CN1913910B - Composition comprising Hovenia dulcis Hoveniae extract, Sanya Aconitum extract, or herbal mixture extracts thereof - Google Patents

Abstract

The present invention relates to a composition comprising hovenia dulcis thunb extract, lindera obstiloba extract or herbal mixture extract thereof as an effective ingredient. Hovenia dulcis thunb extract, lindera obstiloba extract or herbal mixture extract thereof of the present invention is highly effective for improving liver function because it can reduce the levels of GOT, GPT, ALP, BUN and total bilirubin, which are major liver function indices. ALP and BUN are also used as renal function indices, so the decrease in ALP and BUN levels caused by the extract of the present invention indicates that hovenia dulcis thunb extract, lindera obstiloba extract or herbal mixture extract thereof of the present invention can also improve renal function. Hovenia dulcis thunb extract, lindera obstiloba extract or herbal mixture extract thereof of the present invention can also reduce the amount of hydroxyproline in the liver but increase the amount of hydroxyproline in the kidney, suggesting that the above-mentioned extract has excellent anti-fibrosis and kidney-protecting effects. Furthermore, hovenia dulcis thunb extract, lindera obstiloba extract or herbal mixture extract thereof of the present invention can reduce the level of malondialdehyde, an index of lipid peroxidation in liver and kidney tissues, suggesting that the extract has excellent antioxidant effect. Hovenia dulcis thunb extract, lindera obstiloba extract or herbal mixture extract thereof of the present invention promotes cell viability in liver and kidney cell lines, suggesting that the extract has excellent liver and kidney cell protective effects. Therefore, the composition of the present invention can be effectively used not only for antioxidation and anti-fibrosis but also for protecting and improving liver and kidney functions.

Description

Compositions comprising Hovenia dulcis Hovenia extract, Sanya Wuyao extract, or herbal mixture extracts thereof

【Technical field】

The present invention relates to a composition comprising Hovenia dulcis Thunb extract, Lindera obtusiloba extract or herbal mixture extracts as active ingredients. the

【Background field】

As one of the representative adult diseases, liver disease is caused by chronic fatigue caused by stress and liver damage caused by most foreign substances. Compared with foreign countries, the development rate of liver disease is very high in Korea, and in particular, the death rate of liver cancer is the highest in the world, and the death rate of chronic liver disease is the third. Liver disease is the leading cause of death among South Korean adults in their 40s, according to a recent report from the National Statistical Office. In Korea, the deadliest of many liver diseases is viral hepatitis. Meanwhile, in western countries, the death rate caused by liver cirrhosis is 5-10 times higher than that caused by viral hepatitis. the

The liver is an organ showing the most active metabolism among many human organs. Acute or chronic liver damage caused by various causes such as fatty diet, heavy alcoholism, viral infection, toxins including chemicals, nutritional deficiencies, etc. can lead to serious diseases such as fatty liver, hepatitis, jaundice, cirrhosis and liver cancer. In particular, a fatty diet and heavy drinking lead to fatty liver, the accumulation of fat in the liver tissue. At this time, GOT (glutamate-oxaloacetate transaminase), GPT (glutamate-pyruvate transaminase) and γ-GTP (γ-glutamyl transpeptidase) increase in the serum. Due to the liver's enormous buffering capacity, liver disease shows few symptoms in its early stages and only when it becomes severe. the

The liver has functions of maintenance and circulation of blood, regulation of blood flow, and detoxification, and is also known to be involved in mental activities. Due to industrialization, the human body is exposed to pollution and many toxins, so the liver has to overwork to neutralize the toxins. Liver damage caused by mental stress was even worse. If the person's spirit is relaxed, the damaged liver cells can be restored. For the modern dweller, the situation is that there is no spare time for rest. Therefore, excessive mental stress, smoking and alcohol abuse worsen liver damage and lead to a malfunction of detoxification, that is, a malfunction of the immune system, which may be the cause of another disease. the

Cirrhosis is caused by fibrosis of liver tissue, a disease in which the balance between production and degeneration of connective tissue is lost. That is, excessive accumulation of connective tissue in liver tissue, accompanied by necrosis or inflammation. Fibrosis of liver tissue is promoted by overproduction of connective tissue due to the growth and migration of hepatic stellate cells (HSCs) that transform into myofibroblasts ( Gressner et al., Biochem. Biophys. Res. Commun. 151-222, 1988). Cirrhosis is the final stage of every chronic liver disease, through which the liver rapidly loses its general function, manifested by a decrease in hepatic blood flow, decreased blood flow in the liver, dysfunction of metabolic enzymes, qualitative and quantitative changes in blood proteins, Changes in bile outflow, etc. the

Hepatotoxicity inducers are exemplified by carbon tetrachloride (CCl ₄ ), D-galactosamine, lipopolysaccharide (LPS) and bromobenzene, and in particular, carbon tetrachloride is known to induce lipid peroxidation of hepatocytes by To induce liver toxicity (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 is an inducer of hepatotoxicity which has been used in studies of anti-hepatotoxic activity. More precisely, it has been administered to mice or rats, cultured hepatocytes and cultured liver tissue to artificially induce liver toxicity. Carbon tetrachloride is converted into a highly active molecular structure of free radical CCl ₃ · in the endoplasmic reticulum by metabolic enzymes such as cytochrome P450, through which it requires a very strong hepatotoxic effect. Free radical _CCl3 · Oxidizes triglycerides accumulated in the liver and fatty acids on membrane phospholipids by alcohols, leading to acidification and oxidation of lipids, followed by lipid peroxidation. As a result, organic peroxides are produced. Through such lipid peroxidation, fat is accumulated in the liver, protein synthesis is decreased, glycogen is decomposed, and cytoplasmic enzymes in blood vessels are destroyed, resulting in necrosis of liver tissue (Chang IM, et al., Drug and chemicaltoxicology 6 , 443-453, 1983). Free radicals also damage the Golgi body, which affects the proteins released from cells, suggesting that not only the liver but also the kidneys may be damaged.

Antioxidant not only inhibits or prevents lipid peroxidation, but also has hepatoprotective and anti-inflammatory effects. Therefore, antioxidant compounds have been used to protect the liver and hepatocytes against attack by reactive oxygen intermediates. the

Antioxidants isolated from natural sources such as flavonoids, quercetin, silymarin or vitamin E have been reported to have positive effects on lipid peroxidation and liver fibrosis. Also, it has been demonstrated that N-acetylcysteine (NAC) reduces oxidative stress and hepatic fibrosis in the early stages of hepatic fibrosis through its antioxidant activity. Picrorhiza Kurroa (picroside) is another natural antioxidant compound that has hepatoprotective and antifibrotic effects by reducing damage caused by lipid peroxidation and free radicals. the

So far, the treatment methods for liver diseases are mainly divided into two ways; one way is dietary treatment and the other way is drug treatment. In most cases, use both methods together. For drug treatment, various drugs with different functions are used depending on the cause and type of liver disease. For example, using stimulators of hepatocyte replication and/or liver function protection such as ursodeoxycholic acid, silymarin (Biotech.Therapeutics, 4, 263-270, 1993), DDB (diphenyldimethyldicarboxylate (ester ); Biochem.Biophy.Res.Comm., 103,1131-1137,1981), glutathione, glycyrrhizin, etc., antiviral agents such as acyclovir, and immunosuppressive drugs such as corticosteroids, 6-mercaptopurine (6-MP), azathioprine, etc. However, liver disease does not develop from a single cause but from a combination of many factors. Therefore, one drug with a certain function is not enough to treat various liver diseases. Conventional drugs for treating liver diseases have problems of unexpected reactions and other side effects by long-term or large-volume administration. the

At the same time, the kidney has the functions of regulating the concentration of ions and body fluids, discharging waste (urine, uric acid, creatinine, etc.) from the system after eliminating toxins, and removing toxic substances, drugs and metabolites. Products from lipid peroxidation in tissues, malonedialdehyde (MDA) and 4-hydroxynonenal (HNE), are known to be indicators of cellular damage. And superoxide dismutase, which catalyzes lipid peroxidation, is known to be involved in recovery from 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). In addition, alkaline phosphatase (ALP) and blood urine nitrogen (BUN) were also used as diagnostic indicators of renal function. In particular, an increase in BUN indicates a decrease in glomerular filtration rate (GFR) through nitrogen blood. Prerenal azotemia implies abnormal renal function without renal injury, which is caused by hypoperfusion due to congestive heart failure, shock, hypoglycemia, and hemorrhage. Postrenal azotemia, on the other hand, is caused when urine flow is blocked in the lower part of the kidney. In the case of postrenal azotemia, complete recovery is expected to be achieved by elimination of the cause. When azotemia is accompanied by various clinical symptoms and biochemical diseases, it is called uremia. Therefore, uremia is not only a single biochemical disease, but also a syndrome carrying metabolic diseases, endocrine diseases, gastrointestinal diseases, neuromuscular diseases and cardiovascular diseases in addition to elimination disorders. Diseases caused by renal dysfunction are exemplified by acute pyelonephritis, chronic pyelonephritis, renal tuberculosis, UTI, urinary calculus, renal cell carcinoma, and the like. the

Hovenia dulcis is a deciduous broad-leaved tall tree belonging to Rhamnaceae, which is also known as 'Jigumok' in Chinese medicine. In Bon-cho-kang-mok there is an explanation about Hovenia dulcis, which is sweet and pure, non-toxic, makes the five internal organs (heart, liver, spleen, lung and kidney) work stably and calmly, eliminates heat in the chest cavity, Suppresses thirst, neutralizes alcoholism, slows vomiting, detoxifies insects and treats five types of hemorrhoids. It is also known to have hepatoprotective effects. Specifically, it has excellent activities of eliminating bad breath, improving alcoholic hepatitis, fatty liver and cirrhosis, anti-cancer, regulating blood pressure, lowering blood sugar, liver detoxification, and relieving constipation. the

Sanya wuyao is a deciduous broad-leaved tall tree belonging to Lauraceae, which is widely distributed in Korea, Japan, China and Manchuria. The flowers, leaves and stems of Sanya Wuyao exude a unique fragrance. Their stems have been used for medicinal purposes due to their antibacterial effects. Use its fresh buds as a tea. the

After long-term research on natural medicinal compounds with various mechanisms of action and low toxicity, the present inventors have confirmed that Hovenia dulcis Hoveniae extract, Sanya buya extract or herbal mixture extracts have activities in addition to protecting and improving kidney function, It also has anti-oxidation, anti-fibrosis and liver function-improving activities and completed the present invention. the

【Content of invention】

【Technical solutions】

An object of the present invention is to provide a composition comprising Hovenia dulcis Hoveniae extract, Sanya Aconitum extract or herbal mixture extract thereof as active ingredients. the

【Best way】

The present invention provides a compound comprising Hovenia dulcis Hoveniae extract, Sanya Aconitum extract or herbal mixture extract thereof as active ingredients. the

The composition of the present invention includes anti-oxidation composition, anti-fibrosis composition, composition for improving liver function and composition for improving kidney function. the

Hereinafter, the present invention will be described in detail. the

The extraction method of Hovenia dulcis Hoveniae extract of the present invention, Sanya Wuyao extract or herbal mixture extract thereof is as follows. the

Wash Hovenia dulcis or Sanya black medicine with water, then dry it in a dark place. Dried Hovenia dulcis or Sanya Aconitum was placed in a reflex extractor, purified water was added thereto and heated at 100° C. for 90 minutes to obtain a hot water extract. While it was still hot, the hot water extract was filtered through filter paper under reduced pressure. The filtrate was concentrated by using a vacuum evaporator. For long-term storage, dry the solution by using a lyophilizer. In the present invention, the stems, flowers, leaves and seeds of Hovenia dulcis or Sanya aconia are usable. the

The combination ratio of the herbal medicines of the composition of the present invention was determined based on the dry weight of each herbal medicine. To be precise, Hovenia dulcis and Sanya Wuyao are mixed in a ratio of 3:2 to 1:1, more preferably in a ratio of 2:1 to 1:1. Such a ratio is determined in consideration of the effective dose of each herb and its side effects, and when the ratio is out of the above-mentioned range, the effect of the drug drops rapidly or side effects may become a problem. the

In the group administered Hovenia dulcis Hoveniae extract, Sanya Wuyao extract or herbal mixture extract, GOT, GPT, ALP, BUN and total bilirubin, which are all used as indicators of liver function, all decreased, indicating that the present invention The extract has excellent liver function improvement effect. Meanwhile, ALP and BUN are used not only as indicators of liver function but also as indicators of renal function. Therefore, the reduced levels of ALP and BUN indicate that Hovenia dulcis Hoveniae extract, Sanya Wuyao extract or herbal mixture extracts of the present invention also have excellent renal function improving effects. the

The Hovenia dulcis extract of the present invention, the Sanya Wuyao extract or herbal mixture extracts reduce the amount of hydroxyproline in liver tissue, but increase the amount of hydroxyproline in kidney tissue, illustrating that the present invention The extract has excellent anti-fibrosis and renal protection. In particular, the herbal extract extracted from the mixture of Hovenia dulcis Hoveniae and Sanya Aconitum showed higher effects than each individual extract, that is, the mixture extract showed a higher effect on liver tissue than each individual extract. Hydroxyproline was more reduced in kidney tissue, but hydroxyproline was more increased in kidney tissue. the

The Hovenia dulcis extract of the present invention, Sanya Wuyao extract or its herbal mixture extract can reduce the level of malondialdehyde as an indicator of lipid peroxidation in liver and kidney tissues, indicating that the extract of the present invention has extremely Good antioxidant effect. the

The liver cell lines and kidney cell lines treated with Hovenia dulcis Hoveniae extract of the present invention, Sanya Wuyao extract or herbal mixture extracts showed high cell viability, indicating that the extracts of the present invention have excellent hepatocyte and Renal cell protection. the

Therefore, the composition of the present invention can be effectively used for improving and protecting liver and kidney functions and also for anti-oxidation and anti-fibrosis. the

In addition to the Hovenia dulcis extract, Sanya Wuyao extract or herbal mixture extract, the composition of the present invention may additionally include one or more active ingredients having the same or similar functions as the extract of the present invention . the

The Hovenia dulcis extract, Sanya Aconitum extract or herbal mixture extract of the present invention can be administered orally or parenterally and can be used in general forms of pharmaceutical preparations. The herbal mixture extract of the present invention can be prepared for oral or parenteral use by mixing with commonly used fillers, supplements, binders, wetting agents, disintegrants, diluents such as surfactants, or excipients apply. Solid preparations for oral administration are tablets, pills, dusting powders, granules and capsules. These solid formulations are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricating agents such as magnesium stearate, talc, and the like can be used. Liquid preparations for oral administration are suspensions, solutions, emulsions and syrups and the above-mentioned preparations may contain various excipients such as wetting agents, sweetening agents, flavoring agents and preservatives, as well as common simple diluents. Such as water and liquid paraffin. Formulations for parenteral administration are sterile aqueous solutions, water-insoluble excipients, suspensions, emulsions, lyophilizates and suppositories. Water-insoluble vehicles and suspensions may contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Suppositories may contain, in addition to the active compound or compounds, witepsol, polyethylene glycol, Tween 61, cocoa butter, lauryl essence, glycerin, gelatin, and the like. the

A dosage unit may contain, for example, 1, 2, 3 or 4 separate doses or 1/2, 1/3 or 1/4 of a separate dose. Individual doses preferably contain the amount of active compound administered in one administration and which usually corresponds to a full, 1/2, 1/3 or 1/4 of the daily dose. The effective doses of Hovenia dulcis Hoveniae extract, Sanya Wuyao extract or herbal mixture extract of the present invention are respectively 200-600 mg/kg, more preferably 300-400 mg/kg, and the number of administrations is 1-6 per day. Second-rate. the

The composition of the present invention can be administered alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy and biological response modifiers to improve liver and kidney function and obtain antioxidative and antifibrotic effects. the

The composition of the present invention can be added in health foods to improve liver and kidney functions and obtain anti-oxidation and anti-fibrosis effects. At this time, the Hovenia dulcis orientalis extract of the present invention, the Sanya Aconitum extract or herbal mixture extract of the present invention can be added as it is or mixed with other foods or ingredients according to the conventional method. The mixing ratio of the active ingredients is determined according to the purpose of use (prevention, health or therapeutic treatment). In the case of producing food or beverages containing the Hovenia dulcis Hoveniae extract, Sanya Aconitum extract or herbal mixture extract of the present invention, the extract is preferably contained in an amount of 15% by weight of the raw material, more preferably Added at 10% by weight. However, when it is administered for a long time to improve health, the content of the extract may be lower than the above, but the effective dose may contain more than the above, because the extract of the present invention is very safe. the

There is no limitation in applicable foods, which are exemplified by meat, sausage, bread, chocolate, candy, snacks, biscuits, pizza, ramyun, noodles, chewing gum, dairy products including ice cream, soup, beverages, tea, wine, alcoholic Beverages and vitamin complexes, etc. and virtually every health food product in general is included. the

Health beverages including the composition of the present invention, such as other beverages, may additionally include various food flavors or natural sugars and the like. The aforementioned natural sugars may be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xilytole, sorbitol and erythritol. As the sweetener, natural sweeteners such as arrowroot protein and stevia extract or artificial sweeteners such as saccharin and aspartame can be used. The ratio of natural sugar to the composition of the invention is preferably 0.01-0.04g to 100ml, more preferably 0.02-0.03g to 100ml. the

In addition to the ingredients mentioned above, the composition of the present invention may include various nutritional ingredients, vitamins, electrolytes, flavor enhancers, colorants, pectic acid and its salts, arginic acid and its salts, organic Acids, protective colloid viscosifiers, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonators for adding soda, etc. The compositions of the present invention may also include natural fruit juices, fruit drinks and/or fruit pulps which may be added to plant drinks. All mentioned ingredients can be added individually or together. The mixing ratio of those ingredients is not critical actually, but usually each may be 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention. the

【Invention method】

The practice and presently preferred embodiments of the invention are illustrated as shown in the following examples. the

It will be appreciated, however, that those skilled in the art, upon consideration of this specification, may make modifications and improvements within the spirit and scope of the invention. the

<Example 1> Preparation of Hovenia dulcis Hovenia extract

Hovenia dulcis was washed with water, then dried in a dark place. 20 g of dried Hovenia dulcis was placed in a reflex extractor, 1.5 l of purified water was added thereto and heated at 100° C. for 90 minutes. While the hot water extract was still hot, it was filtered through filter paper under reduced pressure. The filtrate was concentrated by using a vacuum evaporator. It was dried by a lyophilizer for long-term storage. the

The concentrated solution was used for animal testing (2 ml/rat/day). the

<Example 2> Preparation of Sanya Wuyao Extract

By using Sanya Agranate, the Sanya Buya extract was prepared in a similar manner as described above in Example 1. The concentrated solution was used for animal testing (2 ml/rat/day). the

<Example 3> preparation of herbal mixture extract

20 g of dried Hovenia dulcis and 10 g of dried Aconiti sativa stems were mixed and its extract was prepared in a similar manner to that described in Example 1. the

The concentrated solution was used for animal testing (2 ml/rat/day) and the 4-fold concentrated solution was used for MTT and NR assays (50 μl/well). the

<Experimental Example 1> Antioxidative, antifibrosis, improvement and protection of liver and kidney function of the extract in rats having liver and kidney damage induced by long-term administration of carbon tetrachloride

Experiments were carried out as follows to study the antioxidant and anti-fibrosis effects of the Hovenia dulcis extract of the present invention, the Sanya wuyao extract or herbal mixture extracts and the improvement and protection of liver and kidney functions in liver and kidney injury models . the

1. Test animals

Twelve-week-old Sprague-Dawley rats (Damul science, Osan, Korea) weighing about 180 to 210 g were used as test animals. The rearing temperature was 23±2°C and the relative humidity was maintained at 60±10%. Feed (Purina feed) and water were provided ad libitum, and the day-night cycle was regulated. the

The animals were acclimatized to the test room for 2 weeks, and then they were divided into 5 groups, ① normal group, ② CCl ₄ treated group, ③ CCl ₄ + herbal mixture extract treated group, ④ CCl ₄ + Hovenia dulcis extract treated group and ⑤ CCl ₄ + Tri Yabuya extract treatment group. Each group consisted of 10 rats. 2. Induction of liver fibrosis (cirrhosis) and kidney injury

Except for the normal group, the experimental group was administered with a mixture of olive oil and CCl ₄ at 1 ml/rat/day, 3 times a week for 4 weeks to induce liver fibrosis (sclerosis) and kidney damage.

Orally administer distilled water to the _CCl4 treatment group at 2ml/rat/day, orally administer the herbal mixture extract prepared in the above-mentioned Example 3 to the _CCl4 +herbal mixture extract treatment group at 2ml/rat/day, at 2ml The Hovenia dulcis extract prepared in the above-mentioned Example 1 was orally administered to the CCl ₄ + Hovenia dulcis extract treatment group per day per rat/day, and the CCl ₄ + Sanya rutabaga extract was also administered at 2 ml/rat/day. The treatment group was orally administered the Sanya Aconitum extract prepared in Example 2 above.

Rats in each group including the normal group were weighed and then anesthetized with ether. Blood was withdrawn from the heart by cardiac puncture, which was left at room temperature for over 2 hours. Next, centrifugation was performed at 3000 rpm for 10 minutes to obtain serum, which was stored at -20°C. Serum was examined to measure levels of GOT, GPT, alkaline phosphatase, BUN, and total bilirubin. the

The liver and kidneys of rats, which had been damaged by artificial induction, were extracted, washed with phosphate buffer (pH 7.0), and then their weights were measured. Portions of liver and kidney tissue were stored at -75°C for use in the measurement of hydroxyproline and malondialdehyde (MDA). the

Weight changes were measured weekly. Study the ears, tail, and feet to detect signs of jaundice. When the rats were sacrificed, the livers were weighed. the

The results were expressed as mean ± SD, and the significance of the difference in the mean values between the control group and the experimental group was checked by Student's t-test. When p<0.005, it was judged to be statistically significant. the

Show the results in Table 1

【Table 1】

^* : p<0.005

As shown in Table 1, compared with the control, the changes in the ratio of body weight to liver weight in the experimental groups treated with the Hovenia dulcis extract, the Sanya Aconitum extract and the herbal mixture extract of the present invention were not significant. obviously. Likewise, changes in the ratio of body weight to kidney weight were also insignificant compared to controls. the

3. Serological and biochemical tests

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

500 μl of AST substrate solution was placed in two falcon tubes, which were heated at 37° C. for 3-5 minutes. In one tube, the substrate solution was diluted with the standard solution, while in the other tube, 100 μl of the serum sample was added. Reactions were induced in two tubes for 60 minutes at 37°C. 100 μl of purified water and 500 μl of a coloring solution (2,3-dinitrophenylhydrazine) were added to each tube, which was then left at room temperature for 20 minutes. 5 ml of 0.4N NaOH was added to each tube, followed by reaction at room temperature for 10 minutes. OD was measured at 505nm. the

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

150 μl of the ALT substrate solution was placed in two falcon tubes, which were heated at 37° C. for 4 minutes. In one tube, the substrate solution was diluted with a standard solution, while in the other tube, 100 μl of serum samples were added and the reaction was induced in both tubes for 30 minutes at 37°C. 100 μl of purified water and 500 μl of a coloring solution (2,3-dinitrophenylhydrazine) were added to each tube, which was then left at room temperature for 20 minutes. 1.5ml of 0.4N NaOH was added to each test tube, followed by reaction at room temperature for 10 minutes. OD was measured at 505nm. the

3) ALP (alkaline phosphatase) measurement

2.0 ml of ALP substrate buffer solution (sodium 2-phenylphosphate) was placed in three test tubes, and reacted at 37° C. for 3 to 5 minutes. 50 μl of serum samples, 50 μl of purified water and 50 μl of standard solutions were added to the test tubes respectively, followed by reaction at 37° C. for 15 minutes. 2.0 ml of colorant was added to each test tube, which was then left at room temperature for 10 minutes. The OD was measured at 570nm within 60 minutes. the

4) BUN (blood urine nitrogen) measurement

20 μl of serum samples, 20 μl of purified water and 20 μl of standard solutions were placed in three test tubes, respectively. 2.0 ml of enzyme solution was added thereto, followed by reaction at 37°C for 5 minutes. 2.0 ml of colorant was added to each tube, which was heated at 37°C for 10 minutes. The OD was measured at 580nm within 60 minutes. the

5) Measurement of total bilirubin

100 μl of serum sample, 100 μl of purified water and 100 μl of standard solution were placed in three test tubes, and 600 μl/tube of a coloring agent for total bilirubin was added thereto. 600 [mu]l of the diazo mixture was added to each test tube, which was left at room temperature for 10 minutes. Add 600 μl of ferring reagent to each tube to induce the reaction. The OD was measured at 600nm within 2 hours. the

Show the results in Table 2

【Table 2】 GOT(IU) GPT(IU) ALP(KA) BUN (mg/□) Total Bilirubin (mg/□) normal group 61.78±6.0 13.2±1.0 63.5 ± 11.2 13.6 ± 1.1 0.2±0.05 CCl ₄ treatment group 241.8±43.1 145.3±38.1 156.9±36.1 23.4 ± 2.1 1.6±0.92 CCl ₄ + herbal mixture extract treatment group 137.6±52.4 ^{* *} 69.6±47.0 ^* 137.0±26.0 1g.04±6.9 1 0.27±0.14 CCl ₄ + Hovenia dulcis extract treatment group 165.1±46.4 ^{* *} 70.9±42.2 ^* 143.1±37.9 18.6±2.1 ^* 0.7±0.04 CCl ₄ + Sanya Wuyao extract treatment 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, compared with the control, in the experimental groups treated with Hovenia dulcis Hoveniae extract, Sanya Aconitum extract and herbal mixture extract of the present invention, respectively, GOT, GPT as liver function index , ALP, BUN and total bilirubin levels were significantly lower. In particular, the levels of those indices were the lowest in the group treated with the herbal mixture extract. the

Therefore, it is proved that the extract of Hovenia dulcis, Sanya wuyao extract and herbal mixture extract of the present invention have excellent liver function improving effects. the

And, ALP and BUN are indexes not only for liver function but also for kidney function, therefore, the reduced level of ALP and BUN caused by the extract of the present invention also means that the Hovenia dulcis extract of the present invention, Sanya buya extract and its herbal mixture extract also have an excellent effect on improving kidney function. the

4. Measurement of hydroxyproline (hyp)

1) Preparation of reagents

①Acetate citrate buffer

50 g of sodium acetate trihydrate, 37.5 g of trisodium citrate and 5.5 g of citric acid monohydrate were mixed in 395 ml of isopropanol. Also, the total volume of the mixture was adjusted to 1 L by adding distilled water. The pH of the mixed solution was also adjusted to 6.0. The final solution was stored at 4°C. the

② Chloramine-T solution

Dissolve 84 mg of Chloramine-T in 10 ml of acetate citrate buffer. the

③Ehrilich's reagent

Mix 10 g of p-dimethylaminobenzaldehyde and 11 ml of 60% perchloric acid to prepare a stock solution. 3 ml of the stock solution were mixed with 8.0 ml of isopropanol to obtain Ehrilich's reagent. The reagents were prepared immediately before use, and the stock solutions were stored at 4°C in the dark. the

2) Measurement of hydroxyproline

0.2 g of liver (or kidney) tissue that was frozen or lyophilized (-50°C, 72 hours) was dispersed in a 10 ml glass vial. 4 ml of 6N HCl was placed in the bottle, followed by homogenization with a homogenizer. Next, hydrolysis was carried out in a dry oven at 110° C. for 10-24 hours, followed by filtration with Whatmann filter paper. Similarly, standard solutions diluted with trans-hydroxyproline 6N HCl at different concentrations of 0, 0.2, 0.4, 0.6, 0.8 and 1.0 μg/50 μl were also hydrolyzed at 110 ° C for 12 to 14 hours. Take 50 μl each of the sample and standard solutions and dry them thoroughly in glass vials to remove HCl. Add 1.2 ml of 50% isopropanol to dissolve the precipitate. 200 µl of chloramine-T solution was added thereto, followed by reaction at room temperature for 10 minutes. Next, 1.2 ml of Ehrilich's reagent was added. Coloration was induced at 50°C for 90 minutes, followed by cooling at room temperature. OD was measured at 558nm by using a spectrophotometer. the

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

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

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

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

The results are shown in Table 3. the

【table 3】

^* : p<0.005

As shown in Table 3, compared with the control, in each of the experimental groups treated with the Hovenia dulcis Hovenia extract, the Sanya Wuyao extract and the herbal mixture extract of the present invention, the hydroxyproline in the liver tissue The content is lower and higher in kidney tissue. In particular, the hydroxyproline content in the liver tissue was significantly lower (50.8%) in the group treated with the herbal mixture extract compared with the control. However, the content of hydroxyproline in the kidney was 5.5% higher than that of the control, 3.0% higher than that of the group treated with the extract of Hovenia dulcis, and 1.2% higher than that of the group treated with the extract of Sanya Aconiti. the

Therefore, it has been confirmed that the Hovenia dulcis extract, the Sanya buya extract and the herbal mixture extract of the present invention have excellent anti-fibrosis and renal protection effects. the

5. MDA (malondialdehyde) measurement

Homogenized tissue samples [0.2 g liver tissue (or kidney tissue) in 1.8 ml of 1.15% KCl] and 200 μl diluted standard substances (0, 4, 8, 16, 32 nmol/200 μl tetramethoxypropane) Put together in a falcon tube. 200 µl of the standard solution diluted with the sample was mixed with 100 µl of 0.2% SDS, followed by reaction at room temperature for 10 minutes. Thereto were added 750 µl of 20% acetic acid and 750 µl of 0.8% thiobarbiturate. The solution was reacted at 95°C for 30 minutes, then cooled in ice. After cooling was completed, 2 ml of n-butanol was added, followed by centrifugation to obtain a supernatant. The OD of the supernatant was measured by a spectrophotometer at 532nm. the

The levels of MDA in serum and tissues were measured as follows. the

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

C × 5 = malondialdehyde content (μmol/ml)

The results are shown in Table 4. the

【Table 4】

^* : p<0.05

As shown in Table 4, compared with the control, the amount of malondialdehyde, lipid Indicator of peroxidation, low in liver and kidney tissues. In particular, in the group treated with the herbal mixture extract, the content in liver tissue was significantly lower (31.0%) and the content in kidney tissue was also very low (13.0%) compared to the control. the

Therefore, it has been confirmed that the Hovenia dulcis extract, the Sanya buya extract and the herbal mixture extract of the present invention have excellent antioxidant and renal protection effects. the

<Experimental Example 2> Cytotoxicity Test

The cell lines used in the present invention are NCTC clone 1469 (hepatic cell line) and vero (kidney cell line), which were purchased from Korea Cell Line Bank (KCLB). the

5 mg of MTT powder was dissolved in 1 ml of phosphate buffer, which was stirred well and then filtered through a 0.4 μm filter to obtain an MTT stock solution. the

4 mg of NR powder was dissolved in 1 ml of triple distilled water, which was then thoroughly stirred and filtered through a 0.4 μm filter to obtain a NR stock solution. the

Purchase trypsin-EDTA containing 0.5% trypsin and 5.3 mM EDTA and make a 10-fold dilution with PBS before use. the

1) Determination of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-brominated diphenyltetrazolium salt]

After removing the medium, add 1 ml of trypsin-EDTA solution and let it stand for 10-20 minutes. Cells were detached from the vessel and then transferred to a 15ml falcon tube. Add 1 ml of medium to the culture flask and shake to detach any remaining cells, which are also placed in falcon tubes. 10 μl of the cell suspension was placed in a hemocytometer and the cells were then counted (2.5×10 ⁴ cells/ml). Cells were seeded on a 96-well plate at 50 μl (1˜2×10 ⁵ cells)/well, and 150 μl of medium (DMEM+10% FBS+antibiotics) was added thereto. Cells were incubated in a 37 °C, 5% _CO2 incubator for 24 h, resulting in cell attachment.

After 24 hours, the medium was carefully removed from the cells attached to the 96-well plate (careful attention was required to keep the medium free from contamination and contamination by the cells). Add 150 μl of the culture medium with 50 μl of the Hovenia dulcis extract prepared in Example 1, 50 μl of the Sanya buya extract prepared in Example 2 and the herbal mixture extract prepared in 50 μl of Example 3, so that The total volume is 200 μl. The solutions were incubated in a 37 °C, 5% _CO2 incubator for 24 h. In this case, the extract should be added later, otherwise the cells will be damaged. Therefore, the medium should be added first and the extract placed therein. After culturing, the 96-well plate was removed to remove the medium. 50 μl/ml of MTT dye (50 μl of stock solution + 950 μl of medium) was added to dilute the solution, which was dispensed in 50 μl per well, followed by further incubation for 4 hours in a CO ₂ incubator. After removing the supernatant, 100 µl of DMSO was added, followed by stirring for 10 minutes. OD was measured at 540nm with an ELISA reader.

Only medium was added to the control and viability was studied. the

2) Determination of NR (neutral red: 3-amino-7-dimethylamino-2-methylphenazine)

After removing the medium, 1 ml of trypsin-EDTA solution was added and left for 10-20 minutes. Cells were detached from the vessel and then transferred to a 15ml falcon tube. Add 1 ml of medium to the culture flask and shake to detach any remaining cells, which are also placed in falcon tubes. 10 μl of the cell suspension was placed in a hemocytometer and the cells were then counted (2.5×10 ⁴ cells/ml). Cells were seeded on a 96-well plate at 50 μl (1˜2×10 ⁵ cells)/well, and 150 μl of medium (DMEM+10% FBS+antibiotics) was added thereto. Cell attachment was obtained by incubating the cells in a 37 °C, 5% _CO2 incubator for 24 h.

After 24 hours, the medium was carefully removed from the cells attached to the 96-well plate (careful attention was required to keep the medium free from contamination and contamination by the cells). Add 150 μl of the culture medium with 50 μl of the Hovenia dulcis extract prepared in Example 1, 50 μl of the Sanya buya extract prepared in Example 2 and the herbal mixture extract prepared in 50 μl of Example 3, so that The total volume is 200 μl. The solution was incubated at 37 °C in a 5% _CO2 incubator for 24 h. In this case, the extract should be added later or the cells will be damaged. Therefore, the medium should be added first and the extract placed therein.

After culturing, the 96-well culture plate was removed to remove the medium. 10 μl/ml of NR dye (10 μl of stock + 990 μl of medium) was added to dilute the solution, which dispensed 200 μl per well, followed by further incubation for 3 hours at 37° C., 5% CO ₂ incubator. After removing the medium, wash the plate with 100 μl of 1% _CaCl2 and 0.5% formaldehyde. Remove the supernatant. Subsequently, 200 µl of 1% acetic acid and 50% ethanol were added, followed by stirring for 10 minutes. OD was measured at 540nm with an ELISA reader.

Only medium was added to the control and viability was studied. the

The results are shown in Table 5. the

 (北枳椇+三丫乌药)  北枳椇提取物   44.60±6.6   42.30±4.1   98.40±4.1   99.00±12  三丫乌药提取物   95.7±10.9   96.7±5.7   98.7±3.8   97.3±3.5 【table 5】

(Hovenia Hoveniae + Sanya Wuyao) Hovenia Hovenia Extract 44.60±6.6 42.30±4.1 98.40±4.1 99.00±12 Sanya Wuyao Extract 95.7±10.9 96.7±5.7 98.7±3.8 97.3±3.5

As shown in Table 5, the extracts of Sanya Wuyao and herbal mixture extracts of the present invention showed high cell viability in hepatic cell lines. In particular, herbal mixture extracts were more effective than Hovenia dulcis extracts in enhancing cell viability in hepatic cell lines. At the same time, the extracts of Hovenia dulcis orientalis of the present invention, the extracts of Sanya Aconiti and herbal mixtures thereof do not impair the cell viability in the kidney cell line. the

Therefore, it has been confirmed that the Hovenia dulcis Hovenia extract, the Sanya Aconitum extract and the herbal mixture extract of the present invention have excellent hepatic and renal protective effects. the

Preparation examples of the composition of the present invention are described below. the

A pharmaceutical composition comprising the Hovenia dulcis Hovenia extract, the Sanya Aconitum extract or the herbal mixture extract thereof of the present invention is prepared as follows. the

<Preparation Example 1> preparation of pharmaceutical composition

1. Preparation of powder

Hovenia dulcis Hovenia extract (or Sanya Wuyao extract or herbal mixture extract) 2g

Lactose 1g

The above ingredients were mixed together, and an airtight bag was filled with the mixture to prepare a powder. the

2. Preparation of tablets

Hovenia dulcis Hovenia extract (or Sanya Aconitum extract or herbal mixture extract) 100mg

cornstarch 100mg

Lactose 100mg

Magnesium stearate 2mg

The above-mentioned ingredients are mixed together, and tablets are prepared by tabletting according to conventional tablet production methods. the

3. Preparation of Capsules

Hovenia dulcis extract (or Sanya Aconitum extract or herbal mixture extract) 100mg

Corn Starch 100mg

Lactose 100mg

Magnesium stearate 2mg

The above-mentioned ingredients are mixed together, and capsules are filled with the mixture according to a conventional capsule production method to prepare capsules. the

<Preparation Example 2> Preparation of food

A food containing the Hovenia dulcis Hovenia extract, the Sanya Aconitum extract or the herbal mixture extract thereof of the present invention is prepared as follows. the

1. Preparation of spices and condiments

Spices and condiments containing 20-95% by weight of the Hovenia dulcis extract of the present invention (or Sanya Aconitum extract or herbal mixture extract) are prepared for improving health. the

2. Preparation of tomato paste and raw materials (source)

Tomato sauce and raw materials are prepared for improving health by adding Hovenia dulcis Hovenia extract (or Sanya aconitum extract or herbal mixture extract) of the present invention to tomato paste and raw materials at 0.2-1.0% by weight. the

3. Preparation of pasta

Add Hovenia dulcis Hovenia extract (or Sanya Wuyao extract or herbal mixture extract) of the present invention to flour at 0.5-5.0% by weight, and use the mixture to prepare bread, cakes, biscuits, crackers, etc. Biscuits and noodles to produce food that improves health. the

4. Preparation of soups and gravy

Preparation of health-improving processed meat, noodle soup and gravy by adding the Hovenia dulcis Hovenia extract (or Sanya Aconitum extract or herbal mixture extract) of the present invention to soup and gravy at 0.1 to 5.0% by weight . the

5. Preparation of Ground Beef

Health-improving ground beef was prepared by adding Hovenia dulcis Hovenia extract (or Sanya Aconitum extract or herbal mixture extract) of the present invention to ground beef at 10% by weight. the

6. Preparation of dairy products

The Hovenia dulcis extract of the present invention (or the extract of Sanya Aconitum or its herbal mixture extract) of the present invention is added to milk at 5-10% by weight, and then it is used to produce health-improving dairy products, including butter and ice cream. the

7. Preparation of cerial

Brown rice, barley, glutinous rice and Job's-tears were gelled by a conventional method, dried and roasted, and then pulverized with a pulverizer to obtain 60-mesh granules. the

Black soybeans, black sesame seeds, Perilla japonica were steamed by a conventional method, dried and roasted, and then pulverized with a pulverizer into 60-mesh granules. the

The Hovenia dulcis extract (or Sanya buya extract or herbal mixture extract) of the present invention is concentrated under reduced pressure in a vacuum concentrator, and then dried by a spray dryer. The dried product was crushed into 60-mesh granules. the

The crops, seeds and dry powder of Hovenia dulcis extract (or Sanya agrariana extract or its herbal mixture extract) were mixed by the following ratios. the

Crops (30% by weight of brown rice, 15% by weight of Job’s tears, 20% by weight of barley),

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

The dry powder (3% by weight) of Hovenia dulcis Hoveniae extract (or Sanya Wuyao extract or its herbal mixture extract),

Ganoderma lucidum (0.5% by weight),

Rehmannia glutinosa (0.5% by weight)

<Preparation Example 3> preparation of beverage

1. Preparation of carbonated beverages

Mix sugar (5-10%), citric acid (0.05-0.3%), caramel (0.005-0.02%) and vitamin C (0.1-1%), add purified water (79-94%) ) to get the syrup. The syrup is sterilized at 85-98° C. for 20-180 seconds, and then mixed with cold water at a ratio of 1:4. Carbon dioxide is injected thereinto at 0.5-0.82%, resulting in the preparation of a carbonated drink containing the Hovenia dulcis Hovenia extract of the present invention (or the Sanya Aconiti extract or herbal mixture extract thereof). the

2. Preparation of healthy drinks

Optional components such as liquid fructose (0.5%), oligosaccharides (2%), sugar (2%), salt (0.5%), water (75%) and Hovenia dulcis extract (or extract or its herbal mixture extract) and mix evenly. After crushing, the mixture is placed in a small container such as a pet or glass bottle, resulting in the preparation of a healthy drink. the

3. Preparation of Plant Juice

Add 5 g of the Hovenia dulcis extract (or Sanya Aconitum extract or herbal mixture extract) of the present invention to 1,000 ml of tomato or carrot juice to prepare a health-improving plant juice. the

4. Preparation of juice

1 g of Hovenia dulcis extract of the present invention (or Sanya buyao extract or herbal mixture extract thereof) is added to 1,000 ml of apple or grape juice to prepare health-improving fruit juice. the

【Industrial applicability】

The Hovenia dulcis extract, Sanya Aconitum extract or herbal mixture extract of the present invention is highly effective for improving liver function because it can reduce GOT, GPRT, ALP, BUN and total bilirubin which are the main indicators of liver function prime level. ALP and BUN are also used as indicators of renal function, so the reduction of ALP and BUN levels caused by the extract of the present invention shows that the extract of Hovenia dulcis of the present invention, the extract of Sanya Wuyao or its herbal mixture extract can also improve kidney function. the

The Hovenia dulcis extract of the present invention, Sanya Wuyao extract or its herbal mixture extract can also reduce the amount of hydroxyproline in the liver but increase the amount of hydroxyproline in the kidney, suggesting that the above-mentioned extracts have excellent anti-fibrotic and renal protective effects. the

In addition, the Hovenia dulcis extract of the present invention, the Sanya Wuyao extract or herbal mixture extracts can reduce the level of malondialdehyde, an indicator of lipid peroxidation in liver and kidney tissues, suggesting that the extracts have extremely Good antioxidant effect. the

The Hovenia dulcis extract, Sanya Wuyao extract or herbal mixture extracts of the present invention promote cell viability in liver and kidney cell lines, indicating that the extracts have excellent liver and kidney cell protection. the

Therefore, the composition of the present invention can not only be effectively used for anti-oxidation and anti-fibrosis, but also can be used for protecting and improving liver and kidney functions. the

Claims (8)

1. An anti-oxidant composition comprising Lindera obtusiloba water extract as an active ingredient. 2. An anti-oxidant composition, which comprises the water extract of Hovenia dulcis Thunb and a herbal mixture of Aconitum arvennata as active ingredients, wherein in the herbal mixture Hovenia dulcis Thunb and A. The dry weight ratio of medicine is 2:1～1:1. 3. An anti-fibrosis composition, which comprises the water extract of Aconiti sativa as an active ingredient. 4. An anti-fibrosis composition, which comprises the water extract of Hovenia dulcis and Sanya aconia as active ingredients, wherein the dry weight of Hovenia dulcis and Sanya aconia in the herbal mixture is The ratio is 2:1 to 1:1. 5. A composition for improving liver function, which comprises the water extract of Aconiti sativa as an active ingredient. 6. A composition for improving liver function, comprising the water extract of Hovenia dulcis Hovenia dulcis and the herbal mixture of Sanya aconia as active ingredients, wherein the dry weight of Hovenia dulcis and Sanya aconia in the herbal mixture is The ratio is 2:1 to 1:1. 7. A composition for improving kidney function, which comprises the water extract of Sanya Aconitum as an active ingredient. 8. A composition for improving renal function, which comprises the water extract of Hovenia dulcis and Sanya aconia as active ingredients, wherein the dry weight of Hovenia dulcis and Sanya aconia in the herbal mixture is The ratio is 2:1 to 1:1.