KR100470150B1 - Meso-dihydroguairetic acid with the activity inhibiting hepatic fibrosis - Google Patents

Abstract

The present invention relates to a meso-dihydroguairetic acid derivative for inhibiting hepatic fibrosis having a structure of formula (1):

Here, R ₁ and R ₂ are each hydrogen or an alkyl group having 1 to 4 carbon atoms, and R ₃ and R ₄ are each independently an alkyl group having 1 to 4 carbon atoms.

The present invention also relates to a novel use of meso-dihydroguairetic acid of formula (1 ') as a hepatic fibrosis inhibitor.

(One')

The present invention also reveals that the Tohubac extract containing the compound (1 ') can be used as an inhibitor of liver fibrosis.

Description

Meso-dihydroguairetic acid with the activity inhibiting hepatic fibrosis

The present invention relates to a meso-dihydroguairetic acid derivative for inhibiting hepatic fibrosis having a structure of the following formula (1) and tofubac extract having hepatic fibrosis inhibitory activity.

(Wherein R ₁ and R ₂ are each hydrogen or an alkyl group having 1 to 4 carbon atoms, and R ₃ and R ₄ are each independently an alkyl group having 1 to 4 carbon atoms.)

Liver cirrhosis is caused by various causes such as excessive intake of alcohol, misuse of drugs, viral hepatitis, cholestasis due to biliary abnormalities. Because it is found, death rate is high and causes social problem. In Korea, liver disease patients, represented by cirrhosis, have been steadily increasing since the late 70s. In particular, Korea is the number one cause of death among men in their 40s (National Statistical Office, Statistical Yearbook of Death, Seoul, 1999).

Liver cirrhosis is caused by the fibrosis of liver tissue. The homeostasis of the production and degradation of connective tissue is lost, and the connective tissue is excessively accumulated, necrosis, or inflammation in the liver tissue. In particular, hepatic stellate cells (HSCs) that have metastasized to myofibroblasts are known to advance the process of fibrosis of the liver by proliferating and moving to produce excess connective tissue (Gressner et al.Biochem. Biophys. Res. Commun. 151, 222. (1988)). Therefore, the development of therapeutic agents for inhibiting liver fibrosis or liver cirrhosis is currently focused on whether the drugs can inhibit the activity and proliferation of HSCs, but there is no development of effective therapeutic agents.

In Korea, there are a large number of patients with liver disease, but there are no treatments, and liver protection agents derived from folk medicines are often used.However, even natural products derived from folk medicines are effective. The amount of the active ingredient is small and side effects may occur due to the other ingredients contained therein. Therefore, it may be necessary to study the structure and the active mechanism by purely separating the natural products and liver substances having hepatoprotective activity and hepatic fibrosis inhibitory activity.

Machilus thunbergii Sieb et Zucc. Is a tree belonging to the family Lauraceae, and its bark is used as a medicinal part. Muscle injury, soil bloating, muscle tremor and leg edema Iii) has been used in the private sector for the treatment of medicinal herbs (information stone, medicinal herbs (medicinal herbs), metabolism (plants), Younglimsa, p 458 (1990)). Tohubak was called and mixed with Magnolia officinalis in appearance, but it is easily identified by physicochemical tests and has no component association. The lignans such as machilin AI, nectandrin A, and B were isolated and reported as the constituents of Tohubak, and in addition to lignans, marinnan, scopoletin, and syringa It has been found to contain syringaresinol, quercetin, and kaempferol (Simomura et al. Phytochem . 26 , 1513, (1987), Shimomura et al., Phytochem . 27 , 634, (1988), Karikome et al., Phytochem. 30 , 315, (1991)).

Therefore, the inventors of the present invention have isolated the liver cells and HSCs of rats cultured primarily from natural products, which have been used for the prevention and treatment of hepatitis and liver cirrhosis in folk medicine or traditional medicine to separate substances having new liver cirrhosis prevention and therapeutic activity from natural products. Was used as a screening system to search for liver protective activity and cirrhosis inhibiting activity. To this end, we induced toxicity in hepatocytes of rats incubated with galactosamine, a hepatotoxic substance known to cause viral hepatitis-like damage in its function and form, and the hepatoprotective activity was primarily searched. . In addition, proliferation inhibitory activity against HSCs induced liver fibrosis was also searched. As a result, it was confirmed that the total methanol extract of Tohubac showed significant hepatocellular protective activity and hepatic fibrosis inhibitory activity. From this, various chromatographic methods were used to prepare meso-dihydroguairetic acid of Formula (1 ′). Isolation and purification identified the structure. Furthermore, the present invention was completed by synthesizing a derivative of the general formula (1 ') and confirming their hepatic fibrosis inhibitory activity.

An object of the present invention is a novel use of meso-dihydroguairetic acid derivative of formula (1) for inhibiting hepatic fibrosis, hepatic fibrosis inhibitor of meso-dihydroguairetic acid of formula (1 ') and liver Tofubak extract having a fibrosis inhibitory activity.

Figure 1 illustrates the process of preparing the Tohubak extract of the present invention,

Figure 2 is a graph showing the effect of GPT release on hepatocytes of rats induced hepatotoxicity of each fraction of the Tohubak extract of the present invention,

3 is a graph showing the effect of GPT release on hepatocytes of rats induced hepatotoxicity of the compound of formula (1 ′) of the present invention,

Figure 4 is a graph showing the inhibitory effect on the proliferation of hepatic radial cells of the compound of formula (1 ') of the present invention.

The present invention provides meso-dihydroguairetic acid derivatives of formula (1) having hepatic fibrosis inhibitory activity:

Here, R ₁ and R ₂ are each hydrogen or an alkyl group having 1 to 4 carbon atoms, and R ₃ and R ₄ are each independently an alkyl group having 1 to 4 carbon atoms (except for meso-dihydroguairetic acid).

The present invention also provides a hepatic fibrosis inhibitor comprising meso-dihydroguairetic acid having a structure of the following formula (1 ') as an active ingredient.

(One')

In addition, the SAT Magnolia extract present invention is prepared by extracting soil Magnolia with C _1-4 alcohol, or re-suspended in water and CH ₂ A soil Magnolia CH ₂ Cl ₂ fraction was Cl ₂ fraction, or to remove it again by column chromatography to provide.

Hereinafter, the present invention will be described in detail.

The present invention provides a new use for use as an inhibitor of hepatic fibrosis of meso-dihydroguairetic acid of the formula (1 ').

(One')

Meso-dihydroguairetic acid of the formula (1 ') having the hepatic fibrosis inhibitory activity of the present invention is isolated and purified in Tofubak.

In another aspect, the present invention provides a thorax extract having hepatic fibrosis inhibitory activity extracted thorax extract with an organic solvent. The organic solvent may be any solvent that can be used conventionally, preferably C _1-4 alcohol, most preferably methanol. In addition to the above process, the present invention provides a water fraction and a dichloromethane fraction in which the crude C _1-4 alcohol extract is suspended in water and then fractionated with dichloromethane (CH ₂ Cl ₂ ).

Specifically, the present invention will be described with an example of the extraction process of Tofu foil.

The Tobacak is ground into a powder, extracted with methanol, filtered, and concentrated under reduced pressure to obtain a Tobacco methanol extract. In addition to this process, the methanol extract is suspended in water and fractionated with dichloromethane (CH ₂ Cl ₂ ) to obtain a water fraction and a dichloromethane fraction. In addition, in the present invention, in order to purely separate the compound having hepatic fibrosis inhibitory activity from the Tohubak extract, the dichloromethane fraction of Tohubak is subjected to silica gel chromatography. The dichloromethane fraction of Tohubak was sequentially increased in polarity, and the fraction having excellent hepatic fibrosis inhibitory activity was separated. Then, silica kel chromatography was performed again with a hexane: ethyl acetate mixed solvent, and recrystallized into a white powder (1 '). To get the compound

The physicochemical and spectroscopic properties of the compound of Formula 1 'were as follows.

(1) Molecular Formula : C ₂₀ H ₂₆ O ₄

(2) the appearance of the substance: white amorphous powder

(3) [α] ²⁵ _D : ± 0 ° (CHCl ₃ ; c , 1.44)

(4) R _f : 0.51 (hexane: ethanol = 3: 1), anis.H ₂ SO ₄ -purple.

(5) IR (KBr) υ (cm-1): 3400, 2360, 1500, 1060.

(6) EIMS ( m / z ) (rel. Int.): 340 [M +] (80), 137 (100), 122 (57), 94 (40), 77 (24).

(7) Hydrogen Nuclear Magnetic Resonance Spectrum ¹ H-NMR (400 MHz, CDCl ₃ )

δ6.83 (2H, d, J = 7.8 Hz, H-5, 5 '), 6.65 (2H, dd, J = 7.8 Hz, 1.7 Hz, H-6, 6'), 6.61 (2H, d, J = 1.7 Hz, H-2, 2 '), 5.50 (2H, s, -OH), 3.84 (6H, S, -OCH3), 2.74 (2H, dd, J = 13.4 Hz, 5.1 Hz, H-7a, 7a '), 2.28 (2H, dd, J = 9.3 Hz, 3.4 Hz, H-7b, 7b'), 1.75 (2H, m, H-8, 8 '), 0.84 (6H, d, J = 6.8 Hz , H-9, 9 ').

(8) Carbon Nuclear Magnetic Resonance Spectrum ¹³ C-NMR (100 MHz, CDCl ₃ )

δ 146.4 (C-3, 3 '), 143.6 (C-4, 4'), 133.8 (C-1, 1 '), 121.7 (C-6, 6'), 114.0 (C-5, 5 ' ), 111.5 (C-2, 2 '), 55.9 (-OCH3), 39.2 (C-7, 7'), 38.9 (C-8, 8 '), 16.2 (C-9, 9').

Or more physicochemical min value based on a reference optical teyita (such Shimomura, Phytochem 26, 1513 (1987 ).) Method of a compound of formula (1 ') as compared to-a-dihydro-old child Lett acid (meso -dihydroguaiaretic acid) I sympathized

(9) Chemical structural formula

Hepatic fibrosis inhibitory activity of the Tohubac extract and the compound of formula (1 ′) isolated and purified therefrom was searched.

In the present invention, galactosamine used for hepatotoxicity is one of the representative toxic substances that cause acute hepatotoxicity, and is known to cause damage similar to viral hepatitis in its function and form (Decker et al . , Pharmacol. Rev. Physiol. Biochem. 71, 77, (1974)). The formation of uridine diphosphate hexosamine and increased synthesis of UDP-N-acetylhexasamine by galactosamine have the effect of capturing uridine and thus UDP-glucose, UDP- in the liver. It dramatically reduces galactose and UDP, reducing the biosynthesis of biopolymers associated with uracil nucleotides such as UDP-glucuronic acid, damaging organelles, and inhibiting RNA and protein synthesis (Wolfender et al . , Phytochem.Anal . 3 , 193 (1992).

In addition, HSCs, which are used to measure liver fibrosis inhibitory activity, store vitamin A in normal liver function, but are rapidly transformed into myofibroblasts due to acute and chronic liver damage, and proliferate rapidly. Collagen, proteoglycan (proteoglycan) And cells that promote the production of extracellular matrix such as hyaluronan and promote liver fibrosis (Friedman et al . , Proc. Natl. Acad. Sci. USA. 82, 8681 (1985); Gressner et al. , supra , 1988). When HSCs are isolated from the liver of rats and cultured for more than two weeks, HSCs are known to metastasize to myofibroblasts and proliferate to increase collagen production. Therefore, in the present invention, the HSCs of the passaged three or more times to obtain the metastasized form of HSCs to measure the inhibition of hepatic fibrosis by measuring the proliferation inhibitory ability of these cells. In other words, the cultured HSCs were administered with Tohubac extract and the compound of Formula (1 ′) at each concentration, and then HSCs using MTT assay (Mosmann, T., J. Immuno. Methods, 65 , 55 (1983)). The extent of proliferation inhibition was determined by measuring survival rate. In addition, the protective activity against hepatotoxicity induced by galactosamine was measured, and for this, a culture solution of hepatotoxicity induced by toxic cells was taken, and the method of Reitman and Frankel using GPT Kit (Reitman and Frankel, Am. J. Clin. , 28 , 56 (1957)) to determine the activity of GPT in the culture. As a result, crude extract of Tohubak, dichloromethane fraction or small fraction separated therefrom and the compound of formula (1 ′) significantly blocked hepatotoxicity, thereby preventing and treating cirrhosis.

The present invention also provides a meso-dihydroguairetic acid derivative of the formula (1) which is a derivative of the compound of the formula (1 ') having hepatic fibrosis inhibitory activity.

Here, R ₁ and R ₂ are each hydrogen or an alkyl group having 1 to 4 carbon atoms, and R ₃ and R ₄ are each independently an alkyl group having 1 to 4 carbon atoms. The compound of formula (1) may be prepared by conventional chemical methods such as methylation in the compound of formula (1 ').

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating cirrhosis of the liver, wherein the extract of the compound of formula (1) and / or Tofubak as an active ingredient is a compound of formula (1 ') or a derivative thereof.

The compound of formula (1 '), the compound of formula (1) or Tohubac extract of the present invention may be formulated by methods known in the pharmaceutical art, and mixed with itself or with a pharmaceutically acceptable carrier, excipient, etc. And granules, powders, tablets, capsules or injections, and the like, and the compounds of formula (1 ') and the compound of formula (1) of the present invention are pharmaceutically acceptable inorganic Or organic salts. They can also be administered orally or parenterally.

The dosage of the compound of formula (1 ′), the compound of formula (1) or Tofubak extract of the present invention may be determined by the absorption rate, excretion rate, active age, sex and condition of the patient, the severity of the disease to be treated, etc. Although appropriately selected according to the above, it is generally preferable to administer to an adult 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg per day. The unit dosage form so formulated may be administered several times at regular time intervals as needed.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for illustrative purposes of the present invention and are not intended to limit the protection scope of the present invention.

Example 1 Extraction and Fraction of Tofubac

7.5 kg of Tobacco was ground into a powder, extracted with 80% methanol at 40 ° C. using an ultrasonic apparatus, filtered, and concentrated under reduced pressure to obtain 800 g of Tobacco methanol crude extract. This Tohubac methanol crude extract was suspended in water and fractionated with CH ₂ Cl ₂ . CH ₂ Cl ₂ fraction of Tohubak was subjected to silica gel column chromatography (100 × 500 mm, 2.5 kg) with increasing polarity sequentially (hexane: ethanol = 10: 1 → ethanol → methanol), and eight fractions (I to Viii) (see FIG. 1).

Example 2

Isolation, Purification and Structure Determination of the Compound of Formula (1 ′) from Tohubac Extract

Of the eight fractions, fractions I and II showing hepatic fibrosis inhibitory activity were combined and subjected to silica gel column chromatography (80 × 400 mm, 1 kg) again (hexane: ethanol = 10: 1) to eight small fractions (i to ⅷ). ) And a white powder (1.5 g) was obtained from the double fraction V.

The physicochemical and spectroscopic properties of the compound of formula (1 ′) separated and purified above are as follows.

(1) Molecular Formula : C ₂₀ H ₂₆ O ₄

(2) the appearance of the substance: white amorphous powder

(3) [α] ²⁵ _D : ± 0 ° (CHCl ₃ ; c , 1.44)

(4) R _f : 0.51 (hexane: ethanol-3: 1), anis.H ₂ SO ₄ -violet.

(5) IR (KBr) υ (cm ^-1 ): 3400, 2360, 1500, 1060.

(6) EIMS ( m / z ) (rel. Int.): 340 [M ⁺ ] (80), 137 (100), 122 (57), 94 (40), 77 (24).

(7) Hydrogen Nuclear Magnetic Resonance Spectrum ¹ H-NMR (400 MHz, CDCl ₃ )

δ6.83 (2H, d, J = 7.8 Hz, H-5, 5 '), 6.65 (2H, dd, J = 7.8 Hz, 1.7 Hz, H-6, 6'), 6.61 (2H, d, J = 1.7 Hz, H-2, 2 '), 5.50 (2H, s, -OH), 3.84 (6H, S, -OCH ₃ ), 2.74 (2H, dd, J = 13.4 Hz, 5.1 Hz, H-7a , 7a '), 2.28 (2H, dd, J = 9.3 Hz, 3.4 Hz, H-7b, 7b'), 1.75 (2H, m, H-8, 8 '), 0.84 (6H, d, J = 6.8 Hz, H-9, 9 ').

(8) Carbon Nuclear Magnetic Resonance Spectrum ¹³ C-NMR (100 MHz, CDCl ₃ )

δ 146.4 (C-3, 3 '), 143.6 (C-4, 4'), 133.8 (C-1, 1 '), 121.7 (C-6, 6'), 114.0 (C-5, 5 ' ), 111.5 (C-2, 2 '), 55.9 (-OCH ₃ ), 39.2 (C-7, 7'), 38.9 (C-8, 8 '), 16.2 (C-9, 9').

Based on the above physicochemical spectroscopic data, the compound of formula (1 ') was identified as meso -dihydroguaiaretic acid in comparison with the literature values.

(9) Chemical structural formula

Example 3 Synthesis of Derivative (2 ') of Compound of Formula (1')

4,4'-dimethyldihydroguairetic acid (2 ') was synthesize | combined by the methylation method which the meso- dihydroguairetic acid of Example 2 is generally used.

Meso-dihydroguairetic acid (20 mg) was added to an appropriate amount of acetone in a circular flask and dissolved, and then, MeI, K ₂ CO ₃ was added thereto, followed by reaction with a heated bath. The reaction solution was concentrated under reduced pressure to obtain 50 mg of the reactant. Silica gel column chromatography was performed to obtain compound 2 '(25 mg).

The physicochemical and spectroscopic properties of the compound of formula (2 ') separated and purified above are as follows.

(1) Molecular formula : C ₂₂ H ₃₀ O ₄

(2) the appearance of the substance: white amorphous powder

(3) [α] ²⁵ _D : ± 0 ° (CHCl ₃ ; c , 1.44)

(4) R _f : 0.65 (hexane: ethanol-3: 1), anis.H ₂ SO ₄ -violet.

(5) IR (KBr) υ (cm ^-1 ): 3400, 2360, 1500, 1060.

(6) EIMS ( m / z ) (rel. Int.): 368 [M ⁺ ] (80), 151 (100), 136 (57), 108 (40), 91 (24).

(7) Hydrogen Nuclear Magnetic Resonance Spectrum ¹ H-NMR (400 MHz, CDCl ₃ )

δ6.79 (2H, d, J = 7.8Hz, H-5, 5 '), 6.65 (2H, dd, J = 7.8Hz, 1.7Hz, H-6, 6'), 6.61 (2H, d, J = 1.7 Hz, H-2, 2 '), 5.50 (2H, s, -OH), 3.82 (6H, S, -OCH ₃ ), 3.84 (6H, S, -OCH ₃ ), 2.74 (2H, dd, J = 13.4Hz, 5.1Hz, H- 7a, 7a '), 2.28 (2H, dd, J = 9.3Hz, 3.4Hz, H-7b, 7b'), 1.75 (2H, m, H-8, 8 ' ), 0.84 (6H, d, J = 6.8 Hz ,. H-9, 9 ').

(8) Carbon Nuclear Magnetic Resonance Spectrum ¹³ C-NMR (100 MHz, CDCl ₃ )

δ 146.4 (C-3, 3 '), 145.6 (C-4, 4'), 133.8 (C-1, 1 '), 121.7 (C-6, 6'), 113.5 (C-5, 5 ' ), 111.5 (C-2, 2 '), 55.7 (2x-OCH ₃ ), 55.9 (2x-OCH ₃ ), 39.2 (C-7, 7'), 38.9 (C-8, 8 '), 16.2 (C-9, 9 ').

Based on the above spectroscopic results, this compound was identified as 4,4'-dimethyldihydroguairetic acid.

Experimental Example 1 Activity Study of Tobacco Extract

Laboratory animals

Sprague-Dawley rats were supplied from the Seoul National University Animal Breeding Farm and were bred in the experimental animal laboratory of the College of Pharmacy, Seoul National University. The environment of the kennel was kept at 22 ± 5 ℃ and the lighting time was fixed from 7 am to 7 pm, and the feed was 23.2% crude protein, 4.0% crude fat, 6.0% crude fiber, 10.0% crude ash and 0.6% crude calcium. Solid feed (Seoul, Samyang) containing 0.4% was used. Rats weighing 200 ± 50g and 500 ± 50g, respectively, were used to separate hepatocytes and HSCs.

Preparation of Sample

Tohubac methanol crude extract and each fraction was dissolved in DMSO (within 0.1% final concentration), diluted with distilled water and passed through a millipore membrane (0.22m, Millex-GV, U.S.A.) to prepare aseptic state.

Hepatocyte Culture in Rats

Hepatocytes of rats were isolated using Berry and Friend's method (Berry and Friend, J. Cell Biol., 43, 506. (1984)) using a slightly modified two-step collagenase perfusion method (Kleiman and Hartin, Anal). Biochem., 94 , 308. (1979); Kang et al., Arch. Pharm. Res. , 21 , 718. (1998)). The isolated hepatocyte suspension was diluted to a concentration of 5 × 10 ⁵ cells / ml and transplanted into a culture vessel pre-coated with collagen to obtain air (95%) and CO ₂ (5%) in a 37 ° C incubator maintained at a constant humidity and temperature. The culture was continued while feeding the mixed gas.

Induction of Toxicity of Hepatocytes by Galactosamine

The isolated hepatocytes were incubated for 1.5 hours after the start of the culture by adding 1.5 mM galactosamine while the culture medium was changed and continued for 14 hours (Kiso et al . , J. Nat. Prod. 46 , 651 (1983).

Hepatoprotective activity measurement

To measure the protective activity against galactosamine-induced hepatotoxicity, Reitman and Frankel's methods (Reitman and Frankel, Am. J. Clin. Pathol., 28) were taken using a GPT kit . , 56 (1957)) was used to determine the activity of GPT in the culture.

HSCs culture and passage

HSCs of rats were isolated by using collagenase and pronase perfusion method and two-step Percoll gradient method that modified liver cell separation method (Vrochides et al., Hepatology , 23 , 1650, (1996) ). The isolated HSCs were cultured by dispensing 0.8 × 10 ⁵ cells / ml in a 60 mm culture dish. Culture medium containing 10% fetal bovine serum and 2mM glutamine in DMEM was used. When the cultured HSCs reached a confluent state, the HSCs cultured using trypsin (0.25%) / 1 mM EDTA were collected and diluted to an appropriate cell concentration for subculture.

Hepatic fibrosis inhibitory activity measurement

Hepatic fibrosis inhibitory activity was determined by measuring the proliferation inhibitory activity of HSCs transferred to myofibroblasts by passage three or more times. That is, 48 hours after the administration of the sample to each concentration of cultured HSCs by using MTT analysis (Mosmann, supra , 1983) to determine the extent of growth inhibition by measuring the survival rate of HSCs.

Statistical processing

The variation from the control was judged by the "ANOVA" test to examine the statistical significance. Statistical significance was determined when the P value was less than 5%.

Experiment result

Tobacco extract of the present invention and its inhibitory effect on the proliferation of HSCs is shown in Table 1, and the effects on hepatocyte protective activity are shown in Table 2 and Table 3.

Fraction (50 mg / ml) % Survival of HSCs Control 100.0 ± 4.0 Methanol crude extract 80.2 ± 15.3 ^* CH ₂ Cl ₂ Fraction 75.1 ± 10.0 ^* Water fraction 107.1 ± 14.0 Small fraction of CH ₂ Cl ₂ Ⅰ 38.8 ± 3.9 ^*** Small fraction of CH ₂ Cl ₂ Ⅱ 55.3 ± 4.0 ^*** Small fraction of CH ₂ Cl ₂ Ⅲ 97.4 ± 4.2 Small fraction of CH ₂ Cl ₂ Ⅳ 92.2 ± 9.1

Small fraction of CH ₂ Cl ₂ Ⅴ 95.9 ± 6.2

Small fraction of CH ₂ Cl ₂ Ⅵ 100.3 ± 2.9 Small fraction of CH ₂ Cl ₂ Ⅶ 85.5 ± 9.6

Small fraction of CH ₂ Cl ₂ Ⅷ 89.4 ± 5.9

(Note) Control group: untreated group, significance: ^* P <0.05, ^*** P <0.001

Small fraction (100 mg / ml) Relative protection rate (%) Control 100.0 ± 9.8 Reference group 0.0 ± 12.3 I 45.3 ± 3.5 ^** Ⅱ 52.2 ± 2.6 ^** Ⅲ 36.7 ± 2.5 ^* Ⅳ 25.5 ± 21.2 Ⅴ 29.5 ± 21.2 Ⅵ 38.4 ± 0.9 ^** Ⅶ 16.7 ± 2.2 Ⅷ 11.2 ± 2.5

(Note) Control group: Galactosamine-treated group, Reference group: Galactosamine-treated group, significance: ^* P <0.05, ^** P <0.01, ^*** P <0.01

Small fraction (100 mg / ml) Relative protection rate (%) Control 100.0 ± 9.8 Reference group 0.0 ± 12.3 Ⅰ 22.0 ± 14.1 Ii 8.4 ± 3.7 Ⅲ -7.4 ± 1.8 Ⅳ -35.5 ± 15.2 Ⅴ 69.3 ± 5.6 ^*** Ⅵ 59.9 ± 16.8 ^** Ⅶ 41.3 ± 5.2 ^** Ⅷ 18.5 ± 1.3

(Note) Control group: Galactosamine-treated group, Reference group: Galactosamine-treated group, significance: ^* P <0.05, ^** P <0.01, ^*** P <0.01

As shown in Table 1 and Table 2, and Figure 2, it was confirmed that the Tofubak methanol crude extract showed a significant inhibitory activity of liver fibrosis, CH ₂ Cl ₂ fraction and water fraction of the Tofubak methanol crude extract was CH ₂ Cl _{The two} fractions showed higher activity, and eight subfractions (I-VIII) of the CH ₂ Cl ₂ fraction showed similar activities to the extent that fractions I and II were similar.

In addition, as shown in Table 3, the small fractions ⅴ ~ 한 further fractionated fractions I and II also showed significant activity.

Experimental Example 2 Activity Study of Compound of Formula (1 ') and Compound of Formula (2')

Hepatoprotective activity measurement

Except for using the compound of formula (1 ') as a sample, the experiment was carried out in the same manner as in Experimental Example 1, the results are shown in Figure 3 below.

As shown in Figure 3, the amount of GPT released from damaged hepatocytes when the compound of formula (1 ') of the present invention was treated in primary cultured hepatocytes of rats induced by toxicity with galactosamine while increasing the concentration from 0.1 μM to 300 μM Significantly reduced concentration-dependently in the range of 0.1 μM to 30 μM, and showed the highest hepatocellular protective activity at 30 μM, restoring GPT to 74.4% of steady state, and at least 50% of hepatocyte protective activity at 1 μM and 10 μM, respectively. Showed.

The compound of formula (2 ') also showed hepatoprotective activity similar to the compound of formula (1'), the highest hepatoprotective activity at 30μM and the GPT value was restored to 50.6% of normal state (not shown).

RNA Biosynthesis Measurement

To investigate the effect on hepatocyte RNA degradation by galactosamine toxicity, [ ³ H] -uridine was added to the culture (1 Ci / ml) and the uridine labeled with radioisotopes was cultured in RNA. The degree of insertion into the was measured. After the culture was removed, the mixture was washed three times with HBSS and 1 ml of 10% trichloroacetic acid was added to precipitate the protein. 1 ml of a mixed solvent of ethanol-ether (3-1, v / v) was added to remove the remaining trichloroacetic acid, and the protein was dissolved in 200 µl of 1N NaOH. 150 μl of the solution was added to 3 ml of aquasol, a scintillation cocktail, and radioactivity was measured (Karner, Biochem. J. 92, 449, (1964)), and the results are shown in Table 4 below. It was.

[ ³ H] -uridine intake (cpm / well) CPM rate Control 620.1 ± 94.5 1.0 Reference group 121.9 ± 22.9 1.5 1 μM 178.3 ± 28.0 2.1 10 μM 221.3 ± 32.5 2.3 30 μM 208.6 ± 41.5 2.5

(Note) [ ³ H] -uridine intake was measured for β-emission using LSC, control group: galactosamine-treated group, reference group: galactosamine-treated group

As shown in Table 4, the compound of Formula (1 ') increased the insertion amount of [ ³ H] -uridine lowered to galactosamine at a concentration of 30 μM by 2.5 times.

Hepatic fibrosis inhibitory activity measurement

In order to investigate the hepatic fibrosis inhibitory effect of the compound of formula (1 ') having significant hepatoprotective activity against galactosamine toxicity using the cultured HSCs, HSCs were isolated and cultured in rats and passaged three times or more. After culturing to obtain HSCs transferred to myofibroblast form, 48 hours after the administration of these compounds by concentration, the survival rate (proliferation rate) was measured by MTT analysis and is shown in FIG. 4.

As a result, the compound of formula (1 ′) showed proliferation inhibitory activity of HSCs in a concentration-dependent manner, and showed cell viability of about 66.5% and 18.6% at concentrations of 50 μM and 100 μM, respectively. That is, at the concentration of 100 μM, 81.4% of the HSCs inhibited the growth, which was more excellent than the silybin (silybin) at the same concentration (29.1% at the concentration of 100μM) showed more excellent inhibition of growth.

Through the above results, the meso-dihydroguairetic acid and derivatives thereof of the present invention, a lignan compound isolated from Tohubak, Tohubac extract, and fractions thereof have a function of protecting hepatocytes from galactosamine-induced toxicity. In addition, it can be used as a preventive and therapeutic agent for new liver cirrhosis by inhibiting the proliferation of HSCs involved in the transition of liver fibrosis in liver cirrhosis, a chronic liver disease.

Claims (5)

Meso-dihydroguairetic acid derivatives having the structure of formula (1) Here, R ₁ and R ₂ are each hydrogen or an alkyl group having 1 to 4 carbon atoms, and R ₃ and R ₄ are each independently an alkyl group having 1 to 4 carbon atoms (except for meso-dihydroguairetic acid). A composition for the prevention or treatment of cirrhosis of the liver comprising a meso-dihydroguairetic acid derivative having a structure of formula (1) as an active ingredient. A composition for preventing or treating liver cirrhosis, including meso-dihydroguairetic acid having the structure of Formula (1 ′): (One') delete Magnolia extract the soil with C _1-4 alcohol, or re-suspended in water and liver cirrhosis, comprising the soil Magnolia CH ₂ Cl ₂ fractions, or the soil magnolia extract prepared by re-isolated by column chromatography in CH ₂ Cl ₂ fraction this prevention Or a therapeutic composition.