg, Roche, GlaxoSmithKline, Merck, Pfizer, Novartis, Boehringer Ingelheim, Eli Lilly, etc.) were used to identify chemicals that antagonize the NK-1 receptor. A number of leading substances have been proposed by synthesizing analogs of the peptidic structure of SP or by organic synthesis of low-molecular weight nonpeptidic drugs. Currently, about 20 organic compounds are in progress in phase I, II, and III clinical trials. therapeutic targets for neurokinin-1 receptor antagonists. Expert Opinion on Emerging Drugs 9: 9-21 (2004).

In Korea, the only low-molecular-weight (MW-370.50) NK-1 receptor antagonist has been developed for the treatment of anesthetics, arthritis, and diabetes in Dong-A. In addition, aprepitant (MK 869) (Formula 2b), a non-peptide NK-1 antagonist recently approved by the US Food and Drug Administration, is an antidepressant developed by Merck. Aprepitant: a neurokinin-1 receptor antagonist for the treatment of chemotherapy-induced nausea and vomiting, which has been shown to be effective in suppressing nausea and vomiting, one of the biggest side effects of chemotherapy. Expert Rev Anticancer Ther 4: 715-724 (2004). As such, it is widely known that NK-1 receptor antagonists exert various effects.

Formula 2

The characteristics of some of the antagonists that have been developed are characterized by species-specific efficacy and due to the structural nature of the receptors, may affect other receptors such as serotonin, noradrenalin, dopamine transforters or monoamine oxidase. There is an advantage in that it shows strongly specificity only for the NK-1 receptor without showing any Mars [Different binding epitopes on the NK1 receptor for substance P and a non-peptide antagonist. Nature 362, 345-348 (1993)].

Nonpeptidic antagonists have a great advantage over the diversity of chemical structures and are more potent than peptide structures. In addition, since it is easy to pass through the BBB (Blood-Brain-Barrier) when used as a neurological disease drug, research into the development of NK-1 receptor antagonists for non-peptide synthetic materials has recently been actively conducted [Pharmacology of CP] -99,994; A nonpeptide antagonist of the tachykinin neurokinin-1 receptor. J Pharmacol Exp Ther 267: 472-479 (1993); A potent non-peptide antagonist of the substance P (NK1) receptor. Science 251: 435 (1991) and Successful virtual screening for a submicromolar antagonist of the neurokinin-1 receptor based on a ligand-supported homology model. J Med Chem 47: 5381-5392 (2004)].

However, it is known in the art that synthetic materials produced by the organic synthesis method are likely to cause adverse effects on the human body. The present inventors have been used as a basis for oriental medicine as a basis for thousands of years of clinical experience and have been used in the treatment or prevention of diseases and have the advantage of low side effects NK-1 receptor antagonist from natural products I would like to try a research to find out.

The development of a substance that interferes with the binding of the substance P and the NK-1 receptor using natural materials has been disclosed by LG H & H Co., Ltd. (Korea Patent Publication No. 2005-0067951). According to this document, it is possible to prevent itching due to sensitive skin diseases, scalp itching or allergic itching due to anti-inflammatory action by inhibiting (interfering) the binding of substance P and NK1 receptor, which is a major mediator of itching, and by inhibiting inflammatory cytokines. An anti-itch or relief composition containing Andrographolide and Andrographis paniculata extract containing andrographolide as an active ingredient is disclosed.

In addition, the Republic of Korea Patent Publication No. 2006-0000127 (2006.01.06) is an itching suppression or alleviation composition that can alleviate the itch caused by sensitive skin diseases, scalp itching, itching caused by allergies, BERBAMINE or BERBAMINE An anti-itch composition is disclosed which contains an extract containing (BERBAMINE). The extract containing BERBAMINE or BERBAMINE may be COPTIS CHINESIS FRENCH. Or SOBERIS AMURENSIS RUPR. From the roots and / or stems of Such anti-itching or alleviating compositions are disclosed to constitute scalp cleansers, hypoallergenic cosmetics, skin protectants against UV, etc., systemic cleansers, face washes, soaps, creams, disposable diapers, atopic baby products, and the like.

In addition, the Republic of Korea Patent Publication No. 2006-0093626 (2006.08.25) provides a composition for inhibiting itching and atopic dermatitis, characterized in that it contains a natural mushroom extract as an active ingredient to suppress the itch or to reduce atopic dermatitis as an active ingredient. do. Situation mushroom extract contained in these compositions inhibits the release of HISTAMINE, which mediates itching, and interferes with the binding of the substance P (SUBSTANCE P) -NK1 receptor, which is a major mediator of itching, thereby not only suppressing itching but also relieving atopic dermatitis It is disclosed that it is very effective.

In addition, the Republic of Korea Patent Registration No. 0544945 (January 13, 2006) is a composition for immuno-promoting comprising a C. nytharis Cordyceps (CORDYCEPS MILITARIS) hot water extract, in detail, the Mita rizona Cordyceps Sinensis hot water extract is a mouse non-implanted solid cancer Significantly increases the number of T-cells, B-cells, and NK-cells in the intestine, resulting in prolonged and anti-tumor effects through enhancement of cell- and humoral mediated immune responses, and increased IL-2 secretion by the spleen By activating the immune function of the host, it is disclosed that it can be usefully used as a pharmaceutical composition and health functional food for immune promotion.

As described above, a number of NK-1 receptor antagonists or anti-tumor effect compositions based on extracts of natural products have been disclosed, but antagonists and SP binding inhibitory activity selective to human NK-1 receptors are known synthetic antagonists. There is no suggestion or initiation for extracts of natural origin that are similar or better than (eg, Aprepitant) sufficient to be used as alternative medicine.

Accordingly, the inventors of the present invention examined the effects of NK-1 receptor antagonism on a number of natural product extracts (for example, 10 species, sulfur, hyangbuja, peony, gold, licorice, longan meat, yellow lotus, raw paper, peppermint, Shiho), surprisingly 1 From the natural product of the species, Buleurum falcatum L., it was found that certain extracts according to the method of the present invention exhibited the desired excellent NK-1 receptor antagonistic effect. In particular, such antagonistic efficacy is more than the efficacy of known synthetic antagonists, it is judged to be sufficient to be used as an alternative medicine, so as a result of earnestly studying the extract of Shiho came to complete the present invention.

In one aspect, the present invention provides a NK-1 receptor antagonistic pharmaceutical composition containing a water fraction of Bupleurum falcatum L. having NK-1 receptor antagonism and excellent in SP binding inhibitory activity.

According to a preferred embodiment, the present invention provides dried branches of Bupleurum falcatum L., which is a natural product whose antagonistic efficacy against the NK-1 receptor is more than that of known synthetic antagonists and without any potential side effects caused by the synthetic material. Ethanol extract of the sections is subjected to sequential polarity treatment of n-hexane, chloroform and ethyl acetate solvents to provide an NK-1 receptor antagonistic pharmaceutical composition containing the final water fraction obtained.

The amount of time lake water fraction contained in this NK-1 receptor antagonist composition ranges from 1 μg to 50 μg / ml.

The NK-1 receptor antagonistic pharmaceutical composition of the present invention may be used in a mixture with other known NK-1 receptor antagonists in addition to the aforementioned lake water fraction and / or contain a pharmaceutically acceptable carrier. It goes without saying that these other NK-1 receptor antagonists and / or pharmaceutically acceptable carriers, as well as do not adversely affect the antagonistic effects of the NK-1 receptor of the lake water fraction.

Shiho is a perennial herb belonging to the Apiaceae family. In oriental medicine, the root is called Shiho, which is used to treat malaria, jaundice, to relieve headaches, colds, fever, pain, and excitement, and to strengthen the liver. Nowadays they are grown in large quantities. The root contains about 3% saponin and fatty oil (see Encyclopaedia Britannica; see http://www.britannica.co.kr). In the present invention, dried branches, roots and the like of Siho can be used. Preferably, sliced dried branches of sea tiger are used.

In detail, the specific extract of Shiho according to the method of the present invention is added to 50% ethanol to the slice of dried branches of Shiho, ultrasonic extraction after filtration, and the filtrate is concentrated under reduced pressure to obtain ethanol X, and then the ethanol X in distilled water Suspension followed by the polar treatment of n-hexane, chloroform and ethyl acetate solvents in sequential order comprises the final fractions of water obtained. This final water fraction shows superior SP binding inhibitory activity than any intermediate extract of the solvent used. This SP binding inhibitory activity is a potency analysis using a known non-peptide antagonist (L-733,060) as a comparison group by concentration, according to the analysis conditions designed by the inventors, the efficacy is similar or superior to the comparison group, It exhibits at least 95% inhibitory activity. Accordingly, the present invention provides an NK-1 receptor antagonistic pharmaceutical composition containing a water fraction of Siho excellent in inhibiting inflammatory liver disease, pain reduction, depression, and cancer cell proliferation.

In addition, BF in five subfractions (BF-W1 to BF-W5) as a reverse phase column chromatography analysis product was refined to obtain an active subfraction which can be more preferably used in terms of efficacy and purity in the final seawater fraction. The SP binding inhibitory activity of the -W4 and BF-W5 subfractions was confirmed to be excellent. Most preferred is the BF-W4 subfraction. Accordingly, in accordance with a preferred aspect of the present invention, there is provided an NK-1 receptor antagonistic pharmaceutical composition containing the fourth and fifth subfractions of reverse phase column chromatography analysis in the above-mentioned water lake fractions.

In another aspect, the present invention provides a step for obtaining an ethanol extract using the dried eggplant slices of Siho, and the extract obtained by treating the extract with hexane, chloroform and ethyl acetate three polarity sequentially It comprises a, NK-1 receptor antagonist relates to a method for producing a lake water fraction excellent in efficacy.

According to an embodiment of this aspect, the step of preparing dried eggplant slices of Shiho, 50% ethanol is added to the shiho, sonicated and filtered, the filtrate is concentrated under reduced pressure to obtain ethanol X, such ethanol X distilled water Suspended in and then fractionated with n-hexane and separated into n-hexane fraction and water fraction, the separated water fraction was further fractionated into chloroform and chloroform (CHCl ₃ ) Separating into a fraction and a water fraction, and separating the separated water fraction into ethyl acetate and separating into an ethyl acetate fraction and a water fraction to obtain this water fraction as the final seawater fraction.

According to a preferred embodiment of this aspect, the ultrasonic extraction is repeated three or more times, particularly preferably five or more times for three hours. The total extract of Shiho extracted as described above is extracted sequentially with each solvent polarity using n-hexane, chloroform, ethyl acetate, and three solvents. The extraction method using each of these solvents is performed as known in the art. can do. For example, the extract was prepared by concentrating 600 g of Siho with 2 L of 50% ethanol at a temperature of 50 ° C. for 3 hours, sonicating three times and suspending it in distilled water, followed by n-hexane, chloroform and ethyl acetate. Each fraction was prepared. Each fraction may be fractionated sequentially by polarity with 2 L of n-hexane, 2 L of chloroform, and 4 L of ethyl acetate.

Human cell cultures in which large amounts of NK-1 receptor are known to measure the NK-1 receptor antagonistic effect of each solvent fraction obtained, specifically the central nervous system such as the amygdala, hippocampus, hypothalamus, striatum and spinal cord of the brain Tissues and cells or peripheral nervous system tissues and cells such as skin, inflammatory cells, digestive system, respiratory system, and cardiovascular system can be used. According to a preferred embodiment, a U-373 MG cell line which is a human glioblastoma-astrocytoma, epithelial-like cell line can be used. Such cell lines can be obtained from Korea Cell Line Bank. In the embodiment of the present invention was used accession number KCLB 30017, which was sold by the Bank of Korea Cell Line.

Analysis of the NK-1 receptor antagonist effect of each solvent extract of Shiho described above was administered to each of the cell line cultures at a concentration similar to that of the comparative group, which is a known outstanding NK-1 receptor antagonist, followed by fluorescence probes. After the combined SP is administered at the same concentration can be carried out by measuring with a fluorophotometer. The fluorescence of the SP to which the fluorescent probe is bound can be obtained by exciting at 485 nm and measuring the emitted light at 528 nm.

In the NK-1 receptor antagonist test, L-733,060, a non-peptide antagonist selective to human NK-1 receptor, is known to be effective in inhibiting inflammatory liver disease, pain reduction, depression and cancer cell proliferation. [Neurokinin-1 receptor antagonists CP-96,345 and L-733,060 protect mice from cytokine-mediated liver injury. J Pharmacol Exp Ther 305: 31-39 (2003)]. As a result of comparison test, total extract (45% inhibition of NK-1 receptor binding capacity of SP) <ethyl acetate fraction (61% inhibition) <water fraction (95% inhibition) in order to inhibit the binding of SP to NK-1 receptor Appears. That is, the water fraction of the final siho had the highest SP binding inhibitory activity, which was superior to the SP binding inhibitory activity against 88% of the receptor at 10 nM concentration of L-733,060, a well-known specific NK-1 receptor antagonist. We think that it is enough to be used as a medicine.

In order to increase the usability as such an alternative drug, refining the time lake water fraction provides an active subfraction that shows similar or higher activity than the above-mentioned comparative group. Accordingly, the present invention provides, as a preferred embodiment, a five-fraction fraction obtained by further analyzing the seawater fraction obtained according to the above-described preparation method by reverse phase column chromatography using a mixed solvent gradient of methanol and water, and each small fraction. It provides a manufacturing method comprising measuring the SP binding inhibitory activity of the fractions to obtain the fourth (BF-W4) and fifth (BF-W5) small fractions excellent in inhibitory activity. In this preparation method, a mixed solvent of methanol and water may be used in a gradient of 100% water, 75% water and 25% methanol, 50% water and 50% methanol, 25% water and 75% methanol and 100% methanol.

Although specific components of the seawater water fraction thus obtained were not confirmed in the present invention, the main components of the seawater water fraction are known to be triterpene-based saikosaponins (The chemical studies on oriental plant drugs XV). . on the constituents of Bupleurum Spp. Chem Pharm Bull 14: 1023-1033 (1966); Saponins isolated from Bupleurum falcatum L. J Chem Soc 20: 2043-2048 (1975); Phenethyl alcohol glycosides and iospentenol glycosides from fruit of Bupleurum falcatum . Phytochemistry 51: 819-823 (1999)], and these psychosaponins are known to be effective in diseases related to anti-inflammatory, acute liver disease, immunomodulation, hyperlipidemia, etc. [Effect of saikosaponin, a triterpene saponin, on apoptosis in lymphocytes: association with c-myc, p53, and bcl mRNA. Br J Pharm 131: 1285-1293 (2000)].

In view of the fact that the SP-binding inhibitory activity of these active fractions is superior to known NK-1 receptor antagonist compounds and natural extracts, it may be used as an alternative to NK-1 receptor antagonist drugs. do. As described above, these seawater fractions or active subfractions can each be used in the concentration range of 1 μg / ml-50 μg / ml in the NK-1 receptor antagonist composition.

In addition, NK-1 receptor antagonist compositions containing such cross-linked water extracts or active subfractions may be formulated to suit the mode of administration of conventional NK-1 receptor antagonists and administered to the subject depending upon the formulation.

Hereinafter, the present invention will be described in more detail with reference to the following examples. Such examples are illustrative of preferred embodiments of the invention and should not be considered as limiting the scope of the invention.

Example

Example 1: Seahawk moisture preparation

3L each of 50% ethanol was added to the siho, and ultrasonic filtration was performed five times for 3 hours, followed by filtration. It was concentrated under reduced pressure to obtain ethanol X (Siho T). Was suspended in ethanol and then in distilled water x n - by fractions with hexane, n - hexane fraction (Shiho H) and can obtain a fraction. The water fraction was again fractionated with chloroform to obtain a CHCl ₃ (Siho C) fraction and a water fraction. This water fraction was partitioned again with ethyl acetate to obtain an ethyl acetate fraction (Siho E) and a water fraction (Siho W).

Example 2: Culture of U-373 MG Cell Line as NK-1 Receptor Source

NK-1 receptor source containing a large amount of NK-1 receptor as a source of human glioblastoma-astrocytoma, epithelial-like cell line U-373 MG cell line Used (Accession Number: KCLB 30017)

This cell line was prepared using a culture medium containing 90% RPMI 1640, 10% fetal bovine serum, 100 IU / mL of penicillin and 100 μg / mL of streptomycin and incubated with air (95%) and CO ₂ ( 5%) of the mixed gas and water were incubated continuously. When the cells were constantly growing, cells were separated from the culture vessel using trypsin, and then diluted to 2 x 10 ⁵ cells / ml and transplanted into the culture vessel. Cell lines were used subcultured once every 7 days.

Example 3: Sample Treatment

Shiho's total extract, each solvent fraction, and L-733,060 (Tocris, USA) were dissolved in DMSO (within 0.1% final concentration), diluted with distilled water to a concentration of 100 mg / ml, and then used as a Millipore membrane filter (0.22). Μm, Millex-GV, USA) to make it aseptic.

Example 4: In vitro NK-1 receptor antagonist effect test

After transplanting U373 MG cells (2x10 ⁵ cells / ml) cultured as described in Example 2 to the culture vessel, the next day NK- which is well known as a comparison group of SP inhibitory activity to measure the effect of NK-1 receptor antagonist One receptor antagonist L-733,060 was treated to culture cells at a concentration of 0 to 100 nM.

After 1 hour, 10 nM of the substance P (SP-ⓕ) to which the fluorescence probe was bound was treated, and after 3 hours, the fluorescence intensity of SP-ⓕ bound to the NK-1 receptor using a fluorophotometer was excited at 485 nm. Emission light was measured at 528 nm.

The concentration of the SP binding inhibitory effect of the comparative group is shown in Figure 1, as the concentration of the sample was confirmed that the SP fluorescence intensity decreased and the SP binding inhibitory activity (%) was increased, thereby NK-1 receptor of this embodiment It has been proven to be effective as an antagonistic effect assay.

Using such an effective assay, cells treated with vehicle only (vehicle) for the SP binding inhibitory activity (%) of each sample and the comparative group of Shiho extract according to the present invention, and cells treated with SP-ⓕ after mediator treatment ( ②) and fluorescence of SP-ⓕ treated cells (③) after sample treatment were obtained as SP binding inhibitory activity (%). At this time, the concentration of each sample was used 50 ㎍ / ㎖, and the comparative group L733,060 was administered at a concentration of 10 nM (4.39 ng / ㎖).

SP binding inhibitory activity (%) was determined according to the following equation and expressed as an average value after repeated three independent experiments:

[Equation 1]

SP binding inhibitory activity (%) = 100-[(②-①) / (③- ①) × 100]

Where ① represents the fluorescence of cells treated with vehicle only, ② represents the fluorescence of SP-ⓕ treated cells after mediator treatment, and ③ represents the fluorescence of SP-ⓕ treated cells after sample treatment. Fluorescence).

As a result, the total extract was divided into three fractions obtained after treatment with n-hexane, chloroform, and ethyl acetate sequentially by solvent polarity, and the NK-1 receptor antagonistic effect was detected. 45% inhibition of binding force) <ethyl acetate (61% inhibition) <water fraction (95% inhibition) in order to inhibit the binding of SP to the NK-1 receptor. The well-known specific NK-1 receptor antagonist L-733,060 exhibited a binding inhibitory activity of SP to 88% of receptors at a concentration of 10 nM (see Figure 2). Through such a comparison, it can be considered that the SP binding inhibitory activity of the lake water fraction according to the present invention is very excellent.

Example 5 Small Fraction Analysis in Water Extracts Showing NK-1 Receptor Antagonistic Effects

As confirmed in Example 4, the water fraction showing the NK-1 receptor antagonistic effect was subjected to C ₁₈ resin reversed phase column chromatography (RP cc) to investigate the active subfraction of the water fraction. First, the water fraction fraction is developed as a solvent, and the mixed solvent of MeOH and water is mixed at various ratios (water 100% → water 75% + methanol 25% → water 50% + methanol 50% → water 25% + methanol 75% → methanol 100% Column chromatography was used to obtain five small fractions (BF-W1 to BF-W5).

The antagonistic effect of each small fraction thus obtained was analyzed in the same manner as in Example 4. As a result of analyzing the activities of the five small fractions, it was confirmed that the inhibitory active ingredients were included in the fourth small fraction BF-W4 and the fifth small fraction BF-W5 in the water lake fraction (see FIG. 3).

The final water fraction of the Seahawk extract obtained by sequentially using four solvents of n-hexane, chloroform, ethyl acetate, and water by solvent polarity was similar or more compared with L-733,160, a known superior NK-1 receptor antagonist. It exhibits excellent SP binding inhibitory activity. Accordingly, the NK-1 receptor antagonist composition containing the seawater fraction of the present invention is useful for various diseases requiring NK-1 receptor antagonists, especially inflammatory liver disease, asthma, arthritis, diabetes, depression, vasodilation, vomiting, pain, It may be usefully used as a natural drug in place of a compound for the treatment or prevention of anxiety and the like.

Claims (7)

An NK-1 receptor antagonist pharmaceutical composition containing a water fraction of Shiho (Bupleurum falcatum L.) having an antagonistic effect against the NK-1 receptor and having excellent SP binding inhibitory activity. The water fraction according to claim 1, wherein the water fraction of Shiho is the final water fraction obtained by sequentially treating the ethanol extracts of Shiho's dried branch slices with n-hexane, chloroform and ethyl acetate solvents in polar order. 1 receptor antagonistic pharmaceutical composition. The NK-1 receptor antagonistic pharmaceutical composition according to claim 1, wherein the water fraction of Shiho is in the range of 1 μg / ml to 50 μg / ml. The NK-1 receptor antagonistic pharmaceutical composition according to claim 1, wherein the SP binding inhibitory activity (%) is comparable or superior to that of the known synthetic antagonist L-733,060 (Tocris, USA). The NK-1 receptor antagonistic pharmaceutical composition according to claim 1, which is useful for the prevention or treatment of inflammatory liver disease, asthma, arthritis, diabetes, depression, vasodilation, vomiting, depression, pain, anxiety. 50% ethanol was added to Shiho, followed by ultrasonic extraction and filtration. The filtrate was concentrated under reduced pressure to obtain ethanol X. The ethanol X was suspended in distilled water, and then fractionated with n-hexane to separate into n-hexane and water fractions. Step, separating the separated water fractions again with chloroform (CHCl ₃ ) and separating them into chloroform fractions and water fractions; and separating the separated water fractions again with ethyl acetate, separating them into ethyl acetate fractions and water fractions, A method for preparing a lake water fraction having excellent NK-1 receptor antagonistic efficacy, comprising the step of obtaining a fraction as a final seawater fraction. The final seawater fraction as described in claim 6 was further purified by reversed phase column chromatography using a mixed gradient solvent of methanol and water to obtain five subfractions, and each subfraction was analyzed by SP through NK-1 receptor antagonistic efficacy analysis. A method for preparing an active subfraction of a water lake fraction having excellent NK-1 receptor antagonistic efficacy, comprising the steps of obtaining fourth and fifth subfractions having excellent binding inhibitory activity.

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