KR100477957B1 - Novel pyrrole derivatives isolated from lycium chinense mill having hepatoprotective activity and composition containing same - Google Patents

Abstract

The present invention relates to a pyrrole derivative compound represented by the following Chemical Formula 1 isolated from Lycium chinense MILL and its use as a hepatoprotectant, and the compounds effectively treat hepatotoxicity by carbon tetrachloride, thereby treating liver diseases such as hepatitis and cirrhosis. It can be used as a pharmaceutical composition useful for prophylaxis.

Where

R ¹ to Each R ³ is independently hydrogen, halogen, or a C _1-4 alkyl group.

Description

Novel pyrrole derivatives having hepatoprotective activity isolated from goji berry and compositions containing them {NOVEL PYRROLE DERIVATIVES ISOLATED FROM LYCIUM CHINENSE MILL HAVING HEPATOPROTECTIVE ACTIVITY AND COMPOSITION CONTAINING SAME}

Hepatitis, one of the most prevalent diseases in Korea, is known to be infected by contact with hepatitis carriers, such as booze, and vaccines have been developed for viral hepatitis B. No specific drug has been developed yet. These hepatitis has been found to cause liver damage by overwork, stress, heavy drinking, smoking, causing chronic hepatitis, cirrhosis and cirrhosis, and eventually develop liver cancer.

The number of hepatic disease patients in Korea has been increasing rapidly since the late 70s. In 1996, the rate of morbidity was 944 people per 100,000 population. Especially noteworthy is that hepatic disease accounts for 26% of the causes of mortality among men in their 40s, with the highest mortality rate (1996 Statistical Yearbook of Statistics). -40 (1997)). The most common cause of mortality among liver diseases is viral hepatitis, but in the West, deaths due to cirrhosis are reported to be 5-10 times higher than viral hepatitis. The liver is a large buffering organ that does not appear well in the early stages of the disease and is found only when it is significantly worse. Cirrhosis is the last step that is common when chronic liver disease progresses. The causes include alcohol, drugs, chemicals, viral hepatitis, metabolic diseases such as biliary tract diseases, autoimmune diseases, etc., but the cause is often unknown. In cirrhosis, overall liver function decreases such as hepatic blood flow decrease, hepatic blood flow decrease, metabolic enzyme function decrease, blood protein quality, quantitative change and bile flow change.

The liver is also known to be involved in blood storage and circulation, blood volume control and defense detoxification in the human body and closely related to mental activity. Our bodies are always exposed to the pollutants and toxic substances caused by industrialization, and our liver is constantly suffering from detoxification. What is more serious is liver damage from 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 and D-galactosamine. Carbon tetrachloride is known to cause hepatotoxicity by causing lipid peroxidation of hepatocytes. Recknagel (Pharmacol. Rev., 19, 145-208. 1967), Alpers, et al. (Mol. Pharmacol., 4, 566-573, 1968) and See Slater (Slater, TF (Eds), Academic Press, London, pp. 243, 1982). Since carbon tetrachloride is toxic by metabolic activation, it selectively acts on the metabolic liver, destroying cytochrome P-450, causing fatty liver, and causing liver necrosis and cirrhosis (Azari et al.). (See Toxicol. Appl. Pharmacol. 112 , 81-89 (1992).) The toxicity mechanism of carbon tetrachloride is a metabolite produced by cytochrome P-450-dependent mixed oxidase, a drug metabolic enzyme present in internal network. Phosphorus methyl trichloride free (CCl ₃ ) binds to cellular proteins and lipids, resulting in lipid peroxidation, resulting in morphological changes in internal network, reduced activity of drug metabolism enzymes, reduced protein synthesis and glucose-6- It is known to cause a decrease in the activity of phosphate and cause fatty liver by inhibiting the release of triglycerides synthesized in the liver [Gravela, E. et al. (See Biochem. J. 178 , 509-515 (1979)).

Hepatotoxicity or protective activity can be determined by measuring various enzymes present in hepatocytes. Glutamic pyruvic transaminase (GPT) and sorbitol dehydrogenase (SDH) are enzymes that are selectively present in hepatocytes rather than other organs. When hepatotoxicity is induced, hepatocytes are ultimately destroyed and cultured in hepatocytes. The amount of enzyme liberated is increased. This fact is widely used to measure hepatotoxicity and protective activity against carbon tetrachloride.

Still, development of treatment for liver disease has not made any progress except for interferon, and it is DDB (dimethyl-4,4'-dimeth), a synthetic derivative of silymarin and Schizandra chinensis isolated from herbal medicine as a therapeutic aid for chronic hepatitis. Methoxy-5,6,5'6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate) and the like are only used. Therefore, it is urgent to develop new medicines that can treat various liver diseases including hepatitis and cirrhosis.

Attempts to find new substances that can prevent or treat human diseases from natural products have been the subject of research by many scientists from the past. In the meantime, more than 50% of the medicines currently being used by these resources are derived from natural products, and 30% of the medicines used in the US are obtained from plants, and 120 drugs are approved by the US FDA. Is about $ 10 trillion. Despite numerous efforts to develop therapeutics for liver disease from natural products, only a handful of examples are currently in use or under clinical trials as therapeutic agents [Phytotheraphy Res., Thabrew, MI et al. 10 , 461-467 (1996)).

Silymarin, a representative hepatic disease treatment agent, developed from natural products, has been isolated from mature fruits and tubules of Silybum marianum, which has been used for the treatment or prevention of hepatic diseases. See Hikino, et al., Planta Med. 50 , 213-218 1984. Silymarin, a mixture of flaborgignan-based substances, such as silybin, isolysin, dihydrosilinine, sillydianin and sillycristine, Activating RNA polymerase in hepatocytes increases protein synthesis to promote regeneration of damaged liver tissue Silymarin is currently used in the treatment of chronic infectious liver disease, cirrhosis and alcoholic liver disease.

Komycin A, a lignan-based substance isolated from the fruit of Schizandra chinensis , which has been used for the prevention of tonic or liver disease in Korean medicine, is synthesized as a derivative of DDB and used as a treatment for chronic hepatitis. In addition to high-mycin A, derivatives such as wuweizisu C, xanthrin D, deoxygomycin and high-mycin N have been reported to restore the hepatotoxicity induced by carbon tetrachloride or galactosamine [Kiso et al. Y. et al. (Planta Med. 50 , 81-85, 1984).

Recently, clinical trials are underway to develop (+)-catechin, known as (+)-cyanninol-3, for the treatment of acute viral hepatitis. Also, in Japan, clinical trials have been conducted to develop glycyrrhizin as a treatment for acute viral hepatitis or chronic hepatitis. See Phytotheraphy Res. 10 , 461-467 (1996) by Thabrew et al. ].

There are a number of cases in which hepatocellular protective components are isolated from plants using hepatocytes of primary cultured rats, which are artificially toxic. Carbon tetrachloride and galactosamine are the most widely used toxic substances. Toxicity was also induced with ionopoa A23187 (see Kiso et. Al., Shoyakugaku Zasshi 39, 218-221 1985).

Kiso Y. (see Planta Med., 49, 222-225 1983) has been shown to induce toxicity with carbon tetrachloride or galactosamine from Curcuma aromatica , which has been used in the civilian treatment for fragrance-free stomach and jaundice. As a screening system, the curcuminoids (curcumin, p -cumeroylferuloylmethane, di- p -coumaroylmethane) having hepatoprotective activity were isolated and reported. In addition, hepatoprotective activity of cinnamic acid derivatives such as coumarin acid and ferulic acid, which are metabolites of curcuminoids, was reported.

Lee MK et al. (Planta Med., 61, 523-526 1995) searched for primary cultured hepatocytes in rats induced by toxicity with carbon tetrachloride from Epimedium koreanum, a civilian tonic. As a system, it has been reported to separate Icarin and Icaridide II having hepatoprotective activity by bioactivity-guided fractionation. Icarin showed hepatoprotective activity equivalent to that of silybin at a low concentration of only one hundredth of the maximum active concentration of silybin, the main component of the silymarin preparation, which is already used for the treatment of liver disease.

Kim SY et al. (J. Nat. Prod., 60, 274-276 1997), as well as Yin Yang Kwak, induces toxicity with carbon tetrachloride from Lycium chinense , which is widely used as a nourishment tonic in oriental medicine or in the private sector. As a screening system for primary cultured hepatocytes of rats, a new serebroside-based substance having hepatoprotective activity was isolated by bioactive component trace fractionation. It has been reported that this substance has hepatoprotective activity which significantly reduces the activity of GPT and SDH released by carbon tetrachloride toxicity in primary cultured hepatocytes. However, there has been no report on the hepatotoxic action of pyrrole derivative compounds isolated from wolfberry.

The present inventors have discovered that pyrrole derivative compounds having a novel chemical structure and separated from goji ( Lycium chinense MILL.) In the process of searching for plants that are expected to be useful for the prevention and treatment of liver diseases for various kinds of natural products. Was found.

Gojija ( Lycium chinense MILL) is a branch and vine shrub, distributed throughout the country, and its mature fruit is called gojija, and its known components have been reported separately, such as carotene and linolenic acid, blood sugar and cholesterol lowering action, blood pressure It has been reported to exhibit a lowering effect (see the publication of Information Suh et al. (Schematics of Illustrated Herbal Medicines (Plant), published by Younglim Inc. p826-828, 1998)).

It is an object of the present invention to provide novel pyrrole derivative compounds.

Still another object of the present invention is to provide a pharmaceutical composition for preventing and treating liver disease, comprising the novel pyrrole derivative as an active ingredient and a pharmaceutically acceptable carrier.

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In accordance with the above object, the present invention provides pyrrole derivative compounds of the formula (1) obtained by a conventional extraction and separation method from the dried Lycium chinense MILL.

Formula 1

Where

R ¹ to Each R ³ is independently hydrogen, halogen, or a C _1-4 alkyl group.

The pyrrole derivative compounds of the present invention can be obtained by a conventional extraction and separation method. First, (1) about 1 to 5 times lower alcohols thereof, for example methanol and ethanol, together with the dried wolfberry are extracted. Ultrasonic extraction or reflux extraction at 30 to 80 ° C. for 1 to 24 hours, concentrated and dried with a thickener to obtain a lower alcohol extract, and the extract was purified by silica gel column chromatography to obtain a polarized fraction. Accordingly, the specific fractions separated can be separated again by reverse phase column chromatography or Sephadex column chromatography to separate pyrrole derivatives of novel chemical structure.

Hepatotoxicity-relieving activity test, liver disease prevention and therapeutic activity test results for the pyrrole derivative compounds obtained through the separation process as described above, the pyrrole derivative compounds are said to resolve the liver toxicity, liver disease prevention and treatment, and liver disease-related enzyme inhibition It was found that the experiment showed significant activity. Therefore, it can be useful for preventing and treating various liver diseases, that is, hepatitis, fatty liver, cirrhosis, as well as hepatotoxicity.

Accordingly, the present invention provides a pharmaceutical composition for treating and preventing liver disease, comprising pyrrole derivative compounds having a novel chemical structure isolated from a wolfberry as an active ingredient and a pharmaceutically acceptable carrier. It also provides a dietary supplement comprising the compounds.

The pyrrole derivative compounds of the present invention may be mixed with a pharmaceutically acceptable carrier as an active ingredient to prepare compositions for the prevention and treatment of various liver diseases, in particular hepatitis and hepatotoxicity. The pharmaceutical composition may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, It may be formulated in unit dosage forms or in multiple dosage pharmaceutical formulations, such as liquid or parenteral formulations.

The pharmaceutical composition containing the pyrrole derivative compounds of the present invention may be parenterally or orally administered according to a desired method, and an amount of 0.01 to 10 g, preferably 1 to 5 g, per 1 kg of body weight as an active ingredient per day It may be administered in several divided doses. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, and severity of disease.

The pyrrole derivative compounds of the present invention may be added to food or beverage for the purpose of preventing various liver diseases. At this time, the amount of the pyrrole derivative compounds in the food or beverage is generally added to 0.1 to 15% by weight, preferably 1 to 10% by weight of the total food weight of the health food composition of the present invention, The amount can be added in the range of 1 to 30 g, preferably 3 to 10 g, based on 100 m1.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the pyrrole derivative compounds as essential ingredients in the indicated ratios, 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 (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of said natural carbohydrate is generally about 1-20g, preferably about 5-12g per 100m1 of the composition of the present invention.

In addition to the above, the composition 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 salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp 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 generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Foods to which the pyrrole derivative compounds of the present invention can be added for development for health foods include various foods, beverages, gums, vitamin complexes, health supplements, and the like.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following Examples are only for illustrating the present invention, and the scope of the present invention is not limited to these.

Reference Example 1 Reagents and Measuring Instruments

Silica gels for column chromatography are Kiesel gel 60 (40-60 μm, 230-400 mesh, product no. 9385, Merck, Germany) and Lychroprep RP-18, 40-63 μm no. 11447, US EM Sciences Sephadex for column chromatography was used Sephadex LH-20 (bead size 25-100 μm, Pharmacia Sweden), XAD-2 resin for column chromatography and XAD-2 resin (Mitsubishi Corporation, Japan). The precoated plates for thin layer chromatography were Kiegel gel 60 F ₂₅₄ (product number 5715, Merck, Germany), RP-18 (product number 15685, Merck, Germany), and celite (Celite 545, Nipponsei Chemical Co., Ltd.). Among the equipment used, the autoclave is manufactured by Nippon Sanyo Co., Ltd. (Sanyo MLS 3000), the analytical balance is manufactured by Swiss METTLER (Mettler AE 50), and the centrifuge is manufactured by DuPont, USA (Technospin R.). ELISA readers are manufactured in the United States (Molecular Devices Emax), Groups Japanese products (ELK la NE), HPLC pump is Hitachi L-6200 product (Japan), Detector HITACHI L-4000 product (Japan), the column is a micro sorbitan probe ^{(Microsorb) R C18 8ψ-299} -C5 Product (Reinin Instruments Inc. USA), the infrared spectrum is Perkin Elmer 1710 spectrometer (USA), the liquid scintillation counter is Wallac 4019 model (United States), Millipore filter is manufactured by the US Millipore company, MS spectrum is VG Trio 2 spectroscopy (UK) and JEOL JMS AX 505 WA spectroscopy (Japan), melting point measuring system Buchi 535 model in Switzerland, NMR spectrum JEOL GSX 400 spectroscopy (400 MHz, USA) and JEOL LA 300 spectroscopy (300 MHz) , USA), and the Burger AMX 500 spectrometer (500 MHz, Germany), pH measurements for Phoenix PMS-500 models (USA), phase contrast microscopes for Olympus CK-2 models (Japan), polarimeters for JASCO DIP-1000 models (Japan ), Sony categorizer is Branson 5200 model (UK), (centrifugal concentrator Bit (Savant) SVC-100H model (USA), UV spectroscopy was used to break Matsumoto (Shimadzu) model UV-2101 (Japan).

Reference Example 2 Preparation of Reagents for Measuring Laboratory Animal and Hepatocellular Protective Activity

Rats (Wista, male, 150-200 g) were fed from Seoul National University experimental animal breeding ground at a temperature of 22 ± 1 ℃ and a humidity of 60 ± 5%, and used to feed and water in a feeding room that changes the contrast every 12 hours. Ingestion was enough. Primary cultured hepatocytes were taken directly from fasted rats from the day before the experiment. L-alanine (A 3534), amphotericin B (A 4888), collagenase type IV (C 5138), Dexamethasone (D 1756), gentamicin sulfate (G 3632), HBSS (H 2387), insulin (I 1881), L-serine (S 5511), trypan blue (T 8154), Waymouth MB 752 / 1 (W1625) used Sigma (St. Louis), urethane (24310-0401) Japan Nippon Seisei Chemical Co., Ltd., fetal bovine serum (Hyclone) (Utah, USA) GPT kits were purchased from Yeongdong Pharm and Silybin (S 0417) from Sigma.

Example 1: 4- [2-formyl-5 (hydroxymethyl) -1 H Separation of -pyrrole-1-yl] butanoic Acid

100 kg of dried wolfberry purchased from Gyeongdong market was extracted with 100 liters of ethanol and ultrasonically extracted 37.5 Kg obtained by ultrasonic extraction at 30 ° C. for 48 hours with 1 liter of water, and then fractionated (fractionated solvent: CHCl ₃ -MeOH = 5). 10 Kg of the non-polar solvent soluble fraction obtained by performing 1, 20 L) was further divided into seven fractions (300 ml each) by C ₁₈ reversed-phase silica gel column chromatography column (elution solvent: methanol, flow rate: 1 ml). 20 g of the third fraction was again subjected to silica gel column chromatography (developing solvent: CHCl ₃ -MeOH = 10: 1-0: 1, column size 4 × 60 cm) to obtain 3 g of the first fraction, which was obtained by separating Sepadex. Perform column chromatography (Sephadex LH-20, eluting solvent: methanol) to collect 20 mg of the fourth fraction obtained by 10 ml aliquots, and repurchase HPLC (Hitachi L-6200, AcCN-H ₂ O = 25: 75, flow rate 2 ml). Per minute) to give the brown powder as a chemical formula (C ₁₀ H ₁₃ O ₄ N ) 2 mg of 4- [formyl-5 (hydroxymethyl) -1 H -pyrrol-1-yl] butanoic acid were obtained.

IR (neat) ν _max ; 3443, 2923, 1715, 1651 cm ^-1

LC-MS ( m / z ); 210 [M-1 ^-]

¹ H-NMR (300 MHz, CD ₃ OD) δ: 9.41, 6.98, 6.26, 4.64, 4.39, 2.31, 2.00.

¹³ C-NMR (100 MHz, CDCl ₃ ) δ: 179.50, 177.30, 141.62, 132.35, 124.50, 110.64, 56.10, 44.54, 29.98, 25.82.

Example 2: 4- [2-formyl-5 (methoxymethyl) -1 H Separation of -pyrrole-1-yl] butanoic Acid

In the separation procedure described in Example 1, 12 g of the fourth fraction obtained from C ₁₈ reversed-phase silica gel was subjected to silica gel column chromatography (developing solvent: CHCl ₃ -MeOH = 10: 1-0: 1, column size 4 × 60 cm). 500 mg of the fourth fraction obtained by fractionation of 100 ml was purified by Sephadex column chromatography (Sephadex LH-20, eluting solvent: methanol), and the second fraction obtained by fractionation of 10 ml was purified by HPLC (developing solvent MeOH-H _2). O = 40: 60, 2 ml / min) Formula (C ₁₁ H ₁₅ O ₄ N) 4- [formyl-5 (methoxymethyl) -1 H -pyrrol-1-yl] butanoic acid 2.2 mg were obtained.

IR (neat) ν _max ; 3438, 2927, 1725, 1659 cm ^-1

LC-MS ( m / z ); 224 [M-1 ^-]

¹ H-NMR (300MHz, CD ₃ OD) δ: 9.44, 6.98, 6.28, 4.49, 4.36, 3.35, 2.29, 2.00

¹³ C-NMR (100 MHz, CD ₃ OD) δ: 181.87, 175.90, 141.91, 134.62, 126.61, 113.59, 67.15, 56.10, 46.79, 33.25, 28.72.

Example 3: 4- [2-formyl-5 (methoxymethyl) -1 H Separation of Pyrrole-1-yl] butanoate

In the separation process described in Example 1, 12 g of the fourth fraction obtained from C ₁₈ reversed-phase silica gel was subjected to silica gel column chromatography (developing solvent: CHCl ₃ -MeOH = 10: 1-0: 1, column size 4 × 60 cm). 600 mg of the fifth fraction obtained by 100 ml aliquots were separated, and Sepadex column chromatography (Sephadex LH-20, eluting solvent: methanol) was performed by fractionation in 10 ml fractions to obtain HPLC (AcCN-H ₂ O = 25). : 75, 2ml / min) separation and purification through the formula (C ₁₂ H ₁₇ O ₄ N brown powder) 4- [formyl-5 (methoxymethyl) -1 H - pyrrole-1-yl] butanoate 1.8 mg were obtained.

IR (neat) ν _max ; 2923, 1737, 1660 cm ^-1

EIMS ( m / z ); 239 [M ⁺ ]

¹ H-NMR (300 MHz, CD ₃ OD) δ: 9.44, 6.98, 6.28, 4.48, 4.36, 3.65, 3.34, 2.35, 2.00.

¹³ C-NMR (100 MHz, CD ₃ OD) δ: 181.94, 175.89, 141.87, 134.67, 126.75, 113.74, 67.12, 59.01, 52.93, 46.60, 32.44, 28.29.

Test Example 1: Effect of Hepatocol Extract Isolated Compounds on the Hepatotoxicity

The hepatotoxicity-relieving effect of the compounds obtained in Examples 1 to 3 was confirmed as follows.

(1) Preparation of experimental material and culture of hepatocyte

Hepatocytes from rats were isolated using a two-step collagenase perfusion method with a slight modification of Barry and Friend's method (see J. Cell. Biol., 43, 506-520 1984) [Kleiman, Anal. Biochem., 94, 308-312 (1979). To obtain hepatocytes, anesthetized with urethane (1 g / kg body weight), the abdomen was disinfected with 70% ethanol and then opened. A 21 gauge catheter was inserted into the portal vein to allow about 50 ml of HBSS solution to perfusion at a rate of 30 ml / min, while at the same time the inferior vena cava was cut under the renal vein to remove blood. After opening the thoracic cavity and inserting an 18 gauge catheter into the relative vein, the inferior vena cava was tied so that the digestive solution was recycled back into the supply vessel through the relative vein. The mixture was recycled for 10-15 minutes with a digestion solution consisting of 95 ml of HBSS and 5 ml of collagenase (50 mg / 5 ml HBSS, 0.05% final concentration) while supplying a mixture of carbon dioxide and oxygen. After the hepatocytes had been digested, the livers were detached, 60 ml of HBSS was added, the liver membranes were opened with scissors, and the liver cells were liberated, followed by filtration with gauze and lens paper. The filtrate was centrifuged at 50 g for 4 minutes and then the supernatant was discarded and again centrifuged under the same conditions as the culture medium to obtain a cell suspension. The hepatocyte suspension thus obtained was diluted to a concentration of 5 × 10 ⁵ cells / ml and transplanted into a culture vessel previously coated with collagen.

As a culture medium, Weymouth MB 752/1 culture [composition: 5% fetal bovine serum, 2.0 mg / ml bovine plasma albumin (fraction V), 10 ^-6 M dexamethasone, 10 ^-7 M insulin, 5.32 x 10 ^-2 M L Serine, 4.09 × 10 ⁻² M L-alanine, 2.67 × 10 ⁻² M sodium bicarbonate, 100 IU / ml penicillin, 100 mg / ml streptomycin, 5 μg / ml amphotericin B or 50 μg / ml gentamicin sulfate ] Was used. Cells were cultured in a 37 ° C. incubator with constant humidity and temperature while continuously supplying a mixture of air (95%) and carbon dioxide (5%).

(2) Elimination of liver toxicity due to carbon tetrachloride

After 1.5 hours of culturing the hepatocytes, the cells were changed to a new culture medium, and further cultured for 24 hours, and then treated with a culture medium containing 6.5 mM carbon tetrachloride for 1.5 hours to induce cytotoxicity. Immediately before administration of carbon tetrachloride, the sample to be searched was administered at each concentration (0.5, 1. 10, 50 μM), incubated for 1.5 hours, and then the culture medium was taken to measure the hepatocellular protective activity. ), Diluted with HBSS, filtered using a Millipore membrane (0.22 μm), and prepared aseptically. The comparative group used silybin, and the control group used no sample. It was.

Cultures of hepatocellularly induced hepatocytes were taken and assayed for the activity of GPT using Reitman-Frankel's method (see Am. J. Cli. Pathol., 28, 56-63 1957) using the GPT kit. The change from the control was determined by the ANOVA test to examine the statistical significance. Statistical significance was determined when the P value was less than 5%.

As shown in Table 1, as a result of detecting the protective activity against hepatocytes of primary cultured rats induced by toxicity of carbon tetrachloride of Compounds 1, 2, and 3, Compound 1 at 0.5 μM to 54.4% and Compound 2 at 10 μM 36.8%, Compound 3 showed hepatoprotective effect of 48.1% at 10μM.

The relative protection rate was calculated from the following equation.

Protective Activity against Hepatic Cells in Primary Cultured Rats Induced Toxicity with Carbon Tetrachloride compound Relative protection rate (%) 0.5 μM 1 μM 10 μM 50 μM One 54.4 49.9 - - 2 n.d. 15.8 36.8 - 3 n.d. 41.7 48.1 39.5 Sillibin - - - 55.5

The compounds of Examples 1 to 3 of the present invention can be used in the form of preparations such as powders, tablets, capsules, injections and solutions according to the methods commonly used pharmaceutically or in combination with excipients used alone or pharmaceutically. have.

Formulation examples are illustrated below.

Preparation Example

[Production of powder]

Example 1 2 g of compound

1g lactose

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

[Production of Tablets]

Example 1 100 mg of compound

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

The tablets were prepared by mixing the above components and tableting according to a conventional method for producing tablets.

[Production of Capsule]

Example 1 100 mg of compound

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After the above ingredients were mixed, the capsules were prepared by filling the gelatin capsules according to a conventional method for preparing gelatin.

[Production of Injection]

Example 1 100 mg of compound

Suitable amount of distilled water for injection

pH adjuster

The injection was prepared by dissolving the active ingredient in distilled water for injection and adjusting the pH to about 7.5 according to the conventional method for preparing an injection, and then filling the whole with a 2 ml ampoule with sterilized water for injection and sterilizing.

In addition, health foods were prepared in the following manner.

[Manufacture of wire]

Brown rice, barley, glutinous rice, and juvenile radish were roasted with alpha by drying in a known manner, and then ground to a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then ground to a powder having a particle size of 60 mesh.

The grains, seeds and compound powders prepared above were blended in the following ratios to form granules.

[Grains: 30% by weight brown rice, 15% by weight barley, 20% by weight of barley,

Seeds: Perilla 7% by weight, Black beans 8% by weight, Black sesame 7% by weight,

Example 1 Compound: 3% by weight, ganoderma lucidum 0.5% by weight, sulfuric acid 0.5% by weight]

The pyrrole derivative compounds of the novel chemical structure of the present invention show excellent hepatotoxicity-reducing effects and are useful as preventive and therapeutic agents for various liver diseases.

1 is an isolation of the active compounds from the wolfberry.

Claims (9)

delete delete delete delete delete delete delete delete 4- [2-formyl-5- (methoxymethyl) -1H-1-yl] butanoic acid or 4- [2-formyl-5 (hydroxymethyl) -1H-pyrrol-1-yl] buta Prevention of hepatitis and liver cirrhosis comprising a compound selected from the group consisting of Noric acid and 4- [2-formyl-5 (hydroxymethyl) -1H-pyrrol-1-yl] butanoate and a pharmaceutically acceptable carrier And therapeutic pharmaceutical compositions.