KR20190068141A - Composition for preventing and treating liver diseases comprising extract of sargassum serratifolium - Google Patents

Abstract

The present invention relates to a composition for protecting a liver and preventing or treating liver diseases containing an extract Sargassum serratifolium, a fraction thereof, or a compound separated from the same as an active component. The extract of Sargassum serratifolium, the fraction thereof, or the compound derived from the same according to the present invention has an excellent effect of protecting the liver against alcohol or tBHP. Therefore, the composition containing the same is the composition for protecting the liver, and preventing, alleviating or treating liver damage or liver diseases, thereby being able to be usefully used as a material of medicine, health functional food and the like.

Description

TECHNICAL FIELD The present invention relates to a composition for prevention or treatment of liver diseases,

The present invention relates to a composition for prevention or treatment of liver diseases or liver diseases comprising extracts of Tootsum matsumana, fractions thereof or a compound isolated therefrom as an active ingredient.

The liver has a variety of functions such as glycogen storage, red cell degradation, plasma protein synthesis and detoxification, as well as important metabolism in the internal organs. The liver is the primary defensive organ to prevent damage caused by ingestion of foreign substances, which can lead to severe diseases caused by viruses and various drugs due to a decrease in liver function. Drugs that are toxic to the liver are also a major cause of liver disease. About half of all severe liver diseases are caused by drugs and most of them are chronic liver diseases.

Korea is the prevalent region of chronic liver disease. Chronic liver disease among the causes of death among 30 ~ Especially, as the aging society is rapidly progressing, aging of the energy metabolism and toxic metabolite metabolism causes a serious deterioration of body function. Loss of chronic liver function is unrelenting, and it loses protection and detoxification of the human body. Therefore, it is necessary to develop health functional foods with safe and no side effects.

On the other hand, ethanol is a major component of alcohol, and its physical and mental effects on the human body are very diverse, and its metabolism and toxicity expression characteristics have been studied. The ingested ethanol is absorbed through the digestive tract and reaches its peak blood concentration between 20 and 120 minutes after ingestion. Absorbed ethanol is metabolized in all organs, including the liver, and some (about 10%) are excreted through the respiratory or urine and sweat. When a small amount of alcohol is consumed in a normal state, ethanol entering the liver is converted into acetate by the action of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the cytoplasm and is excreted through the circulation system out of the hepatocytes . Acute ethanol administration temporarily causes triglyceride accumulation in the liver. Especially, acetaldehyde, which is the first metabolite of ethanol, is highly reactive and toxic than ethanol, and is known to be the main cause of alcoholic liver damage. It is known that acetaldehyde inhibits mitochondrial respiration, inhibits oxidative phosphorylation, combines with membrane proteins and collagen proteins to produce antibodies, immunologically cytotoxic and inhibits the release mechanism of hepatocyte secretory proteins. In addition, acetaldehyde promotes collagen synthesis of myofibroblast, causing hepatic fibrosis and metamorphosis of hepatocytes, and reacting with macromolecules in vivo to form adducts. Neutral fat accumulates in the liver by ethanol. The hepatic lipid accumulation by ethanol is considered to be an important factor in the accumulation of triglycerides, rather than increasing fatty acid synthesis in the liver. According to animal studies, an increase in serum triglycerides due to alcohol administration is believed to be due to increased production of very low density lipoprotein (VLDL) in the liver. When alcohol is administered to the human body by oral or intravenous injection, the VLDL containing triglycerides in the serum is rapidly increased. In addition, even if chronic alcohol is administered to the human body, the serum triglyceride level is increased according to the amount of alcohol administered and the period of administration. The accumulation of triglycerides in the liver reflects hepatic metabolic disturbances and the persistent accumulation of triglycerides eventually results in fibroblast proliferation and hepatocyte damage. As a result of the metabolism of the above-mentioned alcohol, fatty acids are produced so that liver fat accumulates, which is called an 'alcoholic fatty liver'. This alcoholic fatty liver is particularly a chronic disease, and alcoholic hepatitis occurs in 10 to 35% and liver cirrhosis occurs in 8 to 20%.

In addition, non-alcoholic hepatic injury is characterized by liver changes (fatty change, steatosis) and lobular hepatitis (hepatitis), a characteristic feature of alcoholic hepatitis, steatohepatitis) and the like. The pathologic findings of liver involve various spectrum from Non-Alcoholic Fatty Liver (NAFL) to non-alcoholic steatohepatitis (NASH), fibrosis associated with fatty liver and liver cirrhosis. It is used to mean all inclusive. Most of these nonalcoholic liver diseases are accompanied by insulin resistance, obesity, diabetes, and hyperlipidemia. When such complications exist, it is necessary to first perform the treatment. The principle of treatment for nonalcoholic liver disease is improvement of lifestyle such as meal therapy and exercise therapy, but it is a reality that it is difficult to carry out surely. In nonalcoholic steatohepatitis, a more aggressive medication is needed because it is likely to develop into cirrhosis / hepatocellular carcinoma. Although treatment aimed at improvement of oxidative stress and insulin resistance which is thought to be important in the pathogenesis and progression of nonalcoholic fatty liver disease has been attempted, there is no established scientific method.

On the other hand, domestic liver function improvement agents include curcumin as a main component, Ulkum as a main component, and Milk Thistle as a thistle component based on silymarin. The main components are antioxidant vitamins and flavonoids, and products made from dandelion roots and Hovenia fruit are sold have. In particular, currently, the functional raw materials for the improvement of liver function in the KFDA depend on the import of most of the raw materials by Hovenia dulcis, Shiitake mycelia and Milk Thistle 3 items.

Accordingly, the inventors of the present invention have conducted extensive research to develop a raw material for functional food for liver protection derived from seaweed, and as a result, they have discovered that a merteropinoid component is extracted from the sawtooth moth of the seaweed, and the extract and the merotorpinoid component protect the liver And confirming that the effect is excellent, thereby completing the present invention.

Korean Patent No. 10-2015-0182907

Accordingly, an object of the present invention is to provide a pharmaceutical composition or composition for liver protection comprising an extract of Tooth mothballs, fractions thereof or a compound isolated therefrom as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition or food composition for preventing, ameliorating or treating liver diseases comprising extracts of Tootsum matsumai, fractions thereof or a compound isolated therefrom as an active ingredient.

The present invention provides a pharmaceutical composition for liver protection or a food composition comprising an extract of Tooth mothballs, a fraction thereof or a compound isolated therefrom as an active ingredient.

The present invention also provides a pharmaceutical composition or food composition for preventing, ameliorating or treating liver diseases comprising extracts of Tootsumabum, its fractions or a compound isolated therefrom as an active ingredient.

The Sargassum serratifolium extract according to the present invention, its fractions and the compounds derived therefrom are excellent in the effect of protecting hepatocytes against alcohol or tBHP, and thus a composition comprising the extract as an active ingredient can be used for preventing liver damage, As a composition for prevention, improvement or treatment, as a material for medicines, health functional foods and the like.

Fig. 1 shows the results of four fraction chromatograms obtained from fractions of Sawtooth mildew extract.
FIG. 2 is a diagram showing the results of a second fraction chromatogram of the fraction No. 1 among the four fractions obtained from the fraction of Sawtoombaeta extract. FIG.
FIG. 3 is a graph showing the chromatogram results of sagahydroquinoic acid identified by NMR analysis by purifying a fraction obtained by fractionating the first fraction of the four fractions obtained from the fraction of Sawtooth mildew extract.
Fig. 4 shows the results of confirming three peaks of a second fraction chromatogram of the fraction No. 2 in the four fractions obtained from the fraction of the saphena extract.
FIG. 5 is a graph showing the chromatogram results of sagachromenol identified by NMR analysis after purifying the second peak in the second fraction of the second fraction of the four fractions obtained from the fraction of Sawtooth mildew extract .
FIG. 6 is a result of confirming four peaks in a second fraction chromatogram of the third fraction of the four fractions obtained from the fraction of Sawtooth mildew extract.
FIG. 7 is a graph showing the results of chromatographic analysis of sagaquinoic acid identified by NMR analysis after purifying the 4th peak in the second fraction of the second fraction of the four fractions obtained from the fraction of the Sawtoothsmae extract to be.
Fig. 8 is a chart showing cytotoxicity of Hepatospora japonica extract to HepG2 cells.
FIG. 9 shows cytotoxicity of tBHP against HepG2 cells. FIG.
FIG. 10 is a chart for confirming the survival restoration effect of HepG2 cells damaged by tBHP by Saw Mab Bark extract.
11 is a chart showing the cytotoxicity of alcohol to HepG2 cells.
FIG. 12 is a graph showing survival restorative effect of alcohol-damaged HepG2 cells by Saw Mab Bark extract. FIG.
Fig. 13 is a graph showing the inhibitory effect of intracellular reactive oxygen species production upon simultaneous treatment of tBHP and T. manganica extract on HepG2 cells.
Fig. 14 is a graph showing the inhibitory effect of tBHP on the production of lipid peroxides by simultaneous treatment of tBHP and teat mabe extract with HepG2 cells.
FIG. 15 is a graph showing changes in intracellular catalase activity by the extract of Tetranyba princeps moth (Panax obtusa) at the time of simultaneous treatment of tBHP and T. manganica extract with HepG2 cells.
FIG. 16 is a graph showing the change in intracellular superoxide dismutase activity by the extract of Tetranychia molatovii when co-treatment of tBHP and T. manganica extract with HepG2 cells.
FIG. 17 is a graph showing changes in the production of NQO-1, HO-1 and Nrf2 by the extracts of Tetranyba pricklesiae during simultaneous treatment of tBHP and tephritidae with HepG2 cells.
18 shows the cytotoxicity of sago hydraquinoxacic acid isolated from serrate moth on HepG2 cells.
FIG. 19 shows recovery of survival of tBHP-damaged HepG2 cells by sag Hydroquinone acids. FIG.
FIG. 20 is a graph showing the inhibitory effect of tBHP on the production of lipid peroxides by the saphenicum extract in the simultaneous treatment of tBHP and sago hydrocainoic acid isolated from serrate on HepG2 cells.
FIG. 21 is a graph showing changes in the production of NQO-1, HO-1 and Nrf2 by sAg hydroquinone acid during simultaneous treatment of tBHP and sago hydroquinic acid isolated from serrate on HepG2 cells.
FIG. 22 shows the cytotoxicity of sagarchromenol isolated from sawtooth mildew to HepG2 cells.
23 is a graph showing survival restorative effect of sGalcomonol in tGHP-damaged HepG2 cells.
24 is a graph showing the inhibitory effect of tBHP on the production of lipid peroxides by Sawmomycin extracts in the simultaneous treatment of tBHP and sagacromenol isolated from serrate on HepG2 cells.
25 is a graph showing changes in the production of NQO-1, HO-1 and Nrf2 by sAg hydroquinone acid in the simultaneous treatment of tBHP and sagacromenol isolated from the serrate matured HepG2 cells.
26 is a chart showing the cytotoxicity of saga quinoxalic acid isolated from serrate mosaic on HepG2 cells.
FIG. 27 is a graph showing the cell survival recovery effect of sphauquinoxanic acid in BHP-damaged HepG2 cells. FIG.
28 is a graph showing the inhibitory effect of tBHP on the production of lipid peroxides by the saphenic sulphate extract in the simultaneous treatment of tBHP and sago quinolinic acid isolated from the teaspoon of HepG2 cells.
FIG. 29 is a graph showing changes in the production of NQO-1, HO-1 and Nrf2 by sAg hydroquinone acid in the simultaneous treatment of tBHP and sago quinoxaline isolated from serrate on HepG2 cells.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing, ameliorating or treating liver diseases, liver diseases, or the like, comprising an extract of Tooth mackerel, fractions thereof or a compound isolated therefrom as an active ingredient.

The 'Sargassum serratifolium' of the present invention is a seaweed of the algae and is distributed in the southern coast and Jeju region in Korea. The plant has a diameter of 1 to 4 m and a root diameter of 4 to 5 cm, conical and rubbery There is a ring. The present inventors have selected moths belonging to the moth as a material of the composition for the prevention or treatment of liver disease or liver disease, and have found that the moth moth moth extract exhibits remarkably excellent liver protecting activity.

More specifically, the present inventors measured the cytoprotective effect of tBHP and ethanol-induced hepatocyte cell damage in the following examples in order to evaluate the hepatoprotective activity of the extracts of Tohma Namdawa, its fractions or the compounds isolated therefrom As a result, it was confirmed that hepatocyte apoptosis was inhibited depending on the treatment concentration in the test group treated with HepG2 cells and the extract of Sawtooth moth, and the compounds isolated therefrom. The EC50 of the alcohol extract required for 50% recovery of cells damaged by tBHP was found to be 0.28 μg / mL (see FIG. 9), and the EC50 of the ethanol extract was 0.52 μg / mL (see FIG. 12) . The EC50 of sagahydroquinoxanic acid by tBHP was 0.21 [mu] M (see Fig. 19), the EC50 of sagachromenol was 0.59 [mu] M (see Fig. 19), and the EC50 of sagaquinoxanic acid was 0.61 [mu] M (see Fig. 27).

In addition, in the following examples, intracellular lipid peroxide was measured. As a result, the production of peroxide derived from tBHP was decreased depending on the treatment concentration in the experimental group treated with the extract of Sawtooth mussel and the compound isolated therefrom, respectively I could confirm. The inhibitory concentration (EC50) of 50% production of peroxides by the alcohol extract was 0.33 μg / mL, respectively (see FIG. 14). The EC50 of the saga hydroquinoxanic acid was found to be 0.59 [mu] M (see Fig. 20), and the EC50 of the sagachromenol was found to be 2.4 [mu] M (see Fig.

In addition, in the following examples, it was determined whether the tachyhamiae activity induced by tBHP (see FIG. 15) and the superoxide dismutase activity (see FIG. 16) The extract showed 50% scavenging activity (IC50) in the low concentration range (~ 20 μg / ml). These values show a much higher cytoprotective effect than NAC used as a positive control. In addition, we analyzed the expression of NQO-1 and HO-1, which are regulated by Nrf2 and Nrf2, the major transcription factors that have cytoprotective effects against oxidative damage, in order to analyze the mechanism of liver cell protection effect in tBHP. As a result, the concentration of Nrf2 in the cytoplasm decreased while the concentration in the nucleus increased. This means that Nrf2, which is a transcription factor, is transferred to the nucleus by the Sawtooth mulberry extract and is involved in transcription of proteins involved in cell protection. As a result, the concentrations of NQO-1 and HO-1 in the cytoplasm associated with cell protection increased (FIG. 17). Therefore, the composition of the present invention comprising an extract of Tootsuna marijuana, its fractions or a compound isolated therefrom as an active ingredient can be usefully used as a functional material showing hepatocyte protective activity.

In addition, the sawtooth moth extract according to the present invention can be obtained by extracting and isolating from nature using an extraction and separation method known in the art, and the 'extract' defined in the present invention can be obtained by using an appropriate solvent (Sargassum serratifolium), and includes, for example, a crude extract of a saw moth, a polar solvent-soluble extract or a non-polar solvent-soluble extract.

As an appropriate solvent for extracting the extract from the sawtooth mackerel, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used. Examples of the solvent include, but are not limited to, purified water, methanol alcohol having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform (including methanol, ethanol, propanol, isopropanol, and butanol) various solvents such as chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. It is preferable to use ethanol (alcohol), more preferably 95% ethanol.

As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the production method of the sawtooth extract of the present invention, and any known method may be used.

For example, the Saw Mab Bamboo Extract contained in the composition of the present invention can be prepared into a powdery state by an additional process such as vacuum distillation, freeze-drying, spray-drying, or the like, with the primary extract extracted by the hot water extraction or solvent extraction method . Further, the primary extract can be further purified by using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like, You can get it.

Therefore, in the present invention, the teaspoon mallow extract is a concept including all the extract, fraction and purified product obtained in each step of extraction, fractionation or purification, and a diluted solution, a concentrate or a dried product thereof.

Hereinafter, a method of preparing the sawtooth extract according to an embodiment of the present invention will be described in more detail. After the dried sawtooth mats are pulverized, they are subjected to reflux cooling extraction using 50 to 100% ethanol as an extraction solvent to the toothed marmoset powder, followed by filtration and concentration to extract.

The above-described liver protecting composition of the present invention can be used for various purposes and purposes requiring liver protecting activity, and more specifically, it can be used for various purposes such as pharmaceuticals, foods, animal feeds, It can also be used as a food additive and feed additive.

The compounds separated from the sawtooth mabs of the present invention include sagahydroquinoic acid, sagaquinoic acid and sagachromenol. These compounds are represented by Formulas 1 to 3, respectively, in the present invention.

In addition, the liver disease of the present invention can be used for the treatment and / or prophylaxis of non-alcoholic fatty liver disease, alcoholic fatty liver disease, nonalcoholic fatty liver disease, hepatic hepatitis, nonfat liver disease, Liver damage, acute liver injury, and liver cancer.

The sawtooth extract of the present invention, its fractions or the compound isolated therefrom may be contained in the composition of the present invention at a concentration of 0.1 to 1000 μg / ml, but is not limited thereto.

Meanwhile, as a specific aspect of the present invention, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

The composition comprising a pharmaceutically acceptable carrier can 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 are also 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 the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esterol such as ethyl oleate.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories You can have one formulation.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts.

A typical daily dose of a composition comprising an extract, fraction thereof or a compound isolated therefrom according to the present invention as an active ingredient is such that a typical daily dose of a composition comprising fractions or a compound isolated therefrom as an active ingredient Is 0.5 to 2 mg / kg, preferably 1 mg / kg, specifically 200 to 400 mg / kg, preferably 300 mg / kg, once or several times a day. The prophylactic or therapeutic agent of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or two or three times. When the two active ingredients are each monoglyceride, the number of administrations may be the same or different. In addition, the composition of the present invention can be used alone or in combination with other medicines for the prevention or treatment of liver disease. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

In another aspect, the present invention provides a method for preventing or treating liver disease, comprising administering to a subject suspected of having an individual and / or a liver disease requiring it, a fraction thereof or a compound separated therefrom .

In the present invention, the term "individual" means all animals including humans who have already developed or can develop liver disease, and by administering to a subject a composition comprising the extract of Tooth Mothia or the fraction thereof as an active ingredient, The disease can be effectively prevented and treated. The term refers to whole mammals including dogs, cows, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by these examples.

The method of treatment of liver disease of the present invention includes administering a therapeutically effective amount of a teaspoon extract, a fraction thereof, or a compound isolated therefrom. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. It can also be administered once or several times. The optimal amount and dosage interval of the individual doses of the compositions of the present invention will be determined by the nature and extent of the disease being treated, the dosage form, route and site of administration, and the age and health status of the particular patient being treated, It will be apparent to those skilled in the art that the appropriate dosage will be determined. Such dosing can be repeated as often as is appropriate. If side effects occur, dosage and frequency can be altered or decreased depending on the usual clinical practice.

The route of administration of the composition may be administered via any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, or orally, but not limited to, parenteral administration, as desired. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

In another specific embodiment of the present invention, the composition of the present invention may be a quasi-drug composition.

The above-mentioned Sawmill extract or its fractions are as described above for liver disease. More specifically, the composition of the present invention can be added to a quasi-drug composition for the purpose of cell protection or prevention or improvement of liver disease.

The term "improvement" in the present invention means the effect of alleviating the symptom of liver disease by applying the composition of quasi-graft extract or its fractions containing the fraction of the present invention. The term "quasi-drug" in the present invention means a fiber, a rubber product or the like used for treating, alleviating, treating or preventing a disease of a human or an animal, Or products similar to those that are not machinery, preparations used for sterilization, insecticides and similar uses for the prevention of infectious diseases, for the purpose of diagnosing, treating, alleviating, treating, or preventing diseases of human beings or animals Means goods, machinery, or apparatus other than the apparatus, machinery or equipment of the commodity being used and used for the purpose of giving pharmacological effects to the structure and function of humans or animals. The quasi-drugs include external preparations for skin and personal hygiene products.

When the extracts of Tooth moth, extracts thereof or the compounds isolated therefrom of the present invention are used as a quasi-drug additive, the extract can be used as it is or can be used together with other quasi-drugs or quasi-drugs, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The external preparation for skin is not particularly limited, but may be manufactured and used in the form of an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension, a gel or a gel. Such personal care products include but are not limited to soap, cosmetics, wet tissues, tissue paper, shampoo, skin creams, facial creams, toothpastes, lipsticks, perfumes, make-ups, foundations, ball touches, mascaras, eye shadows, A sunscreen lotion, a hair care product, an air freshner gel, or a cleaning gel. Further, another example of the pharmaceutical composition of the present invention is a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment agent or a filter filler.

In another aspect, the present invention provides a feed additive or a feed composition for prevention or improvement of liver disease, liver disease, or the like, comprising a Tohrukwas food extract, a fraction thereof or a compound isolated therefrom.

The extracts of Tooth mothballs, fractions thereof or compounds isolated therefrom are as described above for liver protection and liver disease.

Specifically, the extract of Tooth moth, the fraction thereof or the compound isolated therefrom of the present invention can be added to a feed composition comprising a feed additive or a feed additive for the purpose of preventing or improving liver disease or liver disease.

The term "feed additive" in the present invention means a substance added to feed for various purposes such as nutrient supplementation and weight loss prevention, promotion of digestive utilization of fibrin in feed, improvement of oil quality, prevention of reproductive disorder, improvement of conception rate, . The feed additive of the present invention corresponds to an auxiliary feed in the feed control method and is a mineral preparation such as sodium hydrogencarbonate, bentonite, magnesium oxide and complex mineral, a mineral preparation such as zinc, copper, cobalt and selenium, , Vitamins such as vitamin E, vitamin A, D, E, nicotinic acid and vitamin B complex, protective amino acids such as methionine and lysic acid, protective fatty acids such as calcium salt of fatty acid, probiotics (lactic acid bacteria), yeast culture, Fermented foods and the like, yeast preparations and the like may be further included.

As used herein, the term "feed" means any natural or artificial diet, single meal, or the like component for feeding, ingesting, digesting or suitable for the animal. Specifically, the feed comprising the composition for preventing or treating liver disease according to the present invention as an active ingredient can be manufactured into various types of feeds known in the art, and specifically, a concentrated feed, a forage and / .

Concentrated feeds include seeds containing cereals such as wheat, oats, and corn, and by-products such as rice bran, wheat bran, barley, etc., Which is the by-product of the starch residue, which is the main component of the starch residue, which is the remaining product of starch minerals, sweet potatoes, potatoes, etc., which is a byproduct obtained by concentrating the fresh liquid product obtained from fish meal, fish meal, fish residue, The animal feed such as fish soluble, meat powder, blood meal, wheat meal, skim milk powder, cheese in milk, dried whey in the case of casein production in skim milk, dry yeast, yeast, Chlorella, algae, but not limited thereto.

Roughage includes root vegetables such as wild grasses, grasses and fodder, raw turnip for feed, turnip for feed, feed bit, turnip root, Silage, which is filled with lactic acid and fermented by lactic acid, hay, dried hay, herbage straw, straw of leguminous crops, leaves of leguminous plants, and the like. Special diets include mineral feeds such as fodder, rock salt, urea and its derivatives such as diuretic isobutane, nutrients such as diuretic isobutane, and nutrients that can be supplemented when only natural feed ingredients are mixed or supplemented with diets Feed additives, dietary supplements, which are substances added in small quantities, but are not limited thereto.

The composition for feed comprising a composition for preventing or treating liver disease or liver disease according to the present invention can be produced by a method comprising the steps of preparing a crude extract of Tootsumabum, a fraction thereof or a compound isolated therefrom in a suitable effective concentration range according to various methods for preparing diets known in the art .

The composition for feed according to the present invention is not particularly limited as long as it is an object for liver protection or prevention or treatment of liver disease, and any composition can be applied. For example, it can be applied to any non-human animal such as a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cattle, a sheep, a pig,

Another aspect of the present invention provides a food composition for prevention or improvement of liver disease or liver disease comprising extracts of Tootsuna marijuana, fractions thereof or a compound isolated therefrom as an active ingredient.

The extract, its fractions or the compounds isolated therefrom are as described above for liver disease.

That is, the Sawmill Extract, its fractions or the compounds isolated therefrom may be added to the food composition for the purpose of preventing liver damage or preventing or improving liver disease.

 The food composition may comprise a pharmaceutically acceptable carrier. The food composition of the present invention includes all forms of functional food, nutritional supplement, health food, and food additives, and the food composition of this type is known in the art And can be prepared in various forms according to known conventional methods.

When the extract, fraction or compound of the present invention is used as a food additive, the extract, fraction or compound thereof may be directly added, used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the extract, fractions or compounds thereof are added in an amount of 0.0001 to 1% by weight, specifically 0.001 to 0.1% by weight, of the raw material composition when the food or beverage is produced.

However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. 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 is generally about 0.01 to 0.04 g, and more specifically about 0.02 to 0.03 g per 100 of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. 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 composition of the present invention. In addition, the composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. The proportion of such flesh is not critical but is generally selected in the range of 0.01 to 10 parts by weight per 100 parts by weight of the composition of the present invention. These components can be used independently or in combination.

Example  One. Saw blade  Preparation of extract

The sawtooth moths used in the present invention were naturally occurring natural acids from Gyengjang-gun, Busan-si and Tongyeong-si, Gyeongsangnam-do, and 8 kg of 70% ethanol was added to 2 kg of the powder obtained by natural drying and scouring The mixture was refluxed and cooled at 70 캜 for 3 hours three times, and then debris was removed with an ultrafilter (Seahan Tex, Korea). The filtrate extract was 200 ~ 25g of crude extract as an extracted residue after removing the alcohol at 45 [deg.] C with a vacuum rotary condenser. The thus obtained Saw Moth extract was used as a sample in the following experiment while being stored at 4 ° C in a refrigerator.

Example  2. Saw blade  From the extract Saga Hydroquinoxaline , Saga Chromenol  And separation of the saga quinoxaline acid

2-1. Of saga hydroquinic acid  detach

15 g of the crude extract of Tooth Mab prepared in Example 1 was dissolved in 150 mL of methanol, filtered through a membrane filter, and then 200 μL of Phenomenex C18 (2) (15 μm, 21.2 mm × 250 mm) Separated into four fractions by the attached HPLC (see Figure 1). As a mobile phase of the chromatography, a mixed solvent of methanol (A) and water (B) was used and the flow rate was set to 7 mL. The chromatographic conditions were A / B (90/10) to A / B (94/6) for 33 minutes, A / B (94/6) to A / B / B (100/0) for 10 minutes. Chromatography was performed 50-100 times by automatic sample injection with Autosampler to obtain a large fraction of the first fraction of fraction No. 1 (see FIG. 1) .

The same instrument and column were used to increase the purity of the first fraction obtained in large quantities. The chromatographic conditions were A / B (85/15) to A / B (87/13) for 26 min, A / B (87/13) to A / B / B (100/0) for 6 minutes and the column was equilibrated for 10 minutes at A / B (87:13) concentration. Chromatography was performed by automatically injecting the sample into an autosampler to obtain a second fraction of fraction 1 having a higher purity (see FIG. 2).

One of the second fractions of fraction No. 1 was purified by rechromatography (523 mg) under the same conditions and analyzed with an analytical column (Phenomenex C18 (2), 3 μm, 3 mm × 150 mm) Which was analyzed by NMR and found to be sargahydroquinoic acid (hereinafter referred to as "SHQ") (see FIG. 3). The content of sago hydroquinic acid in the extract of the teaspoon was determined to be 37.6%.

1 H-NMR (CD 3 OD, 400 MHz); (2H, m, H-4), 2.12 (2H, m), 3.25 (2H, d, J = 7.4Hz, H-1), 5.29 7.0 Hz, H-6), 2.08 (2H, m, H-8), 2.50 (2H, dt, J = 7.0 & (2H, m, H-13), 5.07 (1H, tt, J), 5.83 (1H, t, J = 7.3 Hz, S, CH3-17), 1.59 (3H, s, CH3-19), 1.70 (3H, s, CH3-16), 1.56 H-20), 6.38 (2H, br s, H-3 'and H-5'), 2.14 (3H, s, Aromatic-CH3). ≪ 13 > C-NMR (CD3OD, 100 MHz); (C-2), 136.8 (C-3), 40.9 (C-4), 27.6 (C-5), 125.96 (C-8), 28.9 (C-9), 142.7 (C-10), 132.9 (C-11), 36.0 C-15), 25.9 (C-16), 17.8 (C-17), 171.7 (C-18), 15.96 C-2 '), 114.5 (C-3'), 151.4 (C-4 '), 115.7

[Chemical Formula 1]

2-2. Saga of Chromenol  detach

The fraction 2 of FIG. 1 was re-chromatographed using the same HPLC apparatus and column as the fraction 1 of Example 2-1. The chromatographic conditions were as follows: A / B (92.5 / 8.5) to A / B (92.2 / 7.8) for 26 min, A / B / B (100/0) for 6 minutes and the column was equilibrated for 10 minutes at A / B (91.5: 8.5) concentration. Chromatography was performed 50-100 times by automatic sample injection with utosampler to obtain a second fraction of a large fraction of the second fraction (see FIG. 4). As shown in FIG. 4, fraction No. 2 showed three peaks. The second peak (Frc2-2) was further purified to obtain a chromatogram as shown in FIG. 5 (126 mg). The separated product was analyzed by NMR and found to be sargachromenol (SCA) (see FIG. 5).

The content of sagarchromenol in the extract of the sago mushroom was determined to be 6.2%.

1 H-NMR (CD 3 OD, 400 MHz); (1H, d, J = 9.9 Hz, H-3), 6.29 (1H, d, J = 9.9 Hz, H-4) (2H, m, H-2 '), 5.14 (1H, t, J = 6.9 Hz, H (2H, t, J = 7.0 Hz), 2.05 (2H, t, J = 7.5 Hz, H- (2H, t, J = 6.9 Hz, H-9 '), 2.10 (3H, s, H-13 '), 1.56 (6H, brs, H-14' and H-16 '), 1.32 , Aromatic-CH3) < 13 > C-NMR (CD3OD, 100 MHz); (C-3), 124.3 (C-4), 122.6 (C-4a), 111.3 (C-5), 151.3 (C-8), 145.3 (C-8a), 41.9 (C-1 '), 23.7 '), 29.0 (C-6'), 142.5 (C-7 '), 132.9 (C-8'), 36.0 , 133.5 (C-12 '), 25.9 (C-13'), 17.8 (C-14 '), 171.7 C-18 ')

(2)

2-3. Saga cuneo  detach

The fraction No. 3 in Fig. 1 was re-chromatographed using the same HPLC apparatus and column as the fraction No. 1 in Example 2-1. The chromatographic conditions were A / B (93.4 / 6.6) to A / B (93.8 / 6.2) for 20 min, A / B (93.8 / / B (100/0) for 6 minutes and the column was equilibrated for 10 minutes at A / B (93.8 / 6.2) concentration. Chromatography was performed 50-100 times by automatic sample injection with Autosampler to obtain a large fraction of the second fraction of the fraction No. 3 (see FIG. 6). As shown in FIG. 6, the fraction No. 3 showed four peaks. The peak No. 4 (Frc 3-4) was further purified to obtain a chromatogram as shown in FIG. 7 (171 mg). The separated product was analyzed by NMR and found to be sargaquinoic acid (hereinafter referred to as SQA) (see FIG. 7). The content of sagaquinoxanic acid in the extracts of the teaspoon was determined to be 1.9%.

1 H-NMR (CD 3 OD, 400 MHz); (2H, m, H), 3.12 (2H, d, J = 6.9 Hz, H-1), 5.17 (1H, t, J = 7.4Hz, H- (2H, m, H-8), 2.59 (2H, dt, J = 7.2 and 7.2, H-9), 5.84 = 7.3 Hz, H-10), 2.19 (2H, t, J = 7.2 Hz, H-12), 2.12 S, CH3-17), 1.60 (3H, s, CH3-19), 1.62 (3H, s, H-20), 6.42 (1H, m, H-3 '), 6.56 (1H, quin, J = 1.4 Hz, H-5'), 2.12 (3H, d, J = 1.5 Hz, Aromatic- ); (C-2), 140.4 (C-3), 40.7 (C-4), 27.3 (C-5), 125.7 (C-8), 29.0 (C-9), 142.6 (C-10), 132.9

17.8 (C-18), 15.97 (C-19), 16.0 (C-20), 189.4 (C-2 '), 133.0 (C-3'), 188.9 (C-4 '), 133.96 (C-5'), 147.6 -CH3)

(3)

Example  3. The Saw blade  Evaluation of cytotoxicity of extracts

The cytoprotective effect on hepatocyte apoptosis was confirmed by measuring the survival rate of the cells. The survival rate of the cells was measured by MTS, 3- (4,5 - dimethylthiazol - 2 - yl) - 5- (3 - carboxymethoxyphenyl) - (4-sulfophenyl) -2-H-tetrazolium) analysis method (Kim et al., J Agric Food Chem, 2009, 57, 3483-3489). HepG2 cells (5 × 10 ⁴ cells / well) were treated with EMM medium of the present invention prepared in Example 1, and cultured for 24 hours. 95 μl of the medium and 5 μl of the MTS solution were placed in a 96-well plate, reacted for about 1 hour, and the absorbance was measured at 490 nm using a microplate reader. The measurements were repeated three times and expressed as the mean value of the experiment. The results are shown in Fig.

As shown in Fig. 8, the cytotoxicity of the extract of the present invention on the liver cell line (HepG2 cells) did not show up to a concentration of 10.0 μg / mL.

Example  4. The Saw blade  Extract Oxidative  Identification of hepatocyte protective effect against damage

4-1. t- On BHP  Of hepatocyte death

The effects of t-BHP on oxidative damage were analyzed. In order to determine the optimal concentration of t-BHP causing cell damage, the killing effect of hepatocyte on the concentration of t-BHP was measured by the MTS method. The cell viability was measured by the MTS method by treating HepG2 cells (5 x 10 ⁴ cells / well) with 96-well plates on EMEM for 24 hours, and then treated with different concentrations of t-BHP .

As shown in FIG. 9, 50% of the hepatocytes were killed by 0.5 mM t-BHP, and the concentration of t-BHP was 0.5 mM for the cytoprotective effect of oxidative damage.

4-2. t- By BHP  For induced cell damage Saw blade  Identification of hepatocyte protective effect of extract

In order to confirm the liver protection effect of the extracts of Sawtooth mildew, cell viability was measured by MTS assay after treatment with 0.5 mM t-BHP and different concentrations of Sawtooth mildew extract for 24 hours. The results are shown in Fig.

As shown in Fig. 10, it was confirmed that the saphenic acid extract of Toei matsumana inhibited the death of hepatocytes by t-BHP in a concentration-dependent manner. These results demonstrate that the saphenicola extract has a significant cytoprotective effect on hepatocyte injury by t-BHP.

4-3. Determination of hepatocyte killing effect by ethanol

The effect of ethanol on the death of liver cells was analyzed. In order to determine the optimal concentration of ethanol causing cell damage, the death effect of hepatocyte according to ethanol concentration was measured by MTS method. FIG. 11 shows the results of measuring cell viability by the MTS method after culturing HepG2 cells (5 × 10 ⁴ cells / well) on 96-well plates on EMEM medium for one day and treating ethanol at different concentrations for 24 hours.

As shown in FIG. 11, since the survival rate of hepatocytes was slightly decreased by 100 mM of ethanol, the concentration of ethanol for the cytoprotective effect of ethanol was determined to be 100 mM.

4-4. For ethanol induced cell damage Saw blade  Identification of hepatocyte protective effect of extract

Cell viability was measured by MTS assay after treatment with 100 mM ethanol and different concentrations of Sawtooth mabana extract for 24 h to determine the liver protective effect of the extracts. The results are shown in Fig.

As shown in Fig. 12, it was confirmed that the extract of Tetrina miltiflora inhibited the death of hepatocytes by ethanol in a concentration-dependent manner. These results demonstrate that saphenicola extract has a significant protective effect against hepatocyte injury by ethanol.

4-5. t- By BHT  For induced cells Saw blade  Extract ROS  Generation inhibition effect

When cells are treated with t-BHP, the amount of ROS in the cell increases, oxidizing intracellular proteins, DNA and fats, resulting in oxidative damage. In this experiment, treatment of t-BHP with HepG2 cells significantly increased the amount of ROS. However, when t-BHP was treated with Saw mabanum extract, the amount of ROS produced decreased in a concentration-dependent manner (FIG. 13). These results suggest that the extract of Sawatta mashii acts as an antioxidant to reduce the amount of ROS.

4-6. t- By BHT  For induced cells Saw blade  Inhibitory effect of extract on lipid peroxidation

When cells are treated with t-BHP, excess superoxide is produced, and the produced superoxide reacts with intracellular lipids to produce lipid peroxides. The amount of lipid peroxide produced was analyzed by malondialdehyde method. As shown in FIG. 14, it was found that the peroxide generated by the excess of t-BHP was decreased in a concentration-dependent manner by the extract of Sawtooth mildew. This suggests that the antioxidative activity of the extracts of Sawtooth mildew inhibits lipid peroxidation by removing ROS.

4-7. t- By BHT  For induced cells Saw blade  Determination of Antioxidant Enzyme Activity of Extracts.

The cultured liver cell line was treated with t-BHP at 0.5 mM concentration and Sawtooth mildew extract at different concentrations and cultured for 2 hours. The cultured cells were collected, dissolved in a cell lysis buffer, and centrifuged to obtain a cell suspension. The catalase activity was measured by a catalytic assay of cayman. The results are shown in Fig.

As shown in Fig. 15, the catalase activity of the cells treated with t-BHP was high, but the enzyme activity was decreased in a concentration-dependent manner by treatment with the extract of Tetranyba princeps. It is expected that catalase activity will be reduced by removing ROS by the antioxidant activity of the saphenous moth extract.

In addition, the superoxide dismutase activity was measured using a dismutase assay of cayman. The results are shown in Fig.

As shown in FIG. 16, the superoxide dismutase activity of the cells treated with t-BHP was high, but it was found that the enzyme activity was reduced in a concentration-dependent manner by treatment with the extract of Tetranyba princeps. It is expected that catalase and superoxide dismutase activities will be reduced by removing ROS by antioxidant activity of the Sawtooth mildew extract.

4-8. t- By BHT  For induced cells Saw blade  Expression induction of antioxidant enzymes of extracts.

The cultured liver cells were treated with t-BHP at 0.5 mM concentration and Sawtooth mildew extract at different concentrations for 1 hour. The cultured cells were collected, dissolved in a cell lysis buffer, and centrifuged to obtain a cell suspension. The cell suspension was separated into cytoplasmic protein and nuclear protein, and each protein was analyzed by Western blotting to confirm the expression level of Nrf2. In order to confirm the expression level of NQO-1 and HO-1, the cells were cultured for 18 hours under the same conditions as above. Cells were collected and lysed with cell lysis buffer, and centrifuged to obtain a cell suspension. Protein expression of NQO-1 and HO-1 was confirmed by Western blotting using the separated proteins. The results are shown in Fig.

As shown in Fig. 17, NQO-1 and HO-1 in the cytoplasm of the antioxidant enzyme were increased in a concentration-dependent manner by the treatment with the extract of Sawtooth mildew. Expression of NQO-1 and HO-1 is regulated by their transcription factor, Nrf2. Therefore, the concentration of Nrf2 transcription factor in the nucleus was analyzed and the amount of Nrf2 expression was increased in a dose - These results suggest that the Saw mabe extract induces the activation of Nrf2,

And the expression of NQO-1 and HO-1, respectively.

Example  5. The Saw blade  Isolated from the extract Saga Hydroquinoxaline Of hepatocytes against oxidative damage of liver

5-1. The Of saga hydroquinic acid  Cytotoxicity Assessment

HepG2 cells (5 x 10 ⁴ cells / well) were cultured in EMEM medium for 24 hours after treatment with the sagohydroquinolic acid of the present invention prepared in Example 2 above. 95 μl of the medium and 5 μl of the MTS solution were placed in a 96-well plate, reacted for about 1 hour, and the absorbance was measured at 490 nm using a microplate reader. The measurements were repeated three times and expressed as the mean value of the experiment. The results are shown in Fig.

As shown in Fig. 19, the cytotoxicity of the sagas hydroquinoxic acid of the present invention to liver cell line (HepG2 cells) did not show up to a concentration of 4.0 [mu] M. Subsequent experiments were conducted at concentrations below 4.0 μM.

5-2. t- By BHT  For induced cell damage Of saga hydroquinic acid  Confirming hepatocyte protective effect

To confirm the hepatoprotective effect of sagahydroquinoxalic acid, cell viability was measured by MTS assay after simultaneous treatment of 0.5 mM t-BHP and different concentrations of sag hydroquinic acid for 24 hours. The results are shown in Fig.

As shown in Fig. 19, it was confirmed that the sagahydroquinoxalic acid inhibited the death of hepatocytes by t-BHP in a concentration-dependent manner. These results demonstrate that sag Hydrocuneoic acid has a significant cytoprotective effect on hepatocyte injury by t-BHP.

5-3. t- By BHT  For induced cells Of saga hydroquinic acid  Confirmation of lipid peroxidation inhibitory effect

When cells are treated with t-BHP, excess superoxide is produced, and the produced superoxide reacts with intracellular lipids to produce lipid peroxides. The amount of lipid peroxide produced was analyzed by malondialdehyde method. The results are shown in Fig.

As shown in FIG. 20, it was found that the peroxide generated in excess by t-BHP was reduced in a concentration-dependent manner by sag Hydroquinone acid. It can be seen that the antioxidative activity of saga hydroquinone acid inhibits lipid peroxidation by removing ROS.

5-4. t- By BHT  For induced cells Of saga hydroquinic acid  Expression induction of antioxidant enzyme

The cultured liver cell line was treated with 0.5 mM t-BHP and sagar hydroquinic acid at different concentrations for 1 hour. The cultured cells were collected, dissolved in a cell lysis buffer, and centrifuged to obtain a cell suspension. The separated proteins were analyzed by Western blotting to confirm the expression level of pNrf2.

In order to confirm the expression level of NQO-1 and HO-1, the cells were cultured for 18 hours under the same conditions as above, and the cells were collected, dissolved in a cell lysis buffer, and centrifuged to obtain a cell suspension. Protein expression of NQO-1 and HO-1 was confirmed by Western blotting using the separated proteins. The results are shown in Fig.

As shown in FIG. 21, the concentration of NQO-1 in the cytoplasm of the antioxidant enzyme was increased in a concentration-dependent manner up to the concentration applied to the experiment. However, HO-1 showed a concentration-dependent up to 1.0 uM concentration of sag Hydrocuneoic acid Respectively. Expression of NQO-1 and HO-1 is regulated by their transcription factor, Nrf2. Therefore, the expression of pNrf2 transcription factor was analyzed by sAgH hydroquinone acid treatment and the expression of pNrf2 was increased only at the concentration of 2.0 uM.

Example  6. The Saw blade  Isolated from the extract Saga of Chromenol Oxidation Identification of hepatocyte protective effect against red damage

6- 1. Bonn  Invention Saga of Chromenol  Cytotoxicity Assessment

HepG2 cells (5 x 10 ⁴ cells / well) were cultured in EMEM medium for 24 hours after treatment with sagarchromenol of the present invention prepared in Example 2 above. 95 μl of the medium and 5 μl of the MTS solution were placed in a 96-well plate, reacted for about 1 hour, and the absorbance was measured at 490 nm using a microplate reader. The measurements were repeated three times and expressed as the mean value of the experiment. The results are shown in Fig.

As shown in FIG. 22, the cytotoxicity of the sagavatron of the present invention to liver cell line (HepG2 cells) did not show up to a concentration of 4.0 μM. Subsequent experiments were conducted at concentrations below 4.0 μM.

6-2. t- By BHT  For induced cell damage Saga of Chromenol  Liver cell protection effect Sho sign

To confirm the liver protective effect of sagarchromenol, cell viability was measured by MTS assay after treatment with 0.5 mM t-BHP and different concentrations of sagarchromenol for 24 hours. The results are shown in Fig.

As shown in FIG. 23, it was confirmed that sagarchromenol inhibited the death of hepatocytes by t-BHP in a concentration-dependent manner. These results demonstrate a significant cytoprotective effect on hepatocyte injury by sagarchomene t-BHP.

6-3. t- By BHT  For induced cells Saga of Chromenol  Confirmation of lipid peroxidation inhibitory effect

When cells are treated with t-BHP, excess superoxide is produced, and the produced superoxide reacts with intracellular lipids to produce lipid peroxides. The amount of lipid peroxide produced was analyzed by malondialdehyde method. The results are shown in Fig.

As shown in FIG. 24, it was found that the peroxide generated in excess by t-BHP was reduced by 1 to 2 μM of sagacromenol. It can be seen that the antioxidant activity of sagarchromene inhibits lipid peroxidation by removing ROS.

6-4. t- By BHT  For induced cells Saga of Chromenol  Expression induction of antioxidant enzyme

The cultured liver cells were treated with 0.5 mM t-BHP and sagarchromenol at different concentrations for 1 hour. The cultured cells were collected, dissolved in a cell lysis buffer, and centrifuged to obtain a cell suspension. The separated proteins were analyzed by Western blotting to confirm the expression level of pNrf2. In order to confirm the expression level of NQO-1 and HO-1, the cells were cultured for 18 hours under the same conditions as above, and the cells were collected, dissolved in a cell lysis buffer, and centrifuged to obtain a cell suspension. Protein expression of NQO-1 and HO-1 was confirmed by Western blotting using the separated proteins. The results are shown in Fig.

As shown in FIG. 25, antioxidative enzymes NQO-1 and HO-1 were increased in a concentration-dependent manner by sagarchromenol treatment. Expression of NQO-1 and HO-1 is regulated by their transcription factor, Nrf2. As a result, the expression level of pNrf2 was increased in a concentration dependent manner by sagarchromenol treatment. These results suggest that sagarchromene induces the activation of pNrf2 and induces the expression of NQO-1 and HO-1, which are lower proteins thereof.

Example  7. The Saw blade  Isolated from the extract Saga cuneo Oxidation Identification of hepatocyte protective effect against red damage

7-1. The Saga cuneo  Cytotoxicity Assessment

HepG2 cells (5 x 10 4 cells / well) were cultured in EMEM medium for 24 hours after treatment with the saga-quinoxalic acid of the present invention prepared in Example 2 above. 95 μl of the medium and 5 μl of the MTS solution were placed in a 96-well plate, reacted for about 1 hour, and the absorbance was measured at 490 nm using a microplate reader. The measurements were repeated three times and expressed as the mean value of the experiment. The results are shown in Fig.

As shown in FIG. 26, the cytotoxicity of the present sanguaquinoxane to liver cell line (HepG2 cells) did not show up to a concentration of 4.0 μM. Subsequent experiments were conducted at concentrations below 4.0 μM.

7-2. t- By BHT  For induced cell damage Saga cuneo  Confirming hepatocyte protective effect

To confirm the liver protective effect of sagarchromenol, cell viability was measured by MTS assay after treatment with 0.5 mM t-BHP and different concentrations of sagarchromenol for 24 hours. The results are shown in Fig.

As shown in Fig. 27, sagarchromenol enhanced the death of hepatocytes by t-BHP at 0.5 uM, and no change was observed at the concentrations above. These results demonstrate that there is a significant cytoprotective effect on hepatocyte injury by low concentrations of sagarchomonol t-BHP.

7-3. t- By BHT  For induced cells Saga cuneo  Confirmation of lipid peroxidation inhibitory effect

When cells are treated with t-BHP, excess superoxide is produced, and the produced superoxide reacts with intracellular lipids to produce lipid peroxides. The amount of lipid peroxide produced was analyzed by malondialdehyde method. The results are shown in Fig.

As shown in FIG. 28, it was found that the peroxide generated in excess by t-BHP was reduced by 1 to 2 μM of sagacromenol. It can be seen that the antioxidant activity of sagarchromene inhibits lipid peroxidation by removing ROS.

7-4. t- By BHT  For induced cells Saga cuneo  Expression induction of antioxidant enzyme

The cultured liver cells were treated with 0.5 mM t-BHP and sanguinone acid at different concentrations for 1 hour. The cultured cells were collected, dissolved in a cell lysis buffer, and centrifuged to obtain a cell suspension. The separated proteins were analyzed by Western blotting to confirm the expression level of pNrf2. In order to confirm the expression level of NQO-1 and HO-1, the cells were cultured for 18 hours under the same conditions as above. Cells were collected and lysed with cell lysis buffer, and centrifuged to obtain a cell suspension. Protein expression of NQO-1 and HO-1 was confirmed by Western blotting using the separated proteins. The results are shown in Fig.

As shown in FIG. 29, NQO-1 and HO-1 in the cytoplasm, an antioxidant enzyme, were increased in a concentration-dependent manner by saga-quinoxaline treatment. Expression of NQO-1 and HO-1 is regulated by their transcription factor, Nrf2. As a result, the expression level of pNrf2 was increased in a concentration dependent manner by sagarchromenol treatment. These results suggest that sagarchromene induces the activation of pNrf2 and induces the expression of NQO-1 and HO-1, which are lower proteins thereof.

Claims (12)

A pharmaceutical composition for liver protection comprising an extract of Tootsumabum or a fraction thereof as an active ingredient. The composition according to claim 1, wherein the extract is extracted with at least one solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, water and hexane. The composition according to claim 2, wherein the lower alcohol having 1 to 4 carbon atoms is ethanol. A composition for improving liver function or liver function comprising an extract of Tootsum matsumana or a fraction thereof as an active ingredient. A pharmaceutical composition for the prevention or treatment of liver diseases comprising extracts of Tootsum matsumana or a fraction thereof as an active ingredient. 6. The method of claim 5, wherein the liver disease is selected from the group consisting of non-alcoholic fatty liver, alcoholic fatty liver, nonalcoholic fatty liver, fatty liver, nonfat hepatitis, end-stage fibrosis liver disease, nonviral chronic hepatitis, liver cirrhosis, Alcoholic liver damage, acute liver injury, and liver cancer. A food composition for preventing or ameliorating a liver disease comprising extracts of Tootsumabum or a fraction thereof as an active ingredient. A pharmaceutical composition for liver protection comprising at least one compound selected from the group consisting of sagahydroquinoic acid, sagaquinoic acid and sagachromenol as an active ingredient. 9. The composition of claim 8, wherein the compound is a compound separated from the sawmill. 1. A food composition for improving liver function or liver function comprising as an active ingredient at least one compound selected from the group consisting of sagahydroquinoic acid, sagaquinoic acid and sagachromenol. A pharmaceutical composition for the prevention or treatment of liver disease comprising as an active ingredient at least one compound selected from the group consisting of sagahydroquinoic acid, sagaquinoic acid and sagachromenol. A pharmaceutical composition for preventing or ameliorating liver disease comprising, as an active ingredient, at least one compound selected from the group consisting of sagahydroquinoic acid, sagaquinoic acid and sagachromenol.

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