CN105237365B - Extract of solid culture mycelium of antrodia camphorata and application thereof - Google Patents

Abstract

An extract of solid-state cultured mycelium of Antrodia camphorata and use thereof in the preparation of an anti-lung cancer cell metastasis drug. The extract of solid-state cultured mycelium of Antrodia camphorata is 2,3,6-Trimethoxy-4-methylphenol, also known as Leader 1. Leader 1 can effectively inhibit the movement, migration and invasion of lung cancer tumor cells, thereby achieving the effect of anti-lung cancer cell metastasis, and can show excellent physiological activity and mechanism of action against lung cancer cell metastasis.

Description

Extract of Antrodia camphorata solid-state cultured mycelium and its application

technical field

The invention relates to an extract of antrodia camphorata solid-state cultured mycelia, and also relates to the application of the extract of the solid-state cultured mycelium of Antrodia camphorata in the preparation of anti-lung cancer cell metastasis medicine.

Background technique

The formation of tumors will be caused by personal behavior, age, diet, living habits, genetics and environment. According to the data of the Department of Health, malignant tumors are still the top ten causes of death, and the causes of death of cancer patients are usually Because of tumor metastasis, how to effectively control tumor cells and inhibit their metastasis is one of the very important strategies for treating cancer.

Antrodia cinnamomea (Syn. Antrodia camphorata and Taiwanofunguscamphorata) is a very precious medicinal fungus. It has been confirmed in many scientific studies that the methanol extract and hot water extract of Antrodia camphorata mycelium, the methanol extract, ethanol extract and ethyl acetate extract of fruiting body have excellent anti-inflammatory activity, and some studies It is pointed out that the mycelia and fruiting bodies of Antrodia camphorata contain some naphthenic compounds, which have the ability to fight cancer. , still needs to be further understood. Therefore, it generally cannot meet the needs of users in actual use.

Contents of the invention

The main purpose of the present invention is to overcome the above-mentioned problems encountered by the known technology and provide an extract of Antrodia camphorata solid-state cultured mycelium and its application. The extract can inhibit the generation of lung cancer tumor metastasis, and exhibit excellent anti- cancer metastasis.

For reaching above object, the technical scheme that the present invention adopts is: a kind of extract of Antrodia camphorata solid-state culture mycelia, and this extract obtains by following steps:

(A1) provide a dried Antrodia camphorata solid-state culture mycelium;

(B1) Extract the dried Antrodia camphorata solid-state culture mycelium with 10 times the volume of 95% ethanol at 25-40°C.

Another technical scheme adopted in the present invention is: a kind of extract of Antrodia camphorata solid-state cultured mycelia, and the extract is obtained according to the following steps:

(A2) provide a dried Antrodia camphorata solid-state culture mycelium;

(B2) extracting the dried Antrodia camphorata solid-state culture mycelium at 25-40°C with 10 times the volume of 95% ethanol to obtain an ethanol extract;

(C2) concentrating the ethanol extract in vacuo to obtain a concentrated product;

(D2) The concentrated product was layered using 100% by volume ethyl acetate and water to obtain an ethyl acetate extract.

The above-mentioned drying method of drying the solid-state cultured mycelia of Antrodia camphorata is cold air drying.

The present invention also provides the application of the above-mentioned extracts in the preparation of anti-metastatic drugs for lung cancer cells. The extract resists lung cancer cell metastasis by inhibiting lung cancer cell migration or invasion.

The extract inhibits the migration and invasion of lung cancer cells by reducing phosphorylated serine kinase, gelatin decomposing enzyme in matrix metalloproteinase, and increasing the expression of calcium adhesion molecule E and metalloproteinase inhibitor.

The molecule of the above-mentioned extract mentioned in the present invention is 2,3,6-Trimethoxy-4-methylphenol (2,3,6-trimethoxy-4-methylphenol), and its structural formula is as follows:

The present invention also provides a method for resisting lung cancer cell metastasis, which is to make the subject to be tested with the above-mentioned extract or 2,3,6-Trimethoxy-4-methylphenol (2,3,6-trimethoxy-4-methyl phenol) contact.

To sum up, the extract of Antrodia camphorata solid-state cultured mycelium of the present invention has the effect of anti-lung cancer cell metastasis. More specifically, the present invention discloses the physiological activity of the extract of Antrodia camphorata solid-state cultured mycelium in anti-lung cancer cell metastasis. The mechanism of action can be prepared as an anti-lung cancer cell metastasis drug.

Description of drawings

Fig. 1 is a schematic diagram of Leader 1, an extract of Antrodia camphorata solid-state cultured mycelia of the present invention.

Fig. 2 is a schematic diagram showing the effect of Leader 1, an extract of Antrodia camphorata solid-state cultured mycelia, on the cell survival rate of lung cancer cells.

Fig. 3 is a schematic diagram showing the expression of the effect of Leader 1, an extract of Antrodia camphorata solid-state cultured mycelia, on the cell wound healing ability of lung cancer cells.

Fig. 4A and Fig. 4B are schematic diagrams showing the effect of Leader 1, an extract of Antrodia camphorata solid-state cultured mycelia, on the cell migration ability of lung cancer cells in the present invention.

Fig. 5 is a schematic diagram showing the expression of Leader 1, an extract of Antrodia camphorata solid cultured mycelium of the present invention, affecting the cell invasion ability of lung cancer cells.

6A and 6B are schematic diagrams showing the expression of Matrix metalloproteinases (MMP-2, MMP-9) of lung cancer cells affected by Leader 1, an extract of Antrodia camphorata solid-state cultured mycelium of the present invention.

Fig. 7 is a schematic diagram showing the performance of Leader 1, an extract of Antrodia camphorata solid-state cultured mycelium of the present invention, affecting the ability of plasminogen activator in lung cancer cells.

Fig. 8A and Fig. 8B are schematic diagrams showing the expression level of related proteases and protein expression ability of Leader 1, an extract of Antrodia camphorata solid-state cultured mycelia of the present invention, affecting lung cancer cell metastasis.

detailed description

The invention relates to an extract of antrodia camphorata solid-state cultured mycelia and its application. The extract inhibits the metastasis of lung cancer cells and exhibits excellent anti-cancer metastasis function. The extract of Antrodia camphorata solid-state cultured mycelia is 2,3,6-Trimethoxy-4-methylphenol (2,3,6-trimethoxy-4-methylphenol), also known as Leader 1 .

The process of cancer cell metastasis includes cell adhesion (adhesion), invasion (invasion) and migration (migration). The metastasis of cancer cells requires matrix metalloproteinases (matrix metalloproteinases, MMP) to decompose the extracellular matrix (Extracellutar Matrix, ECM), and produce epithelial-mesenchymal transition (Epithelial-Mesenchymal Transition, EMT); It will be expressed in large numbers and promote the occurrence of cancer cell metastasis.

Tumor necrosis factor (TNF) is a small molecular protein (cytokine) secreted by macrophages. Among them, tumor necrosis gene-α (TNF-α) is mainly secreted by monocyte-macrophages. Many previous studies pointed out that TNF-α can promote the invasion and metastasis of lung cancer cells. Accordingly, when used, the present invention uses TNF-α to induce A549 lung cancer cells as a model for exploring the role of the solid-state cultured mycelia in Antrodia camphorata in anti-lung cancer cell metastasis.

The following examples are only examples for understanding the details and connotation of the present invention, but are not intended to limit the patent scope of the present invention.

Embodiment 1: experimental design

[Material]

The solid-state culture mycelium of Antrodia camphorata used in the present invention is the mycelium self-cultivated by Lide Biotechnology Company, Taiwan, China. Fetal Bovine Serum (FBS) was purchased from Gibco BRL (Invitrogen, Grand Island, NY). Dimethyl sulfoxide (DMSO), penicillin, thiazolyl blue tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT), Jim Both Gimsa and Casein were purchased from Sigma-Aldrich (St Louis, MO). All chemicals and solvents used in the present invention are reagent or High Performance Liquid Chromatography (HPLC) grade.

[Cell culture]

In the present invention, human lung cancer tumor A549 cells are selected for tumor cell metastasis experiments. Regarding the cultivation of A549 cells, first replant A549 cells grown in a culture flask (75T Flask) in a 10 cm ² culture dish (dish) at a seeding density of 2×10 ⁵ cells/cm ² , and culture them at 37°C , in a 5% CO ₂ incubator. The culture medium used was Ham's F12 culture medium according to the recommendations of the Bioresources Collection and Research Center (BCRC) and the American Type Culture Collection (ATCC), plus 10% fetal bovine serum.

Embodiment 2: Preparation of the extract Leader 1 of Antrodia camphorata solid-state culture mycelium of the present invention

First the Antrodia camphorata solid-state culture mycelium is dried in a cold-air drying manner, and then at 25° C., 2000 grams of the above-mentioned Antrodia camphorata solid-state culture mycelium are mixed with 95% volume percentage (v/v) of 10 times the volume. Ethanol extraction to obtain the ethanol extract (ACME) of the present invention. The aforementioned drying method can be any drying method known in the art without limitation, such as cold air drying.

The ethanol extract was then concentrated in vaccum to obtain 323.6 g of concentrated product. The resulting concentrated product is then divided into an ethyl acetate separation part and a water-soluble layer with 100% (v/v) ethyl acetate and water, wherein the water-soluble layer is marked as the water-soluble separation part (ACME-water), and The ethyl acetate separation part is the ethyl acetate extract (ACME-EA) of the present invention.

Example 3: Separation of Leader 1

The ethyl acetate extract (ACME-EA) obtained in the above Example 2 was divided into 9 sub-separation parts through the concentration gradient of the mobile phase by column chromatography. Wherein, the stationary phase used in the column chromatography is silica gel (230-400mesh), and the mobile phase is n-hexane/ethyl acetate mixed solution; The n-hexane/ethyl acetate mixture with the concentration ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 30:70, and 0:100 was used as the mobile phase for the The Chromatography.

Then the above-mentioned 4th separation part is purified and separated by high-performance liquid chromatography, using a silica gel column as the stationary phase, and using a n-hexane/ethyl acetate mixed solution with a volume ratio of 77:23 as the mobile phase, at a flow rate 5 ml/min, UV 254nm wavelength conditions, further separation and purification to obtain its constituents, and the highest content of the constituents were analyzed by mass spectrometry to obtain a molecular weight of 198.09, and deduced molecular formula as C ₁₀ H ₁₄ O ₄ . This component was further confirmed by H-NMR and C-NMR spectra and its structural formula is as follows:

Please refer to Figure 1, it is confirmed that this compound is 2,3,6-Trimethoxy-4-methylphenol (Leader1).

Example 4: The test of the extract Leader 1 of Antrodia camphorata solid-state culture mycelium of Example 2 inhibiting the survival rate of lung cancer cells (lung cancer cell line A549)

First, the A549 lung cancer cells were cultured according to the cell culture method described in the above-mentioned Example 1. After washing with trypsin, the cells were placed in a 24-well plate at a seeding density of 2×10 ⁵ cells/cm ² for 24 hours to allow the cells to attach, as shown in the following table As shown in 1, replace the culture medium containing the Leader 1 compound at a concentration of 0, 5, 10, 20 and 40 μM (the group with 0 μM added with DMSO is used as the control group) and remove it after 24 hours of culture, add 5 mg/ml of MTT After acting for 2 hours, MTT is a water-soluble tetrazolium salt (tetrazolium salt), which is light yellow after being dissolved in phosphate buffered saline (PBS), and after being reduced by the dehydrogenase in the mitochondria of the cell, MTT The ring structure will be replaced by water-insoluble blue-purple crystals, and then the cell membrane and blue-purple crystals are dissolved with DMSO, and the absorbance value is measured at a wavelength of 570nm and statistically plotted with sigma plot10.0.

Table I

Please refer to FIG. 2 , which is a schematic diagram showing that the extract of Antrodia camphorata solid-state culture mycelium Leader 1 affects the cell survival rate of lung cancer cells. As shown in the figure: Leader1, the extract of Antrodia camphorata solid-state cultured mycelium obtained in Example 2, has no effect on the viability of lung cancer cells, and there is no difference at a concentration of 0-40 μM, showing that it has no effect on lung cancer cells. No cytotoxic production.

Example 5: The test of the extract Leader 1 of Antrodia camphorata solid-state culture mycelium of Example 2 inhibiting wound healing of lung cancer cells (lung cancer cell line A549)

First, the A549 lung cancer cells were cultured according to the cell culture method described in Example 1 above. After washing with trypsin, the cells were placed in a 12-well dish at a seeding density of 2×10 ⁷ cells/cm ² for 24 hours to allow the cells to attach, and then Draw the line of the wound in each well plate, wash the floating cells slightly with PBS, and add Leader 1 containing concentrations of 0, 5, 10, 20 and 40 μM (the group with 0 μM is treated with DMSO as the control) Group) culture medium, the observation time point is 0, 12, 24 or 48 hours after adding the drug, and take pictures for records.

Please refer to FIG. 3 , which is a schematic diagram of the expression of the effect of the extract Leader 1 of the solid-state cultured mycelium of Antrodia camphorata on the wound healing ability of lung cancer cells. As shown in the figure: Leader 1, the extract of Antrodia camphorata solid-state cultured mycelia obtained in Example 2, exhibited the ability to inhibit and promote wound healing of lung cancer cells, especially at a concentration of 40 μM.

Example 6: Test of the ability of Leader 1, the extract of Antrodia camphorata solid-state cultured mycelium in Example 2, to inhibit the migration of lung cancer cells (lung cancer cell line A549)

The migration ability of lung cancer cells was analyzed using a 24-well transwell kit (Millipore).

First, A549 lung cancer cells were cultured according to the cell culture method described in Example 1 above. After washing with trypsin, the cells were placed in a 6-well plate at a seeding density of 2×10 ⁵ cells/cm ² for 24 hours to allow the cells to attach, and then added After 24 hours in the culture medium containing Leader 1 at a concentration of 0, 5, 10, 20, and 40 μM (the group with 0 μM was treated with DMSO as the control group), the cells were rinsed with PBS, and the cells were washed with trypsin (tripsin). Cells were spun down, put into a centrifuge tube and centrifuged at 1280rpm for three minutes, removed the supernatant, washed slowly with PBS to remove the residual serum on the cells, and then centrifuged at 1280rpm for 3 minutes, added the cells at a density of ^2x105 cells/ ^cm2 containing only 0.1% fetal bovine serum culture medium, get 200 μ l and inject into the upper layer of the through hole, the lower layer uses a medium containing 20% fetal bovine serum (can be added or not added for TNF-α induction), put it in the incubator, and observe the time point: 12 or 24 hours.

Take out the through-hole, slowly moisten the through-hole with secondary water (ddH ₂ O), remove the cells in the upper layer that have not penetrated through the pores with a cotton swab; then soak the through-hole in methanol for 20 minutes, then dry it, soak Stain in Gimsa for 20 minutes, let the through-holes soak in secondary water for several minutes and then dry, observe the cell migration with a microscope, take pictures and record, and use sigma plot10.0 for statistical drawing.

Please refer to FIG. 4A and FIG. 4B , which are schematic diagrams showing the effect of the extract of Antrodia camphorata solid-state cultured mycelium Leader 1 on the cell migration ability of lung cancer cells. As shown in the figure: Leader 1, the extract of Antrodia camphorata solid-state cultured mycelia obtained in Example 2, exhibited the ability to inhibit the migration of lung cancer cells, especially at a concentration of 40 μM.

Example 7: Test of the Invasion Ability of the Extract Leader 1 of Antrodia Camphorata Solid-state Cultured Mycelia of Example 2 to Inhibit the Invasion of Lung Cancer Cells (Lung Cancer Cell Line A549)

The invasive ability of lung cancer cells was analyzed using a 24-well plate set. Matrigel (Matrigel, 3mg/ml-well, BD science) was first injected into the upper layer of the through-hole, and placed in an incubator for 24 hours to solidify for later use.

First, A549 lung cancer cells were cultured according to the cell culture method described in Example 1 above. After dismantling with trypsin, the cells were placed in a 6-well plate at a seeding density of 2×10 ⁵ cells/cm ² for 24 hours to allow the cells to attach, and then added After 24 hours in the medium containing Leader 1 at different concentrations of 0, 5, 10, 20 and 40 μM, the cells were rinsed with PBS, the cells were knocked down with trypsin, put into a centrifuge tube and centrifuged at 1280rpm for 3 minutes, and the upper surface was removed. For the supernatant, slowly wash away the residual serum on the cells with PBS, then centrifuge at 1280rpm for 3 minutes, add the cells to the medium containing only 0.1% fetal bovine serum at a density of ^2x105 cells/ ^cm2 , and inject 200μl into the upper layer of the through-hole , the lower layer uses a medium containing 20% fetal bovine serum, puts it into an incubator, and observes for 12 or 24 hours.

Take out the through-hole, slowly moisten the through-hole with secondary water, and remove the cells in the upper layer that do not penetrate through the pores with a cotton swab; then soak the through-hole in methanol for 20 minutes, then dry it, and soak it in Gimsa for 20 minutes Minutes, soak the through hole in secondary water for a few minutes and then dry it. Use a microscope to observe the cell invasion situation, take pictures and record it, and use sigma plot 10.0 for statistical drawing.

Please refer to FIG. 5 , which is a schematic diagram of the expression of the effect of the extract Leader 1 of the solid-state cultured mycelia of Antrodia camphorata on the cell invasion ability of lung cancer cells. As shown in the figure: Leader 1, the extract of Antrodia camphorata solid-state cultured mycelia obtained in Example 2, exhibited the ability to inhibit and promote the invasion of lung cancer cells, especially at a concentration of 40 μM.

Example 8: Test of the ability of Leader 1, the extract of Antrodia camphorata solid-state cultured mycelium in Example 2, to inhibit the matrix metalloproteinases of lung cancer cells (lung cancer cell line A549)

The hydrolytic enzymes were detected by electrophoresis, and the enzyme activity of gelatinases (gelatinases; MMP-2, MMP-9) in matrix metalloproteinases (MMP) in lung cancer cells was analyzed by gelatin-zymography.

Firstly, the A549 lung cancer cells were cultured according to the cell culture method described in the above-mentioned Example 1. After dismantling with trypsin, the cells were placed in a 12-well dish at a seeding density of 2×10 ⁵ cells/cm ² for 24 hours to allow the cells to attach, and then Rinse twice with PBS, add the medium containing Leader 1 at different concentrations of 0, 5, 10, 20 and 40 μM for 24 hours, replace it with the medium containing 1% fetal bovine serum for 24 hours, and obtain the culture medium with cell culture The culture medium was put into a microcentrifuge tube and centrifuged at 1250 rpm for 10 minutes, and the supernatant was taken and stored at -20°C.

Add an equal amount of supernatant mixture to 6 times matrix metalloproteinase dye (6x MMP dye) to 8% gradient electrophoresis gel well (SDS-PAGE, containing gelatin 1mg/ml), add running buffer (running buffer) Use 80V, 300mA to resolve (resolve) to 45kD matrix metalloproteinase marker (MMP marker) when it reaches the middle of the gel, it will stop working.

Remove the gel and apply it at 35rpm in zymography renaturing buffer, and act twice at room temperature for 30 minutes each time. After the SDS is removed, use zymography developing buffer (zymography developing buffer). buffer) at 35rpm and 37°C for 20-24 hours, removed, stained with coomassie blue R-250 for 30 minutes, soaked in secondary water until the next day, and then put the gel into a digital image analyzer ( Chemi-smart 3000) photographic analysis on the test object plate and statistical mapping with sigma plot 10.0.

Please refer to FIG. 6A and FIG. 6B , which are schematic diagrams showing how the extract Leader 1 of Antrodia camphorata solid-state cultured mycelium of the present invention affects the expression of matrix metalloproteinases in lung cancer cells. As shown in the figure: Leader 1, the extract of Antrodia camphorata solid-state culture mycelia obtained in Example 2, has the ability to inhibit matrix metalloproteinases (MMP-2, MMP-9) in lung cancer cells, especially at a concentration of 40 μM. excellent.

Example 9: Test of the ability of Leader 1, the extract of Antrodia camphorata solid-state cultured mycelium in Example 2, to inhibit lung cancer cells (lung cancer cell line A549) plasminogen activator

The hydrolytic enzyme was detected by electrophoresis, and the urokinase-type plasminogen activator (uPA) in lung cancer cells was analyzed by using casein zymography (Casein-zymography) containing plasminogen (plasminogen). ), the enzyme activity of urokinase-type plasminogen activator will decompose plasminogen into plasmin (plasmin).

First, the A549 lung cancer cell line was cultured according to the cell culture method described in the above-mentioned Example 1. After dismantling with trypsin, the cells were placed in a 12-well dish at a seeding density of 2×10 ⁵ cells/cm ² for 24 hours to allow the cells to attach, and then Rinse twice with PBS, add Leader 1 medium containing different concentrations of 0, 5, 10, 20 and 40 μM for 24 hours, replace with medium containing 1% fetal bovine serum for 24 hours, and obtain the cell culture medium The culture solution was put into a microcentrifuge tube and centrifuged at 1250rpm for 10 minutes, and the supernatant was taken and stored at -20°C.

Add an equal amount of the supernatant mixture to 6 times matrix metalloproteinase staining agent to 8% gradient electrophoresis gel wells (containing plasminogen 15μl/ml and casein 1mg/ml), add running buffer at 80V, 300mA When the matrix metalloproteinase separated to 45kD is labeled in the middle of the gel, it stops working.

Remove the gel and act in zymography refolding buffer at 35rpm, and act twice at room temperature for 30 minutes each time. After removing the SDS, act in zymography developing buffer at 35rpm at 37°C Remove after 20-24 hours, stain with Coomassie Brilliant Blue for 30 minutes, soak in secondary water until the next day, then place the glue on the test plate of a digital image analyzer for photographic analysis and use sigma plot 10.0 for statistical drawing.

Please refer to FIG. 7 , which is a schematic representation of the performance of Leader 1, an extract of Antrodia camphorata solid-state cultured mycelium of the present invention, affecting the ability of plasminogen activator in lung cancer cells. As shown in the figure: Leader 1, the extract of Antrodia camphorata solid-state cultured mycelium obtained in Example 2, exhibited the ability to inhibit and promote plasminogen activator in lung cancer cells, especially at a concentration of 40 μM.

Example 10: The test of the expression of related proteins that Leader 1, the extract of Antrodia camphorata solid-state cultured mycelium in Example 2, regulates the metastasis of lung cancer cells (lung cancer cell line A549)

After obtaining the total protein of the cells, add the same amount of protein into the gel well of 8-10% gradient electrophoresis gel, separate at 80V, 300mA for 240 minutes, separate the proteins according to size at 100V, 2 hours as the condition, After transferring to polyvinylidene difluoride (polyvinylidene difluoride, PVDF) membrane, the vinylidene fluoride membrane was acted with blocking buffer (blocking buffer) for 1 hour. Wherein, the isolation buffer comprises 10% w/v skimmed milk powder in TBS-T buffer, and the TBS-T buffer contains 0.1% Tween 20 TBS buffer.

Then soak the vinyl difluoride membrane in the antibody solution of actin (actin; Cell signaling), the antibody solution of phosphorylated serine kinase (p-Akt or protein kinase B; 1:1000, Cell signaling), the calcium adhesion molecule E ( E-cadherin; 1:1000, Cell signaling), the antibody solution of gelatinolytic enzymes in matrix metalloproteinases (MMP-2,9; 1:1000, Santa Cruz), and the antibody solution of metalloproteinase inhibitors (TIMP-1 ; 1:1000, Cellsignaling), after reacting at 4°C for 12-24 hours, the recovered antibody solution was returned to the -20°C refrigerator, and then washed 3 times with 0.1% TBS-T buffer to wash away non-specificity ( non-specific) bound antibody antigen.

Then soak the vinyl difluoride membrane in the anti-mouse secondary antibody solution (anti-mouse secondary antibodies) and anti-rabbit secondary antibody solution (anti-rabbit secondary antibodies) with horseradish peroxidase (horseradish peroxidase), at room temperature After reacting for 1-2 hours, the antibody solution was recovered, washed three times with 0.1% TBS-T buffer, and then the fluorescence intensity of each group was detected with enhanced chemiluminescene regents (ECL), and β-actin The expression of (β-actin) was used as the control group of the protein, and was statistically plotted with sigma plot10.0.

Please refer to FIG. 8A and FIG. 8B , which are schematic representations of the ability of Leader 1, an extract of Antrodia camphorata solid-cultured mycelium of the present invention, to affect the expression of proteases and proteins related to lung cancer cell metastasis. As shown in the figure: Leader 1, the extract of Antrodia camphorata solid-state cultured mycelium obtained in Example 2, exhibited the ability to inhibit the expression of related proteases that promote lung cancer cell metastasis, and promoted the ability to inhibit the expression of related proteins that promote lung cancer cell metastasis , with significant differences.

Thus, the present invention provides an extract of Antrodia camphorata solid-state cultured mycelium and its application. The extract of the solid-state cultured mycelium of Antrodia camphorata is 2,3,6-Trimethoxy-4-methylphenol (Leader 1), the Leader 1 can effectively inhibit the motility, migration and invasion abilities of lung cancer tumor cells, thereby achieving the effect of anti-metastasis of lung cancer cells, and can exhibit excellent physiological activity and mechanism of action against metastasis of lung cancer cells. In summary, an extract of Antrodia camphorata solid-state cultured mycelium of the present invention and its application can effectively improve various shortcomings in the prior art. The extract of the solid-state cultured mycelium of Antrodia camphorata is 2,3, 6-Trimethoxy-4-methylphenol (Leader 1), can effectively inhibit the ability of lung cancer tumor cells to move, migrate and invade, so as to achieve the effect of anti-metastasis of lung cancer cells, and can exhibit excellent physiological activity and mechanism of anti-metastasis of lung cancer cells , and then make the production of the present invention more progressive, more practical, and more in line with the needs of users, and have indeed met the requirements of the invention patent application, and the patent application is filed according to law.

However, the above descriptions are only preferred embodiments of the present invention, and should not limit the implementation scope of the present invention. Therefore, all simple equivalent changes and modifications made according to the patent scope of the present invention and the contents of the description of the invention shall still fall within the scope covered by the patent of the present invention.

Claims (8)

1. a kind of mycelial extract of antrodia solid state rheology, it is characterised in that the extract is 2,3,6- trimethoxy -4- first Base phenol, its structural formula is as follows：

2. the preparation method of the mycelial extract of antrodia solid state rheology described in claim 1, it is characterised in that the extract is Obtained by with the following steps：

(A1) an antrodia solid state rheology mycelium dried is provided；

(B1) by the antrodia solid state rheology mycelium of the drying at 25-40 DEG C with the ethanol of 95% volumetric concentration of 10 times of volumes Extraction.

3. the preparation method of the mycelial extract of antrodia solid state rheology described in claim 1, it is characterised in that the extract is Follow these steps to obtain：

(A2) an antrodia solid state rheology mycelium dried is provided；

(B2) by the antrodia solid state rheology mycelium of the drying at 25-40 DEG C with the ethanol of 95% volumetric concentration of 10 times of volumes Extract to obtain an ethanolic extract；

(C2) ethanolic extract is concentrated in vacuo to obtain an enriched product；

(D2) the enriched product stratification is extracted with obtaining an ethyl acetate with water using the ethyl acetate of 100% volumetric concentration Thing.

4. the preparation method of the mycelial extract of antrodia solid state rheology as claimed in claim 2 or claim 3, it is characterised in that institute Drying mode is stated for cold air drying.

5. application of the extract in anti-lung cancer cell diversion medicaments are prepared described in claim 1.

6. application as claimed in claim 5, it is characterised in that the extract is by suppressing lung carcinoma cell effect on migration to resist Lung carcinoma cell is shifted.

7. application as claimed in claim 5, it is characterised in that the extract is acted on to resist by suppressing lung carcinoma cell invasion Lung carcinoma cell is shifted.

8. application as claimed in claim 5, it is characterised in that the extract is by reducing phosphorylation serine kinase, base Gelatinolytic enzyme in matter metalloproteinases and increase calcium adhesion molecule E, the performance of metal protease inhibitors are to suppress the lung The effect on migration of cancer cell is acted on invasion and attack.