KR100283297B1 - Pharmaceutical preparations for liver protection which contain sauchinone - Google Patents

Abstract

The present invention discloses a pharmaceutical agent having a hepatocyte protective activity containing a compound selected from the saucinone compounds of the following Chemical Formulas 1, 2, and 3 or an extract of 300 seconds containing them as an active ingredient as an active ingredient.

Formula 1 (Sauchinone) Formula 2 (Sauchinone A) Formula 3 (Sauchinone B)

Description

Pharmaceutical preparations for liver protection which contain sauchinone}

The present invention relates to a pharmaceutical agent having a hepatocellular protective activity containing a compound selected from saucchinone compounds of the following Chemical Formulas 1, 2 and 3, or an extract of 300 sec containing as a main ingredient thereof as an active ingredient.

Formula 1 (Sauchinone) Formula 2 (Sauchinone A) Formula 3 (Sauchinone B)

Hepatic disease is usually caused by acute hepatitis caused by a virus or fatty liver due to continuous drinking of alcohol, and then progresses to chronic hepatitis and then progresses to liver cirrhosis and liver cancer. The number of patients with liver disease in our country has been increasing rapidly since the late 70's. In 1996, the rate of morbidity was 944 people per 100,000 population, and about 9% of deaths that could be classified as death are caused by liver disease. . Particularly noteworthy is that liver disease accounts for 26% of the causes of death among men in their 40s, with the highest mortality rate (Statistics: Statistical Yearbook of Cause of Death, 1996) (National Statistical Office, Seoul, pp 20-40 (1997)). The increase in patients with liver disease is not only a problem in Korea but a global trend, and all over the world are devoted to combating it. However, the development of treatment for liver disease has not yet progressed except for interferon, and DDB (dimethyl-4), a synthetic derivative of silymarin (silymarin) isolated from Maria thistle and lignan components isolated from Schisandra chinensis as a therapeutic aid for chronic hepatitis. , 4'-dimethoxy-5,6,5'6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate) is only used. Therefore, the development of new medicines to treat liver disease is urgent and urgent.

Therefore, in the present invention, a candidate substance that can be developed as a hepatic disease treatment agent has been used for the treatment of hepatic disease in the form of herbal medicine or folk medicine for a long time, or to present separately from natural products that are described as a medicine for treating hepatic disease in medicine. It was. That is, in the present invention, the use of primary cultured hepatocytes induced by toxicity with carbon tetrachloride or galactosamine centered on natural products used for the purpose of treating and preventing tonic or hepatic diseases in oriental medicine or folk medicine as a screening system. As a result, it was confirmed that the total methanol extract of ground and underground of Saururus chinensis Baill. Showed significant hepatoprotective activity. From this, we traced the active component of hepatocellular protective activity and clarified its physicochemical structure. It has been suggested as a candidate material for the development of disease treatment.

Three hundred sec is a perennial herb belonging to the family of three hundred (Saururaceae), and the whole grass or root or leaf is wind poison, diuresis, water species, gonorrhea, hepatitis, pneumonia, venom (便 毒), high blood pressure (高血壓) has been used in the private sector (Cho Bo-seop, Shin Min-kyo): Hyangjedae, Yeonglimsa, Seoul, 812-814 (1990). Three hundred seconds leaves, flowers and roots are white, and the three leaves under the inflorescence (花序) of the upper part is called three hundred seconds (三 白草). Three hundred and four to five species of plants are distributed worldwide in the three hundred genus, and two genus two species of the three hundred sec ., A genus three hundred sec ., A genus of three hundred sec ., And a genus of wild buckwheat ( Houttuynia cordata Thunb.), Which is believed to be naturalized in Japan. do. There are two kinds of genus of three hundred genus plants all over the world, and there are three kinds of three hundred vinegars ( Saururus chinensis ) distributed in Asia and one species ( Saururus cernuus , Lizard tail) in North America (Jae-Gil Kim: Namsan-dang, Seoul, p 174 (1984); Kim, Tae-Jung: Resources Plant of Korea, Seoul National University Press, Seoul, pp 66-67 (1997); Lee Chang-Bok: Korea Plant Book, Hyangmunsa, Seoul, p 252 (1979); Plant Book, Kyohaksa, Seoul, pp 218-219 (1972)).

The three hundred vines are stored in the ancient Chinese medicine medicinal record, Ming-seol, and are collected from the ground in July-September and dried in sunlight. The beauty is high, the sex is cold, and it enters the liver, lungs, and nerves. It is known to have the effects of moist heat, fertilization, disinfection and detoxification, edema, each, jaundice, gonorrhea, and lobster ( It has been used for the treatment of the lower extremities and others (Shin, Jung, Shin, Jung, Shin, 1990). In addition, the three hundred seconds roots are stored in Sinsuboncho, and in July-September, they dig up the underground diameter to remove Ito, soak it in boiling water for a few minutes, and dry it in the sun. Beauty is sentimental and sex is limited. Efficacy in Isu, dehumidification, fever, detoxification, and it has been used to treat various conditions of gonorrhea, gonorrhea, rehabilitation, and improvement. (Jung and Shin, 1990; Nippon Pharmaceutical Method, Nippon River Café, Tokyo, pp. D-481-D-484 (1996) Pharmacy of Japan, 1996).

Studies on the chemical composition of three hundred seconds have been conducted mainly on the flavonoids, alkaloids, amino acids, fatty acids, quinones and essential oils in the ground. It became. The essential oil component of the outpost is methyl-n-nonyl-ketone (methyl-n-nonyl-ketone has been reported as the main ingredient, in addition to alpha-pinene, camphene, linalool, safrol, beta-caryophyllene and humulene (humulene) have been reported (Choe, KH, Yoon, CH and Kwon, SJ: Chemical composition of Saururaceae growing in Korea-on volatile constituents ofSaururus chinensisby GC / MS.Punsok Kwahak One, 259-262 (1988b); Choe, K. H., Kwon, S. J. and Lee K. C .: Chemical composition of Saururaceae growing in Korea-on fatty acids and amino acids ofHouttuynia cordataandSaururus chinensis. Punsok Kwahak 2,285-288 (1989). Flavonoids, one of the main components of the terrestrial sect, are reported to include hyperin, quercetin, isoquarcitrin, and quercitrin (Xu, L., Zhang, X. and Lin, A .: Differential pulsed polarographic determination of flavonoids inSaururus chinensis.Yaowu fenxi zazhi 8, 223-226 (1988); Choe, K. H., Yoon, C. H. and Kwon, S. J .: A study of chemical constituents of Saururaceae growing in Korea-on flavonoid constituents ofSaururus chinensis.Anal. Sci. Technol. 7, 11-15 (1994); Sung, S. H., Kwon, S. H., Cho, N. J. and Kim, Y. C .: Hepatoprotective flavonolglycosides ofSaururus chinensisHerbs.Phytotherapy Res. 11(500-503 (1997)), quinones such as emodin and physcion, and lignans of carpanones have also been reported (Wang, EC, Shih, MH, Liu, MC, Chen, MT and Lee, GH: Studies on constituents ofSaururus chinensis.Heterocycles 43, 969-972 (1996). In addition, aristolactam-based alkaloids, saurolactam, were isolated as cytotoxic components of 300 seconds (Park, SK, Oh, GJ, Bae, CI, Kim, HJ, Han, WS, Chung, SG). and Cho, EW: Studies on the cytotoxic constituents ofSaururus chinensis.Yakhak hoeji 41, 704-707 (1997)), the same series of alkaloids, aristolactam BII, have also been reported. It also contains fatty acids of cupric acid, linoleic acid and lauric acid, alanine, valine, glutamic acid, aspartic acid and leucine ( The presence of amino acids such as leucine, isoleucine and proline was also confirmed.

However, it has not been reported that the sacchinone compound isolated from 300 seconds according to the present invention has hepatocellular protective action.

Accordingly, it is an object of the present invention to provide a pharmaceutical preparation having a hepatocyte protective activity containing as an active ingredient a sautinone compound selected from Chemical Formulas 1, 2 and 3 isolated from 300 seconds.

Still another object of the present invention is to provide a method for preparing the sacchinone of Formula 1, Formula 2 and Formula 3 from three hundred seconds.

In another aspect, the present invention is to provide a pharmaceutical preparation having a hepatocellular protective activity containing as an active ingredient three hundred seconds extract containing the compounds of the formula (1), (2) and (3) as a main component.

1 is a ¹³ C- ¹ H COSY spectrum (300 MHz, CDCl ₃ ) of Compound 1 of the present invention,

2 is an HMBC spectrum (300 MHz, CDCl ₃ ) of Compound 1 of the present invention,

3 is a NOE differential spectrum (300 MHz, CDCl ₃ ) of Compound 1 of the present invention,

4 is ¹³ C- ¹ H COSY spectrum (300MHz, CDCl ₃ ) of the compound 2 of the present invention,

5 is an HMBC spectrum (300MHz, CDCl ₃ ) of the compound 2 of the present invention,

6 is a NOE parallax spectrum (300 MHz, CDCl ₃ ) of the compound 2 of the present invention,

7 is a NOESY spectrum (500 MHz, CDCl ₃ ) of the compound 3 of the present invention,

FIG. 8 is a graph of the effects of Succinone (Compound 1), Succinone A (Compound 2), and Succinone B (Compound 3) on GPT activity in primary cultured rat hepatocytes induced by toxicity with carbon tetrachloride ( In this case, Ctrl is the value of hepatocytes not treated with carbon tetrachloride and its Ctrl value is 24.98 ± 3.42 IU / L Ref is the value of hepatic cells treated with carbon tetrachloride and its Ref value is 109.62 ± 10.34 IU / L The protection percentage (%) was calculated by the formula (Ref value-sample value / Ref value-Ctrl value) x 100, and significant differences from Ref values were p <0.05 ^* , p <0.01 ^** , p <0.001 ^*** , and the Sily value is that of the Silybin 100 μM treatment group.)

FIG. 9 shows the effects of Succinone, Succinone A, and Succinone B (50 μM) on the MDA content in primary cultured rat hepatocytes induced toxicity with carbon tetrachloride (where Ctrl is the effect of hepatic cells not treated with carbon tetrachloride). The MDA's Ctrl value is 0.89 ± 0.05μM / mg protein, Ref is carbon tetrachloride-treated hepatocytes, and the Ref value of MDA is 1.65 ± 0.35μM / mg protein, and the protective percentage (%) is (Ref value-sample value / Ref value-Ctrl value) was calculated by the formula of x100, and the significant difference from the Ref value is expressed as p <0.01 ^** .,

FIG. 10 is the effect of Succinone, Succinone A and Succinone B (50 μM) on the activity of GSH Px and GSSG-R in primary cultured rat hepatocytes induced toxicity with carbon tetrachloride (where Ctrl is carbon tetrachloride Values of untreated hepatocytes, the Ctrl values of GSH Px and GSSG-R are 2.21 ± 0.0015 μmol / mg prot./min and 82.4 ± 7.74nmol / mg prot./min, respectively, and Ref is carbon tetrachloride-treated hepatocytes The Ctrl values of GSH Px and GSSG-R are 0.89 ± 0.17μmol / mg prot./min and 56.6 ± 7.58nmol / mg prot./min, respectively, and the protective percentage (%) is (Ref value-sample). Value / Ref value-Ctrl value) x100, and a significant difference from the Ref value is expressed as p <0.05 ^* ),

FIG. 11 is a graph showing the effects of Succinone, Succinone A, and Succinone B on the activity of GPT in primary cultured rat hepatocytes induced by toxicity with galactosamine (GalN) (where Ctrl is Galacto The value of hepatocytes not treated with samine, the Ctrl value of GPT was 45.06 ± 3.55 IU / L Ref was the value of hepatocytes treated with galactosamine, and the Ref value of GPT was 97.24 ± 7.62 IU / L The percentage of protection was calculated by the formula (Ref value-sample value / Ref value-Ctrl value) x 100, and the significant difference from the Ref value is expressed as p <0.05 ^* , p <0.01 ^** ).

Hereinafter, the present invention will be described in detail.

Hepatocyte protective activity screening method

In the process of generating a substance with hepatoprotective activity from natural products, the first requirement is the establishment of an appropriate screening method to search for substances with such activity. It is known to be physiologically and biochemically similar to human liver cells in order to find substances that have hepatocyte protective activity from natural products. Hepatocytes of primary cultured rats are widely used as a screening system. Hepatocytes of primary cultured rats retained their original function (Jones, CA, Moore, BP, Cohen, GM, Fry, JR and Bridges, JW: Studies on the metabolism and excretion of benzo (α) pyrene in isolated adult rat hepatocytes.Biochem. Pharmacol.27, 693-702 (1978)), in particular, possesses the drug metabolizing enzyme, which is one of the important functions of the liver, and has the advantage that it can be used for research by replacing experimental animals. However, since the primary cultured cells do not undergo the same metabolism, excretion, and accumulation processes as in the body, and their interactions and effects with other organs are excluded, the experimental results using them cannot be directly accepted as a result in vivo. Have In order to detect hepatocyte protective activity in natural products, it is common to first artificially induce toxicity to liver cells of rats cultured first, and then measure the extent to which natural products recover damage caused by toxicity. Several methods of artificially inducing toxicity in hepatocytes have already been developed and applied (Kiso, Y., Tohdin, M. and Hikino, H .: Assay method for antihepatotoxic activity using ionophore A23187-induced cytotoxicity in primary cultured hepatocytes.Shoyakugaku zasshi 39,218-221 (1985); Kiso, Y., Kawakami, Y., Kikichi, K. and Hikino, H .: Assay methods for antihepatotoxic activity using complement-mediated cytotoxicity in primary cultured hepatocytes.Planta Med. 53, 241-247 (1987)).

In the present invention, carbon tetrachloride and galactosamine were used to induce hepatotoxicity, respectively. 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, S). , Mata, HP, Reid, LL, Gandlofi, AJ and Brendel, K .: Further examination of the selective toxicity of CCl ₄ in rat liver slices.Toxicol.Appl . Pharmacol. 112 , 81-89 (1992)). Toxic mechanisms of carbon tetrachloride is the ER (endoplasmic reticulum) in the presence drug-metabolizing enzyme, cytochrome P-450 a metabolite produced by the dependent mixed oxidase (cytochrome P-450 dependent mixed oxidase) to the trichloro methyl radicals (· CCl ₃ ) Binds to cellular proteins and lipids, resulting in lipid peroxidation, resulting in morphological changes in endoplasmic reticulum, decreased activity of drug metabolism enzymes, reduced protein synthesis and glucose-6-phosphate It is known to cause a decrease in the activity of the liver and to cause fatty liver by inhibiting the release of triglyceride synthesized in the liver (Gravela, E., Alvano, E., Dianzani, M., Polia, G. and Slater , TF: Effects of carbon tetrachloride on isolated hepatocytes.Biochem. J. 178 , 509-515 (1979); Groot, H. and Noll, T .: The crucial role of low steady oxygen partial pressures in haloalkane free-radical media .. ted lipid peroxidation Biochem Pharmacol 35 , 15-26 (1986); Slipes, IG, Kroshna, G. and Gillett, JR:... Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane Life Sci 20, 1541-1548 (1977) Wolf, CR, Harrelson, WG, Mastainczyk, WM and Rhipot, RM: Metabolism of carbon tetrachloride in hepatic microsomes and reconstituted monooxygenase system and its relationship to lipid peroxidation. Mol. Pharmacol . 18 , 553- (1980)).

Galactosamine, another toxin that causes hepatotoxicity, is known to impair liver function or morphology, similar to viral hepatitis (Decker, K and Keppler, P .: Galactosamine hepatitis; Key role of the nucleotide deficiency period in the pathogenesis of the cell injury and cell death.Pharmacol . Rev. Physiol.Biochem . 71 , 77-86 (1974)). Galactosamine inhibits the synthesis of RNA and protein in the liver, which leads to the production of uridine diphosphate hexosamine and UDP-N-acetylhexasamine (UDP-N) by galactosamine in the liver. Increasing synthesis of -acetylhexosamine results in the capture of uridine resulting in a sharp decrease in UDP-glucose, UDP-galactose and UDP in the liver. Thus, biosynthesis of biopolymers associated with uracil nucleotides such as UDP-glucuronic acid is compromised and organelles are damaged (Matsuo, R. and Ryschi, UK: The role of Kupffer cells in complement activation in D -galactosamine / lipopolysaccharide-induced hepatic injury of rat. Acta. Med. Okayama 46, 345-352 (1992)).

Hepatotoxicity or protective activity can be determined by measuring various enzymes present in hepatocytes. GPT (glutamic pyruvic transaminase) and sorbitol dehydrogenase (SDH) are enzymes that are selectively present in hepatocytes rather than other organs. When hepatotoxicity is induced, hepatocytes are ultimately destroyed, increasing the amount of enzyme released into the culture medium in hepatocytes. do. This fact has been widely used to measure hepatotoxicity and protective activity by carbon tetrachloride or galactosamine. In addition, hepatocellular protective mechanism against carbon tetrachloride-induced toxicity is due to enzymes involved in metabolism and detoxification in hepatocytes, namely glutathione-S-transferase (Habig, WH, Pabst, MJ and Jakoby, WB: Glutathione S-transferases. J. Biol. Chem.249, 7130-7135 (1974)), glutathione reductase (Carlberg, I. and Mannervik, B .: Purification and characterization of the flavoenzyme glutathione reductase from rat liver.J. Biol. Chem. 250, 5475 (1975)), glutathione peroxidase (Flohe, L. and Gunzler, W. A .: Assays of glutathione peroxidase.Method Enzymol. 105, 114-121 (1984)), superoxide dismutase (McCord, J. M. and Fridovich, J .: Superoxide dismutase.J. Biol. Chem. 244, 6049-6055 (1969)) and the activity of catalase (Fridovich, I .: Biological effects of the superoxide radical.Arch. Biochem. Biophys. 247, 1-11 (1986)). The amount of glutathione (Hissin, P. J. and Hill, R .: A fluorimetric method of determination of oxidized and reduced glutathione on tissue.Anal. Biochem. 74(214-226 (1976)) and the amount of malondialdehyde due to lipid peroxidation (Ohkawa, Y., Ohisi, N. and Yagi.K .: Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction.Anal. Biochem. 95, 351-358 (1979)). The mechanism of action is determined by measuring the degree of biosynthesis of RNA and protein in hepatocytes, along with the activity of GPT and SDH, which are primarily released in culture, to determine the recovery effect on hepatotoxicity induced by galactosamine ( Karner, A .: Regulational of the rate of systhesis of messenger ribonucleic acid by growth hormone.Biochem. J. 92, 449-457 (1964).

Extraction and fractionation of three hundred seconds

Following the method outlined in Scheme 1, extraction and fractionation from three hundred seconds yield an n-hexane fraction.

Scheme 1. Autumnal collection of three hundred seconds.

Ten kilograms of three hundred seconds were extracted with hot water three times with 80% MeOH for 2 hours and concentrated under reduced pressure to obtain a total of 1 kg.

Three hundred seconds was suspended in distilled water X (1.0kg) and then, CH ₂ Cl ₂ fraction and concentrated under reduced pressure to give the CH ₂ Cl ₂ fraction and water fraction. Was re-suspended in 90% MeOH to CH ₂ Cl ₂ fraction was concentrated, and then, n-hexane (hexane) to 90% MeOH fraction fraction (120g) and the n-fractions (60g) were obtained with hexane.

Isolation of Compound 1

Of the 25 fractions obtained by silica gel column chromatography of n -hexane extracts of 300 seconds, the crude crystals formed in fraction 8 were recrystallized with MeOH to obtain 80 mg of white amorphous crystals. Examination of the ¹ H NMR and ¹³ C NMR spectral data of this material revealed that it was a mixture of several materials with similar backbones. In order to separate it into a single substance, a semi-preparative HPLC was carried out with a mixed solvent of methanol: acetonitrile: water (MeOH: Acetonitrile (AcCN): H ₂ O = 15: 60: 25) to give Rt 24.08 min. The compound was aliquoted and concentrated under reduced pressure to separate compound 1 (56 mg). This compound was colored brown with anisealdehyde-H ₂ SO ₄ .

Structure Determination of Compound 1

C ₂₀ H ₂₀ O ₆

[α] D; + 95.0 ° (c, 0.63; CHCl 3)

R _f ; 0.35 ( n- hexane: EtOAc = 5: 1)

UV (CHCl ₃ ) λ _max (log ε); 299.5 (2.55), 254.2 (2.74) nm

IR (neat) ν _max ; 2916, 1676, 1664, 1418, 1433, 1321, 1241, 1184, 1155, 979, 926, 892, 756 cm ^-1

EIMS (m / z) (rel. Int.); 356 [M ⁺ ] (100), 270 (13), 257 (18), 205 (26), 175 (57), 151 (39), 138 (23)

HR EIMS; m / z 356.1262 (calcd for C ₂₀ H ₂₀ O ₆ m / z 356.1260)

¹ H NMR (300 MHz, CDCl ₃ )

δ 6.82 (1 H, s, H-6), 6.38 (1 H, s, H-3), 5.90 (1 H, d, J = 1.40 Hz, aromatic -OCH ₂ O-), 5.87 (1 H, d, J = 1.40 Hz, aromatic -OCH ₂ O-), 5.65 (1 H, s, aliphatic -OCH ₂ O-), 5.60 (1 H, s, aliphatic -OCH ₂ O-), 5.57 (1 H, s, H-3 '), 3.03 (1 H, d, J = 5.40 Hz, H-7), 2.52 (1 H, td, J = 11.93, 3.47 Hz, H-1'), 2.48 (1 H, d, J = 5.40 Hz, H-6 '), 2.44 (1 H, m, H-8), 1.92 (1 H, m, 7'-H _ax ), 1.88 (1 H, m, H-8' ), 1.64 (1 H, m, 7'-H _eq ), 1.22 (3 H, d, J = 7.26 Hz, H-9), 0.71 (3 H, d, J = 7.39 Hz, H-9 ')

¹³ C NMR (100 MHz, CDCl ₃ )

δ 199.69 (C-2 '), 168.66 (C-4'), 146.72 (C-5), 145.70 (C-2), 143.26 (C-4), 115.73 (C-1), 105.54 (C-6 ), 101.34 (C-3 '), 101.24 (aromatic -OCH ₂ O-), 100.42 (C-5'), 99.50 (C-3), 98.19 (aliphatic -OCH ₂ O-), 37.60 (C-6 '), 37.55 (C-1'), 35.06 (C-7), 34.82 (C-8), 33.45 (C-8 '), 25.27 (C-7'), 21.29 (C-9), 20.92 ( C-9 ')

Based on the above spectral data, Compound 1 was estimated as Sauchinone and was identified by comparison with literature values.

Isolation of Compound 2

Compound 1 having Rt 26.78 min was separated by preparative HPLC during the separation of Compound 1 , and concentrated under reduced pressure to obtain Compound 2 (16 mg). This compound developed brown color in anisealdehyde-H ₂ SO ₄ .

Structure Determination of Compound 2

C ₂₀ H ₂₀ O ₆

[α] _D ; + 32.5 ° (c 0.32, CHCl ₃ )

R _f ; 0.35 (n-hexane: EtOAc = 5: 1)

UV (CHCl ₃ ) λ _max (log ε); 298.5 (2.12), 253.0 (2.54) nm

IR (neat) ν _max ; 2918, 1668, 1478, 1346, 1240, 1163, 936, 888, 755 cm ^-1

EIMS (m / z) (rel. Int.); 356 [M ⁺ ] (100), 298 (11), 270 (18), 257 (19), 205 (11), 175 (34), 151 (20), 138 (16)

HR EIMS; m / z 356.1262 (calcd for C20H20O6 m / z 356.1260)

¹ H NMR (300 MHz, CDCl ₃ )

δ 6.73 (1 H, d, J = 0.60 Hz, H-6), 6.53 (1 H, s, H-3), 5.93 (1 H, s, aromatic -OCH ₂ O-), 5.91 (1 H, s, aromatic -OCH ₂ O-), 5.74 (1 H, s, aliphatic -OCH ₂ O-), 5.61 (1 H, s, H-3 '), 5.52 (1 H, s, aliphatic -OCH ₂ O 2.68 (1 H, m, H-1 '), 2.30 (1 H, td, J = 2.73, 13.74 Hz, 7'-H _eq ), 2.20 (1 H, dd, J = 6.60, 12.50 Hz , H-6 '), 2.09 (1 H, d, J = 9.69 Hz, H-7), 1.55 (1 H, m, H-8), 1.45 (1 H, m, H-8'), 1.14 (3 H, d, J = 5.70 Hz, H-9), 1.09 (1 H, m, 7'-H _ax ), 1.05 (3H, d, J = 6.06 Hz, H-9 ')

¹³ C NMR (100 MHz, CDCl ₃ )

δ 197.81 (C-2 '), 163.81 (C-4'), 145.86 (C-5), 145.83 (C-2), 143.00 (C-4), 126.00 (C-1), 105.00 (C-5 '), 104.83 (C-6), 101.38 (C-3'), 101.24 (aromatic -OCH ₂ O-), 100.94 (C-3), 98.19 (aliphatic -OCH ₂ O-), 45.10 (C-1 '), 42.34 (C-6'), 41.54 (C-7), 36.56 (C-8), 32.72 (C-8 '), 32.04 (C-7'), 19.85 (C-9 '), 18.32 (C-9)

Based on the above spectral data, Compound 2 is a new substance that is first separated from nature, and it was found that the stereoisomer of Sauchinone was already reported in 300 seconds. Compound 2 was named Succino A.

Isolation of Compound 3

Compound 1 having Rt 20.84 min was separated by preparative HPLC during the separation of compound 1 , and concentrated under reduced pressure to obtain compound 3 (8 mg). This compound developed brown color on anisealdehyde-H ₂ SO ₄ .

Structure Determination of Compound 3

C ₂₀ H ₂₀ O ₆

[α] _D ; + 15.2 ° (c, 0.30; CHCl ₃ )

R _f ; 0.35 (n-hexane: EtOAc = 5: 1)

UV (CHCl ₃ ) λ _max (log ε); 300.1 (2.32), 254.5 (2.51) nm

IR (neat) ν _max ; 2916, 1651, 1477, 1187, 1039, 915 cm ^-1

EIMS (m / z) (rel. Int.); 356 [M +] (89), 270 (9), 257 (8), 205 (42), 175 (100), 147 (17), 138 (9)

HR EIMS; m / z 356.1262 (calcd for C ₂₀ H ₂₀ O ₆ m / z 356.1260)

¹ H NMR (300 MHz, CDCl ₃ )

δ 6.76 (1 H, s, H-6), 6.34 (1 H, s, H-3), 5.91 (1 H, s, aromatic -OCH ₂ O-), 5.88 (1 H, s, aromatic -OCH ₂ O-), 5.61 (1H, s, aliphatic -OCH ₂ O-), 5.60 (1H, s, aliphatic -OCH ₂ O-), 5.57 (1 H, d, H-3 '), 3.18 (1 H , dd, J = 10.74, 1.45 Hz, H-7), 2.81 (1 H, t, J = 8.52 Hz, H-6 '), 2.60 (1 H, td, J = 12.71, 2.94 Hz, H-1 '), 2.22 (1 H, dd, J = 14.39, 6.33 Hz, H-8), 1.98 (1 H, m, 7'-H _eq ), 1.50 (1 H, m, H-8'), 1.20 (3 H, d, J = 7.56 Hz, H-9), 1.09 (1 H, m, 7'-H _ax ), 0.68 (3 H, d, J = 7.05 Hz, H-9 ')

¹³ C NMR (100 MHz, CDCl ₃ )

δ 199.53 (C-2 '), 168.26 (C-4'), 146.49 (C-5), 144.95 (C-2), 143.26 (C-4), 118.77 (C-1), 100.73 (C-5 '), 107.33 (C-6), 101.24 (aromatic -OCH ₂ O-), 98.19 (aliphatic -OCH ₂ O-), 100.31 (C-3), 99.50 (C-3'), 42.16 (C-6 '), 39.45 (C-1'), 36.13 (C-7), 35.22 (C-8), 32.72 (C-8 '), 30.78 (C-7'), 24.54 (C-9), 23.58 ( C-9 '),

Based on the above spectral data, Compound 3 was another stereoisomer of Sauchinone, which was already separated and reported in 300 seconds as a new substance that is first separated from nature. Compound 3 was named Sauchinone B.

Hepatoprotective activity was measured using the sautinone compound prepared as above.

Laboratory animals

Rats (Wistar, male, 150-200 g) were supplied from Seoul National University's experimental animal breeding ground at a temperature of 22 ± 1 ℃ and a humidity of 60 ± 5%, changing their contrast at 12 hour intervals. Intake. Primary cultured hepatocytes were taken directly from starved and fasted rats one day before the experiment.

Hepatocyte Culture in Rats

Hepatocytes from rats were isolated using a two-step collagenase perfusion method modified slightly from Berry and Friend's method (1984) (Kleiman, HK and Hartin, SR: Preparation of collagen substrates for cell attachment.Anal. Biochem. 94 , 308-312 (1979)). To obtain hepatocytes, rats were anesthetized with urethane (1 g / kg body weight), and the abdomen was disinfected with 70% ethanol and then opened. A 21 gauge catheter was intubated into the portal vein to allow about 50 ml of HBSS solution to flow through at a rate of 30 ml / min, and at the same time, the inferior vena cava was cut under the renal vein to remove blood. The chest tube was then opened, an 18 gauge catheter was inserted into the relative vein, and the inferior vena cava was tied up to allow recirculation of the digestive solution back into the supply vessel through the relative vein. A mixed gas of CO ₂ and O ₂ 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, final conc. 0.05%). After the liver cells were digested, the liver was removed, HBSS 60 ml 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 resulting hepatocyte suspension was diluted to a concentration of 5 × 10 ⁵ cells / mL and transplanted into a culture vessel previously coated with collagen.

Culture medium was Waymouth MB 752/1 medium, 5% fetal bovine serum, 2.0 mg / ml bovine serum albumin (fraction V), 10 ^-6 M dexamethasone (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 mg / ml streptomycin, 50 μg / ml gentamycin sulfate were used.

Culture environment

The cells were cultured in a 37 ° C. incubator with constant humidity and temperature while continuously supplying a mixture of air (95%) and CO ₂ (5%).

Induction of Hepatic Cell Toxicity by Carbon Tetrachloride

After 1.5 hours of culturing the hepatocytes, the cells were changed to a new medium, followed by further culture for 24 hours, and then treated with a medium containing 6.5 mM carbon tetrachloride for 1.5 hours to induce cytotoxicity (Kiso, Y., Tohdin, M. and Hikino, H .: Assay method for antihepatotoxic activity using carbon tetrachloride induced cytotoxicity in primary cultured hepatocytes.Planta Med. 49, 222-225 (1983)). The hepatoprotective activity of the sample was measured immediately before administration of carbon tetrachloride for each concentration of the sample to be searched and incubated for 1.5 hours, and then the culture solution was taken to measure the hepatoprotective activity.

Induction of Hepatotoxicity by Galactosamine

After 1.5 hours of culturing the hepatocytes, the culture medium was changed to a culture medium containing 1.5 mM galactosamine, followed by incubation for 14 hours to induce cytotoxicity, and then to a new culture medium. Hepatoprotective activity was measured by incubating at each concentration for 24 hours and then taking the culture medium (Kiso, Y., Tohdin, M. and Hikino, H .: Assay method for antihepatotoxic activity using galactosamine induced cytotoxicity in primary cultured hepatocytes J. Nat. Prod. 46 , 651-654 (1983b).

Preparation of Sample

The sample was dissolved in HBSS buffer or dissolved in dimethylsulfoxide (DMSO, final conc., 0.1% or less) or 99.9% ethanol (final conc., 1% or less) and diluted with HBSS. It was prepared aseptically by filtration using a millipore membrane (0.22 μm).

Hepatoprotective activity measurement

(1) Activity measurement of GPT (glutamic pyruvic transaminase)

Reitman, Frankel, SA: Colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase using a GPT kit from a culture of hepatocellularly induced hepatocytes Am. J. Cli. Pathol. 28 , 56-63 (1957)) measured the activity of GPT.

(2) Determination of activity of SDH (Sorbitol dehydrogenase)

Cultures of hepatic cells induced cytotoxicity were taken from Gerlach (Gerlach, U .: Sorbitol dehydrogenase, In Methods of Enzymatic Analysis , Bergmeyer, HV (ed), Verlagp Chemic, Weinhein, pp 761-765 (1965) The activity of SDH was measured by slightly modifying)). To 650 µl of the culture solution, 200 µl of 0.2 M triethanolamine HCl buffer (pH 7.4) and 50 µl of NADH (23.3 mg / 4 ml deionized water) were added and left at room temperature for 30 minutes. Subsequently, 150 μl of 4.0 M fructose was added thereto, mixed well, and the change of UV absorbance at 365 nm was measured. The change in absorbance for 1 minute was measured and expressed in SDH units.

(3) Preparation of hepatocyte supernatant

After removing the culture medium from the culture vessel, 3 ml of 0.1 M sodium phosphate buffer (pH 7.6) was added to prepare a cell suspension. The cell suspension thus prepared was homogenized for 15 seconds and then centrifuged at 12500 × g for 15 minutes to prepare hepatocyte supernatant (Gibson, GG and Skelf, P .: Techniques and experiments illustrating drug metabolism, In Introduction to drug metablism . Gibson, GG and Skelf, P. (eds), Chapman and Hall, New York, pp 239-271 (1988)).

(4) Determination of the amount of total glutathione (GSH + GSSG)

The total amount of GSH in hepatocytes was determined by Hisin, PJ and Hill, R .: A fluorimetric method of determination of oxidized and reduced glutathione on tissue.Anal.Biochem . 74 , 214-226 (1976 )). The supernatant obtained in (3) was used as a sample to measure the amount of GSH. To the 1 ml cuvette, 700 μl of 0.3 mM NADPH, 100 μl of 6 mM DTNB and 200 μl of hepatocyte supernatant were added, followed by 10 μl of pre-warmed 50 units / ml GSSG-R, followed by well mixing, immediately at 412 nm. Absorbance change was measured. That is, the amount of total glutathione was measured at 10-second intervals for the portion where the absorbance increased linearly, and was expressed in terms of the amount of product the mg protein produced per minute.

(5) Determination of glutathione disulfide (GSSG) amount

The amount of GSSG in hepatocytes was determined using Hissin, PJ and Hill, R .: A fluorimetric method of determination of oxidized and reduced glutathione on tissue.Anal . Biochem. 74 , 214-226 (1976). It was measured by. The supernatant obtained in (3) was used as a sample solution to measure the amount of GSSG. 4 μl of 2-vinylpyridine was added to 200 μl of hepatocyte supernatant, followed by reaction at 25 ° C. for 30 minutes, and the amount of GSSG in hepatocytes was measured in the same manner as in (4).

(6) Determination of activity of GSH Px (Glutathione peroxidase)

GSH Px activity in hepatocytes was measured using the method of Flohe et al. (Flohe, L. and Gunzler, WA: Assays of glutathione peroxidase. Method Enzymol. 105 , 114-121 (1984). The hepatocyte supernatant obtained from was used as a sample for measuring the activity of GSH Px: 100 μl of 10 mM GSH, 100 μl of 2 mM NADPH, and 650 μl of 0.1 M potassium phosphate buffer (pH 7.2) in a 1 ml cuvette. 6 µl of 100 units / ml GSSG-R were added, and 50 µl of supernatant of hepatocytes was added and mixed immediately, and then the change in absorbance was measured at 340 nm. Was measured at intervals of 10 minutes for 2 minutes and the mg protein was expressed in terms of the amount of product produced per minute.

(7) Measurement of activity of glutathione disulfide reductase (GSSG-R)

The activity of GSSG-R in hepatocytes was determined by Carlberg et al. (Carlberg, I. and Mannervik, B .: Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem. 250 , 5475- ( 1975). The supernatant obtained in (3) was used as a sample solution for measuring the activity of GSSG-R. 100 ml of 10 mM glutathione disulfide, 100 µl of 1 mM NADPH and 600 µl of 0.1 M potassium phosphate buffer (pH 7.4) were added to a 1 ml cuvette, 200 µl of the hepatocyte supernatant was added well, and the absorbance was immediately changed at 340 nm. Measured. That is, the activity of the enzyme was measured by converting the portion where the absorbance was linearly measured at 10-second intervals for 2 minutes and converted to the amount of the product produced by the mg protein per minute.

(8) Activity measurement of glutathione-S-transferase (GST)

The activity of GST in hepatocytes is determined by methods of Habig et al. (Habig, W. H., Pabst, M. J. and Jakoby, W. B .: Glutathione S-transferases. J. Biol. Chem.249, 7130-7135 (1974)). The supernatant obtained in (3) was used as a sample solution for measuring GST activity. After mixing 100 μl of 10 mM, 10 μl of 0.1 M 1-chloro-2,4-dinitrobenzene and 500 μl of 0.1 M potassium phosphate buffer (pH 7.4) and 390 μl of distilled water, 50 μl of hepatocyte supernatant was mixed well. The change in absorbance was immediately measured at 340 nm. That is, the activity of the enzyme was measured by converting the portion where the absorbance was linearly measured at 10-second intervals for 2 minutes and converted to the amount of the product produced by the mg protein per minute.

(9) Determination of the activity of catalase

Catalase activity in hepatocytes was measured using a method of Fridovich et al. (Fridovich, I .: Biological effects of the superoxide radical. Arch. Biochem. Biophys. 247 , 1-11 (1986)). The supernatant obtained in (3) was used as a sample solution for measuring the activity of catalase. To a cuvette containing 150 µl of 19 mM H ₂ O ₂ (in DW), add a mixture of 825 µl of 0.1 M potassium phosphate buffer and 25 µl of hepatocyte supernatant. The decomposition rate of H ₂ O ₂ was measured in terms of the amount of product that the mg protein produced per minute by measuring the portion where the absorbance was linearly increased at 240 nm at 10 second intervals for 2 minutes. .

(10) Determination of SOD (Superoxide dismutase) activity

The activity of SOD in hepatocytes can be determined by McCord and Fridovich et al. (McCord, JM and Fridovich, J .: Superoxide dismutase. J. Biol. Chem. 244 , 6049-6055 (1969)). 100 ml of hepatocyte supernatant were added with 0.2 ml of reagent A (0.2 mM hydroxylamine and hypoxanthine), 500 µl of distilled water and 200 µl of reagent B (xanthine oxidase), followed by 30 minutes at 37 ° C. Then, 200 μl of reagent C (300 ㎍ / ㎖ sulfanic acid, 5 ㎍ / ㎖ N-α-naphthylethylenediamine, 16.7% acetic acid) was added thereto, and allowed to stand at room temperature for 20 minutes, followed by measurement of absorbance at 550 nm. Converted to.

(11) lipid peroxidation measurement

The degree of lipid peroxidation was measured by Logani method (1972) using 1,1,3,3-tetraethoxypropane as a standard. 1 ml of thiobarbituric acid (pH 3.5, 0.5%) was added to 200 μl of hepatocyte supernatant, heated at 100 ° C. for 1 hour, and allowed to stand at room temperature for 10 minutes, followed by measurement of absorbance at 535 nm (Recknagel , RO and Goshal, EA: New data on the question of lipoperoxidation in carbon tetrachloride poisoning.Exp. Mol.Pathol . 5 , 108-112 (1966); Ohkawa, Y., Ohisi, N. and Yagi.K .: Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction.Anal . Biochem. 95 , 351-358 (1979)).

(12) RNA biosynthesis measurement

The degree of RNA biosynthesis of hepatocytes was determined by adding [ ³ H] -uridine to the culture and measuring the extent to which radioisotope-labeled uridine was bound to the RNA of the cultured cells. [ ³ H] -uridine was added to the culture at a concentration of 1 μCi / ㎖ and then the culture was removed after 24 hours. Washed three times with HBSS and 1 ml of 10% trichloroacetic acid was added to precipitate the protein. A mixed solvent of 1 ml ethanol-ether (3-1, v / v) was added to remove the remaining trichloroacetic acid, and the protein was dissolved in 200 μl of 1 N NaOH. 150 μl of the solution was added to 3 ml of aquasol, a scintillation cocktail, and radioactivity was measured using a liquid scintillation counter (Karner, A .: Regulational of the rate of systhesis). of messenger ribonucleic acid by growth hormone.Biochem . J. 92 , 449-457 (1964)).

(13) Protein Quantitation

The amount of protein was determined by Lowy's method (Lowry, OH, Rosebrough, NJ, Farr, AL and Randall, RJ: Protein measurement with the foline phenol reagent.J. Biol. ) Using bovine serum albumin as a standard . Chem. 193, was measured at 500 nm to the 265-275 (1951)).

Statistical processing

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%.

Experiment result

1) Determination of the structure of compound 1 (sauchinone)

Compound 1 was a white colorless powder and showed strong absorption in the short wavelength of UV lamp on TLC. It was dark brown by an anisealdehyde-H ₂ SO ₄ color reagent. UV spectra showed strong absorption bands at 250 and 295 nm and were estimated by the lignan skeleton (Hattori et. Al. , 1987), and the molecular formula of C ₂₀ H ₂₀ O ₆ was determined from the high resolution EIMS data. In IR, characteristic conjugated carbonyl groups were observed at 1676 cm ^-1 and 1664 cm ^-1 . Carbonyl carbon (δ 199.69), 2 methylenedioxy carbons (δ 101.24, 98.19), 3 oxygen—from ¹ H NMR, ¹³ C NMR, DEPT, ¹ H- ¹ H COZY and ¹³ C- ¹ H COZY spectra Oxygen-binding aromatic carbons (δ 146.72, 145.70, 143.26) and the presence of two aromatic protons (δ 6.82, δ 6.38) in the para position with each other were confirmed. In addition, two methyl group signals (δ 21.29, 20.92), one methylene signal (δ 30.78) and five methine signals (δ 37.60, 37.55, 35.06, 34.82, 33.45) were observed (FIG. 1).

In the HMBC spectrum, 199.69 (C) carbonyl carbon with long range coupling with a signal of δ 5.57 (H-3 ′; 100.42 (CH)) and C-3 ′ of 101.34 (C) were observed. Methylenedioxy group [δ 5.60, 5.65; 98.19 (CH _2)] of the methylene proton and remote coupling methylene dioxide in time in C-4 'and 100.42 (C) C-5 in the' observed for compound 1 is β, γ position δ 168.66 (C) to (methylenedioxy It was confirmed that the group) had a substituted alpha, beta-unsaturated carbonyl moiety (α, β-unsaturated carbonyl moiety). In addition, it can be seen that H-6 'and H-1' are remotely coupled with C-3 'and C-5', respectively (FIG. 2).

Also in the HMBC spectrum Para each otherpara)In position Aromatic moiety with 1, 2, 4, and 5 position substitutions correlated with two aromatic protons and oxygen bond-aromatic at δ 146.72 (C), 145.70 (C), 143.26 (C) Carbon was observed, δ 101.24 (CH₂; δ 5.87 and 5.90) confirm the presence of typical methylenedioxy groupsOneIt was confirmed that this 2-oxy-4,5-methylenedioxy group had a substituted aromatic moiety.

^{In 1} H NMR, the signals δ 1.22 ( J = 7.26 Hz) and 0.71 ( J = 7.39 Hz) due to two methyl groups coupling with the vicinal protons were observed and ¹ H- ¹ H, ¹³ The structure of subunit C can be inferred from the C- ¹ H, HMBC spectra. Each of these subunits has a long range ¹³ C- of H-7 and H-6 '. Combinations were possible through ¹ H correlation. In the HMBC spectrum, a signal of δ 3.03 corresponding to H-7 was observed for long distance coupling with C-8, 9 ', and 6', so that the skeletons of subunits B and C could be connected to C-7. In addition, H-6 'is remotely coupled with C-1, 3', and 5 'and H-3' is remotely coupled with C-6 ', 4', 2 ', and 5'. C could be combined. In addition, 4 ° carbon (C-5 ′) observed at δ 101.34 was determined as carbon bound to oxygen bound to subunit B.

A B C

The stereogenicity of 1 ', 6', 7, 8, 8 'of Compound 1 was determined via NOE parallax spectra. When Iridinated δ 3.03, H-7 of Compound 1 , H-6 '(δ 2.48, 13.45%) and H-9 (δ 1.22, 7.28%) showed relatively strong NOE effects. H-6 (δ 6.38, 2.95%) also showed NOE effect. When δ 0.71 (H-9 ') is validated, H-1' (δ 2.52, 10.43%) and H-8 (δ 2.44, 6.03%) show NOE effects. It was determined to have a cis, trans ( cis, trans) spatial structure (Fig. 3).

compoundOneof Compound to determine the stereochemistry of 5 'carbonOneHigh pressure H using platinum oxide as a catalyst₂React under gas to compound1aGot. compound1aof^OneDecoupling experiments were performed in H NMR. When δ 4.19 is irradiated to saturate the spin states of H-4 ′, the spin coupling with two adjacent H-3 ′ disappears. As a result, δ 3.06 and 2.66 were newly generated by reduction by observing the doublet doublet signal of δ 3.06 and 2.66 changed to the doublet signal.1aIt was confirmed that the H-3 '. compound1aof^OneΔ 5.08 (aliphatic -OC) when δ 4.19, the newly generated H-4 ', is validated by H NMRHaOnly HbO-, 1.91%) and 3.06 (3'-Ha, 7.41%) signals showed a positive NOE effect, indicating that H-4 'has a different spatial arrangement than H-6'. . Compound from the above dataOneThe spatial structure of 5'-H was determined.

From the above data, Compound 1 was estimated to be the four launch non-lignan of carboxylic propanone series literature value (Wang, EC, Shih, MH , Liu, MC, Chen, MT and Lee, GH:. Studies on constituents of Saururus chinensis Heterocycles 43 969-972 (1996)).

2) Determination of the structure of compound 2 (sauchinone A)

Compound 2 was a white colorless powder and showed strong absorption in the short wavelength of UV lamp on TLC. It was dark brown by anisealdehyde-H ₂ SO ₄ color reagent. UV spectra showed strong absorption bands at 250 and 295 nm to estimate the lignan skeleton (Hattori et. Al. , 1987), and the molecular formula of C ₂₀ H ₂₀ O ₆ was determined from high resolution EIMS. Compound 2 was assumed to be the stereoisomer of Compound 1 because it has the same molecular weight as Compound 1 and the same functional group in EIMS, IR and the like.

Carbonyl carbon (δ 197.81), 2 methylenedioxy carbons (δ 101.24, 98.19), 3 oxygen- from ¹ H NMR, ¹³ C NMR, DEPT, ¹ H- ¹ H COZY and ¹³ C- ¹ H COZY spectra The presence of bound aromatic carbons (δ 145.86, 145.83, 143.0) and two aromatic protons (δ 6.73; s, δ 6.53; s) at para positions with each other were confirmed. In addition, δ 1.22 ( J = 7.26 Hz), 0.71 ( J = 7.39 Hz) signals with two methyl groups coupling with adjacent protons were observed, two methyl group signals (δ 19.85, 18.32) and one methylene signal (δ). 32.72) and five methine signals (δ 45.10, 42.34, 41.54, 36.56, 32.04) were observed (FIG. 4).

Δ 197.81 (C) carbonyl carbon with δ 5.61 (H-3 ′; 101.38 (CH)) in the HMBC spectrum and C-6 ′ (δ 45.10, CH) and C-5 ′ (δ 105.00, C) was observed and C-4 '(δ 163.81, C) and C-5' (δ 105.00, C), which are remotely coupled with methylene protons of methylenedioxy groups (δ 5.60, 5.65; 98.19 (CH ₂ )) Observing the same as the compound 1 , it was confirmed that the alpha, beta-unsaturated carbonyl moiety (α, β-unsaturated carbonyl moiety) in which the methylenedioxy group was substituted at β and γ positions. In addition, it can be seen that H-6 'and 1' are remotely coupled with C-3 'and 5', respectively (Fig. 5).

Also in the HMBC spectrum Para each otherpara) In position 1, 2, 4, 5 substituted aromatic moieties correlated with two aromatic protons and oxygen bond-aromatic carbons of δ 145.86 (C), 145.83 (C) and 143.70 (C) Δ 101.24 (CH₂; compounds of typical methylenedioxy groups at δ 5.52)OneIn the same manner as the 2-oxy-4,5-methylenedioxy group (2-oxy-4,5-methylenedioxy group) was confirmed to have a substituted aromatic moiety (aromatic moiety).^OneΔ 1.09 (7.3), 1.05 (7.3) signals were observed in H NMR by two methyl groups coupling with adjacent protons.^OneH-^OneH,^OneH-¹³C, compound through HMBC spectrum2CompoundOneIt was confirmed that the stereoisomer of (Fig. 6).

Was "When come retardation for the δ 2.20 H-1, H-6 of Compound (2) for determining the solidity of Compound 2 (7.85%), the beam of a relatively strong NOE effect, δ 1.14 to (H-9) In the case of IRR, NO-6 effects of H-6 (δ 6.73, 3.34%) and H-7 (δ 2.09, 1.79%) were observed, indicating that H-7, 6 ', and 1' were trans and cis. It was determined to have a spatial structure of. Based on this data, cis (cis) of the compound 2 Compound 1, it was confirmed that the trans (trans) stereoisomer than having different three-dimensional structure. Compound 2 is the stereoisomer of sachinone, which is reported for the first time.

3) Determination of the structure of compound 3 (sauchinone B)

Compound 3 was a white colorless powder and showed strong absorption in the short wavelength of UV lamp on TLC. It was dark brown by anisealdehyde-H ₂ SO ₄ color reagent. UV spectrum was 250, was assumed the lignan skeleton showing strong absorption band at 295 nm (Hattori et. Al. , 1987), and a molecular formula of Compound (3) from the fractional capacity was determined EIMS C ₂₀ H ₂₀ O _6. Compound 3 had the same molecular weight as that of Compounds 1 and 2, and the fragmentation pattern of EIMS and the spectral data of UV and IR were almost identical to each other, which resulted in another stereoisomer of Compounds 1 and 2 .

Carbonyl carbon (δ 199.53), 2 methylenedioxy carbons (δ 101.24, 98.19), 3 oxygen—from ¹ H NMR, ¹³ C NMR, DEPT, ¹ H- ¹ H COZY and ¹³ C- ¹ H COZY spectra The presence of bound aromatic carbons (δ 145.49, 144.95, 143.26) and two aromatic protons (δ 6.76; s, δ 6.34; s) substituted in the para position with each other were confirmed. In addition, δ 1.20 ( J = 7.26 Hz) and 0.68 ( J = 7.39 Hz) signals with two methyl groups coupling with the vicinal protons were observed, two methyl signals (δ 24.54, 23.58) and one Methylene signal (δ 30.78) and four methine signals (δ 42.16, 39.45, 36.13, 32.72) were observed to confirm that compound 3 is a stereoisomer of compound 1 .

NOESY experiments determined the stericity of compound 3 . Δ which is H-7 of compound 3

3.18 showed NOE effects with H-6 '(δ 2.81), H-8 (δ 2.22), H-9 (δ 1.20) and H-6 (δ 6.76), while δ 2.81 (H-6') H-1 ', the NOE effect on (δ 2.60) and H-9 (δ 1.20) was observed H-7, 6' was determined to have a spatial structure of a 1 'cis (cis), cis (cis) (FIG. 7).

Compound 3 is a compound in which 1 'and 7' double bonds are reduced in carpanone which has already been separated from plants. Brophy et al. (Brophy, GC, Mohandas, J., Slaytor, M., Sternhell, S. , Watson, TR and Wison, LA: Novel lignans from a Cinnamomum sp. From Bougainville.Tetrahedron Lett. 59 , 5159-5162 (1969) This is the first time that it has been identified.

4) Sauuccinon, Sauuccinon A And Hepatoprotective Activity of Succinone B

Sauuccinon, Sauuccinon A The hepatoprotective activity of Succinone B was investigated using hepatocytes of primary cultured rats induced by toxicity with carbon tetrachloride or galactosamine.

Hepatotoxicity Induced by Carbon Tetrachloride in Primary Cultured Hepatocytes and Succinone and Succinone A And the hepatoprotective effect of Succinone B was determined primarily by measuring the activity of GPT released into the culture. Induced toxicity of hepatic cells with carbon tetrachloride significantly increased the amount of GPT released into the culture due to damage to the cell membrane. At this time Sauchinnon, Sauchinnon A And when Succinone B was treated with increasing concentrations from 5 μM to 100 μM, they significantly reduced the level of GPT increased by carbon tetrachloride in a concentration dependent manner (FIG. 8). Sauuccinon, Sauuccinon A And Succinone B showed similar hepatocyte protective activities of 70.1, 78.2, and 75.9% at 100 μM, respectively, and the activity was almost similar to that of silybin used as a positive control.

Hepatotoxicity by carbon tetrachloride is a metabolite produced by cytochrome P-450 dependent mixed oxidase, a methyl trichloride free group (CCl).₃) Is known to be due to lipid permeate produced by binding to cell membrane lipids. Lipid peroxidation increased when carbon tetrachloride induced toxicity in hepatocytes of primary cultured rats as an indicator of MDA, and decreased activity of intracellular drug metabolism enzymes was associated with the amount of GSH, an antioxidant It can be measured by the change in the activity of Px, GSSG-R, SOD and catalase. Therefore, Sauuccinon, Sauuccinon A And the mechanism of hepatoprotective activity of sacchinone B may be regarded as a restorative effect on lipid peroxidation and reduced activity of intracellular antioxidants and antioxidant enzymes by carbon tetrachloride toxicity.

Succinone, Succinone A for Lipid Peroxidation And the amount of production of MDA was measured using the TBA method to determine the recovery effect of Succinone B (FIG. 9). As a result, Sauuccinon, Sauuccinon A And Saucinone B significantly reduced the lipid peroxidation of carbon tetrachloride-induced lipid peroxidation by reducing MDA production to 69.8%, 78.2% and 75.4% compared to the toxic group when administered at 50 μM concentration, respectively. And it was found.

Also Sauuccinon, Sauuccinon A And Saucinone B significantly restored the total GSH (GSH + GSSG) levels to 90.4, 98.0 and 91.7% levels, respectively, at a concentration of 50 μM. In particular, the amount of GSH rapidly reduced by the toxicity of carbon tetrachloride was maintained at almost normal state (Table 1).

Table 1. Effect of Succinone, Succinone A and Succinone B (50 μM) on the total GSH and GSSG content in rat hepatotoxicity induced by carbon tetrachloride.

total GSH ^c) ± SD GSSG ± S.D. reduced GSH ± S.D. GSSG / total GSH Ctrl. ^a) 19.62 ± 0.40 8.75 ± 1.82 10.87 ± 2.21 0.45 Ref. ^b) 2.74 ± 2.03 2.05 ± 2.26 0.70 ± 0.23 0.75 Sauchinone 18.00 ± 4.46 ^** 8.07 ± 2.16 ^* 9.93 ± 2.30 ^** 0.45 Sauchinone A 19.13 ± 3.57 ^*** 10.28 ± 0.03 ^** 8.84 ± 3.59 ^* 0.54 Sauchinone B 18.22 ± 3.15 ^*** 8.16 ± 2.74 ^* 10.07 ± 0.41 ^*** 0.45

^a) Ctrl. is the value of hepatocytes which were not challenged with CCl ₄ .

^b)Ref. is the value of hepatocytes which were challenged with CCl₄.

^c) GSH: μM / mg protein / min.

Significantly different from Ref. value: p <0.05 ^* , p <0.01 ^** , p <0.001 ^** _.

In addition, using GSH as a substrate, the activity of GSH Px, an antioxidant enzyme that eliminates peroxy radical (ROO ·), was significantly increased to 50.1, 64.9, and 48.5% compared to the control group. However, for the GSSG-R, which plays an important role in maintaining the amount of GSH in hepatocytes under oxidative stress by reducing GSSG using NADPH to produce GSH, Succinone and Succinone A And Saucinone B did not show a significant recovery effect (FIG. 10). In addition, there was no recovery effect on GST, which plays a major role in the detoxification of hepatocytes, and a significant recovery effect on SOD, another antioxidant enzyme, but no recovery effect on catalase (Table 2).

Table 2. Effect of Succino, Succino A and Succino B (50 μM) on SOD and Catalase Activity in Primary Cultured Rat Hepatocytes Induced by Carbon Tetrachloride.

SOD ^c) ± SD Catalase ^d) ± SD Ctrl. ^a) 27.36 ± 1.84 701.2 ± 22.2 Ref. ^b) 15.32 ± 2.52 401.5 ± 51.9 Sauchinone 23.64 ± 2.31 ^** 467.3 ± 51.8 Sauchinone A 17.22 ± 1.08 354.5 ± 62.7 Sauchinone B 20.76 ± 1.23 ^* 588.3 ± 41.1 ^**

^a)Ctrl. is the value of hepatocytes which were not challenged with CCl₄.

^b) Ref. is the value of hepatocytes which were challenged with CCl ₄ .

^C) SOD: units / mg protein

^d) Catalase: μM / mg protein / min

Significantly different from Ref. value: p <0.05 ^* , p <0.01 ^** .

All three substances in the diastereoisomer relationship showed similar hepatoprotective activity, but slightly different results were observed in the measurement of antioxidant activity. This result is presumed to be due to the difference in three-dimensional structure of the three materials.

From the above results, hepatocellular protective activity against carbon tetrachloride toxicity of sautinone, sautinone A and sautinone B maintains the amount of GSH and decreases MDA by increasing the activity of GSH Px, thereby reducing lipid peroxidation. Presumably due to a mechanism of suppressing

Hepatotoxicity Induced by Galactosamine in Primary Cultured Hepatocytes and Succinone and Succinone A And the hepatoprotective effect of Succinone B was determined primarily by measuring the activity of GPT released into the culture. When galactosamine was induced to induce hepatocyte toxicity, damage of the cell membrane significantly increased the amount of GPT released into the culture. At this time Sauchinnon, Sauchinnon A And Succinone, Succinone A when Succinone B was treated with increasing concentrations from 5 μM to 100 μM. And Saucinone B significantly reduced the level of GPT increased by galactosamine in a concentration dependent manner (FIG. 11). Succinone showed 34.4% at 10 μM, and Succinone A and B showed 50.7 and 62.5% hepatocyte protective activity at 100 μM.

Since the toxic mechanism of galactosamine is to lower the biosynthesis of high molecular materials in vivo, the radioisotope-labeled uridine binds to the cultured cells after [ ³ H] -uridine is added to the culture medium of primary cultured hepatocytes (incorporation). ) And the effect on the RNA biosynthesis of these hepatocytes (Table 3).

TABLE ³ Effect of Succino, Succino A and Succino B on [ ³ H] -uridine capture ^a) .

Conc. (μM) DPM ± S.D. (arbitary unit) DPM ratio ^d) Ctrl. ^b) Ref. ^c) 12820.3 ± 854.92 199.9 ± 271.3 1.0 Sauchinone 102550100 2473.8 ± 390.12579.7 ± 293.02599.8 ± 273.92532.1 ± 161.8 1.11.21.21.2 Sauchinone A 102550100 2264.5 ± 243.92491.8 ± 357.62138.0 ± 292.52633.5 ± 366.0 1.01.11.01.2 Sauchinone B 102550100 2862.3 ± 153.6 ^** 2945.3 ± 323.1 ^* 3200.1 ± 494.0 ^* 4292.1 ± 298.0 ^*** 1.31.31.52.0

^a) [ ³ H] -uridine (final concentration, 1 μCi / mL) was incorporated into the hepatocytes and counted β-emmission with LSC.

^b) Ctrl. is the value of hepatocytes which were not challenged with GalN.

^c) Ref. is the value of hepatocytes which were challenged with GalN.

^d) DPM ratio is calculated as the DPM value of compounds treated / DPM value of GalN-treated.

Significantly different from Ref. value: p <0.05 ^* , p <0.01 ^** , p <0.001 ^*** .

Primary cultured hepatocytes induced by galactosamine were [³The degree of incorporation of H] -uridine was significantly reduced. At this time Sauchinnon, Sauchinnon A And when Saucinone B was treated with increasing concentrations from 10 μM to 100 μM, Sauchinone and Sauchinone A were trapped in hepatocytes [³H] -uridine did not affect the amount, but Succinone B was trapped in hepatocytes at a concentration of 100 μM [³The amount of H] -uridine was significantly doubled. However, these results indicate that Succino, Succino A for galactosamine toxicity. And hepatocyte protective activity of sacchinone B is insufficient to explain the recovery of hepatocyte RNA biosynthesis. McMillan et al. (McMillan, J. M. and Jollow, D. J .: Galactosamine hepatotoxicity; Effect of galactosamine on glutathione resynthesis in rat primary hepatocyte cultures.Toxicol. Appl. Pharmacol. 115, 234-240 (1992) reported that galactosamine has a function of depleting GSH by inhibiting the GSH biosynthesis process induced by methionine in hepatocytes of primary cultured rats. Based on these findings, Succinone and Succinone A on galactosamine toxicity And the hepatoprotective activity of Succinone B may be due to a pathway that activates the antioxidant defense mechanism of hepatocytes by restoring the reduced GSH biosynthesis by galactosamine rather than by direct action of these three substances on RNA biosynthesis. It allows you to.

Acute Toxicity Test

Three hundred seconds is a herbal medicine that has been used by whales and is known to have little toxicity. In order to confirm this, three days after administration of Succinone, Succinone A and Succinone B prepared according to the method of the present invention to rats (Wistar, male, 150-200g) in an amount of 1000 mg / kg, respectively As a result of the observation, there was no death.

Thus, it was confirmed that the sautinone compound had little toxicity.

As described above, the sautinone compounds isolated from the three hundred seconds according to the present invention have the following effects.

1. Succinone A and Succinone B isolated from three hundred seconds are novel stereoisomers of Succinone which are first reported in nature.

2. Succinone, Succinone A, and Succinone B significantly reduced the amount of GPT released from the primary cultured hepatocytes of rats induced by toxicity with carbon tetrachloride. And the mechanism of inhibiting lipid peroxidation by reducing the production of MDA by increasing the activity of GSH Px.

3. Succinone, Succinone A, and Succinone B showed hepatocellular protective activity by significantly reducing the amount of GPT released from primary cultured hepatocytes in rats induced by galactosamine toxicity.

4. It showed the protective activity of blocking or restoring both hepatotoxicity by carbon tetrachloride or galactosamine of sautinone, sautinone A and sautinone B.

As described in detail above, the sautinone compound according to the present invention has excellent hepatocyte protective activity and has little toxicity, and thus can be used as a protective and therapeutic agent of hepatocytes.

Meanwhile, as described in detail above, the n-hexane fraction separated according to the method of the present invention (Scheme 1) contains the compounds of Formulas 1, 2 and 3 as a main component, and thus the n-hexane fraction Of course, it can be usefully used as a hepatocellular protective and therapeutic agent.

The hepatocyte protective agent of the present invention may be administered 1 to several times daily with 0.1 mg to 1000 mg / kg as a sacchinone compound.

The sautinone compound of the present invention or the extract containing the same as a main component is usually a pharmaceutical preparation commonly used in combination with a pharmaceutically acceptable excipient, for example, an injection, a liquid, a syrup, a tablet, a capsule, and the like. It can be formulated to prepare a pharmaceutical formulation.

Hereinafter, the present invention will be described in more detail as a preparation example of a hepatoprotective agent containing a sautinone compound according to the present invention as an active ingredient.

Formulation Example

Formulation Example 1

Succinone (Formula 1) 100 mg

Appropriate sterile distilled water for injection

pH adjuster

Succinone is dissolved in distilled water for injection, adjusted to pH 7.6 with a pH adjuster, and then the total amount is 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2

Saucinone A (Formula 2) 2 mg

Appropriate sterile distilled water for injection

pH adjuster

Succinone A is dissolved in sterile distilled water for injection, adjusted to pH 7.2 with a pH adjuster, the total amount is 2 ml, and filled into 2 ml ampoules to prepare an injection.

Formulation Example 3

Succinone B 200 mg

Lactose 100mg

Starch 100mg

Magnesium stearate proper amount

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

Formulation Example 4

Succinone (Formula 1) 10mg

Lactose 100mg

Starch 50mg

Magnesium stearate proper amount

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

Formulation Example 5

Succinone A (Formula 2) 100 mg

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate appropriate amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 6

Succinone B 5 mg

Lactose 100mg

Starch 93mg

Talc 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 7

Succinone 1000mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

Formulation Example 8

1000 mg n-hexane extract

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

The pharmaceutical preparation containing the sautinone compound according to the present invention or n-hexane extract containing it as an active ingredient as an active ingredient has little toxicity and is expected to be useful as a new hepatocyte protective agent because it has excellent hepatocyte protective activity. do.

Claims (2)

It is prepared by containing at least one compound selected from the compounds of formulas (1), (2) and (3) as an active ingredient, and adding a pharmaceutically acceptable excipient thereto and formulating it in a conventional method for preparing a pharmaceutical formulation. Pharmaceutical preparations having hepatocellular protective activity. Formula 1 (Sauchinone) Formula 2 (Sauchinone A) Formula 3 (Sauchinone B) The extract obtained by boiling hot water with methanol and concentrated under reduced pressure was suspended in distilled water, partitioned with CH ₂ Cl ₂ , concentrated under reduced pressure, suspended in methanol and partitioned with n -hexane to give the compounds of Formulas 1, 2 and 3 A pharmaceutical preparation having hepatocellular protective activity prepared by obtaining an n -hexane fraction containing as a main component, and adding to the fraction a pharmaceutically acceptable conventional excipient and formulating it by a conventional method for preparing a pharmaceutical preparation. Formula 1 (Sauchinone) Formula 2 (Sauchinone A) Formula 3 (Sauchinone B)