CN102232940A - Cyclohexenone compound from Antrodia antrodia for inhibiting the growth of colorectal cancer tumor cells - Google Patents

Abstract

The invention relates to a new application of a compound and a pharmaceutical composition for inhibiting the growth of colorectal cancer tumor cells, wherein 4-hydroxy-2, 3-dimethoxy-6-methyl-5 (3, 7, 11-trimethyl-2, 6, 10-dodecatriene) -2-cyclohexenone (4-hydroxy-2, 3-dimethoxy-6-methyl-5 (3, 7, 11-trimethyl-dodeca-2, 6, 10-trienyl) -cyclohexenone) is obtained by separating and purifying antrodia camphorata extract, and the cyclohexenone compound can be applied to inhibiting the growth of colorectal cancer tumor cells and can also be applied to the pharmaceutical composition for inhibiting the growth of colorectal cancer tumor cells.

Description

Cyclohexenone compound from Antrodia antrodia for inhibiting the growth of colorectal cancer tumor cells

technical field

The present invention relates to a new application of a compound, in particular to a use of a compound isolated and purified from the extract of Antrodiacinnamomea to inhibit the growth of colorectal cancer tumor cells. the

Background technique

The large intestine includes the cecum, colon and rectum, which is the last checkpoint of the human digestive system. The normal colorectal mucosa will cause benign cells to proliferate on the inner wall of the large intestine due to many factors to form adenoma (i.e. deflated flesh), and even further become colorectal cancer. Colorectal cancer, also known as colorectal cancer, is one of the most common malignant tumors in the world. In recent years, in Taiwan, due to the westernization and refinement of Chinese people's life and diet, as well as the increase in animal fat and protein intake Increasing year by year and decreasing dietary fiber intake, the incidence rate of colorectal cancer is increasing year by year, and now it has become the third cause of cancer death. the

The main symptoms of colorectal cancer include bloody stools, abnormal bowel movements, abdominal distension, anemia, and even abdominal lumps. It is not easy to have subjective symptoms when colorectal cancer occurs in the early stage, but when you feel that you have prostatic symptoms, it is not the stage of early cancer, which makes the treatment difficult. In addition, colorectal cancer generally occurs in patients over the age of 50, but studies have pointed out that the average age of colorectal cancer patients in Taiwan is lower than that of foreign countries, and the trend of rejuvenation is obvious, and the prognosis of colorectal cancer for young people is relatively poor, which is different from that of young people. Human cancers are usually poorly differentiated and highly malignant cancers. At the same time, it is also related to the fact that cancers in young people are often terminal cancers when they are first diagnosed. the

The treatment of colorectal cancer is mainly based on surgical resection, and for patients with spread of cancer cells, radiation therapy and chemotherapy are supplemented. One of the main functions of chemotherapy is to interfere with the progress of the cancer cell cycle, affect the synthesis of DNA, RNA and protein, and achieve the purpose of inhibiting the proliferation of tumor cells. However, radiotherapy and chemotherapy are often accompanied by other side effects that cause physical discomfort. On the other hand, the liver and lung are the most likely sites for colorectal cancer to develop remote metastasis. Once colorectal cancer has remote metastasis, even compound chemotherapy and cancer cell target therapy will have limited effect on most patients with remote metastasis. And its prognosis is poor. Therefore, it is imperative to develop effective and less side effect therapeutic substances for clinical application. the

Antrodia cinnamomea, also known as camphor mushroom, camphor mushroom, camphor mushroom, camphor mushroom or red camphor, is a unique medicinal mushroom in Taiwan, which belongs to the order Aphyllophorales, Any of perennial mushrooms of the family Polyporaceae. Because Antrodia camphorata only parasitizes on the inner wall tissue of the hollow core wood trunk of Taiwan’s unique conservation class Cinnamomum camphorata in nature, coupled with artificial illegal logging, the number of wild Antrodia camphorata that can only grow in it is even rarer, and due to the natural state The fruiting body of Antrodia camphorata grows very slowly, so wild Antrodia camphorata is rare and expensive. the

The fruiting body of Antrodia cinnamomea is perennial, sessile, corky to woody, with a strong camphor tree aroma, and its shape is varied, with plate, bell, horseshoe or tower shape. It is flat and bright red when it is born, and then its periphery will show a radial and reverse roll shape, and it will expand and grow around, and the color will also change to light reddish brown or light yellowish brown, with many pores, and it is the medicine of Antrodia camphorata. Use the most valuable parts. the

In Taiwanese folk medicine, Antrodia Cinnamomea has the effects of dispelling wind and promoting qi, removing blood stasis and promoting blood circulation, warming the middle and eliminating accumulation, detoxifying, reducing swelling, and sedating and relieving pain, and is regarded as a good antidote. It has a detoxification effect, and has auxiliary therapeutic effects on improving liver and stomach dysfunction and blood circulation diseases. Antrodia camphorata, like common edible and medicinal mushrooms, has many complex components. The known physiologically active components include: triterpenoids, polysaccharides (such as β-D-glucan), Adenosine (adenosine), vitamins (such as vitamin B, niacin), protein (including immunoglobulin), superoxide dismutase (superoxide dismutase, SOD), trace elements (such as: calcium, phosphorus, germanium), nucleic acid, Sterols and blood pressure stabilizing substances (such as antodia acid), etc. These physiologically active ingredients are considered to have various effects such as anti-tumor, increasing immunity, anti-allergy, anti-bacteria, anti-hypertension, lowering blood sugar and lowering cholesterol, and Helps protect the liver and treat liver-related diseases. the

Most of the research on the composition of Antrodia camphorata focuses on macromolecular polysaccharides (polysaccharides) and small molecules of triterpenoids (triterpenoids) and sterols (steroids). The sugar composition exists in its fruiting bodies and mycelia, but after spectral analysis, they all contain physiologically active β-D-glucans (β-D-glucans); triterpenoids are composed of 30 carbon elements Combined into a general term for hexagonal or pentagonal natural compounds, the bitterness of Antrodia camphorata mainly comes from triterpenoids, which are also the most studied ingredients. The triterpenoids obtained from fruiting bodies are antrocin, 4,7-dimethoxy-5-methyl-1,3-benzodioxocycle (4,7-dimethoxy-5-methy-1,3- benzodioxole) and 2,2′,5,5′-tetramethoxy-3,4,3′,4′-bis-methylenedioxy-6,6′-dimethylbiphenyl (2,2 ', 5, 5'-teramethoxy-3, 4, 3', 4'-bi-methyl enedioxy-6, 6'-dimethylbiphenyl) (Chiang et al., 1995), with ergostane (ergostane) as the skeleton New triterpenoids antcin A, antcin B, antcin C antcin E, antcin F, methyl antcinate G and methyl antcinate H (Cherng et al., 1995, 1996). The fruiting body also contains compounds with ergosterane as the backbone, including Zhankuic acid A, B and C zhankuic acid D and zhankuic acid d E (Chen and Yang, 1995; Yang 1996), a new compound with lanostane as the backbone. Compound 15α-acetyl-dehydrosulphurenic acid (15α-acetyl-dehydrosulphurenic acid), dehydroeburicoic acid and dehydrasulphurenic acid. the

Although it can be known from many current experiments that the extract of Antrodia Cinnamomea has the aforementioned effects, and its components have been analyzed one after another, but no specific research has been published on which active ingredients in the extract can contribute to the anticancer effect of Antrodia Cinnamomea. The relevant active ingredients need to be clarified by further experimental research. Therefore, if the ingredients contained in the extract can be found to effectively inhibit tumor growth, it will be beneficial to the research on the mechanism of Antrodia Cinnamomum anticancer, and the application of Antrodia Cinnamomea to cancer such as colorectal cancer Treatment and prevention are of great help. the

Contents of the invention

In order to understand which components in the Antrodia camphorata extract have the effect of suppressing cancer, the present invention separates and purifies the compound with the following structural formula (1) from the Antrodia camphorata extract;

Among them, X is oxygen (O) or sulfur (S), Y is oxygen or sulfur; R ₁ is hydrogen (H), methyl (CH ₃ ) or (CH ₂ )m-CH ₃ , R ₂ is hydrogen , methyl or (CH ₂ )m-CH ₃ , R ₃ is hydrogen, methyl or (CH ₂ )m-CH ₃ , m=1-12; n=1-12.

As in the compound of formula (1) structural formula, preferred is the compound of formula (2) as shown below:

The compound of formula (2), its chemical name is 4-hydroxyl-2,3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2,6,10-dodeca Triene)-2-cyclohexenone (4-hydroxy-2,3-dimethoxy-6-methy-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2- enone), the molecular formula is C ₂₄ H ₃₈ O ₄ , the appearance is light yellow powder, and the molecular weight is 390.

In the present invention, the compounds of formula (1) and formula (2) are isolated and purified from Antrodia camphorata water extract or organic solvent extract, and the organic solvent can include alcohols (such as methanol, ethanol or propanol), esters (such as ethyl acetate ), alkanes (such as hexane) or haloalkanes (such as methyl chloride, ethyl chloride), but not limited thereto, among which alcohols are preferred, and ethanol is more preferred. the

With the aforesaid compound, the present invention applies it to inhibiting the growth of tumor cells, so that it can be further applied in the pharmaceutical composition for treating cancer, so as to enhance the therapeutic effect of cancer. The scope of application of the compound of the present invention includes the growth inhibition of colorectal cancer tumor cells, by inhibiting the rapid growth of these tumor cells, thereby inhibiting the proliferation of tumors, and delaying the deterioration of tumors. Among them, the preferred compound is 4-hydroxyl-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodeca Carbotriene)-2-cyclohexenone. the

On the other hand, in the present invention, the compound of formula (1) or/and formula (2) can also be used as a component of a pharmaceutical composition for inhibiting the growth of colorectal cancer tumor cells. In addition to the effective dose of the compound of formula (1) or/and formula (2), the aforementioned pharmaceutical composition may also include a pharmaceutically acceptable carrier. Carriers can be excipients (such as water), fillers (such as sucrose or starch), binders (such as cellulose derivatives), diluents, disintegrants, absorption enhancers or sweeteners, but are not limited thereto . The pharmaceutical composition of the present invention can be produced according to the general known pharmaceutical preparation methods, and the formula (1) or/and formula (2) active ingredient dosage is mixed with more than one carrier to prepare the required dosage form. Tablets, powders, granules, capsules or other liquid preparations may be included, but not limited thereto. the

Embodiments of the present invention will be further described below. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone who is familiar with this art will not depart from the spirit and scope of the present invention Some changes and modifications can be made, so the protection scope of the present invention shall be determined by the claims. the

Detailed ways

The extracted water extract or organic solvent extract of Antrodia camphorata can be further separated and purified by high performance liquid chromatography, and then each fraction is tested for its anticancer effect. Finally, component analysis was carried out on the fractions with anti-cancer effects, and the components that may have anti-cancer effects were further tested for their inhibitory effects on colorectal cancer cells. Finally, it was found that the compound of formula (1)/formula (2) in the present invention has the effect of inhibiting the growth of colorectal cancer tumor cells. the

For the convenience of describing the present invention, the 4-hydroxyl-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10- Dodecatrienyl)-2-cyclohexenone compound will be described. In addition, to confirm that 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclo The inhibitory effect of hexenone compounds on tumor cell growth, in the present invention, the cell survival rate of colorectal cancer tumor cells is carried out according to the anti-tumor drug screening mode of the National Cancer Institute (National Cancer Institute, NCI) with the MTT analysis method in the present invention. test. As confirmed by these tests, 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodecatriene)-2 - Cyclohexenone for colorectal cancer tumor cells: the WiDr cell line can reduce its survival rate, and in contrast, it can also reduce the concentration required for the half-inhibition rate of growth (ie, IC50 value), so 4-hydroxyl-2 , 3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone, used in colorectal cancer Cell growth inhibition, and further can be used in the treatment of colorectal cancer. The foregoing implementation manner is described in detail as follows:

Example 1:

4-Hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone separate

About 100 grams of Antrodia mycelium, fruiting bodies or the mixture of the two are placed in a conical flask, and an appropriate proportion of water and alcohols (70% to 100% alcohol aqueous solution) is added, wherein the alcohols are relatively Preferably it is ethanol, stir and extract at 20-25°C for at least 1 hour, then filter with filter paper and 0.45μm filter membrane, collect the filtrate to obtain the extract of Antrodia camphorata. the

The Antrodia camphorata extract collected above was analyzed by using a high performance liquid chromatography (High Performance Liquid chromatography) with an RP 18 chromatographic tube (column), and methanol (A) and 0.1% to 0.5% acetic acid aqueous solution ( B) as mobile phase (mobile phase) (the solution ratio is: 0-10 minutes, B ratio is 95%-20%; 10-20 minutes, B ratio is 20%-10%; 20-35 minutes, The proportion of B is 10%-90%; 35-40 minutes, the proportion of B is 10%-95%), eluting at a speed of 1ml per minute, and simultaneously analyzing with an ultraviolet-visible light full-wavelength detector. the

Collect and concentrate the eluate from 25 minutes to 30 minutes to obtain a light yellow powdery solid product, which is 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11 - Trimethyl-2,6,10-dodecatrienyl)-2-cyclohexenone. After analysis, its molecular formula is C ₂₄ H ₃₈ O ₄ , its molecular weight is 390, and its melting point (mp) is 48°C to 52°C. The nuclear magnetic resonance (NMR) analysis values are as follows: ¹ H-NMR (CDCl ₃ ) δ (ppm): 1.51, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.68, 4.05, 5.07 and 5.14 . ¹³ C-NMR (CDCl ₃ ) δ (ppm): 12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 39.71, 39.81, 4.027, 43.34, 59.22, 60.59, 120.97, 123.84, 124.332, 131 , 135.92, 138.05, 160.45 and 197.12.

Example 2:

In vitro activity test against colorectal cancer tumor cells

In order to further test the inhibitory effect of the compound found in Example 1 on tumor cells, this example will first take the isolated compound in Example 1 according to the anti-tumor drug screening mode of the National Cancer Institute (NCI) in the United States. 4-Hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone compound , added to the culture medium containing human colorectal cancer tumor cells WiDr, to test the viability of tumor cells. Wherein, the test of cell viability can be analyzed by the known MTT analysis method, and the colorectal cancer tumor cell line WiDr is a human colorectal cancer cell line (human colon adenocarcinoma cell line). the

MTT assay is a common analytical method used to analyze cell proliferation, percent of viable cells, and cytotoxicity. Among them, MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) is a yellow dye, which can be absorbed by living cells and absorbed by succinate tetrazolium reductase (succinate) in mitochondria Tetrazolium reductase) is reduced to formazan, which is water-insoluble and blue-purple. Therefore, the survival rate of cells can be judged and calculated by whether formazan is formed or not. the

First, human colorectal cancer cells WiDr were cultured in RPMI 1640 medium containing 10% fetal bovine serum, which also contained 100 IU/ml of penicillin and 100 mg/ml of streptavidin Streptomycin, and cultured in 5% CO ₂ , 37°C for 24 hours. The proliferated cells were washed once with PBS, and the cells were treated with 1 times trypsin-EDTA, then centrifuged at 1,200 rpm for 5 minutes, the cells were pelleted and the supernatant was discarded. Then add 10ml of new culture medium, shake slightly to resuspend the cells, and then divide the cells into 96-well microplates. During the test, 30, 10, 3, 1, 0.3, 0.1 and 0.03 μg/ml of Antrodia camphorata extract were added to each well as a control group (total extract without purification and separation); and in each well Add 30, 10, 3, 1, 0.3, 0.1 and 0.03 μg/ml of 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2, 6,10-dodecatrienyl)-2-cyclohexenone was used as the test group and cultured at 37° C. under 5% CO ₂ for 48 hours. Thereafter, 2.5 mg/ml of MTT was added to each well in a dark environment, and after 4 hours of reaction, 100 μl of lysis buffer was added to each well to terminate the reaction. Finally, the absorbance value was measured with an enzyme immunoassay analyzer at a wavelength of 570nm to calculate the survival rate of the cells, and calculate the concentration required for the half-inhibition rate of growth (ie, the IC ₅₀ value). The results are shown in Table 1.

Table 1: In vitro test results on the survival rate of colorectal cancer tumor cells

It can be seen from Table 1 that by 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodecatriene) - The effect of 2-cyclohexenone, its IC ₅₀ value for WiDr human colorectal cancer tumor cells is 0.92 μg/ml, which is lower than the IC ₅₀ value (not shown in the table) measured by the Antrodia camphorata extract mixture of the control group Therefore, it can be confirmed that 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodeca Triene)-2-cyclohexenone can indeed be used to inhibit the growth of colorectal cancer tumor cells.

In summary, the present invention is isolated from 4-hydroxyl-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodeca Triene)-2-cyclohexenone compound can effectively inhibit the growth of colorectal cancer tumor cells. On the other hand, since the cyclohexenone compound of Antrodia antrodia is a naturally extracted substance, when it is used to inhibit colorectal cancer, it will not cause discomfort to patients or produce other side effects such as toxicity and complications, and it can also be used in combination with chemotherapeutic agents , to reduce the dosage of chemotherapy drugs and reduce the side effects caused by these chemotherapy drugs; in addition, it can also be prepared into a pharmaceutical composition for the treatment of colorectal cancer, wherein, the pharmaceutical composition contains effective doses of Antrodia antrodia cyclohexenone compound In addition, a pharmaceutically acceptable carrier may also be included. Carriers can be, but are not limited to, excipients (such as water), fillers (such as sucrose or starch), binders (such as cellulose derivatives), diluents, disintegrants, absorption enhancers or sweeteners . The pharmaceutical composition of the present invention can be manufactured according to the generally known pharmaceutical preparation method, and the active ingredient dose of Antrodia cyclohexenone compound is mixed with more than one carrier to prepare the required dosage form, which can include lozenges , powder, granule, capsule or other liquid preparations, but not limited thereto. So as to achieve the purpose of treating colorectal cancer tumor disease. the

Claims (16)

1. A compound with the following structural formula is used for preparing the application of the medicine that suppresses colorectal cancer tumor cell growth:

Among them, X is oxygen (O) or sulfur (S), Y is oxygen or sulfur; R ₁ is hydrogen (H), methyl (CH ₃ ) or (CH ₂ ) _m -CH ₃ , R ₂ is hydrogen , methyl or (CH ₂ ) _m -CH ₃ , R ₃ is hydrogen, methyl or (CH ₂ ) _m -CH ₃ , m=1-12; n=1-12. 2. The application as claimed in claim 1, wherein the compound is 4-hydroxyl-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10 -Dodecatriene)-2-cyclohexenone (4-hydroxy-2,3-dimethoxy-6-methy-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl)- cyclohex-2-enone). 3. The application as claimed in claim 2, wherein the compound is isolated from the extract of Antrodia camphorata. 4. The application according to claim 3, wherein the compound is isolated from the water extract of Antrodia camphorata. 5. The application as claimed in claim 3, wherein the compound is isolated from the organic solvent extract of Antrodia camphorata. 6. The application according to claim 5, wherein the organic solvent is selected from the group consisting of esters, alcohols, alkanes and haloalkanes. 7. The application according to claim 6, wherein the alcohols are ethanol. 8. The application according to claim 1, wherein the colorectal cancer tumor cells are WiDr cell lines. 9. A pharmaceutical composition for inhibiting the growth of colorectal cancer tumor cells, comprising an effective dosage of the compound according to claim 1 and a pharmaceutically acceptable carrier. 10. The pharmaceutical composition of claim 9, wherein the compound is 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6 , 10-dodecatriene)-2-cyclohexenone (4-hydroxy-2,3-dimethoxy-6-methy-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl )-cyclohex-2-enone). 11. The pharmaceutical composition as claimed in claim 10, wherein the compound is isolated from the extract of Antrodia camphorata. 12. The pharmaceutical composition as claimed in claim 11, wherein the compound is isolated from the water extract of Antrodia camphorata. 13. The pharmaceutical composition as claimed in claim 11, wherein the compound is isolated from the organic solvent extract of Antrodia camphorata. 14. The pharmaceutical composition as claimed in claim 13, wherein the organic solvent is selected from the group consisting of esters, alcohols, alkanes and haloalkanes. 15. The pharmaceutical composition as claimed in claim 14, wherein the alcohol is ethanol. 16. The pharmaceutical composition as claimed in claim 9, wherein the colorectal cancer tumor cells are WiDr cell lines.