KR102150548B1 - 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 for preventing or treating colitis comprising a complex herbal extract and sulfasalazine as an active ingredient, and more specifically, a mixed extract of dermis and siho; And it relates to a pharmaceutical composition for preventing or treating colitis comprising sulfasalazine (Sulfasalazine) or a pharmaceutically acceptable salt thereof as an active ingredient.
The pharmaceutical composition according to the present invention can effectively prevent, delay, ameliorate or treat colitis, particularly inflammatory bowel disease (IBD) or irritabel bowel syndrome (IBS), as well as high doses of sulfasalazine. (sulfasalazine) The risk of side effects caused by long-term administration is remarkably low, so it can be very useful in developing colitis prevention or treatment.

Description

A composition comprising mixture of the herb extract and sulfasalazine for preventing or treating colitis comprising a mixed herbal extract and sulfasalazine as active ingredients

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

Colitis is a disease in which inflammation occurs in the large intestine, and includes Inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), etc. Representative of Inflammatory bowel disease (IBD) The causes of ulcerative colitis (UC) and Crohn's disease (CD), which are diseases, have not yet been clearly identified, and can cause severe chronic diarrhea and bloody diarrhea along with abdominal pain. It is difficult to cure and improves and worsens. Ulcerative colitis is a disease in which soreness (erosion) or ulcers are continuously formed on the mucous membrane of the large intestine, and bloody stools, mucous stools, diarrhea and abdominal pain occur, and in severe cases, fever, weight loss, and anemia. In addition, ulcerative colitis can occur in any part of the gastrointestinal tract, Crohn's disease is a disease in which lesions such as ulcers occur discontinuously in any part of the digestive tract from the mouth to the anus. , Diarrhea, bloody stool, and in severe cases, symptoms such as fever, bleeding, weight loss, general malaise, anemia, etc. Ulcerative colitis and Crohn's disease differ in lesions and inflammatory symptoms, but have similar patterns in many ways. Because it can be seen, the distinction between the two diseases is often not clear.

Conventionally, the incidence of ulcerative colitis and Crohn's disease has been known to be high in Westerners, but recently, the number of patients in East such as Korea is increasing rapidly due to changes in lifestyles such as eating habits. However, there are reasons for which the cause is unclear, so no fundamental treatment has been established. For this reason, it is not aimed at complete treatment, but a situation in which drugs that relieve symptoms and maintain such a condition as long as possible are used. As drugs for such symptomatic therapy, mainly aminosalicylic acid preparations, adrenal cortical steroids, immunosuppressants, etc. are used, but various side effects have been reported. For example, salazosulfapyridine, which is often used as an aminosalicylic acid, has been reported to have side effects such as nausea, vomiting, loss of appetite, rash, headache, liver injury, leukocytosis, abnormal red blood cells, proteinuria, and diarrhea. In addition, adrenocorticosteroids are generally used by oral administration of prednisolone, enema, suppositories, intravenous injections, etc., but have strong side effects such as gastric ulcer or femoral head necrosis caused by long-term use. However, since discontinuation of medication recurs symptoms, these drugs are compelled to continue to be used. Therefore, there is a need to develop a therapeutic agent for intestinal diseases such as ulcerative colitis and Crohn's disease that is excellent in effect and does not cause side effects. Irritable bowel syndrome (IBS) is also a chronic abdominal disease whose cause is not clear. Currently, there is no fundamental treatment for IBS, and symptomatic therapy is being performed for the purpose of alleviating each type of symptoms. For example, for Hari-type IBS, anticholinergic drugs that have antispasmodic action to inhibit contraction of smooth muscle are used, salt laxatives for constipation-type IBS, and drugs for replacement-type IBS are difficult to control. have.

In order to treat such colitis, research has been actively conducted to develop a composition capable of treating colitis from natural products with few side effects. For example, Korean Registered Patent No. 1466308 discloses a composition for treating colitis comprising an extract of Jimo as an active ingredient, and Korean Patent No. 1526467 discloses a composition for treating colitis comprising an extract of cornus milk as an active ingredient. .

However, these extracts derived from natural products have limitations in that the preventive or therapeutic effects of colitis are not satisfactory. Therefore, there is an urgent need to develop a new type of composition that has few side effects and has a very excellent effect of preventing or treating colitis.

Accordingly, the present inventors have conducted extensive research to develop a new type of Western-Oriental complex composition that exhibits very excellent colitis prevention or treatment effect and has few side effects, and as a result, the mixed extract of dermis and Shiho and sulfasalazine are effective. The present invention was completed by discovering that the pharmaceutical composition containing as a component exhibits remarkably superior colitis preventing or treating effects compared to those administered with each substance alone.

Accordingly, an object of the present invention is a mixed extract of dermis and siho; And sulfasalazine (Sulfasalazine) or a pharmaceutically acceptable salt thereof to provide a pharmaceutical composition for preventing or treating colitis comprising as an active ingredient.

In order to achieve the object of the present invention described above, the present invention is a mixed extract of dermis and siho; And it provides a pharmaceutical composition for preventing or treating colitis comprising sulfasalazine (Sulfasalazine) or a pharmaceutically acceptable salt thereof as an active ingredient.

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

The present invention is a mixed extract of dermis and siho; And it provides a pharmaceutical composition for preventing or treating colitis comprising sulfasalazine (Sulfasalazine) or a pharmaceutically acceptable salt thereof as an active ingredient.

According to an embodiment of the present invention, the composition of the present invention exhibited an effect of inhibiting the expression of oxidative stress-related proteins, inflammation-related proteins, and necrosis-related proteins in colon tissues of an animal model of ulcerative colitis. It was confirmed to exhibit an effect of increasing the expression of. On the other hand, this effect of the composition of the present invention was found to be superior compared to the group administered with the same dose of sulfasalazine alone, and it was confirmed that a synergistic effect was exhibited by the combined administration of the herbal extract and sulfasalazine.

In the present invention, the dermis (Citrus Unshiu Peel) means that the peel of the mature fruit of Citrus unshiu Markovich is dried. As a scented dry placebo, it is used for gastritis and indigestion, and is known for its efficacy as an antitussive, antitussive, and expectorant.

In the present invention, the Shiho (Bupleurum falcatum (BF)) is a perennial plant of the parsley family and grows in mountains or fields, has a peculiar smell, and tastes slightly bitter. Since ancient times, the roots have been used as medicines for chills, antipyretic, anticancer, pleurisy, seawater, capillary blood, heart, morale, upper limit, ripening, fever, pain relief, ground damage and malaria, and the component is about 2% of fatty oil (stearic acid). , oleic acid, linolic acid, linolenic acid, etc.), and adonitiol as a sugar. In addition, as saponin glycosides, saikosaponin a, seiko saponin b, seiko saponin c, seiko saponin d, seiko saponin e, seiko saponin f (of which saikosaponin b is a secondary product in saikosaponin d), and it Corresponding sapogenin E, sapogenin F, and sapogenin G are known. As a sterol component, α-spinasterol (α-spinasterol; about 70%), stigmasterol, Δ7-stigmastenol, and Δ22-stigmastenol are contained. (Park Jong-hee, Herbal Medicine Encyclopedia (Top), Book Publishing Shinil Sangsa, pp.497-499, 2002).

The composition comprising the mixed extract of the dermis and Shiho may be a mixture of the extract of the dermis and Shiho alone, or may be a complex extract extracted after mixing the dermis and Shiho. In the present specification, a mixture of the single extract or a complex 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 dermis and Shiho extract containing as an active ingredient. There is no limitation on the shape or properties of the extract, and may be a solution, a concentrate, or a solid or powder from which the solvent used to prepare the extract.

In the present invention, the mixed extract may be a mixture of Siho and the dermis alone extract in a weight ratio of 1: 1 to 10. In the present invention, when the mixed extract contains a mixture of the extract of dermis and siho alone, the extracts of siho and dermis may be mixed in a weight ratio of 1: 1 to 10 for each. In addition, in the present invention, the mixed extract may be extracted at the same time after mixing siho and dermis in a weight ratio of 1: 1 to 10 based on dry weight. That is, in the present invention, when the mixed extract includes a complex extract of dermis and siho, siho and dermis may be mixed in a weight ratio of 1: 1 to 10 and then extracted. More preferably, the weight ratio of the siho and the dermis may be 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 using 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 is water, a lower alcohol having 1 to 4 carbon atoms (e.g., methanol, ethanol, propanol and butanol), or a water-containing lower alcohol, propylene glycol, 1, 3-butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane, and may be selected from the group consisting of a mixture thereof, and water, alcohol, or mixtures thereof. It is desirable. When using water as the extraction solvent, it is preferable that water is hot water. In addition, when alcohol is used as the 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. In addition, when a hydrous alcohol is used as the extraction solvent, the alcohol content is preferably 40 to 90%, and more preferably 40 to 70%.

On the other hand, in the present invention, it is obvious to those skilled in the art that the mixed extract can obtain an extract exhibiting substantially the same effect even when using not only the above-described extraction solvent but also other extraction solvents. In addition, in the present invention, the mixed extract includes not only the extract by the above-described extraction solvent, but also an extract obtained through another conventional extraction method, or an extract that has undergone purification and fermentation processes. For example, decompression by carbon dioxide, extraction by supercritical extraction by high temperature, extraction by extraction by ultrasonic wave, separation by using an ultrafiltration membrane having a constant molecular weight cut-off value, various chromatography (size, charge, hydrophobicity or affinity) Active fractions obtained through various purification and extraction methods additionally carried out, such as those prepared by separating according to sex) or by fermentation products using natural or various microorganisms are also included in the extract of the present invention.

The extraction method by the supercritical extraction method by the decompression by carbon dioxide and the high temperature refers to a supercritical fluid extraction method.In general, the supercritical fluid is a liquid and a liquid that is possessed when a gas reaches a critical point under high temperature and high pressure conditions. It has gaseous properties and chemically has a polarity similar to that of non-polar solvents. Due to this property, supercritical fluids are used for extraction of oil-soluble substances (J. Chromatogr. A. 1998;479:200-205). Carbon dioxide becomes a supercritical fluid having both liquid and gaseous properties as the pressure and temperature reach a critical point due to the operation of the supercritical fluid device, and as a result, the solubility of the oil-soluble solute increases. When the supercritical carbon dioxide passes through an extraction container containing a certain amount of a sample, the fat-soluble substance contained in the sample is extracted and released into the supercritical carbon dioxide. After extracting the fat-soluble substance, if the sample remaining in the extraction container is passed through the supercritical carbon dioxide containing a small amount of co-solvent again, components that were not extracted with 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 supercritical carbon dioxide or a mixed fluid in which a co-solvent is additionally mixed with carbon dioxide. As such a co-solvent, one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane, and diethyl ether may be used.

In addition, the ultrasonic extraction method is an extraction method that uses energy generated by ultrasonic vibration, and the ultrasonic waves can destroy the insoluble solvent contained in the sample in the aqueous solvent, and due to the high local temperature generated at this time, Since the kinetic energy of the reactant particles is increased, sufficient energy required for the reaction is obtained, and high pressure is induced by the impact effect of ultrasonic energy to enhance the mixing effect of the material contained in the sample and the solvent, thereby increasing the extraction efficiency. The extraction solvent that can be used in the ultrasonic extraction method may be one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane, and diethyl ether. The extracted sample may be vacuum filtered to recover the filtrate, then removed with a vacuum evaporator, and freeze-dried to obtain an extract through a conventional extract preparation method. In addition, the mixed extract according to the present invention may also include an extract that has undergone a fermentation process.

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

[Formula 1]

On the other hand, when sulfasalazine is administered at a high dose for a long period of time, it has been reported that side effects such as nausea, vomiting, loss of appetite, rash, headache, liver injury, leukocyte reduction, abnormal red blood cells, proteinuria, and diarrhea appear. Accordingly, the present inventors intend to develop a new type of pharmaceutical composition in which the above-described side effects are reduced while maintaining the excellent pharmacological effect of the sulfasalazine, and according to an embodiment of the present invention, a mixed herbal medicine of dermis and shiho in the sulfasalazine In the case of compounding the extract, it was confirmed that even if sulfasalazine was administered at a low dose, the same or better preventive or therapeutic effect of colitis appeared as if administered at a high dose.

The present invention includes not only sulfasalazine represented by Chemical Formula 1, but also pharmaceutically acceptable salts thereof, and possible solvates, hydrates, racemates, or stereoisomers that may be prepared therefrom.

In particular, sulfasalazine represented by Chemical Formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, butin-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, me Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, Maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is a conventional method, for example, by dissolving sulfasalazine represented by Formula 1 in an excessive amount of an aqueous acid solution, and this salt is dissolved in a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation using. In addition, the mixture may be dried by evaporating a solvent or an excess of acid, or may be prepared by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt can be made using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).

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

In the present invention, the colitis refers to a state in which inflammation has occurred in the large intestine due to bacterial infection or pathological fermentation of intestinal contents, and is a concept including infectious colitis and non-infectious colitis. Specific types of colitis that can be prevented or treated through the pharmaceutical composition according to the present invention include inflammatory bowel disease or irritable colitis syndrome, but are not limited thereto. The inflammatory bowel disease is, for example, ulcerative colitis, or Crohn's disease. In addition, colitis that can be prevented or treated through the pharmaceutical composition according to the present invention includes both acute colitis and chronic colitis. The acute colitis refers to an acute inflammation of the large intestine or colon, and the mucous membrane is damaged due to the inflammation, leading to symptoms of mucous diarrhea or blood blood. In the present invention, acute colitis includes not only general acute infectious colitis, but also acute pseudomembranous colitis and acute ulcerative 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. In the above, "pharmaceutically acceptable" is a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans, and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness or similar reactions. Say.

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 substances used for oral administration of the peptide preparation. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include 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 formulations may be referred to as those described in the following literature (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, intestinal, topical, sublingual or rectal administration Can be

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

In the case of a formulation for oral administration, the composition of the present invention may be formulated using a method known in the art as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. I can. For example, in oral preparations, tablets or dragees can be obtained by mixing the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing into a granule mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, gelatin, and polyvinylpyrrolidone may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In the case of a formulation for parenteral administration, it can be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, a formula generally known for all pharmaceutical chemistry.

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 that is administered for a long time in multiple doses. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 1,000 mg per 1 kg of the patient's body weight per day. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the age, weight, health condition, sex, disease severity, diet and excretion rate of the patient, as well as the formulation method, route of administration and number of treatments. Therefore, considering these points, those of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

On the other hand, the term "prevention" as used in the present invention means any action of suppressing or delaying a disease by administering a composition containing the mixture of the present invention. In addition, the term "treatment" as used in the present invention refers to any action in which symptoms of a disease are improved or cured by administration of a composition comprising a mixture of the present invention.

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

The pharmaceutical composition according to the present invention can effectively prevent, delay, ameliorate or treat colitis, particularly inflammatory bowel disease (IBD) or irritabel bowel syndrome (IBS), as well as high doses of sulfasalazine. (sulfasalazine) The risk of side effects caused by long-term administration is remarkably low, so it can be very useful in developing colitis prevention or treatment.

1 is a result showing the change in body weight of each administration group at the start and end of the experiment.
2 is a graph (A) or visual observation (B) by measuring the length of the large intestine of an animal at the end of the experiment.
3 is a view showing a graph of quantifying the expression of oxidative stress-related proteins (NOX4, Rac1, and p47 ^phox ) in colon tissue after Western analysis.
4 is a view showing a graph of quantifying the expression of inflammation-related proteins (p-ikB and NF-kB) in colon tissues after Western analysis.
5 is a view showing a graph of quantification of the expression of inflammation-related proteins (COX-2, iNOS, TNF-α, and IL-1b) in colon tissue after Western analysis.
6 is a diagram showing a graph showing a quantification of the expression of MCP-1 and ICAM-1 proteins in colon tissue after Western analysis.
7 is a view showing a graph of quantifying the expression of antioxidant-related proteins (SOD-1 and Catalase) in colon tissue after Western analysis.
8 is a diagram showing a graph of quantification of the expression of necrosis-related proteins (Bax and Caspase 3) and anti-necrosis-related protein (Bcl-2) in colon tissue after Western analysis.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, terms or words used in the specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventors appropriately explain the concept of terms in order to explain their own invention in the best way. Based on the principle that it can be defined, it should be interpreted as a meaning and concept consistent with the technical idea of the present invention. Therefore, the configuration of the embodiments described in the present specification is only the most preferred embodiment of the present invention, and does not represent all the technical spirit of the present invention, and various equivalents and modifications that can replace them at the time of application It should be understood that there may be. In addition, throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

Hereinafter, the present invention will be described in detail.

<Test method>

1. Test substance

Dextran Sulfate Sodium (DSS; molecular weight; 36,000-50,000) used in this experiment was purchased from MP Biomedical, and sulfasalazine and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma aldrich (St. Louis, MO). Nitrocellulose membranes were purchased from Amersham GE Healthcare (Little. Chalfont, UK), NOX4, Bac1, p47 ^phox , phosphor-inhibitor of nuclear factor kappa B α ((p-IκB α), nuclear factor-kappa B (NF-κBp65). ), superoxide dismutase (SOD), catalase, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), monocyte chemotactic protein-1 (MCP-1), intracellular adhesion molecule-1 (ICAM-1), tumor Necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 beta), Bax, histone, β-actin and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Mono anti- Caspase-3 (BioVision, Mountain View, CA, USA) Protease inhibitor mixture DMSO, solutionethylenediaminetetraacetic acid (EDTA) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka. Japan), 2′,7 '-Dichlorofluorescein diacetate (DCF-DA) and Dihydrorhodamine 123 were purchased from Molecular Probes (Eugene, OR, USA) and ECL Western Blotting Detection Reagents were purchased from GE Healthcare. The BCA protein assay kit for protein quantification was Thermo Scientific (Rockford). , IL, USA) I bought it at.

2. Manufacturing method of herbal extract

The dermis used in the experiment was extracted using 50% ethanol, and Shiho was extracted with hot water.

Specifically, the dermis was extracted at room temperature by adding 50% ethanol 10 times the weight of the dermis. After that, the 50% ethanol extract was filtered with Whatman (No. 1) filter paper, concentrated under reduced pressure with a rotary evaporator, and stored frozen until the experiment.

Siho was extracted with hot water for 2 hours after adding water 10 times the weight of the siho, heated in a water heater, and after extraction was completed, it was filtered with Whatman (No. 1) filter paper, concentrated under reduced pressure with a rotary evaporator, and then freeze-dried. The powder obtained after drying in (Eyela, model FDU-2000, Japan) was used in the experiment.

3. Establishment of colitis animal model and Experimental group Furtherance

(1) Colitis animal model

Experimental animals were supplied with 8-week-old male BALB/c mice from Orient Bio Co., Ltd. (Seongnam, Gyeonggi-do) and used for experiments after being adapted to the laboratory environment for 1 week. The conditions of the animal breeding room were controlled by a conventional system, with a temperature of 22±2℃, humidity of 50±5%, and a light: dark cycle of 12 hours. The feed was sufficiently supplied with solid feed (over 22.1% crude protein, 8.0% crude fat, 5.0% or less crude fiber, 8.0% or less crude ash, 0.6% or more calcium, 0.4% or more phosphorus, Samyang Corporation, no antibiotics added) and water.

The colitis-inducing group, excluding the normal group, induced ulcerative colitis by supplying mice with water dissolved in 5% DSS for 7 days.

(2) Composition of experimental group

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

1) Normal: A group administered only a solvent to normal animals that did not cause colitis.

2) Solvent-administered group (vehicle): A group in which only a solvent was administered to an animal that caused colitis.

3) 30 Sulfa group: A group administered with sulfasalazine at 30 mg/kg to animals inducing colitis.

4) 60 Sulfa group: A group administered with sulfasalazine at 60 mg/kg to animals inducing colitis.

5) Jinsi 30 + 30 Sulfa group: A group that co-administered 30 mg/kg of dermis and Siho's 5:1 (weight ratio) mixed extract + sulfasalazine 30 mg/kg to an animal model that caused colitis.

6) Jinshi 60 group: a group administered with a mixed extract of dermis and Siho 5:1 (weight ratio) 60 mg/kg to an animal model that caused colitis.

Each test substance was orally administered once daily to the animals classified according to the experimental group. Drug administration was performed for a total of 7 days, and after the experiment was completed, each animal was anesthetized and sacrificed, and organs and blood were removed, and the following experiment was performed.

4. blood Oxidative Stress analysis

The change in the amount of ROS produced in the serum obtained by centrifuging the blood obtained by collecting blood using cardiac puncture on the last day of administration at 4,000 rpm for 10 minutes was analyzed by Ali et al. After mixing 25mM DCFH-DA according to the method of, the calculated value measured for 30 minutes was calculated using a fluorescence photometer using a 530nm emission wavelength and 485nm 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 microscopic evaluation, adipose tissue was cut to separate the middle compartment. This compartment was embedded in paraffin and then fixed in 10% neutral-buffered formalin, cut into 2 mm sections and stained with H/E for microscopic evaluation. The stained slices were observed with an optical microscope and analyzed using software (i-Solution Lite software program, Innerview Co.).

6. Western Blotting

Buffer A (buffer A; 100mM Tris-HCl (pH 7.4), 5mM Tris-HCl (pH 7.5), 2mM MgCl2, 15mMCaCl2, 1.5Msucrose and 0.1M DTT, added protease inhibitor cocktail) to obtain the cytoplasmic protein of colon tissue. And pulverized with a tissue grinder (Bio Spec Product, USA), and then a 10% NP-40 solution was added. After standing on ice for 20 minutes, centrifugation was performed at 12,000 rpm for 2 minutes to separate the supernatant containing cytoplasm. Rinse twice in buffer A with 10% NP-40 added to obtain nuclear protein, and 100 μl of buffer C (buffer C; 50mM HEPES, 50mM KCl, 0.3mM NaCl, 0.1mM EDTA, 1mM DTT, 0.1mM PMSF and 10%). glycerol) was added and resuspended, and vortex was performed 3 times every 10 minutes. After centrifugation at 12,000 rpm for 10 minutes at 4°C, a supernatant containing nuclei was obtained and stored frozen at -80°C. Cytoplasmic proteins NOX4, Bac1, p47 ^phox , phosphor-inhibitor of nuclear factor kappa B α ((p-IκB α), nuclear factor-kappa B (NF-κBp65), superoxide dismutase (SOD), catalase, inducible nitric oxide synthase ( iNOS), cyclooxygenase-2 (COX-2), monocyte chemotactic protein-1 (MCP-1), intracellular adhesion molecule-1 (ICAM-1), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL -1 beta), Bax, histone, and β-actin were electrophoresed (8-13% sodium dodecylsulfate polyacrylamide gel; SDS-PAGE), and the acrylamide gel was transferred to a nitrocellulose membrane. Each primary antibody was treated on the prepared membrane, overnight at 4°C, washed 5 times every 6 minutes with PBS-T, and secondary antibodies used for each treated primary antibody ( After 1 hour reaction at room temperature using PBS-T diluted 1:3000), it was washed 5 times every 6 minutes with PBS-T, and ECL (enhanced chemiluminescence) solution was used by GE Healthcare (Arlington Heights, IL) , USA), Sensi-Q2000 Chemidoc (Lugen Sci Co., Ltd., Seoul, Korea) after confirming the protein expression by sensitization, the band is ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan) ) Was quantified using the program.

7. Statistical processing

All results were expressed as mean±standard error (mean±S.E). For the significance test, a one-way analysis of variance (ANOVA) according to Dunnett's multiple comparison test (SPSS 18.0 for Windows, SPSS Inc., U.S.A.) was performed. When P<0.05, it was judged to have statistical significance.

<Experiment result>

1. Animal weight and colon length

The body weight and colon length during the experiment were measured in FIGS. 1 and 2 below. Using a digital mass meter, body weight was measured at a constant time every day during the process of DSS feeding and composition administration after fasting. As a result of comparing the body weight between the normal group, the DSS control group, the sulfa30 group, the sulfa60 group, and the composition administration group (herbal medicine complex extract + sulfasalazine), the body weight at the end of the experiment in the vehicle control group treated with 5% DSS When looking at the change in weight before this experiment, it was significantly (p<0.001) decreased, but the test substance administration group did not show significance, but the weight loss was suppressed compared to the DSS control group (Fig. 1).

After 7 days of DSS salary, the dissection was performed and the length from the cecum to the rectum was measured and photographed. The results are presented in Figs. 2A (Length of the large intestine) and Fig. 2B (visual pictures). The large intestine (vehicle control) of the mice inducing ulcerative colitis with 5% DSS was significantly shorter than that of the normal group (p<0.001). Although the test substance administration group did not show any significance, it was found that the damage to the colon was improved compared to the vehicle control group.

On the other hand, when sulfasalazine was not administered in combination and only a mixed extract (60 mg/kg) of dermis and siho was administered alone, in order to compare the change in colon length, separate from the above experiment (see Figs. 2, A and B) and the normal group (Normal) , For the solvent-administered group (Vehicle) and Jinsi 60 group, an additional experiment was performed under the same conditions as the above experiment, and after 7 days of DSS salary, the dissection was performed to measure the length from the cecum to the rectum and take a picture. Length) and Fig. 2D (gross photograph). It was found that the group administered with only the mixed extract of dermis and siho without sulfasalazine did not show any significance in the change in the length of the colon compared to the control group, and no clinical signs of improving the inflammation and damage of the colon compared to the control group. Could

As described above, weight loss occurred due to the induction of colitis and the length of the colon was shortened, but administration of the test substance suppressed weight loss compared to the control group, and showed the effect of improving the shortening of the colon length due to damage to the colon.

2. Analysis of oxidative stress-related proteins in colon tissue

To control the Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase ) is a variety of protein enzymes that play a major role in the generation of O ^2- in particular, the cells in one of the ROS that are generated caused by the Ab peptiedes and generates a large amount of ROS, It is well characterized in macrophages. Its major pathological role is degeneration and necrosis in acute and chronic inflammation in inflammatory cells, epithelial cells, collagen, and elastin. NOX4, p47 ^phox , and Rac1 belong to the subunits of NADPH oxidase. Accordingly, the present inventor attempted to evaluate the oxidative stress improvement effect of the composition according to the present invention by measuring the expression levels of the proteins belonging to the subunit of NADPH oxidase in colon tissue.

The results are shown in FIG. 3.

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

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

3. In the colon tissue NF -κB and related inflammatory protein analysis

One of the most common nuclear transcription factors regulating gene expression, including cell differentiation and growth, inflammatory response, and cell adhesion, is NF-kB. NF-kB is normally present in the cytoplasm in an inactivated state by binding with inhibitor kappa B (IkB), which is an inhibitory protein, then rapidly phosphorylates IkB by stimulation of ROS to separate NF-kB from IkB and become an activated free dimer. As it exists, it is transferred to the nucleus. It plays a central role in mediating inflammation and helps regulate the expression of various early reaction genes involved in inflammation. Genes regulated by NF-kB are COX-2 and iNOS, and cytokines such as TNF-a and IL-1b also exist. Accordingly, the present inventors tried to determine how the expression of the proteins that mediate inflammation is changed by the treatment of the composition according to the present invention.

The results are shown in FIGS. 4 and 5.

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

As shown in Figure 5, the expression of COX-2, iNOS, TNF-α, and IL-1 b in the colon tissue of the vehicle control group was significantly increased compared to the normal group, but the expression of the proteins due to administration of the composition according to the present invention It was confirmed that this remarkably decreased. On the other hand, the effect of the composition according to the present invention was found to be significantly superior to sulfasalazine 60 mg / kg.

Through these results, it was found that the composition according to the present invention prevented the decrease in the expression of p-IκB in the colon tissue and showed an anti-inflammatory effect through inhibition of the activity of NF-κBp65. In addition, 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 the intranuclear migration of NF-κBp65. Was judged. The effect of the composition according to the present invention was much better than that of sulfasalazine 60 mg/kg, and it was confirmed that the mixed herbal extract and sulfasalazine exhibited a synergistic effect.

4. In the colon tissue MCP -1 protein expression analysis

MCP-1 mediates the release of monocytes and macrophages from blood vessels and attachment to target organs. MCP-1 plays a very central role in many inflammatory conditions, and is known to regulate recruitment of these cells by being expressed in various inflammation-related cells such as monocytes and macrophages. On the other hand, the degree of ICAM-1 expression in endothelial cells increases with endothelial cell damage. ICAM-1 is a cell surface glycoprotein that regulates the interaction with immune cells and is known to be upregulated at the site of inflammation. Accordingly, the present inventors tried to confirm how the expression of the proteins is changed by the treatment of the composition according to the present invention.

As shown in Figure 6, it was confirmed that the expression of MCP-1 and ICAM-1 was significantly increased in the vehicle control group compared to the normal group, and the expression level was reduced to the level of the normal group in the composition administration group according to the present invention. I could confirm that. It was confirmed that this effect of the composition according to the present invention is equivalent to or greater than that of the sulfasalazine 60 mg/kg treatment group.

5. Analysis of expression of antioxidant protein in colon tissue

The increase in free radicals including O ^{2 -} produced by ROS accumulates in large amounts even in cells. SOD interacts with superoxide radicals to form hydrogen peroxide (H2O2), which is metabolized to water by catalase. Catalase is a heme protein that protects tissues from the harmful effects of highly reactive hydroxyl radicals. Accordingly, the present inventor attempted 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. 7.

As shown in FIG. 7, the vehicle control group significantly reduced the expression of antioxidant proteins in the colon tissue compared to the normal group, and the composition-treated group according to the present invention showed a pattern in which the expression of the proteins recovered to the level of the normal group. . On the other hand, this effect of the composition according to the present invention was found to be equal to or more than sulfasalazine 60 mg / kg.

6. Analysis of expression of necrosis-related proteins in colon tissue

It is known that when tissue necrosis occurs, the expression of proteins such as Bax and Caspase 3, which are necrosis factors, increases in the corresponding tissue, and the amount of bars of Bcl-1, an anti-necrosis protein, decreases. Accordingly, the present inventor attempted to determine how the expression of necrosis-related proteins in colon tissues changes by treatment with the composition according to the present invention.

The results are shown in FIG. 8.

As shown in FIG. 8, in the Vehicla control group, the expression of necrosis-related proteins (Bax, Caspase 3) was significantly increased compared to the normal group, but the effect of reducing the expression of the protein by treatment with the composition according to the present invention Appeared. In addition, in the case of Bcl-2, an anti-necrosis-related protein, the vehicle-administered group was significantly reduced compared to the normal group, but the protein expression level showed a tendency to recover again in the group administered the composition according to the present invention. On the other hand, it was confirmed that this effect of the composition according to the present invention is equal to or higher than that of the sulfasalazine 60 mg/kg treatment group.

As described above, it was found that the composition containing the mixed herbal extract + sulfasalazine according to the present invention has remarkably excellent preventive or therapeutic effects of colitis compared to the same dose of sulfasalazine alone treatment group. That is, it was confirmed that, according to the combined administration of both Western and oriental medicine, it was possible to reduce side effects due to the administration of high doses of the Western medicine and at the same time exert an excellent therapeutic effect for colitis. It is the first disclosure through invention.

Preferred embodiments of the present invention described above are disclosed to solve the technical problem, and those of ordinary skill in the art to which the present invention belongs will be able to make various modifications, changes, additions, etc. within the spirit and scope of the present invention. , Such modifications and changes should be viewed as falling within the scope of the following claims.

Claims (6)

Mixed extract of dermis and shiho; And sulfasalazine (Sulfasalazine) or a pharmaceutical composition for the prevention or treatment of colitis consisting of a pharmaceutically acceptable salt thereof.
The composition of claim 1, wherein the extract is extracted with any one solvent selected from the group consisting of water, 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 siho: dermis 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 1:0.5-3.
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 of claim 5, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's Disease.

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