KR20180119508A - A composition comprising mixture of the herb extract and sulfasalazine for preventing or treating colitis - Google Patents

Abstract

The present invention relates to a composition comprising a mixed herb extract and sulfasalazine as effective components for preventing or treating colitis, and more specifically, to a pharmaceutical composition comprising a mixed extract of Scutellaria baicalensis and Bupleurum falcatum and sulfasalazine or pharmaceutically acceptable salt thereof as effective components for preventing or treating colitis. The pharmaceutical composition of the present invention is not only capable of effectively preventing, delaying, ameliorating, or treating colitis, especially inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), or the like, but also has extremely low risk of side effects due to the long-term administration of a high dose of sulfasalazine, and thus can be very useful for the development of a preventive or therapeutic agent for colitis.

Description

[0001] The present invention relates to a composition for preventing or treating colitis, comprising a mixed herbal extract and sulfasalazine as an active ingredient,

The present invention relates to a composition for preventing or treating colitis, comprising a mixed herbal medicine extract and sulfasalazine as an active ingredient, and more particularly, to a composition for preventing or treating colitis, And a pharmaceutical composition for preventing or treating colitis, comprising Sulfasalazine or a pharmaceutically acceptable salt thereof as an active ingredient.

Colitis is an inflammatory disease of the large intestine that includes inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), etc. Representative examples of inflammatory bowel disease (IBD) The causes of ulcerative colitis (UC) and Crohn's disease (CD) are still unclear, and they can cause severe chronic diarrhea and hemorrhagic diarrhea along with abdominal pain, and it is difficult to cure, Ulcerative colitis is a disease in which erosions (erosions) and ulcers are continuously formed in the mucous membranes of the large intestine, and blood, stool, diarrhea, and abdominal pain occur. In severe cases, ulcerative colitis causes fever, weight loss, Ulcerative colitis may occur in any part of the gastrointestinal tract, and Crohn's disease may be present in the digestive tract from the mouth to the anus In addition to abdominal pain, diarrhea, and stool, severe symptoms include fever, low blood pressure, weight loss, general malaise, and anemia. Ulcerative colitis and Crohn's disease Although there is a difference in lesion and inflammation symptoms, it is common that the distinction between two diseases is not clear because of similarity in several aspects.

In the past, the incidence of ulcerative colitis and Crohn's disease was known to be high in westerners, but recently, the number of patients has increased rapidly in Korea and other countries due to changes in lifestyle such as eating habits. However, there is a reason why the cause is unclear, and fundamental treatment is not established. Because of this, it is not the goal of complete treatment, but rather the use of medicines that relieve symptoms and maintain these conditions for as long as possible. Aminosalicylic acid preparations, adrenocorticosteroids, immunosuppressants and the like are mainly used as medicines for such symptomatic therapy, but various side effects have been reported. For example, salazosulfapyridine, which is frequently used as an aminosalicylic acid preparation, has been reported to have side effects such as nausea, vomiting, anorexia, rash, headache, liver damage, leukocytosis, abnormal red blood cells, proteinuria and diarrhea. Adrenocortical steroids are generally used for oral administration of prednisolone, enema, suppository, intravenous injection, etc. However, side effects such as gastric ulcer necrosis caused by gastric ulcer or long-term use are strong. However, since discontinuation of the medication recurs, the medicines must be continuously used. Therefore, there is a demand for the development of therapeutic agents for intestinal diseases such as ulcerative colitis and Crohn's disease which are excellent in efficacy, safe, and do not cause side effects. Irritable bowel syndrome (IBS) is also a chronic abdominal disorder that is not clearly defined. Currently, there is no fundamental treatment for IBS, and symptomatic therapy for each type of symptom relief is under way. For example, for hari-type IBS, anticholinergic agents having a gentle action to suppress smooth muscle contraction are used. In contrast, constipation-type IBS is difficult to control with a salt-substituting agent, and substitutional IBS is difficult to control with drugs. Basically, have.

In order to treat such colitis, researches are actively conducted to develop a composition capable of treating colitis from a natural product having few side effects. For example, Korean Patent Registration No. 1466308 discloses a composition for treating colitis, which comprises asparagine extract as an active ingredient, and Korean Patent No. 1526467 discloses a composition for treating colitis, which comprises an extract of corn oil as an active ingredient .

However, these extracts derived from natural products have limitations in that the preventive or therapeutic effect of colitis is not satisfactory. Therefore, it is urgently required to develop a new type of composition which is excellent in the prevention or treatment effect of colitis even though it has few side effects.

Accordingly, the present inventors have conducted intensive studies to develop new forms of herbal and herbal compositions which exhibit a very good colitis prevention or therapeutic effect and have few side effects. As a result, they have found that a combination of golden and herbal mixed extracts and sulfasalazine as active ingredients Of the present invention exhibits a remarkably excellent colitis prevention or therapeutic effect as compared with the case where the pharmaceutical composition containing each substance alone is administered. Thus, the present invention has been completed.

Accordingly, an object of the present invention is to provide a mixed extract of gold and citrate; And a pharmaceutical composition for preventing or treating colitis, which comprises Sulfasalazine or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the object of the present invention, And a pharmaceutical composition for preventing or treating colitis, which comprises Sulfasalazine or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a mixed extract of gold and citric acid; And a pharmaceutical composition for preventing or treating colitis, which comprises Sulfasalazine or a pharmaceutically acceptable salt thereof as an active ingredient.

According to one embodiment of the present invention, the composition of the present invention has an effect of inhibiting the expression of oxidative stress-related protein, inflammation-related protein and necrosis-related protein in the colonic tissue of the ulcerative colitis animal model, And the expression of the < RTI ID = 0.0 > On the other hand, this effect of the composition of the present invention was superior to that of the single dose group of sulfasalazine alone, and thus it was confirmed that the synergistic effect of the combined administration of the herbal medicine mixed extract and the sulfasalazine was exhibited.

In the present invention, the gold (Skullcap, Scutellaria baicalensis) is a root of a perennial herbaceous plant such as a spiny lentil which is removed as it is. Gold contains more than 35% of flavonoids such as Baikal, Baikal and Wigonin. It is known that this ingredient has anti-cancer, antiviral and antioxidant effects as well as anti-inflammatory action (Kim, EN et al., 2008) and gold as a herbal medicine exhibits various pharmacological effects such as chewing and detoxification in oriental medicine et al., 2006).

In the present invention, the Bupleurumfalcatum (BF) is a perennial herb that grows in mountainous areas and has a peculiar smell and a bit of taste. The roots have been used for medicines such as chills, fever, jaebaek, pleuritis, seawater, capillitis, brackishness, fraud, upper limit, wings, fever, , oleic acid, linolic acid, linolenic acid, and the like), and contains adonitiol as a sugar. In addition, as a saponin glycoside, saikosaponin a, Seiko saponin b, Seiko saponin c, Seiko saponin d, Seiko saponin e, Seiko saponin f (among them saikosaponin b is a secondary product from saikosaponin d) Corresponding sapogenins E, sapogenin F, sapogenin G are known. Examples of the sterol component include α-spinasterol (about 70%), stigmasterol, Δ7-stigmastenol and Δ22-stigmastenol (Park Jong-hee, Illustrated Book of Chinese Medicine, Award, Shinil Publishing Co., pp.497-499, 2002).

The composition containing the mixed extract of Gold and Seeds may be a mixture of gold and Sei alone extract, or may be a complex extract obtained by mixing Gold and Sei. In the present specification, a mixture or a complex extract of the single extract may be collectively referred to as a mixed extract. The composition of the present invention is not limited to the mixing method of the extract of Gold and Seiho as an active ingredient. There are no limitations on the form or properties of the extract, and it may be a solution, a concentrate, or a solid or powder from which the solvent used for the preparation of the extract is removed.

In the present invention, the mixed extract may be a mixture of Sepharose and a single extract of gold at a weight ratio of 1: 1 to 10. In the present invention, when the mixed extract contains a mixture of a golden extract and a single extract of Shiho, the mixture of Shiho and Golden extract may be mixed at a weight ratio of 1: 1 to 10. In addition, in the present invention, the mixed extract may be obtained by mixing Seiho and gold at a weight ratio of 1: 1 to 10, and then extracting them simultaneously, on a dry weight basis. That is, in the present invention, when the mixed extract contains a complex extract of gold and Sepharose, the extract may be obtained by mixing Sephia and Gold at a weight ratio of 1: 1 to 10. More preferably, the weight ratio of silver to gold is 1: 2 to 8, more preferably 1: 3 to 7, and most preferably 1: 4 to 6.

In the present invention, the mixed extract may be prepared by a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in the preparation of the mixed extract using the solvent extraction method includes water, a lower alcohol having 1 to 4 carbon atoms (for example, methanol, ethanol, propanol and butanol), or a mixture of water and lower alcohols, propylene glycol, May be selected from the group consisting of water, alcohols, or mixtures thereof, such as, but not limited to, acetonitrile, methylene chloride, isopropanol, . When water is used as the extraction solvent, the water is preferably hot water. When an alcohol is used as an extraction solvent, the alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, and the lower alcohol is more preferably selected from methanol or ethanol. When a functional alcohol is used as an extraction solvent, the alcohol content is preferably 40 to 90%, more preferably 40 to 70%.

It will be apparent to those skilled in the art that the present invention can provide extracts having substantially the same effects not only in the above-mentioned extraction solvents but also in other extraction solvents. In addition, the mixed extract of the present invention includes not only the extract obtained by the above-mentioned extraction solvent, but also the extract obtained through other conventional extraction methods, and the extract obtained through purification and fermentation processes. For example, decompression by carbon dioxide, extraction by supercritical extraction with high temperature, extraction by extraction using ultrasound, separation by ultrafiltration membrane with constant molecular weight cutoff value, various chromatography (size, charge, hydrophobic or affinity And the active fraction obtained through various purification and extraction methods, such as those extracted by fermentation products using natural or various microorganisms, are also included in the extract of the present invention.

The supercritical fluid extraction refers to supercritical fluid extraction. Generally, the supercritical fluid is extracted from the liquid and the liquid when the gas reaches the critical point at high temperature and high pressure. (J. Chromatogr. A. 1998; 479: 200-205). In addition, it has been reported that the supercritical fluid has a polarity similar to that of a nonpolar solvent. Carbon dioxide is a supercritical fluid with both liquid and gaseous nature, with the operation of supercritical fluid equipment reaching its critical pressure and temperature, resulting in increased solubility in fat-soluble solutes. When supercritical carbon dioxide passes through an extraction vessel containing a certain amount of sample, the lipophilic substance contained in the sample is extracted into supercritical carbon dioxide. When the supernatant carbon dioxide containing a small amount of cosolvent is passed through the sample remaining in the extraction vessel after extracting the lipid-soluble substance, components that were not extracted by pure supercritical carbon dioxide can be extracted. The supercritical fluid used in the supercritical extraction method of the present invention can effectively extract an active ingredient by using a mixed fluid in which a cosolvent is further mixed with supercritical carbon dioxide or carbon dioxide. These co-solvents may be used alone or in admixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether.

In addition, the ultrasonic extraction method is an extraction method using energy generated by ultrasonic vibration. Ultrasonic waves can destroy an insoluble solvent contained in a sample in a water-soluble solvent. Due to the high local temperature, Since the kinetic energy of the reactant particles is increased, sufficient energy required for the reaction is obtained. By inducing the high pressure by the shock effect of the ultrasonic energy, the mixing effect of the substance contained in the sample and the solvent is enhanced, thereby increasing the extraction efficiency. As the extraction solvent which can be used for the ultrasonic extraction method, one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether can be used. The extracted sample is recovered by vacuum filtration, and the filtrate is recovered, and the extract is removed by a vacuum evaporator and freeze-dried to obtain an extract. In addition, the mixed extract according to the present invention may also include fermented extract.

In the present invention, the sulfasalazine represents 2-hydroxy-5 - [[4- (2-pyridinylamino) sulfonyl] azo] benzoic acid having the structure represented by the following formula (1). The sulfasalazine is an aminosalicylic acid preparation and is a drug frequently used for ulcerative colitis.

[Chemical Formula 1]

On the other hand, sulfasalazine has been reported to exhibit side effects such as nausea, vomiting, anorexia, rash, headache, hepatic injury, leukocytosis, abnormal red blood cells, proteinuria and diarrhea. Accordingly, the present inventors have developed a new type of pharmaceutical composition in which the above-mentioned side effects are reduced while maintaining excellent pharmacological effects of the sulfasalazine. According to one embodiment of the present invention, It has been confirmed that when the extract is formulated, even when the sulfasalazine is administered at a low dose, the effect of preventing or treating colitis is equivalent to or higher than that administered by a high dose.

The present invention includes not only sulfasalazine represented by the above formula (1), but also pharmaceutically acceptable salts thereof, possible solvates, hydrates, racemates, or stereoisomers thereof.

In particular, the sulfasalazine represented by the formula (1) of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention may be prepared by dissolving sulfasilazane represented by the formula (1) in an excess amount of an aqueous acid solution and dissolving the salt in a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile ≪ / RTI > It is also possible to prepare the mixture by evaporating a solvent or an excess acid in the mixture, or by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

The weight ratio of the sulfasalazine or the pharmaceutically acceptable salt thereof to the herbal medicine mixed extract contained in the pharmaceutical composition of the present invention may be 1: 0.5 to 3, preferably 1: 0.5 to 2, more preferably 1: : 0.5 to 1.5, and most preferably 1: 0.5 to 1.

In the present invention, the colitis is a condition in which inflammation occurs in the large intestine due to bacterial infection or pathological fermentation of intestinal contents, and includes infectious colitis and non-infectious colitis. Examples of the specific colitis that can be prevented or treated through the pharmaceutical composition according to the present invention include, but are not limited to, inflammatory bowel disease or irritable colitis syndrome. Such inflammatory bowel diseases include, for example, ulcerative colitis, Crohn's disease, and the like. In addition, the colitis that can be prevented or treated through the pharmaceutical composition according to the present invention includes both acute colitis and chronic colitis. Acute colitis is an acute inflammation of the colon or colon. The mucous membrane is damaged due to inflammation, and mucous diarrhea or hemorrhagic symptoms are mainly seen. Acute colitis in the present invention includes acute pseudomembranous colitis and acute ulcerative colitis as well as general acute infectious colitis.

When the composition is provided in the form of a pharmaceutical composition, it may further contain one or more pharmaceutically acceptable carriers, excipients or diluents in addition to the mixture of the present invention. The term "pharmaceutically acceptable" as used herein means a non-toxic composition which is physiologically acceptable and which, when administered to humans, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, .

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration to peptide preparations. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and preparations can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI >

The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route as described above.

In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa., 1995, which is a commonly known formulary for all pharmaceutical chemistries.

 The total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the total preferred dose of the pharmaceutical composition of the present invention may be from about 0.01 μg to 10,000 mg, and most preferably from 0.1 μg to 1,000 mg per kg body weight of the patient per day. However, the dosage of the pharmaceutical composition may be determined depending on various factors such as the formulation method, administration route and frequency of treatment, as well as the patient's age, body weight, health condition, sex, severity of disease, diet and excretion rate, It will be possible to determine the appropriate effective dose of the composition of the present invention by those of ordinary skill in the art in view of this point. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The term "prevention" as used herein, on the other hand, means any action that inhibits or delays disease by administration of a composition comprising the mixture of the present invention. The term "treatment ", as used herein, refers to any action that improves or alleviates a symptom of a disease upon administration of a composition comprising a mixture of the present invention.

The effect of the composition according to the invention is illustrated in the following examples.

The pharmaceutical composition according to the present invention not only can effectively prevent, delay, ameliorate, or treat colitis, especially inflammatory bowel disease (IBD) or irritabel bowel syndrome (IBS), but also sulfasalazine ), The occurrence of side effects due to long-term administration is remarkably low, which can be very useful for development of a preventive or therapeutic agent for colitis.

FIG. 1 shows the weight change of each administration group at the start and end of the experiment.
Fig. 2 is a graph (A) or a visual observation (B) of the colon length of an animal at the end of the experiment.
FIG. 3 is a graph showing a quantitative analysis of the expression of oxidative stress-related proteins (NOX4, Rac1 and p47 ^phox ) in colon tissues by Western analysis.
FIG. 4 is a graph showing the expression of inflammation-related protein (NF-kB) in colon tissues by Western analysis and quantification thereof.
FIG. 5 is a graph showing the quantification of the expression of inflammation-related proteins (COX-2, iNOS, TNF-α and IL-1b) in colon tissues by Western analysis.
FIG. 6 is a graph showing Western analysis of the expression of antioxidative-related proteins (SOD-1 and Catalase) in colon tissues and quantification thereof.
FIG. 7 is a graph showing a quantitative analysis of necrotic-related protein (Caspase 3) expression in colon tissues after Western analysis. FIG.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately The present invention should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention. Accordingly, it is to be understood that the constituent features of the embodiments described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the inventive concepts of the present invention, so that various equivalents, And the like. Also, throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements, not excluding other elements, unless specifically stated otherwise.

Hereinafter, the present invention will be described in detail.

<Experimental Method>

1. Experimental material

Sulfasalazine and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma aldrich (St. Louis, Mo.) in MP biomedicals and Dextran Sulfate Sodium (DSS; Nitrocellulose membranes were purchased from Amersham GE Healthcare (Little Chalfont, UK) and catalyzed by NOx4, Bac1, p47 ^phox , NF-κBp65, superoxide dismutase, catalase, inducible nitric oxide synthase (iNOS) , cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 beta), histone and β-actin and secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mono anti-caspase-3 (BioVision, Mountain View, CA, USA). Protease inhibitor mixture DMSO and solutionethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). In addition, 2 ', 7'-Dichlorofluorescein diacetate (DCF-DA) and Dihydrorhodamine 123 were purchased from Molecular Probes (Eugene, OR, USA) and ECL Western Blotting Detection Reagents from GE Healthcare. The BCA protein assay kit for protein determination was purchased from Thermo Scientific (Rockford, IL, USA).

2. Production method of herbal medicine extract

The gold used in the experiment was extracted with 50% ethanol and the extract was extracted with hot water.

Specifically, 50% ethanol of 10 times as much as the weight of gold was added to the gold and extracted at room temperature. The 50% ethanol extracts were then filtered through Whatman (No. 1) filter paper, concentrated under reduced pressure on a rotary evaporator, and stored frozen until the experiment.

After the extraction was completed, it was filtered with a Whatman (No. 1) filter paper, concentrated under reduced pressure using a rotary evaporator, and then dried in a freeze dryer (Eyela, model FDU-2000, Japan) was used for the experiment.

3. Establishment of an animal model of colitis and Experimental group  Furtherance

(1) colitis animal model

Male BALB / c mice were received from Orient Bio (Gyeonggi Province, Seongnam) for 8 weeks and were adapted to the laboratory environment for 1 week and used in the experiment. The conditions of animal breeding room were controlled by conventional system at temperature of 22 ± 2 ℃, humidity of 50 ± 5% and light cycle of dark cycle for 12 hours. Feeds were fed with solid feed (more than 22.1% crude protein, less than 8.0% crude fat, less than 5.0% crude protein, 8.0% crude protein, 0.6% calcium or more, 0.4% or more, samyangsa, no antibiotics) and water.

Colitis inducible group except normal group was fed with water containing 5% DSS for 7 days to induce ulcerative colitis.

(2) Experimental composition

The colitis animal model or the normal animal established according to the above method was classified as follows. Each experimental group included 9 animals.

1) Normal: Normal animals that did not cause colitis were treated with only solvent.

2) Solvent-injected vehicle (vehicle): only the solvent was administered to the animal that caused the colitis.

3) 30 Sulfa group: Sulphasalazine administered at 30 mg / kg to the colitis-causing animals.

4) 60 Sulfa group: Sulphasalazine administered at 60 mg / kg to the colitis-causing animals.

5) Huangshi 30 + 30 Sulfa group: An animal model that induced colitis was co-administered 30 mg / kg of Sulfasalazine 30 mg / kg with 5: 1 (weight ratio)

6) Hwangsik 60 group: An animal model that induced colitis was administered 60 mg / kg of 5: 1 (weight ratio) mixed extract of golden and citrus.

Each experimental material was orally administered to the divided animals once per day according to the experimental group. The drug administration was carried out for a total of 7 days. After completion of the experiment, each animal was anesthetized, sacrificed, organs and blood were extracted and the following experiment was conducted.

4. Blood Oxidative  Stress analysis

On the last day of administration, blood was collected by cardiac puncture and the blood was centrifuged at 4,000 rpm for 10 min. , 25mM DCFH-DA was mixed, and the calculated value was measured for 30 minutes using a fluorescence photometer at 530 nm and 485 nm excitation wavelength every 5 minutes from 0 minutes . S. F. Ali, C. P. LeBel, and S. C. Bondy, "Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity," Neurotoxicology, vol. 13, no. 3, pp. 637-648, 1992.

5. Histological analysis

For evaluation of the microscopy, the adipose tissue was cut to separate the intermediate compartments. This compartment was embedded in paraffin and fixed in 10% neutral-buffered formalin, cut into 2 mm sections and stained with H / E for microscopic evaluation. Dyeing slices were observed with an optical microscope and analyzed using software (i-Solution Lite software program, Innerview Co.).

6. Western blotting

To obtain the cytoplasmic proteins of the colon tissues, a buffer A (buffer A; 100 mM Tris-HCl (pH 7.4), 5 mM Tris-HCl (pH 7.5), 2 mM MgCl ₂ , 15 mM CaCl ₂ , 1.5M sucrose and 0.1M DTT, ) Was added, followed by pulverization with a tissue grinder (Bio Spec Product, USA) and 10% NP-40 solution was added. The mixture was allowed to stand on ice for 20 minutes and then centrifuged at 12,000 rpm for 2 minutes to separate the supernatant containing cytoplasm. 50 mM HEPES, 50 mM KCl, 0.3 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and 10% buffer C (Buffer C) were rinsed twice in Buffer A supplemented with 10% NP- glycerol) and resuspended, followed by 3 voltexes every 10 minutes. After centrifugation at 12,000 rpm for 10 min at 4 ° C, the supernatant containing the nuclei was obtained and stored at -80 ° C for freezing. (P-IKBA), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase, Gpx1 2, phosphor-p38 (p-p38), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), monocyte chemotactic protein 1, MCP-1, intracellular adhesion molecule-1, tumor necrosis factor alpha, interleukin-1 beta, Bax, Bcl2, caspase 3, And nuclear protein (NF-κBp65), c-fos, nuclear factor-erythroid 2-related factor 2 (Nrf2) and histone were electrophoresed (8-13% sodium dodecylsulfate polyacrylamide gel; SDS-PAGE) The prepared membrane was treated with each primary antibody and incubated at 4 ° C overnight. The membrane was then incubated with PBS-T The cells were washed five times every 6 minutes and reacted for 1 hour at room temperature using a secondary antibody (diluted 1: 3000 with PBS-T) used for each primary antibody treated. And then exposed to GE Healthcare (Arlington Heights, Ill., USA) and sensitized to Sensi-Q2000 Chemidoc (Lugen Sci Co., Ltd., Seoul, Korea) After confirming expression, the band was quantified using ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan) program.

7. Statistical processing

All results were expressed as mean ± standard error (mean ± S.E). One-way analysis of variance (ANOVA) was performed according to Dunnett's multiple comparison test (SPSS 18.0 for Windows, SPSS Inc., U.S.A.) for significance testing. P <0.05 was considered statistically significant.

<Experimental Results>

1. Animal weights and colon length

1 and 2, the body weight and the colon length during the experimental period were measured. Using a digital mass meter, the body weight was measured at predetermined time intervals after the fasting and during the administration of the DSS and composition. As a result of comparing the body weights between the normal group, the DSS control group, the sulfa30 group, the sulfa60 group and the composition administration group according to the present invention (herbal medicine combination extract + sulfasalazine), in the vehicle control group treated with 5% DSS, (P < 0.001), but the weight reduction was inhibited more than the DSS control group (Fig. 1).

DSS was dissected 7 days later and the length from the cecum to the rectum was taken and photographed. The results are shown in Fig. 2A (colon length) and Fig. 2B (gross photograph). The colon (vehicle control) of the mice that induced ulcerative colitis with 5% DSS was significantly shorter than the normal group (p <0.001). There was no significant difference in the experimental group, but it was found to improve the damage of the colon compared to the vehicle control group.

On the other hand, in the case of administering only the mixed extract of Gold and Seicho (60 mg / kg) alone without using the sulfasalazine in combination, the normal colon (Normal) , Solvent-applied vehicle (Vehicle), and Huangshi 60-treated group were subjected to further experiments under the same conditions as those described above, and after dissection 7 days after DSS administration, the lengths from the cecum to the rectum were taken and photographed. Length) and 2D (gross photograph). It was found that the group treated with only the mixed extract of Golden and Seicho without the combination of sulfasalazine did not show any significant change in the length of the colon compared to the control group and no clinical symptoms that improved the inflammation and damage of the colon compared to the control group I could.

As described above, although weight loss was observed due to colitis induced and the length of the colon was shortened, administration of the test substance suppressed weight loss compared to the control group, and the colonic length shortening caused by the damage of the colon was improved.

2. Analysis of Oxidative Stress-Related Proteins in Colonic Tissue

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) is one of the causes of ROS produced by Ab peptiedes. It plays a major role in the production of intracellular O ^2- and regulates various protein enzymes that produce large amounts of ROS. It is well characterized in macrophages. Its major pathological role is degeneration and necrosis in acute and chronic inflammation sites in inflammatory cells, epithelial cells, collagen, and elastin. NOX4, p47 ^phox , and Rac1 belong to the subunits of NADPH oxidase. Accordingly, the present inventors evaluated the oxidative stress-improving effect of the composition of the present invention by measuring the expression levels of the proteins belonging to the subunit of NADPH oxidase in the colon tissue.

The results are shown in Fig.

As shown in FIG. 3, expression of NOX4, p47 ^phox and Rac1 protein in the DSS control group was significantly increased ( P <0.001) compared with the normal group. Meanwhile, the NOX4, p47 ^phox and Rac1 protein expression was significantly inhibited in the group administered with the composition according to the present invention, and this effect was more remarkable than the sulfasalazine 60 mg / kg administration group.

As described above, it was found that the composition according to the present invention improves the oxidative stress by reducing NOX4, p47 ^phox and Rac1 protein expression in ulcerative colitis mice.

3. In the colon tissue NF -KB and related inflammatory proteins

One of the most common nuclear transcription factors that regulate gene expression, including cell differentiation and growth, inflammatory response, and cell adhesion, is NF-kB. NF-kB is normally inactivated by binding to the inhibitor kappa B (IkB), which is inactivated by the inhibitory protein, and rapidly phosphorylated IkB by ROS stimulation, separating NF-kB from IkB and activating the free dimer As it exists, it is transferred to the nucleus. Plays a central role in mediating inflammation and plays a role in regulating expression of various early response genes involved in inflammation. The genes regulated by NF-kB are COX-2 and iNOS, and cytokines such as TNF-a and IL-1b are also present. Accordingly, the present inventors tried to determine how the expression of the proteins mediating the inflammation changes by the treatment of the composition according to the present invention.

The results are shown in Figs. 4 and 5.

As shown in FIG. 4, the expression of NF-kB was significantly increased (p <0.05) in the vehicle control group compared with the normal group, and the expression of the protein was significantly decreased in the composition administration group according to the present invention . However, in the group treated with sulfasalazine, the decrease tendency of the protein was not observed at all.

As shown in FIG. 5, expression of COX-2, iNOS, TNF-α and IL-1b in the colonic tissues of the vehicle control group was significantly increased compared with that of the normal group. However, As shown in FIG. On the other hand, the effect of the composition according to the present invention was significantly better than sulfasalazine 60 mg / kg.

 These results indicate that the composition according to the present invention has an anti-inflammatory effect by inhibiting the activity of NF-κBp65 in the colon tissue. Inhibition of the activity of NF-κBp65 regulates the expression of inflammatory mediators (COX-2, iNOS) and inflammatory cytokines (TNF-α, IL-1 b) caused by intracellular migration of NF-κBp65 . The effect of the composition according to the present invention was much better than that of sulfasalazine (60 mg / kg), indicating that the mixed herbal extract and sulfasalazine exhibited a synergistic action.

4. Analysis of Antioxidant Protein Expression in Colonic Tissue

The increase in free radicals, including O2- produced by ROS, accumulates in large amounts in cells. SOD interacts with the superoxide radical to form hydrogen peroxide (H2O2), which is metabolized by Catalase to water. Catalase is a hemeprotein that protects tissues from the harmful effects of highly reactive hydroxyl radicals. Accordingly, the present inventors tried to determine how the expression of the antioxidant proteins in the colon tissue was changed by treatment with the composition according to the present invention.

The results are shown in Fig.

As shown in FIG. 6, the expression of antioxidant protein in the vehicle control group was significantly reduced in the colon tissue as compared with the normal group, and the expression of the proteins was recovered to the normal group level in the composition-treated group according to the present invention . On the other hand, this effect of the composition according to the present invention was found to be equal or higher compared to sulfasalazine 60 mg / kg.

5. Expression of Necrotizing Proteins in Colonic Tissue

When tissue necrosis occurs, caspase 3 protein expression, which is a necrotic factor, is known to be increased in the tissue. Accordingly, the present inventors have investigated how the expression of necrosis-related protein changes in the colon tissue by treatment with the composition according to the present invention.

The results are shown in Fig.

As shown in FIG. 7, in the Vehicla control group, the expression of the necrosis-related protein (Caspase 3) was significantly increased compared with the normal group, but the expression of the protein was decreased by the treatment of the composition according to the present invention . On the other hand, this effect of the composition according to the present invention was confirmed to be more than that of the sulfasalazine 60 mg / kg treated group.

As described above, it was found that the composition containing the mixed herbal medicine extract + sulfasalazine according to the present invention is remarkably superior in the prevention or treatment effect of colitis compared to the group treated with the same amount of sulfasalazine alone. That is, it has been confirmed that, according to the combined administration of both drugs and herbal preparations, it is possible to reduce adverse effects caused by administration of a high dose of the two preparations and to exert an excellent therapeutic effect on colitis. The invention is first disclosed.

While the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, Such modifications and changes are to be considered as falling within the scope of the following claims.

Claims (6)

Mixed extract of golden and citrus; And a pharmaceutical composition for preventing or treating colitis, comprising Sulfasalazine or a pharmaceutically acceptable salt thereof as an active ingredient. The composition according to claim 1, wherein the extract is one selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, hexane, ethyl acetate, and a mixed solvent thereof. The pharmaceutical composition according to claim 1, wherein the weight ratio of Citrate: Gold in the mixed extract is 1: 1 to 10. The pharmaceutical composition according to claim 1, wherein the weight ratio of the mixed extract: sulfasalazine or a pharmaceutically acceptable salt thereof is 1: 0.5 to 3. 5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the colitis is inflammatory bowel disease or irritable colitis syndrome. The pharmaceutical composition according to claim 5, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

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