TWI551291B - A pharmaceutical or food composition of antrodia cinnamomea extract reducing drug-resistance in cancer cells - Google Patents

Description

Anthraquinone mycelium extract with reduced drug resistance of cancer cells and pharmaceutical or food composition thereof

The present invention relates to an anthrax extract of Antrodia camphorata, the extract thereof and a pharmaceutical or food composition thereof having reduced drug resistance of cancer cells.

Cancer is one of the common diseases in modern people. It ranks first in the top ten causes of death every year. About one in four people will suffer from cancer. The prevention and treatment of cancer has become an issue that modern people cannot ignore. Our body is made up of countless cells that make new cells through mitosis, allowing us to grow, replace old cells, or repair damaged cells. Such a mechanism is controlled by genes, and if there is a problem in gene regulation, it is easy to cause cytopathic lesions, which in turn leads to abnormally proliferating cells leading to cancer. These cytopathic factors accumulate as we receive the external environment and stress, and some people inherit the damaged genes. Normal cells will divide and replicate in an orderly manner with gene regulation. When they are out of control, they will continue to replicate and accumulate in the body into a mass that becomes a tumor. Tumors can be divided into two types: benign and malignant. Malignant tumors are also called cancers. Cells of benign tumors do not spread to other parts of the body. Malignant tumors have the ability to metastasize because they have cancer cells. When it is first formed, it will remain in the original part, so it is called "in situ cancer." But as the situation worsens, it destroys the surrounding tissues and spreads to other organs, turning into invasive cancers. When the tumor spreads from its original location to other parts of the body, it is called secondary cancer or metastasis. Cancer (metastasis).

The way cancer is treated depends on the type of cancer, where it occurs, and whether it spreads to other parts of the body. Common treatments include surgery, chemotherapy, radiation therapy, etc.; sometimes combined to achieve better results. The use of oral or intravenous anticancer drugs to eliminate cancer cells or inhibit the growth of cancer cells has always been the mainstream of chemical drugs, and the targets are often malignant tumors that have been metastasized or cannot be eradicated.

Cancers found in the early stage can be treated with surgical resection, but when the cancer is advanced, it will be transferred to other organs via blood and lymph. At this time, local treatment alone cannot control the cancer cells. Local treatment alone cannot usually completely control cancer. Cells require systemic treatments such as chemotherapy, hormonal therapy or a new generation of targeted therapies. Currently used chemotherapeutic drugs are paclitaxel such as paclitaxol and docetaxel, purine analogs such as gemcitabine, topoisomerase II inhibitor such as campto (CPT-11), third generation platinum derivatives such as oxaliplatin, and combinations of liposome such as liposomal adriamycin. Significant progress has been made in improving the efficacy of chemotherapy and reducing side effects. In addition to killing cancer cells, the purpose of chemotherapy can also reduce the volume of tumors. The use of chemotherapy in the terminal patients can reduce the pain of patients, and combined with surgery and radiation therapy to reduce the risk of recurrence.

In the 1970s, it was found that clinically cancer patients who were treated with chemotherapy had reduced sensitivity to various chemotherapeutic drugs, called multidrug resistance (MDR), which was an important cause of chemotherapy failure. There are many mechanisms involved in the transformation of cancer cells into drug-resistant processes. Currently known mechanisms include: increasing drug excretion, changing drug metabolism pathways or products, cell cycle checkpoint changes, and apoptotic pathways. (cell apoptosis) regulation of disorders and changes in gene repair mechanisms. Multi-drug resistant Cancer cells, a large number of transmembrane transporters, called P-glycoprotein (P-gp), P-gp protein is ATP-dependent efflux pump, a large number of expression in intestinal epithelial cells and liver. Many different structures of the drug are carried out from the cells via the P-gp protein, thereby reducing the concentration of intracellular drugs. In cancer cells, when a chemotherapy drug stimulates a cancer cell, the cancer cell expresses a large amount of P-gp protein to expel the drug, thereby invalidating the chemotherapy drug. Although the role of P-gp protein in multidrug resistance in cancer cells has been extensively studied, it has been shown to be associated with multiple drug resistance caused by chemotherapeutic drugs, and other mechanisms that cause multiple drug resistance in cancer cells have been discovered, but still cannot be Solve the problem of clinical failure of chemotherapy due to multiple drug resistance. The therapeutic index of most anticancer drugs is very narrow. Once the sensitivity of cancer cells to anticancer drugs is slightly reduced, clinically, due to toxicity considerations, the dose cannot be increased, thus causing treatment interruption.

The type of apocynum lucidum is also known as the oyster mushroom, the oyster, the scorpion scorpion, and Taiwan has a yin and yang counterpart. The body of A. camphorata is perennial and has a strong aroma of eucalyptus, which is quite different from general ganoderma lucidum. Its appearance is plate-shaped or bell-shaped. In the plate shape, the surface is orange-red (yellow) color, the whole surface has micropores, and the bottom layer of the plate has a pale yellow-white cork, whereby the cork is attached to the inner wall of the hollow material in the burdock tree. The bell-shaped type, the sub-solid layer (clock face) is also orange (yellow) color, full of micropores (4-5 bacteria/mm), with spore taste very bitter, fresh orange-red, then It becomes orange-brown or brown, and the bell body is dark green-brown. The spores were observed under a microscope, and the shape was a smooth, colorless transparent micro-bend shape.

Wild anthrax grows on the hollow inner wall of the burdock trunk, and this characteristic causes many burdocks to fall. According to the literature, Antrodia camphorata is the only wood rot fungus found in burdock trees. The disease is brown and decayed, so it is brown rot fungus. However, the pathogenicity of Antrodia is not strong, so Burdock trees rarely die. Although Antrodia camphorata is a pathogen for Burdock, because Antrodia is expensive and exceeds the economic value of Burdock, it is not important that the pathogen of Burdock is not important.

The cultivation of Antrodia camphorata, the technique of artificial cultivation, still needs to be worked hard. Therefore, it is still obtained by means of deep mountain collection. But collecting anthocyanin is not an easy task, because the first place is to find the origin of the burdock tree. The common difficulty is the burdock tree and the lynx, which are very similar and difficult to distinguish. At present, the most direct method has been proposed by Fujita Anji. The dried oil is mainly Saforle and fifteen aldehydes, so there is the taste of scutellaria in SARS, and the sirloin is terpineol (d -terpinenol) is the main, but has the taste of camphor oil, which can distinguish the burdock and the cockroach; the second difficulty is to find the trunk with hollow holes from the large woods, which is quite difficult. If there is an ointment in the cavity, it can be collected regularly.

Anthraquinone fruiting bodies have been considered effective in the treatment of detoxification, hypertension, itching, diarrhea, allergies, and liver cancer. Because Antrodia camphorata is rich in triterpenoids, superoxide dismutase (SOD), adenosine, polysaccharides, β-D-glucan, β-D-glucan, Vitamins, which are rich in triterpenoids and polysaccharides, are believed to inhibit cancer cell proliferation to a certain extent, and have the ability to kill cancer cells, especially for liver cancer, stomach cancer, breast cancer, and cervical cancer. . At present, many studies have found that triterpenoids have anti-inflammatory, analgesic, poisonous tumor cells, induce tumor cell apoptosis, and anti-hypoxia, and also have the effect of improving immunity. The β-D-glucan can enhance the immune function by stimulating macrophages, T lymphocytes, B lymphocytes and natural killer cells to achieve anti-tumor effects.

In view of the fact that wild amaranth is sent to the burdock, the burdock belongs to the first-class wood species of the conservation class, and the hollow burdock is not easy to obtain; in addition, cancer is the top ten cause of death, and the chemotherapy process is often Because of the drug resistance of cancer cells, the efficacy of chemotherapy is not good; therefore, the inventor of the present invention has long conceived and researched to provide a method for cultivating the active substance of the mycelium extract of Ganoderma lucidum in liquid state, and proves that the liquid cultured Antrodia camphorata The active substance of the filament extract has the effect of reducing the drug resistance of the cancer cells, which is an important subject to be solved by the present invention.

In view of the foregoing problems and deficiencies, the present invention aims to provide a pharmaceutical composition for reducing cancer drug resistance, comprising an effective dose of an anthraquinone mycelium extract or an active substance of an anthraquinone mycelium, thereby reducing cancer cell pairs. Drug resistance of chemotherapy drugs.

An object of the present invention is to provide a use of an extract of Antrodia camphorata for extracting a medicine, wherein the mycelium extract of Antrodia camphorata is extracted from Antrodia camphorata (CCRC35396 strain), and the medicine is Paclitaxel, and The Antrodia camphorata mycelium extract reduces the resistance of cancer cells to the drug, and the drug resistance is the MDR protein expression amount.

For the purpose of the foregoing invention, the mycelium extract of Antrodia camphorata is prepared by the following steps: Step 1: The mycelium of Antrodia camphorata is subjected to ultrasonic wave shaking with an organic solvent, and then shaken to obtain mycelium extract of Antrodia camphorata. a liquid; the organic solvent is methanol; the volume ratio of the mycelium of Antrodia camphorata to the organic solvent is 1:5-20; Step 2: concentrated to remove the organic solution in the mycelium extract of A. niger, and then Ethyl acetate and water were layered, and the ethyl acetate layer was taken to remove ethyl acetate to obtain a crude extract of Antrodia camphorata mycelium; Step 3: The crude extract of the mycelium of A. angustifolia obtained in the step 2 is purified by colloidal column chromatography and extracted with n-hexane-ethyl acetate to obtain an active substance of the mycelium of Antrodia camphorata.

For the purpose of the foregoing invention, in the step 3, the colloidal column is a silicone or Sephadex LH-20 colloidal column, and the ratio of the n-hexane-ethyl acetate is 95:5 to 0:100.

For the purpose of the foregoing invention, the effective dose of the mycelium extract of Antrodia camphorata is 20 μg/ml.

For the purpose of the foregoing invention, the cancer cells include cervical cancer cells or liver cancer cells.

For the purpose of the foregoing invention, the volume ratio of the Antrodia camphorata to the organic solvent in the step 1 is 1:10.

For the purpose of the foregoing invention, the effective dose of the Paclitaxel is 0.5 μg/ml.

Another object of the present invention is to use an anthraquinone active substance for preparing a medicine, wherein the anthraquinone active substance comprises 4-Acetyl antroquinonol, Antrodin or Antroquinonol, the medicine is Paclitaxel, and the Antrodia camphorata active substance reduces cancer cell pairs The drug resistance of the drug, which is the amount of MDR1 protein expression.

For the purpose of the foregoing invention, the effective dose of the Paclitaxel is 0.5 μg/ml.

For the purpose of the foregoing invention, the effective dose of the active ingredient of the Antrodia camphora is 10 μg/ml.

In summary, the characteristics of the present invention include: 1. Extraction method of mycelium extract of Antrodia camphorata; 2. Mycelium extract of Antrodia camphorata can inhibit the expression of multidrug resistance protein MRD1 of cancer cells; 3. Antrodia camphorata The filamentous active substance expresses the multidrug resistance protein MRD1 which inhibits cancer cells.

The first picture is the effect of the mycelium extract of Antrodia camphorata on the expression of MDR1.

Figure 2 is a schematic diagram showing the effect of mycelium active substance on the expression of MDR1.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

All of the technical and scientific terms described in this specification, unless otherwise defined, are intended to be understood by those of ordinary skill in the art.

Source of strain

The strain A.camphorata CCRC-35396 used in the present invention was purchased from the Food Industry Development Research Institute.

Example 1

Fermentation of Antrodia camphorata mycelium

The CCRC-35396 strain was inoculated into a 2 liter Hinton Erlenmeyer flask containing 1 liter of medium containing 1.0% glucose, 0.5% soy flour, 0.5% peptone, 0.01% magnesium sulfate (MgSO4), 0.01 % defoamer (KM-72 antifoam), adjust the pH to The mixture was shaken at 28 ° C for 10 days at pH 4.0. The cultured mycelium was transferred to a 200 liter fermenter in a sterile chamber containing 120 liters of the above medium and fermented for 12 days at 28 °C.

Example 2

Antrodia camphorata mycelium extraction

The medicinal solution of the mycelium mycelium obtained by fermentation is freeze-dried, and ultrasonically shaken with 10 times of 100% methanol at 15-25 ° C for 1 hour, and the extraction may be repeated several times and then shaken overnight by 120 rpm. The obtained extract is concentrated under reduced pressure to remove methanol, and the residual methanol extract is separated with ethyl acetate and water, and the ethyl acetate layer extract is concentrated under reduced pressure to remove the solvent, and then separated by silica gel column chromatography. The active substance of the mycelium of Antrodia camphorata was extracted with n-hexane-ethyl acetate (95:5→0:100), and a total of 12 parts were obtained. According to the polarity from small to large, it is labeled as ACME (A~K), and after reconstitution, it is stored at 4 °C.

Example 3

Purification of active substances of Antrodia camphorata mycelium

The mycelium mycelium active material layer ACME-G was mixed with ACME-H, further separated by silica gel and Sephadex LH-20 column chromatography to obtain the compound 4-Acetyl antroquinonol B, Antrodin C, Antroquinonol, and stored after reconstitution. At 4 ° C. The pure compound obtained was confirmed by HPLC against a standard.

Example 4

Human cancer cell strain culture

Add 1 ml of medium to each well of a 6-well flat-bottomed cell culture dish containing 5×10 ⁴ cells, and incubate in a 5% carbon dioxide and 37° C. environment for 24 hours to allow the cells to be attached to the culture plate; The effects of the active substances of Antrodia camphorata on the expression of MDR1 in human liver cancer HepG2 cells were detected by the pure substances in human cervical HeLa cells and Antrodia camphorata.

The mycelium active substance of Antrodia camphorata is dissolved in Dimethyl sulfoxide (DMSO), and the concentration of DMSO is not more than 0.1% when tested to avoid its toxicity affecting cell growth; the cells are treated with the active substance of Antrodia camphorata Protein extraction was carried out for 24 hours; the experimental group was combined with the chemotherapeutic drug paclitaxel for the mycelium active substance of Antrodia camphorata.

Example 5

MDR1 protein detection

Protein extraction step: all steps are performed on ice, the cell culture medium is removed, washed with phosphate buffer solution, and a lysis buffer containing a protease inhibitor is added to break the cells and avoid protein hydrolysis.轻轻 Scrape the cells gently, use a micropipette to repeatedly aspirate to break the cells, collect the broken cell fluid into a centrifuge tube, centrifuge at 12000-13000 rpm, 4 ° C for 20 minutes, and extract the supernatant for protein quantification in the West. The amount of MDR1 expression was detected by western blot.

Protein quantification step: the sample was added to Bradford reagent, and the absorbance at 595 nm was detected by enzyme immunoassay (ELISA READER); the bovine serum albumin (BSA) was used as a standard curve to push back the sample. Protein concentration.

Western ink dot method: the quantitative protein sample was separated by 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular weight of MDR1 protein was 141KD; transfer After that, the MDR1 one-stage antibody was used for identification, and the MDR1 expression amount was analyzed by tableting, and the β-actin expression amount was used for internal quantitative control to correct the expression amount of MDR1.

Example 6

Effect of Mycelium Extract of Antrodia Camphorata on the Expression of MDR1

To detect the effect of mycelium extract of Antrodia camphorata on the expression of MDR1 in human cervical cancer Hela cells, Hela cells were treated with mycelium extract of Antrodia camphorata for 24 hours, and then treated with paclitaxel for 48 hours, low concentration (0.1 μM) The paclitaxel treatment increased the MDR1 expression of HeLa cells by 11.3% (Fig. 1). The high concentration (0.5 μM) paclitaxel treatment increased the MDR1 expression of HeLa cells by 44.6% ( p <0.01); the mycelium mycelium extract treatment In HeLa cells (with no paclitaxel), the stratified ACME-F of Antrodia camphorata active substance increased the expression of MDR1 but there was no significant difference. ACME-G and ACME-H reduced the expression of MDR1 in HeLa cells, ACME- H reduced the expression of MDR1 in HeLa cells by 29% ( p <0.05); ACME-F, ACME-G, ACME-H combined with paclitaxel treatment significantly reduced the MDR1 expression of HeLa cells ( p <0.01), among which ACME- The inhibitory effect of G was the most significant, and the MDR1 expression decreased from 144.61% to 10.43% ( p < 0.001).

Example 7

To detect the effect of the active substance treatment of Agaricus blazei on the expression of MDR1 in human hepatoma HepG2 cells, the treatment of HepG2 cells with Antroquinoneol for 24 hours can effectively reduce the MDR1 expression (Fig. 2), followed by chemotherapy. The drug paclitaxel treated human hepatocellular carcinoma HepG2 cells for 48 hours. The low concentration of paclitaxel (0.1 μM) increased MDR1 expression in Hep G2 cells by 8.7%, while the high concentration of paclitaxel (0.5 μM) increased MDR1 expression in Hep G2 cells by 34.2%. The treatment of human hepatocellular carcinoma HepG2 cells with 4-Acetyl antroquinonol B increased the expression of MDR1 by 45.8% ( p < 0.05), and Antrodin C and Antroquinonol significantly decreased the expression of MDR1 ( p <0.001); The filamentic active substance was combined with paclitaxel, and the pure compounds of the three anthraquinones significantly decreased the expression of MDR1 ( p <0.01). Among them, the effect of Antroquinonol was the most significant, and the amount of MDR1 decreased from 134.116% to 9.34% ( p <0.001). .

The mycelium mycelium active substance of the invention is obtained by fermentation of natural medicinal fungi, and has the effect of reducing the resistance of cancer cells compared with chemical synthetic drugs. The detailed description of the present invention is intended to be illustrative of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. In the scope of patents.

In summary, this case is not only innovative in terms of technical thinking, but also has the above-mentioned effects of reducing the resistance of cancer cells. It has fully complied with the statutory invention patent requirements of novelty and progressiveness, and applied for it according to law, and you are requested to approve this article. Invention patent application, in order to invent invention, to the sense of virtue.

Claims (10)

The use of an extract of Antrodia camphorata mycelium for preparing a medicine, wherein the mycelium extract of Antrodia camphorata is extracted from Antrodia camphorata (CCRC35396 strain), the medicine is Paclitaxel, and the mycelium of Antrodia camphorata The extract reduces the resistance of the cancer cells to the drug, which is the amount of MDR1 protein expression. The use of the mycelium extract of Antrodia camphorata as described in claim 1 for the preparation of a medicament, which is prepared by the following steps: Step 1: Take the mycelium of Antrodia camphorata to organic The solvent is oscillated by ultrasonic wave and shaken to obtain an extract of mycelium of Antrodia camphorata; the organic solvent is methanol; the volume ratio of the extract of Antrodia camphorata to the organic solvent is 1:5-20; Step 2: decompression The organic solution in the extract of the mycelium of Antrodia camphorata is concentrated and removed, and then layered with ethyl acetate and water to obtain an ethyl acetate layer, and ethyl acetate is removed to obtain a crude extract of Antrodia camphorata mycelium; Step 3: The crude extract of the mycelium of Antrodia camphorata obtained in the step 2 was purified by colloidal column chromatography and extracted with n-hexane-ethyl acetate to obtain an extract of Antrodia camphorata mycelium. The use of the mycelium extract of Antrodia camphorata described in claim 2 for the preparation of a pharmaceutical product, wherein in the step 3, the colloidal column is a tannin or Sephadex LH-20 colloidal column, the n-hexane-acetic acid The ester ratio is from 95:5 to 0:100. The use of the mycelium extract of Antrodia camphorata as described in claim 1 for the preparation of a pharmaceutical product, wherein the effective dose of the mycelium extract of Antrodia camphorata is 20 μg/ml. The use of the mycelium extract of Antrodia camphorata as described in claim 1 for the preparation of a medicament, wherein the cancer cell comprises a cervical cancer cell or a liver cancer cell. The use of the mycelium extract of Antrodia camphorata as described in claim 2 for the preparation of a pharmaceutical product, wherein the volume ratio of the mycelium of Antrodia camphorata to the organic solvent in step 1 is 1:10. The use of the mycelium extract of Antrodia camphorata as described in claim 1 for the preparation of a medicament, wherein the effective dose of the Paclitaxel is 0.5 μg/ml. The use of an active ingredient for the preparation of a medicinal preparation, wherein the active substance of the genus Antrodia campanum comprises 4-Acetyl antroquinonol, Antrodin or Antroquinonol, the medicine The strain is Paclitaxel, and the anthraquinone active substance reduces the resistance of the cancer cell to the drug, and the drug resistance is the MDR1 protein expression amount. The use of the preparation of a pharmaceutical preparation according to the invention of claim 8, wherein the effective dose of the Paclitaxel is 0.5 μg/ml. The use of the medicinal preparation for preparing an active medicinal preparation according to the invention of claim 8 wherein the effective dose of the active ingredient of the scorpion is 10 μg/ml.