WO2009026794A1

WO2009026794A1 - Cyclohex-enone compounds from antrodia camphorata for protecting liver

Info

Publication number: WO2009026794A1
Application number: PCT/CN2008/070413
Authority: WO
Inventors: Sheng-Yun Liu; Wu-Che Wen; Mao-Tien Kuo
Original assignee: Golden Biotechnology Corporation
Priority date: 2007-08-29
Filing date: 2008-03-05
Publication date: 2009-03-05
Also published as: CN101376624A; CN101376624B

Abstract

The invention discloses acyclohex-enone compounds from antrodia camphorata, in particular 4-hydroxy-2,3-dimethoxy-6-methy-5[3,7,11-trimethyl-dodeca-2,6,10- trienyl]-cyclohex-2-enone, which can protect hepatic tissue effectively. In the invention, the acyclohex-enone compounds from antrodia camphorata for protecting liver have the effects of relieving the injure and fibrosis of liver induced by chemical liver injure, decreasing the concentration of ALT and AST, increasing the concentration of GSHPx and CAT, decreasing the injure of hepatic cell induced by free radical, improving the antioxidative effect of hepatic tissue.

Description

Antrodia camphora cyclohexenone compound for liver protection

Technical field

The invention relates to a compound for protecting liver, in particular to a kind of

(Antrodia camphorata) Extracted and purified cyclohexenone compound which can be used to protect the liver and slow the degree of liver damage and fibrosis. Background technique

The liver is the most important and most important metabolic organ in the human body. It plays an important role in the metabolism of sugar, lipids, proteins, vitamins, hormones, bile and other substances. At the same time, the liver also has secretion, excretion, biotransformation and so on. Function; The liver is also an important barrier organ of the body. Its detoxification function has an important protective effect on the body. Therefore, when the liver function is damaged, it will cause metabolic disorders and affect other organs' functions, which is serious and life-threatening. The incidence of liver disease among Chinese people is very high. According to the statistics of the Department of Health of the Executive Yuan, chronic liver disease and cirrhosis are the sixth leading cause of death, and the number of deaths per year is more than 3,000, and the number is still rising. Therefore, it is imperative to develop substances that can effectively protect the liver and can be used to prevent or treat liver-related diseases.

Antrodia camphorata (also known as Antrodia camphorata, burdock or red locust, etc.) is a perennial fungus belonging to the genus Aphyllophorales and Polyporaceae. It is a unique species of fungi in Taiwan and only grows. On the inner wall of the hollow decayed heartwood of Cinnamoum kanehirai Hay in Taiwan. Due to the extremely rare distribution of burdock trees, artificially slashed, the number of wild burdocks that can grow in the middle of it is even rarer. And because its fruiting bodies grow quite slowly, the growth period is only between June and October, so the price is very expensive.

The fruiting body of Antrodia camphorata is perennial, sessile, with cork to wood. It has a strong aroma of eucalyptus, and its morphology varies, and it has a plate shape, a bell shape, a horseshoe shape or a tower shape. It is flat and bright red at the beginning of life, and then it radiates and rewinds around it, and grows to the periphery. The color also changes to reddish brown or yellowish brown, and there are many fine pores, and it is medicinal The most valuable part.

In Taiwanese folk medicine, Niobium has the functions of detoxification, alleviating diarrhea, anti-inflammatory, treating liver-related diseases and anti-cancer. Antrodia camphorata has many complex ingredients, such as triterpenoids and polysaccharides. It is known to have many complex components. (polysaccharides such as β-D-glucan), adenosine, vitamins (such as vitamin B, nicotinic acid), proteins (including immunoglobulins), superoxide dismutase (SOD), trace amounts Elements (such as calcium, phosphorus, strontium), nucleic acids, sterols, and blood pressure stabilizing substances (such as antodia acid), which are considered to have anti-tumor, immune-enhancing, anti-allergic, anti-pathogenic, anti-hypertensive , lowering blood sugar, lowering cholesterol, protecting the liver and fighting fatigue.

Among the many components of Antrodia camphorata, triterpenoids are the most studied. Triterpenoids are a general term for a combination of thirty carbon elements into hexagonal or pentagonal natural compounds. The bitter taste of Antrodia camphorata is mainly derived from triterpenoids. In 1995, Chemg et al. found that three extracts of triterpenoids based on ergostane were found in the extract of Antrodia camphorata fruit bodies: antcin A, antcin B and antcin C (Chemg, IH, and Chiang, HC 1995. Three new triterpenoids from Antrodia cinnamomea. J. Nat. Prod. 58:365-371). Chen et al. and E. sinensis extracted three kinds of triterpenoids such as zhankuic acid A, zhankuic acid B and zhankuic acid C (Chen, C. Η·, and Yang, SW 1995. New steroid acids from Antrodia) Cinnamomea, ― a fungus parasitic on Cinnamomum micranthum. J. Nat. Prod. 58: 1655- 1661 ). In addition, in 1995, Chiang et al. also found three other new triterpenoids, sesquiterpene lactone and two bisphenol derivatives, from the fruit extract, which is antrocin, 4, 7-dimethoxy-5-indenyl-1,3-benzodioxane

(4,7-dimethoxy-5-methy-l,3-benzodioxole) with 2,2',5,5'-tetradecyloxy-3,4,3',4'-bis-arylenedioxy —6,6'-Dimercaptobiphenyl (2,2 ^, ,5,5'-teramethoxy-3,4,3',4 ^, -bi-methylenedioxy- 6,6'- dimethylbiphenyl ) ( Chiang, H. C, Wu, DP, Chemg, IW, and Ueng, CH 1995. A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. Phytochemistry. 39:613-616). In 1996, Chemg et al. found four new triterpenoids in the same way: antcin E, antcin F, methyl antcinate G, methyl antcinate H (Chemg, IH, Wu, DP, and Chiang, HC 1996. Triteroenoids from Antrodia cinnamomea. Phytochemistry. 41 :263-267 ); and Yang et al. found two new compounds, zhankuic acid D, zhankuic acid E, and lanostane, which are based on ergot. A new compound for the skeleton: 15 α -Acetyl-dehydrosulphurenic acid ( 15 a -acetyl-dehydrosulphurenic acid )

( dehydroeburicoic acid ) and dehydrated lycopene acid ( dehydrasulphurenic acid X Yang, SW, Shen, Y. C, and Chen, CH 1996. Steroids and triterpenoids of Antrodia cinnamomea- a fungus parasitic on Cinnamomum micranthum. Phytochemistry. 41 : 1389-1392 ).

Although it is known from many experiments at present, the extract of Antrodia camphorata has the aforementioned effects, and its components are gradually analyzed. However, what kind of active ingredients in the extract can promote the liver-protecting effect of Antrodia camphorata, and no specific The relevant active ingredients are subject to further experimental research to clarify, so if we can find out the true effective liver-protecting ingredients contained in the extract, it will be beneficial to the research on the liver-related mechanism of Niuzhizhi and the treatment of liver-related diseases. There is great help with prevention. Summary of the invention

In order to clarify why the component of the extract of Antrodia camphorata has the effect of protecting the liver, the present invention separates and purifies the compound of the formula (1) from the extract of Antrodia camphorata;

Wherein X is oxygen (0) or sulfur (S), Y is oxygen or sulfur; is hydrogen (H), sulfhydryl (C3⁄4) or (CH ₂ ) _m -CH ₃ , R ₂ is hydrogen, fluorenyl Or (CH ₂ ) _m -CH ₃ , R ₃ is hydrogen, fluorenyl or (CH ₂ ) _m -CH ₃ , m = 1-12; n = 1-12.

Among the compounds of the formula (1), a compound of the formula (2) shown below is preferred:

a compound of formula (2) having the chemical name 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6, 10-dode carbon) Trienyl-2-cyclohexenone (4-hydroxy-2,3-dimethoxy-6-methy-5(3,7,11-trimethyl-dodeca-2,6, 10-trienyl)-cyclohex-2- Enone ), the molecular formula is C ₂₄ H ₃₈ 04, It has a pale yellow powder and a molecular weight of 390.

The cyclohexenone compound of the formula (1) and the formula (2) in the present invention is isolated and purified from an aqueous extract of Antrodia camphorata or an organic solvent extract, and the organic solvent may include an alcohol (for example, decyl alcohol, ethanol or propanol), an ester ( For example, ethyl acetate), an alkane (e.g., hexane) or a halogenated alkane (e.g., chlorodecane, ethyl chloride), but not limited thereto, preferably an alcohol, and more preferably an ethanol.

By the cyclohexenone compound of the above formula (1), formula (2), the present invention is applied to liver protection and prevention and slowing of liver damage, and after feeding liver injury induced by carbon tetrachloride, feeding anthraquinone Cyclohexenone compounds help to slow the damage and fibrosis of rat liver tissue, and reduce alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in rat plasma. The concentration, in turn, achieves the efficacy of protecting the liver; this, as well as the concentration of catalase (CAT), in order to reduce the damage caused by free radicals to liver cells and the oxidative stress they bear, and improve the resistance of liver tissue Oxidation ability.

On the other hand, in the present invention, the compound of the formula (1) and/or the formula (2) can also be used as a component of a pharmaceutical composition for treating liver damage, thereby improving symptoms caused by liver damage in a mammal such as a human. The aforementioned pharmaceutical composition may further comprise a pharmaceutically acceptable carrier in addition to an effective amount of a compound of the formula (1) and/or formula (2). The carrier may be, but is not limited to, an excipient such as water, a filler such as sucrose or starch, a binder such as a cellulose derivative, a diluent, a disintegrant, an absorption enhancer or a sweetener. . The pharmaceutical composition of the present invention can be produced according to a conventional pharmacy preparation method, and the active ingredient dose of the formula (1) and/or the formula (2) is mixed with one or more carriers to prepare a desired dosage form, and the dosage form is prepared. These may include lozenges, powders, granules, capsules or other liquid preparations, but are not limited thereto.

The embodiments of the present invention will be further described with reference to the accompanying drawings, which are set forth to illustrate and not to limit the scope of the present invention, without departing from the spirit and scope of the invention. In the meantime, the scope of the present invention is defined by the appended claims. DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an example of the present invention, in the case of carbon tetrachloride-induced liver injury, anthraquinone cyclohexenone compound The effect on the weight of the rat. In the figure *: control group; Δ: negative control group (20% carbon tetrachloride); □: positive control group (20% carbon tetrachloride + milk thistle); country: treated with carbon tetrachloride and fed 300 Mg/kg experimental group of anthraquinone cyclohexenone compound; A: an experimental group treated with carbon tetrachloride and fed with 1000 mg/kg of anthraquinone cyclohexenone compound; ·: treated with carbon tetrachloride and fed 3000 mg/ An experimental group of kg of anthraquinone cyclohexenone compound;

Fig. 2 is a graph showing the effect of the anthraquinone cyclohexenone compound on the food intake of rats in the case of liver injury induced by carbon tetrachloride in the examples of the present invention. In the figure *: control group; Δ: negative control group (20% carbon tetrachloride); □: positive control group (20% carbon tetrachloride + milk thistle); country: treated with carbon tetrachloride and fed 300 Experimental group of mg/kg anthraquinone cyclohexenone compound; A: experimental group treated with carbon tetrachloride and fed with 1000 mg/kg of anthraquinone cyclohexenone compound; * : treated with carbon tetrachloride and fed 3000 mg/ An experimental group of kg of anthraquinone cyclohexenone compound;

Fig. 3 is a view showing the results of pathological changes of the liver surface of rats after the liver injury induced by carbon tetrachloride and the administration of anthraquinone cyclohexenone compound in the present invention. Figure (A) is the control group (only corn oil is given); Figure (B) is the negative control group (20% carbon tetrachloride is given); Figure (C) is the positive control group (20% carbon tetrachloride and 200%) Mg/kg silybum); Figure (D) is a low-dose anthrax ring cyclohexenone compound experimental group (20% carbon tetrachloride and 300 mg/kg anthraquinone cyclohexenone compound); Figure (E) is medium Dosage of Antrodia camphora cyclohexenone compound experimental group (administer 20% carbon tetrachloride and 1000 mg/kg anthraquinone cyclohexenone compound); Figure (F) is a high dose of Antrodia camphora cyclohexenone compound experimental group (giving 20% four Carbon chloride and 3000 mg/kg of anthraquinone cyclohexenone compound); Fig. 4 is a result of pathological changes of liver tissue damage in rats after liver injury induced by carbon tetrachloride according to an example of the present invention. Figures (A) and (B) show the results of vigorous mitosis in swollen hepatocytes and hepatocytes after staining with hematoxylin-eosin staining (H&E stain) (magnification 200 X and 400 X) Fig. (C) shows the vacuolar degeneration of liver tissue after staining with collagen staining (MT stain), and the liver fibrosis divides the liver parenchyma into many nodules, resulting in observation of cirrhosis ( The magnification is 100 X).

Fig. 5 is a view showing the pathological changes of liver fibrosis stained by collagen staining (MT stain) before and after liver injury induced by carbon tetrachloride in the Example of the present invention. Figure (A) shows normal liver tissue; Figure (B) shows the degree of fibrotic lesions and collagen hyperplasia in liver tissue after treatment with carbon tetrachloride; Figure (C) shows the center of the liver after carbon tetrachloride treatment Vein and portal vein Incompletely septum; Figure (D) After treatment with carbon tetrachloride, the liver tissue forms a complete septum and intersects with each other, and divides the liver parenchyma into many nodules, but the septum is still thin; E) After treatment with carbon tetrachloride, the septum of the liver tissue becomes thicker and forms cirrhosis; Figure (F) shows the liver fibrosis and green collagen after treatment with carbon tetrachloride. The magnification of FIGS. 5A to 5E is 40x, and the magnification of FIG. 5F is 100x. detailed description

First, the mycelium, fruiting body or a mixture of the two is taken from Antwdia camphorata, and extracted by water or an organic solvent by a conventional extraction method to obtain an aqueous extract of Antrodia camphorata or an organic solvent extract. Wherein, the organic solvent may include an alcohol (for example, decyl alcohol, ethanol or propanol), an ester (such as ethyl acetate), an alkane (such as hexane) or a halogen (for example, chlorodecane, ethyl chloride), but Not limited to this. Among them, preferred are alcohols, and more preferably ethanol.

The extracted aqueous extract of Antrodia camphorata or the organic solvent extract can be further separated and purified by high-performance liquid chromatography, and then each fraction is subjected to biochemical tests related to liver protection. Finally, the composition of the liver-protecting liquid is analyzed, and the components that may produce liver-protecting effects are subjected to biochemical experiments such as the effect of slowing liver damage. Finally, it was found that the compound of the formula (1) / formula (2) of the present invention has an effect of slowing liver damage to protect the liver.

For convenience of description of the present invention, 4-hydroxy-2,3-didecyloxy-6-mercapto-5 (3,7,11-trimethyl-2,6,10-) of the formula (2) will be exemplified below. The dodecanetriene)-2-cyclohexenone compound will be described. Further, in order to confirm 4-hydroxy-2,3-dimethoxy-6-mercapto-5(3,7,11-tridecyl-2,6,10-dodecatriene)-2-ring The hexenone compound has a liver-protecting effect. In the present invention, after the chronic liver injury is induced in the rat by the chemical substance carbon tetrachloride (CC1 ₄ ), different doses of the anthraquinone cyclohexenone compound are administered, and the degree of liver damage is detected. , degree of liver fibrosis, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations, and glutathione (GSH), glutathione peroxidase (GSHPx) , liver enzymes such as catalase (CAT), superoxide dismutase (SOD), liver antioxidant enzymes, etc., to determine the liver-protecting ability of the anthraquinone cyclohexenone compound.

From these liver tissue observations and biochemical test values, 4-hydroxy-2,3-dimethoxy-6-nonyl-5 (3,7,11-tridecyl-2,6,10-dode carbon) was confirmed. Triene)-2-cyclohexenone compound has reduced Effects of liver injury and fibrosis caused by academic liver injury, and alanine aminotransferase

Inflammation of liver function such as (ALT) and aspartate aminotransferase (AST) is a concentration in the blood, and also promotes glutathione peroxidase (GSHPx) and catalase in the liver.

The content of (CAT) is to reduce the damage caused by free radicals to liver cells and the oxidative stress they bear, and to improve the antioxidant capacity of liver tissues, thereby achieving the purpose of protecting the liver. The foregoing embodiment is described in detail: 3⁄4 under: Example 1:

4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone Separation

Put about 100 grams of Astragalus membranaceus mycelium, fruiting body or a mixture of the two into a triangular conical flask, add appropriate proportion of water and alcohol (for example, more than 70% aqueous alcohol solution, at 20~25 °C The mixture was stirred for at least 1 hour, and then filtered through a filter paper and a 0.45 μηι filter to collect the extract.

The collected Antrodia camphorata extract was analyzed by high performance liquid chromatography using a column of RP18, and decyl alcohol (A) and 0.1% to 0.5% aqueous acetic acid solution. (B) as the mobile phase (the ratio of the solution is: 0-10 minutes, B ratio is 95% to 20%; 10-20 minutes, B ratio is 20% to 10%; 20-35 minutes, The B ratio is 10% to 90%; 35 to 40 minutes, B ratio is 10% to 95%), and is eluted at a rate of 1 ml per minute, and analyzed by an ultraviolet-visible full-wavelength detector.

The extract is concentrated and concentrated for 25 minutes to 30 minutes to obtain a pale yellow powdery solid product, which is 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11 - Tridecyl-2,6,10-dodecatriene)-2-cyclohexenone. After analysis, the molecular formula is C ₂₄ H ₃₈ 0 ₄ , the molecular weight is 390, and the melting point (mp ) is 48 ° C to 52 ° C. The nuclear magnetic resonance (NMR) analysis values are as follows:

1.51, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 111, 2.25, 3.68, 4.05, 5.07 and 5.14. ¹³ C-NMR (CDCl ₃ ) 5 (ppm): 12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 39.71, 39.81, 4.027, 43.34, 59.22, 60.59, 120.97, 123.84, 124.30, 131.32, 135.35 , 135.92, 138.05, 160.45 and 197.12. Example 2:

Liver test of burdock ring cyclohexenone compound

Because the main factors causing liver disease are viral, alcoholic and chemical, and the pathological phenomenon in the animal model of the mouse is consistent with the human body, it is chemical liver injury. Therefore, the evaluation method is for chemistry. Assessment of liver function in patients with liver injury; experimental model of chronic liver injury induced by carbon tetrachloride (CC1 ₄ ), and biochemical values of liver injury and liver tissue sections were examined to investigate the treatment of anthraquinone cyclohexenone compound The effect on chronic liver injury in rats. Among them, the toxicity of carbon tetrachloride can cause hepatocyte necrosis, and further develop into fibrosis of liver. If it continues to be administered, it will cause irreversible cirrhosis. The principle of inducing liver damage is due to four Carbon chloride can be activated by liver microsomal enzymes to form trichloromethyl radical (CC1 ₃ - ), which is combined with protein to cause protein synthesis to be blocked, and cause lipid catabolism, causing triglyceride in liver cells. Accumulation, in addition to the peroxide formed by the trichlorodecane radical, can also lead to lipid peroxidation and damage to the liver cell membrane, resulting in enzyme exudation in the liver and cytopathic necrosis. Therefore, the liver injury model of carbon tetrachloride is a simulation of human liver cirrhosis, and thus can be applied to evaluate the therapeutic effect of a drug or a food component for treating liver diseases such as liver fibrosis. The test steps are detailed below:

(1) Establishment of an animal model of liver injury induced by carbon tetrachloride

The experimental animals were five-week-old rats (Sprague-Dawley, SD) purchased from Lesco Biotech Co., Ltd., and the rats were indwelled in the animal breeding room for two weeks after purchase, and the body weight was similar (about 220 g). To 270 g) healthy adult rats were tested. Before the experiment, the rats were divided into six groups, 12 rats in each group. The body weight of the rats was recorded for each dose, which was used as the basis for the subsequent experimental doses. The grouping experiments shown in Table 1 were used as the control group and the negative control. Groups, positive control groups, and three groups of experimental groups of 300 mg/kg, 1000 mg/kg, and 3000 mg/kg, respectively. Among them, silymarin can alleviate the inflammatory reaction in the liver and reduce the degree of liver damage. It can also reduce the toxicity of the liver by neutralizing the toxicity of toxic substances or competing with the toxic substances. The amount of absorption of the substance; and the anti-oxidant effect of the milk thistle can protect the liver cells from free radical damage. Because of its many different liver damage studies, including a large number of animal experiments and some clinical experiments, it has been shown to have a very good function of protecting the liver. Therefore, it is recognized as a therapeutic drug for liver diseases in modern medicine, and The animal model of liver injury was also used as a drug in the positive control group. Table 1. Experimental animal group and its feeding quality and dosage

Group 20% (v/v) burdock

Carbon tetrachloride cyclohexenone compound

A (control group) 0 0 0

B (negative control group) 2 ml/kg BW 0 0 *, ,,

C (positive control group) 2 ml/kg BW 0 200 mg/kg group

Real D 2 ml/kg BW 300 mg/kg 0

Test E 2 ml/kg BW 1000 mg/kg 0

Group F 2 ml/kg BW 3000 mg/kg 0

Group A in Table 1 is a control group, which is a gastric tube perfusion with corn oil (purchased from Sigma chemical co.); Group B to F is filled with 20% of corn oil by gastric tube perfusion (v/v) ) Carbon tetrachloride (purchased from Japan's Shijiujiu), each rat was given a dose of 2 ml/kg according to its body weight, and gastric perfusion was given 2 days a week (Tuesday, Thursday afternoon). Week; group C was prepared by gastric tube perfusion with physiological saline to prepare 200 mg / kg dose of milk thistle; group D to F was separately perfused with gastric tube by the preparation of Example 1 and prepared with physiological saline 300 mg /kg, 1000 mg/kg and 3000 mg/kg doses of Antrodia camphorata 4-hydroxy-2,3-didecyloxy-6-mercapto-5 (3,7,11-tridecyl-2,6 , 10-dodecatriene)-2-cyclohexenone compound, and the amount of food in the C to F group was 10 ml/kg of the above substance per body weight, and was given 5 days a week (am) Gastric tube perfusion was continued for 8 weeks. The results of the measured rat body weight and rat feeding during the experiment are shown in Fig. 1 and Fig. 2.

Figure 1 is a control group and oral administration of 20% (v/v) carbon tetrachloride and administration of silymarin or burdock ring. Figure 1 shows that there is no significant difference between the groups in the initial body weight, 20% by oral administration. (v/v) After one week of carbon tetrachloride treatment, the body weight of the rats in the control group and the experimental group showed a downward trend compared with the control group without carbon tetrachloride treatment; and at the eighth week, The final body weight measured by the negative control group given the milk thistle or the burdock cyclohexenone compound was 366.9 g, which was significantly lower than the final body weight of the control group of 448.7 g, indicating that the treatment of carbon tetrachloride caused the rat body. The lesion, in turn, causes the rat to lose weight. In addition, the positive control group of oral milk thistle showed a final body weight of 375.8 g, and the weight loss was less than that of the negative control group. This indicates that the body weight of the rat decreased due to the influence of carbon tetrachloride, but the effect of the milk thistle Rats did not lose weight; instead, they were fed different concentrations of Antrodia camphorata In the experimental group of ketone compounds, when the feeding doses were 300 mg/kg, 1,000 mg/kg and 3,000 mg/kg, respectively, the rats weighed were 386.6 g, 365.1 g and 355.0 g, respectively. The mg/kg of Antrodia camphora cyclohexenone compound can effectively alleviate the weight loss caused by carbon tetrachloride.

Figure 2 is a control group and oral administration of 20% (v/v) carbon tetrachloride and administration of silymarin or burdock ring. It can be seen from Fig. 2 that the feed consumption of rats in each group increased at the first 1-5 weeks after treatment, but after the fifth week, the feed consumption of each group began to decrease and showed a downward trend, which was induced by carbon tetrachloride. Rat liver injury affects the food intake of experimental animals.

(2) Effects of carbon tetrachloride on liver, kidney and spleen weight

Rats were sacrificed at the end of the eighth week, and the liver, kidney and spleen were weighed by laparotomy, and the measured liver, kidney and spleen weights were compared to the total weight of the rats from which the organs were obtained. The ratio (%) of these organs to the total weight of the rats was converted to observe the effects of carbon tetrachloride treatment and administration of anthraquinone cyclohexenone on the weight of liver, kidney and spleen. In addition, each experimental group The data are expressed as mean and standard deviation (mean±SD). One-way analysis of variance (ANOVA) is used. When the variance analysis results show significant differences, the LSD method is further used. Multiple comparisons were performed, or the Student Mest statistical program was used to compare the differences between the experimental group and the control group, the positive control group, and the negative control group of the Antrodia camphora ketene compound, and /?<0.05 was significant. Differences, the results are shown in Table 2.

Table 2. Feeding of anthraquinone cyclohexenone compound after liver injury induced by carbon tetrachloride

Effect on the weight of experimental animals (%)

Group / dose spleen kidney liver

(mg/kg) (%) (%) (%) Control group 0.16±0.03 0.62±0.06 2.68±0.33 Negative control group (20% CCL ₄ ) 0.25±0.08* 0.76±0.11* 3.30±0.54* Positive control group ( Milk thistle silymarin) 0.22±0.05* 0.71±0.08* 3.43±0.63*

Enone compound

300 0.20±0.05* 0.68±0.06* 3.29±0.35

1,000 0.22±0.06* 0.69±0.13 3.20±0.47*

3,000 0.30±0.10* 0.73±0.08* 3.65±0.53* ^{The D} data is the mean value and the standard machine difference (Mean ± SD ).

* The data of each treatment group and control group were compared by Student's i-test, and the value of less than 0.05 was considered to be significant.

a The data of each treatment group and the negative control group were compared by Student's i-test, and the value of less than 0.05 was considered to be significant.

As can be seen from Table 2, the percentage (%) of liver, kidney and spleen weight and total body weight measured by carbon tetrachloride-treated negative control group without silymarin or burdock hexacene ketene compound were significant ( /? < 0.05 ) Higher than the control group, this shows that carbon tetrachloride has caused liver, kidney and spleen lesions, and the weight of these organs has increased; the liver, kidney and spleen weight indicated by the positive control group of oral milk thistle It was higher than the control group due to the influence of carbon tetrachloride, but the weight of these organs was still lower than that measured by the negative control group due to the liver protection effect of the milk thistle itself. In addition, compared with the control group, the percentage of liver, kidney and spleen weight (%) increased in the experimental group fed with different concentrations of Antrodia camphora, but the weight of these organs was still lower than the negative control. The weight measured by the group was low. The results showed that feeding the rats with the anthraquinone cyclohexenone compound can effectively reduce the damage of the liver, kidney and spleen caused by carbon tetrachloride, so that the weight of these organs does not last. rising.

(3) Liver histopathological observation

When the rat liver was removed by laparotomy, the pathological changes of the liver surface were observed with the naked eye, and liver tissue sections were taken. According to the anatomical location of the liver, the five hepatic lobules were cut into half of the liver tissue and frozen in a freezing rejection at -80 °C for subsequent detection of antioxidant enzyme activity. The remaining liver tissue was fixed in a 10% neutral formalin solution for one week. After roughing and paraffin embedding, tissue sections of 2 μηι thickness were prepared and stained with hematoxylin-eosin (Hematoxyline). -eosin satin, H&E) observed fat accumulation, inflammation, cell necrosis and fibrosis, and special staining of collagen fibers (Masson's trichromc, MT) stained reticular fibers and collagen fibers to assess the extent of liver fibrosis, in general Various histopathological changes of liver injury after the above pathological staining were observed under an optical microscope (Opticphot-2, Nikon, Tokyo, Japan).

The assessment of chronic liver injury is based on the literature published by Jonker et al. (Jonker, AM, Dijkhuis, F. W" Boes, A., Hardonk, MJ and Grand J. 1992. Immunohistochemical study of extracellular matrix in acute galactosamine hepatitis in rats. Hepatology The method described in 15(3): 423-31.), the degree of inflammation of the liver cells, vacuolar degeneration, liver Semi-quantitative analysis of cell necrosis and bile duct hyperplasia; assessment scores are 0 to 4 grades, where grade 0 represents no lesion (-, none), grade 1 represents mild (+, slight), grade 2 represents mild (+ + , mild ), level 3 represents moderate ( + + + , moderate ) and level 4 represents significant ( + + + + , remarkable ) „

For semi-quantitative analysis of liver fibrosis, it can be based on Gabriele (Gabriele, B. 1997. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 26: 643-649.) and Wang ( Wang, GS, Eriksson, L. C, Xia, L., Olsson, J. and Stal, P. 1999. Dietary iron overload inhibits carbon tetrachloride-induced promotion in chemical hepatocarcinogenesis: effects on cell proliferation, apoptosis, and antioxidation. J Hepatol. 30(4): 689-98.) et al., the method of liver fibrosis is divided into the following 0 to 4 grades: Grade 0 represents normal liver tissue without any liver fibrosis, Grade 1 represents collagen hyperplasia, but no septum is formed (radial fibrosis in the central vein or portal vein), and grade 2 represents an incomplete septum between the central vein and the portal vein (in this case) Level 3 does not meet each other. Level 3 represents the formation of a complete septum, which intersects each other and divides the liver parenchyma into many nodules. However, the septum is still thin and grade 4 represents the formation of a complete septum, and the septum becomes thicker, that is, complete cirrhosis. The results are shown in Figures 3 to 5 and Table 3.

Fig. 3 is a view showing the observation of liver surface pathological changes in the control group and the experimental group of the control group and the carbon tetrachloride-induced liver injury and the administration of the milk thistle or the burdock. As can be seen from the figure, the liver surface of the control group was smooth and complete (please refer to Fig. 3A); the negative control group treated with carbon tetrachloride showed yellowish liver color, rough surface, hard touch and unevenness, and appeared Swelling (see Figure 3B); a positive control group treated with carbon tetrachloride and feeding silymarin, and an experimental group treated with carbon tetrachloride and fed with different concentrations of anthraquinone cyclohexenone compound, although the liver surface was There was swelling and surface lesions due to the influence of carbon tetrachloride (please refer to Figure 3C to Figure 3F), but the degree was slightly lower than that of the negative control group, which was fed with the 3000 mg/kg dose of Antrodia camphora cyclohexanone compound experimental group. The swelling and pathological condition of the liver surface is the slightest (see Figure 3F). This result indicates that the anthraquinone cyclohexenone compound can effectively improve the symptoms of liver surface damage caused by carbon tetrachloride. Table 3. Feeding of anthraquinone cyclohexenone compound after liver injury induced by carbon tetrachloride

Results of histopathological evaluation of liver in experimental animals

Group/dose degree of liver damage"" Liver fibrosis degree control group ¹² - - - - 0.0 12 - - - - 0.0 Negative control group (20% CCL ₄ ) - - 2 3 4 3.2* - - 2 3 4 3.2" Positive control group (silymarin) - 2 1 4 3 2.8* - 2 1 5 3 2.8*

Enone compound

5 6 1.8* ^a

300 - - 1 1.8* ^a - 6 4 1 1

1,000 - 3 6 1 1 2.0* ^a 2 1 4 3 1 2.0* ^a

3,000 - 2 6 1 2 2.3* ^a - 2 4 3 2 2.5*

^D. Assessment of chronic liver injury According to the method of Jonker et al. (1992), the degree of inflammation of hepatocytes, vacuolar degeneration, hepatocyte necrosis and bile duct hyperplasia were semi-quantitatively analyzed. The evaluation score is from 0 to 4 levels, where 0 is no significant lesion ( none ). 1 slight ( slight ) , 2 mild ( mild ) , 3 moderate ( moderate ) and 4 significant ( remarkable ) .

²⁾ Pathological scores of chronic liver injury in rats = total number of lesions only total number of moles.

³⁾ Semi-quantitative analysis of liver fibrosis, according to Gabriele et al (1997) and Wang et al (1999), the liver fibrosis is divided into 0~4 grades, 0 is normal liver tissue, no liver Fibrosis, 1 is the proliferation of collagen, but does not form a septum (radial fibrosis in the central vein or portal area), 2 between the central vein and the portal vein, forming an incomplete septum (this septum) There is no meeting with each other), 3 In order to form a complete septum, the middle meets and divides the liver parenchyma into many nodules, but the septum is still thin and 4 is a complete septum and the septum becomes thicker, forming a complete liver. hardening.

⁴⁾ Rats have fibrotic pathological scores = total number of lesions

^{5) The} number of lesions produced in rats.

* The data of each treatment group and control group were compared by Student's -test, and the value of less than 0.05 was considered to be significant.

Please also refer to Figure 4 and Table 3. Figure 4 shows the pathological changes of liver tissue damage after liver injury induced by carbon tetrachloride. As can be observed from Figure 4, after treatment with carbon tetrachloride, The tissue sections of the liver cells are swollen and cause vigorous mitosis of the liver cells.

The number of Kuffer's cells will also increase (Fig. 4A and Fig. 4B), and in severe cases, vacuolization will occur, and the fibrotic liver will divide the liver into many nodules to form cirrhosis (Fig. 4C). At the same time, according to the chronic liver injury assessment method published by Jonker et al., the chronic liver injury induced by carbon tetrachloride was the most obvious, mainly distributed in 3 moderate ( + + + ) and 4 significant ( + + + + ) level, pathological scores up to 3.2. Compared with the negative control group treated with carbon tetrachloride and feeding the milk thistle, the degree of chronic liver injury was mild, mainly distributed at 3 moderate (+ + + ) level, and the pathological score was 2.8; The experimental group of carbon tetrachloride treated and fed with different concentrations of Antrodia camphora cyclohexanone compound can improve the degree of chronic liver injury, and the dose of Antrodia camphora cyclohexenone compound is 300 mg/kg and 1000 mg/kg. The group effect was most significant, mainly distributed in 1 mild ( + ) grade and 2 mild ( + + ) grades. Compared with the negative control group, the pathological scores were reduced to 1.8 and 2.0, respectively.

Please also refer to Figure 5 and Table 3. Figure 5 shows the pathological changes of liver tissue fibrosis after hepatic injury induced by carbon tetrachloride. It can be observed from Fig. 5 that there is no fibrosis in normal liver tissue (see Fig. 5A), and the liver at this time belongs to grade 0 in the degree of fibrosis published by Gabriele and Wang et al; After carbon treatment, liver tissue has varying degrees of fibrotic lesions. Among them, the collagen that proliferates but does not form a septum (see Figure 5B) belongs to grade 1 in the degree of fibrosis, when the central vein and portal area The liver that forms the incomplete septum between the two (see Figure 5C) is Grade 2 in the degree of fibrosis, if the intact septum is formed and the middle meets and divides the liver parenchyma into many nodules, but this septum The liver, which is still very thin (see Figure 5D), is grade 3 in the degree of fibrosis, and once the formed septum becomes thicker, it becomes cirrhosis (see Figure 5E), where the liver is fibrosis. Grade 4 in the degree, and collagen of liver tissue was observed to be green in the results of Masson's trichromc (MT), showing the condition of liver fibrosis (see Fig. 5F). The tissue sections of the control group and the experimental group were observed according to the above-mentioned results of the degree of tissue lesions and evaluated. It was found that the degree of liver fibrosis was most obvious in the negative control group treated with carbon tetrachloride, mainly distributed in grade 3 and grade. 4, pathological scores up to 3.2. Compared with the negative control group, the positive control group treated with carbon tetrachloride and feeding silymarin had a milder degree of liver fibrosis with a pathological score of 2.8; treated with carbon tetrachloride and fed with different concentrations of Antrodia camphorata The experimental group of the hexenone compound can effectively reduce the degree of liver fibrosis, The effect of the group of 300 mg/kg of Antrodia camphora cyclohexenone compound was the most significant, mainly distributed in grades 1 to 2. Compared with the negative control group, the pathological product decreased to 1.8.

From the above results, it can be seen that when the rat liver injury is induced by carbon tetrachloride, the feeding of the anthraquinone cyclohexenone compound can effectively reduce the degree of liver damage and liver fibrosis, and the concentration of the anthraquinone cyclohexenone compound protection. The effect of liver tissue was better than that of the positive control group administered with silymarin, and the efficacy of the anthraquinone cyclohexenone compound with a feeding dose of 300 mg/kg was the best.

(4) Liver function pointer biochemical numerical test

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important enzymes in various organs of the human body such as liver, heart and muscle, and are used to participate in the synthesis of important amino acids in the body. Under normal circumstances, these enzymes maintain a stable low amount in serum, wherein the normal value of alanine aminotransferase is below 40 U/L, and the normal value of aspartate aminotransferase is below 50 U/L. When the cells of the device are inflamed, due to changes in cell permeability or destruction of the cells themselves, the alanine aminotransferase and aspartate aminotransferase in the cells are released, so that the concentration of these enzymes in the serum is increased, so the alanine The activity of transaminase and aspartate aminotransferase is related to liver inflammation and hepatocyte damage, and can be used as one of the indicators for evaluating liver diseases.

Rats were sacrificed at the end of the eighth week, and liver biochemical function was examined by blood sampling in the abdominal aorta. Place 5 ml of blood into a blood cell serum separation tube and centrifuge at 775 xg for 15 minutes. Take 0.5 ml of serum to a serum biochemical analyzer (Express plus automatic clinical chemistry analyzer, Chiron diagnostics corporation, OH, USA) and use the detectable valley. Liver enzyme activity and cholesterol (Bayer diagnostics, Cat No. E33940), such as ALT; Bayer diagnostics, Cat No. E36941, glutamate aminotransferase (AST; Bayer diagnostics, Cat No. E37041) The biochemical values of these liver damages were measured by commercial reagents. In addition, the data of each experimental group were expressed as mean and standard deviation (mean±SD), and analyzed by one-way analysis of variance (ANOVA). When the difference analysis results show that there is a significant difference, the LSD method is used for multiple comparisons, or the independent M test (Student Mest) statistical program is used to compare the experimental group with the control group and the control group. The difference between the group and the negative control group, and /? <0.05 was significant, and the results are shown in Table 4. Table 4. Feeding of Antrodia camphora cyclohexenone after liver injury induced by carbon tetrachloride

Effect of liver biochemical function index on serum of experimental animals

Control group 50.7±9.1i) 110.9±25.1 63.8±12.2 Negative control group (20% CCL ₄ ) 453.4±201.8* 470.4±310.1* 66.7±8.7 Positive control group (silymarin) 180.8±94.6* a 249.5±105.0* _a 69.5 ±14.3 牛樟芝环

Enone compound

300 226.6±138.7* _a 323.4±189.0* 63.7±15.1

1,000 181.7±78.4* a 283.2±144.2* 69.6±14.8

3,000 206.0±98.4* a 311.7±99.6* 62.1±16.2

^{The D} data were averaged by MS-Excels method and the standard machine difference (Mean±SD).

a The data of each treatment group and the negative control group were compared by Student's i-test, and the value of less than 0.05 was considered to be significant. It can be observed from Table 4 that the negative control group treated with carbon tetrachloride has higher values of glutamate propionate transaminase (ALT), glutamate aminotransferase (AST) and cholesterol than the control group. The liver is damaged by carbon tetrachloride, resulting in an increase in liver damage enzyme activity in plasma, and a slight increase in cholesterol content; a positive control group treated with carbon tetrachloride and fed with milk thistle compared to the control group The glutamate transaminase (ALT), glutamate aminotransferase (AST) and cholesterol values were higher than the negative control group, indicating that the milk thistle has reduced carbon tetrachloride. The effect of the degree of hepatocyte injury; in addition, the experimental group treated with carbon tetrachloride and fed with different concentrations of Antrodia camphora cyclohexenone compound, glutamate propionic acid transaminase (ALT), glutamate aminotransferase (AST) and Although the value of cholesterol was higher than that of the control group, it was significantly lower (;? < 0.05) than the negative control group, especially the group with the feeding dose of 1000 mg/kg of anthraquinone cyclohexenone compound, compared with the negative control group. , its value drops The most significant (respectively ALT: 181.7 Disabled 78.4 U / L and AST: 283.2 ± 144.2 U/L), it can be seen that the administration of rat concentration of each dose (300 mg / kg, 1000 mg / kg and 3000 mg / kg) of anthraquinone cyclohexenone compound can effectively alleviate the caused acid propionic acid Release of liver enzymes such as aminotransferase (ALT) and glutamate aminotransferase (AST).

(5) Liver antioxidant enzyme analysis

Antioxidant enzymes are glutathione (GSH), glutathione peroxidase (GSHPx), catalase (CAT), superoxide dismutase (superoxide dismutase). , SOD ), etc., through the antioxidant system, can build a strict line of defense in the organism, in order to prevent the oxidation of cells in organ tissues and reduce the oxidative stress when free radicals increase in the body. ). In this study, the effects of antioxidant enzymes in the liver on the liver's antioxidant capacity were evaluated by detecting the liver damage induced by carbon tetrachloride and feeding the anthraquinone cyclohexenone compound.

The liver in the above-mentioned frozen rejection at -80 ° C was taken out, and immersed in a phosphate buffered saline solution (pH 7.4 ) containing 0.16 mg/ml heparin to remove red blood cells for homogenization. The tissue was disrupted and 10 ml of buffer (50 mM phosphate, pH 6-7, containing 1 mM EDTA) was added to each gram of tissue, and the homogenizer (Brinkmann Polytron homogenizer, setting 5, PT 10 probe) was used. After crushing for 1 minute, at 4. After centrifugation at 10,000 g for 15 minutes at C, the supernatant was taken and subjected to BCA protein assay kit (Pierce, IL, USA) to MAX ELISA Reader (Quant, Bio-Tek Instrument, Vermont, USA). ), the absorbance was measured at 550 nm, and the protein content (mg/ml) was determined. Next, the antioxidant enzymes in the liver homogenate were detected by a commercial kit (Cayman Chemical, Company, MI. USA): Glutathione Assay Kit (Cat No. 703002), glutathione peroxidation Glutathione Peroxidase Assay Kit (Cat No. 703102), Catalase Assay Kit (Cat No. 707002) and Superoxide Dismutase Assay Kit (Cat No.706002), respectively The enzyme contents were determined by ELISA Reader at 405 nm, 340 nm, 540 nm, and 450 nm. In addition, when using these commercial kits to detect these enzymes, in addition to superoxide dismutase (SOD), other enzymes are required to carry out the deproteinization step, adding 10% of the sample volume to the sample to be tested (w/ v) Metaphosphoric acid (MPA), shaken evenly, shake at room temperature for 5 minutes, centrifuge (2,000 xg, 2 minutes), take the supernatant to check the enzyme concentration, and the average of each experimental group data And standard deviation ( meaniSD ) Representation, one-way analysis of variance (ANOVA), when significant differences are found through the results of the variance analysis, further comparisons are performed using the LSD method, or by independent m-test (Student Mest) Statistical procedures were used to compare the differences between the experimental group and the control group, the positive control group and the negative control group, and the results were as shown in Table 5.

Table 5. Feeding of anthraquinone cyclohexenone compound after liver injury induced by carbon tetrachloride

Effects of antioxidant enzymes on the liver of experimental animals

Group / Dose GSH GSHPx Catalase SOD

(mg/kg) (uM/mg protein) (nmol/min/ml) (U/mg protein) (U/mg protein) Control group β ΐ ^.δ ¹ 11.4±2.8 8.9±1.4 0.65±0.22 Negative control group (20% CCL ₄ ) 31.5±6.5 9.9±2.5 9.6±1.6 0.80±0.41 Positive control group (silymarin) 30.5±4.1 9.6±3.0 9.2±1.4 0.68±0.23

Enone compound

300 30.3±3.2 9.6±1.7 9.1±1.0 0.47±0.17* ^a

1,000 25.2±1.8* ^a 11.2±3.4 10.1±1.4 ^# 0.52±0.28

3,000 26.7±4.3* ^a 10.6±3.1 9.8±0.9 0.73±0.23

GSH: Glutathion activity ( mM/g protein ); GSHPx: Glutathion peroxidase activity ( nmol/min/ml ) One unit is defined as 25 ° C per One minute is defined as the amount of enzyme that will cause the oxidation of 1 nmol of NADPH to NADP+ per minute at 25 °C; Superoxide dismutase (CATalase and SOD): U/mg protein (protein)

D data is the mean and standard machine difference (mean±SD),

a The data of each treatment group and the negative control group were compared by Student's i-test, and the value of less than 0.05 was considered to be significant. It can be observed from Table 5 that the negative control group treated with carbon tetrachloride has no significant difference in the measured concentrations of various antioxidant enzymes in the liver compared with the control group; Feeding water There was no significant difference in the concentration of antioxidant enzymes in the positive control group of the planthopper compared with the control group and the negative control group. Compared with the control group, the negative control group and the positive control group, the feeding dose was 300 mg/kg. In the experimental group of cyclohexenone compounds, the concentration of superoxide dismutase (SOD) enzyme exhibited was significantly lower (/?<0.05) than other groups, and the feeding doses were 1000 mg/kg and 3000 mg/kg. In the experimental group, the content of glutathione peroxidase (GSHPx) and catalase (CAT) was slightly higher than other groups, of which glutathione peroxidase (GSHPx) was used. Hydrogenase (CAT) is a water that can decompose hydrogen peroxide (H ₂ O ₂ ) into cells that are not toxic to cells, so as to reduce the damage of peroxide to tissues. Therefore, it can be seen from these results that anthraquinone cyclohexenone compound By increasing the concentration of glutathione peroxidase (GSHPx) and catalase (CAT) in the liver, the damage caused by free radicals to hepatocytes and the oxidative stress they undergo can be reduced, and the resistance of liver tissue can be enhanced. Oxidation ability.

In summary, the present invention is isolated from 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-dode carbon) of Antrodia camphorata Triene)-2-cyclohexenone compound, which can effectively alleviate liver tissue damage and liver fibrosis caused by chemical liver injury, and can reduce alanine aminotransferase (ALT) and aspartate aminotransferase in blood ( AST) and other indicators of liver function inflammation and damage, while also increasing the content of glutathione peroxidase (GSHPx) and catalase (CAT) in the liver to reduce the damage caused by free radicals to liver cells. Its oxidative stress enhances the antioxidant capacity of liver tissue, thereby slowing the liver damage of mammals such as humans, thereby achieving the purpose of protecting the liver. On the other hand, because the anthraquinone cyclohexenone compound is a naturally extracted substance, it is used for treating liver damage or protecting the liver, and does not cause discomfort or other side effects such as toxicity, complications, etc. It is resistant to free radicals such as hydrogen peroxide (3⁄40 ₂ ), so it can be prepared into health foods, foods and beverages, etc., to prevent liver damage and improve human health. In addition, it can also be used to protect the human body. The ketene compound is prepared as a pharmaceutical composition for mitigating liver damage caused by a chemical substance, wherein the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier in addition to an effective amount of the anthraquinone cyclohexenone compound. The carrier may be an excipient (such as water), a filler (such as sucrose or starch), a binder (such as a cellulose derivative), a diluent, a disintegrant, an absorption enhancer or a sweetener, but is not limited thereto. . The pharmaceutical composition of the present invention can be produced according to a conventional pharmacy preparation method, and an active ingredient dose of an antrodia camphora cyclohexenone compound is mixed with one or more carriers to prepare a desired dosage form, and the dosage form may include a tablet. , powders, granules, capsules or other liquid preparations, But not limited to this. In order to prevent and treat liver damage in mammals such as humans and protect the liver.

Claims

Claim

1. An anthraquinone cyclohexenone compound for protecting the liver, having the following structural formula:

Wherein X is oxygen (0) or sulfur (S), Y is oxygen or sulfur; is hydrogen (H), methyl (CH ₃ ) or (CH ₂ ) _m -CH ₃ , R ₂ is hydrogen, A Or (CH ₂ ) _m -CH ₃ , R ₃ is hydrogen, methyl or (CH ₂ ) _m -CH ₃ , m = 1-12; n = l-12.

The burdock cyclohexenone compound for liver protection according to claim 1, wherein the compound is isolated from an organic solvent extract of Antrodia camphorata.

The burdock cyclohexenone compound for liver protection according to claim 2, wherein the organic solvent is selected from the group consisting of esters, alcohols, alkanes or halothanes.

The burdock cyclohexenone compound for liver protection according to claim 3, wherein the alcohol is ethanol.

The burdock cyclohexenone compound for liver protection according to claim 1, wherein the compound is obtained by separating an aqueous extract of Antrodia camphorata.

The burdock cyclohexenone compound for liver protection according to claim 1, wherein the compound is 4-hydroxy-2,3-dimethoxy-6-methyl-5 (3,7) , 11-trimethyl-2,6,10-dodecatriene)-2-cyclohexanylbutyric acid (4-hydroxy-2,3-dimethoxy-6-methy-5(3,7,ll- Trimethyl-dodeca- 2,6,10-trienyl)-cyclohex-2-enone ).

The burdock cyclohexenone compound for liver protection according to claim 1 or 6, wherein the compound prevents and slows the degree of damage and fibrosis of the liver of the mammal.

The burdock cyclohexenone compound for liver protection according to claim 7, wherein the compound is used in a concentration of 300 mg/kg to 3000 mg/kg.

The burdock cyclohexenone compound for liver protection according to claim 7, wherein the mammal is a human.

The burdock cyclohexenone compound for liver protection according to claim 7, wherein the liver damage is caused by a chemical substance.

The burdock cyclohexenone compound for liver protection according to claim 10, wherein the chemical substance is carbon tetrachloride (ecu)o

The burdock cyclohexenone compound for liver protection according to claim 7, wherein the 4 conjugate is alanine aminotransferase (ALT) and aspartate aminotransferase (a). Aspartate aminotransferase, AST) increases in concentration and protects the liver.

The burdock cyclohexenone compound for liver protection according to claim 7, wherein the compound increases glutathione peroxidase (GSHPx) and catalase in the liver by increasing glutathione peroxidase (GSHPx) and catalase , CAT), while slowing the damage caused by free radicals to the liver.

14. A pharmaceutical composition for liver protection comprising at least an effective amount of a compound according to claim 1 and a pharmaceutically acceptable carrier.

A pharmaceutical composition for liver protection comprising at least an effective amount of the compound according to claim 6 and a pharmaceutically acceptable carrier.