TWI644669B - Use and pharmaceutical composition for liver fibrosis prevention and/or treatment - Google Patents

Abstract

The invention provides a pharmaceutical composition for preventing and/or treating liver fibrosis and a use thereof, and specifically discloses an extract of Antrodia camphorata and a medical application thereof, wherein the extract of Antrodia camphorata is 5-methyl-benzo[1,3] Dioxo-4,7-diol (5-methyl-bcnzol [1,3]dioxolc-4,7-diol, MBDD). It has been experimentally confirmed that the extract of Antrodia camphorata of the present invention can inhibit the activity of stellate cells, thereby achieving the effect of preventing and/or treating liver fibrosis. Accordingly, the present invention discloses novel medical applications of extracts of Antrodia camphorata, and further provides an alternative to the prevention and/or treatment of liver fibrosis in the field.

Description

Medicine composition for preventing and/or treating liver fibrosis and use thereof

The present invention relates to a method and a pharmaceutical composition for preventing and/or treating liver fibrosis; more specifically, to a method and a pharmaceutical composition for preventing and/or treating liver fibrosis comprising an extract of Antrodia camphorata.

The liver is an important organ in the human body, which is composed of parenchymal cells and non-parenchymal cells (Roberts et al., 2007). Parenchymal cells, also known as hepatocytes, account for 60% of the cells in the whole liver and 80% of the liver. Non-parenchymal cell lines are composed of other biologically important cells, including sinusoidal endothelial cells, kupffer cells, Hepatic Stellate Cells, and liver killer cells. Each of these cells accounts for 3 to 20% of the amount (Malarkey et al., 2005, Santi-Rocca et al., 2009).

The stellate cells, also known as Ito Cells, are located in the space between the cavernous blood vessels of the liver and the hepatocytes (Space of Disse, aka perisimusoidal space). In the normal functioning state of the liver, the stellate cells are in a stationary phase, which stores oil and fat and accounts for 90% of the body's vitamin A. When the liver is inflamed, the stellate cells are activated by growth factors or inflammatory cytokines, start to proliferate or make collagen, disperse oil, release vitamin A, and produce and release cytokines to affect peripheral cells.

Hepatic fibrosis is caused by the accumulation of excessive extracellular matrix (ECM) when the liver is chronically damaged. Causes such as chronic alcohol consumption, type B or hepatitis C virus infection, nonalcoholic steatohepatitis and genetic defects may be associated with fibrosis, resulting in changes in liver structure leading to liver disease. Problems with the blood supply to the cells, loss of liver function, and ultimately may even lead to cirrhosis and hepatocellular tumors (Farazi and DePinho, 2006).

Antrodia camphorata is a medicinal fungus, classified as Polyporaceae, Antrodia. The fruiting body shape of Antrodia camphorata is varied, and it has a plate shape, a bell shape, a horseshoe shape or a tower shape; it is bright red at the time of birth, and then gradually turns into a light reddish brown, light brown or yellowish brown. Antrodia camphorata is endemic to Taiwan, and is commonly found in the hollow heartwood of the trunk of the evergreen broad-leaved large arbor arborvitae at an altitude of 200-1500 meters in Taiwan.

In the early literature, Antrodia camphorata can be used for detoxification, treatment of abdominal pain, and itching of the skin. With the improvement of extraction and analysis technology, the application of the ingredients of Antrodia camphorata in the field of medicine has gradually developed. In previous studies, it has been known that Antrodia camphorata contains a compound 5-methyl-benzo[1,3]dioxoline-4,7-diol (5-methyl-benzol[1,3]dioxole-4,7- Diol, MBDD), and MBDD has potential for cancer treatment. Furthermore, studies have indicated that MBDD has antioxidant activity and can scavenge free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH). The peroxide has a weak inhibitory effect. In addition, in the mechanism of nitric oxide induced by lipopolysaccharide, the experimental results show that MBDD has a reduced nitric oxide production effect on macrophage cell line RAW 264.7, but it is speculated that this excellent effect may be highly cytotoxic with MBDD. Related to activity.

In addition, some studies have also pointed out that the extract of Antrodia camphorata has the effect of inhibiting the production of reactive oxygen species, thereby inhibiting inflammation. should. However, it is currently known that all medical applications for Antrodia camphorata do not mention the efficacy of preventing and treating liver fibrosis. Since the physiological mechanism of the inflammatory reaction involves the in vivo pathways of members such as cyclooxygenase, prostaglandin, interferon and active oxides, the main cells involved are mononuclear spheres and macrophages; the mechanism of fibrosis is the excessive deposition of connective tissue. The main participating cells are stellate cells, which are essentially different from the perspective of physiological mechanisms. Therefore, even though it is known that Antrodia camphorata has the potential to inhibit the inflammatory reaction, the effect of the constituents of Antrodia camphorata on the stellate cells of the liver is not clear, and the effect of Antrodia camphorata on the prevention and treatment of liver fibrosis is not yet clear.

One of the objects of the present invention is to provide a method for treating and preventing liver fibrosis, thereby preventing the occurrence of liver cirrhosis and liver cancer.

Another object of the present invention is to develop the medical use of Antrodia camphorata and thereby enhance its industrial value.

In order to achieve the aforementioned object, the present invention provides a use of a compound of the following formula (I) for the preparation of a pharmaceutical composition for preventing and treating liver fibrosis;

Preferably, the compound of formula (I) is 5-methyl-benzo[1,3]dioxo-4,7-diol (5-methyl-benzol[1,3]dioxole-4,7- Diol, MBDD).

Preferably, the compound of formula (I) is isolated from the extract of Antrodia camphorata.

Preferably, the compound of formula (I) has an activity of inhibiting stellate cells.

Preferably, the pharmaceutical composition comprises an effective amount of the compound of formula (I) and a pharmaceutically acceptable additive.

Preferably, the pharmaceutical composition is a lozenge, capsule, injection, powder, granule or drink.

Preferably, the pharmaceutically acceptable additive comprises: a carrier, an excipient, a preservative, a diluent, a filler, a binder, a disintegrant, an absorption enhancer, a sweetener, or a combination thereof.

In still another aspect, the present invention provides a use of a compound of the following formula (II) for the preparation of a pharmaceutical composition for preventing and treating liver fibrosis;

Preferably, the compound of formula (II) is 2,4-dimethoxy-6-methylbenzene-1,3-diol.

Preferably, the compound of formula (II) is prepared by isolating an extract of Antrodia camphorata.

Preferably, the compound of formula (II) has an activity of inhibiting stellate cells.

Preferably, the pharmaceutical composition comprises an effective amount of the compound of formula (II) and a pharmaceutically acceptable additive.

Preferably, the pharmaceutical composition is a lozenge, capsule, injection, powder, granule or drink.

Preferably, the pharmaceutically acceptable additive comprises: a carrier, a excipient, and an anti-drug a humic agent, a diluent, a filler, a binder, a disintegrant, an absorption enhancer, a sweetener, or a combination thereof.

The invention further provides a pharmaceutical composition for preventing and treating liver fibrosis comprising an effective amount of a compound of the formula (I) and / or (II), and a pharmaceutically acceptable carrier;

Preferably, the compound of formula (I) is 5-methyl-benzo[1,3]dioxo-4,7-diol, and the compound of formula (II) is 2,4-dimethoxy. -6-methylbenzene-1,3-diol.

Preferably, the effective amount is 0.02 to 2.0 g / 60 kg body weight / day.

Preferably, the pharmaceutical composition is a lozenge, capsule, injection, powder, granule or drink.

Preferably, the pharmaceutically acceptable additive comprises: a carrier, an excipient, a preservative, a diluent, a filler, a binder, a disintegrant, an absorption enhancer, a sweetener, or a combination thereof.

Preferably, the pharmaceutical composition comprises from 5 to 95% by weight of the compound of formula (I) and/or formula (II); wherein the aforementioned % by weight is based on the total weight of the pharmaceutical composition.

The present invention provides a use of an extract of Antrodia camphorata for preparing a pharmaceutical composition for preventing and treating liver fibrosis, wherein the extract of Antrodia camphorata is prepared by a preparation method; wherein the preparation method comprises the following steps: (a) obtaining an Antrodia camphorata (b) soaking the burdock in a 95 to 100 vol% ethanol to obtain a first burdock Zhi extract.

Preferably, the step (a) comprises pulverizing the Antrodia camphorata into a powder.

Preferably, step (b) is carried out at 25 to 65 °C.

Preferably, the step (b) further comprises drying the first A. angustifolia extract to obtain the first A. angustifolia extract in a powder form.

Preferably, the HPLC chromatogram of the extract of the first Antrodia camphorata is 1.66 minutes, 2.38 minutes, 4.11 minutes, 4.42 minutes, 7.53 minutes, 8.21 minutes, 16.48 minutes, 17.19 minutes, 19.89 minutes, 25.79 minutes, 31.49 minutes. And a signal peak at a residence time of 38.81 minutes; or a map as shown in the first figure.

Preferably, the step (b) further comprises a step (c) of obtaining a second extract of Antrodia camphorata, wherein the step (c) is: using the aqueous solution of ethanol as a flushing solution to pass the first extract of Antrodia camphorata The second A. angustifolia extract is obtained by a styrene resin column; wherein the aqueous ethanol solution is 95 to 100% by volume of ethanol and water in a volume ratio of 1:1.

Preferably, the styrene resin column has a pore volume of 1 to 2 mL/g. Preferably, the pores of the styrene resin column have a size of 200 to 300 angstroms.

Preferably, the step (c) further comprises drying the second Antrodia camphora extract to obtain the second extract of the Antrodia camphorata in a powder form.

Preferably, the second Antrodia camphora extract has the following characteristics: (i) a high performance liquid chromatogram of the second extract of Antrodia camphorata at 1.68 minutes, 2.38 minutes, 4.10 minutes, 4.41 minutes, 6.46 minutes, 9.58 minutes, 17.19 Minutes and 19.90 minutes of residence time have signal peaks; or have maps as shown in Figure A; (ii) positive ion mode liquid chromatography tandem mass spectrometry analysis of the second Antrodia camphorata extract Chromatograms at 1.80 minutes, 2.26 minutes, 7.38 minutes, 8.73 minutes, 10.24 minutes, 14.08 minutes, 18.64 minutes, 19.50 minutes, 25.06 minutes, 30.16 minutes, 32.55 minutes, 34.88 minutes, 38.54 minutes, 48.46 minutes, 57.09 minutes, 63.79 Minutes, 68.37 minutes, 69.50 minutes, and 70.01 minutes with a signal peak at the residence time; or with a map as shown in Figure B; (iii) Negative ion mode liquid chromatography tandem mass spectrometry chromatography of the second Antrodia camphorata extract The spectra are at 2.35 minutes, 4.18 minutes, 7.72 minutes, 11.27 minutes, 12.89 minutes, 19.29 minutes, 23.13 minutes, 24.99 minutes, 27.36 minutes, 27.99 minutes, 33.78 minutes, 42.40 minutes, 42.53 minutes, 42.76 minutes, 43.12 minutes, 43.61 minutes, 43.84 minutes, 44.90 minutes, 48.21 minutes, 59.30 minutes, 61.97 minutes, 64.98 minutes, and 71.28 minutes of residence time having a signal peak; or having a map as shown in FIG. C; and/or (iv) the second Antrodia camphorata extract The chromatogram of the ultraviolet mode liquid chromatography tandem mass spectrometry was performed at 1.25 minutes, 5.91 minutes, 10.22 minutes, 19.29 minutes, 23.09 minutes, 28.98 minutes, 29.21 minutes, 29.42 minutes, 32.50 minutes, 38.18 minutes, 42.93 minutes, 44.52 minutes, 48.37 minutes, 54.01 minutes, 59.40 minutes, and 66.76 minutes have a signal peak at the residence time; or have a map as in the second panel D.

Preferably, after the step (c), the method further comprises the following steps to obtain a third extract of Antrodia camphorata: (d) using an aqueous methanol solution as a flushing solution, and passing the second extract of Antrodia camphorata through monohydroxypropyl dextran The third A. angustifolia extract is obtained from a gel resin column; wherein the aqueous methanol solution is 95 to 100% by volume of methanol and water in a volume ratio of 1:1.

Preferably, the step (d) further comprises a drying step to obtain the third type of Antrodia camphorata extract in powder form.

Preferably, after the step (d), the method further comprises the following steps to obtain a fourth extract of Antrodia camphorata: (e) using an aqueous methanol solution as a flushing solution, and passing the third extract of Antrodia camphorata through a carbon 18 reverse phase column And the fourth calf Zhizhi extract; wherein the aqueous methanol solution is 95 to 100% by volume of methanol and water in a volume ratio of 1:1.

Preferably, the step (e) further comprises a drying step to obtain the fourth extract of the Antrodia camphorata in a powder form.

Preferably, after the step (e), the following step (f) is further included to obtain a fifth extract of Antrodia camphorata and a sixth extract of Antrodia camphorata: (f) performing thin layer chromatography on the fourth extract of Antrodia camphorata, using An aqueous methanol solution is used as a developing solution to obtain the fifth extract of Antrodia camphorata and the extract of the sixth Antrodia camphorata; wherein the aqueous methanol solution is 95 to 100% by volume of methanol and water in a volume ratio of 1:1.

Preferably, the chromatographic profile of the extract of the fifth Antrodia camphorata by positive ion mode liquid chromatography tandem mass spectrometry is 1.16 minutes, 1.96 minutes, 2.62 minutes, 5.49 minutes, 6.31 minutes, 8.25 minutes, 9.66 minutes, 13.07 minutes, Signals at 13.71 minutes, 15.93 minutes, 18.30 minutes, 21.30 minutes, 23.02 minutes, 23.73 minutes, 25.49 minutes, 25.98 minutes, 30.17 minutes, 31.02 minutes, 33.24 minutes, 35.29 minutes, 35.83 minutes, 38.37 minutes, and 39.93 minutes of residence time Peak; or has a map as in Figure A of Figure 3.

Preferably, the chromatogram of the sixth extract of the Antrodia camphorata extract by positive ion mode liquid chromatography tandem mass spectrometry is 1.67, 2.95, 5.11, 5.60, 7.25, 9.44, 10.45, 12.72, 12.90, 15.50, 17.52, 18.10, 18.54, 19.81, 22.14, 23.62, 21.96, 25.38, 26.52, 28.36, 30.23, 31.01, 31.26, 35.18, 37.48, 38.98, 39.80 minutes with a signal peak at the residence time; or with a map as shown in Figure A.

Preferably, the Antrodia camphorata extract may comprise 5-methyl-benzo[1,3]dioxo-4,7-diol having the following formula (I) and/or 2 having the following formula (II), 4-dimethoxy-6-methylbenzene-1,3-diol:

Preferably, the effective amount of the first Antrodia camphorata extract for controlling liver fibrosis is 0.02 to 2.0 g/60 kg body weight/day.

Preferably, the effective amount of the second Antrodia camphorata extract for controlling liver fibrosis is 0.02 to 2.0 g/60 kg body weight/day.

Preferably, the effective amount of the extract of the fifth Antrodia camphorata in preventing and treating liver fibrosis is 0.02 to 2.0 g/60 kg body weight/day; and/or the effective amount of the extract of the sixth Antrodia camphorata in preventing and treating liver fibrosis is 0.02 to 2.0g/60kg body weight/day.

The invention further provides a pharmaceutical composition for preventing and treating liver fibrosis, comprising: an extract of Antrodia camphorata and a pharmaceutically acceptable carrier; wherein the extract of Antrodia camphorata is selected from the group consisting of the following extracts: the first An Antrodia camphora extract, the second Antrodia camphora extract, the third Antrodia camphora extract, the fourth Antrodia camphora extract, the fifth Antrodia camphorata extract, and the sixth Antrodia camphorata extract.

Preferably, the Antrodia camphorata extract may comprise 5-methyl-benzo[1,3]dioxo-4,7-diol having the following formula (I) and/or 2 having the following formula (II), 4-dimethoxy-6-methylbenzene-1,3-diol:

Preferably, the pharmaceutically acceptable additive comprises: a carrier, a excipient, and an anti-drug a humic agent, a diluent, a filler, a binder, a disintegrant, an absorption enhancer, a sweetener, or a combination thereof.

Preferably, the pharmaceutical composition comprises from 5 to 95% by weight of the Antrodia camphorata extract; wherein the foregoing weight percentage is based on the total weight of the pharmaceutical composition.

Preferably, the pharmaceutical composition is a lozenge, capsule, injection, powder, granule or drink.

In summary, the present invention provides a use of a compound extracted from Antrodia camphorata for the preparation of a pharmaceutical composition for preventing and treating liver fibrosis, and a pharmaceutical composition containing the same. The invention successfully uses the compound extracted from Antrodia camphorata for novel medical application for preventing/treating liver fibrosis, and contributes to the industrial value of the re-medicine field of Antrodia camphorata.

The first figure shows a high performance liquid chromatography analysis of the extract of the first Antrodia camphorata.

Figure 2A shows a high performance liquid chromatography analysis of the extract of A. angustifolia.

Figure B shows the chromatogram of the second Antrodia camphorata extract by positive ion mode liquid chromatography tandem mass spectrometry.

Figure C shows the chromatogram of the negative ion mode liquid chromatography tandem mass spectrometry analysis of the second Antrodia camphorata extract.

Figure 2D shows the chromatogram of the second A. angustifolia extract by ultraviolet mode liquid chromatography tandem mass spectrometry.

The third panel A shows the chromatogram of the positive extractive liquid chromatography tandem mass spectrometry analysis of the extract of the fifth Antrodia camphorata.

Figure 3B shows a chromatogram of 5-methyl-benzo[1,3]dioxo-4,7-diol by positive ion mode liquid chromatography tandem mass spectrometry.

Figure IIIC shows the mass spectrum of the extract of the fifth Antrodia camphorata by positive ion mode liquid chromatography tandem mass spectrometry.

Figure 3D shows the ¹ H NMR spectrum of the extract of the fifth Antrodia camphorata.

Figure 4A shows the chromatogram of the sixth extract of Antrodia camphorata by positive ion mode liquid chromatography tandem mass spectrometry.

Figure 4B shows a chromatogram of 2,4-dimethoxy-6-methylbenzene-1,3-diol by positive ion mode liquid chromatography tandem mass spectrometry.

Figure 4C shows the mass spectrum of the sixth extract of Antrodia camphorata by positive ion mode liquid chromatography tandem mass spectrometry.

Figure 4D shows the ¹ H NMR spectrum of the extract of the sixth Antrodia camphorata.

The fifth panel shows the results of the Sirius red staining quantification method of Experiment 4.

The term "control" as used herein is an abbreviation for prevention and treatment. As used herein, "prevention" refers to the protection of a body from the onset of a disease, discomfort, or harmful condition. As used herein, "treatment" refers to the process of eliminating, terminating, or reducing a disease, discomfort, or deleterious condition in a body.

As used herein, "a compound having an activity of inhibiting stellate cells" means that the compound has a negative influence on the growth of stellate cells. The negative effect can be exemplified by, but not limited to, inhibiting the proliferation of stellate cells, inhibiting the production of fibrillar proteins by stellate cells, or even killing stellate cells.

One aspect of the present invention relates to a medical use of an extract of Antrodia camphorata for use in the preparation of a pharmaceutical composition for preventing and treating liver fibrosis. The extract of Antrodia camphorata is obtained by the following steps: obtaining an Antrodia camphorata; immersing the Antrodia camphorata in an aqueous alcohol solution to obtain a first extract of Antrodia camphorata Take things.

In a preferred embodiment of the invention, the preparation of the extract of Antrodia camphorata is carried out using a fruiting body of Antrodia camphorata. In a preferred embodiment, the Antrodia camphorata is pulverized, and the pulverization method may be dry grinding or wet milling. In a preferred embodiment, the aqueous alcohol solution is an aqueous ethanol solution.

In a possible embodiment of the invention, the concentration of the aqueous alcohol solution may be from 50 to 100% by volume; preferably from 95 to 100% by volume. In a possible embodiment, the Antrodia camphorata is soaked in the aqueous alcohol solution at a specific temperature; preferably, the specific temperature may be 25 to 65 °C. In a preferred embodiment, the agitation is carried out while soaking the Antrodia camphorata. In a possible embodiment, the soaking time is 2-6 days; preferably 3-5 days.

In a preferred embodiment of the invention, the first Antrodia camphora extract is passed through a resin. Next, the resin is washed with an aqueous alcohol solution to obtain a second extract of Antrodia camphorata. In a preferred embodiment, the resin may be a styrenic resin. In a preferred embodiment, the styrenic resin may have a pore volume ranging from 1 to 2 mL/g. In a preferred embodiment, the styrenic resin has a pore size of from 200 to 300 angstroms. In a preferred embodiment, a commercially available styrene resin such as, but not limited to, DIAION HP 20 (manufactured by Mitsubishi Chemical Corporation), Amberlite XAD-4 (Rohra & Haas), and D101 (Tianjin) may be used. Nankai Hecheng Technology Co., Ltd.). The DIAION HP 20 (manufactured by Mitsubishi Chemical Corporation) has a pore volume of 1.3 mL/g and a pore size of 260 angstroms.

In a preferred embodiment, the alcohol used in the aqueous solution of the alcohol for extraction is ethanol. In a possible embodiment of the invention, the concentration of the aqueous alcohol solution may be from 50 to 100% by volume; preferably from 95 to 100% by volume. In a preferred embodiment, the ratio of water to alcohol in the aqueous alcohol solution for extraction It can be from 2:1 to 1:2; more preferably 1:1.

In a preferred embodiment of the invention, the second Antrodia camphora extract is passed through a resin. Next, the resin is extracted by an aqueous alcohol solution to obtain a third Antrodia camphorata extract.

In a preferred embodiment, the resin is a hydroxypropyl dextran gel resin. In a preferred embodiment, a commercially available hydroxypropyl dextran gel resin such as, but not limited to, SEPHADEX LH-20 (manufactured by GE Healthcare Life Sciences) may be used. .

In a preferred embodiment, the aqueous alcohol solution comprises water and an alcohol, wherein the alcohol may be methanol.

In a preferred embodiment, the third Antrodia camphora extract is passed through a carbon 18 reverse phase column. Next, the column is washed with an aqueous alcohol solution to obtain a fourth A. angustifolia extract. In a preferred embodiment, the aqueous alcohol solution is obtained from the third extract of Antrodia camphorata as described in the foregoing paragraph, and details are not described herein.

In a preferred embodiment, a commercially available carbon 18 reverse phase column can be used, such as, but not limited to, Agilent, XDB-C18, 5 [mu]m, 4.6 x 150 mm.

In a preferred embodiment of the present invention, the fourth extract of Antrodia camphorata is further subjected to thin layer chromatography using an aqueous alcohol solution as a developing solution to obtain a fifth extract of Antrodia camphorata and a extract of the sixth Antrodia camphorata. . In a preferred embodiment, the aqueous alcohol solution can be referred to the extract used in the step of obtaining the third extract of Antrodia camphorata and the extract of the fourth Antrodia camphorata, and details are not described herein again. In a preferred embodiment, a commercially available thin layer chromatography sheet such as, but not limited to, a thin layer of tantalum film (Merck, Germany) may be used.

In a preferred embodiment of the invention, the Antrodia camphorata extract obtained according to the above steps is dried. Those who have the usual knowledge in the field can use the collar This drying procedure is carried out in a manner known in the art, for example: drying and drying.

Another aspect of the present invention is directed to a pharmaceutical composition comprising the extract of Antrodia camphorata and an additive of the present invention. As used herein, "effective amount" refers to an amount sufficient to produce the aforementioned prophylactic and/or therapeutic effects. Based on the in vitro cell culture experiment, the aforementioned effective amount is defined as "μg/ml" based on the total volume of the cell culture fluid used in each culture. Based on animal model experiments, the aforementioned effective amount is defined as "g/60 kg body weight/day". In addition, the effective amount of data obtained by in vitro cell culture experiments can be converted to a reasonable effective amount for animal use by the following formula:

◆Generally (Reagan-Shaw et al., 2008), 1 "μg/ml" unit (based on an effective amount in vitro cell culture experiments) can be equivalent to 1 "mg/kg body weight/day" units (based on The effective amount obtained by the mouse model experiment, and based on the known metabolic rate of the mouse is six times that of humans, the effective dose of human can be further determined.

◆ Therefore, if an effective amount is 500 μg/ml based on the in vitro cell culture experiment, the effective amount used in the mouse can be calculated as 500 mg/kg body weight/day (ie, 0.5 g/kg body weight/day). Further, depending on the difference in the aforementioned metabolic rate, the effective amount for human use can be calculated as 5g/60kg body weight/day.

According to the examples described in the following paragraphs, an effective amount of 2.0 to 200 μg/ml is determined based on the in vitro cell culture experiment, so a reasonable effective dose for human use should be 0.02 to 2.0 g/60 kg body weight/day.

In a preferred embodiment, the additive of the pharmaceutical composition may be selected from the group consisting of a carrier, an excipient, a preservative, a diluent, a filler, a binder, a disintegrant, an absorption enhancer, a sweetener, or combination. The carrier can be water, ethanol, polyol, carboxymethyl Cellulose, vegetable oil, benzyl ester or a combination thereof. The excipient can be selected from the group consisting of sodium citrate, calcium carbonate, calcium phosphate, or a combination thereof. The preservative extends the shelf life of the pharmaceutical composition, such as benzyl alcohol, parabens. The diluent can be selected from the group consisting of water, ethanol, propylene glycol, glycerin, or a combination thereof. The filler may be selected from the group consisting of lactose, nougat, high molecular weight ethylene glycol or a combination thereof. The binder may be selected from the group consisting of sucrose, gelatin, gum arabic or combinations thereof. The disintegrant may be selected from the group consisting of potato starch, tapioca starch, citrate or a combination thereof. The absorption enhancer can be selected from the group consisting of dimethyl hydrazine (DMSO), laurel, propylene glycol, glycerin, polyethylene glycol, or a combination thereof. The sweetener may be selected from the group consisting of Acesulfame K, aspartame, saccharin, sucralose, neotame or a combination thereof. In addition to the additives listed above, other additives may be selected according to requirements without affecting the medical effects of the extract of Antrodia camphorata.

The following examples are presented to further illustrate the advantages of the invention, but are not intended to limit the scope of the invention.

Experiment 1: Preparation of the first Antrodia camphora extract and the second Antrodia camphorata extract of the present invention.

(1) Preparation of extracts.

The scale is taken from the dry or wet milled burdock (Kejie Biotechnology Co., Ltd.), and the 10 times volume of 95 vol% ethanol is added to the burdock, and stirred at a temperature of 30 ° C for 3-5 days to obtain a first burdock Extracts. Next, the first A. angustifolia extract is concentrated by suction filtration to remove as much ethanol as possible from the extract. The concentrated first burdock extract is placed in a moisture-proof box and dried to obtain a powdered form of the first Antrodia camphora extract. The yield was calculated to be 18.53% based on the weighing result.

For the first Antrodia camphorata extract, chromatographically by styrene resin Get the second extract of Antrodia camphorata. First, 20 times by weight of styrene resin particles (DIAION HP 20, manufactured by Mitsubishi Chemical Corporation) of the first A. angustifolia extract was weighed, and the resin particles were immersed in 95% by volume of ethanol overnight. Next, the ethanol-soaked resin pellets were poured into an open column, and the column was rinsed with an aqueous ethanol solution (95 vol% ethanol: water = 1:1). The 10-fold amount of the aqueous ethanol solution was used to re-dissolve the powdered form of the first Antrodia camphora extract. The re-dissolved first A. angustifolia extract was poured into the column, and eluted with 5 times by volume of the aqueous solution of the styrene resin particles as a solvent. The elution obtained from the column was collected, that is, the second extract of Antrodia camphorata in this example. The second Antrodia camphorata extract is concentrated, and then placed in a moisture-proof box for drying to obtain a powder type of the second Antrodia camphora extract. The yield was calculated to be 70.36% based on the weighing result.

(2) HPLC analysis.

Next, the first Antrodia camphora extract and the second Antrodia camphorata extract were analyzed by high performance liquid chromatography (HPLC). The test solution is reconstituted with methanol, pure water or a mixed solution of isopropyl alcohol and acetonitrile (mixing ratio of about 1:1). After the test solution was formulated to have a concentration of 5 mg/mL, it was centrifuged at 13,500 rpm for 10 minutes. The supernatant of the test solution was taken as a sample. The specific conditions of the HPLC used in this example are as follows:

Pump: Spectra SYSTEM P1000.

Autoinjector: Spectra SYSTEM AS1000.

Detector: FINNIGAN SURVEYOR PDA Plus.

Column (stationary phase): Agilent, XDB-C18, 4.6 mm x 150 mm, 5 μm.

Detector: PDA (ultraviolet light (UV) 254 nm).

Injection volume: 5 μl.

Flow rate: 0.8 mL/min.

Solvent (mobile phase) gradient:

The HPLC chromatogram of the first Antrodia camphorata extract was as shown in the first figure, which was 1.66 minutes, 2.38 minutes, 4.11 minutes, 4.42 minutes, 7.53 minutes, 8.21 minutes, 16.48 minutes, 17.19 minutes, 19.89 minutes, 25.79 minutes, 31.49 minutes. There is a signal peak at the 38.81 minute residence time.

The HPLC chromatogram of the second Antrodia camphorata extract is as shown in the second panel A, and has a signal peak at the residence times of 1.68 minutes, 2.38 minutes, 4.10 minutes, 4.41 minutes, 6.46 minutes, 9.58 minutes, 17.19 minutes, and 19.90 minutes. .

(3) LC/MS analysis.

Further, the first A. angustifolia extract and the second A. angustifolia extract were obtained by liquid chromatography tandem mass spectrometry (LC/MS) analysis. The analyte to be detected is reconstituted with methanol or pure water. After the test solution was prepared to have a concentration of 10 mg/mL, it was centrifuged at 13,500 rpm for 10 minutes. The supernatant of the test solution was taken as a sample. The specific conditions of the LC/MS used in this embodiment are as follows:

Mass spectrometer: Autosampler (Thermo AS3500).

Detector: UV 6000LP and LCQ Fleet mass.

Pump: Thermo P1000.

Column: Thermo Hyper Carb, 4 mm x 100 mm, 5 μm.

Detector: ESI mass (positive ion mode or negative ion mode) / UV 254 nm.

Injection volume: 10 μl.

Flow rate: 1.0 mL/min.

gradient:

The chromatogram of the second Antrodia camphorata extract by positive ion mode liquid chromatography tandem mass spectrometry is shown in Figure B, which is 1.80 minutes, 2.26 minutes, 7.38 minutes, 8.73 minutes, 10.24 minutes, 14.08 minutes, 18.64. There are signal peaks at the residence times of minutes, 19.50 minutes, 25.06 minutes, 30.16 minutes, 32.55 minutes, 34.88 minutes, 38.54 minutes, 48.46 minutes, 57.09 minutes, 63.79 minutes, 68.37 minutes, 69.50 minutes, and 70.01 minutes.

The chromatogram of the second Antrodia camphorata extract by negative ion mode liquid chromatography tandem mass spectrometry is shown in Figure 2, which is 2.35 minutes, 4.08 minutes, 7.72 minutes, 11.27 minutes, 12.89 minutes, 19.29 minutes, 23.13 minutes. , 24.99 minutes, 27.36 minutes, 27.99 minutes, 33.78 minutes, 42.40 minutes, 42.53 minutes, 42.76 minutes, 43.12 minutes, 43.61 minutes, 43.84 minutes, 44.90 Signal peaks are available at minutes, 48.21 minutes, 59.30 minutes, 61.97 minutes, 64.98 minutes, and 71.28 minutes of residence time.

The chromatogram of the second A. angustifolia extract by ultraviolet mode liquid chromatography tandem mass spectrometry is shown in Figure D, at 1.25 minutes, 5.91 minutes, 10.22 minutes, 19.29 minutes, 23.09 minutes, 28.98 minutes, 29.21 minutes. There are signal peaks at 29.42 minutes, 32.50 minutes, 38.18 minutes, 42.93 minutes, 44.52 minutes, 48.37 minutes, 54.01 minutes, 59.40 minutes, and 66.76 minutes of residence time.

Experiment 2: Preparation of the third Antrodia camphorata extract of the present invention.

(1) Preparation of extracts.

The third Antrodia camphora extract was obtained from the second Antrodia camphorata extract through a hydroxypropyl dextran gel. First, hydroxypropyl dextran gel particles (SEPHADEX LH-20, manufactured by Singular Asia Medical Equipment Co., Ltd.) were weighed and poured into an open column, and the column was washed with an aqueous methanol solution. Next, the powdered second extract of Antrodia camphorata was re-dissolved with another aqueous methanol solution. The re-dissolved second A. angustifolia extract is poured into the open column and eluted with the aqueous methanol solution as a solvent. The effluent obtained from the column was collected, that is, the third extract of Antrodia camphorata in this example. The third A. angustifolia extract is placed in a moisture-proof box and dried to obtain a powder type third A. angustifolia extract.

Experiment 3: Preparation of the fourth Antrodia camphorata extract of the present invention.

(1) Preparation of extracts.

The fourth calf was obtained from the third extract of Antrodia camphorata through a carbon 18 reversed phase silica gel (C18 reversed phase silica gel; Agilent, XDB-C18) Zhi extract. First, the column was washed with an aqueous methanol solution as a solvent until the column was filled with the aqueous methanol solution. Next, the third A. angustifolia extract in powder form is reconstituted with a small amount of methanol, introduced into the column, and then extracted with the aqueous methanol solution.

The effluent obtained from the column was collected, that is, the fourth Antrodia camphora extract of the present example. The obtained A. angustifolia extract was placed in a moisture-proof box to be dried and weighed.

Experiment 4: Preparation of the fifth and sixth Antrodia camphorata extracts of the present invention.

(1) Preparation of extracts.

The fifth and sixth Antrodia camphorata extracts of the present invention were isolated from the fourth Antrodia camphorata extract by Thin Layer Chromatography. The fourth A. angustifolia extract in powder form was reconstituted with a trace amount of 100% by volume of methanol. The fourth A. angustifolia extract was reconstituted on a 20×20 cm thin layer chromatography sheet (TLC aluminium sheets, silica gel 60 F254, MercK) in an aqueous methanol solution (100% by volume methanol:=1:1). Two ribbons were observed by irradiating the developed thin layer chromatography with a short-wave 254 nm ultraviolet lamp. The two ribbons on the chromatogram were cut out, soaked in methanol for 10 minutes, and the components on the ribbon were dissolved in methanol. The methanol was then filtered by suction filtration. Next, the above-described step of immersing the ribbon in methanol was repeated three times to obtain two fractions, that is, the fifth and sixth Antrodia camphorata extracts of the present example.

(2) LC/MS analysis.

The fifth and sixth Antrodia camphorata extracts were analyzed by liquid chromatography tandem mass spectrometry. The experimental conditions are as set out in the previous experiment 1, and will not be repeated here.

Positive ion mode liquid chromatography tandem mass spectrometry of the extract of the fifth Antrodia camphorata The chromatogram of the analysis is as shown in the third panel A, which is 1.16 minutes, 1.96 minutes, 2.62 minutes, 5.49 minutes, 6.31 minutes, 8.25 minutes, 9.66 minutes, 13.07 minutes, 13.71 minutes, 15.93 minutes, 18.30 minutes, 21.30. There are signal peaks at the residence times of minutes, 23.02 minutes, 23.73 minutes, 25.49 minutes, 25.98 minutes, 30.17 minutes, 31.02 minutes, 33.24 minutes, 35.29 minutes, 35.83 minutes, 38.37 minutes, and 39.93 minutes. The chromatogram shown in the third panel A is highly consistent with the chromatogram of the standard (5-methyl-benzo[1,3]dioxo-4,7-diol) (Fig. B). . The mass spectrum of the component having a residence time of 9.66 minutes in the third panel A is shown in Fig. C, and it is expected that the component is 5-methyl-benzo[1,3]dioxo-4,7-diol.

The chromatogram of the sixth Antrodia camphorata extract by positive ion mode liquid chromatography tandem mass spectrometry is shown in Figure 4A, which is 1.67 minutes, 2.95 minutes, 5.11 minutes, 5.68 minutes, 7.25 minutes, 9.44 minutes, 10.45 minutes, 12.72 minutes, 12.98 minutes, 15.50 minutes, 17.52 minutes, 18.10 minutes, 18.54 minutes, 19.81 minutes, 22.14 minutes, 23.62 minutes, 24.86 minutes, 25.38 minutes, 26.52 minutes, 28.36 minutes, 30.23 minutes, 31.01 minutes, 31.26 minutes There are signal peaks at 35.18 minutes, 37.48 minutes, 38.98 minutes, and 39.86 minutes of residence time. The chromatogram shown in Figure 4A is highly consistent with the chromatogram of the standard (2,4-dimethoxy-6-methylbenzene-1,3-diol) (Fig. B). . The mass spectrum of the component having a residence time of 12.98 minutes in the fourth panel A is shown in Figure 4C, and it is expected that the component is 2,4-dimethoxy-6-methylbenzene-1,3-diol.

(3) NMR analysis.

Further, the components of the fifth A. angustifolia extract retention time of 9.66 minutes and the retention time of the sixth A. angustifolia extract were 12.98 minutes were separately analyzed by a mass spectrometer. For the fifth Antrodia camphorata extract, a Bruker DMX-500 SB Spectrometer was used, and an electromagnetic wave of 500 MHz was applied and NMR measurement was carried out using deuterated methanol (CD ₃ OD) as a solvent.

The NMR spectrum of the component of the fifth A. angustifolia extract retention time of 9.66 minutes is shown in the third figure D, and is determined by the following chemical shift value as 5-methyl-benzo[1,3]dioxo-4. 7-diol.

For the sixth Antrodia camphorata extract, a Varian UNITY INOVA 500 spectrometer was used, and an electromagnetic wave of 500 MHz was applied and NMR measurement was carried out using deuterated chloroform (CDCl ₃ ) as a solvent. The NMR spectrum of the composition of the sixth A. angustifolia extract retention time of 12.98 minutes is shown in the fourth figure D, and is determined as 2,4-dimethoxy-6-methylbenzene-1 by the following chemical shift value. 3-diol.

Experiment 5: Experiment on the activity of Antrodia camphorata extract against anti-fibrosis

[MTT cell poisoning test]

First, rat stellate cells (HSC-T6) were cultured in a cell culture flask (75T), and the culture flask was placed in an incubator having a carbon dioxide concentration of 5% and a temperature of 37 ° C for 2 to 3 days. Next, the cells were collected with 0.5% trypsin and the collected liquid was centrifuged. The cells were seeded in a 96-well plate (1.5-2.0 x 10 ⁴ cells/well), and the cells were then cultured in an incubator at a carbon dioxide concentration of 5% and a temperature of 37 °C. On the next day, cell viability was determined by adding MTT (3-(4,5-Dimcthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction method for 24 or 48 hours after adding different concentrations of the extract of Antrodia camphorata. After adding 12 μl of MTT reagent (5 mg/ml) to each well in a 96-well plate, it was placed in an incubator at 37 ° C for 2.5 to 3.0 hours. The MTT reagent was removed and 100 μl of lysis buffer was added. The composition of the dissolution buffer was 50% N,N-dimethylformamide (N,N-DMF) and 20% sodium dodecyl sulfate (SDS). The 96-well plate was placed in an incubator at 37 ° C until the purple crystals in the cells were dissolved. Finally, the absorbance at 570 nm was measured using a microplate spectrometer. The cytotoxicity ratio of the experimental group to which the sample of the extract of Antrodia camphorata was added was calculated relative to the untreated control group. Table 1 below shows the IC ₅₀ concentration of the extract of Antrodia camphorata of the present invention on rat stellate cells.

[Sirius red staining quantitative method]

The effect of the first A. angustifolia extract and the second A. angustifolia extract on the intracellular fiber protein content of rat stellate cells was examined by Picro-Sirius red (PSR) staining and spectrophotometric analysis. Rat stellate cells were treated with different concentrations (25, 50 and 100 μg/ml) of Antrodia camphorata extract for 24 hours. Next, the cells were fixed with methanol and placed in a refrigerator at -20 ° C overnight. After rinsing the cells twice with phosphate buffer solution, add the appropriate amount. The 0.1% PSR stain was left to stand at room temperature for 3 hours. After the excess dye was washed three times with 0.1% formic acid, 100 μl of 0.1 N sodium hydroxide was added to each well to dissolve the fibrin-bound dye. Finally, the absorbance at 540 nm was measured with a Dynex Technologies MRX spectrophotometer. The proportion of fibrin in the cells of the experimental group was calculated relative to the untreated control group.

From the fifth graph, the relative fiber protein content (relative COL content) of each experimental group was shown based on the group to which the extract of the present invention was not administered. The results showed that the stellate cells administered the first Antrodia camphorata extract or the second Antrodia camphorata extract of the present invention contained lower fibrin. Especially in the experimental group of 100 μg/ml, the content of fibrin in stellate cells was only about 30% of that of the cells.

Experiment 6: Preparation of a pharmaceutical composition containing Antrodia camphorata extract

The extract of Antrodia camphorata and the additive of the present invention are taken in the proportions shown in Table 2, and thoroughly mixed in a sterile water to prepare a pharmaceutical composition of the present invention in a liquid dosage form. The formulated pharmaceutical composition was placed at 4 ° C until use.

Claims (6)

Use of a compound of the following formula (I) for the preparation of a pharmaceutical composition for preventing and treating liver fibrosis; The use according to claim 1, wherein the compound of the formula (I) is obtained by isolating an extract of Antrodia camphorata. The use according to claim 1, wherein the compound of the formula (I) has an activity of inhibiting stellate cells. The use of claim 1, wherein the pharmaceutical composition comprises an effective amount of the compound of formula (I) and a pharmaceutically acceptable additive. The use according to claim 4, wherein the pharmaceutical composition is a tablet, capsule, injection, powder, granule or drink. The use of claim 4, wherein the pharmaceutically acceptable additive comprises: a carrier, an excipient, a preservative, a diluent, a filler, a binder, a disintegrant, an absorption enhancer, a sweetener, or combination.