KR100919625B1 - The composition comprising the extract or purified extract of Magnolia cortex for preventing and treating fatty liver diseases, and a method for preparing purified extract - Google Patents

Abstract

The invention Magnolia (Magnolia cortex) extract, fractions or extracts thereof as an active ingredient, the composition for the prevention and treatment of fatty liver disease, and more specifically, the extract of the thick extract extracted with water, alcohol or a mixture thereof as a solvent, fractions thereof and Extraction tablets increase the contents of S-adenosyl-L-methionine and glutathione in the liver to inhibit liver damage and to inhibit triglyceride accumulation in liver tissue. By confirming that it can be usefully used as a composition for the prevention and treatment of fatty liver disease.

Description

The composition comprising the extract or purified extract of Magnolia cortex for preventing and treating fatty liver diseases, and a method for preparing purified extract }

The invention Magnolia (Magnolia cortex) The present invention relates to a composition for the prevention and treatment of fatty liver disease containing the extract, fractions or extracts thereof as an active ingredient.

The liver is a large organ located in the upper, right abdominal cavity of the body and weighs about 1.5 kg in adults. The normal liver is visually reddish and the surface is smooth. The shape and size of the liver will change as the liver develops. As a representative example, if you drink a lot of alcohol to accumulate oil in the liver, the size of the liver is increased, and the oily color is yellowish. Conversely, as cirrhosis develops, the size of the liver decreases and the surface becomes rugged.

The liver is a very important organ that has a variety of functions. First, the liver functions to properly process the metabolism of various nutrients in our body. Foods absorbed from the intestine are properly changed in the liver for use in various tissues of our bodies, and nutrients are used in the various tissues, and the remaining wastes are transported back to the liver for disposal. Second, the liver functions to store some of the nutrients our bodies need. Third, bile acids, which are essential for the absorption of nutrients from the intestine, are made and released into the intestine through the biliary tract. If these bile acids are not produced, they will not be absorbed by various nutrients, resulting in malnutrition. Fourth, the body functions to make substances that are absolutely necessary for proper functioning (albumin, coagulation protein, cholesterol, etc.). Finally, it is the action of detoxifying alcohol (alcohol), drugs, and various toxins in our body (Eugene Brainward, et al. Harrison 's principles). of internal medicine . 15 th edition . 2001).

The mortality rate of liver disease in Korea is very high, with 23.5 deaths per 100,000 population (37.8 males, 9.0 females), the number one cause of death in the 40s (41.1 / 100,000), and the second cause of death in the 50s (72.4 / 100,000). In fact, liver disease is the leading cause of death among middle-aged Koreans. In particular, alcoholic liver disease occurs when a person drinks 40 to 80 g of alcohol for about 10 years and can appear in most of the overactive drinkers (two hops containing a bottle of soju, 90 g alcohol; 1,000 cc beer, 40 g alcohol). The majority of chronic overdrinkers (90-100%) have fatty liver, alcoholic hepatitis in 10-35%, and cirrhosis in 8-20%. Alcoholic fatty liver, etc., is recognized as a disease that is naturally cured by nutritional control after this week, and there is no special treatment. However, the fatal liver patients have a 30% chance of developing cirrhosis within 10 years. Percentage of alcoholic cirrhosis patients is 5 years survival rate is less than 40%, chronic liver disease caused by alcohol is an important part of the mortality rate of liver disease in Korea. In addition, Korea is a prevalent area of hepatitis B, which is a serious problem for national health, with the mortality rate from chronic liver disease and liver cancer accounting for 10% of the total cause of death (National Statistical Office, Statistical yearbook of death cause).

Hug ( Magnolia Cortex on Japanese Thick Magnolia obovata Thunberg , Magnolia officinale Rehder et Wils and Machilus thunbergii Sieb . Et Zucc ), and the hulk contains 1% of essential oils, and the components of the essential oils include β-odesmol, β-eudesmol, γ-eudesmol and α-β-pinene ) Is known. Phenol Magnolol, Honokiol, Alkaloid Magnocurarine, Magnoflorine, Michelarbine, Anonaine, Liriodenin may also contain (liriodenine) (jeongboseop et al., illustrates hyangyak Dictionary,, pp.470, 1989 per Younglim).

The development of alcoholic liver disease is a complex action of several processes. The key part is that alcohol intake increases the permeability of endotoxin in the intestine, which stimulates and activates kupffer cells, macrophages in the liver. When activated, the cooper cells in the liver release various cytokines and free radicals, and these inflammatory mediators attack the liver cells, resulting in a complex damaging effect (Michael D, et al., Alcohol Research & Health. Vol . 27, No. 4 , 2003). Thus, inhibiting the activation of macrophages in the liver can reduce the symptoms of several alcoholic liver diseases, including fatty liver (Jarvelainen HA et al., Journal of Hepatology , 32 (6): 900-910, 2000).

S-adenosyl-L-methionine (SAM) is a cysteine precursor and is essential for the synthesis of glutathione, which plays many important functions in the liver and has a major antioxidant activity in the liver. In particular, it is extremely important for detoxifying the toxicity of free radicals produced by alcohol, and it is an essential substance in the methylation process in the body, and detoxifies the toxin through methylation (Charles S Lieber., Am J Clin Nutr . 76, Nov, 2002). It is also involved in the methylation of phospholipids, restoring the function of damaged cell membranes, thereby promoting the transport of fat from the liver, thus affecting fatty liver formation (Hirata F, Science 209 : 1082-1090, 1980; Hirata F et al. ., Proc Natl Acad Sci USA 75: 1718-1721, 1978). Excessive intake of ethanol causes metabolic abnormalities of SAM in the liver and decreases its content. Increasing SAM can prevent fatty liver caused by ethanol (Anthony J et al., Alcohol Clin Exp Res , Vol 21, No 6, p 1100-1102, 1997).

Considering Korea's unique drinking culture, it is necessary to treat early alcoholic liver disease properly in order to reduce the mortality rate of liver disease. However, the treatment of alcoholic liver disease has not been developed in Korea. Therefore, in recent years, efforts have been made to find a substance that can treat alcoholic fatty liver disease.

Therefore, the inventors of the inventor ( Magnolia) officinalis REHD. et WILS.) extract, fractions or extracts thereof increase the contents of S-adenosyl-L-methionine and Gluthathione in the liver to inhibit liver damage, The present invention was completed by confirming that the triglyceride accumulation inhibitory effect can be usefully used as a composition for preventing and treating fatty liver disease.

An object of the present invention is to provide a composition for the prevention and treatment of fatty liver disease, containing a thick extract, fractions or extracts thereof as an active ingredient.

In order to achieve the above object, the present invention provides an agent for the prevention and treatment of fatty liver disease, which comprises a thick extract extracted with water, alcohol or a mixture thereof as a solvent as an active ingredient.

In addition, the present invention provides an agent for preventing and treating fatty liver disease, which contains the ethyl acetate fraction of the hoobac extract as an active ingredient.

In addition, the present invention provides a drug for preventing and treating fatty liver disease comprising the active fraction obtained by adsorbing the thick ethyl acetate fraction to silica gel column chromatography and applying a step concentration gradient consisting of n-hexane: ethyl acetate as an active ingredient. to provide.

In addition, the present invention provides a method for producing a thick extract extract tablets by purifying the thick extract.

The present invention also provides an agent for preventing and treating fatty liver disease, which contains the thick extract extracted tablet prepared by the above method as an active ingredient.

In addition, the present invention provides a health food for the prevention and improvement of fatty liver disease, containing a thick extract, fractions or extracts thereof as an active ingredient.

Thicken extract, fractions or extracts thereof of the present invention is to increase the content of S-adenosyl-L-methionine, glutathione in the liver to inhibit liver damage, liver tissue By confirming the effect of inhibiting the accumulation of triglycerides in the inside can be usefully used as a fatty liver prevention and treatment, and health food.

1 is a diagram showing the tissue of the liver through H & E staining, when the ethanol extract of the hunchus in chronic ethanol administration rats,

Figure 2 is a diagram showing the liver tissue through Oil Red O staining, when the ethanol extract of the hunchus in chronic ethanol administration rats,

3 is a graph showing the TLC analysis of the thick ethanol extract tablets.

Hereinafter, the present invention will be described in detail.

The present invention provides an agent for preventing and treating fatty liver disease, which contains the pear extract, ethyl acetate fraction thereof and the active fraction thereof as an active ingredient.

The thick extract of the present invention, the ethyl acetate fraction and the active fraction thereof can be prepared by the following method.

1) dried grommets ( Magnolia cortex) by adding water, alcohol or a mixture thereof to obtain a thick extract;

2) dispersing the thick extract in water and then partitioning with ethyl acetate to obtain an ethyl acetate fraction; And

3) adsorbing the ethyl acetate fraction on silica gel column chromatography to obtain an active fraction obtained by applying a concentration gradient consisting of n-hexane: ethyl acetate,

In the manufacturing method, the thick of step 1) ( Magnolia cortex is Japanese nobular ( Magnolia) obovata Thunberg , Magnolia officinale Rehder et Wils , Machilus thunbergii Sieb . et Zucc ) is characterized in that any one selected from the group consisting of, can be used without limitation, such as cultivated or commercially available.

In the above production method, the alcohol of step 1) is preferably a C ₁ to C ₄ lower alcohol, more preferably lower alcohol is ethanol or methanol, most preferably ethanol. It is preferable to extract water, alcohol or mixtures thereof by adding 2 times to 10 times the weight of the thickness after extracting the dried thick gourd during extraction, and more preferably extracting it by adding 3 times to 5 times but not limited thereto. Do not. The extraction temperature is preferably 30 ° C to 100 ° C, more preferably 80 ° C to 100 ° C, but is not limited thereto. The extraction time is preferably 1 hour to 3 hours, more preferably 2 hours to 3 hours, but is not limited thereto. The extraction method may be any of cold needle, ultrasonic extraction or reflux cooling extraction method, it is preferable to use a reflux cooling extraction method. The number of extraction is preferably 1 to 5 times, more preferably 4 times, but is not limited thereto. After extracting the thick gourd by the above method, the obtained extract was filtered and concentrated under reduced pressure to obtain a thick gourd extract.

In addition, the present invention provides a method for producing a thick extract extract tablets by purifying the thick extract.

1) dried grommets ( Magnolia cortex) by adding water, alcohol or a mixture thereof to obtain a thick extract;

2) obtaining the filtrate after suspending the thick extract of step 1) in an alcohol or an aqueous alcohol solution;

3) step 2) adding distilled water to the filtrate and mixing and adjusting the pH to 2.0 to 6.0 to obtain a precipitate produced;

4) It provides a method for producing a thick extract extract comprising the step of dissolving the precipitate of step 3) in an alcohol or an aqueous alcohol solution and then concentrated under reduced pressure to obtain a thick extract extract.

In the above production method, the thick gourd extract of step 1) is the same as the preparation method of the thick gourd extract described above.

In the above production method, the alcohols of steps 1), 2) and 4) are preferably C ₁ to C ₄ lower alcohols, more preferably lower alcohols are ethanol or methanol, most preferably ethanol.

In the above production method, pH 2.0 to 6.0 of step 3) is the pH is most preferably 4.0, the pH of the adjustment can be used both acetic acid, hydrochloric acid, sulfuric acid or organic acid, it is preferable to use acetic acid.

The present invention also provides an agent for preventing and treating fatty liver disease, which contains the thick extract extracted tablet prepared by the above method as an active ingredient.

The thick extract extract tablet of the present invention is analyzed by thin layer chromatography (Thin-Layer Chromatography, TLC), it characterized in that the content of the Honokiol (magnolol) component and magnolol (Honokiol) compared to the thick extract It is effective in preventing and treating fatty liver.

In the present invention, the fatty liver is any one selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, nutrient fatty liver, hunger fatty liver, obese fatty liver, diabetic fatty liver.

In the present invention, the effect of hakbak extract, fractions or extracts thereof on the production of cytokines and active oxygen components in the treatment of lipopolysaccharide (LPS) which is a kind of ethanol or endotoxin in rodent macrophages.

In the present invention, in order to determine the effect of the bakbak extract, fractions or extracts thereof on reducing cell viability by ethanol or LPS, rodent macrophages were treated with LPS and ethanol, respectively, and bakbak extracts, fractions or extracts thereof were used. After treatment with different concentrations, the absorbance was measured with an ELISA reader. As a result, it was found that cell viability by LPS and ethanol was increased as the concentration of the extract of gourd extract, its fraction or extract tablet was increased.

In addition, in the present invention, in order to determine the effect of the extract after extract, its fractions or extracts on the production of TNF-α, rodent macrophages were treated with LPS and ethanol, respectively, and the concentration of the extract after extract, its fractions or extracts After different treatments, the TNF-α production inhibition rate was measured using the TNF-α kit. As a result, it was found that the higher the concentration of thick extract, fraction or extract treatment, increased the inhibition rate of TNF-α production.

In addition, in the present invention, LPS and ethanol in rodent macrophages in order to determine the effect of the extract, extracts or purified tablets on the active oxygen species (ROS), nitrogen monoxide (NO) and NADPH oxidase activity After treatment with each of the thick extract, fractions or extracts thereof was treated at different concentrations, the absorbance was measured with an ELISA reader. As a result, it was found that the higher the concentration of thick extract, fraction or extract treatment, inhibited the production of ROS and NO, and also increased the rate of inhibition of NADPH oxidase activity.

In addition, in the present invention, in order to determine the effect of the pear extract, fractions or extracts thereof on the production of peroxide anion, rodent macrophages were treated with LPS and ethanol, respectively, and the concentration of the pear extract, fractions or extracts thereof was determined. After the treatment was performed differently, the amount of cytochrome was reduced by adding a cytochrome c solution, and the amount of anion peroxide produced was measured by using a microplate reader. As a result, it was found that the higher the concentration of the thick extract, fractions thereof or the extraction tablet treatment, the higher the rate of inhibition of peroxide production.

Therefore, it can be seen that the thick extract, fractions or extracts thereof of the present invention inhibit liver damage caused by alcohol intake.

In addition, in the present invention, the effect of the extract of bakbak, fractions or extracts thereof on the activity of ALT (S-GPT) (ALanine Transaminase (Glutamate Pyruvate Transaminase)) enzyme activity in blood of experimental animals was measured. In general, ALT is reported to be slightly elevated in alcoholic fatty liver (Tomita K. et al., Alcohol Clin Exp Res . 29 (12 Suppl): p. 240-245, 2005). After administration of the thick extract, fraction or extract of the present invention together with ethanol to the blood of male wister rats, respectively, was measured by the method of Reitman-Frankel using an ALT kit. It was. As a result, the group treated with extract, fraction or extract tablets of the extract was similar in ALT enzyme activity compared to the group treated with S-Adenosyl-L-methionine (SAM), which is known to inhibit fatty liver. It was found that the more inhibited, the higher the concentration of the extract, fractions or extracts thereof, the higher the inhibitory effect.

       In addition, in the present invention, in order to determine the effect of hoobak extract, fractions or extracts thereof on the fatty liver formation by the ethanol and the accumulation of triglycerides in liver tissue in the experimental animal, the liver of the experimental animal is extracted and the present invention together with ethanol Thick extract, its fractions or extracts were administered, respectively, and observed through staining and kits. As a result, it was confirmed that the hufa extract, fractions or extracts treated group significantly reduced the fatty liver findings by ethanol and triglyceride accumulation in liver tissue. Therefore, it was found that the thick extract of the present invention, fractions or extracts thereof may have an effect of treating alcoholic fatty liver in animal experiments.

In addition, in the present invention, in order to determine the effect of the extract, extracts or tablets thereof on the change of S-Adenosyl-L-methionine (SAM) in the laboratory animals, liver of the experimental animals is extracted The concentration of SAM was measured through HPLC after the administration of the thick extract, fractions or extracts thereof of the present invention together with ethanol. As a result, it was confirmed that the group treated with thick extract, fractions thereof, or extract tablets increased the concentration of SAM. S-adenosyl-L-methionine (SAM) is a cysteine precursor and is essential for the synthesis of glutathione, which plays many important functions in the liver and has a major antioxidant activity in the liver. In particular, it is extremely important to detoxify the toxicity of free radicals produced by alcohol, and it is an essential substance in the methylation process in the body, and serves to detoxify toxins through methylation. Therefore, it was found that the extract of habakkuk, fractions or extracts thereof showed an effect of inhibiting liver damage by increasing the concentration of SAM in liver tissues lowered by ethanol.

In addition, in the present invention, in order to determine the effect of the extract of the bakbak, fractions or extracts thereof on the change of glutathione in the experimental animal, extract of the liver of the experimental animal and ethanol extract of the present invention, fractions thereof Alternatively, the concentrations of glutathione were measured using HPLC separation and fluorometric detection after administration of the extracts. As a result, it was confirmed that thick extract, fractions or extracts thereof increased the concentration of glutathione. Glutathione is one of the strongest antioxidants and protects the liver from free radicals produced from the administration of ethanol. Therefore, it could be seen that the thick extract, fractions or extracts thereof may inhibit hepatotoxicity by ethanol by increasing the concentration of glutathione.

The prophylactic and therapeutic agent for fatty liver disease of the present invention preferably comprises 0.1 to 50% by weight of the extract, fractions or extracts thereof based on the total weight of the composition. However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

The prophylactic and therapeutic agent for fatty liver disease of the present invention may optionally contain one or more in a groat extract, fractions or extracts thereof, and in addition to the above components, suitable carriers, excipients and the like conventionally used in the manufacture of pharmaceutical compositions and It may further comprise a diluent.

The thick extract, fractions or extracts thereof of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical formulations.

      The prophylactic and therapeutic agents for fatty liver disease of the present invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be additionally included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate (eg, starch extract, fractions or extracts thereof). It is prepared by mixing calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

      Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose. The effective dose of the therapeutic agent of the present invention is 0.0001 to 10 g / kg, preferably 0.0001 g to 5 g / kg, may be administered 1 to 6 times a day.

In addition, the present invention provides a health food for preventing and improving fatty liver disease, comprising a thick extract, fractions or extracts thereof as an active ingredient prepared by the manufacturing method.

In addition, the present invention provides a health food for preventing and improving fatty liver disease, comprising a thick extract, characterized in that it contains a large amount of Honokiol (Honokiol) and magnolol (magnolol) as an active ingredient or fractions thereof. do.

The thick extract of the present invention, fractions or extracts thereof may be added as is or used in combination with other foods or food ingredients, and may be suitably used according to conventional methods. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the pharmaceutical compositions of the present invention are added in an amount of up to 15 parts by weight, preferably up to 10 parts by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

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

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the thick extract of the present invention, fractions or extracts thereof of the present invention, various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers , Preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the thick extract of the present invention, fractions or extracts thereof may contain the flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is usually 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.

       Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Formulation Examples.

      However, the following Examples, Experimental Examples, and Formulation Examples specifically illustrate the present invention, and the content of the present invention is not limited to Examples, Experimental Examples, and Formulation Examples.

< Example  1> Preparation of Thick Extract

<1-1> Preparation of Thicken Ethanol Extract

      1.2 kg of dried dried gourd, which was purchased from University of Korea, Iksan-si, Jeollabuk-do, was cut into pieces, and 5l of ethanol was added thereto, followed by extraction with reflux cooling at 100 ° C for 2 hours. The reflux cooled extract was filtered using filter paper (Advantec no. 2 filter paper), and then the filtrate was concentrated under reduced pressure using an evaporator (EYELA, N-1000, Japan), followed by drying. Obtained.

<1-2> Preparation of a water extract of a thick pepper

In the manufacturing method of <1-1>, 42.5 g of water extract of hoobak was obtained by the same method except that water was used instead of ethanol.

<1-3> Preparation of a thick extract methanol extract

In the preparation method of <1-1>, 50.2 g of methanol extract of thick was obtained by the same method except that methanol was used instead of ethanol.

< Example  2> Thick ethyl acetate Fraction  Produce

The thick ethanol extract (48.3 g) prepared in Example <1-1> was dispersed in water, and then partitioned into ethyl acetate, and the ethyl acetate soluble portion (26.5 g) was concentrated under reduced pressure to obtain ethyl acetate fraction (15.4 g). Obtained.

< Example  3> Thick ethyl acetate From fractions  activation Fraction  Produce

The fraction of Example 2 (15 g) was obtained from Bang et al., Arch . Pharm . Res . 23: 46-49, 2000) to obtain an active fraction (8.3 g) obtained by adsorption on silica gel column chromatography and applying a step concentration gradient consisting of n-hexane: ethyl acetate.

< Example  4> From Thick Extract Extraction tablet  Produce

Thick ethanol extract (48.3 g) of Example <1-1> was dissolved in ethanol (250 ml), filtered to remove insoluble matters, distilled water (1000 ml) was added to the filtrate and mixed, and then acetic acid was added dropwise to pH 4.0. The precipitate was then produced. The precipitate was filtered to remove the filtrate, and the obtained residue (precipitate) was dissolved in ethanol (500 ml) and filtered again to concentrate the filtrate under reduced pressure to give a thick extract extract (19.3 g).

< Example  5> thick Extraction tablet  Ingredient analysis

2 mg each of the thick extract extract tablets obtained in <Example 4>, a standard of honokiol and magnolol (retained after separation and identification of the herbal medicine laboratory of Wonkwang University College of Pharmacy, respectively), known as the main ingredient of the thick pepper, were each accurately taken, and methanol (1 ml). After dissolving in the form of these samples, these samples were observed through thin layer chromatography (TLC). TLC used silica gel 60 F ₂₅₄ (manufactured by Merck) as a carrier, hexane-acetone (3: 1) as a developing solvent, and ultraviolet 254 nm as a detector. As a result, it was found that the thick extract extract contained honokiol and magnolol (see FIG. 3).

< Experimental Example  1> Thick extract, its Fraction  And Extraction tablet  Measure the effect on cell viability

<1-1> Cell Culture

Rodent macrophages, RAW264.7 cells (American Type Culture Collection; purchased from ATTC) were cultured in RPMI1640 medium (GIBCO BRL) containing 10% FBS, 100 U / ml penicillin, 100 mg / ml streptomycin. . Cells were cultured in a 37 ° C., 5% CO 2 incubator and cultured cells were tested by dispensing 1 ml (2 × 10 ⁵ / ml) cells into 24-wells.

<1-2> RAW  264.7 cells LPS  process

After dispensing 1 ml (2 × 10 ⁵ / ml) cells into the 24-wells, the cells were stabilized in a CO ₂ cell incubator for at least 12-16 hours until the cells were completely attached to the 24-wells and shaped properly. The cells were then treated with 25 ng / ml LPS or treated with LPS and 1 μg / ml, 5 μg / ml, 10 μg / ml, 50 μg / ml thick extract, fractions or extracts thereof.

<1-3> RAW  Ethanol Treatment of 264.7 Cells

After dispensing 1 ml (2 × 10 ⁵ / ml) cells into the 24-wells, the cells were stabilized in the CO2 cell culture incubator for at least 12-16 hours until the cells were completely attached to the 24-wells and shaped properly. 125 mM ethanol was then treated with the cells or ethanol and 1 μg / ml, 5 μg / ml, 10 μg / ml, 50 μg / ml thick extract, fractions or extracts thereof. The ethanol was purchased from Merck, and when used for cell treatment, 99.8% of ethanol was dehydrated to obtain a purity close to 100%.

<1-4> RAW  Measuring Cell Viability of 264.7 Cells

Experiments were performed as follows using the method described in Monks, A. et al., J. Natl . Cancer Inst . , 83, 1991 to determine the viability of RAW 264.7 cells. Namely, MTT (3- [4,5-dimethylthiazole-2-yl] -2,5-diphenyl-tetrazolium bromide) (Sigma) after reagent treatment according to the method of <1-2> and <1-3>. Chemical) solution (5 mg / ml in PBS) was added so that the final concentration to 100 μg / ml. Determination of cell viability was followed by shaking for 15 minutes by adding DMSO (Sigma Chemical Co., Ltd.) to dissolve insoluble purple formazan produced by living cells after 4 hours of MTT treatment, followed by shaking at 540 nm with an ELISA analyzer. It was determined by measuring the absorbance at. Only the control medium was added to the sole medium solution, the cell viability was calculated according to the following equation (1).

As a result, as shown in Table 1 to Table 2, the survival rate of RAW 264.7 cells treated with thick extract extract was treated with LPS and treated with 86.4 ± 1.4% (P <0.001) and ethanol. Was 70.2 ± 2.4% (P <0.01), and the treatment of thick extract and fraction also inhibited the decrease in survival rate.

Experimental group Cell survival rate (%) Control 100 ± 3.2 LPS treatment group 53.4 ± 0.4 a LPS + Thick ethanol extract (1 μg / ml) 59.3 ± 2.4 LPS + Thick ethanol extract (5 µg / ml) 70.7 ± 0.9 b LPS + Thick ethanol extract (10 µg / ml) 85.0 ± 0.7 b LPS + Thick ethanol extract (50 µg / ml) 86.4 ± 1.4 b LPS + Thick ethanol extract (1 μg / ml) 52.3 ± 2.4 LPS + Thick ethanol extract (5µg / ml) 66.7 ± 0.8 b LPS + Thick ethanol extract (10 µg / ml) 75.0 ± 0.5 b LPS + Thicken Ethanol Extract (50µg / ml) 81.4 ± 7.4 b LPS + Thick ethyl acetate fraction (5 μg / ml) 68.4 ± 1.2 b LPS + thick active fraction (5 μg / ml) 72.4 ± 1.1 b a: P <0.001; There was a statistically significant difference compared to the control group b: P <0.001; There is a statistically significant difference compared to the LPS treatment group.

Experimental group Cell survival rate (%) Control 102 ± 2.5 Ethanol treatment group 50.0 ± 4.4 a Ethanol + Thick ethanol extract (1 µg / ml) 40.8 ± 1.1 Ethanol + Thick ethanol extract (5 µg / ml) 45.7 ± 1.9 Ethanol + Thickened Ethanol Extract Purification (10µg / ml) 53.9 ± 1.8 Ethanol + Thick ethanol extract (50 µg / ml) 70.2 ± 2.4 b Ethanol + Thicken Ethanol Extract (1µg / ml) 35.8 ± 1.0 Ethanol + Thicken Ethanol Extract (5µg / ml) 49.4 ± 1.1 Ethanol + Thicken Ethanol Extract (10µg / ml) 55.2 ± 1.8 Ethanol + Thicken Ethanol Extract (50µg / ml) 65.2 ± 2.1 b Ethanol + Thick ethyl acetate fraction (5 µg / ml) 62.7 ± 1.1 Ethanol + thick active fraction (5 μg / ml) 64.7 ± 1.3 a: P <0.001; There is a statistically significant difference compared to the control group b: P <0.01; Statistically significant difference compared to ethanol treatment group.

< Experimental Example  2> RAW  264.7 cells TNF -α production inhibition rate measurement

TNF-α kit (R & D) was used to measure the inhibition rate of TNF-α production during the treatment of the extracts, fractions or extracts thereof in RAW 264.7 cells of Experimental Examples <1-2> and <1-3>. The measurement was carried out according to the kit manual.

As a result, as shown in Tables 3 to 4, the TNF-α production inhibition rate of RAW 264.7 cells treated with the thick ethanol extract was treated with LPS and 77.5 ± 1.6 (P <0.001) and ethanol. The group treated with 30.1 ± 2.4 (P <0.001) also showed increased inhibition of TNF-α production when treated with thick extract or fraction.

Experimental group % Inhibition of TNF-α production Control - LPS treatment group - LPS + Thick ethanol extract (1 μg / ml) 69.5 ± 1.8 a LPS + Thick ethanol extract (5 µg / ml) 66.2 ± 0.7 a LPS + Thick ethanol extract (10 µg / ml) 75.7 ± 0.6 a LPS + Thick ethanol extract (50 µg / ml) 77.5 ± 1.6 a LPS + Thick ethanol extract (1 μg / ml) 54.5 ± 1.2 a LPS + Thick ethanol extract (5µg / ml) 60.2 ± 0.6 a LPS + Thick ethanol extract (10 µg / ml) 70.7 ± 0.4 a LPS + Thicken Ethanol Extract (50µg / ml) 72.5 ± 1.1 a LPS + Thick ethyl acetate fraction (5 μg / ml) 66.0 ± 1.6 a LPS + thick active fraction (5 μg / ml) 68.2 ± 1.2 a a: P <0.001; There is a statistically significant difference compared to the LPS treatment group.

Experimental group % Inhibition of TNF-α production Control - Ethanol treatment group - Ethanol + Thick ethanol extract (1 µg / ml) 3.5 ± 1.2 Ethanol + Thick ethanol extract (5 µg / ml) 12.1 ± 0.4 a Ethanol + Thickened Ethanol Extract Purification (10µg / ml) 25.9 ± 5.0 a Ethanol + Thick ethanol extract (50 µg / ml) 30.1 ± 2.4 a Ethanol + Thicken Ethanol Extract (1µg / ml) 3.0 ± 1.2 Ethanol + Thicken Ethanol Extract (5µg / ml) 11.2 ± 0.4 a Ethanol + Thicken Ethanol Extract (10µg / ml) 20.8 ± 5.0 a Ethanol + Thicken Ethanol Extract (50µg / ml) 26.1 ± 2.4 a Ethanol + Thick ethyl acetate fraction (5 µg / ml) 13.2 ± 0.4 a Ethanol + thick active fraction (5 μg / ml) 14.6 ± 0.8 a a: P <0.001; Statistically significant difference compared to ethanol treatment group.

< Experimental Example  3> RAW  Free radicals of cells ROS ) Inhibition of production

In order to measure the inhibitory rate of ROS generation, the cells were treated with 1 mM dichlorofluorine when RAW 264.7 cells of Experimental Examples <1-2> and <1-3> were treated with thick extract, fraction or extract. After treatment with diacetate (dichlorofluorescin diacetate, DCFH-DA) and left for 30 minutes at 37 ℃. After treatment with different concentrations of TNF-α, fluorescence was measured by setting the excitation filter 485nm, emission filter 530nm and chamber temperature 37 ℃ in the Elisa reader at regular intervals. Rosenkranz AR, et al., J Immunol Methods . 156: 39-45, 1992).

As a result, as shown in Tables 5 to 6, the inhibitory rate of reactive oxygen production of RAW 264.7 cells treated with thick ethanol extract was 55.2 ± .1.5% (P <0.001) in the group treated with LPS. ) And ethanol showed 37.0 ± 0.3% (P <0.001), and the inhibitory rate of reactive oxygen production was also increased when the extract and fraction were treated.

Experimental group ROS generation inhibition rate (%) Control - LPS treatment group - LPS + Thick ethanol extract (1 μg / ml) -1.4 ± 0.5 LPS + Thick ethanol extract (5 µg / ml) 19.1 ± 1.2 a LPS + Thick ethanol extract (10 µg / ml) 29.7 ± 1.9 b LPS + Thick ethanol extract (50 µg / ml) 55.2 ± 1.5 b LPS + Thick ethanol extract (1 μg / ml) -0.4 ± 0.5 LPS + Thick ethanol extract (5µg / ml) 10.1 ± 1.1 a LPS + Thick ethanol extract (10 µg / ml) 24.2 ± 1.7 b LPS + Thicken Ethanol Extract (50µg / ml) 40.2 ± 1.5 b LPS + Thick ethyl acetate fraction (5 μg / ml) 18.2 ± 1.1 a LPS + thick active fraction (5 μg / ml) 19.2 ± 1.5 a a: P <0.01; There was a statistically significant difference compared to the LPS treatment group. B: P <0.001; There is a statistically significant difference compared to the LPS treatment group.

Experimental group ROS generation inhibition rate (%) Control - Ethanol treatment group - Ethanol + Thick ethanol extract (1 µg / ml) -9.4 ± 0.2 a Ethanol + Thick ethanol extract (5 µg / ml) -2.8 ± 1.0 b Ethanol + Thickened Ethanol Extract Purification (10µg / ml) 13.8 ± 0.7 c Ethanol + Thick ethanol extract (50 µg / ml) 37.0 ± 0.3 c Ethanol + Thicken Ethanol Extract (1µg / ml) -6.4 ± 0.2 a Ethanol + Thicken Ethanol Extract (5µg / ml) -0.8 ± 1.2 b Ethanol + Thicken Ethanol Extract (10µg / ml) 10.8 ± 0.5 c Ethanol + Thicken Ethanol Extract (50µg / ml) 32.2 ± 1.1 c Ethanol + Thick ethyl acetate fraction (5 µg / ml) 5.8 ± 0.7 b Ethanol + thick active fraction (5 μg / ml) 6.2 ± 0.5 b a: P <0.01; There was a statistically significant difference compared to the ethanol treatment group. B: P <0.05; There was a statistically significant difference compared to the ethanol treated group. C: P <0.001; Statistically significant difference compared to ethanol treatment group.

< Experimental Example  4> RAW  Nitric oxide (264.7 cells) NO ) Inhibition of production

In order to know the inhibition rate of nitric oxide (NO) production, the amount of NO produced was measured when the raw 264.7 cells of Experimental Examples <1-2> and <1-3> were treated with thick extract, fraction or extract. NO production was measured by the amount of nitrite released from the cells. After the same amount of grease reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride, 2.5% phosphoric acid) was mixed in the medium for 10 minutes, the absorbance was measured at 540 nm (Ridnour LA, et al. , Anal Biochem . 281: 2000, 223-9).

As a result, as shown in Table 7 and Table 8, the inhibition rate of nitric oxide (NO) production in RAW 264.7 cells treated with thick ethanol extract tablets was 80.2 ± 0.6% (P <0.001) in the group treated with LPS. And 67.0 ± 0.3% (P <0.001) in the group treated with and ethanol, the treatment of the extract and fractions also increased the inhibition rate of nitrogen monoxide production.

Experimental group NO production inhibition rate (%) Control - LPS treatment group - LPS + Thick ethanol extract (1 μg / ml) 6.3 ± 1.7 LPS + Thick ethanol extract (5 µg / ml) 26.2 ± 1.5 a LPS + Thick ethanol extract (10 µg / ml) 38.3 ± 1.3 a LPS + Thick ethanol extract (50 µg / ml) 80.2 ± 0.6 a LPS + Thick ethanol extract (1 μg / ml) 5.0 ± 1.2 LPS + Thick ethanol extract (5µg / ml) 22.2 ± 1.5 a LPS + Thick ethanol extract (10 µg / ml) 30.3 ± 1.1 a LPS + Thicken Ethanol Extract (50µg / ml) 68.4 ± 0.6 a LPS + Thick ethyl acetate fraction (5 μg / ml) 24.6 ± 0.8 a LPS + thick active fraction (5 μg / ml) 25.4 ± 1.5 a a: P <0.001; There is a statistically significant difference compared to the LPS treatment group.

Experimental group NO production inhibition rate (%) Control - Ethanol treatment group - Ethanol + Thick ethanol extract (1 µg / ml) 2.3 ± 0.7 Ethanol + Thick ethanol extract (5 µg / ml) 16.2 ± 1.5 a Ethanol + Thickened Ethanol Extract Purification (10µg / ml) 28.3 ± 1.3 a Ethanol + Thick ethanol extract (50 µg / ml) 67.0 ± 0.3 a Ethanol + Thicken Ethanol Extract (1µg / ml) 5.0 ± 1.2 Ethanol + Thicken Ethanol Extract (5µg / ml) 2.2 ± 0.5 a Ethanol + Thicken Ethanol Extract (10µg / ml) 10.3 ± 1.1 a Ethanol + Thicken Ethanol Extract (50µg / ml) 28.4 ± 0.6 a Ethanol + Thick ethyl acetate fraction (5 µg / ml) 24.6 ± 0.8 a Ethanol + thick active fraction (5 μg / ml) 25.4 ± 0.5 a a: P <0.01; Statistically significant difference compared to ethanol treatment group.

< Experimental Example  5> RAW  264.7 cells NADPH  Oxidase activity measurement

In order to measure the inhibition rate of NADPH oxidase activity in the treatment of extracts of thick and thick extracts, their fractions or extracts in RAW 264.7 cells of Experimental Examples <1-2> and <1-3>, the cells were buffered in A after the reagent treatment. After dissolution using a solution (5μM lucigenin, Protease inhibitor), the protein is quantified and measured by reacting a certain amount with B buffer (500μM lucigenin, 100μM NADPH, Protease inhibitor) (Konjeti R, et al., Cancer Research 63, 5636-5645, September 1, 2003).

As a result, as shown in Tables 9 to 10, NADPH oxidase activity inhibition rate of RAW 264.7 cells treated with thick ethanol extract tablets was 36.1 ± 9.3% (P <0.01) and ethanol in the group treated with LPS. In the group treated with 62.5 ± 0.6% (P <0.001), the inhibitory rate of oxidase activity was also increased when the extract and fraction were treated.

Experimental group Inhibition rate of NADPH oxidase activity (%) Control - LPS treatment group - LPS + Thick ethanol extract (1 μg / ml) 0.8 ± 0.7 LPS + Thick ethanol extract (5 µg / ml) 9.0 ± 0.4a LPS + Thick ethanol extract (10 µg / ml) 23.5 ± 0.5 a LPS + Thick ethanol extract (50 µg / ml) 36.1 ± 9.3 b LPS + Thick ethanol extract (1 μg / ml) 0.8 ± 0.2 LPS + Thick ethanol extract (5µg / ml) 7.2 ± 0.4a LPS + Thick ethanol extract (10 µg / ml) 20.1 ± 0.5 a LPS + Thicken Ethanol Extract (50µg / ml) 31.2 ± 9.3 b LPS + Thick ethyl acetate fraction (5 μg / ml) 12.7 ± 0.8a LPS + thick active fraction (5 μg / ml) 14.2 ± 0.5a a: P <0.001; There was a statistically significant difference compared to the LPS treatment group. B: P <0.01; There is a statistically significant difference compared to the LPS treatment group.

Experimental group NADPH oxidase activity inhibition rate (%) Control - Ethanol treatment group - Ethanol + Thick ethanol extract (1 µg / ml) 31.3 ± 1.4 a Ethanol + Thick ethanol extract (5 µg / ml) 33.0 ± 1.0 a Ethanol + Thickened Ethanol Extract Purification (10µg / ml) 56.4 ± 0.1 a Ethanol + Thick ethanol extract (50 µg / ml) 62.5 ± 0.6 a Ethanol + Thicken Ethanol Extract (1µg / ml) 21.3 ± 1.1 a Ethanol + Thicken Ethanol Extract (5µg / ml) 26.0 ± 1.0 a Ethanol + Thicken Ethanol Extract (10µg / ml) 46.4 ± 0.1 a Ethanol + Thicken Ethanol Extract (50µg / ml) 52.5 ± 0.6 a Ethanol + Thick ethyl acetate fraction (5 µg / ml) 35.0 ± 1.1 a Ethanol + thick active fraction (5 μg / ml) 37.2 ± 1.0 a a: P <0.001; statistically significant difference from ethanol treated group.

< Experimental Example  6> RAW  264.7 Cell Peroxide Production Inhibition Rate Measurement

In order to measure the inhibition rate of anion peroxide generation in the treatment of the extracts, fractions or extracts thereof of the RAW 264.7 cells of Experimental Examples <1-2> and <1-3>, cytochrome was added to the cell culture after reagent treatment. ), The amount of cytochrome reduction was measured by adding a solution containing a microplate reader to calculate the amount of anion peroxide produced (Zsuzsa Varga, et al., Phytother . Res . 15, 608-612). , 2001).

As a result, as shown in Table 10 to Table 11, 40.1 ± 0.1% (P <0.001) and ethanol were suppressed in the group treated with LPS as the inhibition rate of the production of peroxide anion in RAW 264.7 cells treated with thick ethanol extract. In the group treated with 63.4 ± 1.5% (P <0.001), the treatment of thick extract and fraction also increased the rate of anion peroxide production.

Experimental group Peroxide anion production inhibition rate (%) Control - LPS treatment group - LPS + Thick ethanol extract (1 μg / ml) -1.1 ± 0.7 LPS + Thick ethanol extract (5 µg / ml) 5.7 ± 0.2 a LPS + Thick ethanol extract (10 µg / ml) 12.2 ± 0.2 b LPS + Thick ethanol extract (50 µg / ml) 40.1 ± 0.1 b LPS + Thick ethanol extract (1 μg / ml) 0.1 ± 0.5 LPS + Thick ethanol extract (5µg / ml) 3.2 ± 0.2 a LPS + Thick ethanol extract (10 µg / ml) 10.2 ± 0.1 b LPS + Thicken Ethanol Extract (50µg / ml) 28.4 ± 0.8 b LPS + Thick ethyl acetate fraction (5 μg / ml) 6.7 ± 0.7 a LPS + thick active fraction (5 μg / ml) 8.4 ± 0.5 a a: P <0.01; There was a statistically significant difference compared to the LPS treatment group. B: P <0.001; There is a statistically significant difference compared to the LPS treatment group.

Experimental group Peroxide anion production inhibition rate (%) Control - Ethanol treatment group - Ethanol + Thick ethanol extract (1 µg / ml) 38.0 ± 0.4 a Ethanol + Thick ethanol extract (5 µg / ml) 42.1 ± 1.5 a Ethanol + Thickened Ethanol Extract Purification (10µg / ml) 46.2 ± 1.5 a Ethanol + Thick ethanol extract (50 µg / ml) 63.4 ± 1.5 a Ethanol + Thicken Ethanol Extract (1µg / ml) 32.0 ± 1.4 a Ethanol + Thicken Ethanol Extract (5µg / ml) 37.1 ± 1.5 a Ethanol + Thicken Ethanol Extract (10µg / ml) 41.2 ± 1.1 a Ethanol + Thicken Ethanol Extract (50µg / ml) 53.4 ± 1.2 a Ethanol + Thick ethyl acetate fraction (5 µg / ml) 45.3 ± 0.6 a Ethanol + thick active fraction (5 μg / ml) 46.7 ± 0.8 a a: P <0.001; There is a statistically significant difference compared to the LPS treatment group.

< Experimental Example  7> Thick Extract, Toothpaste Fraction  And Extraction tablet  Blood ALT (s- GPT Determination of the effect on enzyme activity

<7-1> Preparation of Experimental Animal

      Male wister rats (170-190 g) were purchased from central laboratory animals (Seoul, South Korea). Before the experiment, water and standard feed were freely ingested, and during the experiment, a Lieber-Decarli liquid feed purchased from Daiets, USA was used. The lighting time was maintained for 12 hours (lighting from 8 am to 8 pm) in a kennel meeting GLP facility standards and managed according to the guidelines for animal breeding.

<7-2> Group Classification and Administration Method of Experimental Animals

Healthy Wister rats with a weight of about 180 g were selected and sampled for a week-long stabilization period for environmental adaptation in the laboratory. -Decarli) liquid feed was used. Group classification and administration were divided into six groups as follows.

Group 1 (normal group): Only diet (Standard Lieber-Decarli Control Diet) was given.

    Group 2 (group treated with Thick Extract Tablets): The Thick Sample Tablets were suspended in 10% PEG400 solution at 45 mg / kg for the last 2 weeks with diet (Standard Lieber-Decarli Control Diet) only. It was.

Group 3 (ethanol-administered group): ethanol was given a diet (Standard Lieber-Decarli Ethanol Diet) 36% of the total calories for 4 weeks.

Group 4 (group administered 15 mg / kg thick extract extract to ethanol group): 15 mg / thick extract extract sample was given for 4 weeks with a diet (Standard Lieber-Decarli Ethanol Diet) which accounted for 36% of the total calories of ethanol. The solution was suspended orally in 10% PEG400 solution at kg concentration for the last 2 weeks.

Group 5 (group administered with 45 mg / kg of HT extract sample to ethanol group): 45 mg of HT extract sample with 4% diet (Standard Lieber-Decarli Ethanol Diet) which accounts for 36% of the total calories of ethanol. Suspended in 10% PEG400 solution / kg concentration was administered orally for the last two weeks.

Group 6 (positive dose group): Intramuscular injection of SAM at 20 mg / kg for the last 2 weeks while feeding for 4 weeks with the diet (Standard Lieber-Decarli Ethanol Diet), which accounted for 36% of the total calories of ethanol.

Group 7 (group administered with 45 mg / kg thick ethanol extract sample to ethanol group): 15 mg / kg concentration of thick ethanol extract sample was given for 4 weeks with the diet (Standard Lieber-Decarli Ethanol Diet) which accounts for 36% of the total calories of ethanol. Suspended in 10% PEG400 solution was administered orally for the last two weeks.

Group 8 (45 mg / kg thick ethyl acetate fraction fraction in ethanol group): 45 mg thick thick ethyl acetate fraction sample for 4 weeks with diet (Standard Lieber-Decarli Ethanol Diet) accounting for 36% of the total calories of ethanol. Suspended in 10% PEG400 solution / kg concentration was administered orally for the last two weeks.

Group 9 (Group of 45 mg / kg thick active fraction sample in ethanol group): 45 mg / kg of thick active fraction sample for 4 weeks with a diet (Standard Lieber-Decarli Ethanol Diet) which accounts for 36% of the total calories of ethanol. The solution was suspended orally in 10% PEG400 solution for the last two weeks.

<7-3> ethanol administration Rat  Blood ALT (s- GPT Effect on Enzyme Activity

In order to measure the effect of the extract of the hooves, fractions and extracts thereof on the blood ALT (s-GPT) enzyme activity of rats using the experimental animals classified in <7-2>, blood was collected from the abdominal aorta of rats. Blood was left at room temperature for one hour, then centrifuged at 3000 rpm for 15 minutes to obtain serum and stored at minus 70 ° C. until used in the experiment. ALT was measured by the method of Reitman-Frankel using the Youngdong Pharmaceutical kit (Reitman, S. et al., American Journal of Clinical Pathology 28, 56, 63, 1957).

As a result, as shown in Table 13, the ALT of the ethanol-administered group was increased to 52.5 u / l, 45% (P <0.01) compared to the administration group, but administered 15 mg / kg (low concentration) of thick ethanol extract tablets with alcohol. In one group, it was reduced to 46.3 u / l and in the case of 45 mg / kg (high concentration) thick ethanol extract tablets, it was reduced to 38.4 u / l. In addition, it was confirmed that also reduced the ALT efficacy activity in the hupak extract and fraction administration group.

Military number ALT (s-GPT) enzyme activity (u / l) One 36.2 ± 2.2 2 41.6 ± 0.9 3 52.5 ± 1.6 a 4 46.3 ± 1.6 b 5 38.4 ± 8.9 c 6 41.3 ± 1.1 c 7 48.2 ± 1.4 b 8 45.4 ± 2.8 c 9 48.4 ± 4.2 c Note: a: P <0.01; Statistically significant difference compared to group 1. b: P <0.05; Statistically significant difference compared to group 3. c: P <0.01; Statistically significant difference compared to group 3.

< Experimental Example  8> Chronic Ethanol Administration Rat  Effect on fatty liver formation

In order to measure the effect of the hull extract tablets on the fatty liver formation of rats using the experimental animals classified in the above <7-2>, the livers of the experimental animals were extracted and immersed in a part of the frozen sample preservation solution (OST compound). Immediately after freezing, frozen sections were made and then stained with Oil Red O to specifically stain fat (see FIG. 2). In addition, a part of the extracted liver tissue was soaked in 4% formaldehyde, paraffin sections were made and H & E stained (see FIG. 1), and the results were read (Jens Werner, et al., Am J Physiol). Gastrointest Liver Physiol 283: 65-73, 2002).

As a result, as shown in FIGS. 1 and 2, clear fatty liver findings were observed in the ethanol-administered group, but it was confirmed that the fatty liver findings by ethanol were significantly reduced in the thick extract extract-treated group.

< Experimental Example  9> Chronic Ethanol Administration Rat  Measurement of Triglyceride Accumulation Inhibitory Effect in Liver Tissue

In order to confirm the liver tissue staining results of the test animals classified in the above <7-2>, triglyceride concentration in the liver was measured using a kit (cat # TR0100) of Sigma Chemicals. It is shown in Table 14 below.

As shown in Table 14 below, the triglyceride concentration of liver tissue in the ethanol-administered group was 63.2mg / g, which was 99% higher than the administration group (P <0.001). In the administered group, 27.1 mg / g was reduced by 57.2% compared to the alcohol group, and in the 15m / kg (low concentration) group, 36.9 mg / g was decreased by 41.7% compared to the alcohol group. In addition, triglyceride concentrations were also decreased in the hubac extract and fraction administration groups.

Military number  Triglyceride concentration (mg / g) One  31.7 ± 1.8 2  35.0 ± 0.7 3  63.2 ± 1.7 a 4  36.9 ± 5.7 b 5  27.1 ± 3.1 b 6  19.5 ± 1.8 b 7  40.3 ± 2.5 b 8  31.2 ± 3.4 b 9  34.1 ± 3.1 b a: P <0.001; There was a statistically significant difference compared to group 1 b: P <0.001; Statistically significant difference compared to group 3.

< Experimental Example  10> Chronic Ethanol Administration Rat  Liver s Adenosylmethionine (S- adenosyl -L-methionine; SAM ) Change measurement

Effects of Thick-leaf Extract, Fractions, and Extracts of S-adenosyl-L-methionine (SAM) in Rat Liver Using Experimental Animals Categorized in <7-2> For measurement, the livers of the test animals were shaken with 4 times the amount of 1M perchloric acid, and then centrifuged at 10000 g for 10 minutes, and the supernatant was separated and measured using a UV detector of HPLC. (She QB, et al., Biochem Biophys Res Commun . 205: 1748-1754, 1994).

As a result, as shown in Table 15 below, the concentration of SAM in liver tissue was 69.4 nmol / g by ethanol administration, which was 15.3% (P <0.05) lower than that of the control group. ), The concentration of SAM was increased by 12.1% compared to the ethanol group, and 24.2% was increased in the 15 mg / kg (low concentration) group compared to the ethanol group. In addition, the concentration of SAM was also increased in the hupak extract and fraction administration group. Therefore, it was confirmed that the extract, extracts and tablets of the bakbak showed the effect of inhibiting liver damage by increasing the concentration of SAM in the liver tissue lowered by ethanol.

Military number  SAM (nmol / g) One  82.0 ± 1.4 2  86.2 ± 2.5 3  69.4 ± 1.9 a 4  86.2 ± 3.4 b 5  77.8 ± 2.3 c 6  90.2 ± 2.3 d 7  80.1 ± 2.4 b 8  76.5 ± 2.1 c 9  80.8 ± 2.3 c a: P <0.05; Statistically significant difference compared to group 1 b: P <0.01; Statistically significant difference compared to group 3. c: P <0.05; There is a statistically significant difference compared to group 3. d: P <0.001; Statistically significant difference compared to group 3.

< Experimental Example  11> Chronic Ethanol Administration Rat  Liver Of glutathione  Content change measurement

In order to determine the effect of the extract, the fractions and the extracted tablets of glutathione in the liver of rats using the experimental animals classified in the above <7-2>, the livers of the laboratory animals were 4 After shaking with 1 M of perchloric acid in the culture, it was centrifuged at 10000 g for 10 minutes, and the supernatant was separated and measured using HPLC separation / fluorometric detection method. (Kim, et al., Food and Chemical Toxicology 40, 545-549, 2002).

As a result, as shown in Table 16, administration of ethanol reduced the glutathione concentration in liver tissue 57.4% (P <0.001) compared to the administration group, but in the group administered with ethanol and thick ethanol extract tablets administered only ethanol Glutathione concentration was increased by 1.23 times in 45 mg / kg (high concentration) group and 1.18 times in 15 mg / kg (low concentration) group. In addition, the concentration of glutathione was also increased in the hufa extract and fraction administration groups.

Military number  Glutathione (mg / g) One  7.1 ± 0.2 2  4.8 ± 0.2 3  3.0 ± 0.4 a 4  8.0 ± 0.4 b 5  7.9 ± 0.2 b 6  11.3 ± 0.5 b 7  7.5 ± 0.3 b 8  7.9 ± 0.1 b 9  8.0 ± 0.4 b a: P <0.001; Statistically significant difference compared to group 1. b: P <0.001; Statistically significant difference compared to group 3.

Examples of preparations for the compositions of the present invention are illustrated below.

< Formulation example  1>: Preparation of pharmaceutical preparation

1. Preparation of powder

2 g of extracted tablet of Example 4

1 g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

100 mg of extracted tablet of Example 4

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

100 mg of extracted tablet of Example 4

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Manufacture of rings

1 g extract of Example 4

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

5. Preparation of Granules

150 mg of the extracted tablet of Example 4

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

< Formulation example  2>: Manufacture of food

Foods containing the pak extract of the present invention was prepared as follows.

1. Preparation of Cooking Seasonings

20 to 95 parts by weight of the extract of Example <1-1> of the present invention was prepared for cooking spices for health promotion.

2. Preparation of Tomato Ketchup and Sauce

0.2 ~ 1.0 parts by weight of the extract of Example <1-1> of the present invention was added to tomato ketchup or sauce to prepare tomato ketchup or sauce for health promotion.

3. Manufacturing of Flour Foods

0.5 to 5.0 parts by weight of the extract of Example <1-1> of the present invention was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

4. Preparation of soups and gravy

0.1 to 5.0 parts by weight of the extracted tablet of Example 4 of the present invention was added to soups and broth to prepare meat products for health promotion, soups of noodles and broths.

5. Preparation of Ground Beef

10 parts by weight of the extracted tablet of Example 4 of the present invention was added to the ground beef to prepare a ground beef for health promotion.

6. Manufacture of Dairy Products

5-10 parts by weight of the extracted tablet of Example 4 of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

7. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The extract of Example 1-1 of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and ground to a particle size of 60 mesh using a grinder to obtain a dry powder.

The dry powders of the grains, seeds and extracts of Example <1-1> prepared above were prepared by blending in the following ratios.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Dry powder (3 parts by weight) of the extract of Example <1-1>,

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

8. Preparation of Health Supplements

1000 mg of extracted tablet of Example 4

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

< Formulation example  3>: Manufacture of beverage

1. Manufacture of health drinks

1000 mg of extract of Example <1-1>

       Citric acid 1000 mg

       100 g oligosaccharides

       Plum concentrate 2 g

       1 g of taurine

       Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and intended use.

2. Preparation of Vegetable Juice

5 g of the extract of Example 1-1 of the present invention was added to 1,000 ml of tomato or carrot juice to prepare a vegetable juice for health promotion.

3. Preparation of Fruit Juice

1 g of the extract of Example <1-1> of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice for health promotion.

Claims (16)

Fatty liver disease prevention and treatment agent containing Magnolia cortex extract extracted with water, C ₁ to C ₄ lower alcohol or a mixture thereof as a solvent. The method of claim 1, wherein the Magnolia cortex is Magnolia obovata Thunberg or Magnolia officinale Rehder et Wils . delete According to claim 1, wherein the lower alcohol is fatty liver disease prevention and treatment agent, characterized in that ethanol or methanol. Fatty liver disease prevention and treatment, comprising the ethyl acetate fraction of the extract of claim 1 further extracted with ethyl acetate as an active ingredient. The ethyl acetate fraction of claim 5 is further adsorbed on silica gel column chromatography and a fatty liver disease prevention and treatment comprising an active fraction obtained by applying a step concentration gradient consisting of n-hexane: ethyl acetate as an active ingredient. 1) adding the dried grommets to water, C ₁ to C ₄ lower alcohols or mixtures thereof to obtain a grommets extract; 2) obtaining the filtrate after suspending the thick extract of step 1) in a C ₁ to C ₄ lower alcohol or an aqueous alcohol solution; 3) step 2) adding distilled water to the filtrate and mixing and adjusting the pH to 2-6 to obtain a precipitate produced; And 4) The method of preparing a thick extract extracted tablet comprising the step of dissolving the precipitate of step 3) in C ₁ to C ₄ lower alcohol or an aqueous solution of alcohol and then concentrating the filtered filtrate under reduced pressure. delete 8. A process according to claim 7, wherein the pH of step 3) is 4.0. 8. The method according to claim 7, wherein the pH of the step 3) is adjusted to 6.0 by adding acetic acid. A fatty liver disease prevention and treatment agent comprising a thick extract extract according to the manufacturing method of claim 7 as an active ingredient. 12. The method for preventing and treating fatty liver disease according to claim 11, wherein the thick extract extract has an increased content of Honokiol and magnolol compared to the thick extract of claim 1. The method according to claim 1, 5, 6 or 11, wherein the fatty liver disease is any one selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, nutritional fatty liver, hunger fatty liver, obese fatty liver, diabetic fatty liver. Fatty liver disease prevention and treatment agent characterized in that. For preventing and improving fatty liver disease, comprising a thick extract which is extracted with water, ethanol or a mixture thereof as a solvent, an ethyl acetate fraction thereof, an active fraction of the fraction, or an extract purified by the method of claim 7 as an active ingredient. Health food. The health food for preventing and improving fatty liver disease according to claim 14, wherein the thick extract extract has an increased content of Honokiol and magnolol compared to the thick extract of claim 1. The fatty liver according to claim 14 or 15, wherein the fatty liver disease is any one selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, trophic fatty liver, hunger fatty liver, obese fatty liver, diabetic fatty liver. Health food for disease prevention and improvement.