KR20090059231A - Composition comprising oroxylin a for treating and preventing liver disease - Google Patents

Abstract

A composition for preventing and treating liver disease is provided to ensure the effects of DNA fragmentation suppression, caspase-3 and caspase-8 activity inhibition, poly(ADP-ribose)polymerase inhibition and JNK phosphorylation inhibition. A composition for treating and preventing liver disease comprises an oroxylin A of the chemical formula A as an active ingredient. The oroxylin A suppresses the liver cell damage by bilestagnation. The liver disease is selected from autoimmunity liver disease, drug attractancy liver disease, adrenoleukodystrophy, infection liver disease, congenital metabolic liver disease, bile congestion, acute hepatitis, permanent exposure, cirrhosis, steatosis or liver cancer. The composition is contained in a ratio of 0.1-50 weight% based on total weight. A health functional food containing the oroxylin A for preventing liver disease and improving cure thereof is used in a form of powder, granule, tablet, capsule and beverage.

Description

Composition comprising Oroxylin A for treating and preventing liver disease}

The present invention relates to a composition for the treatment and prophylaxis of liver disease, which contains an organoline A as an active ingredient.

[Reference 1] Lissoos et al ., J Clin. Gastroentero., 15 (1), pp 63-67, 1992

2 Patel et al., Toxicol Appl. Pharmacol., 142 (1), pp 116-22, 1997

Gores et al., J. Hepatol., 37 (3), pp 400-410, 2002

[4] Boulares et al., J. Biol. Chem. ,, 277, pp 372-378, 2002

Czaja, M.J., Liver Physiol. 284, pp G 875 879, 2003

6 Marderstein et al., Surgery. 134, pp280284, 2003

7 Gumpricht et al., J. Biol. Chem. 280, pp1055610563,2005

8, Park et al., Planta Med. 72, pp661664, 2006

9 Hu Y et al., Biochem Biophys Res Commun. 351 (2), pp 521-7, 2006

10. Huang WH et al., Biosci Biotechnol Biochem. 70 (10), pp 2371-8, 2006

[11] Kim JY et al., J Toxicol Environ Health A. 65 (5-6), pp 373-81, 2002

12 Suresh Babu K et al., Bioorg Med Chem Lett. 15 (17), pp3953-6, 2005

Document 13 Kim DH et al., Neurobiol Learn Mem. 87 (4), pp 536-46, 2007

Document 14 Tomimori T, Miyaichi Y, Kizu H., Yakugaku Zasshi. 102 (4), pp 388-91, 1982

15 Seglen et al., Exp. Cell. Res., 82, pp 391-398, 1973

16 Lian et al., Basic Clin. Pharmacol. Toxicol., 96, pp. 495-502, 2005

The liver is a long-lasting silencer that has excellent buffering effects, and some parts of the liver are damaged and unable to perform their functions. Because that is not easy to treat. In addition, there is almost no treatment system. In Korea, more than 15,000 people die from liver disease every year, which causes social problems with high mortality and difficulty of early diagnosis.

The liver is a very important organ that is located between the digestive system and the systemic circulatory system, which functions to defend the whole body from ex vivo substances entering the digestive system. Since the extracellular substance, once in vivo, passes through the liver, the liver is more at risk of being exposed to many toxic substances besides nutrients, which is more likely to be damaged than other organs.

The liver is an organ with good regenerative capacity, and in case of some hepatocellular damage, it is restored to normal enough. However, if it is continuously damaged, hepatic cells are completely destroyed, and the damaged parts cannot be recovered normally. As the connective tissue accumulates excessively, scars form in the liver tissue and liver function is not restored to normal (Lissoos et al. , J Clin.Gastroentero., 15 (1), pp 63-67, 1992). The causes of liver damage can vary greatly, including alcohol, bile, and viruses, but if the damage persists and becomes a chronic liver disease (CLD), the inflammatory condition persists regardless of the cause, and the extracellular matrix (ECM). Qualitative and quantitative change of).

Hepatocellular injury is a common phenomenon in liver disease, leading to necrosis or self-killing of hepatocytes. In stressful conditions such as acute liver failure, major cellular structures (mitochondria and cytoskeleton proteins), macromolecules (DNA, lipids, enzyme proteins) and metabolic pathways are damaged and eventually lead to necrosis. Hepatic cells have been reported to induce self-killing rather than necrosis when the stress is relatively weak, that is, when the mitochondrial function cannot be completely irreversibly damaged. It has been reported that bile salts induce hepatocellular apoptosis through various pathways, among which oxidative stress has been mentioned (Patel et al., Toxicol Appl. Pharmacol. 142 (1), pp116-22, 1997).

In general, self-killing does not cause an inflammatory response, so cell self-killing has little effect on physiological changes, but hepatocyte self-killing has been reported to be very different from this general condition. If hepatic cell death is severe, several enzyme levels in the hepatocytes are increased in blood, and the removal by the phagocytes is insufficient, eventually causing an inflammatory response and destroying the normal structure of the liver tissue. Self-killing has been reported to play an important role in many diseases known to be induced by necrosis in the past. In liver diseases caused by alcohol, viruses, and cholestasis, severe liver damage and hepatic insufficiency have been reported due to hepatocellular self-killing (Gores et al., J. Hepatol. 37 (3), pp400-410, 2002). .

Cholestasis is a feature found in several chronic progressive liver diseases that eventually lead to liver failure, cirrhosis and eventually death. During bile stagnation, hepatic damage is induced by the accumulation of hydrophobic bile salts such as chenodeoxycholate and deoxycholate in hepatocytes. Clinical observations showed that hepatic cell death was more important than hepatocellular necrosis. An in vitro model that induces hepatocyte self-killing by treating 20-100 μM of hydrophobic bile salts such as glycochenodeoxycholate (GCDC) in primary cultured hepatocytes is a hepatocyte caused by endogenous toxins. It is useful for self-killing studies. Hydrophobic acids such as GCDC induce self-killing by inducing Fas oligomerization. Fas oligomerization cleaves and activates procaspase-8 into caspase-8, resulting in downstream caspase or effector caspase; caspase-3, -6 , -7) self-killing is typically induced by activating caspase-3.

Caspase-3 activated during autokilling is inactivated by cleaving 116-kDa poly (ADP-ribose) polymerase (PARP) to sizes 89- and 24-kDa. PARP is known as an enzyme necessary for DNA repair damaged by external stress (Duriez and Shah, Biochem Cell Biol., 75, 337-49, 1997). PARP is inactivated by cleavage by the caspase-3 active form, and endonuclease is activated to induce DNA fragmentation in the nucleus. (Boulares et al., J. Biol. Chem. 277, pp372-378, 2002).

Mitogen-activated protein kinases (MAPKs) are known to mediate intracellular signal transduction by various environmental stresses, and JNK along with ERK and p38 have been reported as representative MAPKs. JNK phosphorylation is reported to be an important pathway in hepatocyte self-killing. Although detailed pathways to self-killing after JNK phosphorylation are not clearly suggested, JNK phosphorylation (activation) is observed in hepatocytes induced by self-killing, and when JNK phosphorylation is inhibited, hepatic cell self-killing is suppressed. Hepatocellular self-apoptosis is recognized as a mechanism (Czaja, MJ, Liver Physiol. 284, ppG875879, 2003; Marderstein et al., Surgery 134, pp280284, 2003; Gumpricht et al., J. Biol. Chem. 280, pp1055610563, 2005 Park et al., Planta Med. 72, pp661664, 2006).

Among the liver tissues, hepatocytes that perform the main functions of the liver, such as nutrient absorption and metabolism, storage, and plasma protein synthesis, as well as cooper cells, macrophages in the liver, and excessive synthesis of connective tissues during liver injury As a result, hepatic stellate cells (HSC), endothelial cells, concave cells, and the like, which cause hepatic fibrosis, are present. Hepatocytes make up more than 90% of all the cells that make up the liver tissue, and they are the parenchymal cells that perform the major functions of the liver. This persistence results in hepatic fibrosis / curing. Therefore, the inhibition and prevention of hepatocellular damage may be the most important part in the prevention and treatment of liver disease.

The benefits of oroxylin A include anticancer activity (Hu Y et al., Biochem Biophys Res Commun. 351 (2), pp521-7, 2006), antioxidant and anti-inflammatory action (Huang WH et al., Biosci Biotechnol Biochem. 70 (10), pp2371-8, 2006), CYP2C9 inhibitory activity (Kim JY et al., J Toxicol Environ Health A. 65 (5-6), pp373-81, 2002), antimicrobial activity (Suresh Babu K et al. , Bioorg Med Chem Lett. 15 (17), pp3953-6, 2005), anti- forgetful action (Kim DH et al., Neurobiol Learn Mem. 87 (4), pp536-46, 2007).

Therefore, the present inventors conducted a study to find a substance having an effect that can effectively protect and prevent hepatocellular damage, and as a result, 5,7-dihydroxy-6- as a component belonging to the flavonoid system isolated from Scutellaria baicalensis Georgi Finding out that Oxylin A, a chemical name of 5,7-dihydroxy-6-methoxyflavone, can be used for the treatment and prevention of liver disease by preventing bile acid-induced self-killing of liver cells. The present invention has been completed.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment and prophylaxis of liver disease, which contains an organoline A represented by the following structural formula (1) as an active ingredient.

(1)

(One)

The aurocillin A provides a pharmaceutical composition for the treatment and prevention of liver disease, characterized in that it has the effect of inhibiting hepatocellular damage caused by cholestasis.

The liver disease is autoimmune liver disease, drug-induced liver disease, alcoholic liver disease, infectious liver disease, congenital metabolic liver disease, cholestatic liver disease, acute hepatitis, chronic hepatitis, cirrhosis, fatty liver, liver cancer, preferably alcoholic Liver disease, cholestatic liver disease, acute hepatitis, chronic hepatitis or cirrhosis.

Oroxylin A according to the present invention can be used by any method well known in the art prepared by the extraction method or chemical synthesis method, and commercially available, and the method or material is not particularly limited.

Hereinafter, the method for obtaining the compound of the present invention will be described in detail.

Oroxylin A of the present invention is chopped dried gold to about 1 to 30 times, preferably about 10 to 20 times the dry weight of water, C ₁ to C ₄ lower alcohol or a mixed solvent thereof, preferably Extraction method such as heating reflux extraction, cold leaching extraction, ultrasonic extraction, etc., preferably heating reflux extraction for 1 to 12 hours, preferably 2 to 5 hours with water, concentrated under reduced pressure by filtration under reduced pressure to obtain a crude extract First step; A second step of dissolving the crude extract in a solvent of butanol, chloroform and methanol, preferably in chloroform; A third step of dividing the chloroform extract obtained in the second step into three fractions by performing column chromatography; The first of the fractions of the third step may be subjected to column chromatography through the production process of the fourth step of separating the oroxylin A can be obtained the auroxylin A of the present invention.

The present invention provides a pharmaceutical composition for the treatment and prevention of hepatic diseases, which contains an auxilin A obtained by the above manufacturing process as an active ingredient.

The pharmaceutical composition for liver protection and treatment and prevention of liver damage of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

The pharmaceutical composition comprising the compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The compositions containing the compounds according to the invention are each formulated in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injectable solutions according to conventional methods. Can be used. Carriers, excipients and diluents which may be contained in the composition containing the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound of the present invention is preferably administered at a concentration of 1 to 2,000 mg / kg body weight per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a dietary supplement for the prevention and improvement of liver disease, which contains an organoline A as an active ingredient.

The health functional food may be prepared in powder, granule, tablet capsule or beverage form.

It may also be added to foods or beverages for the purpose of preventing and improving liver disease. At this time, the amount of the compound in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition is added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be.

The health beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as essential ingredients in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compounds of the present invention may contain 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 so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

As mentioned above, the oroxylin A of the present invention inhibits DNA fragmentation and PARP cleavage and active expression of caspase induced by 100 μM GCDC and is also known to play a major role in hepatocyte self-killing. By inhibiting phosphorylation, it was confirmed that it has a hepatocyte protective activity from self-killing. In addition, the above axolin A has the effect of inhibiting the hepatocyte cell death by hydrophobic bile can be used as an active ingredient for the treatment and prevention of liver diseases.

Hereinafter, the present invention will be described in detail by the following reference examples, examples and experimental examples. However, the following Reference Examples, Examples, and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Reference Examples, Examples, and Experimental Examples.

Example  One. Aurocillin  Separation and structural identification of A

1-1. Gold Crude extract  Produce

600 g of dried golden powder was purchased from Gyeongdong Market, powdered, heated to reflux for 3 hours with 6 L of water, and vacuum filtered to recover the supernatant. This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure (EYELA, N-1000, Japan) to obtain 1 L of golden water extract.

1-2. Gold Nonpolar Solvent Soluble Extracts  Produce

1 L of the golden crude extract obtained in Example 1-1 was distributed three times using 800 ml of normal butanol, and the solvent fraction was concentrated under reduced pressure to obtain 21.1 g of normal butanol soluble part. The obtained butanol soluble part was dissolved in 1 L of 60% methanol, partitioned three times with 800 ml of chloroform, and then concentrated under reduced pressure to obtain 11.5 g of chloroform soluble part and 9.1 g of 60% methanol soluble part.

1-3. From gold extract Aurocillin  Separation of A

11.5 g of the chloroform soluble part obtained in the above Example 1-2 was hung on a silica gel (900 g) column and then eluted with a mixed solvent of dichloromethane and methanol (30: 1-20: 1-10: 1) to give a mixing ratio. Accordingly divided into three fractions (A, B, C). Fraction A (5.4 g) was subjected to silica gel (200-300 mash, 500 g) column chromatography using chloroform and methanol (40: 1) as a mobile phase to obtain an aoxineline A (120 mg). As a result, it was confirmed that it is an aurocillin A of yellow plate crystal having the following physical properties (Tomimori T, Miyaichi Y, Kizu H., Yakugaku Zasshi. 102 (4), pp 388-91, 1982).

Melting point: 218-219 ° C .;

¹ H-NMR spectrum (DMSO- d ₆ , 400 MHz): 3.75 ppm (3H, s, OCH ₃ ), 6.63 ppm (1H, s, H-3), 6.97 ppm (1H, s, H-8), 8.10-7.52 ppm (5H, m, H-2'-6 '), 12.95 ppm (1H, s, 5-OH);

¹³ C-NMR spectrum (DMSO- d ₆ , 62.5 MHz): 182.5 (C-4), 163.4 (C-2), 157.9 (C-7), 153.0 (C-9), 152.8 (C-5), 132.2 (C-4 '), 131.7 (C-1'), 130.9 (C-6), 129.4 (C-3 ', C-5'), 126.6 (C-2 ', C-6'), 104.9 (C-3), 104.5 (C-10), 94.6 (C-8), 60.2 (OCH ₃ ).

Reference Example  1. Isolation and Culture of Hepatocytes from Rats

In order to separate hepatocytes from rats, experiments were performed according to Saglan et al. (Seglen et al., Exp. Cell. Res. 82, pp391-398, 1973).

Rats were anesthetized by anesthesia, intubated into the portal vein, 5% CO ₂ And 95% O ₂ in the Ca ^{2 +,} Mg ^{2 +} a 37 ℃ into blowing a mixed gas - within the liver send Hank's balanced salt solution ^{(Ca 2 +, Mg 2 +} -free Hank's balanced salt solution) without flowing to the tube Blood was removed. Thereafter, 0.05% collagen type IV (collagenase type IV) and 1 mM CaCl ₂ solution were flowed. The liver tissue was removed from the body, and then made a hepatocyte suspension, centrifuged at 50 × g for 2 minutes, the precipitated hepatocytes were taken, washed twice with a balanced salt solution of Ca ^{2 +} , Mg ^{2 +} -free Hank, and then with type 1 collagen. 10% (v / v) fetal bovine serum (FBS, Gibco BRL, USA), 10 ^-8 M insulin, 50 U / ml penicillin and 50 U / ml streptomycin (Sigma) in coated culture vessels. , USA) added Williams media E (Williams'Medium E; WME, Gibco BRL, USA) as a culture medium cell number 1 × 10 ⁶ The cells were allowed to have a number of cells / ml and cultured at 5% CO ₂ and 37 ° C. After 2 hours of incubation, the cells were washed with Hank's balanced salt solution to remove non-adherent hepatocytes, replaced with fresh culture, and then incubated for 16 hours at 5% CO ₂ , 37 ° C., and then used in the following experiment.

Reference Example  2. Primary culture  Hepatocellular apoptosis in hepatocytes apoptosis ) Induction and Aurocillin  A treatment

Hepatic cells were treated with glycochenodeoxycholate (GCDC), a hydrophobic bile salt that exhibits hepatotoxicity in bile ducts, to form autologous death by forming conditions such as bile ducts in isolated and cultured hepatocytes as in Reference Example 1. do. After 10 minutes pretreatment of hexane A with auroxillin A was added to 100 μM GCDC and incubated for 4 hours. Oroxylin A was dissolved in dimethylsulfoxide (DMSO), and GCDC was dissolved in Hank's balanced salt solution (HBSS), filtered through a 0.22 μm filter, and then added to the culture. The experimental group was set as shown in Table 1 below.

Group 1 DMSO-containing culture treatment Group 2 Treatment of medium containing 100 μM GCDC and DMSO Group 3 Treatment of Cultures Containing 100 μM GCDC and Oroxylin A (40 μM)

Experimental Example  One. DNA  Fragmentation Aurocillin  Measure the protective effect of A

 The protective effect of oroxylin A on hepatic DNA fragmentation was performed in the hepatocyte experimental group treated as in Reference Example 2 as follows.

DNA in hepatocytes was extracted and quantified using a commercially available Wizard Genomic DNA Purification Kit (Promega, USA), followed by electrophoresis of 30 μg of DNA on a 2% agarose gel at 50 mV for 40 minutes. Fragmentation was confirmed and the results are shown in FIG. 1.

From the experimental results, it was confirmed that DNA fragmentation of hepatocytes was induced upon 100 μM GCDC treatment, but induction of DNA fragmentation was inhibited in hepatocytes treated with oroxylin A and 100 μM GCDC (see FIG. 1).

Experimental Example 2. Determination of the inhibitory effect of oroxylin A on caspase-3 and caspase-8 activity

Lian et al., Basic Clin to determine the inhibitory effect of caspase-3 (caspase-3) and caspase-8 (caspase-8) of the auroxylin A obtained in Example 1 Pharmacol.Toxicol. 96, pp495-502, 2005) was performed as follows.

Caspase activity was determined using commercially available caspase-3 and caspase-8 activity colorimetric assay kits (R & D system Inc., USA), as in Reference Example 1. Cultured hepatocytes were measured for the experimental group treated with GCDC and aeroxolin A at 40 μM for 4 hours, the results are shown in Table 2 below.

Military number Caspase-3 activity (%) Caspase-8 activity (%) One 100.0 ± 6.7 100.0 ± 12.4 2 447.8 ± 53.2 ^a 180.3 ± 25.2 ^a 3 132.8 ± 11.8 ^{a, b} 107.7 ± 21.6 ^b [Note] ^a P <0.05, statistically significant difference compared to group 1 ^b P <0.05, statistically significant difference compared to group 2

From the above results, the activity of caspase-3 and caspase-8 was significantly increased by GCDC treatment. However, when GCDC and oroxylin A were treated together, caspase-3 was significantly higher than that of GCDC treatment alone. And it was confirmed that the activity of caspase-8 was inhibited. As a result, it was confirmed that oroxylin A significantly inhibited the activation of hepatocyte caspase-3 and caspase-8 induced by GCDC.

Experimental Example  3. Immunoblot  Used Caspase -3 and Caspase -8 active expression and poly ( ADP Ribose) Polymerase ( PARP ) For split Aurocillin  Inhibitory effect of A

In order to confirm the inhibitory effect of apoptosis A on the hepatocytes obtained in Example 1, the active type increase inhibition of caspase-3 and caspase-8 and the inhibition of PARP cleavage by an immunooblot assay Using the method of Lian et al. (Lian et al., Basic Clin. Pharmacol. Toxicol. 96, pp495-502, 2005), the following immunoblot assay was performed.

As described in Reference Example 1, immunotransfer was performed on the experimental group treated with GCDC and oroxylin A for 4 hours in the isolated cultured hepatocytes. Drug-treated hepatocytes were dissolved in lysis buffer (10 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP-40, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM Dithiothreitol, 1 μg / ml aprotinin, was dissolved and centrifuged at 14000 rpm and 4 ° C. for 20 minutes. After centrifugation, the protein concentration in the upper layer was measured using a Bio-Red DC Protein Test Kit (Bio-Rad Laboratories, USA), and 50 μg of protein was measured in 12% (w / v) SDS gel (sodium dodecyl sulfate polyacrylamide gel). Electrophoresis at 120 V for 1.5 h. By electrophoresis, proteins in gels were separated according to molecular weight size, and these proteins were transferred to nitrocellulose membrane (Millipore, USA) at 30 mA for 16 hours. In case of PARP cleavage, the transferred nitrocellulose membrane was blocked with 5% skim milk, and then 100 times diluted mouse anti-PARP antibody (PARP antibody; Cell Signaling Technology, Beverly, MA, USA) was used as the primary antibody. The reaction was carried out at 4 ° C. for 16 hours. The reaction was performed for 1 hour using a 2,000-fold dilution of horseradish peroxidase-bound anti-mouse immunoglobulin G (horseradish peroxidase (HRP) -conjugated anti-mouse IgG; Santa Cruz, USA) as a secondary antibody.

In case of caspase-3 active type, the transferred nitrocellulose membrane was blocked with 5% skim milk, and then 200 times diluted rabbit anti-caspase-3 polyclonal antibody (Santa cruz) was used. It was used as a secondary antibody and reacted at room temperature for 2 hours. In case of caspase-8 active, the transferred nitrocellulose membrane was blocked with 5% skim milk, and then 1000 times diluted rabbit caspase-8 antibody (Polyclonal anti-caspase-8; StressGen Biotechnologies, Canada) was used. It was used as a secondary antibody and reacted at room temperature for 4 hours. The reaction was performed for 1 hour using a 2,000-fold dilution of horseradish peroxidase-coupled anti-rabbit immunoglobulin G (horseradish peroxidase (HRP) -conjugated anti-rabit IgG; Santacruz) as a secondary antibody. Color development was confirmed by exposure to X-ray films (Agfa, EC) using an enhanced chemiluminescence (ECL) western blotting detection system kit (Amersham, USA) according to the user manual. The results are shown in FIG.

From the experimental results, in the GCDC treated hepatocyte group, the caspase-8 active form (44 kD), the caspase-3 active form (17 kDa) and the cleaved PARP (85 kDa) were significantly higher than the normal group. It was confirmed that the increase. In contrast, the active caspase-8 (44 kD) and caspase-3 (17 kDa) and cleaved PARP (85 kDa) were significantly reduced in the group treated with GCDC and oroxylin A compared with the GCDC alone group. Could confirm. From the above results, it was confirmed that oxicillin A inhibited hepatocyte self-killing by bile salts (see FIG. 2).

Experimental Example  4. c- jun - NH2 - termial kinase  ( JNK Phosphorylation inhibitory effect

In order to determine the inhibitory effect of JNK phosphorylation of the axoline A obtained in Example 1 using the method of Park et al. (Park et al., Planta Med. 72, pp661-4, 2006) as follows: (immunoblot assay) was performed.

Hepatocellular proteins obtained using the hepatocellular experimental group separated and cultured as in Reference Example 1 were prepared as in Experimental Example 3, and 100 μg of the protein in each experimental group was obtained at 200 V on a 12% (w / v) SDS gel, respectively. Electrophoresis was performed for a time. Proteins in the gel were separated by electrophoresis according to the molecular weight, and transferred to the nitrocellulose membrane (Millipore, USA) for 16 hours at 30 mA. The nitrocellulose membrane was blocked with 5% skim milk and reacted at 4 ° C. for 16 hours using a 1000-fold diluted phosphoro-JNK polyclonal antibody (Cell Signaling Technology) as the primary antibody. , Horseradish peroxidase (HRP) -conjugated anti-rabbit IgG conjugated with horseradish peroxidase, diluted 2000-fold, was used as a secondary antibody for 1 hour. . The enhanced chemiluminescence (ECL) western blotting detection system kit (KPL) was used for luminescence reaction, and the degree of luminescence was confirmed by exposure to X-ray film (Agfa, EC) according to the user's manual. The results are shown in FIG.

From the experimental results, it was confirmed that 100 μM GCDC induced phosphorylation of JNK, which plays a major role in hepatocyte self-killing, but that JNK phosphorylation was remarkably suppressed by the treatment with oroxylin A (see FIG. 3).

Examples of the pharmaceutical compositions containing the compounds of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation Example 1 Preparation of Powder

Oroxylin A 20 mg

Lactose 100 mg

Talc 10 mg

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

Formulation Example 2 Preparation of Tablet

Oroxylin A 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation Example 3 Preparation of Capsule

Oroxylin A 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Oroxylin A 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Oroxylin A 10 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, lemon juice is added in an appropriate amount, the above components are mixed, and then purified water is added to adjust the total volume to 100 ml, and then sterilized by filling into a brown bottle. do.

Formulation Example 6 Preparation of Health Functional Food

Oroxylin A 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinamide 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

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

Formulation Example 7 Preparation of Healthy Drink

Oroxylin A 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

900 ml of purified water whole

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

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

1 is a diagram showing the effect of inhibiting the hepatocellular self-apoptosis of the axocillin A by inhibiting the hepatocellular DNA fragmentation by hydrophobic bile salt treatment according to the present invention,

Figure 2 shows the expression of active caspase-8, active caspase-3 and poly (ADP-ribose) polymerase cleaved form by hydrophobic bile salt treatment is inhibited by oroxylin A according to the present invention. Is a diagram showing the inhibitory effect of hepatocyte self-apoptosis,

3 is a diagram showing that phosphorylation of c-jun-NH2-terminal kinase in hepatocytes induced by hydrophobic bile salt treatment is inhibited by oroxylin A of the present invention.

Claims (6)

A pharmaceutical composition for the treatment and prevention of liver disease containing Oroxylin A as an active ingredient. [Claim 2] The pharmaceutical composition for treating and preventing liver disease according to claim 1, wherein the oroxylin A has an action of inhibiting hepatocellular damage caused by cholestasis. The method of claim 1, wherein the liver disease is autoimmune liver disease, drug-induced liver disease, alcoholic liver disease, infectious liver disease, congenital metabolic liver disease, cholestatic liver disease, acute hepatitis, chronic hepatitis, cirrhosis, fatty liver or Pharmaceutical composition characterized in that the disease is selected from liver cancer. The pharmaceutical composition of claim 1, wherein the compound comprises 0.1 to 50% by weight based on the total weight of the composition. Health functional food for the prevention and improvement of liver disease, which contains Oroxylin A as an active ingredient. 6. The dietary supplement of claim 5 in the form of a powder, granule, tablet capsule or beverage.

Images