KR100924216B1 - Hepatoprotective composition comprising pectolinarin or pectolinarigenin - Google Patents

Abstract

The present invention relates to a composition for protecting liver comprising fentorinarin or fentorariginine. Fectorinarin and Fectorinarigenin reduce the concentration of AST, ALT, ALP and LDH in D-galactosamine-induced liver damage and lower the amount of malondialdehyde, which is an indicator of lipid peroxide, to inhibit lipid oxidation In addition, by increasing the concentration of GSH, GR, GCS, GST and SOD, hepatoprotective and antioxidant effects are excellent. Therefore, the fentorinarin and fentorariginine of the present invention can be usefully used as a medicine for liver protection and health food.

Description

Hepatoprotective composition comprising pectolinarin or pectolinarigenin}

The present invention relates to a composition for protecting liver comprising fentorinarin or fentorariginine.

The liver is located between the digestive system and the circulatory system of the human body to protect the body from toxic substances from the outside and is responsible for metabolism of in vitro substances. Since the in vitro material that enters the living body once passes through the liver, the liver is more likely to be damaged than other organs because of the high risk of exposure to many toxic substances besides nutrients. The liver is a good organ with good regenerative capacity and recovers to normal enough for some damage. However, continuous damage caused by excessive ingestion of alcohol, abuse of chemicals, viral hepatitis, and biliary secretion can lead to a decrease in liver function, as well as complete destruction of liver tissue and inability to restore normal damage. And eventually develops liver diseases such as hepatitis, cirrhosis and liver cancer. Liver disease is a disease with high mortality due to the absence of pain or subjective symptoms in the early stages, and since it is found only at the end, it cannot be treated in a timely manner.

In addition, our bodies are constantly exposed to pollutants and toxic substances due to industrialization, and our liver is constantly suffering from detoxification. What is more serious is liver damage caused by mental stress. If you have mental rest, damaged hepatocytes will be repaired, but in an urgent modern society, you will not be able to afford mental rest, which will increase your liver damage by mental stress, heavy drinking, and smoking. It can also cause illness.

Caustic agents causing hepatotoxicity include carbon tetrachloride (CCl ₄ ), D-galactosamine (GalN), lipopolysaccharide (LPS), and bromobenzene. D-galactosamine is a well known hepatotoxic substance and has been used as a model for liver damage in vivo and in vitro. In vivo, D-galactosamine has been studied to cause liver damage similar to hepatitis caused by viruses and hepatitis caused by drugs (Keppler et al., 1968; MacDonald et al., 1987). This hepatotoxicity inhibits RNA and protein synthesis by loss of uridine nucleotides and accumulation of UDP hexoseamine in hepatocytes (Decker and Keppler, 1974; Keppler et al. 1968). In rats, Kuffer cells are activated by low concentrations of endotoxins, which are associated with D-galactosamine toxicity (Galanos et al., 1979; Kasravi et al., 1995; Stachlewitz et al., 1999). . In particular, D-galactosamine increases TNF-α sensitivity in the liver and contributes to liver damage (Leist et al. 1997).

In addition, the antioxidant effect, which is a function that inhibits or prevents lipid peroxide, has been reported to have a hepatoprotective effect and an anti-inflammatory action, and thus, an antioxidant compound is used against an attack by a reactive oxygen intermediate.

It is used for the purpose of protecting the liver and liver cells.

Therefore, many studies have been conducted on substances capable of exhibiting a hepatoprotective effect by antioxidant mechanisms, and many studies have been attempted to search for herbal medicines. The search for new substances that can prevent and treat human diseases from herbal medicines has been the subject of research by many scientists from the past. The development of these natural resources has led to more than 50% of the drugs currently in use, derived from natural products, and more than 120 approved drugs from the US FDA, with a market size of about $ 10 trillion. There have been many researches and developments of natural products with anti-hepatotoxic activity, but only a few cases have been used or tested in actual treatments.

Meanwhile, thistle ( Cirsium) setidens ) is a perennial plant belonging to the family Asteraceae, also called gondre or gondalbi . Korean thistle is a wild plant that grows at an altitude of 700 meters above sea level of Taebaek Mountain. It is widely distributed in temperate and cold regions of the northern hemisphere such as Southeast Asia such as Korea, Japan and China, as well as the Mediterranean coast and southwestern North America. Young leaves and stems are edible and rich in protein, calcium and vitamin A. Pharmacological effect is diuretic, detoxification, anti-inflammatory, hemostatic action, pneumonia, high blood pressure, enteritis, nephritis is also used. In particular, the fresh juice of Korean thistle leaves is known to be good for neuralgia and arthritis. The main active ingredient of Korean thistle is pectolinarin, Cirsium A pektori Naryn separated from subcoriaceu m has been demonstrated that the analgesic and anti-inflammatory effect (Martinez-Vazquez M, Ramirez Apan TO, Lastra AL, Bye R. A comparative study of the analgesic and anti-inflammatory activities of pectolinarin isolated from Cirsium subcoriaceum and linarin isolated from Buddleia cordata.Planta Med. 1998; 64: 134-137.), Linaria Factorinirin isolated from reflexa Desf showed anticancer effects in cell lines such as COR-L23, Caco-2 and C32 (Tundis R, Deguin B, Loizzo MR, Bonesi M, Statti GA, Tillequin F, Menichini F. Potential antitumor agents: flavones and their derivatives from Linaria reflexa Desf.Bioorg Med Chem Lett. 2005; 15: 4757-4760.), Cirsium Pectolinerin isolated from japonicum has been shown to have anticancer effects in S180 and H22 mice (Liu S, Luo X, Li D, Zhang J, Qiu D, Liu W, She L, Yang Z. Tumor inhibition and improved immunity in mice treated with flavone from Cirsium japonicum DC.Int Immunopharmacol. 2006; 6: 1387-1393. and Liu S, Zhang J, Li D, Liu W, Luo X, Zhang R, Li L, Zhao J. Anticancer activity and quantitative analysis of flavone of Cirsium japonicum DC.Nat Prod Res. 2007; 21: 915-922.).

As mentioned above, although the pharmacological activity of the factorinirin is known, the hepatoprotective effect is not known yet, and there is no research on this.

While the present inventors have studied the hepatoprotective activity of fentorinarin and its glycoside, fentorariginine, the concentrations of AST, ALT, ALP and LDH in hepatitis induced by D-galactosamine By reducing the, inhibiting lipid oxidation, increasing the concentrations of GSH, GR, GCS, GST and SOD confirmed that hepatoprotective and antioxidant effects are excellent and completed the present invention.

The present invention is to provide a composition for protecting liver comprising fentorinarin or fentorariginine.

The present invention provides a composition for protecting liver, which comprises a factorinarin represented by the following formula (1).

In another aspect, the present invention provides a composition for liver protection comprising the fentorariginine represented by the following formula (2).

Compositions of the present invention include pharmaceutical compositions and food compositions.

Hereinafter, the present invention will be described in detail.

The effective ingredients of fentorinarin and fentorariginine of the composition of the present invention can be obtained by all conventional methods, commercially available reagents can be used, and extracted and separated from the herbal medicines. In the present invention can be obtained by extracting and separating the fentorinarin and fentorariginine in the following method.

First, the Korean thistle leaf is added to water, alcohol or a mixed solvent of water and alcohol to extract reflux (3 times). The extract was filtered, concentrated under reduced pressure, and lyophilized to obtain Korean thistle leaf extract in powder form. The mixed solvent of water and alcohol may be selected from 10 to 100% methanol or 10 to 100% ethanol, preferably 10 to 100% methanol. The Korean thistle leaf methanol extract is suspended in distilled water and extracted three times with chloroform. The chloroform soluble fraction is dried under vacuum to give the chloroform fraction. The remaining aqueous layer is then partitioned and extracted three times with butanol and the butanol soluble fraction is dried in vacuo to give a butanol fraction.

The Korean thistle leaf butanol fraction was subjected to silica gel column chromatography (SiO ₂ , Art No. 7734, Merck, Germany, 280 g, 5 x 55 cm) using a solvent of chloroform: methanol: water 7: 3: 1. To separate into three fractions. The second fraction is dried in vacuo and recrystallized from methanol to give compound 1 and identified as factorininarin.

In addition, the factorinirin is added to 5% sulfuric acid in a 50% methanol solution, hydrolyzed under reflux, and then partitioned and extracted three times with chloroform. The chloroform fraction is washed with distilled water, dried and concentrated, and then the residue is dissolved in methanol to obtain Compound 2, which is identified as pectoriginalinine.

Fectorinarin and Fectorinarigenin reduce the concentration of AST, ALT, ALP and LDH in D-galactosamine-induced liver damage and lower the amount of malondialdehyde, which is an indicator of lipid peroxide, to inhibit lipid oxidation In addition, by increasing the concentration of GSH, GR, GCS, GST and SOD, hepatoprotective and antioxidant effects are excellent. Therefore, the fentorinarin and fentorariginine of the present invention can be usefully used as a medicine for liver protection and health food.

The composition of the present invention may contain at least one known active ingredient having hepatoprotective effect together with atorcrinein or atorcrineignin.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as an antioxidant, buffer And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dosage of the fentorinarin or fentorariginine is about 5 to 20 mg / kg, preferably about 10 to 20 mg / kg, and more preferably administered once or several times a day.

The composition of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for liver protection.

The composition of the present invention may be added to health food for the purpose of liver protection. In the case of using the fentorinarin or fentorinariginin of the present invention as a food additive, the fentorinarin or fentorariginine can be added as it is or used in combination with other foods or food ingredients, and can be suitably 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 manufacture of foods or beverages, the fentorinarin or fentorariginine of the present invention is added in an amount of 15 wt% or less, preferably 10 wt% or less with respect to 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. Natural carbohydrates described above are glucose, monosaccharides such as fructose, malsaccharides, disaccharides such as sucrose, and polysaccharides such as dextrin, 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, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is 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  One  : From Korean Thistle Leaf Extract Factory  Produce

1. Preparation of Korean Thistle Leaf Extract

Korean thistle leaves were collected from Jinbu-myeon, Pyeongchang-gun, Gangwon-do. Korean thistle leaves were dried and shredded. 628 g of shredded Korean thistle leaves were placed in a round bottom flask, and 3 L of methanol was added thereto, followed by extraction three times for 5 hours under reflux. The extract was filtered, filtered and concentrated under reduced pressure, and then lyophilized to obtain 128 g of Korean thistle leaf methanol extract in powder form. 108 g of the Korean thistle leaf methanol extract was suspended in 800 ml of distilled water, and 800 ml of chloroform was added thereto to extract three times. The chloroform soluble fraction was dried under vacuum to give 44 g of chloroform fraction. The remaining aqueous layer was then extracted three times with 800 ml of butanol, and the butanol soluble fraction was dried under vacuum to give 6 g of butanol fraction.

2. Korean thistle leaf Butanol  From fractions Factory  detach

The Korean thistle leaf butanol fraction obtained in 1 was subjected to silica gel column chromatography (SiO ₂ , Art No. 7734, Merck, Germany, 280 g, 5 x 55 cm using a solvent of chloroform: methanol: water 7: 3: 1). ) Into three fractions. The second fraction was dried in vacuo and recrystallized from methanol to give compound 1 as a white amorphous powder. The physicochemical properties of the compound 1 are as follows and were identified as factorinarin as a result of comparative analysis with that of the published literature.

Compound 1: Factorinarin

1) Appearance of the substance: white amorphous powder,

2) Melting Point: 250 ~ 253 ℃

3) ¹ H NMR (500MHz, pyridine- d ₅ ): 6.86 (1H, s, H-1), no peak (H-8), 8.04 (2H, d, J = 9.0 Hz, H-2 ', 6 '), 7.25 (2H, d, J = 9.0 Hz, H-3', 5 '), 4.05 (3H, s, H-6), 3.71 (3H, s, H-4'); Sugar moieties, Glc-5.73 (1H, d, J = 7.5 Hz, H-1 "), 4.34 (1H, m, H-2"), 4.34 (1H, m, H-3 "), 4.08 (1H, m, H-4 "), 4.27 (1H, m, H-5"), 4.72 (1H, dd-like, H _a- 6 "), 4.14 (1H, dd-like, H _b -6"), Rha-5.47 (1H, brs, H-1 '''), 4.62 (1H, dd, J = 1.5, 9.0 Hz, H-2'''), 4.51 (1H, dd, J = 3.0, 9.0 Hz, H-3 '''), 4.16 (1H, dd-like, H-4'''), 4.27 (1H, d, J = 6.5 Hz, H-5 '''), 1.55 (3H, d, J = 6.5 Hz, H-6 ''');

4) ¹³ C NMR (125.5 MHz, pyridine- d ₅ ) δ Genin 164.5 (C-2), 104.1 (C-3), 153.0 (C-5), 133.9 (C-6), 157.6 (C-7) , 95.0 (C-8), 122.8 (C-1 '), 128.6 (C-2', 6 '), 115.0 (C-3', 5 '), 162.9 (C-4'), 60.6 (6- OCH ₃ ), 55.2 (4′-OCH ₃ ); Glc-102.3 (C-1 "), 74.5 (C-2"), 78.3 (C-3 "), 71.1 (C-4"), 77.5 (C-5 "), 67.4 (C-6"); Rha-102.2 (C-1 '''), 71.9 (C-2'''), 72.7 (C-3 '''), 73.8 (C-4'''), 69.6 (C-5 ''' ), 18.3 (C-6`` ').

3. From factory narin Of factorinariginin  Produce

2 g of factorinarin obtained in 2 was added to 200 ml of 5% sulfuric acid in a 50% methanol solution and hydrolyzed under reflux for 3 hours. After the reaction solution was cooled, the mixture was extracted three times with 200 ml of chloroform. The chloroform fraction was washed twice with distilled water, dried and concentrated. The residue was taken up in methanol to give compound 2. The physicochemical properties of the compound 2 are as follows, and as a result of comparative analysis with that of the published literature was identified as pectorinariginin [Do, JC, Jung GY, Son GH, Isolation of pectolinarin from the aerial parts of Cirsium nipponicum, Kor. J. Pharmacogn. 25: 73-75 (1994).].

Compound 2: Factorinarigenin

1) Appearance of material: yellow orange powder,

2) Melting Point: 200 ~ 205 ℃.

Experimental Example  One  : According to the present invention Factorinarin  And Of factorinariginin Liver protection  Effect and antioxidant effect

In order to determine the effects of ectorinarin and fentorariginine according to the present invention on the hepatoprotective and antioxidant effects, the following experiment was performed.

1. Experimental Animal

The experimental animals were distributed by Biolink Co., Ltd. for Sprague-Dawley male rats weighing 200 ± 10g and kept at a constant condition (temperature: 20 ± 2 ℃, humidity: 40-60%, contrast cycle: 12 h) for 2 weeks. Adapted to breeding. Experimental animals were divided into 12 groups and 9 animals were assigned to each group, and only water was fasted for 24 hours before the experiment.

2. D- To galactosamine (GalN)  by Liver damage  Induction and Sample Dosing

Liver damage was induced by intraperitoneal administration of 400 mg / kg of D-galactosamine (Sigma, St. Louis, MO, USA) dissolved in physiological saline to the experimental animals 24 hours prior to the last 24 hours of sample administration.

The normal group and one liver injury induction group were administered with 1 ml of physiological saline, respectively, the six liver damage induction groups, Korean thistle leaf methanol extract prepared in Example 1, Korean thistle leaf butanol fraction, Korean thistle leaf chloroform The fractions were dissolved in a 10% Tween 80 solution prepared with physiological saline, and 100 mg / kg and 200 mg / kg, respectively. / Kg and 20 mg / kg were dissolved in 1 ml of saline and orally administered for 2 weeks using oral jonde.

Protein concentration was determined by using the Bovine serum albumin (Sigma chemical Co., St. Louis, MO, USA) by the Lowery method, the statistical results of the results obtained in the experiment was expressed as mean ± standard deviation. Statistical analysis was performed using Duncan's multiple rage test, and the significance was expressed as p <0.05.

3. Collection of Samples and Enzyme  Produce

After the animal was lightly anesthetized with CO ₂ , the abdominal midline was opened, blood was collected from the abdominal aorta, left at room temperature for 60 minutes, and centrifuged at 1,000 xg for 15 minutes to separate serum. The isolated serum was used to measure the enzyme activity of aspartate (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH).

The liver is perfused with physiological saline to remove blood from the tissue, extracted, washed with physiological saline, and then freed of blood and other foreign substances in the liver, and then added with 4 times 0.1M phosphate buffer solution (pH 7.4) per 1 g of liver tissue. The cold phase was triturated with a glass Teflon homogenizer. The ground liquor was centrifuged at 1,000 xg for 10 minutes using a refrigerated centrifuge, and the supernatant from which the nucleus and uncrushed portions were removed was centrifuged at 10,000 xg for 20 minutes using a high-speed centrifuge. The supernatant was further centrifuged at 105,000 xg for 60 minutes to obtain a cytosolic fraction, which was obtained by GSH (glutathione), GST (glutathione-S-transferase), GR (glutathione reductase), and GCS (γ-glutamylcysteine synthetase). And SOD (superoxide dismutase) enzyme activity was measured. All experiments below were run at 4 ° C.

4. Determination of enzyme activity- Liver protection  effect

4-1. AST ( aspartate ) And ALT ( alanine transaminase ) Measurement

Aminotransferase is an enzyme that transfers amino groups and is used to diagnose and diagnose clinically when the liver is damaged. In particular, when cell membrane damage occurs, intracellular AST and ALT flow out of the cell. Therefore, this level in the blood is very important for the diagnosis of liver disease.

The AST and ALT enzyme activities were measured using Asan Pharmaceutical Kit prepared according to the method of Reitman and Frankel (1957) [Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol. 1957; 28: 56-63.]. 1 ml each of AST substrate solution (0.0266 mg / ml L-aspartic acid, 0.29 mg / ml α-ketoglutamic acid) and ALT substrate solution (0.0178 mg / ml DL-alanine, 0.29 mg / ml α-ketoglutamic acid) 37 It left for 5 minutes at ° C. 0.2 ml of serum was added to the AST substrate solution and ALT substrate solution, and the reaction was carried out at 37 ° C. for 60 minutes and ALT for 30 minutes, followed by 1 ml of stop solution (0.19 mg / ml 2,4-dinitrophenylhydrazine). 1 ml of 0.4N NaOH was added thereto, mixed, and left to stand at room temperature for 10 minutes. The absorbance was measured at 505 nm and the enzyme activity was calculated according to the standard calibration curve and expressed in Karmen units per 1 ml of serum.

4-2. ALP ( alkaline phosphatase ) Measurement

ALP is a hydrolase that removes phosphate groups from nucleotides and proteins. It is primarily produced in the liver and brain and is an enzyme that can recognize the condition of the liver.

ALP enzyme activity was measured with an Asan pharmaceutical kit prepared according to the method of Roos (1966) [Roos K. A simplified AutoAnalyzer procedure for the determination of serum alkaline phosphatase activity. Clin Chim Acta. 1966; 13: 403-405.]. 2 ml of 0.05 M carbonate buffer (pH 10.0) containing 1 mM phenyl phosphate was left at 37 ° C. for 3 minutes. After adding 0.05 ml of serum to the ALP substrate solution, 1 ml of a stop solution (0.19 mg / ml 2,4-dinitrophenylhydrazine) and 1 ml of 0.4N HCl were added, mixed, and left to stand at room temperature for 10 minutes. The absorbance was measured at 500 nm and the activity was calculated according to the standard calibration curve, expressed in Karmen units per 1 ml of serum.

4-3. LDH ( lactate dehydrogenase ) Measurement

LDH is a general indicator of the acute and chronic progression of tissue damage and is an important enzyme for the diagnosis of organs, tissues and cells.

LDH enzyme activity was measured by Asan Pharmaceutical kit prepared according to the method of Kim et al. (2005) [Kim NY, Lee MK, Park MJ, Kim SJ, Park HJ, Choi JW, Kim SH, Cho SY, Lee JS. Momordin Ic and oleanolic acid from Kochiae Fructus reduce carbon tetrachloride-induced hepatotoxicity in rats. J Med Food. 2005; 8: 1777-1783.]. Solution A [23.1 mg / ml lithium acetate, 24.2 mg / ml tris (hydroxymethyl) aminomethane] and Solution B [57.4 mg / ml NAD, 0.034 mg / ml 1'-methoxy-5'-methylphenazinium Methyl sulfate (1'-methoxy-5'-methylphenazinium methylsulfate; phenazine methosulfate (PMS))] was mixed in the same volume and left for 5 minutes at 37 ℃. This was mixed with a homogeneous solution (100-300 mL protein) and left at 37 ° C. for 10 minutes, and then 0.4N HCl was added to stop the reaction. The absorbance was measured at 570 nm and the activity was calculated according to the standard calibration curve.

Table 1 shows the AST, ALT, ALP and LDH enzyme activities of the fentorinarin and fentorariginine of the present invention in liver damage induced by D-galactosamine.

※ There is no significant difference between the values following the same letter (p <0.05).

As shown in Table 1, in the liver damage induced by D-galactosamine, the fentorinine of the present invention has inhibition rates of AST enzyme activity of 16.5% and 31.7% at 10 mg / kg and 20 mg / kg, respectively, and ALT enzyme inhibition rate. Were 23.3% and 32.3%, and the inhibition rate of ALP enzyme activity was 14.5% and 31.5%, and the inhibition rate of LDH enzyme activity was 39.6% and 53.2%. In addition, the inhibition rate of AST, ALT, ALP, and LDH enzyme activity of pectorinaligin was weaker than that of pectorinarin, but showed almost the same effect.

5. Determination of Enzyme Activity-Antioxidant Effect

Oxidative stress is the primary cause of hepatocellular injury when D-galactosamine is administered. Inactive oxygen species (ROS) indirectly in vivo (Yoshikawa et al., 1982) and in vitro (Quintero et al., 2002). An increase was reported. In particular, the increase of reactive oxygen species may affect the antioxidant defense system that oxidizes intracellular lipids or contains enzymes related to GSH as well as GSH synthesis. D-galactosamine has been reported to decrease GSH levels by inhibiting GSH synthesis, resulting in a decrease in enzymes involved in GSH synthesis and an increase in enzymes involved in GSH degradation (McMillan and Jollow 1992).

5-1. Measurement of Lipid Peroxide Content

According to the method of Ohkawa et al. (1979), the degree of oxidation of lipids was measured using thiobarbituric acid reactive substances (TBARS) [Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979; 95: 351-358. To 0.4 ml of 10% homogenate containing 0.9% NaCl, 1.5 ml of 8.1% SDS, 1.5 ml of 20% acetate buffer (pH 3.5) and 1.5 ml of 0.8% TBA solution were added. Heated. After the reaction solution was cooled to room temperature, 5.0 mL of n-butanol-pyridine (15: 1) was added thereto, and the n-butanol-pyridine layer was measured by measuring the amount of TBARS at 532 nm. It is expressed as protein.

5-2. GSH ( glutathione ) Measurement

The method of Gaitonde et al. (1967) was modified to measure the degree of change from nonprotein binding-SH to cysteine-SH [Gaitonde MK. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J. 1967; 104: 627-633. 10% trichloroacetic acid was added to the homogenate and centrifuged. 0.5 ml of acetic acid and 0.5 ml of ninhydrin reagent were added to the supernatant, followed by heating and cooling for 10 minutes. Then immediately 3 ml of ethanol was added to determine the amount of GSH at 412 nm.

5-3. GST ( glutathione -S- transferase ) Measurement

GST enzyme activity was measured according to the method of Habig and Jakoby (1981) [Habig WH, Jakoby WB. Glutathione S-transferases (rat and human). Methods Enzymol. 1981; 77: 218-231. The reaction solution was made up to 2.5 ml of 0.1 M sodium phosphate buffer (pH 7.4) and 0.1 ml of post-mitochondrial supernatant (10% wt / vol) to a total volume of 3.0 ml. The absorbance was measured at 340 nm and the enzyme activity was calculated using the nanomole absorption coefficient (9.6 × 10 ³ / M / cm) of 1-chloro-2,4-dinitrobenzene.

5-4. GR ( glutathione reductase ) Measurement

GR enzyme activity was measured according to the method of Mize and Langdon (1962) [Mize CE, Langdon RG. Hepatic glutathione reductase. I. Purification and general kinetic properties. J Biol Chem. 1962; 237: 1589-1595.]. Mix 3.5 ml reaction solution [0.1 M potassium phosphate buffer (pH 7.5), 0.94 mM EDTA, 4.6 mM oxidized glutathione, 0.16 mM NADPH] and homogenate (100-300 ml protein) at 37 ° C. The reaction was carried out for 10 minutes. Absorbance was measured at 340 nm and calculated as the change in NADPH.

5-5. GCS (γ- glutamylcysteine synthetase ) Measurement

GCS enzyme activity was measured according to the method of Richman and Meister (1975) [Richman PG, Meister A. Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem. 1975; 250: 1422-1426.]. Mix 3.5 mL of reaction solution [0.1M Tris-HCl buffer (pH 8.0), 8.9 mM L-glutamic acid, 0.94 mM EDTA, 3.2 mM MgCl ₂ , 1.35 mM ATP] and homogenate (100-300 mL protein) 37 It was made to react at 30 degreeC. Absorbance was measured at 600 nm and calculated with a standard calibration curve.

5-6. SOD ( superoxide dismutase ) Measurement

SOD enzyme activity was measured according to the methods of Marklund S. and Marklund G. (1974). Eur J Biochem. 1974; 47: 469-474. 1 ml of cytochrome c solution (0.2 M potassium phosphate buffer (pH 8.6), 0.1 mM EDTA) was left in an ice bath for 20 minutes. 0.5 ml of basic DMSO or non-basic DMSO was mixed and left at 37 ° C. for 30 minutes. Reduced cytochrome c was measured at 550 nm, and activity was defined as 1 unit that 50% inhibition of reduced cytochrome c was suppressed.

In the liver damage induced by D-galactosamine, the contents of lipid peroxides of fentorinarin and fentorariginine of the present invention are shown in FIG. 1, and GSH, GST, GR, GCS and SOD enzyme activities are shown in Table 2. .

※ There is no significant difference between the values following the same letter (p <0.05).

As shown in FIG. 1, it can be seen that the factorinarin and the factorinariginin of the present invention lower the amount of malondialdehyde, an indicator of lipid peroxide in liver damage induced by D-galactosamine, thereby inhibiting lipid oxidation. .

In addition, as shown in Table 2, in the liver damage induced by D-galactosamine, the fentorinarine and fentorarigenin of the present invention activates the enzymes GR and GCS at 10 mg / kg and 20 mg / kg, respectively, to D-galacto Inhibition of GSH by samine was inhibited. In addition, the Fectorinarin of the present invention increased the GST activity of 34.4% and 44.6%, SOD activity of 40.7% and 50.0% at 10mg / kg and 20mg / kg, respectively. In addition, the GST and SOD enzyme activity of efectorariginine was weaker than that of efectorinarin, but showed almost the same effect.

Therefore, it can be seen that the fentorinarin and fentorariginine of the present invention have excellent hepatoprotective and antioxidant effects in liver damage induced by D-galactosamine.

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

Formulation example  One  : Preparation of Pharmaceutical Formulations

1. Preparation of powder

2 g of fentorinarin (or fentorariginine)

1g lactose

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

2. Preparation of Tablets

Pectorinarin (or Pectorinarigin) 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

Pectorynarine (or Pectoryriginin) 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

1. Preparation of Cooking Seasonings

20% to 95% by weight of fentorinarin (or fentorrigininin) was prepared for health promotion cooking seasoning.

2. Preparation of Tomato Ketchup and Sauce

Health-promoting tomato ketchup or sauce was prepared by adding 0.2-1.0 wt% of fentorinarin (or factorinariginin) to tomato ketchup or sauce.

3. Manufacturing of Flour Foods

0.5 to 5.0% by weight of fentorinarin (or fentorariginine) was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture to prepare health-promoting foods.

4. Preparation of soups and gravy

0.1-5.0% by weight of fentorinarin (or fentorariginine) was added to soups and gravy to prepare health-producing meat products, soups of noodles and gravy.

5. Preparation of Ground Beef

Health promoting ground beef was prepared by adding 10% by weight of fentorinarin (or fentorariginine) to the ground beef.

6. Manufacture of Dairy Products

5-10% by weight of fentorinarin (or fentorariginine) was added to milk, and the milk was used to prepare various dairy products such as butter and ice cream.

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 After sterilizing at 20 ° C. for 20 seconds to 180 seconds, the mixture was mixed with cooling water at a ratio of 1: 4, and then 0.5 to 0.82% of carbon dioxide was injected to prepare a carbonated beverage containing fentorinarin (or fentorariginine) of the present invention.

2. Manufacture of health drinks

Instant sterilization by homogeneous mixing of ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%) After that, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare healthy drinks.

3. Preparation of Vegetable Juice

Health supplement vegetable juice was prepared by adding 5 g of factorinarin (or factorinariginin) to 1,000 ml of tomato or carrot juice.

4. Preparation of Fruit Juice

Health supplement fruit juice was prepared by adding 1 g of ectorinarin (or ecterinariginin) to 1,000 ml of apple or grape juice.

Fectorinarin and Fectorinarigenin reduce the concentration of AST, ALT, ALP and LDH in D-galactosamine-induced liver damage and lower the amount of malondialdehyde, which is an indicator of lipid peroxide, to inhibit lipid oxidation In addition, by increasing the concentration of GSH, GR, GCS, GST and SOD, hepatoprotective and antioxidant effects are excellent. Therefore, the fentorinarin and fentorariginine of the present invention can be usefully used as a medicine for liver protection and health food.

Figure 1 is a diagram showing the content of lipid peroxide of the fentorinarin and fentorariginine of the present invention in liver damage induced by D-galactosamine.

Claims (8)

A composition for the prevention and treatment of hepatotoxicity, comprising fentorariginine represented by the following formula (2). <Formula 2>

[Claim 2] The composition for the prevention and treatment of hepatotoxicity according to claim 1, wherein the factorinariginin is obtained by acid hydrolysis of factorinarin. [Claim 2] The composition for preventing and treating hepatotoxicity according to claim 1, wherein the hepatotoxicity is induced by D-galactosamine. The composition for preventing and treating hepatotoxicity according to any one of claims 1 to 3, wherein the composition is a pharmaceutical composition. The composition for the prevention and treatment of hepatotoxicity according to any one of claims 1 to 3, wherein the composition is a food composition. delete delete delete

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