KR101899443B1

KR101899443B1 - beverage composition for protecting liver

Info

Publication number: KR101899443B1
Application number: KR1020160036298A
Authority: KR
Inventors: 홍재희; 홍성은; 홍성민; 홍기성; 오준석; 이현주; 김성연; 장보윤
Original assignee: 동부생약 영농조합법인
Priority date: 2016-03-25
Filing date: 2016-03-25
Publication date: 2018-09-18
Also published as: KR20170113809A

Abstract

The present invention relates to a beverage composition for liver protection, which has a high content of polyphenols and flavonoids and thus has an excellent antioxidative activity, thereby reducing the increase of ALT, AST and lipid peroxidation induced by hepatotoxic substances, &Lt; / RTI >
The beverage composition for liver protection of the present invention contains a complex extract obtained by extracting a mixture of Hovenia dulcis, Cucumis sativus L., and Sanchenoides.

Description

Beverage composition for protecting liver < RTI ID = 0.0 >

The present invention relates to a beverage composition for liver protection, which has a high content of polyphenols and flavonoids and thus has an excellent antioxidative activity, thereby reducing the increase of ALT, AST and lipid peroxidation induced by hepatotoxic substances, &Lt; / RTI >

The liver is responsible for metabolism and storage of nutrients, regulation and protection of blood flow, and detoxification of harmful substances. In particular, most drugs and chemicals that enter the body from outside are metabolized in the liver, so the liver has a high activity of an enzyme system (cytochrome P450) that can detoxify these substances.

When drugs and chemicals are introduced into hepatocytes, the cytochrome P450 enzyme in the cell converts these compounds to water-soluble derivatives, allowing them to be easily excreted outside the body. However, such biotransformation may lead to the process of converting an incoming compound into a more reactive substance, rather than attacking the cell to induce toxicity (Park BK, et al., Annu. Rev. Pharmacol. Toxicol , &Lt; / RTI > 45: 177-202, 2005). Due to this action, reactive oxygen species (ROS) can be overproduced and free radicals, which are highly reactive, attack the organelles and constituent molecules in the cell to induce oxidative stress, It can cause disease.

Due to the unique metabolism, the liver is likely to be exposed to oxidative stress by drugs and chemicals.

Liver disease is frequently caused by excessive stress, drinking, smoking and drugs, and it is a societal concern because it poses a serious risk to the human body. Liver disease generally involves a series of processes in which liver fibrosis progresses to liver cirrhosis and finally develops into liver cancer. Liver diseases caused by viral liver disease, alcoholic liver disease, liver disease caused by drug toxicity, and accumulation of fat in liver tissue Autoimmune liver disease due to abnormalities in the immune system, metabolic diseases caused by excessive accumulation of toxic substances, and the like.

In order to distinguish between liver-related diseases, it is necessary to comprehensively analyze several biochemical tests. For this purpose, a number of test items are collectively referred to as liver function tests. Major tests include AST, ALT, ALP, and GGT. In addition, there are many cases where the total protein, albumin, lactate dehydrogenase (LDH) and ammonia are added to the test.

AST (aspartate aminotransferase, alanine aminotransferase) and alanine aminotransferase (ALT) are enzymes present in hepatocytes, which are released into the blood and increase in blood levels when hepatocytes are damaged. In the early stages of acute hepatocellular damage, ASTs with higher concentrations in hepatocytes increase more than ALTs, but after 24-48 hours, the longer half-life ALTs are higher. However, alcoholic hepatitis AST is further increased. ALT is more common in chronic hepatocellular damage. In addition, there may be a chronic increase in drug use, nonalcoholic fatty liver, and obesity.

On the other hand, the search for new substances capable of preventing and treating human diseases from natural products has been studied from the past.

Korean Patent No. 10-0477957 (a novel pyrrole derivative having a liver protecting activity isolated from Goji) and a composition containing the same, Korea Patent No. 10-0633851 A composition for prevention and treatment of liver disease or liver disease containing an acid-garlic extract as an active ingredient) and Korean Patent No. 10-1106499 (a food composition for liver protecting effect including a hinoki extract).

In Korea and other Asian countries, natural products have been used to treat various kinds of diseases. Natural product preparations have been tested for their physiological activity based on their long experience and are widely used for low side effects. In particular, since the complex of a natural product is composed of various natural substances rather than a single substance, a complex effect of various components can exhibit a wide range of effects, and the efficacy can be changed depending on the compounding ratio and the extraction method.

1. Korean Patent Registration No. 10-0477957 (Novel pyrrole derivatives having liver-protecting activity isolated from goji, and compositions containing the same) 2. Korean Patent Registration No. 10-0633851 (a composition for prevention or treatment of liver diseases or liver diseases containing an acid-garlic extract obtained by heat-drying treatment as an active ingredient) 3. Korean Patent No. 10-1106499 (Food Composition for Liver Protection Effect Containing Extract of Lycopersicon esculentum)

The present invention has been completed as a result of studying the liver protecting activity against natural products used for a long time as a medicinal herb.

The present invention is characterized by high content of polyphenols and flavonoids due to the combined effects of various natural products, and thus has excellent antioxidant activity, thereby reducing the increase of ALT, AST and lipid peroxidation induced by hepatic toxic substances, It is an object of the present invention to provide a composition.

To achieve the above object, the beverage composition for liver protection of the present invention contains a complex extract obtained by extracting a mixture of Hovenia dulcis, Mucilage, and Sophora.

The mixture further contains Fenugreek mushroom, Arugacea, Gugija, Ogapi, Dermis and Saururus chinensis.

The complex extract is extracted by adding at least one extraction solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol or a mixture thereof to the mixture.

The liver protection beverage composition is characterized by reducing the increase of ALT, AST and lipid peroxidation induced by hepatotoxic substances in hepatocytes.

The mixture further comprises cotton seeds and rhusin.

The present invention has high antioxidant activity because of the high content of polyphenols and flavonoids due to the combined effects of various natural products. Accordingly, it is possible to provide a beverage composition for protecting the liver which has a high effect of liver protecting activity by reducing the increase of ALT, AST and lipid peroxidation induced by the liver toxic substance and is useful for health.

Therefore, the present invention can be utilized variously in the field of health food for liver function.

1 is a graph showing the radical scavenging performance of a sample,
FIG. 2 is a graph showing changes in body weight of an experimental animal during an experimental period of an acute liver injury induction model,
FIG. 3 is a graph showing changes in the spleen and liver weight of experimental animals during the experimental period of the acute liver injury induction model,
FIG. 4 is a graph showing the results of changes in blood ALT and AST activity in an acute liver injury induction model,
FIG. 5 is a graph showing the GSH content and lipid peroxidation measurement results in liver tissue of the acute liver injury induction model,
FIGS. 6 and 7 are graphs showing lipid content in blood and liver tissues of an acute liver injury induction model. FIG.
8 is a graph showing changes in the body weight of the experimental animals during the experimental period of the chronic liver injury induction model,
FIG. 9 is a graph showing changes in the spleen and liver weight of experimental animals during the experimental period of the chronic liver injury induction model,
10 is a graph showing the results of blood ALT and AST activity changes in the chronic liver injury induction model,
11 is a graph showing the GSH content and lipid peroxidation measurement results in the liver tissue of the chronic liver injury induction model,
FIGS. 12 and 13 are graphs showing the results of lipid content in blood and liver tissues of the chronic liver injury-induced model. FIG.

Hereinafter, the beverage composition for liver protection according to a preferred embodiment of the present invention will be described in detail.

The beverage composition for liver protection of the present invention contains, for example, a combined extract obtained from a mixture of Hovenia dulcis, Cucumber mackerel, and Sachiko.

For example, the mixture can be obtained by mixing 30 to 50 parts by weight of buttercups and 30 to 50 parts by weight of anchovy powder with respect to 100 parts by weight of hinoki fruit.

In addition, the mixture may further contain fungus mushroom, arthaxis, gugija, ogapi, dermis, and saururus. For example, the mixture may comprise from 80 to 120 parts by weight of fenugreek mushroom, from 70 to 90 parts by weight of arum jugular bean, from 30 to 50 parts by weight of parsley, from 30 to 50 parts by weight of sophora, 15 to 20 parts by weight of the raw materials, 5 to 15 parts by weight of the dermis and 2 to 8 parts by weight of the raw materials.

Thus, since the present invention is composed of natural materials used for a long time as a herb medicine, the liver protecting effect can be improved by the combined effect of various ingredients.

The hinoki tree is also called the tree of the earth tree. It is 10 ~ 17m high and its bark is black gray. Leaves are 8 ~ 15cm long, ovate, wide oval or oval. There are three coarse veins on the leaf, with serrate on the edge. The white flowers bloom in June ~ July as the euphorbiae. It is bosunghwa (兩性花). The fruit that can be obtained from September to October is brownish, 8mm in diameter, and is shaped like a chicken's claw. Three seeds of fruit each contain one seed. Seeds are dark brown and glossy. By the time when the fruit is ripe, the stem (果莖) becomes thick and rugged. It has a soft scent, it has a sweet taste, and it tastes like a food. It is said that there is an effect of drinking alcohol in the main course gangmyeol, and juice is said to solve the nausea and to stop the nausea.

The medicinal value of fungus mushroom was first recorded in the Compendium of Materia Medica (Compendium Medica). According to the above-mentioned document, when regularly taking the fungus mushroom, it gives a refreshing feeling, maintains a proper weight, promotes longevity, and prevents unnecessary aging. Polysaccharide peptide (PSP) derived from fungus mushroom has immunomodulatory, anticancer, and hepatoprotective activities and has been used as an immunomodulator and anticancer agent in cancer patients.

It is a perennial plant belonging to the family Asteraceae. It is a perennial plant belonging to the family Asteraceae. It is composed of essential oil, coumarin, chromone, flavonoids, caffeic acid, aromatic oxycarbonic acid, It has been known to contain vitamins and has been used for the treatment and prevention of various diseases such as antivirals, blood pressure strengthening, vasodilatory action, analgesic and antipyretic action, cholecystitis, jaundice, genomes, indigestion, menstrual disorders, . The major pharmacological components of the mugwort are scoparone (6,7-dimethoxycumarine) and betaine. Scoparone is a coumarin derivative. It is present in the ground part of the mugwort, , Is used as a diuretic and antioxidant activity is known to be high.

Butterflies have long been known to have the ability to break down toxic substances with perennial plants that grow in wells and rice fields that do not dry well. Especially, it is effective to loosen the hangover after drinking the watercress. In other words, it is known that it releases alcoholic digestion, neutralizes digestion by detoxification, and excretes harmful debris from alcohol by diuretic action. In one room, the juice of buttercups is good for jaundice, small bowel disease, bowel disease, nervous breakdown, and it is effective to prevent the obesity by drinking about one cup of buttercup juice every day.

Sancheong is the tinnitus of beech. Sancheong is a plant that grows in damp valleys of highlands and grows to 10 ~ 15m in height. The branches are honeycomb, so they are named as the honeycomb. Sancheong is known to be cured in liver, liver cirrhosis, hepatitis, and leukemia in the private sector.

Gugija is a fruit of Gugija that belongs to the genus Gugija and has a unique aesthetic sensation. It has carotene, vitamin B1, B2 and C, nicotinic acid, linoleic acid, betaine, pisalene and β- sistosterol) and various inorganic ingredients, it has been recognized as an effective medicament for prevention and treatment of adult diseases, prevention of adult diseases, protection of eyesight, improvement of mental concentration, improvement of mental concentration and amenorrhea, and has been used for many years In modern medicine, it is widely used as an additive to drink ingredients.

Ogphy is also called Ogalli, which uses mainly roots and shells as herbal medicine, and its shell contains acanthoside, tannic acid, about 7%, essential oil, vitamin A and C, and oil.

The dermis is called the skin of the mandarin. It is usually collected from October to November and dried. Ingredients include volatile essential oils, hesperidin (flavone glycoside), citric acid, and vitamin B1.

Saururus chinensis is a kind of perennial herbaceous perennial plant of the perennial plant. The rootstock is white and extends sideways in the mud. The height of the stem is 50 ~ 100cm, white flowers bloom in June ~ August. It grows mainly in wetlands and is called Saururus chinensis because its roots, leaves and flowers are white. It is known to be effective against jaundice, hepatitis, etc., and is distributed in Korea, Japan, and China.

The present invention extracts a complex extract from a mixture of a mixture of the above-mentioned Hovenia dulcis, Fengji mushroom, Artemisiawis, Minari, Sanchenoides, Gugija, Ogaki, Dermis and Saururus.

In another embodiment, the present invention may further comprise white rice and Rhus verniciflua in the mixture. For example, the mixture may contain 80-120 parts by weight of fenugreek mushroom, 70-90 parts by weight of arthropod, 30-50 parts by weight of parsley, 30-50 parts by weight of sophorae, 5-15 parts by weight of guacamole, 10 to 20 parts by weight of an oregano, 5 to 15 parts by weight of a dermis, 2 to 8 parts by weight of a syrup, 5 to 15 parts by weight of a cauliflower, and 5 to 15 parts by weight of a rhusan.

The white moth is a plant of the genus Imperata cylindrica ) flowers. Belt is a perennial plant that grows in the sunny grasses of the foothills and fields. The young flowers with silver color are called 삘 ギ 삘 비 비 비....................

Rhus chinensis ) is a tree root of the pine tree rootstock, which grows in the mountainous region. It is also known as a larch tree, a pine tree, an oyster tree, a horn tree, a fire tree, and a salt tree. It belongs to the lacquer tree, but unlike other lacquer trees, it is not toxic. The thick branches are sparse and the small branches have yellowish brown hairs. Leaves are alternate phyllotaxis of 7-13 small leaves. Small leaf is ovate with coarse sawtooth and brown hairs on the back. The fruit is yellowish red, yellowish brown, covered with yellowish brown hairs, ripened in October, and the white substance appears like salt on the surface of the fruit.

Various methods are applicable as an extraction method for extracting a complex extract from a mixture. For example, the mixture may be extracted with an extraction solvent. As the extraction solvent, at least one selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, or a mixture thereof may be used. As the lower alcohol having 1 to 4 carbon atoms, methanol, ethanol and the like can be used. As the polyhydric alcohol, butylene glycol, propylene glycol, pentylene glycol and the like can be used. Mixtures of water and lower alcohols, mixtures of water and polyhydric alcohols, mixtures of lower alcohols and polyhydric alcohols, or mixtures of water and lower alcohols and polyhydric alcohols can be used as the mixture.

As an example of the extraction, an extracting solvent may be added in an amount of 2 to 20 times by weight, followed by hot water extraction at a temperature of 10 to 150 DEG C for 1 to 36 hours, or cold extraction or hot extraction. Further, a reflux cooling extraction method, an ultrasonic extraction method, or the like can be used. In addition to the above-described extraction method, an extract obtained through a conventional purification process is also included. For example, an active fraction obtained through various purification methods, such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies, etc., is also included in the extract. It goes without saying that the extract obtained by using the above-mentioned extraction solvent can be obtained in the form of powder through filtration, concentration under reduced pressure or freeze-drying, spray drying or the like.

The beverage composition for liver protection of the present invention may be composed of 100% of the combined extract. In addition, a known food additive may be added to the beverage composition for liver protection of the present invention to contain the complex extract in an amount of 10 to 90% by weight.

Examples of known food additives include sugars such as monosaccharides, disaccharides, polysaccharides and sugar alcohols and flavorings such as tau martin, stevia extract, saccharin and aspartame, and nutrients, vitamins, edible electrolytes, flavors, colorants, , Cheese, chocolate, etc.), pectic acid, alginic acid, organic acid, protective colloid thickener, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonating agent, purified water and the like.

The prepared liver protection beverage composition is filled into a packaging container. It is preferable to use a pouch, a spout pouch, or a PET container to maintain the simplicity and quality of the packaging in the distribution process.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples.

(Example)

Mung bean and sancheong were used in Dongguk Herbal Medicinal Cooperatives Corporation, and Hovenia dulcis, Fungus mushroom, Aruginosugi, Gugija, Ogapi, Dandelion and Saururus were purchased from Yeongdong Herbal Farming Association.

100 parts by weight of fenugreek fruit were blended with 100 parts by weight of fenugreek mushroom, 80 parts by weight of arum jugular bean, 40 parts by weight of parsley, 40 parts by weight of saponin, 10 parts by weight of gugija, 15 parts by weight of ogapi, , And 5 parts by weight of Saururus chinensis was added 10 times by weight of water, followed by hot extraction at 120 ° C for 7 hours, followed by filtration to obtain a combined extract.

The inorganic components of the combined extracts were pretreated by dry digestion method.

0.5 g of the sample was converted to white whiteness at 600 ° C, and then 10 ml of diluted hydrochloric acid (2-fold dilution) was added thereto, followed by filtration. The mixture was evaporated to dryness on a water bath. 10 ml of diluted hydrochloric acid was added thereto, The filtrate was used as the analytical sample. The quantitative determination of each inorganic component was carried out using an atomic absorption spectrophotometer (Perkin Elmer AAnalyst 400). Standard concentrations of each element were adjusted to 0.1, 0.5 and 1.0 ppm, and standard calibration curves were prepared and analyzed.

The inorganic components of the combined extract are shown in Table 1 below.

Item Content (ppm) K 2016.20 ± 0.97 Ca 36.76 ± 0.33 Mg 190.78 ± 1.04 Na 220.07 + - 0.61

Referring to Table 1, the major inorganic components were potassium, and Mg, Na and Ca were detected in that order. The content of potassium was 2,016.20 ppm and the content of K was the highest.

Free sugar content of the combined extracts was analyzed by the method of Wilson et al. That is, 5 g of the sample was added with distilled water, stirred, and centrifuged at 100 mL for centrifugation (50,000 rpm, 30 min). The supernatant was taken and filtered (Whatman No. 2). The filtrate was purified by Sep-pak C18, then filtered through a 0.45 μm membrane filter (Millipore Co, USA) and analyzed by HPLC. The content was calculated by the external standard method.

The free sugar of the combined extract is shown in Table 2 below.

Item Content (mg / 100g) Fructose 194.54 Glucose 500.52 Sucrose 1284.44

Referring to Table 2, the free sugar contents were sucrose, glucose, and fructose in the order of sucrose, and 1284.44 mg / 100 g, respectively.

The organic acid content of the combined extracts was analyzed according to the method of Paler and List. That is, 5 g of the sample was added with distilled water, stirred, and centrifuged at 100 mL for centrifugation (50,000 rpm, 30 min). The supernatant was taken and filtered (Whatman No. 2). The filtrate was purified by Sep-pak C18, then filtered through a 0.45 μm membrane filter (Millipore Co, USA) and analyzed by HPLC. The content was calculated by the external standard method.

The organic acids of the complex extract are shown in Table 3 below.

Item Content (mg / 100g) Oxalic acid 266.26 Malic acid 45.09

As a result of the organic acid analysis, the total of two kinds of organic acids was detected, and oleic acid was high as 266.26 mg / 100 g.

Free amino acid analysis of the combined extracts was carried out by Ohara and Ariyosh (00). In other words, 5 g of the sample was treated with the pretreatment method of free sugar, and 25 mg of sulfasalicylic acid was added to 10 L of the filtrate. The mixture was allowed to stand at 4 ° C. for 4 hours and then centrifuged (50,000 rpm, 30 min) The filtrate obtained by filtration with a membrane filter was filtered through a ㎛ membrane filter and then subjected to HPLC analysis using an AccQ-Tag reagent. Content was calculated by external standard method by integrator.

The free amino acids of the combined extract are shown in Table 4 below.

Item Content (mg / 100g) Aspartic acid 15.91 ± 0.00 Serine 13.14 ± 0.21 Glutamic acid 43.43 + - 0.17 Glycine 6.45 + 0.07 Histidine 34.35 ± 0.06 Arginine 210.58 ± 1.80 Threonine 59.75 ± 0.63 Alanine 33.80 ± 0.19 Proline 50.02 + - 0.95 Tyrosine 17.99 ± 0.30 Valine 5.16 + 0.04 Methionine 8.24 + 0.07 Lysine 11.97 + - 0.47 Isoleucine 8.12 ± 0.09 Leucine 16.83 + - 0.82 Phenylalanine 15.25 + - 0.50 TAA ¹ ⁾ 551.00 EAA ² ⁾ 99.92 EAA / TAA (%) 18.13

1) Total free amino acid

2) Total essential amino acid (Thr. + Val. + Met. + Ile. + Leu. + Phe. + His. + Lys.)

Referring to Table 4, 16 free amino acids were detected in the free amino acids, and the total content was 551.00 mg%. Among them, essential amino acids were 99.92 mg% and major free amino acids were glutamic acid, arginine, threonine and so on.

L (lightness), a (+ redness / - green degree) and b (+) degrees were measured using a colorimeter (TOKYO DENSHOKU, SP-80, Japan) with a sample of 2 cm in diameter and 1 cm in height. Yellow color value) and expressed as a mean value by repeatedly measuring 9 times per each sample. The standard color plates used were L = 92.59, a = -0.19, and b = + 2.04.

The turbidity was expressed as an average value after three repeated measurements using an absorbance meter (U-1800, Hitachi Japan) at 650 nm.

The pH was measured using a pH meter (Mettler-Toledo, Switzerland) and then repeated three times.

The turbidity, chromaticity and pH of the combined extract are shown in Table 5 below.

Item
value Turbidity 1.104 + 0.13 pH 4.16 ± 0.01
Chromaticity
L 16.52 + 0.04 a 1.81 ± 0.18 b 3.46 0.10

Referring to Table 5, turbidity and chromaticity measurement result showed that dark brown color was observed due to the characteristic of hot water extraction, and L value indicating lightness was low in chromaticity. Also, the turbidity measurement was also low. The pH value was 4.16, which was similar to that of other natural materials.

The total polyphenol content of the combined extracts was determined by adding 75 μl of distilled water and 25 μl of Folin-Ciocalteu phenol reagent reagent to 25 μl of the diluted sample, reacting for 6 minutes and then adding saturated Na ₂ CO ₃ And the mixture was allowed to stand at room temperature for 90 minutes. The absorbance at 765 nm was measured, and the content of polyphenol was calculated by comparing the absorbance of the standard curve with the standard curve of gallic acid.

The total flavonoid content of the compound extract was obtained by adding 25 μL of the sample and 100 μL of water and 10 μL of the NaNO 2 solution and allowing to stand for 5 minutes. 15 μL of an AlCl ₃ 6H ₂ O solution was added and the mixture was allowed to stand for 6 minutes. 50 μL of NaOH and 50 μL of distilled water were added to the reaction solution, and the absorbance at 510 nm was measured. The total flavonoid content of the extracts was expressed in μg CE (catechin equivalent) / g after the standard calibration curve was prepared using catechin as a reference material.

Total polyphenols and total flavonoids of the combined extracts are shown in Table 6 below.

Item Content (ppm) Total polyphenol 1487.4 ± 2.11 Total flavonoid 2380.83 + - 2.43

Total polyphenol content was 1487.4 ppm and total flavonoid content was 2380.83 ppm.

The radical scavenging activity of the complex extract is shown in FIG.

The electron donating ability (EDA) was measured by the Blois method. 60 μL of 0.45 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 120 μL of each sample solution, and the solution was allowed to stand for 15 minutes, and the absorbance at 517 nm was measured. The electron donating ability was expressed by the absorbance reduction ratio of the sample solution addition group and the no addition group.

Inhibition rate (%) = (1 - absorbance of sample addition group / absorbance of no addition group) x 100

Referring to FIG. 1, it was confirmed that the radical scavenging activity was increased in proportion to the concentration of the complex extract.

1. Experimental Method

(1) Test animals

Six-week-old SD RAT obtained from Orient Co. was tested. After 1 week of adaptation period, group separation was performed by random method according to body weight range and then used in this experiment. The animals used in the experiment had no abnormal symptoms during the acquisition and purification period. During the refinement and experiment period, the breeding environment was set at 23 ℃, 55% relative humidity, 12 hours of illumination time and 150 ~ 300 lux of illumination. The sterilized solid feed and water for laboratory animals supplied by Orient Bio Co., Respectively. Experimental animals were individually cultured in a polycarbonate cage to the extent that they did not interfere with the observation of each animal.

(2) Sample material

The compound extracts of the above examples were used as test substances for administration to experimental animals, and silymarin and liver keeper (Chunho food) were used as comparative substances for comparison with test substances.

Three kinds of test materials were prepared at different concentrations. That is, the combined extract was diluted in distilled water and divided into a first test substance having a concentration of 50 mg / kg, a second test substance having a concentration of 100 mg / kg, and a third test substance having a concentration of 200 mg / kg.

The silymarin was diluted with distilled water and adjusted to a concentration of 50 mg / kg. The liver keeper was diluted with distilled water and adjusted to a concentration of 200 mg / kg.

Experimental animals were divided into 7 groups. (Control group), a positive control group (syrinmarin 50 mg / kg), a comparative control group (liver keeper 200 mg / kg), a first test group (star of star 50 mg / kg) (star of star 100 mg / kg) and the third test group (star of star 200 mg / kg).

The positive control group was the group administered with silymarin, the comparative control group was the group administered with the liver keeper, the first test group was the group to which the first test substance was administered, the second test group was the group to which the second test substance was administered , The third test group is the group to which the third test substance was administered. And the negative control group was the group that caused only liver damage.

(2) Acute liver injury induction model

The test group was orally administered at a dose of 10 ml / kg for 4 days based on the body weight of the test animal once a day. The positive control group was orally administered with 10 ml / kg of silymarin for 4 days based on the body weight of the experimental animal once a day, and the control group was administered with 10 ml / kg of liver keeper for 4 days based on the weight of the experimental animal once a day Orally.

CCl ₄ was diluted in corn oil to a concentration of 0.75 ml / kg for the induction of acute hepatic injury on the fourth day of experiment, and 10 ml per kg of body weight was orally administered to all but the normal group.

(3) Chronic liver injury induction model

CCl ₄ was diluted in corn oil to a concentration of 0.75 ml / kg to induce chronic liver injury, and 10 ml / kg of body weight per kg of body weight was orally administered for 14 days, except for the normal group .

From the 7th day after starting carbon tetrachloride administration, 10 ml / kg of test substance was orally administered to the test group 4 hours before the carbon tetrachloride injection, 10 ml / kg of the silymarin was administered to the positive control group and 10 kg / The experiment was carried out.

(4) Blood and organ harvesting

After completion of the experiment, the rats were anesthetized with ethyl ether, and the spleen and liver were extracted after hemolysis. The collected blood was centrifuged at 3,000 rpm for 15 minutes at 4 ° C and serum was separated. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol and triglyceride (TG) The liver and spleen were weighed and total glutathione (GSH), malondialdehyde (MDA), total cholesterol and triglyceride (TG) were measured.

(5) Measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

ALT, and AST were measured by the Reitman and Frankel (1957) method. The separated serum was incubated at 37 ° C for 30 minutes and 60 minutes, then added with 500 ml of 24-DNPH, left at room temperature for 20 minutes, added with 5 ml of 0.4 N NaOH, reacted for 30 minutes and absorbance was measured at 520 nm.

(6) Determination of Glutathione Content in liver

Glutathione was measured by the Griffith (1979) method. In 2 g of liver tissue, 1 M HClO ₄ containing 2 mM EDTA 5 ml of the solution was added and homogenate was prepared by pulverizing with Polytron (IKA-WERKE). The homogenate was centrifuged at 5,000 rpm for 5 minutes, and the supernatant was taken and diluted with phosphate buffer to make the concentration of glutathione fall within the standard calibration curve and used as a sample for total glutathione determination. To the Microcuvette, 0.2 ml of sample or glutathione standard solution was added to 0.7 ml of 0.3 mM NADPH solution and 0.1 ml of 6 mM DTNB solution, and the mixture was allowed to stand at room temperature for 4 minutes. The glutathionereductase (50 units / ml) was added to the mixture, and the change in absorbance at 412 nm for about 1 minute was measured to determine the slope change.

(7) Malondialdehyde measurement

Malondialdehyde (MDA) was measured by Draper (1990) method using thiobarbituric acid reaction. Tetraethoxypropane was used as the standard solution. SDS (8.1%, 0.2ml), thiobarbituric acid (0.67%, 1.5ml), acetic acid (pH 3.5, 1.5ml) and 0.6ml of water were added to the liver homogenate at 95 ℃ After 1 hour of reaction, the reaction mixture was cooled on ice, mixed with 5 ml of n-buthanol, centrifuged at 4,000 rpm for 10 minutes, and the supernatant was measured at 532 nm.

(8) Measurement of triglyceride and cholesterol content

Serum triglyceride and total cholesterol concentrations were measured using an automatic analyzer (SPOTCHEM EZ, AP-4430, ARKRAY). Lipids were extracted by adding a solvent (chloroform: methanol = 2: 1) 10 times the weight of liver tissue according to the method of Folch et al. (1957). The extracted lipids were dissolved again in chloroform solution and the total cholesterol and triglyceride contents were measured by the same method as analyzed in serum.

2. Experimental results

(1) Results of acute liver injury induction model

end. Weight change

The results of the weighing of the experimental animals are shown in Fig.

Referring to Fig. 2, no difference between the groups was observed.

I. Long-term weight change

The results of measuring the spleen and liver weight of the experimental animals are shown in FIG.

Referring to FIG. 3, the spleen weight was increased by 15% and the liver weight by 30% according to the administration of CCl ₄ . The administration of the second test substance (100 mg / kg) among the test substances (9 kinds of starches) showed a spleen weight reduction of 13% and a liver weight loss of 11% compared with the negative control. It has an efficacy similar to that of a commercialized product, liver keeper (spleen weight: 13%, liver weight: 13% reduction).

All. Changes in serum ALT and AST activity

FIG. 4 shows the results of ALT and AST activity changes in blood.

Referring to FIG. 4, the negative control group showed a significant increase of 7.3 times in ALT and 6.5 times in AST compared to the normal group. And a concentration-dependent decrease in the test group relative to the negative control group. In the third test group compared with the negative control group, AST was significantly decreased by 40.5% and the blood concentration was decreased by 38.2%. In the control group, ALT and AST were decreased to 31.6% and 30.5%, respectively.

la. Measurement of GSH content and lipid peroxidation in liver tissue

FIG. 5 shows GSH content and lipid peroxidation measurement results in liver tissues.

Referring to FIG. 5, the negative control group was significantly decreased by 38% as compared with the normal group. In the test group, GSH content decreased by CCl ₄ -induced liver injury was significantly increased in the first, second and third test groups by 6.1, 20.6 and 28.0%, respectively. And 20.6% in the conventional control group.

The expression of MDA in the group treated with CCl ₄ was significantly increased by 263.1% compared to the normal group. The MDA content increased by CCl ₄ -induced liver injury in the test group was significantly decreased by 7.6, 21.2, and 26.7% in the first, second and third test groups, respectively. And a decrease of 19.5% in the control group.

hemp. Lipid content in blood and liver tissue

6 and 7 show the lipid content in the blood and liver tissues.

Referring to FIGS. 5 and 6, the contents of triglyceride in blood and liver tissues of the group treated with CCl ₄ were increased 2.7-fold and 2.2-fold, respectively, compared with the normal group. In the test group, the concentration of triglyceride in serum and liver increased by CCl ₄ -induced liver damage. In the third test group, 32.7% (blood) and 29.4% ), Respectively. And 24.0% (in the blood) and 27.0% (in the liver) in the control group, respectively.

The serum and liver cholesterol levels of the group treated with CCl ₄ were increased 1.3 and 3.3 times, respectively, compared to the normal group. In the test group, the concentration of cholesterol in serum and liver increased by CCl ₄ -induced liver damage in a dose-dependent manner. In the third test group, 34.7% (blood), 40.8% (liver) Respectively. And 30.8% (in the blood) and 25.8% (in the liver) in the control group, respectively.

(2) Results of chronic liver injury induction model

end. Weight change

The change in body weight is shown in Fig.

Referring to FIG. 8, there was a significant increase in body weight in all groups compared to the negative control group.

I. Long-term weight change

The long-term weight change is shown in Fig.

Referring to FIG. 9, the spleen weight increased by 35.2% and the liver weight by 62.6% according to administration of CCl ₄ . In the third test group, the spleen weight decreased by 22.8% and the liver weight by 19.5%, respectively. The spleen weight and the liver weight were decreased by 20% and 10%, respectively, compared with the control group.

All. Changes in serum ALT and AST activity

The results of ALT and AST activity changes in blood are shown in Fig.

Referring to FIG. 10, the ALT and the AST of the negative control group were significantly increased by 12.4 times and 10.0 times, respectively. And a concentration-dependent decrease in the test group relative to the negative control group. ALT concentration was significantly decreased in 78.2% of ALT and 80.4% in AST in 200mg / kg of CCl ₄ group. In the control group, ALT decreased to 64.1% and AST decreased to 44.1%.

la. Change in GSH content and measurement of lipid peroxidation in liver tissue

Changes in GSH content and measurement of lipid peroxidation in liver tissues are shown in Fig.

Referring to FIG. 11, the CCl ₄ administration group was significantly decreased by 46.8% compared with the normal group. In the test group, GSH content decreased by CCl ₄ -induced liver damage was 55.8, 67.6 and 82.7% in the first, second and third test groups, respectively. And 66.2% in the control group.

The expression of MDA in the group treated with CCl ₄ was significantly increased by 431.7% compared to the normal group. The MDA levels increased by CCl ₄ -induced liver injury in the test group were significantly decreased by 8.6, 16.6, 30.4% in the first, second and third test groups, respectively. And a decrease of 12.5% in the control group.

hemp. Lipid content in blood and liver tissue

The results of lipid content in blood and liver tissues are shown in Figs. 12 and 13. Fig.

12 and 13, the content of triglyceride in the blood and liver tissues of the group treated with CCl ₄ was increased 1.8-fold and 2.6-fold, respectively, compared with the normal group. In the test group, the concentration of triglyceride in serum and liver increased by CCl ₄ - induced liver injury in a dose - dependent manner. In the test group, 47.1%, (in the blood) and 23.9% Tissue) significantly decreased. Compared with 8.5% (in the blood) and 2.1% (in the liver) in the control group.

The serum and liver cholesterol levels of the group treated with CCl ₄ were increased 1.2 and 4.0 times, respectively. In the test group, the concentration of cholesterol in blood and liver increased by CCl ₄ - induced liver injury in a dose - dependent manner. In the third test group, 9.2%, (in the blood) and 38.7% Of the total). Compared with 4.3% (in the blood) and 8.0% (in the liver) in the control group.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation. Accordingly, the true scope of protection of the present invention should be determined only by the appended claims.

Claims

The present invention relates to a method for producing a hinoki fruit, which comprises 80 to 120 parts by weight of fenugreek mushroom, 70 to 90 parts by weight of arum japonica, 30 to 50 parts by weight of parsley, 30 to 50 parts by weight of sophorae, 5 to 15 parts by weight of ginger, 10 to 20 parts by weight of dermis, 5 to 15 parts by weight of dermis, and 2 to 8 parts by weight of 3 to 3 seconds,
AST, and lipid peroxidation induced by hepatotoxic agents in hepatocytes.

delete

The beverage composition for liver protection according to claim 1, wherein the complex extract is extracted by adding at least one extraction solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, or a mixture thereof to the mixture.

delete

The beverage composition for liver protection according to claim 1, wherein the mixture further comprises white marsh and rhus wood.