KR20170011236A - Pharmaceutical composition comprising artemisia sp extract or eupatilin for prevention or treatment of ischemia reperfusion injury - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for prevention and treatment of ischemia-reperfusion injury comprising as an active ingredient, ephathiline or an extract of mugwort containing it. The present invention also relates to a pharmaceutical composition for prevention and treatment of ischemia-reperfusion injury comprising as an active ingredient a pharmaceutical salt of phytheline or a pharmaceutically acceptable salt thereof. Which can be useful for the prevention or treatment of ischemia-reperfusion injury by mitigating tissue damage and protecting cells from inflammation and cytotoxicity, which are frequent complications after temporary blood closure during organ transplantation such as kidney transplantation or surgical treatment, ≪ / RTI >

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating ischemic reperfusion injury comprising, as an active ingredient,

The present invention relates to a pharmaceutical composition for prevention and treatment of ischemia-reperfusion injury or damage to organs such as kidney or heart, which comprises extract of Artemisia sp. Containing Eupatilin compound or oil tillin as an active ingredient will be.

During surgical procedures such as organ transplantation and surgery for the treatment of cardiovascular diseases, blood supply to specific tissues can be restricted. In such cases, blood circulation is blocked in organs that require blood supply such as heart, brain, and kidney Ischemic damage usually occurs according to. In addition, if the blood flow is suddenly increased in the ischemic state in which oxygen is not supplied by ischemic reperfusion, a lot of activated oxygen or a strong oxidative substance derived therefrom may be generated and the cells and tissues may be damaged. Renal Ischemia Reperfusion Injury (IRI) is a major complication following renal transplantation. After renal transplantation, the subsequent reperfusion after the blood supply to the kidney is temporarily stopped induces an acute inflammation response and oxidative tissue damage (JV Bonventre, A. Zuk. Ischemic acute renal failure: an inflammatory disease, Kidney Int, 2004; 66: 480-5). In particular, tubular cell apoptosis caused by acute inflammatory reaction or oxidative damage is one of the major causes of renal failure (R. Bonegio, W. Lieberthal, Role of apoptosis in the pathogenesis of acute renal failure. Opin Nephrol Hypertension 2002; 11: 301-8). Renal ischemia-reperfusion injury (IRI) causes complex interactions between biochemical, cytological, vascular endothelial, and tissue-specific factors, such as ischemia, Although apotosis and subsequent restoration are induced, restoration is important or paradoxical to potentially induce inflammatory reactions to prevent further damage, further exacerbating the damage already occurring (Z. Aydin, AJ van Zonneveld , JW de Fijter, et al., New horizons in prevention and treatment of ischaemic injury to kidney transplants, Nephrol Dial Transplant 2007; 22: 342-6). Ischemic reperfusion injury may occur as sudden increase in intracellular calcium concentration due to sudden increase in blood flow in oxygen deficiency state. Increased intracellular calcium may mediate damage to mitochondria, where ATP reacts with the released material to produce free radicals (oxygen radicals), which are recognized by the body as inflammation and attack by white blood cells Many active oxygen can be generated and lead to cell damage.

Ischemic reperfusion injury is more likely to occur when blood is reintroduced at a faster rate, particularly when ischemic reperfusion injury occurs when blood flow resumes after cardiac surgery or organ transplant surgery. Although it has been reported that strong antioxidants can be prevented by pre-administration of strong antioxidants when such ischemic reperfusion injury is predicted, strong antioxidants have been used limitedly due to limitations in effectiveness against toxicity or prevention of specific tissue damage, However, there has been no development of a drug that directly prevents or treat ischemic reperfusion injury. However, there has been no development of a drug that directly prevents or treat ischemic reperfusion injury. In particular, inflammation and tubular cell necrosis after ischemia-reperfusion injury (IRI) It is necessary to develop a new method for restoring the function of the kidney while suppressing the function of the kidney.

On the other hand, mugwort has been widely used as a medicine for moxibustion in the past, and has been widely used as a medicine for moxibustion (Leptospira) , Have been used in the treatment of liver disease. Recently, stilene tablets containing ethanol as a main ingredient have been developed and marketed as medicines for the treatment of gastrointestinal diseases. Artemisia argyi , A. princeps , A. asiatica or A. montana have been mixed with mugwort, which is mainly used as a leaf , and Artemisia iwayomogi) and sacheolssuk (A. capillaris), Firefly wormwood (A. annua), jebissuk (A. japonica) also each injinho, Chungho, such as lagoons to come is important to use traditional medicines Artemisia Mugwort plants belonging to the genus have been utilized as important plant resources as raw materials for moxibustion, food and traditional medicinal materials. In the literature on the physiological activity of the extracts of mugwort plant extracts, kidney toxicity of Artemisia deserti has been reported (Avicenna J. Phytomed 2014, 4 (6), 371-376), Artemisia pallens (Pharm Biol. 2015, 53 (4), 571-581), Artemisia indica , A. campestris , and A. afra have been reported to mitigate kidney damage by acetaminophen overdose of methanol extract Diabetic kidney damage mitigation efficacy of the extracts has been reported (J. Ethnopharmacol. 2014, 151 (1), 618-623; Pathol Res Pract. 2012, 208 (3), 157-162; Evid Based Complement Alternat Med, 2013, 2013 , 990 (7), 101486), A. campestris has been reported to be effective in the prevention of oxidative tissue damage induced by administration of galactose Have been reported (Gen Physiol Biophys, 2013, 32 (4), 577-588). However, the effect of preventing and treating ischemia-reperfusion renal damage in extracts of Artemisia princeps including the leaves has not been known so far, and it has been known that epothilamine, which is an active ingredient contained in the mugwort extract used as a leaf, The present inventors completed the present invention by first confirming.

Korean Registered Patent No. 761,705

The inventors of the present invention have found that it is possible to prevent renal tubular cell necrosis and to inhibit the inflammatory reaction while extracting toxic natural substance-derived substances, resulting in prevention of ischemia-reperfusion injury or recovery of inflammatory response and renal function after ischemia-reperfusion injury The present invention has been completed by confirming a substance or a composition derived from a natural product for a new use.

Accordingly, another object of the present invention is to provide a pharmaceutical composition for prevention and treatment of ischemia-reperfusion injury comprising as an active ingredient a pharmaceutical salt of epothilamine or a pharmaceutically acceptable salt thereof.

It is also an object of the present invention to provide a pharmaceutical composition for preventing and treating ischemia-reperfusion injury comprising Artemisia sp. Extract containing epothiline as an active ingredient.

Another object of the present invention is to provide a composition for health functional foods for prevention and improvement of ischemia-reperfusion injury diseases including oil tillin.

Another object of the present invention is to provide a composition for health functional foods for prevention and improvement of ischemia-reperfusion injury diseases including Artemisia sp. Extract containing oil tillin .

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing and treating ischemia-reperfusion injury comprising mugwort extract as an active ingredient.

In one embodiment of the present invention, the extract may be extracted with an organic solvent which is water, a C1 to C4 alcohol, or a mixture thereof.

In one embodiment of the present invention, the C1 to C4 alcohols may be one or more selected from the group consisting of methanol, ethanol, propanol, isopropanol, and butanol.

In one embodiment of the present invention, the ischemia-reperfusion injury occurs after the occlusion of the artery, or may be an ischemia-reperfusion injury resulting from transient closure of the vessel in a surgical procedure.

In one embodiment of the present invention, the tissue to be damaged may be at least one of kidney, heart, liver, or intestine.

In one embodiment of the present invention, the ischemia-reperfusion injury may be acute ischemia-reperfusion injury generated after renal transplantation.

In one embodiment of the invention, the composition may be to block cellular necrosis by increasing the expression of the HO-1 protein.

In addition, the present invention provides a pharmaceutical composition for preventing and treating ischemia-reperfusion injury comprising as an active ingredient a pharmaceutical salt of phytolin or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, the oil palate may be extracted from Artemisia .

In one embodiment of the present invention, the ischemia-reperfusion injury occurs after occlusion of the artery temporarily or may be an ischemia-reperfusion injury generated after organ transplantation.

In one embodiment of the present invention, the organ may be any of the following: kidney, heart, liver or bowel.

In one embodiment of the present invention, the injury caused by the organ transplantation may be an acute ischemia-reperfusion injury resulting from the transplantation of the kidney.

In addition, the present invention provides a composition for health food for preventing and improving ischemia-reperfusion injury disease comprising mugwort extract or milk tilline as an active ingredient.

In one embodiment of the present invention, the < RTI ID = 0.0 > yaffatiline < / RTI >

The present invention relates to a pharmaceutical composition for preventing or treating kidney IRI by suppressing an inflammatory reaction, reducing tubular necrosis and protecting cells from cytotoxic effects, The composition of the present invention containing the active ingredient can be usefully used as a preventive or therapeutic agent for renal diseases due to acute ischemia-reperfusion injury.

1 is a schematic representation of the experimental design of Example 1. Fig.
FIG. 2 shows the effect of mugwort extract on ischemia-reperfusion injury (IRI) as a numerical value of the following main factors. (a) serum creatinine, (b) blood urea nitrogen (BUN), and (c) tubular damage score.
FIG. 3 shows the effect of mugwort extract on ischemia-reperfusion injury (IRI) in comparison with the gastric surgery group and the solvent alone group. (a) Immunostaining of HO-1, (b) HO-1 expression.
Figure 4 shows the efficacy of the mugwort extract to attenuate iNOS and cell necrosis induced by IRI. (a) Western blot results and graphs of iNOS, (b) Western blot results and graphs of Bcl-2.
Figure 5 shows the effect of isofilament on ischemia-reperfusion injury (IRI) by numerical values of the following main factors: (a) neutrophil gelatinase-associated lipocalin (NGAL), (b) kidney injury molecule-1 KIM-1), (c) serum creatinine, and (d) blood urea nitrogen (BUN).
* Sham (fake surgery group), IRI (solvent alone treatment group) and Eupatilin (yupatiline treatment group)
FIG. 6 shows the result of staining tissues with H & E to evaluate the efficacy of mitomycin C for the treatment of renal tissue injury-induced damage. (a) immunohistochemical staining photographs, (b) tubular damage scores.
* Sham (fake surgery group), IRI (solvent treatment group) and Eupatilin (treatment group)
Figure 7 shows data on the expression of HSP70 protein in the kidney. (a) immunohistochemical staining photographs, (b) Western blot results and graphs of HSP70.
* Sham (fake surgery group), IRI (solvent treatment group) and Eupatilin (treatment group)
Figure 8 shows data on the expression rates of various factors in the kidney. Western blot results and graphs of (a) iNOS, (b) Western blot results and graphs of Bcl-2, (c) Western blot results and graphs of BAX, (d) Western blot results and graphs of Cleaved caspase 3.
* Sham (fake surgery group), IRI (solvent treatment group) and Eupatilin (treatment group)

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition comprising Artemisia sp. ) Extract as an active ingredient for the prevention and treatment of ischemia-reperfusion injury. In terms of pharmacological activity of mugwort, it is known that it has anticancer activity, inhibition of platelet aggregation, and antifungal activity. In the present invention, the mugwort extract or mugwort may be commercially available, purchased from nature or cultivated.

Preferably, the ischemia-reperfusion injury according to the present invention occurs after a temporary occlusion of an artery or an ischemia-reperfusion injury resulting from transient closure of a blood vessel in an organ transplantation, and the target organ is a kidney, heart, It can be either. In particular, the present invention provides a method for treating acute renal ischemia reperfusion injury renal injury.

As used herein, the term " extract " means that it is used in the art as a crude extract as described above, but broadly includes fractions obtained by further fractionating the extract. That is, the mugwort extract includes not only those obtained by using the above-mentioned extraction solvent, but also those obtained by further applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained by the purification method is also included in the mugwort extract of the present invention.

The extract may be extracted with a solvent which is water, a C1 to C4 alcohol, or a mixture thereof. Preferably, a polar solvent or a non-polar solvent may be used. Suitable polar solvents are (i) water, (ii) alcohols (methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol) , (iii) acetic acid, (iv) dimethyl-formamide (DMFO), and (v) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- But are not limited to, pentane, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2- chloropropane, toluene, 1- chloropropane, chlorobenzene, benzene, diethyl ether, diethylsulfide, Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF. More preferably, the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane, and more preferably from isopropanol or ethanol.

The mugwort extract may be, but is not limited to, those produced by a process comprising the following steps:

1) extracting the mugwort seedlings or the top part by adding an extraction solvent;

2) cooling the extract of step 1) and filtering; And

3) Concentrating the filtered extract of step 2) under reduced pressure and drying.

In the above method, the mugwort of step 1) may be used without limitation such as cultivated or commercially available. The mugwort may be, but not limited to, an outpast of the Artemisia plant containing the oil tillin as an ingredient, or a top or bottom portion of the Artemisia plant.

The mugwort may be extracted by a conventional method such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, pressure extraction, subcritical extraction, supercritical extraction, and ultrasonic extraction. To 5 times, and more specifically, may be repeated three times. However, the present invention is not limited thereto. The extraction solvent may be added to dried mugwort 0.1 to 10 times, preferably 0.3 to 5 times. The extraction temperature may be 20 to 40, but is not limited thereto. In addition, the extraction time may be 12 to 48 hours, but is not limited thereto.

In this method, the vacuum concentration of step 3) may be by using a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. The drying may be, but not limited to, vacuum drying, vacuum drying, boiling, spray drying or lyophilization.

The present invention also provides a pharmaceutical composition for the prevention and treatment of ischemia-reperfusion injury comprising eupatilin as an active ingredient.

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is a compound represented by the following formula (1).

≪ Formula 1 >

Yaffa tiline is a species of Artemisia sp. Such as Artemisia argyi , A. princeps , A. asiatica or A. montana . As a component contained in plants, it is preferable that the present tilapine is extracted or isolated from Artemisia sp. Extracts for the separation of the < RTI ID = 0.0 > lymphocytes < / RTI > can be extracted from various organs of natural, hybrid, and variant plants and extracted from plant tissue cultures as well as roots,

In the present invention, ischemia-reperfusion injury refers to a disease caused by damage caused by any one of the following ischemic diseases.

1) acute myocardial ischemia;

2) central nervous system (CNS) ischemia due to thrombosis or embolism or cardiac arrest in the intracranial artery, accompanied by a permanent or temporary interruption of blood flow in some cerebral artery regions or the entire brain;

3) a surgical procedure that temporarily closes any arterial portion as in renal cancer resection;

4) in vitro culture, preservation and re-transplantation procedures for organs that require transplantation such as kidney, heart, liver, or bowel;

5) all other ischemic diseases characterized by reduced blood flow, interruption and restoration of oxygen / nutrient input to subsequent tissues;

6) hypoxic state of brain tissue capable of restoring normal oxygenation concentration according to subsequent regular medical treatment such as carbon monoxide poisoning or drowning;

7) other tissue damage leading to a time of severe anatomical and functional lesions with hypoxia, ischemia, or trauma that may cause death by cell suicide or child action;

8) Brain damage due to chronic trauma (CTE).

Preferably, the ischemia-reperfusion injury according to the present invention is an ischemia-reperfusion injury generated after temporal occlusion of an artery or a temporal occlusion of a blood vessel, and the organs may be any one of a kidney, heart, . In particular, the present invention is a major target disease of renal ischemia reperfusion injury renal injury occurring after renal transplantation or surgical operation.

The inventors of the present invention have demonstrated the protective effect of the mugwort extract against ischemia-reperfusion injury through the following examples. The administration of Mugwort extract decreased the blood urea nitrogen (BUN) level, serum creatine level and renal tubular injury in the blood. Western analysis showed that the mugwort extract significantly increased the expression of HO-1 and the expression of B-cell lymphoma protein (Bcl-2). In addition, the expression level of inducible oxidase (iNOS) was decreased after 48 hours of IRI. HO-1 is an enzyme with cytoprotective effect in response to oxidative stress. Its major function is to break down Heme into biliverdin, iron and cannon monoxide. Overexpression of HO-1 results in a marked decrease in cytotoxicity and damage induced by oxidative stress. Mugwort extract increases renal function and prevents cell necrosis by increasing HO-1 protein expression. Therefore, one of the main active mechanisms of the protective effect against the IRI damage of the mugwort extract of the present invention is the activity of inducing the expression of HO-1 in the mugwort extract and the major components contained therein.

In addition, the present inventors have demonstrated the protective effect of oil tillin against ischemia-reperfusion injury through the following examples. The use of tiline significantly reduced the levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in the urine, and the blood urea nitrogen (BUN) and serum creatinine levels and renal tubular injury. Western analysis showed that the presence of inducible oxidase synthase (iNOS) and Bcl-2-associated X (Bcl-2) significantly increased the levels of heat shock protein 70 (HSP 70) and B- protein (BAX) and caspase-3 levels. In addition, the administration of yupatiline showed renal protection effect on kidney IRI through inhibition of inflammation and inhibition of tubular necrosis.

From the above test results, it was proved that the mugwort extract and the milk tilline showed the kidney protective effect without harmful action in the animal model of the kidney IRI, and the kidney function was remarkably improved in the mugwort extract or the oil tilline treated animals compared with the control group . Influences of ITP on IGF-I and IGF-I levels were not statistically significant. In addition, the effect of bovine tiline on renal ischemia-induced tubular cell necrosis is very effective. The effect of Bcl-2 on the expression of Bcl-2 is directly influenced by kidney epithelial cells at the mitochondrial level, Bcl-2 upregulation and downregulation of BAX and caspase-3 protein expression showed cytoprotective activity.

HSPs can constitute a family of stress proteins and exhibit cytoprotective effects against the external environment that causes cell disorders such as IRIs. HSP70 (molecular weight: 70) is a molecule that protects against oxidative stress and acts as a protector of antioxidants. HSP70 exhibits anti-cell necrosis activity through down regulation of BAX gene expression, and overexpression of HSP70 in cells Is known to induce a marked reduction in stress induced disorders, cell necrosis and cytotoxicity. Overexpression of HSP70 was observed in the troponin-treated group and renal IRI was significantly restored, which suggested that HSP70 was a regulator of the activity of the associated tropamin-associated cytotoxic activity.

Therefore, as described above, the mugwort extract or milk tilline inhibits inflammation, reduces tubular necrosis and cytoprotective activity, resulting in protection of body tissue and life from kidney IRI.

The mugwort extract of the present invention or milk tilline is contained as an active ingredient in an amount of 0.1 to 99.9% by weight based on the total weight of the composition of the present invention, and may include a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The present invention also provides a method for treating ischemia-reperfusion injury disease comprising administering a pharmaceutically effective amount of the composition according to the present invention to an individual suffering from ischemia-reperfusion injury.

The ischemia-reperfusion injury is an ischemia-reperfusion injury occurring after temporary occlusion of an artery or ischemia-reperfusion injury resulting from temporary occlusion of a blood vessel in organ transplantation, and the organ is one or more selected from the group consisting of kidney, heart, But not limited to.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, and 0.001 to 10 mg / kg, but is not limited thereto. The dose may vary depending on the weight, age, sex, health condition, diet, administration period, method of administration, rate of elimination, severity of disease, etc. of the particular patient.

The composition can be administered orally or parenterally at the time of clinical administration and can be administered orally or parenterally in the case of parenteral administration by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine injection, intracerebral injection, And may be used in the form of a general pharmaceutical preparation.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The subject is a vertebrate animal, specifically a mammal, and more specifically, an experimental animal such as a mouse, a rabbit, a guinea pig, a hamster, a dog, or a cat, and more specifically an ape-like animal such as a chimpanzee or a gorilla have.

In the present invention, "administering" means introducing a predetermined substance into a patient in any appropriate manner, and the administration route of the substance can be administered through any conventional route so long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal. In addition, the pharmaceutical composition may be administered by any device capable of moving the active substance into the target cell.

In addition, the present invention provides a composition for health food for preventing and improving ischemia-reperfusion injury diseases including mugwort extract.

In addition, the present invention provides a composition for health food for preventing and improving ischemia-reperfusion injury disease containing as an active ingredient, It can be extracted from various organs of natural, hybrid, and variant plants and extracted from plant tissue cultures as well as roots, shoots, stems, and leaves.

In the present invention, ischemia-reperfusion injury (IRI) refers to a disease caused by damage caused by any one of the following ischemic diseases.

1) acute myocardial ischemia;

2) central nervous system (CNS) ischemia due to thrombosis or embolism or cardiac arrest in the intracranial artery, accompanied by a permanent or temporary interruption of blood flow in some cerebral artery regions or the entire brain;

3) a surgical procedure that temporarily closes any arterial portion as in renal cancer resection;

4) in vitro culture, preservation and re-transplantation procedures for organs that require transplantation such as kidney, heart, liver, or bowel;

5) all other ischemic diseases characterized by reduced blood flow, interruption and restoration of oxygen / nutrient input to subsequent tissues;

6) hypoxic state of brain tissue capable of restoring normal oxygenation concentration according to subsequent regular medical treatment such as carbon monoxide poisoning or drowning;

7) other tissue damage leading to a time of severe anatomical and functional lesions with hypoxia, ischemia, or trauma that may cause death by cell suicide or child action;

8) Brain damage due to chronic trauma (CTE).

Preferably, the ischemia-reperfusion injury according to the present invention is an ischemia-reperfusion injury occurring after temporal occlusion of an artery, or an ischemia-reperfusion injury resulting from temporary occlusion of a blood vessel during organ transplantation or surgical treatment, Heart, liver or bowel. Particularly, the present invention is an injury resulting from renal ischemia reperfusion injury caused by renal transplantation.

The functional food of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be selected from the range of 0.01 to 0.04 part by weight, specifically about 0.02 to 0.03 part by weight per 100 parts by weight of the health food of the present invention.

In addition to the above, the functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the functional food of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

Best Mode for Carrying Out the Invention Hereinafter, the functions and effects of the present invention will be described in more detail through specific embodiments of the present invention. It is to be understood, however, that these embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention.

≪ Example 1 >

Kidney in Animal IRI cause

8 week old male C57BL / 6 mice (body weight: 22-25) were purchased from DaeHanBioLink Co., Ltd, Eumseong, Korea. All experiments complied with the guidelines for the use and care of laboratory animals issued by the NIH (Publication No. 85-23, Rev. 1985) and were approved by the Ethics Committee of KIST (Gangneung, Korea). Mice are in standard laboratory conditions (temperature 22 ° C ± 2 ° C, relative humidity 55%). The animals were fed with basic mouse feed and tap ad libitum . And clamped on both renal pedicle with microvascular clamp for 30 minutes. The reperfusion began after the vessel clamp was removed. Occlusion and referpusion were identified as changes in kidney color and were observed to change to a slightly paler shade or slightly red blush. During the process, the animals were given a good supply of water with warm saline. A warm pad was used to maintain a constant body temperature (37 ° C). Renal reperfusion was observed after the clamp was removed. The sham operation was also performed in a similar fashion except for the extension clamp. Blood and kidney tissues were collected 48 hours after reperfusion. Both kidneys were isolated and frozen in nitrogen solution and stored at -80 ° C until further analysis. Blood samples were collected from the lower vena cava (Figure 1).

Experimental group of aeyeop extract to animals, comparison of the sham surgery group and the control group

The isopropanol extract of the leaf plants (Richwood Trading Co., Ltd., Seoul, South Korea) was dissolved in an organic solvent of 5% hydroxypropyl methylcellulose. Thirty animals were randomly divided into three groups of 10 animals each. False surgical sham and IRI groups (control, solvent treated group) were administered only once per day. The drug treatment group (lady extract) was administered intragastrically once daily (100 mg / kg body weight (BW)) 4 days before IRI with a solution containing a lye extract.

Numerical measurement in serum

Blood samples were collected from the inferior vena cava. Blood urea nitrogen (BUN) and serum creatinine levels were measured with a Kovas C702 analyzer (Roche, Germany). Data are expressed as means ± SD (* P < 0.05 Aetemisia vs. solvent treated group (IRI), sham operation group (sham)

In solvent-treated mice (IRI group), IRI caused kidney dysfunction. After 2 days of IRI, BUN and serum creatinine levels were significantly elevated (FIG. 2). On the other hand, after 2 days of IRI, BUN and serum creatinine values were lower in comparison to the control group, and renal dysfunction was weakened in the aflatoxin group (Fig. 2).

Histological and immunohistochemical analysis

For histopathological examination, the kidneys administered in the animal experiment of Example 1 were collected, cut into tubules, fixed in 10% formalin and embedded in paraffin. A 5 μm cut was prepared and stained with hematoxylin and eosin (H & E). To observe changes in kidney at 48 hours after IRI, the cut pieces were measured using a semi quantitative scale specially designed for this purpose. The ratio of tubules within the corticomedullary junction showing loss of cell necrosis and brush border was measured on a scale of 1 to 4. One whole deep coronal fragment was observed under a microscope and graded according to the extent of tubular necrosis based on the proportion of kidney tissue. Higher scores indicate more serious damage. (4 points = maximum value, 0 = normal height, 1 = minimal cell necrosis, <5% relativity, 2 = weak cell necrosis, 5% to 25% relativity, 3 = severe cell necrosis, 25 to 75% relativity ; And 4 = severe cell necrosis,> 75% relativity).

In the results, the lobule extract weakened the histological damage. As shown in FIG. 3, the IRI group demonstrated a wide range of tubular necrosis within the corticomedullary junction after 2 days of IRI and loss of brush board, cast formation, and tubular dilation (Fig. 3, p < 0.05), while the severity of tubulointerstitial injury was as shown in Fig. No tubular injury was observed in the fake surgery group (Fig. 3).

Analysis of protein expression

The kidneys were washed twice with ice buffer 1M Tris-HCl buffer, pH 7.5, protease inhibitor, 25 mM sodium fluoride, 10 mM sodium Orthovanadate, 0.5 mol / L ethylenediaminetetraacetic acid (EDTA) 1% Triton X-100; GenDEPOT, Barker, TX, USA) and then centrifuged at 14,000 rpm for 20 minutes. Protein concentrations were measured using a Bradford protein assay (Bio-Rad, Hercules, USA). 20 μg of the protein extract was separated from 10% to 15% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE; Bio-Rad, USA) and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, USA). Membranes were sealed with 5% milk in Tris-buffered saline and Tween 20 (TBST) buffer (10 mM Tris-base, 100 mM NaCl, and 0.1% Tween-20, pH 8.0) (1: 200 dilution, Santa Cruz Biotechnology, Inc.), glyceraldehyde 3-phosphate dehydrogenase (GAPHD), B-cell lymphoma (Bcl- Santa Cruz, Calif., USA). The membranes were again detected with a goat anti-rabbit (1: 1,000) or goat anti-mouse (1: 1,000) horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA). Protein bands were identified using chemiluminescent Substrate (SuperSignal West Pico Chemiluminescent Substrate, Pierce, Rockford, IL., USA) and blot images were collected for quantitative analysis.

Treatment of the leaf extract increases the expression of the Heme oxygenase-1 (HO-1) protein in the kidney. To elucidate the mechanism of cytoprotective effect induced by the extracts of lobules, we examined the protein expression using immunohistochemical staining and Western blotting. HO-1 is an enzyme that has antioxidant and cytoprotective activity against oxidative stress. Cells treated with lobules showed strong immunohistochemical staining of HO-1 enzyme when compared to cells in the fake surgery group and the IRI group (Fig. 3). In addition, the treatment with lobule extract increased the expression level of HO-1 (Fig. 3b, P < 0.01). This demonstrates that the induction of HO-1 by the leaf extract is correlated with the cytoprotective effect against IRI.

The lobule extract increases the expression of the Bcl-2 protein in the kidney and reduces the expression of the iNOS protein. Treatment of the lobule extract reduced IRI-induced iNOS protein expression (Fig. 4, p < 0.05). Intracellular responses that occur during early cell necrosis involve mitochondrial changes mediated by anti-apoptotic Bcl-2 proteins. Therefore, Bcl-2 expression can be assessed in western blot analysis, which is to determine the mechanism by which the lobule extract inhibits cellular necrosis after IRI. Immunoreactivity of Bcl-2 was remarkably increased after treatment with a lobule extract (Fig. 4, P < 0.05). The density of the Western blot band was quantified by Fujifilm Multi-Gauge Imaging (Fujifilm, Japan) software and normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The data are expressed as mean ± SEM (* P <0.05 lobule extract versus fake surgery group and solvent treated group (IRI))

&Lt; Example 2 >

Kidney in Animal IRI cause

Gt; IRI &lt; / RTI &gt; induction in the animal of Example 1 above.

For experimental animals Yaffatilin  Group of administration, Fake surgery.  And comparison with control group

Yupatiline was obtained from DS Chang (Kyung Hee University, Korea) and dissolved in 5% hydroxyl methylcellulose solvent. Thirty animals were randomly divided into three groups of 10 animals each. Fake surgery group and IRI group (control group, solvent treatment group) were administered only once a day with solvent. The drug treatment group (tropamine) was intragastrically administered once daily (10 mg / kg, body weight (BW)) 4 days before IRI with a solvent containing empirical tiline. It protects the kidney function of ischemic reperfusion injury (IRI).

Serum and urine NGAL  And KIM -1 numerical measurement

Blood samples were separated from the lower vena cava and blood urea nitrogen and serum creatinine levels were measured with a Kobas C702 analyzer (Roche, Germany). The urinary neutrophil gelatinase-associated lipocalin (NGAL) and renal molecule-1 (KIM-1) levels were measured by ELISA (R & D Systems, USA) according to the manufacturer's guidelines. Urine samples were collected at 6 hours and 1 day after IRI, and urine neutrophil gelatinase-associated lipocalin (a) and kidney injury molecule-1 (KIM-1) (b) levels were measured. Blood samples were collected two days after IRI and serum creatine (c) and blood urea nitrogen (BUN) (d) levels were measured. Data are presented as means ± SD (* P &lt; 0.05 yfastiline vs. solvent treated group (IRI))

As shown in FIG. 5, in the solvent treated mice (IRI group), IRI caused renal dysfunction. The urinary NGAL and KIM-1 levels significantly increased after 6 hours and 24 hours after IRI. After 2 days of IRI, BUN and serum creatinine levels were significantly elevated. Conversely, kidney dysfunction was attenuated in the efalathrin group. After 2 days of IRI, BUN and serum creatinine values were lower than control. (P <0.05, Fig. 5b, KIM-1 p <0.05, Fig. 5c, BUN p <0.05, Fig. 5d; creatinine p <0.05). As a result, it was found that the administration of the tilapine weakens IRI-induced renal dysfunction. Data were expressed as mean ± SEM and evaluated by one-way analysis of variance (ANOVA) via Bonferroni's post-hoc correction (SPSS software, version 15.0; SPSS Inc, USA). p value <0.5: Significant

Histological and immunohistochemical analysis

For histopathological examination, the kidneys administered in the animal experiment of Example 1 were collected, cut into tubules, fixed in 10% formalin and embedded in paraffin. A 5 μm cut was prepared and stained with hematoxylin and eosin (H & E). To observe changes in the kidney, the cut pieces were measured using a semi quantitative scale specially designed for this purpose. The ratio of tubules within the corticomedullary junction showing loss of cell necrosis and brush border was measured on a scale of 1 to 4. One whole deep coronal fragment was observed under a microscope and graded according to the extent of tubular necrosis based on the proportion of kidney tissue. Higher scores indicate more serious damage. (4 points = maximum value, 0 = normal height, 1 = minimal cell necrosis, <5% relativity, 2 = weak cell necrosis, 5% to 25% relativity, 3 = severe cell necrosis, 25 to 75% relativity ; And 4 = severe cell necrosis,> 75% relativity).

Mouse kidneys were collected 2 days after IRI and evaluated for tubular damage score. The kidneys were cut and stained with hematoxylin and eosin (H & E) to assess renal tubular damage (a, b). Data are presented as means ± SD (* P < 0.05 yfastiline vs. solvent treated group (IRI))

In Fig. 6, the IRI group demonstrated a significant (but not significant) reduction in tubular necrosis and brush board loss, cast formation, and tubular dilation within the corticomedullary junction 2 days after IRI, (Fig. 6, p < 0.05). In contrast, the &lt; RTI ID = 0.0 &gt; paclitaxel &lt; / RTI &gt; No tubular injury was observed in the fake surgery group. The treatment of yupatiline also showed an attenuation of renal dysfunction when observed anatomically. The group treated with the &lt; RTI ID = 0.0 &gt; lymphocytes &lt; / RTI &gt; significantly weakened IRI-induced pathological damage.

Analysis of protein expression

The kidneys were crushed in ice buffer (1M Tris-HCl buffer, pH 7.5, containing protease inhibitor; 25 mM NaF, 10 mM NaV; 0.5 mol / EDTA; and 1% Triton X-100) and incubated at 14,000 rpm for 20 min And centrifuged. Protein concentrations were measured using a Bradford protein assay (Bio-Rad, Hercules, USA). 20 μg of the protein extract was separated from 10% to 15% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE; Bio-Rad, USA) and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, USA). The membranes were sealed with 5% milk in Tris-buffered saline and Tween 20 (TBST) buffer (10 mM Tris-base, 100 mM NaCl, and 0.1% Tween-20, pH 8.0) Bcl-2-associated X protein (BAX), caspase-3, cleaved caspase-3 (Cell Signaling Technology Inc., USA) ) And inducible nitric oxide synthase (iNOS) (1: 1,000 dilution, BD Transduction Laboratory, San Diego, CA, USA). The membranes were again detected with a goat anti-rabbit (1: 1,000) or goat anti-mouse (1: 1,000) horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA). Protein bands were identified using the EZ-Capture ST imaging system (Atto, Japan) and blot images were collected for quantitative analysis.

Treatment with the Ephatonil tillin increases expression of the HSP70 protein in the kidney.

In order to elucidate the mechanism of the effect of the tiline induced cytoprotection, we examined the protein expression using immunohistochemical staining and Western blotting. HSP 70 is an enzyme with antioxidant and cytoprotective activity against oxidative stress. The high expression level of HSP 70 was observed in the kidney cells of the pravastatin group and the fake surgical group (Fig. 7A, B). The lymphocyte-treated cells showed strong HSP70 immunohistochemistry when compared to the cells of the fake surgery group and the IRI group (Fig. 7A). In addition, the treatment with efalil tillin increased the expression level of HSP70 (Fig. 2B, P < 0.05). This shows that induction of HSP70 by epothilone is correlated with cytoprotective effect on IRI. The data are expressed as mean ± SD (* P <0.05 yfastiline vs. fake surgery group and solvent treated group (IRI))

It increases expression of the Bcl-2 protein in the kidney and reduces the expression of iNOS, BAX and caspase-3 proteins.

Treatment of the yupatiline reduced IRI-induced iNOS protein expression (Fig. 8A, p < 0.05). Intracellular responses that occur during early cell necrosis involve mitochondrial changes mediated by anti-apoptotic Bcl-2 proteins. Therefore, Bcl-2 expression can be assessed in western blot analysis, which identifies mechanisms by which ephtaliline inhibits cellular necrosis after IRI. Immunoreactivity of Bcl-2 was significantly increased after treatment with efavatiline (Fig. 8B, P < 0.05). In addition, the efaviren tilline markedly attenuated the levels of BAX and caspase-3 after 48 hours of IRI (Fig. 5C, P < 0.05). The density of the Western blot band was quantified by Atto EZ-capture ST imaging software and normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± SEM (* P <0.05 yfastilin vs. fake surgery group and solvent treated group (IRI)).

&Lt; Preparation Example 1 &

Manufacture of Powder

0.1 g of lobster extract

Lactose 1.5 g

Talc 0.5 g

The above ingredients were mixed and filled in an airtight container to prepare powders.

&Lt; Preparation Example 2 &

Manufacture of tablets

0.1 g of lobster extract

Lactose 7.9 g

Crystalline cellulose 1.5 g

0.5 g of magnesium stearate

After mixing the above ingredients, tablets were prepared by direct tableting method.

&Lt; Preparation Example 3 &

Preparation of capsules

0.1 g of lobster extract

Corn starch 5 g

4.9 g of carboxycellulose

&Lt; Preparation Example 4 &

Manufacture of Powder

0.1 g of &lt; RTI ID = 0.0 &

Lactose 1.5 g

Talc 0.5 g

The above ingredients were mixed and filled in an airtight container to prepare powders.

&Lt; Production Example 5 &

Manufacture of tablets

0.1 g of &lt; RTI ID = 0.0 &

Lactose 7.9 g

Crystalline cellulose 1.5 g

0.5 g of magnesium stearate

After mixing the above ingredients, tablets were prepared by direct tableting method.

&Lt; Production Example 6 &

Preparation of capsules

0.1 g of &lt; RTI ID = 0.0 &

Corn starch 5 g

4.9 g of carboxycellulose

After mixing the above components to prepare a powder, the powder was filled in a hard capsule according to a conventional preparation method of a capsule to prepare a capsule. Each component was dissolved in purified water in accordance with a conventional method for producing a liquid agent, and the lemon flavor was added in an appropriate amount, followed by mixing the above components. Then, purified water was added to adjust the total volume to 100 ml, and the solution was filled in a brown bottle and sterilized to prepare a liquid preparation.

&Lt; Production Example 7 >

 Manufacture of health food

100 mg of yupatiline

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 mg of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

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

&Lt; Production Example 8 &

Manufacture of health drinks

1000 mg of yupatiline

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to the conventional health drink manufacturing method, and the mixture was heated at 85 for about 1 hour with stirring. The solution thus prepared was filtered and sterilized in a 2-liter sterile container. The resulting solution was refrigerated, And used for manufacturing.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (9)

A pharmaceutical composition for preventing and treating ischemia-reperfusion injury comprising Astemisia sp. Extract as an active ingredient. The method according to claim 1,
Wherein the extract is extracted with an organic solvent which is water, a C1 to C4 alcohol or a mixture thereof. 3. The method of claim 2,
Wherein the C1 to C4 alcohol is at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, and butanol. The method according to claim 1,
Wherein said ischemia-reperfusion injury is ischemia-reperfusion injury that occurs after temporary occlusion of the artery or after transient occlusion of the vessel in a surgical procedure. 5. The method of claim 4,
Wherein the tissue to be damaged is at least one of kidney, heart, liver or intestine. 5. The method of claim 4,
Wherein said ischemia-reperfusion injury is an acute ischemia-reperfusion injury occurring after renal transplantation. A pharmaceutical composition for the prevention and treatment of ischemia-reperfusion injury comprising eupatilin or a pharmaceutically acceptable salt thereof as an active ingredient. 8. The method of claim 7,
Wherein said oil tillin is extracted from Artemisia . A composition for a health food for prevention and improvement of ischemia-reperfusion injury diseases containing Artemisia sp. Extract or oil tillin as an active ingredient.

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