KR100190449B1 - Compounds having liver cell-protective activities and pharmaceutical compositions containing them as effective ingredients - Google Patents

Abstract

The present invention provides a compound of formula (I) having a hepatocyte protective activity and a pharmaceutical composition containing the compound of formula (I) as an active ingredient.

(Ⅰ)

Description

Compound having hepatoprotective activity and pharmaceutical composition containing the compound as an active ingredient

Figure 1 shows the action of the compounds of formula (I) of the present invention on the activity of GPT and SDH, which are effluxed from primary cultured rat hepatocytes induced by galactosamine,

2 shows the action of the compound of formula (I) on RNA synthesis in primary cultured rat hepatocytes induced by galactosamine,

3 shows the action of the compound of formula (I) on the inhibition of actinomycin D-induced RNA synthesis in galactosamine-induced toxic primary rat rat hepatocytes,

4 shows the action of the compound of formula (I) on the activity of GPT and SDH released from primary cultured rat hepatocytes induced by carbon tetrachloride (CCI4),

FIG. 5 shows the action of the compound of formula (I) on the MDA content in primary cultured rat hepatocytes induced by carbon tetrachloride.

6 shows the action of the compound of formula (I) on the activity of SOD in primary cultured rat hepatocytes induced by carbon tetrachloride;

7 shows the action of the compound of formula (I) on the cytochrome P-450 content in primary cultured rat hepatocytes induced by carbon tetrachloride,

8 shows the action of the compound of formula (I) on the activity of GST in primary cultured rat hepatocytes induced by carbon tetrachloride;

FIG. 9 shows the action of the compound of formula (I) on hydroxyproline in rat hepatocytes induced by long-term culture of carbon tetrachloride.

The present invention relates to a compound of formula (I) having a hepatocyte protective activity and a pharmaceutical composition containing the compound of formula (I) as an active ingredient.

(Ⅰ)

The compound of formula (I) has a chemical name of 1-O- (β-D-glucopyranosyl)-(2S, 3R, 4E, 8z) -2- (N-palmitoyl) -octadeca spinga-4, 8-diene [1-0-glucopyranosyl)-(2S, 3R, 4E, 8Z) -2- (N-palmitoyl) -octadeca sphinga-4,8-dienine].

Countries around the world are speeding up the development of treatments for various liver diseases, including hepatitis and cirrhosis. Hepatic disease is the highest cause of death among males in their 40s. It is understood that hepatic disease is usually caused by acute hepatitis caused by a virus, and a large part of the disease is chronically developed and developed into cirrhosis and liver cancer. The incidence of hepatitis caused by viruses and the spread of hepatitis carriers are increasing worldwide, and it is estimated that more than 4 million hepatitis carriers are in Korea. Therefore, eradication of hepatitis virus is recognized as an important problem not only in medical terms but also socially. However, only vaccines to prevent hepatitis B have been developed so far, but no treatment has been developed except for interferon. Currently, it is not easy to develop a drug that works specifically for the hepatitis virus, and indirect treatments such as reviving damaged hepatocytes, enhancing immune function and liver function of the human body have been attempted.

The development of a soy sauce protector having an excellent effect is urgent and urgent. There are many ways to create new materials for new drug development, such as by chemical synthesis, extraction of active ingredients from natural products, and creation of new materials by biotechnology. There is a growing interest. This is because natural products have been used for a long time as herbal or folk medicine, so a lot of information on medicinal natural products is accumulated.

The first step in the development of a hepatoprotectant from natural products is the establishment of an appropriate screening method to search for substances with hepatoprotective activity. Currently, although the experimental animal has been used for the activity study, when using the conventional animal for the study of the biological activity of a substance isolated from natural resources, if the amount of the substance to be studied is extremely small, more than 90% of the bioactive substance is metabolized in the liver to improve the kidneys. Because it is excreted through, research is almost impossible. Moreover, it is unthinkable to separate the active ingredient while tracking the biological activity. Primary cultured hepatocytes presented as a solution to this problem are known to be biochemically similar to human hepatocytes (Groot, H. and Nool, T., Exp. Mol. Pathol. 100, 25 (1980)). Search for natural products with hepatoprotective activity by using carbon tetrachloride and galactosamine, which are the representative hepatotoxic agents, in livers of primary cultured rats, respectively, as a screening system to search for substances with hepatoprotective activity. Goji berry extract was found to have significant hepatocellular protective activity.

Goji is a mature fruit of the Goji tree belonging to the Solanaceae family, and has already been reported to have blood sugar and cholesterol lowering, blood pressure lowering and hepatoprotective effects (Murav'ev, MA and Vasilenko, Y, K., Farmatsiya 32 (1), 17 (1983)), it is used as a tonic tonic in Chinese medicine and in the private sector. Ingredients of wolfberry are about 30 kinds of volatile components (Sannai, A., Fujimori, T. and), including dehydro-α-cyperone and solvetirone, which are the unique ingredients of wolfberry. Kato, K., Agric. Biol. Chem. 47, 10, 2397 (1983)), various steroid components including zeaxanthin, β-sitosterol, a polyene alcohol, and Betaine has been reported (Nishiyama, R., Nippon Shokuhin Kogyo Gakkaishi 10, 517 (1963)); Itoh, T., Ishii, T. and Tamura, T., Phytochem. 17 (5), 971 (1978); Jeong, T. M. Yang, M. S. and Nah, H. H., Kor. J. Agri. Chem. 21, 1, 51 (1978). However, no specific pharmacological action of certain components of the wolfberry has been reported yet.

Therefore, the present inventors collected the mature fruit from summer to autumn, and then extracted with a solvent mixture of CHCl ₃ and MeOH 3: 1 mixed with the sun-dried goji until the flesh is soft, fractions according to the polarity of the solvent By dividing and searching for hepatoprotective activity of each fraction, it can be seen that ethyl acetate fraction has significant hepatoprotective activity. Thus, the ethyl acetate fraction was subjected to column chromatography to obtain the compound of formula (I), and then the compound of formula (I) was obtained from NMR, IR and mass spectral data, and the cerev having a sphingadiene skeleton. 1-O- (β-D-glucopyranosyl)-(2S, 3R, 4E, 8Z) -2- (N-palmitoyl) -octadecasperga-4,8-die of cerebroside family It was identified as nin (Fujno, Y., Ohnishi, M. and Ito, S., Lipids 20 (6), 337 (1985)), and it was confirmed that this is a new compound that is reported for the first time in nature. 1-O- (β-D-glucopyranosyl)-(2S, 3R, 4E, 8Z) -2- (N-palmitoyl) -octadecasinga-4,8-dieneine was found in primary cultured rats. Glutamic pyruvic transferase (GPT) and sorbitol dehydrogenase (SDH), which are freed into the culture medium, were significantly reduced when carbon tetrachloride induced artificial toxicity. The compound is also used as an indicator of antioxidant activity in hepatocytes of primary cultured rats induced by toxicity with carbon tetrachloride (Eollner, H, in; Handbook of enzyme inhibitors (Weller, MG. Ed) P248, VCH, Veragsgesells chaft Weinheim ( 1989) significantly reduced the activity of superoxide dismutase (SOD) and significantly reduced the synthesis of collagen, which is known to increase in cirrhosis. However, the activity of glutathione-S-transferase (GST), a marker of enzymes involved in liver detoxification (Chasseud, LF, Adv. Cancer.Res. 29. 175 (1979)) and drugs in hepatocytes Metabolic enzymes (Wrighton, SA and Stevens, JC, Crit. Rev. Toxicol. 22 (1), 1 (1992)) had no effect on the amount of cytochrome P-450. In addition, this compound significantly reduced GPT and SDH values released from the culture of hepatocytes of primary cultured rats that caused artificial toxicity with galactosamine, another hepatotoxic substance, and hepatotoxicity induced by galactosamine. Significantly increased the synthesis of RNA in the hepatic cell was found to have a protective activity.

The present inventors have conducted a long research to identify a substance having hepatocellular protective activity from the components of goji. As a result, the substance represented by the above structural formula (I) exists in the components of the ball game field, and the surprising fact that the compound of the structural formula (I) has excellent hepatocyte protective activity was found to complete the present invention.

It is therefore an object of the present invention to provide a novel substance represented by formula (I) having hepatocellular protective activity.

Another object of the present invention is to provide a pharmaceutical composition containing the novel substance represented by the formula (I) as an active ingredient.

Next, the isolation and identification of the compound of formula (I) of the present invention will be described in detail as examples.

(Example 1)

Ingredients-Goji, the fruit of Lycium chinensis Miller, was purchased by Cheongyang Agricultural Cooperative as Cheongyang Mountain, and was used by an honorary professor at Hankuk University of Seoul.

Experimental Animals-Rats (Wistar, male, 150-200 g) were supplied from a laboratory animal breeding center at Seoul National University to maintain an environment of temperature 22 ± 1 ℃ and humidity 60 ± 5%, and change the contrast every 12 hours. The animals were bred to the full intake of water and starved for an empty stomach a day before the experiment.

Instrument-IR spectra were measured by KBr disc method using a Perkin Elmer 1710 spectrophotometer. NMR spectra were measured using a JEOL GSX 400 spectrometer as an internal standard of trimethylsilane (TMS). FAB mass spectra were measured with a VG Ttio-2 spectrometer. Silylation is the same amount of Tris-Sil N, O-bis Trimethylsilyl acetamide (BSA) and sample solution dissolved in dimethylformamide (DMF) and mixed well. Next, the reaction was carried out at 73 ° C. for 1 hour. GC was measured using a GC 353 (GL Science) and a DB-1 column (30mX0.25mm, ID), He (flow rate: 30ml / min) as the mobile gas, column temperature is 240 ℃, injector temperature 250 ℃, detector temperature was carried out at 250 ℃. GC-MS used Shimadzu QP-1000 as an EI mode, using a DB-5 (Lichrosorb ^® ) column, He (flow rate: 30 ml / min) as a moving gas, and the column temperature was 300 at 200 ° C. It was performed with 5 ° C. increase per minute to 1 ° C. After TLC development, the colorants were sprayed with anisealdehyde / H ₂ SO ₄ , and then heated on a hot plate to identify the components. In biochemical studies of the active ingredient, the absorbance was measured by Shimadzu 2101 UV specrophotometer.

Extraction and Separation-40 kg of dried wolfberry was extracted with a mixed solvent of CHCl ₃ and MeOH 3: 1 in an extractor equipped with a reflux condenser and concentrated under reduced pressure to obtain 2.8 kg. This wolfberry extract (2.8 kg) was suspended in water, and then sequentially partitioned into n-hexane and ethyl acetate depending on the polarity to obtain 252.6 g of n-hexane fraction and 199.7 g of ethyl acetate fraction. Silica gel column chromatography was performed on ethyl acetate fractions that showed hepatoprotective activity against hepatocytes of primary cultured rats induced artificial toxicity with carbon tetrachloride and galactosamine. As the outflow solvent, a polar solvent was gradually increased, starting with a 30: 1 mixture of CHCl ₃ and MeOH. Each fraction received 150 fractions, and each fraction was TLC to collect all the similar patterns to obtain 16 (A → O) fractions. As a result of searching the activity of each fraction, it was found that the active substance was contained in the J and K fractions. J, K fractions (28.0 g) were mixed with CHCl ₃ , MeOH and H ₂ O from 100: 4: 1 to 6: 5: 1, and the silica gel column chromatography was performed using the lower layer as an outflow solvent. Fractions (JK-1 → JK-7) were obtained. Among them, JK-4 fraction (0.8 g), which was an active fraction, was chromatographed with a effluent solvent using a mixed solvent of CHCl ₃ and MeOH 2: 1 with Sephadex LH20 to obtain 50 mg of amorphous white powder.

TLC system; Silica gel plates (5715, Merck), solvent conditions; CHCl ₃ : MeOH: H ₂ O (15: 4: 1, lower layer) R _f value; 0.47

Compound (I)-white amorphous power; 1-O- (β-D-glucopyranosyl)-(2S, 3R, 4E, 8Z) -2- (N-palmitoyl) -octadeca spinga-4,8-dienine, C ₄₀ H ₇₅ NO ₈ ; IR (KBr) ν max (cm ⁻¹ ); 3290, 1622 (secondary amide), 3390 (OH), 1000-1100 (glycosidic CO bond); FAB mass (m / z); 698 (M + H) ⁺ , 536 (M + HC ₆ H ₁₀ O ₅ ) ^{+ 1} H-, ¹³ C-NMR (CDCl ₃ : CD ₃ OD = 2: 1, 400 MHz, 100 MHz): See Table 1.

Hydrolysis of compound (I); 3 mg of the compound of formula (I) was dissolved in 2 ml of a solvent mixed with 2N HCl and MeOF in 1: 1 and reacted at 100 ° C. for 1 hour for hydrolysis. The reaction was partitioned with CHCl ₃ , and then the remaining aqueous layer GC was performed under the conditions described above to confirm glucose in comparison with the title product.

Methanolysis of Compound (I) (Brady, RO and Koval, GJ, J. Biol. Chem. 233, 26 (1958)): 5 mg of the compound of Formula (I) was dissolved in 2 ml of 5% HCl-MeOH. The reaction was carried out at 80 ° C. for 10 hours. The reaction mixture was partitioned with n-hexane and concentrated to give n-hexane X, which was subjected to GC / MS under the conditions described above to confirm methyl palmitate.

Acetylation of Compound (I) (Manas, C., Archana, B, and Baruna, AK, J. natl. Prod. 57 (3), 393 (1994)); Structure (Ⅰ) was added to an equal volume of acetic anhydride was dissolved in 5mg compound of 200㎕ pyridine and then diluted with distilled water and then allowed to stand for overnight and 3ml of CHCl ₃ to CHCl ₃ fraction obtained by fractionation of a Sephadex LH 20 column, CH ₂ Chromatography was carried out with a solvent mixed with Cl ₂ and MeOH 3: 1, and pentaacetate of the compound of formula (I) was isolated and subjected to Fab mass spectrometry.

The results are shown in Table 1 below.

TABLE 1 ¹ H-, ¹³ C-NMR chemical shifts and assignments for compound Ⅰ.

*, # 'assigments with the same superscript may be reversed.

Solvent; CDCl ₃ : CD ₃ OD = 2: 1

¹ H-NMR; 400 MHz, ¹³ C-NMR; 100 MHz.

Structure (Ⅰ) long aliphatic chains at 2860, 2920 cm ^-1 from the IR spectrum of the compound (long aliphatic chains), at 3290 and 1622cm ^-1 2 primary amide (secondary amide), a hydroxyl group and 1000-1100 at 3390cm ^-1 The presence of a sugar CO group was confirmed at cm ⁻¹ . From the ¹ H-NMR and ¹³ C-NMR data in Table 1, amide linkages, long aliphatic chains and sugars were identified to determine the compound as a glycolipid family (Kawano, Y. Higuchi). , R., Isobe, R. and Komori, T., Liebigs Ann. Chem. 19 (1988)). From the positive FAB mass data, the peaks of the molecular ions peak m / z 698 and the m / z 536 peaks corresponding to the fructose were identified. In addition, m / z 908 (M ⁺ + H) and non-sugar part m / z 578 (M ⁺ + H-tetraacetyl hexose) corresponding to the molecular ions of pentaacetate produced by acetylation of the compound were observed. ^From the δ 3.8-4.8 peaks on ¹ H-NMR, it was possible to estimate the presence of protons of sugar, and the anomeric signals can be confirmed at δ 4.27 (d, J = 7.8 Hz). The anomeric proton was found to be β-anomer. ^The δ 103.7, 74.1, 77.8, 70.7 and 62.0 peaks on the ¹³ C-NMR spectrum indicate that the sugar is β-glucopyranoside. In addition, by using GC, the sugar obtained by hydrolysis was compared with the title product, and the sugar bound to the compound was confirmed by glucose. In addition, δ 53.8 of ¹³ C-NMR data suggests adjacent carbon next to a nitrogen atom, and δ 176.4 indicates an amide carbonyl group. The presence of two aliphatic chains was estimated by the signal of δ 1.27 on ¹ H-NMR and the signal of two terminal methyls of δ 0.99 (t, J = 6.9 Hz). In addition, it was found that two double bonds exist from signals of ¹³ C-NMR 129.1, 134.1, and 131.1 ppm. From the above spectrum data, it was found that the compound is a cerebside of spinga-4,8-diene type. The double bonds of C-4 and C-5 are also represented by the large vicinal couplings constant of ¹ H-NMR (J _4.5 = 16.0) and the chemical shift δ33 of carbon located next to the double bond. We confirmed that the configuration of is trans. The olefinic protons of C-8 and C-9 are equivalent, and the adjacent carbon atoms are present at δ28.0, 28.3, so the geometry of the C-8 and C-9 double bonds is (cis) was confirmed. This fact indicates that when the arrangement of double defects of C-4 and C-5 is trans, the carbon shift of carbon adjacent to the double bond is shown at δ32-33, and the double bond of C-8 and C-9 In the case of the cis configuration, reference is made to the fact that the carbon shift of the carbon adjacent to the double bond occurs at δ 27-28 (Stothers, JB, Carbon-13 NMR spectroscopy Academic press. Cerebrosides usually present in mature seeds are 4-trans, 8-trans-sphingadiene (4-trans, 8-trans-sphingadiene) 7-8 times more than 4-trans, 8-cis-sphingadiene And 4 times more dihydroxy base than trihydroxy base (Ohnishi, M and Fujino, Y., Agric. Biol, Chem. 45 (5), 1283, ( 1981). On the other hand, ceramides are mainly present in the form of trihydroxy bases, and the geometry has been reported to be relatively less likely to exist as four- or eight-transforms (Masao, O. and Fujino, Y., Lipids 17). (1), 803 (1982)). CH ₂ adjacent to the acyl group had ¹³ C-NMR shift at δ 36.0 and ¹ H-NMR shift at δ 2.15, respectively, and were found to be between 25.1, 29.7, 29.8-30.3 ppm in ¹³ C-NMR data. As an existing signal, it could be assumed that it is a straight acyl chain without side chains. The relative stereochemistry of C ₂ / C ₃ is based on the 2S, 3R-forms, which are biosynthesized by chemical shifts of C-2 (δ53.8) and C-7 (δ72.5) from ¹ C-NMR data. Almost identical to the D-erythro configuration, N-octadecanoul-D-erythro-sphingosine (δ54.7, δ73.1) Since most sphingosines in nature exist in the D-form, they could be assumed to be D-sphingadiene (Sarimentos, F., Schwarzmann, G. and Sandhoff, K., Eur J. Biochem. 146, 59 (1985)). In addition, it was confirmed by GC / MS that the fatty acid obtained by methanolysis of this compound was icosanoate.

Based on the above spectral data and compared with the spectral data in the literature, the compound was identified as 1-O- (β-D-glucopyranosyl)-(2S, 3R, 4E, 8Z) -2- (N-palmitoyl ) -Octateca spinga-4,8-dienine was identified and it was confirmed that this is a new compound that is reported for the first time in nature.

The cerebrosandroids are found in the brain (Cuzner, ML and Davison, AN, Biochem. J. 106, 29 (1968)), kidney (Makita, A. and Yamakawa, T., J. Biochem (Tokyo), 51, 124 ( 1962), milk (Morrison, WR and Hay, JD, Biochim, Biophys. Acta 212, 460 (1970)). Oyster (Hayashi, A. and Matsubara, T., Biochim, Biophy. Acta 248, 306 (1971)). Sea cucumbers (Igaki, M., Togawa, K. and Higuchi, R., Liebigs Ann.chem, 653 (1994)), several plants (Carter, HE, Hedry, RA, Nojima, S., Stanacev, NZ and Ohno, KJ Biol. Chem. 236, 1912 (1961); Rouser, G., Kritchevsky, G., Simon, G. and Nelson, GJ, Lipids 2, 379 (1967)), and yeast (Minek, S., IIda, M. and Tsutsum, t., J. Ferment.Bioeng. 78 (4), 327 (1994)). Cerebrosides are composed of various sugars, fatty acids and sphingosine, and since their derivatives are many, it is known that their pure separation is very difficult. Since serebrosides are associated with lipidoses such as Gaucher's disease, biochemical studies (Inoue, K. and Nojima, S., Proteins, Nuscleic acid and Enzyme 16, 565 (1971)) Although much has been done, not much pharmacological research has been conducted. Soya-cerebroside I and II were isolated from the seeds of Glycine max Merril (Leguminosae) (Shibuya, H. and Kawashima, k., Chem. Pharm. Bull. 38) 11), 2933 (1990)). They are reported to have Ca ²⁺ ionophoretic activity. In order to exhibit this activity, the double bond of the sphingosine base and the sugar portion are necessary, and the non-sugar portion of ceramide is said to be involved in sustaining the action. Cerebrosides isolated from Bomkyoo, Alfalta, soybean, rice bran and oat bran are known to exhibit inhibitory effects on stressful gastric ulcers (Okuyama, E. and Yamazaki, M., Chem. Pharm Bull.31 (7), 2209 (1983)), this action is characterized by the presence of heterogeneous sugars, such as gluco-galacto-serebrosides, or when hydroxyl groups Although the activity is reported to be strong, the relationship between the structure of cerebroside and antiulcer activity has not been thoroughly studied. 1-O- (β-D-glucopyranosyl) -2-N-2'-hydroxypalmitoyl-sphinga-4,8-diene (1-O- (β-D-glucopyranosyl) -2- Intraperitoneal injections of N-2'-hydroxypalmitoyl-sphinga-4,8-diene have been reported to delay the onset of progression caused by tromorine (Boiron-Sargueil, F., Heape, A., Garbay, B. and Cassagne, C., Nerrosce. Lett. 186, 21 (1995)). There are many studies on glycosyl sphingolipids in animal tissues, but in fungi, only a few species, such as Aspergillus oryzae, have been identified. Thoma, B. and Richard, R., Liebigs Ann. Chem. 669 (1988)). Deformin, a new cerebroside compound isolated from the yeast Candida deformans, has been reported to induce fruiting of fungi (Zhao, H., Zhao, S. and Guilaume, D., J. Natl. Prok. 57 (1), 138 (1994)). However, there have been no published studies on the hepatoprotective effects of serebrosides.

Hereinafter, the hepatoprotective action of the compound of formula (I) of the present invention will be described in detail as an experimental example.

Experimental Example

Culture of hepatocytes (Berry, MN and Friend, DS, J. Cell. Biol. 43, 506 (1969); Berry, MN, Edward, AM and Barritt, GJ, Laboratory technique in biochemistry and molecular biology., In Burdon, RH , Knippenberg, P. H, (Eds.), Vol. 21, Elsevier, Amsterdam, New York, p. 15 (1991); Kleiman, HK and Hartin, SR, Anal.Biochem. 94, 308 (1979))- Hepatocytes were isolated from liver of rats by a two-stage collagenase perfusion method with slightly modified methods of Berry and Friend. The isolated hepatocytes were transplanted into a culture vessel pre-coated with collagen at a concentration of 5 × 10 ⁵ cells / ml and cultured while supplying mixed air (95% air / 5% CO ₂ ) in an incubator maintained at a constant humidity and temperature. . Cultures were Waymouth's MB 752/1 medium, 5% fetal bovine serum, 2.0 mg / ml bovine serum albumin (fraction V), 10 ^-6 M dexamethasone, 10 ^-7 M Insulin, 5.32 × 10 ^-2 M L-serine, 4.09 × 10 ^-2 M L-alanine, 2.67 × 10 ^-2 M NaHCO ₃ , 100 IU / ml penicillin, 100 IU / One consisting of ml streptomycin, 5 μg / ml amphotericin B and 50 μg / ml gentamycin sulfate was used.

Induction of hepatotoxicity by carbon tetrachloride (Kiso, Y., Tohkin, M. and Hikino, H., Planta Med. 49, 222 (1983))-Hepatocytes were cultured for 24 hours and treated with 10 mM CCl ₄ for 1.5 hours. Caused toxicity.

Induction of hepatocellular toxicity by galactosamine (Hoppe, A. and Seyler, Z., Physiol. Chem. Biol. 351, 101 (1970))-After incubating the hepatocytes for 1.5 hours and changing to a medium containing 1.5 mM galactosamine Treatment for 14 hours resulted in cytotoxicity.

Determination of glutamic pyruvic transaminase (GPT) activity (Reitman, S. and Frankel, S., Am. J. Cli. Pathol. 28, 56 (1957))-by taking a culture of hepatocellularly induced hepatocytes The activity of glutamic pyruvic transferase was measured using the method of Reitman-Frankel.

Determination of sorbitol dehydrogenase (SDH) activity (Gerlach, U. Sorbital dehydrogenase in Methods of Enzymatic Analysis.HU Berhmeyer, Ed, p.761 (1965)). The activity of sorbitol dehydrogenase was measured by a slightly modified method of Gerlach).

Measurement of lipid peroxide (Asakawa, T. and Matsushita, S., Lipids 14 (4), 401 (1980))-The degree of lipid peroxidation was determined by 1,1,3,3-tetraethoxypropane (1,1,3, 3-tetraethoxypropane) was used as a standard and measured by Masuage method.

Determination of Superoxide Dismutase Activity (Mccord, JM and Frivich, J., J. Biol. Chem. 244, 6049 (1969))-The activity of superoxide dismutase in hepatocytes is McCord. It was measured by the method of and Pridivich (Fridivich).

Glutathione-S-transferase activity assay (Habig, WH, Pabst, MJ and Jakoby, WB, J. Biol. Chem. 249, 7130 (1974))-Glutathione-S-transferase in hepatocytes The activity of was measured using a method such as Habig (Habig).

Measurement of cytochrome P-450 content (Omura, R. and Sato R., J. Biol. Chem. 239, 2370 (1964))-The cytochrome P-450 content in hepatocytes was measured by the method of Omura et al. It was.

Determination of 4-Hydroxyproline content (Switzer, BR, J. Nutr. Biochem. 2, 229 (1991))-4-Hydroxyproline content in liver cells cultured for 21 days was well (Jall It measured by the method, such as).

^[3 H] - uridine combination (incorporation) Measurement (Karner, A., Biochemical J. 92 , 449 (1964).) Of ^{([3 H] -uridine) -} RNA biosynthesis is ^[3 H] - uridine Was added to the culture medium and the degree of binding of the radioisotope-labeled uridine to the RNA of the hepatocytes was measured by Karner et al.

Protein Determination (Lowry, OH, Rosebrough, NJ, Farr, AL and Randall, RJ, J. Biol. Chem. 193, 265 (1951))-The amount of protein is standardized by bovine serum albumin. ), And the like.

Statistical Treatment—Variation from the control was determined by ANOVA test to examine statistical significance. Statistical significance was determined when the P value was less than 5%.

The results of the above experiments are shown in FIGS. 1 to 9 as follows, and the corresponding experiments and evaluation methods are described together.

1 shows the action of the compounds of formula (I) of the present invention on the activity of GPT and SDH effluxed from primary cultured rat hepatocytes induced by galactosamine. After an hour and a half of plating, the isolated rat hepatocytes were exposed to a medium containing 1.5 mM galactosamine (1.5 ml). Some cultures were used as controls without treatment. Galactosamine attack (challenge) after 14 hours to remove the culture medium and, Intralipos ^ⓡ

To the fresh medium containing the compound of formula (I) (final concentration of 1%) was added and further incubated for 19 hours. The culture medium was removed, fresh medium was added, and further cultured for 5 hours. GPT and SDH activity in the medium was saved. n = 3.

Control ( ³ Control) is the value for hepatocyte cultures not attacked by galactosamine.

Controls for GPT and SDH were 5.0 ± 0.1 IU / L and 0.5 ± 0.013 Unit / ml, respectively. ^B Reference is the value for untreated hepatocytes attacked by galactosamine.

Baseline values for GPT and SDH were 35.0 ± 1.2 IU / L and 29.8 ± 0.2 Unit / ml, respectively.

^C protection is

× 100

Calculated as

Significantly different from control, effective p 0.05 ^* , p 0.01 ^** .

Figure 2 shows the action of the compound of formula (I) on RNA synthesis in primary cultured rat hepatocytes induced by galactosamine. After an hour and a half plate culture, the isolated rat hepatocytes were exposed to a medium containing 1.5 mM galactosamine (1.5 ml). Some cultures were used as controls without treatment. After galactosamine attack 14 hours to remove the culture medium and, ^[3 H] in Intralipos ^ⓡ - uridine containing a sample compound of (2μCi / ml of medium) + formula (Ⅰ) (final concentration, 1%), fresh medium Was added and incubated for 24 hours. Incorporation of [ ³ H] -uridine in the cells was quantified. n = 3.

Control ( ³ Control) is for hepatocyte cultures not challenged with galactosamine. The control value of [ ³ H] -uridine binding in the cells averaged 2217 ± 47 cpm / 7.5 × 10 ⁵ cells.

Significantly different from control, effective p 0.05 ^* , p 0.01 ^** .

3 shows the action of the compound of formula (I) on the inhibition of actinomycin D-induced RNA synthesis in primary cultured rat hepatocytes induced by galactosamine.

The level of nomenclature and significance is as described in the description of FIG.

The control is the value for hepatocyte cultures not attacked with galactosamine.

In the cell ^[3 H] - control values for uridine coupling ⁽³ Control value) was the average of 1685 ± 134 cpm / 7.5 × 10 5 cells.

The average ^b control value for [ ³ H] -uridine binding in cells treated with actinomycin D (10 μg / ml) was 176 ± 9 cpm / 7.5 × 10 ⁵ cells.

Baseline values are for untreated hepatocytes challenged with galactosamine.

The ^c Reference value for [ ³ H] -uridine binding in the cells averaged 639 ± 31 cpm / 7.5 × 10 ⁵ cells.

In cells treated with actinomycin D (10 μg / ml), the ^d reference value for [ ³ H] -uridine binding averaged 132 ± 21 cpm / 7.5 × 10 ⁵ cells.

Significantly different from control, effective p 0.05 ^* , p 0.001 ^*** .

4 shows the action of the compound of formula (I) on the activity of GPT and SDH released from primary cultured rat hepatocytes induced by toxicity by carbon tetrachloride (CCl ₄ ). Intralipos ^ⓡ rat hepatocytes isolated after 1 day of plate culture

Was exposed to medium (3.0 ml) containing 10 mM CCl ₄ / ethanol (0.03 ml) or 10 mM CCl ₄ / ethanol + Structural Formula (I) sample compound (final temperature, 1%). Some cultures were used as controls without treatment. After 90 minutes of CCl ₄ challenge, the culture medium was removed, fresh medium was added and incubated for another 5 hours. GPT and SDH activity of the medium was quantified. n = 3.

Control ( ³ Control) is the value for hepatocytes not attacked with CCl ₄ . Controls for GPT and SDH were 10.8 ± 0.5 IU / L and 2.8 ± 0.1 Unit / ml, respectively.

^B Reference is the value for untreated hepatocytes challenged with CCl ₄ . Baseline values for GPT and SDH were 50.8 ± 9.2 IU / L and 48.8 ± 0.2 Unit / ml, respectively.

^C protection is

× 100

Calculated as

Significantly different from control, effective p 0.05 ^* , p 0.01 ^** , p 0.001 ^*** .

5 shows the action of the compound of formula (I) on the MDA content in primary cultured rat hepatocytes induced by carbon tetrachloride.

MDA content was quantified in the cells. n = 3.

Experimental process, nomenclature and significance level are as described in the description of FIG.

The control value for the content of MDA ^(a Control value) was a mean 80.6 ± 5.5 pM / mg protein.

The ^b Reference value for MDA content was 114.5 ± 6.6 pM / mg protein on average.

Significantly different from control, effective p 0.05 ^* .

Figure 6 shows the action of the compound of formula (I) on the activity of SOD in primary cultured rat hepatocytes induced by carbon tetrachloride.

The activity of SOD was quantified in the cells. n = 3.

Experimental process, nomenclature and significance level are as described in the description in FIG.

The control value for the SOD activity ^(a value Control) was the average of 2.03 ± 0.11 unit / mg protein.

The ^b Reference value for SOD activity averaged 8.69 ± 0.04 unit / mg protein.

Significantly different from control, effective p 0.05 ^* , p 0.01 ^** , p 0.001 ^*** .

FIG. 7 shows the action of the compound of formula (I) on cytochrome P-450 content in primary cultured rat hepatocytes induced by carbon tetrachloride. Cytochrome P-450 content was quantified in the cells. n = 3.

Experimental process, nomenclature and significance level are as described in the description of FIG.

The control value for the cytochrome P-450 content ^(a value Control) was the average of 1969.7 ± 40.4 pM / mg protein.

The ^b reference value for the cytochrome P-450 content was an average of 344.2 ± 28.4 pM / mg protein.

8 quantifies the activity of GST in primary cultured rat hepatocytes induced by carbon tetrachloride. n = 3.

Experimental procedures, nomenclature and significance levels are as described in FIG.

The control value for the SGT activity ^(a value Control) was the average of 19.3 ± 3.9 nM / min / mg protein.

The ^b reference value for GST activity averaged 5.3 ± 0.3 nM / min / mg protein.

9 shows the action of the compound of formula (I) on hydroxyproline in rat hepatocytes induced by toxicity of carbon tetrachloride cultured for a long period of time. Medium was changed daily and preincubated for 21 days, then primary rat hepatocytes were exposed to CCl ₄ with or without compounds, samples as described in the description of FIG. After 90 minutes of CCl ₄ challenge, the culture medium was replaced and the cultures were incubated for another 5 hours. The hydroxyproline content was then quantified in the cells. n = 3.

Naming and significance levels are as described in the description of FIG.

Control values for hydroxyproline content ^(a value Control) was the average of 0.228 ± 0.01 ㎍ / mg protein.

The ^b Reference value for hydroxyproline content was 0.867 ± 0.02 μg / mg protein on average.

Significantly different from control, effective p 0.05 ^** , p 0.01 ^** .

Consider the above results.

Hepatoprotective activity of the compound of formula (I) is determined by inducing toxicity with carbon tetrachloride and galactosamine in primary cultured rat hepatocytes or by measuring the extent to which the compound of formula (I) blocks or recovers. saw.

Galactosamine causes toxicity by inhibiting protein and RNA synthesis in hepatocytes. Once galactosamine is toxic to hepatocytes, galactosamine is known to resemble the function and morphology of hepatocytes when infected with a virus. The activity of the compound of formula (I) on GPT and SDH efflux and RNA synthesis in hepatocytes induced by galactosamine was investigated. When hepatocytes are toxic by galactosamine, GPT and SDH in hepatocytes are leaked into the culture medium, which significantly increases the GPT and SDH values in the culture medium. However, inducing hepatotoxicity with galactosamine and then acting the compound of formula (I) from 1μM to 10μM significantly reduced the GPT value due to galactosamine toxicity. That is, the compounds of formula (I) recovered hepatotoxicity up to 38.2%, 59.3% and 79.2% at steady state, respectively, at 1, 5 and 10 μM concentrations (FIG. 1). In addition, the compound of formula (I) also significantly reduced the SDH value in the excretion solution due to galactosamine. That is, the compounds of formula (I) restored hepatotoxicity up to 42.5%, 60.4% and 69.9% at steady state at 1, 5 and 10 μM concentrations, respectively (FIG. 1). As a result, it was found that the compound of formula (I) showed significant hepatoprotective activity by decreasing GPT and SDH values in culture medium increased by galactosamine in a concentration-dependent manner.

When the compound of formula (I) acted on hepatocytes induced by galactosamine, the RNA synthesis in hepatocytes was significantly increased (FIG. 2). Thus, the compound of formula (I) is known to inhibit RNA synthesis ( Benzamin, YMT, Amy, MSB and Pui, KC, Cancer research 50, 5987 (1990)) In order to determine whether RNA promotes synthesizing by blocking the action of actinomycin D, the compound of formula (I) and actino Mycin D was administered simultaneously. Actinomycin D has a very high affinity for the Gpc region of DNA and is inserted into this site and inhibits RNA synthesis by inhibiting transcription of γRNA genes in mammals in particular (Perry, RP, Exp. Cell Res. 29, 400 (1963)). It was found that the compound of formula (I) did not decrease RNA synthesis by blocking the action of actinomycin D (FIG. 3). From these results, it can be inferred that the compound of formula (I) shows hepatocellular protective activity by increasing the synthesis of RNA reduced by galactosamine by increasing the transcription of the γRNA gene.

Carbon tetrachloride is metabolized to CCl ₃ and CClCC by cytochrome P-450 dependent mixed function oxidase, a hepatic metabolic enzyme. These highly reactive free radicals bind to lipids and proteins in cells, leading to peroxidation of lipids, altering the endoplasmic reticulum morphology, reducing protein synthesis, and triglycerides synthesized in the liver. It is known to cause fatty liver by inhibiting the outflow of trihlyceride (Azri, S., Mata, HP, Reid, LL, Gandlofi, AJ and Brendel, K., Toxicol. Appl. Pharmacol. 112, 81 (1992)). In order to examine how the compounds of formula (I) act on hepatotoxicity induced by carbon tetrachloride, the activities of GPT and SDH, which are primarily detected, were measured. When hepatocytes were toxic by carbon tetrachloride, GPT and SDH were released into the culture, which significantly increased the GPT and SDH values in the culture. However, it was found that increasing the compound of formula (I) from 1 μM to 25 μM significantly reduced the GPT value released into the culture as a result of treating carbon tetrachloride toxicity. That is, the compounds of formula (I) blocked the toxicity by carbon tetrachloride at 1, 5, 10 and 25 μM to maintain 49%, 60%, 77% and 96% of steady state, respectively (FIG. 4). In addition, even when the SDH value was measured, the treatment of the compound of formula (I) from 1 μM to 25 μM decreased the SDH value in the culture medium increased by toxicity by carbon tetrachloride, resulting in 38% of the steady state at 1, 5, 10 and 25 μM, respectively. It can be seen that it is maintained at 57%, 66% and 80% (FIG. 4). From these results, it can be seen that when 25 μM of the compound of formula (I) has an excellent hepatocellular protective activity of blocking toxicity to a nearly steady state level.

Production of malondialdehyde (MDA) using thiobarbituric acid (TBA) method to investigate the effect of the compound of formula (I) on lipid peroxidation induced by carbon tetrachloride Was measured. In the intestinal state, MDA production was 80.599 + 5.50 pM / mg protein, but MDA production increased to 114.499 + 6.60 pM / mg protein when poisoned by carbon tetrachloride. However, treatment of compounds of formula (I) with different concentrations from 1 μM to 10 μM was found to significantly reduce the amount of MDA produced due to toxicity by carbon tetrachloride. That is, the compounds of formula (I) block carbon tetrachloride toxicity at 1, 5, and 10 μM, decreasing to 105.74 + .23 pM / mg protein, 89.53 + 4.3 pM / mg protein, and 83.43 + 6.2 pM / mg protein, respectively. I was. From these results, it was found that the compound of formula (I) inhibits lipid peroxidation by decreasing carbon dioxide tetrachloride increased MDA production in a concentration-dependent manner. It can be seen that it is maintained (figure 5). This is due to the oxidative damage recently found in cyclic relapsing experimental allergic encephalomyelitis of glycerophospho lipids and sphingolipids present in myelin sheath. It was confirmed that it is associated with the report that inhibits increased TBA formation. Thus, the effect of the compound of formula (I) on the activity of SOD used as a method of searching for homeostasis was measured. SOD activity was 2.03 + 0.12 unit / mg protein in normal liver cells, but increased to 8.69 + 0.04 unit / mg protein when poisoned with carbon tetrachloride. However, it was found that treatment of compounds of formula (I) with different concentrations from 1 μM to 10 μM significantly reduced SOD activity due to carbon tetrachloride toxicity. That is, the compounds of formula (I) block the toxicity of carbon tetrachloride at 1, 5, and 10 μM, resulting in levels of 7.67 + 0.32 unit / mg protein, 3.75 + 0.05 unit / mg protein, and 2.25 + 0.13 unit / mg protein, respectively. Reduced. From these results, it can be seen that the compound of formula (I) reduced the SOD activity increased by carbon tetrachloride in a concentration-dependent manner, thereby almost blocking the toxicity at a concentration of 10 μM or more, thereby maintaining the level at a steady state (6). Degree).

Mixed function oxidation involving cytochrome P-450 is the most widely known enzymatic reaction among drug metabolism reactions. When the terminal oxidase component of the electron transport system present in the microsome complexes or reduces with carbon monoxide, the maximum absorbance value is obtained at 450 nM. In hepatocytes toxic by carbon tetrachloride, the amount of cytochrome P-450 was reduced to about 1/6 of the normal hepatocytes (1969.7 + 40.44 pM / mg protein) (344.24 + 28.41 pM / mg protein). In order to investigate how the compound of formula (I) acts on the mixed function oxidation involving cytochrome P-450, the concentration was increased from 1 μM to 10 μM. However, the compound of formula (I) has little effect on the mixed function oxidation in which cytochrome P-450 is involved, and this compound is a metabolic system of cytochrome P-450 lowered by carbon tetrachloride. It could be assumed that it does not show hepatoprotective activity by activating (Fig. 7).

Glutathione-S-transferase is a protein present in the cytoplasm of the liver and is an enzyme that plays an important role in metabolism and excretion of foreign substances in vivo. That is, glutathione-S-transferase is an enzyme that metabolizes and detoxifies toxic substances in the body, and an increase in activity of glutathione-S-transferase indicates an increase in detoxification in vivo. Therefore, it can be said that a substance that increases the activity of glutathione-S-transferase has a hepatoprotective effect. Toxicity induced by carbon tetrachloride is known to reduce the activity of glutathione-S-transferase in hepatocytes. Carbon tetrachloride induced toxicities in glutathione-S-transferase activity in hepatocytes by about a quarter (5.331 + 0.303 nM / min / mg protein) compared to normal (19.328 + 3.880 nM / min / mg protein). . In order to examine how the activity of the compound of structural formula (I) was examined, the concentration was increased from 1 μM to 10 μM. However, the compound of formula (I) has little effect on the activity of glutathione-S-transferase, which increases the activity of glutathione-S-transferase lowered by carbon tetrachloride to increase hepatocyte protective activity. It could be assumed that it does not represent (Fig. 8).

If the liver is injured or inflamed by a toxic substance, leading to hardening of the liver tissue, functional and structural changes will occur in the tissue (Hendriks, HFJ, Brouwer, A. and Leeuw, AM, Exp. Cell Res. 160, 138 (1985)). That is, a characteristic change occurs in a specific matrix in the epithelial tissue of the liver containing collagen. Therefore, hydroxyproline, which is contained in a large amount in collagen, has an important meaning as an index for measuring the presence, amount, and metabolism of collagen. Hepatocytes are known to increase collagen synthesis by increasing the amount of hydroxyproline under certain culture conditions. In other words, hepatocytes directly isolated from liver of rats contained fresh vitamin C while maintaining the insulin concentration at 10 ^-6 M. The composition of Waymouth'S MB752 / 1 medium was changed and cultured for 2 weeks or more. In vitro models are developed to study. In order to determine how the compound of formula (I) affects cirrhosis in liver cells cultured for a long time, the concentration of 4-hydroxyproline was significantly reduced at 10 μM when the concentration of 1 μM to 10 μM was treated. Furthermore, when toxic to hepatocytes cultured under these conditions with carbon tetrachloride, collagen synthesis is further increased, so that the inhibitory effect of collagen synthesis can be judged more clearly. Induced toxicity of carbon tetrachloride in hepatocytes was increased approximately four-fold (0.0867 + 0.0016 μg / mg protein) compared to (0.0228 + 0.0013 μg / mg protein) when cultured in steady state. To determine how compounds of formula (I) act on the amount of 4-hydroxyproline, block the toxicity by carbon tetrachloride at 1, 5, and 10 μM to reduce the amount of 4-hydroxyproline, respectively, at 0.0535 + 0.006 μg / mg. protein, 0.0512 + 0.0021 μg / mg protein, and 0.0391 + 0.0050 μg / mg protein (FIG. 9). These results suggest that the compound of formula (I) has a significant effect of inhibiting liver hardening by reducing the amount of 4-hydroxyproline increased by carbon tetrachloride in a concentration-dependent manner.

From the above results, the compound of formula (I) is mainly involved in the early stages of antioxidant activity to activate the physiological action of hepatocytes inhibited by carbon tetrachloride, promote RNA synthesis in hepatocytes, and inhibit the synthesis of hydroxyproline. Hepatoprotective activity can be a basic clue to the treatment of cirrhosis. In recent years, the biosynthesis of extracellular matrix of liver is known to be more active in fat storing cells than in hepatocytes. Therefore, in order to elucidate the effect on the collagen biosynthesis of the compound of formula (I), the study on the effect on the synthesis of 4-hydroxyproline in adipose storage cells should be carried out.

Experimental Example 2: Acute Toxicity Test

1. Oral administration

ICR-based mice (25 ± 5g) were divided into 5 groups of 10 rats and observed for toxicity for 2 weeks after oral administration of the compounds of formula (I) of the present invention at doses of 1, 5, 25, 125, and 625 mg / kg, respectively. As a result, there was no death in the 5th group.

2. Intraperitoneal administration

ICR-based mice (25 ± 5g) were divided into four groups of 10 animals, and the compounds of the structural formula (I) of the present invention were intraperitoneally administered at doses of 0.2, 2, 20, and 200mh / kg, respectively, and observed for toxicity for 24 hours. Results There were no deaths in all four groups.

From the above results, it was confirmed that the compound of formula (I) of the present invention has almost no acute toxicity.

Therefore, the compounds of the present invention can be used as a prophylactic and therapeutic agent having a useful hepatoprotective function.

The compound of formula (I) of the present invention is formulated into a pharmaceutical preparation which can be mixed in a dosage of 1.0 to 100 mg per day with excipients or adjuvants which are commonly used pharmaceutically, and can be administered orally or parenterally by conventional pharmaceutical methods. It can be used as medicine or food.

Next, the preparation example of the preparation of this invention is illustrated as a preparation example.

Formulation Example 1 Preparation of Tablet

Structural Formula (I) Compound 10mg

Lactose 20mg

Starch 20mg

Magnesium stearate proper amount

The above ingredients are compressed into 50 mg tablets according to a conventional method for producing tablets.

Formulation Example 2: Preparation of Capsule

10 mg of compound of formula (I)

Lactose 20mg

Starch 19mg

Talc 1mg

Magnesium stearate proper amount

The above-mentioned ingredients are filled in 50 mg of gelatin capsules according to a conventional method for producing a capsule to prepare a capsule.

Formulation Example 3: Preparation of Injection

Structural Formula (I) Compound 2mg

Melting aid amount

Appropriate sterile distilled water for injection

According to the conventional method for preparing an injection, the total volume of the above component is 2 ml, the ampoule is filled in 2 ml, sealed, and sterilized to prepare an injection.

Preparation Example 4 Preparation of Syrup

100 mg of compound of formula (I)

5 g of sugar

5 g of sugar

Purified water

50 ml total

The above ingredients were added to purified water, stirred well, and then filled into 50 ml brown glass bottles and sterilized to prepare a syrup.

Formulation Example 5 Preparation of Ointment

1.0 g of compound of formula (I)

Diethanolamine 1.5 g

Carboxy vinyl polymer 1.5g

5.0 g of hydroxyethyl cellulose

Ethanol 20.0 g

5.0 g of polyvinylpyrrolidone

Propylene Glycol 30.0g

The ointment is prepared according to the above-described method for producing a conventional ointment.

Formulation Example 6 Preparation of Cream

1.0 g of compound of formula (I)

Isopropyl myristate / isopropyl palmitate 5.0g

/ Isopropyl Sterate Mixture

4.0 g of palmitic acid and mono- and di-glyceride mixtures

0.5 g of high molecular weight crosslinked polyacrylic acid

Cetyl Palmitate 4.0g

0.11 g of 45% (intensity) sodium hydroxide

Glycerin 3.0g

Benzyl Alcohol 3.0g

100 ml whole with decontamination

A cream agent is manufactured according to the above-mentioned method of manufacturing a cream agent.

Claims (2)

A compound of formula (I) (Ⅰ) A pharmaceutical formulation wherein the compound of formula (I) is formulated into a conventional pharmaceutical formulation according to a conventionally used pharmaceutical preparation together with conventionally used excipients and auxiliaries. (Ⅰ)