KR100792029B1 - Composition comprising the extract of Purple-Fleshed sweet potato for treating and preventing liver diseases - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of liver disease, and more particularly to a composition for the prevention and treatment of liver disease containing a purple sweet potato pigment extract as an active ingredient. The composition of the present invention inhibits the activity of the enzymes of cytochrome P450 (P450 1A1, P450 2E1), a hepatic metabolism enzyme involved in the metabolism of liver-causing chemicals such as acetaminophen, and induces liver damage. By increasing the detoxifying enzyme that promotes the release of the substance through incorporation of metabolites of the substance, it can be usefully used for the prevention and treatment of liver disease.

Purple sweet potato pigment extract, liver disease

Description

Composition comprising the extract of Purple-Fleshed sweet potato for treating and preventing liver diseases}

1 is a diagram showing the activity of ALT and AST to protect the liver damaged by purple sweet potato pigment extract caused by liver damage causing substances (acetaminophen),

Figure 2 is a diagram showing the lipid peroxidation level of the experimental animal treated with purple sweet potato pigment extract,

Figure 3 is a diagram showing the amount of glutathione in the liver tissue of the experimental animal treated with purple sweet potato pigment extract,

Figure 4 is a diagram showing the activity of cytochrome P450 1A1 enzyme in microsomes isolated from liver of experimental animals treated with purple sweet potato pigment extract,

5 is a diagram showing the activity of cytochrome P450 2E1 enzyme in microsomes isolated from liver of experimental animals treated with purple sweet potato pigment extract,

6 is a diagram illustrating Western blot using microsomes isolated from livers of experimental animals treated with purple sweet potato pigment extract,

7 is a diagram showing the effect of purple sweet potato pigment extract on the activity of cytochrome P450 1A1 enzyme in extracellular activity test (in vitro) in microsomes isolated from the liver of normal laboratory animals,

8 is a diagram showing the effect of inhibiting the activity of cytochrome P450 1A1 enzyme of purple sweet potato pigment extract in extracellular activity experiment in microsomes isolated from livers of experimental animals treated with beta-naphthaflavones that induce cytochrome P450 1A1 enzyme. ,

9 is a diagram showing the effect of inhibiting the enzyme activity of purple sweet potato pigment extract on the activity of cytochrome P450 2E1 enzyme in extracellular activity test in microsomes isolated from the liver of normal experimental animals,

 10 is a diagram showing the effect of inhibiting the activity of cytochrome P450 2E1 enzyme of purple sweet potato pigment extract in extracellular activity experiment in microsomes isolated from liver of pyridine-treated experimental animals inducing cytochrome P450 2E1 enzyme,

11 is a diagram showing glutathione-S-transferase enzyme activity in microsomes isolated from liver treated with purple sweet potato pigment extract.

The present invention relates to a composition for preventing and treating liver diseases, and more particularly, to a composition for preventing and treating liver diseases containing purple sweet potato pigment extract as an active ingredient.

Studies on hepatoprotective activity mainly focus on two pathways. One is toxic hepatoxins such as carbon tetrachloride (CCl ₄ ), acetaminophen, thioacetamide, tacrine, and rubratoxin B and hydrogen peroxide (H ₂ O ₂ ). In addition to detoxification, the body's antioxidant defenses such as superoxide dismutase, catalase, and glutathione peroxidase are evaluated together, and the other is released by liver damage. Serum glutamate pyruvate transaminase (GPT, alanine amino transferase; ALT) and serum glutamate oxaloacetate transaminase (GOT, aspartate alanine amino transferase; AST) To evaluate the level and activity of enzymes. Free carbon tetrachloride (CCl ₄ ) metabolites associated with the Cytochrome P450 system, such as trichloromethyl free radicals (CCl ₃ ·) and trichloromethyl peroxy radicals (CCl ₃ OO ·) Scavenging and / or inhibitory activity against free radicals may be associated with hepatoprotective activity in organelles and cell membranes against oxidation (Hsiao et al., J. Agric. Food Chem., 51 , pp 3302-3308, 2003; Song et al., J. Agric.Food Chem., 51 , pp 1571-1577, 2003).

Acetaminophen is currently widely used as a standard hepatotoxic agent to verify the effectiveness of new hepatoprotectants or therapeutics (DJ Jollow et al., J. Pharmacol. Exp. Ther. 187 , pp 195-202, 1973; JR Mitchell et al., J. Pharmacol.Exp. Ther. 187 , pp 185-194, 1973). Acetaminophen is an effective antipyretic and analgesic when used in therapeutic doses, but it is known to cause fatal liver damage when taken in overdose (BH Rumack, Hepatology 40 , pp 10-15, 2004; WM Lee, Hepatology 40 , pp 6-9, 2004). Fatal injuries following an overdose of acetaminophen are explained by secondary liver damage characterized by extensive necrosis of hepatocytes (D. Zakin et al., Hepatology , WB Saunders Company, Philadelphia, PA, pp 759-762, 1990). This toxicity occurs when acetaminophen is metabolized to N-acetyl-p-benzoquinone imine (NAPQI), a by-product activated by cytochrome P450 in the liver, and this metabolite causes damage and necrosis of liver tissue.

When acetaminophen is administered at therapeutic doses, NAPQI is effectively detoxified by GSH (glutathione), but when administered in excess, intracellular GSH is depleted and NAPQI is not excreted and toxic effects in organs, especially liver tissues, in the human body. (JD Gibson et al., Chem. Res. Toxicol. 9 , pp 580-585, 1996). This results in hepatic central necrosis, the most characteristic histological indicator of acetaminophen-induced hepatotoxicity, causing degeneration of hepatocytes and the appearance of inflammatory cells (D. Zakin et al., Hepatology , WB Saunders Company, Philadelphia, PA, pp 759-762, 1990). In addition, alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activity, which indicate general cell necrosis, are elevated as hematological indicators of liver damage. Furthermore, when acetaminophen is overdosed when human body cells are depleted of GSH, an excess of NAPQI, a metabolite of acetaminophen, is produced, leading to more powerful liver damage (JD Gibson et al., Chem. Res. Toxicol). . 9, pp 580-585, 1996) . In normal conditions, the concentration of human GSH remains constant, but in special pathological conditions, the concentration of GSH drops rapidly, increasing the human's susceptibility to toxic substances (EY Park et al., Chem. Biol. Interact. 155 , pp 82 -96, 2005). Similarly, hepatic damage due to viral infection is also amplified when the biological GSH concentration decreases. This fact is supported by findings that involve liver tissue necrosis in patients with chronic HBV or HCV infection accompanied by a decrease in biological GSH (T. Tanyalcin et al., Hepatol. Res. 18 , pp 104-109, 2000).

In the prior art in the art, in general, N-acetylcysteine (NAC) is almost the only drug used for the treatment of acetaminophen toxicity. Therefore, it is urgent to develop other alternative drugs that are more efficient than NAC.

On the other hand, purple sweet potato (Purple-Fleshed Sweet Potato) was artificially bred in 1993 using a variety of dried varieties of processed varieties of dry brown rice and purple pigment containing Yamakawamurasaki as a copy. Evaluation of production system (MI9308-11, purple sweet potato for coloring material and breeding (1994. Kinnosuke Odake, Japan, etc.) technical report 41 (4), 287 ~ 293) In the test and regional adaptation test (Mokpo 29), multiplicity was confirmed and breeding was carried out with three new varieties: breeding in 1998, Borami in 2000 and Shinjami in 2001. These new varieties have all the constituents of wild sweet potatoes and are similar in size to regular sweet potatoes, while yielding more than twice the yield of wild sweet potatoes.

The main active ingredient of this purple sweet potato is an anthocyanin, a flavonoid pigment ( J Agric. Food Chem. 51 , pp 628-633, 2003; J Nat. Prod. 62 , p 802, 1999; J Med. Food. 4 pp 211-218, 2001), anti-diabetic ( J Agric Food Chem . 49 , pp 1948-1951, 2001), and cancer lett. In press pp 1-12, 2005. . The pigment extract of purple sweet potato contains about 3.8% of the weight of the body, and not only the epidermal layer but also the entire flesh has a dark purple color.The functional pigment content is 10-60 times higher than that of the berry, and 5-7 times higher than that of the grape. It is excellent in value not only as a pigment source but also as a functional food material.

Therefore, the present invention is to find a material for preventing liver damage from the safe and non-toxic natural products and to treat liver cirrhosis or liver disease caused by chronic liver damage, the purple sweet potato pigment extract causes liver damage such as acetaminophen introduced into the liver Enzyme activity of glutathione-S-transferase which inhibits the activity of certain cytochrome P450 1A1 and 2E1 enzymes involved in the metabolism of chemicals and promotes the in vitro release of toxic chemical metabolites formed by the metabolism of these chemicals The present invention was found to inhibit hepatotoxicity with an effect of increasing the amount of.

An object of the present invention is to provide a pharmaceutical composition and health functional food containing the purple sweet potato pigment extract effective for liver disease as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of liver disease containing a purple sweet potato pigment extract as an active ingredient.

The purple sweet potato includes varieties of Jami (mokpo 29), Borami (mokpo 35), Shinjami (mokpo 38).

The extract includes extracts available for polar solvents such as water, methanol, ethanol and the like, and mixed solvents thereof.

The liver disease includes autoimmune liver disease, drug-induced liver disease, alcoholic liver disease, infectious liver disease, congenital metabolic liver disease, acute hepatitis, chronic hepatitis, cirrhosis, liver cirrhosis, fatty liver, liver cancer.

Hereinafter, a method of obtaining the purple sweet potato pigment extract will be described in detail.

The purple sweet potato extract of the present invention is chopped raw purple sweet potato, about 1 to 20 times the weight (kg), preferably about 3 to 10 times the water, C ₁ to C ₄ lower alcohol or a mixture thereof Solvent, preferably water or ethanol, chilled, hot water extraction, ultrasonic for about 1 to 10 days, preferably about 2 to 5 hours, at an extraction temperature of 20 ° C. to 100 ° C., preferably 50 ° C. to 100 ° C. Extraction, extraction obtained by reflux cooling extraction method can be obtained by filtration, concentrated under reduced pressure or dried.

The extract was diluted 20 times (w / v) in 100% water and passed through a tube filled with HP-20 resin to allow the pigment to bind to HP-20 resin, and a purple pigment bound to HP-20 resin. After re-extracting with C ₁ to C ₄ lower alcohol or a mixed solvent thereof, preferably water or ethanol, pure purple sweet potato pigment extract may be obtained through reduced pressure, concentration and drying. The purchased purple pigment powder was quantitatively taken and measured at absorbance of 532 nm to prepare a calibration curve. It is preferable to use the calibration curve to measure the pigment content of the purple pigment extract to use a purity of 98% or more.

The present invention also provides a purple sweet potato pigment extract obtained by the above method as an active ingredient, and provides a pharmaceutical composition effective for the prevention and treatment of liver disease.

The composition of the present invention preferably contains 0.01 to 99.9% of purple sweet potato pigment extract, and more preferably 0.1 to 90%. However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

The composition comprising the purple sweet potato pigment extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be used. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , 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 extracts of the present invention vary depending 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, and may be appropriately selected by those skilled in the art.

However, for the desired effect, the extract of the present invention is preferably administered at 10 to 1,000 mg / kg, preferably 10 to 100 mg / kg. Administration may be administered once a day or may be divided several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. 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 health functional food comprising a purple sweet potato pigment extract showing an effect of preventing liver disease as an active ingredient. Examples of foods to which the purple sweet potato pigment extract may be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods, and may be used in the form of powders, granules, tablets, capsules, or beverages. Can be.

The purple sweet potato pigment extract of the present invention is a drug that can be used with confidence even when taken for a long time for the purpose of prevention because there is little toxicity and side effects.

The extract of the present invention may be added to food or beverage for the purpose of preventing liver disease. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

The health beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated ratio, 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 extract of the present invention includes 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 adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Preparation of Purple Sweet Potato Extract

1-1. Preparation of Purple Sweet Potato Extract

100 g of Mokpo No. 29 purple sweet potato meaty whole provided by Hamyang-gun, Gyeongnam, was chopped and immersed in 20% ethyl alcohol to obtain an extract at room temperature, and extracted two more times by adding ethyl alcohol. The filtrate was mixed. The ethanol extract was reduced pressure, concentrated and dried to give 12.3 g of the total extract.

1-2. Preparation of Purple Sweet Potato Pigment Extract

The crude extract of Example 1-1 was diluted 20 times (w / v) in 100% water, and then passed through a tube filled with HP-20 resin to enable dye to bind to HP-20 resin. The purple pigment bound to HP-20 resin was extracted again using 70% ethyl alcohol, and then 2.5 g of pure purple sweet potato pigment extract was obtained through reduced pressure, concentration and drying.

Reference Example  1. Experimental Animals and Treatment

The experimental animals were 25-30 g of male mice, 8 weeks old, which were purchased from Korea Biolink Co., Ltd. (SPF), and the Institute of Cancer Research (ICR). Intake freely, the temperature of the kennel was maintained at 21 ~ 24 ℃, relative humidity 40 ~ 80%. In addition, the light in the kennel was adjusted to repeat day and night every 12 hours.

Acetaminophen (APAP), alanine amino transferase (ALT) and aspartate amino transferase (AST) kit, Thiobarbituric acid (TBA), phthaldialdehyde ( O-Phthaldiadehyde, phenylmethoxyl sulfonylfloride and glutathione (GSH) were purchased from Sigma chemical Co.

Experimental Example 1. Hepatoprotective effect of purple sweet potato pigment extract against a substance causing liver toxicity

1-1. Administration of Purple Sweet Potato Pigment Extract and Induced Liver Damage by Chemicals

To the experimental animals (ICR Mouse) purple sweet potato pigment extract was dissolved in physiological saline (0.85% NaCl) at 200, 400, 800 mg / kg was administered orally for 7 days. The control group was orally administered only physiological saline (0.85% NaCl). Each experimental group used 6 animals. After pretreatment with purple sweet potato pigment extract, acetaminophen (APAP, 400 mg / kg) was intraperitoneally induced to cause artificial liver damage. After 18 hours, mice were anesthetized with ether, and blood and liver were collected.

1-2. Determination of Alanine Amino Transferase (ALT) and Aspartate Amino Transferase (AST) Activities in Blood

The amount of alanine amino transferase (ALT) and aspartate amino transferase (AST) activity released into the blood upon liver injury was measured by Reitman-Frankel ( J Ethnopharmacol. 66 , pp 263-269, 1999; J Ethnopharmacol. 66 , pp 355-361, 1999; J Nutr. Biochem. 11 , pp 509-514, 2000). After 7 days of oral administration of purple sweet potato pigment extract, acetaminophen was treated by intraperitoneal administration, and after 18 hours, serum from the heart was reacted with substrate solution of ALT and AST for 30 minutes at 37 ° C. for pyruvate. After the production, the color developing solution was added, and after 20 minutes, 0.4 N-sodium hydroxide (NaOH) was mixed to give a color development. The absorbance was measured at 505 nm. The results are shown in Table 1.

division Control Acetaminophen (APAP) Purple Sweet Potato Pigment Extract (mg / kg) 0 200 400 800 Serum ALT (Karmen Units / ml) 3 ± 1 372 ± 44 93 ± 12 37 ± 10 15 ± 5 Serum AST (Karmen Units / ml) 36 ± 14 470 ± 52 131 ± 21 82 ± 27 37 ± 8

Table 1 and Figure 1 shows the results of the measurement of alanine aminotransferase (ALT) and aspartate amino transferase (AST) activity released from liver cells to serum by liver damage caused by acetaminophen, pre-treated with purple sweet potato pigment extract The alanine aminotransferase (ALT) and aspartate amino transferase (AST) activities, which are indicators of liver damage, decreased depending on the concentration of purple sweet potato pigment extract compared to the group treated with acetaminophen alone alone, especially purple sweet potato pigment extract. The pretreatment group with 800 mg / kg of showed a similar degree of hepatoprotective activity (reduction of ALT and AST activity) as the control.

1-3. Lipid Peroxidation in Liver

To determine the liver damage protection effect of purple sweet potato pigment extract against lipid peroxidation produced by acetaminophen, 100 mg of liver was added to 10 ml of buffer (Tris-HCl), and then crushed by a homogenizer and then degraded peroxide lipids. The absorbance at 532 nm was measured at 532 nm of the thiabbituric acid pigment produced by heating the product of malon-dialdehyde (MDA) with thiobarbituric acid (2-Thiobarbituric acid (TBA) for 20 minutes at 100 ° C. . The results are shown in Table 2.

division Control Acetaminophen (APAP) Purple Sweet Potato Pigment Extract (mg / kg) 0 200 400 800 Hepatic Lipid Peroxidation (MDA, nmol / g liver) 2.1 ± 0.32 7.6 ± 0.72 5.5 ± 0.42 4.2 ± 0.42 2.3 ± 0.33

Table 2 and FIG. 2 shows the concentration of lipid peroxidation in concentration-dependently compared with the group treated with acetaminophen alone as a result of measuring the level of lipid peroxidation induced by acetaminophen. Pre-treatment with purple sweet potato pigment extract at 800 mg / kg showed hepatoprotective activity (inhibition of lipid peroxidation levels) similar to the control.

Experimental Example  2. by liver damage Reduced  Glutathione ( Glutathione , GSH Recover volume

The extracted liver is washed with distilled water, dried for a while, weighed, and then the amount of glutathione is measured using a portion of the liver. In this method, 100 mg of the extracted liver is added to 0.02 M EDTA, and the tissue is crushed using a Homogneizer. After centrifugation of this aqueous solution at 12000 rpm for 30 minutes, only 100 ul of the supernatant was taken. Into this supernatant, 1.8 ml of phosphate-EDTA buffer and 100 ul of phthaldialdehyde (O-Phthaldiadehyde) are added, and after 15 minutes, the absorbance is measured at 412 nm with a spectrophotometer. The results are shown in Table 3.

division Control Acetaminophen (APAP) Purple Sweet Potato Pigment Extract (mg / kg) 0 200 400 800 GSH (μmole / g liver wt.) 8.1 ± 0.88 3.6 ± 0.42 5.2 ± 0.45 6.5 ± 0.71 8.3 ± 0.93

Table 3 and FIG. 3 shows the results of measuring the amount of glutathione (GSH) in liver tissue to confirm the effect of increasing the amount of glutathione loss lost by acetaminophen detoxification activity in liver tissue. The pretreatment was increased in a concentration-dependent manner compared to the acetaminophen-only group, especially when pre-treated with 800 mg / kg of purple sweet potato pigment extract, it was similar to the amount of glutathione in the control group.

Experimental Example  3. Hepatotoxicity  Related to metabolic activity of triggers Cytochrome P450  ( cytochrome P450 Pharmacokinetic Enzyme Activity

3-1. Microsome Isolation after Treatment of Experimental Animals for In Vivo Activity

 After 100 days of oral administration of purple sweet potato pigment extract at 200, 400, 800 mg / kg for 7 days in 10 ml homogenizer buffer, the tissue is crushed using a homogenizer. After centrifugation of this aqueous solution for 30 minutes at 9,000 xg 4 ° C, only the supernatant was transferred to an ultracentrifuge tube again, and centrifuged at 100,000 xg for 4 hours at 1 ° C. After ultracentrifugation, the supernatant cytosol was transferred to a new microcentrifuge tube, and the precipitated pellet was mixed with 10% glycerol in a storage buffer (homogenizer solution). ) 1 ml was added and suspended well using a pipette and then transferred to a new microcentrifuge tube and stored at -80 ° C until use.

3-2. EROD (7-ethoxyresorufin-), a specific enzymatic reaction to cytochrome P450 1A1 metabolic enzyme O -dealkylase Enzyme Activity

2 mg / ml bovine serum albumin, 10 μm Dicoumarol, NADPH degeneration system (5 mM glucose-6 phosphate) in 0.1 M potassium phosphate (pH 7.4) glucose-6-phospate), 5 unit / ml glucose-6-phosphate dehydrogenase, 10 μm NADPH, 2.5 μm ethoxyresorufin and microsome ) The sample was mixed and reacted at 37 ° C. for 20 minutes. Resorufin formed after the reaction was measured by a fluorometer at an emission wavelength of 550 nm and an emission wavelength of 585 nm. The concentration of protein in microsome samples was determined by Lori et al . ( J Ethnopharmacol. 66 , pp 263-269, 1999; J Nutr. Biochem. 14 , pp 452-458, 2003; Toxicon. 42 , pp 367-372 , 2003). The results are shown in Table 4.

division Control Purple Sweet Potato Pigment Extract (mg / kg) 200 400 800 EROD activity (nmole / mg protein / min) 14.34 ± 0.96 12.26 6.48 4.14

 Table 4 and Figure 4 is to investigate the effect of acetase P450 1A1 in the liver involved in the production of metabolites that cause liver damage of liver damage chemicals, including acetaminophen (Acetaminophen) introduced into the liver, acetaminophen In order to measure the activity of the enzyme P450 1A1, which converts it to N-acetyl-p-benzoquinone imine, its activity, EROD (ethoxyresorufin O-deethylase), an enzyme reaction specific to P450 1A1, was measured. The groups treated with sweet potato pigment extract decreased the EROD activity in the liver depending on the treatment concentration.

3-3. Cytochrome P450  ( cytochrome P450 ) 2 E1  Determination of AH (aniline hydroxylase) enzyme activity, a specific enzymatic reaction to drug metabolism enzymes

Aniline (5-20 mM), 10 μm NADPH, NADPH regenerating system and microsome samples were mixed in 0.1 M potassium phosphate buffer (pH 7.4) and then a 37 ° C. shacking water incubator for 20 minutes, and then the reaction was terminated by adding 20% TCA and mixed with 5% phenol and 2.5 N sodium hydroxide (NaOH), followed by 2.5 N sodium carbonate (Na ₂ CO ₃ ) for 30 minutes. The color was developed while standing at room temperature. Absorbance was measured at 630 nm and activity was calculated from the hydroxyline aline (4-hydroxyaniline) calibration curve. The results are shown in Table 5.

division Control Purple Sweet Potato Pigment Extract (mg / kg) 200 400 800 AH activity (nmole / mg protein / min) 11.34 ± 1.2 6.23 ± 0.71 4.16 ± 0.33 2.65 ± 0.32

Table 5 and Figure 5 is a study of the effect on the metabolic enzyme P450 2E1 in the liver involved in the production of metabolites that cause liver damage of liver damage chemicals, including acetaminophen introduced into the liver, acetaminophen In order to measure the activity of the enzyme P450 2E1, which converts it to N-acetyl-p-benzoquinone imine, the activity of AH (aniline hydroxylase), an enzyme specific to P450 2E1, was measured. The groups treated with the extract decreased the AH enzyme activity in the liver depending on the treatment concentration.

3-4. Investigation of Cytochrome P450 2E1 Metabolic Enzyme Expression

To investigate the effect of purple sweet potato pigment extract on the liver metabolism enzyme cytochrome P450 2E1 expression, the microsome isolated from liver treated with purple sweet potato pigment extract was analyzed by using an antibody against cytochrome P450 2E1. Western blots were performed. The results are shown in FIG. FIG. 6 shows the effects of metabolic enzyme P450 2E1 enzyme expression in the liver involved in the production of metabolites causing liver damage of liver damage chemicals including acetaminophen introduced into the liver. It was confirmed that the expression of the enzyme P450 2E1 converting to the chain acetylbenzoquinone imine decreased depending on the treatment concentration of the purple sweet potato pigment extract. Beta-actin (β-actin) was used as a control for P450 2E1 protein expression.

3-5. Treatment of experimental animals for extracellular activity in vitro

The experimental animals were treated with SPF (Sprague-Dawley; SD) purchased from Daehan Biolink Co., Ltd. (Sprague-Dawley; SD) at 8 weeks old, and rats of normal rats were treated with no normal group and P450 1A1 in liver. Enzyme induction was beta-napthaflavone (β-napthaflavone) and P450 2E1 induction was pyridine (pyridine) three days intraperitoneally. On day 4, rats were anesthetized with ether, and livers were extracted to separate microsomes in the liver in the same manner as above and stored at −80 ° C. until use.

3-6. Determination of 7-ethoxyresorufin-O-dealkylase (EROD) enzyme activity, a specific enzymatic response to cytochrome P450 1A1 metabolic enzyme in extracellular activity experiment

The experiment was performed in the same manner as the method for measuring the EROD activity in the intracellular activity experiment. The results are shown in Table 6 and FIG.

division Control Purple Sweet Potato Pigment Extract (㎍ / ㎖) 200 400 800 EROD activity (nmole / mg protein / min) 6.34 ± 0.67 5.33 ± 0.61 3.26 ± 0.36 2.55 ± 0.32

Table 6 and Figure 7 is to investigate the effect on the activity of the enzyme metabolic enzyme cytochrome P450 1A1 in the liver involved in the production of metabolites that cause liver damage of liver damage chemicals, including acetaminophen introduced into the liver In order to measure the activity of the enzyme P450 1A1, which converts to acetaminophen metabolite, N-acetyl-p-benzoquinone imine, the microsomal isolated from normal liver was treated with purple sweet potato pigment extract and then P450 1A1. The EROD (ethoxyresorufin O-deethylase) activity, a specific enzyme reaction, was measured. The groups treated with purple sweet potato pigment extract decreased EROD enzyme activity depending on the treatment concentration.

division Beta-napthaflavone induced hepatic microsomes Purple Sweet Potato Pigment Extract (㎍ / ㎖) 0 200 400 800 EROD activity (nmole / mg protein / min) 36.42 ± 2.13 19.25 ± 0.52 15.37 ± 0.11 10.74 ± 3.21

Table 7 and FIG. 8 show hepatic metabolizing enzyme cytochrome P450 1A1 enzyme by purple sweet potato pigment extract in extracellular activity test on microsomes isolated from livers of experimental animals treated with beta-naphthaflavones that induce cytochrome P450 1A1 enzyme. As a result of investigating the effect on activity, EROD (ethoxyresorufin O-deethylase) activity, an enzyme specific to P450 1A1, was measured, and the group treated with purple sweet potato pigment extract decreased EROD enzyme activity depending on the treatment concentration.

3-7. Determination of AH (aniline hydroxylase) enzyme activity, a specific enzyme response to cytochrome P450 2E1 drug metabolism enzyme in extracellular activity experiment

 The experiment was performed in the same manner as the method for measuring AH activity in the intracellular activity experiment. The results are shown in Table 8 and FIG.

division Control Purple Sweet Potato Pigment Extract (㎍ / ㎖) 200 400 800 AH activity (nmole / mg protein / min) 10.32 ± 1.21 6.21 ± 0.72 5.34 ± 0.63 3.93 ± 0.44

Table 8 and Figure 9 is to investigate the effect on the activity of the enzyme metabolic enzyme cytochrome P450 2E1 in the liver involved in the production of metabolites that cause liver damage of liver damage chemicals, including acetaminophen introduced into the liver In order to measure the activity of the enzyme P450 2E1, which converts it to acetylbenzoquinone imine, a metabolite of acetaminophen, P450 2E1 was treated with purple sweet potato pigment extract in microsomes isolated from normal livers. As a result of measuring AH (aniline hydroxylase) activity, an enzyme specific to, the groups treated with purple sweet potato pigment extract decreased AH enzyme activity depending on the treatment concentration.

division Pyridine-induced hepatic microsomes Purple Sweet Potato Pigment Extract (㎍ / ㎖) 0 200 400 800 AH activity (nmole / mg protein / min) 28.37 ± 3.92 13.72 ± 2.12 10.28 ± 1.75 7.31 ± 0.84

Table 9 and FIG. 10 are enzymes for hepatic metabolizing enzyme cytochrome P450 2E1 enzyme activity by purple sweet potato pigment extract in extracellular activity experiment on microsomes isolated from liver of pyridine-treated experimental animals that induce cytochrome P450 2E1 enzyme. As a result of measuring the effect of AH (aniline hydroxylase) activity, an enzyme specific to P450 2E1, the groups treated with purple sweet potato pigment extract decreased AH activity depending on the treatment concentration.

Experimental Example 4 Investigation of Glutathione-S-Transferase (GST) Enzyme Activity Changes Related to Extracorporeal Release of Hepatotoxic Agents

Glutathione-S-Transferase enzyme activity was measured after dissolving the cytosol of the liver stored at -80 ° C by ultracentrifugation at 4 ° C. 10 μl of cytosol, 0.1 μl of 0.1M potassium phosphate, 50 μl of 10mM GSH, 50 μl of 10mM diene (cDNB; 1-chloro-2,4-dinitrobenzene) were mixed and reacted at 37 ° C. Absorbance was measured at 340 nm with a spectrophotometer at 5 minute intervals for 15 minutes. After 15 minutes, the reaction was stopped by standing at 4 ° C. for 5 minutes, and the protein was quantitated by the method of Lori et al . ( J Ethnopharmacol. 66 , pp 263-269, 1999; J Nutr. Biochem. 14 , pp 452-458, 2003; Toxicon. 42 , pp 367-372, 2003). After subtracting the 5-minute quantitative value from the 15-minute quantitative value, the activity of glutathione-S-transferase in the cytosol was calculated. The results are shown in Table 10.

division Control Purple Sweet Potato Pigment Extract (mg / kg) 200 400 800 GST activity (nmole / mg protein / min) 98 ± 10.2 224 ± 33.2 316 ± 43.4 268 ± 62.4

Table 10 and FIG. 11 show in vitro excretion through the conjugation reaction of metabolites and liver glutathione that cause liver damage of liver damage chemicals including acetaminophen introduced into the liver. We investigated the effect of purple sweet potato pigment extract on the activity of the Phase II metabolic enzyme glutathione-S-transferase in the liver, and treated with purple sweet potato pigment extract. Glutathione-S-Transferase enzyme activity in the liver was increased depending on the concentration.

Based on the above results, it was confirmed that the hepatocellular damage caused by acetaminophen was recovered by the purple sweet potato pigment extract through serum enzymes (ALT, AST) and intracellular lipid peroxidation levels. In addition, the purple sweet potato pigment has an enzymatic activity of glutathione-S-transferase, a phase 2 metabolic enzyme that prevents liver damage associated with the promotion of extracellular release of acetyl-para-benzoquinoneimine, a hepatotoxic substance produced by acetaminophen. It was confirmed that it was increased by the extract.

Based on these metabolic pathways, the recovery mechanism of purple sweet potato pigment extract was investigated by cytochrome P450 enzymes and glutathione deficiency effects. As a result, the enzyme activity of P450 1A1 and P450 2E1 in microsomes was reduced in a concentration-dependent manner. It was confirmed that this, it can be assumed that the production of acetylbenzoquinone imine by acetaminophen reduced the inhibition of liver toxicity.

A separate detoxification process involves the detoxification of glutathione with acetylbenzoquinone imine. In the case of purple sweet potato pigment extract, the amount of glutathione was increased in a concentration dependent manner. However, inhibition of the enzyme activity of P450 1A1 and P450 2E1 inhibited acetylbenzoquinone immunization and decreased glutathione and acetyl-para-benzoquinone imine bonds, thus reducing the amount of glutathione present in the liver.

Formulation example  1. Preparation of Injection

Purple sweet potato pigment extract of Example 1 ............ 10 mg

Sodium metabisulfite ... 3.0 mg

Methylparaben ............... 0.8 mg

Propylparaben .................................. 0.1 mg

Sterile distilled water for injection ..............

The above ingredients are mixed and made into 2 ml by a conventional method, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

Purple sweet potato pigment extraction of Example 1 ............... 200 mg

Lactose ......................................... 100 mg

Starch ......................................... 100 mg

Magnesium Stearate ...............

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 3 Preparation of Capsule

 Purple sweet potato pigment extract of Example 1 ............. 10 mg

Lactose ......................................... 50 mg

Starch ..................................... 50 mg

Talc ........................................ 2 mg

Magnesium Stearate ...............

Capsules are prepared by mixing the above ingredients and filling into gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 4 Preparation of Liquid

 Purple sweet potato pigment extract of Example 1 ........ 1000 mg

Sugar ......................................... 20 g

Isomerized sugar ............................................... 20 g

Lemon scent ...

Purified water was added to adjust the total volume to 1000 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 5 Preparation of Healthy Food

Purple sweet potato pigment extract of Example 1 .......... 1000 mg

Vitamin Blend ........................

Vitamin A Acetate ............... 70 μg

Vitamin E ..................................... 1.0 mg

Vitamin B1 ..................... 0.13 mg

Vitamin B2 ..................... 0.15 mg

Vitamin B6 ......................................... 0.5 mg

Vitamin B12 ......................... 0.2 mg

Vitamin C ......................................... 10 mg

Biotin ......................................... 10 μg

Nicotinamide ......................................... 1.7 mg

Folic Acid ... 50 mg

Calcium Pantothenate ......... 0.5 mg

Mineral mixture ........................

Ferrous Sulfate ......... 1.75 mg

Zinc Oxide ............... 0.82 mg

Magnesium Carbonate ................................... 25.3 mg

Potassium monophosphate ......................................... 15 mg

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 mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 6 Preparation of Healthy Drink

Purple sweet potato pigment extract of Example 1 .......... 1000 mg

Citric Acid ......................................... 1000 mg

Oligosaccharide ......................... 100 g

Plum concentrate ........................... 2 g

Taurine ......................................... 1 g

Purified water is added to the whole .......... 900 ml

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 mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the purple sweet potato pigment extract of the present invention exhibits an action of restoring hepatocytes damaged by an external substance such as acetaminophen, and is a composition for the prevention or treatment of liver disease, which is composed of non-toxic natural medicines and Can be used as a dietary supplement.

Claims (7)

Acute hepatitis or chronic acute hepatitis induced by acetaminophen containing crude pigment extract prepared by extracting purple sweet potato (mokpo 29) with 20% ethanol as an active ingredient Pharmaceutical composition for the prevention and treatment of hepatitis. Acute hepatitis or chronic induced by acetaminophen containing crude extract prepared by extracting purple sweet potato (mokpo 29) with 20% ethanol as an active ingredient Health functional food for the prevention and improvement of hepatitis. delete delete delete delete delete

Images