KR100536856B1 - Composition comprising herbal mixture extract - Google Patents

Abstract

The present invention relates to a composition containing a mixed herbal medicine extract as an active ingredient, the mixed herbal medicine extract includes a liana extract and ginger tree extract.

The mixed herbal medicine extract of the present invention is GOT, GPT, ALP, BUN, the total bilirubin amount of liver function indicators appear low, it is excellent in liver function improving effect. In addition, ALP, BUN levels are used as diagnostic indicators of renal function as well as liver function, it can be seen that the herbal extracts of the present invention are also excellent in renal function improving effect.

In addition, the mixed herbal medicine extract of the present invention, the amount of hydroxyproline is low in the liver tissue and high in the kidney tissue, it is excellent in the antifibrotic effect and kidney protection effect.

In addition, the mixed herbal medicine extract of the present invention has a low amount of malondialdehyde, which is an indicator of lipid peroxidation, in liver and kidney tissues, and thus has an excellent antioxidant effect.

In addition, the mixed herbal medicine extract of the present invention shows a high cell survival rate in hepatocytes and renal cell lines, it can be seen that the hepatocyte protection and renal cell protection effect is excellent.

Therefore, the composition of the present invention can be usefully used not only for antioxidant, antifibrotic, improving liver function but also for protecting kidney and improving kidney function.

Description

Composition comprising herbal mixture extract as an active ingredient

The present invention relates to a composition containing a mixed herbal medicine extract as an active ingredient, the mixed herbal medicine extract includes a liana extract and ginger tree extract.

Liver disease, one of the representative adult diseases, is a disease caused by damage to the liver by stress chronic fatigue and most exogenous substances. At present, the incidence of liver disease in Korea is considerably higher than that of foreign countries. Recently, the mortality rate of liver cancer is the world's number one, and the third case of chronic liver disease is also the third. According to a recent report by the National Statistical Office, liver disease is the leading cause of death among people in their 40s. The highest mortality rate among liver disease in Korea is viral hepatitis, but death from cirrhosis is reported to be 5 to 10 times higher in the West than viral hepatitis.

Liver is the most active organ of human body organs in vivo. It is caused by various causes such as excessive intake of food or alcohol containing fat ingredients, infection of virus, harmful substances such as various medicines, and malnutrition. Chronic disorders occur, and fatty liver, hepatitis, jaundice, cirrhosis, liver cancer, and the like can be caused. In particular, excessive fat intake or excessive alcohol intake leads to fatty liver in which lipids accumulate in liver tissue. ) And so on. The liver is a large buffering organ that does not show up well in the early stages of the disease, but only after it has worsened.

In addition, the liver is known to be closely related to mental activity and to the blood storage and circulation, blood flow control and defense detoxification in the human body. Our bodies are always exposed to the pollutants and toxic substances of industrialization, and our liver is suffering from constant detoxification. Moreover, what is serious is liver damage from mental stress. If you have a mental rest, damaged hepatocytes will be repaired, but in a modern day, you cannot find a room for mental rest. Therefore, mental stress, heavy drinking, and smoking will cause liver damage, which will cause your body to fail to detoxify your body. It can also cause other diseases.

Liver cirrhosis is a result of fibrosis of liver tissue, a condition in which homeostasis of connective tissue production and degradation is lost. Excessive accumulation of connective tissue in the liver tissue is accompanied by necrosis or inflammation. In particular, hepatic stellate cells (HSCs) that have been metastasized to myofibroblasts are known to advance the process of fibrosis of liver by proliferating and moving to produce excess connective tissue (Gressner et al., Biochem. Biophys. Res. Commun. 151-222, 1988). Liver cirrhosis is the last step that is common when chronic liver disease progresses, and overall liver function decreases such as hepatic blood flow decrease, hepatic blood flow decrease, metabolic enzyme function decrease, blood protein qualitative and quantitative change and bile flow change. do.

Hepatotoxic agents include carbon tetrachloride (CCl ₄ ), D-galactosamine, lipopolysaccharide (LPS), and bromobenzene. (Recknagel et al., Pharmacol. Rev. 19: 145-208, 1967; Alpers et al., Mol. Pharmacol. 4: 566-573, 1968; Slater TF, Biochem. J. 222: 1-15, 1994) . Carbon tetrachloride, which causes hepatotoxicity, is an agent that artificially causes hepatotoxicity by directly administering to hepatocyte culture, liver tissue culture, and mouse or rat to test for hepatotoxicity protection. Carbon tetrachloride has a strong hepatotoxic effect by converting into a molecular structure of CCl ₃ ·, a highly reactive free radical in the endoplasmic reticulum by metabolic enzymes such as cytochrome P450, which has a complex function. CCl ₃ ·, a free radical, causes oxidation of fatty acids in hepatic triglycerides or membrane phospholipids accumulated by alcohols, leading to rancidity of lipids, binding to oxygen, and forming organic peroxides through lipid peroxide reactions. Lipid peroxidation leads to the accumulation of fat in the liver, reduced protein synthesis, glycogen breakdown and the destruction of cytoplasmic enzymes in blood vessels, resulting in necrosis of liver tissue (Chang IM, et al., Drug and chemical toxicology 6, 443-453, 1983). These free radicals are known to damage the Golgi apparatus and thus damage the kidneys as well as the liver by adversely affecting protein release from cells.

In addition, the antioxidant effect, which is a function that inhibits or prevents lipid peroxidation, has been reported to have a hepatoprotective effect and an anti-inflammatory action, and therefore, an antioxidant compound is overlooked in response to an attack by a reactive oxygen intermediate. It is used for the purpose of protecting hepatocytes.

Antioxidants such as flavonoids, quercetin, silymarin or vitamin E extracted from natural products have been reported to be effective in lipid peroxidation and liver fibrosis. N-acetylcysteine (NAC) Antioxidant activity is known to inhibit hepatic fibrosis and oxidative stress in the early stages of hepatic fibrosis. Picrorhiza Kurroa (kutkin) is also reported to be a natural product with antioxidant effects that help to protect liver and anti-fibrotic as well as hepatoprotective effect by reducing damage caused by lipid peroxidation and free radicals.

Currently, the commonly used treatment methods for liver disease are largely divided into diet and pharmaceutical therapy, and in most cases, these two methods are used in combination. In the therapy for liver disease, drugs with various mechanisms of action may be used depending on the cause and type of liver disease, for example, ursodeoxycholic acid, silymarin (Silymarin; Biotech. Therapeutics , 4, 263-270, 1993), DDB (biphenyl dimethyl dicarboxylate; Biochem. Biophy.Res. Comm. , 103, 1131-1137, 1981), hepatocellular rejuvenating agents such as glutathione, glycyrrhizin and liver function Pharmaceutical agents such as adjuvants, antiviral agents such as acyclovir, immunosuppressants such as corticosteroids, 6-mercaptopurine (6-MP), azathioprine and the like are commonly used. have. However, since liver disease is not caused by only one cause but is caused by the complex action of several factors, it is possible to expect a high enough therapeutic effect for all liver diseases by using drugs with only one mechanism of action. There is no. In addition, the known currently used liver disease treatment drug has a disadvantage in that a sudden action occurs or when a large amount or long-term administration occurs.

On the other hand, the kidney regulates the amount of water (body fluid) and ions in the body properly to release waste products (urea, uric acid, creatinine, etc.) into urine, and toxins, drugs, and metabolites detoxify after excretion. Do it. Malondialdehyde (MDA) and 4-hydroxynonenal (HNE), the breakdown products of lipid peroxidation in tissues, are known as indicators of cell damage and are superoxide dismutases that catalyze lipid peroxides. (superoxide dismutase) has been reported to be involved in repairing cell damage (Slater TF. Biochem. J., 222, 1-15, 1994; Esterbauer H, Schauer RJ, Zollner H., 1994; Free Radical Biology & Medicine 11 , 81-128, 1992). Alkaline phosphatase (ALP) and blood urea nitrogen (BUN) are used in clinical practice as diagnostic indicators of renal metabolism. In particular, an increase in BUN levels is indicated by a decrease in glomerular filtration rate (GFR) due to azotemia. For reference, prerenal hypertension is a result of hypoperfusion in the kidney, such as congestive heart failure, shock, hypovolemia, and bleeding. Postrenal hypertension occurs when the flow of urine is blocked in the lower kidney, but can be recovered if the cause is corrected. Hypertension is called uremia if it is accompanied by various clinical symptoms, symptoms and biochemical abnormalities. Thus, uremia is not just a biochemical abnormality, but a clinical syndrome, which causes not only abnormal renal excretion but also metabolic, endocrine dysfunction, gastrointestinal, neuromuscular, and cardiovascular events. Diseases caused by abnormal kidney function include acute pyelonephritis, chronic pyelonephritis, renal tuberculosis, urinary tract infections (UTI), urinary stones, and kidney cancer (Renal cell). cancer).

On the other hand, Hovenia dulcis Thunb. Is a buckthorn and deciduous broad-leaved arbor, and is called 'earth tree' in Chinese medicine. In the herbaceous tree, the barn tree is sweet, calm and non-toxic, softens and lubricates the intestines, removes heat from the chest, quenches thirst, quenches alcohol, removes vomiting, eliminates detoxification and eliminates five types of hemorrhoids. It is said to heal. In addition, it is known to have a protective effect on the liver, and it has been found to have an excellent effect on the removal of bad breath, alcoholic hepatitis, fatty liver, cirrhosis, especially anti-cancer effect, blood pressure control, hypoglycemia, liver detoxification, constipation.

Ginger (Lindera obstiloba) is a deciduous broad-leaved tree belonging to the family Lauraceae, distributed in Japan, China, and Manchuria, including Korea. Ginger tree flower and leaf stem has a characteristic scent, so the stem is used for medicinal and antibacterial effect is known. In addition, sprout is used as raw material of tea.

Therefore, the present inventors have various mechanisms of action, and while searching for drugs with low toxicity in natural products, there are anti-oxidation, anti-fibrosis, and liver function as well as renal protection and renal function improvement in mixed herbal medicines of lianas and ginger trees. Confirmed and completed the present invention.

The present invention is to provide a composition containing a mixed herbal extract of the fern and ginger tree extract as an active ingredient.

The present invention provides a composition containing a mixed herbal extract of the fern and ginger tree extract as an active ingredient.

The composition of the present invention includes a composition for improving liver function and a composition for improving kidney function.

Hereinafter, the present invention will be described in detail.

The composition ratio of the herbal ingredients in the composition of the present invention is based on the dry weight of each, using the ratio of the bark tree and ginger tree 3: 2 ~ 1: 1, preferably of the barn tree and ginger tree The ratio is used in combination of 2: 1 to 1: 1. Such a composition ratio is found in consideration of the effective dose and side effects of each herbal ingredient, and if it is out of the range of the ratio, there is a possibility that the effect is drastically reduced or side effects.

Extraction method of the mixed herbal medicine contained in the composition of the present invention is as follows.

Wash the barn and ginger trees with water and dry them in the shade. Dry bark and ginger wood in a weight ratio of 2: 1 by weight in a reflux extractor, and then put the bottled water and heated at 100 ℃ for 90 minutes to extract hot water. The hot water extract was filtered under reduced pressure with a filter paper when hot, and the filtrate was concentrated using a vacuum evaporator. When using for a long time, use a freeze dryer to dry. In the present invention, the stems, flowers, leaves, seeds, etc. of the barn and ginger trees can be used.

The mixed herbal medicine extract of the present invention is GOT, GPT, ALP, BUN, the total bilirubin amount of liver function indicators appear low, it is excellent in liver function improving effect. In addition, ALP, BUN levels are used as diagnostic indicators of renal function as well as liver function, it can be seen that the herbal extracts of the present invention are also excellent in renal function improving effect.

In addition, the mixed herbal medicine extract of the present invention, the amount of hydroxyproline is low in the liver tissue and high in the kidney tissue, it is excellent in the antifibrotic effect and kidney protection effect.

In addition, the mixed herbal medicine extract of the present invention has a low amount of malondialdehyde, which is an indicator of lipid peroxidation, in liver and kidney tissues, and thus has an excellent antioxidant effect.

In addition, the mixed herbal medicine extract of the present invention shows a high cell survival rate in hepatocytes and renal cell lines, it can be seen that the hepatocyte protection and renal cell protection effect is excellent.

Therefore, the composition of the present invention can be usefully used not only for antioxidant, antifibrotic, improving liver function but also for protecting kidney and improving kidney function.

The composition of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the mixed herbal medicine extract.

The mixed herbal extracts can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical formulations. That is, the mixed herbal extract of the present invention may be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, humectants, disintegrating agents, and surfactants are usually used. Or using excipients. Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, which may be used in mixed herbal extracts, including at least one excipient such as starch, calcium carbonate, sucrose, lactose and gelatin, and the like. Are mixed to prepare. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, 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. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, 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, glycerol and gelatin may 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 mixed herbal medicine extract is 200 to 600 mg / kg, preferably 300 to 400 mg / kg, and may be administered 1 to 6 times a day.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, chemotherapy and biological response modifiers to improve liver and kidney function.

The composition of the present invention can be added to health food for the purpose of improving liver function and kidney function. When the mixed herbal medicine extract of the present invention is used as a food additive, the mixed herbal medicine extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverage, the mixed herbal extract of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for health and hygiene or health control, 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 above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example  : Preparation of Mixed Herbal Extracts

The barn and ginger trees were washed with water and dried in the shade. 20 g of dry bark and 10 g of ginger stem were placed in a reflux extractor, and 1.5 liter of bottled water was added and heated at 100 ° C. for 90 minutes to extract hot water.

The hot water extract was filtered under reduced pressure with a filter paper when hot. The filtrate was concentrated using a vacuum evaporator. When used for a long time, it was dried using a lyophilizer.

The concentrate was used for animal experiments (2ml / rat / day), was used to concentrate 4 times in MTT, NR assay (50 μl / well).

Comparative Example 1  : Manufacture of Rhododendron Extract

Instead of the mixed herbal medicines were used in the same manner as in the above example using a hut tree.

Comparative Example 2  : Preparation of Ginger Tree Extract

Ginger tree was used to prepare in the same manner as in the above example.

Experimental Example 1  : Antioxidant, Antifibrotic, Liver Function and Kidney Protection, and Kidney Function Effect on Liver and Kidney Damage Induced by Long-term Administration of Carbon Tetrachloride in Rats

In order to investigate the effects of antioxidant, antifibrotic, liver function and kidney protection, kidney function improvement effect on liver damage and kidney damage of the mixed herbal medicine extract of the present invention was performed as follows.

1. Experimental Animal

The experimental animals used 12-week-old Sprague-Dawley rats (multi-body science, Osan, Korea) with a weight of about 180 ~ 210g, and kept a breeding environment of 23 ± 2 ℃ and 60 ± 10% relative humidity. Feed (purina feed) and drinking water were supplied freely, and night and day.

The experimental animals adapted to the laboratory environment for 2 weeks were divided into 5 groups: ① normal group, ② CCl ₄ administration group, ③ CCl ₄ + mixed herbal medicine administration group, ④ CCl ₄ + hollyhock extract administration group, ⑤ CCl ₄ + ginger tree extract administration group. Ten animals were assigned to each group.

2. Induced liver fibrosis (hardening) and kidney damage

Liver fibrosis (curing) and kidney damage were induced in rats of the experimental group except the normal group by administering a mixture of olive oil and CCl ₄ 1 ml / rat / day three times a week for 4 weeks.

CCl ₄ administration group to distilled water, CCl ₄ + mixed herbal medicine administration group to the mixed herbal medicine extract prepared in the above Example, CCl ₄ + hut tree extract administration group to the bark tree extract prepared in Comparative Example 1, CCl ₄ + ginger tree In the extract administration group, 2 ml / rat / day of the ginger tree extract prepared in Comparative Example 2 was orally administered.

The rats of each group, including the normal group, were weighed and anesthetized with ether, collected from the heart by heart puncture, left at room temperature for at least 2 hours, and centrifuged at 3000 rpm for 10 minutes to obtain serum. Store at -20 ° C. The stored serum was used to determine transaminase (GOT and GPT) and alkaline phosphatase, BUN and total bilirubin values, serum biochemical tests.

In addition, livers and kidneys of rats induced liver fibrosis (curing) and kidney damage were extracted, washed with phosphate buffer (pH 7.0), and weighed. Some of the liver and kidney tissues were stored at -75 ° C and used for the measurement of hydroxyproline and malondialdehyde (MDA).

Changes in body weight were measured weekly. The presence of jaundice in the ears, tail and feet was checked and the weight changes during slaughter were observed.

The experimental results were expressed as mean ± standard deviation, and when testing the difference between the control group and the experimental group, the student's t- test was used to determine statistically significant difference when the P value was less than 0.005.

The results are shown in Table 1.

Weight (g) Liver weight (g) (Internal weight / weight) * 100 (%) Normal 200.8 ± 8.6 6.3 ± 0.3 3.2 ± 1.3 CCl ₄ administration group 167.3 ± 19.4 12.9 ± 1.9 7.7 ± 0.7 CCl ₄ + group of herbal medicines 190.7 ± 10.3 11.7 ± 1.6 ^* 6.7 ± 0.8 ^* CCl ₄ + hawthorn extract group 173.2 ± 17.3 13.4 ± 1.6 7.0 ± 0.6 CCl ₄ + ginger extract group 181.8 ± 16.1 12.5 ± 1.2 6.9 ± 0.5 ^*

*: p <0.005

As shown in Table 1, the ratio change of the weight and liver weight of the mixed herbal medicine administration group of the present invention was 13% significantly lower than the control group.

3. Serum Biochemical Test

1) GOT (AST) measurement (using EMBIEL kit)

500 μl of AST substrate solution was added to two falcon tubes and warmed at 37 ° C. for 3-5 minutes. One test tube was diluted with a standard solution, and the other test tube was added with 100 µl of serum sample and reacted at 37 ° C. for 60 minutes. 100 µl of purified water and 500 µl of a color developing solution (2,3-dinitrophenylhydrazine) were added to each test tube, and allowed to stand at room temperature for 20 minutes. 5 ml of 0.4 N NaOH was added to each test tube and reacted again at room temperature for 10 minutes, and then the absorbance was measured at 505 nm.

2) GPT (ALT) measurement (using EMBIEL kit)

150 μl of ALT substrate solution was added to two Pelcon test tubes and warmed at 37 ° C. for 4 minutes. One test tube was diluted with a standard solution, and 100 μl of serum sample was added to the other test tube and allowed to react at 37 ° C. for 30 minutes. 100 µl of purified water and 500 µl of a color developing solution (2,3-ditrophenylhydrazine) were added to each test tube, and the mixture was left at room temperature for 20 minutes. 1.5 mL of 0.4 N NaOH was added to each test tube and reacted again at room temperature for 10 minutes, and then the absorbance was measured at 505 nm.

3) Alkakine phosphatase (ALP) measurement

2.0 ml of ALP substrate buffer (-2-sodium phenyl phosphate) was added to three test tubes and warmed at 37 ° C. for 3-5 minutes. 50 μl of serum sample, 50 μl of purified water, and 50 μl of standard solution were added to each test tube, and the mixture was warmed at 37 ° C. for 15 minutes. 2.0 ml of color reagent was added to each test tube and allowed to stand at room temperature for 10 minutes, and then absorbance was measured at 570 nm within 60 minutes.

4) Measurement of Blood urea nitrogen (BUN)

20 μl of serum sample, 20 μl of purified water, and 20 μl of standard solution were added to three test tubes, 2.0 ml of enzyme solution was added to each of the tubes, and the mixture was reacted at 37 ° C. for 5 minutes. 2.0 ml of color reagents were added to each test tube, mixed and warmed at 37 ° C. for 10 minutes, and then absorbance was measured at 580 nm within 60 minutes.

5) Determination of total bilirubin amount

100 μl of serum sample, 100 μl of purified water and 100 μl of standard solution were added to each of three test tubes, and 600 μl of the total bilirubin color reagent was added. 600 microliters of diazo mixture was added to each test tube, and the mixture was left to stand at room temperature for 10 minutes. After reacting by adding 600 µl of ferring reagent to each test tube, the absorbance was measured at 600 nm within 2 hours.

The results are shown in Table 2.

GOT (IU) GPT (IU) ALP (KA) BUN (mg / ㎗) Total bilirubin amount (mg / dl) Normal 61.78 ± 6.0 13.2 ± 1.0 63.5 ± 11.2 13.6 ± 1.1 0.2 ± 0.05 CCl ₄ administration group 241.8 ± 43.1 145.3 ± 38.1 156.9 ± 36.1 23.4 ± 2.1 1.6 ± 0.92 CCl ₄ + mixed herbal medicine group 137.6 ± 52.4 ^** 69.6 ± 47.0 ^* 137.0 ± 26.0 18.04 ± 6.91 0.27 ± 0.14 CCl ₄ + hawthorn extract group 165.1 ± 46.4 ^** 70.9 ± 42.2 ^* 143.1 ± 37.9 18.6 ± 2.1 ^* 0.7 ± 0.04 CCl ₄ + ginger extract group 144.5 ± 5.7 ^** 74.4 ± 17.6 ^* 138.2 ± 14.8 18.4 ± 3.6 -

*: p <0.05, **: p <0.005

As shown in Table 2, GOT, GPT, ALP, BUN, and total bilirubin levels of liver function indicators of the mixed herbal medicine group of the present invention were significantly lower than those of the control group, and lower than those of the lone tree alone group and the ginger tree alone group. appear.

Therefore, the mixed herbal medicine extract of the present invention can be seen that the effect of improving liver function.

In addition, ALP, BUN levels are used as diagnostic indicators of renal function as well as liver function, it can be seen that the herbal extracts of the present invention are also excellent in renal function improving effect.

4. Determination of the amount of hydroxyproline (hyp)

1) Preparation of Reagent

Acetate citrate buffer

50 g of sodium acetate trihydrate, 37.5 g of trisodium citrate, 5.5 g of citric acid monohydrate, and 395 ml of isopropanol were added to distilled water to make 1 L of total, followed by pH 6.0 Made and stored at 4 ℃ was used.

② Chloramine-T Solution

84 mg of chloramine-T was used dissolved in 10 ml of acetate citrate buffer.

③ Ehrilich's reagents

10 g of p-dimethylaminobenzaldehyde and 11 ml of 60% perchloric acid were mixed to make a stock solution. 3 ml of stock solution was taken and mixed with 8.0 ml of isopropanol. The eririch reagent was prepared and used immediately before use, and the stock solution was shielded and stored at 4 ° C.

2) Determination of the amount of hydroxyproline

After weighing 0.2 g of freeze-dried (-50 ° C., 72 hours) or frozen liver tissue (or kidney tissue) in a 10 ml glass bottle, 4 ml of 6N HCl was added and homogenized with a homogenizer, After hydrolysis in a drying oven for 24 hours, it was filtered using Whatman filter paper. At this time, the standard solution diluted with 0, 0.2, 0.4, 0.6, 0.8, 1.0 μg / 50 μl of trans-hydroxyproline 6N HCl was hydrolyzed at 110 ° C. for 12 to 14 hours as in the sample. Each sample and standard solution were taken in a 50 μl glass bottle and completely dried to remove hydrochloric acid. Thereafter, 1.2 ml of 50% isopropanol was added to dissolve the precipitate, 200 µl of chloramine-T solution was added thereto, and the mixture was reacted at room temperature for 10 minutes. After 90 minutes of development at 50 ° C. and cooling at room temperature, the absorbance was measured at 558 nm using a spectrophotometer.

The concentration of hydroxyproline in liver tissue (or kidney tissue) was calculated by the following formula.

C [concentration of hydroxyproline of 0.2 g of liver tissue (or kidney tissue)] =

[HA (absorbance of sample) / SA (absorbance of standard solution diluted with 1.0 μg / 50 μl of trans-hydroxyproline 6N HCl)] × 80

C × 5 = amount of hydroxyproline / g liver tissue (or kidney tissue)

The results are shown in Table 3.

Amount of hydroxyproline (μg / g) Liver tissue Kidney tissue Normal 997.7 ± 87.8 918.2 ± 78.7 CCl ₄ administration group 2201.6 ± 16.0 825.6 ± 57.6 CCl ₄ + group of herbal medicines 1094.3 ± 186.7 870.4 ± 74.3 CCl ₄ + hawthorn extract group 1118.3 ± 255.0 ^* 850.2 ± 57.6 CCl ₄ + ginger extract group 1110.1 ± 201.0 ^* 860.4 ± 69.3

*: p <0.005

As shown in Table 3, the amount of hydroxyproline of the mixed herbal medicine administration group of the present invention was significantly lower in liver tissue 50.8% than in the control group, in the kidney tissue 5.5% than the control group, only 3.0% compared to the fir tree alone group, It was 1.2% higher than Ginger alone.

Therefore, it can be seen that the mixed herbal medicine of the present invention has an excellent antifibrotic effect and kidney protective effect.

5. Measurement of MDA (Malondialdehyde)

Homogenized tissue sample (0.2 g liver tissue (or kidney tissue) in 1.8 ml of 1.15% KCl) and 200 µl of diluted standard (0, 4, 8, 16, 32 nmol / 200 µl tetramethoxypropane) It was added to a Pelcon test tube. 100 µl of 0.2% SDS was added to 200 µl of the diluted standard solution and the mixture was reacted at room temperature for 10 minutes. 750 µl of 20% acetic acid and 750 µl of 0.8% thiobarbiturate were added and mixed. Thereafter, the mixture was reacted at 95 ° C. for 30 minutes and cooled in ice. After confirming complete cooling, 2 ml of n-butanol was added and centrifuged to measure the absorbance of the supernatant using a spectrophotometer at 532 nm.

Serum and tissue MDA concentrations were calculated as follows.

C [concentration of malondialdehyde in 0.2 g of liver tissue (or kidney tissue) (or 200 μl serum)] = [HA (absorbance of sample) / SA (absorbance of standard solution (8 μmol / 1.15% KCl 200 μl)] × 80

C × 5 = malondialdehyde amount (μmol / ml)

The results are shown in Table 4.

Amount of malondialdehyde (μmol / ml) Liver tissue Kidney tissue Normal 89.6 ± 3.7 142.3 ± 12.7 CCl ₄ administration group 151.3 ± 27.7 181.4 ± 24.4 CCl ₄ + group of herbal medicines 105.9 ± 12.9 ^* 161.7 ± 13.2 CCl ₄ + hawthorn extract group 130.7 ± 25.9 168.3 ± 19.8 CCl ₄ + ginger extract group 121.3 ± 17.6 156.6 ± 14.7 ^*

*: p <0.05

As shown in Table 4, the amount of malondialdehyde of the mixed herbal medicine-administered group of the present invention was significantly lower in liver tissue than in the control group by 31.0%, and in kidney tissue by 13.0%. Malondialdehyde is an indicator of lipid peroxidation.

Therefore, it can be seen that the mixed herbal medicine of the present invention has excellent antioxidant and kidney protective effects.

Experimental Example 2  : Toxicity Test

The cell lines used in this experiment were NCTC clone 1469 (liver cell line) and vero (renal cell line), and were purchased from Korea Cell Line Bank (KCLB).

MTT stock solution was prepared by dissolving 5 mg of MTT powder in 1 ml of phosphate buffer solution, shaking well, and then filtering with a 0.4 μm filter.

NR stock solution was prepared by dissolving 4 mg of NR powder in 1 ml of tertiary distilled water, shaking well, and then filtering with a 0.4 μm filter.

Trypsin-EDTA was sold containing 0.5% trypsin, 5.3 mM EDTA, which was used diluted 10-fold with PBS.

1) MTT [3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] analysis

After removing the medium, 1 ml of trypsin-EDTA solution was added and left for 10-20 minutes to separate the cells from the container and placed in a 15 ml Pelcon tube. In order to collect all remaining cells in the culture flask, 1 ml of medium was added, shaken, and the remaining cells were collected and placed in the Pelcon tube. 10 μl of cell suspension was placed in a hemocytometer and the number of cells was counted (2.5 × 10 ⁴ cells / ml). 50 μl of cells (1 ~ 2 × 10 ⁵ cells) were put into 96 well plate and 150μl of medium (DMEM + 10% FBS + antibiotic) was added to the cells and left for 24 hours in a 37 ° C., 5% CO ₂ incubator. Was attached.

After 24 hours, the medium was carefully removed from the cells attached to the 96 well plates (this prevents the adhered cells from sticking to the bottom and attention to contamination). 50 μl of the mixed herbal medicine extract prepared in Example or the halibut tree extract prepared in Comparative Example 1 or the ginger tree extract prepared in Comparative Example 2 was added to 150 μl of the medium so that the total volume was 200 μl. Incubated for 24 hours in a% CO ₂ incubator. At this time, since the damage to the cell is added to the medicinal herbs first put the medium and then treated the medicinal herbs. After incubation, the 96 well plate was taken out to remove the medium. 50 μl / ml of MTT dye (50 μl of reservoir solution + 950 μl of medium) was added thereto, diluted to 50 μl in each well, and incubated for 4 hours in a CO ₂ incubator. After removing the supernatant, 100 μl of DMSO was added, followed by shaking for 10 minutes, and then OD (absorbance) was measured at 540 nm with an ELISA reader.

Survival was calculated using only the medium as a control.

2) NR (Neutral Red: 3-amino-7-dimethylamino-2-methyl phenazine) analysis

After removing the medium, 1 ml of trypsin-EDTA solution was added and left for 10-20 minutes to separate the cells from the container and placed in a 15 ml Pelcon tube. In order to collect all remaining cells in the culture flask, 1 ml of medium was added, shaken, and the remaining cells were collected and placed in the Pelcon tube. 10 μl of cell suspension was added to the hemocytometer and the number of cells was counted (2.5 × 10 ⁴ cells / ml). 50 μl of cells (1 ~ 2 × 10 ⁵ cells) were put into 96 well plate and 150μl of medium (DMEM + 10% FBS + antibiotic) was added to the cells and left for 24 hours in a 37 ° C., 5% CO ₂ incubator. Was attached.

After 24 hours, the medium was carefully removed from the cells attached to the 96 well plates (this prevents the adhered cells from sticking to the bottom and attention to contamination). 50 μl of the mixed herbal medicine extract prepared in Example or the halibut tree extract prepared in Comparative Example 1 or the ginger tree extract prepared in Comparative Example 2 was added to 150 μl of the medium so that the total volume was 200 μl. Incubated for 24 hours in a% CO ₂ incubator. At this time, since the damage to the cell is added to the medicinal herbs first put the medium and then treated the medicinal herbs.

After incubation, the 96 well plate was taken out to remove the medium. 10 μl / ml of NR dye (10 μl of reservoir solution + 990 μl of medium) was added thereto, diluted to 200 μl in each well, and then incubated for 3 hours in a 37 ° C., 5% CO ₂ incubator. After the medium was removed again, 100% of 1% CaCl ₂ , 0.5% formaldehyde was added and washed. After removing the supernatant, 200 μl of 1% acetic acid and 50% ethanol was added thereto, followed by shaking for 10 minutes, and then OD (absorbance) was measured at 540 nm with an ELISA reader.

Survival was calculated using only the medium as a control.

The results are shown in Table 5.

Hepatocyte Line (NCTC) Renal Cell Line (Vero) MTT (%) NR (%) MTT (%) NR (%) Mixed Herbal Medicine 105.70 ± 12.9 109.00 ± 5.3 96.20 ± 4 106.90 ± 7.4 Hawthorn extract 44.60 ± 6.6 42.30 ± 4.1 98.40 ± 4.1 99.00 ± 12 Ginger tree extract 95.7 ± 10.9 96.7 ± 5.7 98.7 ± 3.8 97.3 ± 3.5

As shown in Table 5, the mixed herbal medicine of the present invention showed a higher cell survival rate in hepatocytes than the bark extract. In addition, neither the mixed herbal medicine nor the liana extracts showed any risk for survival in kidney cell lines.

Therefore, it can be seen that the mixed herbal medicine of the present invention has excellent hepatoprotective and kidney protective effects.

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

Formulation Example 1  : Preparation of Pharmaceutical Formulations

1. Preparation of powder

Mixed Herbal Extract 2g

1g lactose

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

2. Preparation of Tablets

Mixed Herbal Extract 100mg

Corn Starch 100mg

Lactose 100mg

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

Mixed Herbal Extract 100mg

Corn Starch 100mg

Lactose 100mg

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.

Formulation Example 2  : Manufacture of food

Food products containing the mixed herbal medicine extract of the present invention were prepared as follows.

1. Preparation of Cooking Seasonings

20 to 95% by weight of the mixed herbal medicine extract of the present invention was prepared for health promotion cooking seasoning.

2. Preparation of Tomato Ketchup and Sauce

0.2 ~ 1.0% by weight of the mixed herbal medicine extract 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 ~ 5.0% by weight of the mixed herbal medicine extract of the present invention was added to the flour, and using the mixture to prepare bread, cakes, cookies, crackers and noodles to prepare health foods.

4. Preparation of soups and gravy

0.1 to 5.0% by weight of the mixed herbal medicine extract of the present invention was added to soups and broths to prepare meat products for health promotion, soups of noodles and broths.

5. Preparation of Ground Beef

10% by weight of the mixed herbal medicine extract of the present invention was added to ground beef to prepare ground beef for health promotion.

6. Manufacture of Dairy Products

5 to 10% by weight of the mixed herbal medicine extract of the present invention was added to milk, and various milk 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 using a grinder.

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 mixed herbal medicine extract of the present invention was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

It was prepared by combining the dry powder of the grains, seeds and mixed herbal medicines prepared in the following ratio.

Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),

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

Dry powder (3% by weight) of the mixed herbal medicine extract,

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

Formulation Example 3  : Preparation of Beverages

1. Preparation of Carbonated Drinks

5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 Sterilizing for 20 ~ 180 seconds at ℃ and mixed with a cooling water in a ratio of 1: 4 and then injected with 0.5 ~ 0.82% carbon dioxide gas to prepare a carbonated beverage containing the mixed herbal medicine extract of the present invention.

2. Manufacture of health drinks

Instant sterilization by homogeneously mixing subsidiary ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and mixed herbal extracts , Packed in a small packaging container such as plastic bottles to prepare a healthy beverage.

3. Preparation of Vegetable Juice

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

4. Preparation of Fruit Juice

1 g of the mixed herbal medicine extract of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

The mixed herbal medicine extract of the present invention is GOT, GPT, ALP, BUN, the total bilirubin amount of liver function indicators appear low, it is excellent in liver function improving effect. In addition, ALP, BUN levels are used as diagnostic indicators of renal function as well as liver function, it can be seen that the herbal extracts of the present invention are also excellent in renal function improving effect.

In addition, the mixed herbal medicine extract of the present invention, the amount of hydroxyproline is low in the liver tissue and high in the kidney tissue, it is excellent in the antifibrotic effect and kidney protection effect.

In addition, the mixed herbal medicine extract of the present invention has a low amount of malondialdehyde, which is an indicator of lipid peroxidation, in liver and kidney tissues, and thus has an excellent antioxidant effect.

In addition, the mixed herbal medicine extract of the present invention shows a high cell survival rate in hepatocytes and renal cell lines, it can be seen that the hepatocyte protection and renal cell protection effect is excellent.

Therefore, the composition of the present invention can be usefully used not only for antioxidant, antifibrotic, improving liver function but also for protecting kidney and improving kidney function.

Claims (8)

A pharmaceutical composition for improving liver function, which contains an herbal extract of a medicinal herb extract and a ginger extract as an active ingredient. The pharmaceutical composition for improving liver function according to claim 1, wherein the hut extract and the ginger extract are 2: 1 to 1: 1. A food composition for improving liver function, which contains a mixed herbal extract of the bark extract and the ginger extract as an active ingredient. According to claim 3, wherein the liver function extract and the liver function improvement food composition, characterized in that the mixture of ginger extract 1: 2: 1 to 1. A pharmaceutical composition for improving renal function, comprising a herbal extract of a hawthorn tree extract and a ginger tree extract as an active ingredient. The pharmaceutical composition for improving kidney function according to claim 5, wherein the extract of the liana and the extract of ginger is 2: 1 to 1: 1. A food composition for renal function improvement, comprising a herbal extract of a hawthorn tree extract and a ginger tree extract as an active ingredient. The method according to claim 7, characterized in that the food composition for improving kidney function, characterized in that the compound of the bark and the extract of ginger from 2: 1 to 1: 1.