WO2008089674A1

WO2008089674A1 - Cyclohexenone extractant of antrodia camphorata

Info

Publication number: WO2008089674A1
Application number: PCT/CN2008/070071
Authority: WO
Inventors: Sheng-Yun Liu; Wu-Che Wen; Wan-Ling Tsou; Mao-Tien Kuo; Chun-Chou Chang; Chun-Hung Huang; Ya-Ying Li; Ka-Hang Fok
Original assignee: Golden Biotechnology Corporation
Priority date: 2007-01-18
Filing date: 2008-01-10
Publication date: 2008-07-31
Also published as: RU2422431C2; IL199882A0; RU2009131324A; UA97135C2; IL199882A; CN101225066A; CN101225066B

Abstract

The present invention relates to a novel compound and its use, especially to 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone), which is separated from an extractant of Antrodia camphorate, and its use is in inhibiting the growth of tumour cells. The so-called compound of the present invention is found in Antrodia camphorate for the first time, and it may be used to inhibit the growth of tumour cells of breast cancer, liver cancer and prostate cancer. Meanwhile, the so-called compound may be used as a component of a pharmaceutical composition for inhibition of the growth of tumour cells above, or as that compound has the activity of anti-oxidation, it can further be used for prevention of cardiovascular disease or be added into health protection potables and foodstuff.

Description

Cyclohexenone extract of Antrodia camphorata

Technical field

This invention relates to a novel compound, and more particularly to a compound isolated and purified from an extract of Antrodia camphorata and its use in inhibiting tumor cell growth. Background technique

Antrodia camphorata, also known as Antrodia camphorata, burdock mushroom, red peony root, red peony root, scorpion or scorpion scorpion, is a unique species of fungi in Taiwan. It grows only in the mountainous area of Taiwan at an altitude of 450-2000 meters. The inner wall of the hollow decayed heartwood of Cinnamoum kanehirai Hay is thus grown from the inner surface of the trunk. Burdock trees are mainly distributed in mountainous areas such as Taoyuan and Nantou. Because Burdock is a very rare species of conservation trees in Taiwan, combined with artificial logging, the number of wild Antrodia camphorata that can grow in it is even rarer. The growth is quite slow, and the growth period is only between June and October, so the price is very expensive.

The fruiting body of Antrodia camphorata is perennial, sessile, woody to woody, and its shape is varied, with plates, bells, horseshoes or towers. It is flat at the beginning and is attached to the surface of the wood. The leading edge is then slightly curled and raised in a plate-like shape (slab-like) or as a stalactite. The top surface of the burdock is brown to dark brown, with inconspicuous wrinkles, shiny, flat and blunt edges, and its ventral surface is orange-red or partially yellow with many fine pores.

In addition, Antrodia camphorata has a strong astringent aroma, which fades to yellow and white after drying, and tastes extremely bitter. It is used by the folks as an anti-drug, liver-protecting and anti-cancer herb. Antrodia camphorata is a common medicinal oyster mushroom with many complex components. Among the known physiologically active ingredients are: polysaccharides (such as β-glucan), triterpenoids (triterpenoids), superoxide. Superoxide dismutase (SOD), adenosine (adenosine), protein (containing immunoglobulin), vitamins (such as vitamin B, niacin), trace elements (such as calcium, phosphorus and strontium), nucleic acids, lectins , amino acids, sterols, lignin, and blood pressure stabilizing substances (such as antodia acid), etc. These physiologically active ingredients are considered to have anti-tumor, immunity, anti-allergy, inhibition of platelet aggregation, anti-virus, anti-bacterial, anti- High blood pressure, lowering blood sugar, lowering cholesterol and protecting the liver. Among the many components of Antrodia camphorata, triterpenoids are the most studied. Triterpenoids are a general term for the combination of thirty carbon elements into hexagonal or pentagonal natural compounds. The bitterness of Antrodia camphorata is mainly derived from triterpenoids. In 1995, Cherng et al. found that three extracts of triterpenoids based on ergoline ( _er g _0S tane ) were found in the fruit extract of Antrodia camphorata: antcin A, antcin B and antcin C ( Cherng, IH, And Chiang, HC 1995. Three new triterpenoids from Antrodia cinnamomea. J. Nat. Prod. 58:365-371 ). Chen et al. found three kinds of triterpenoids such as zhankuic acid A, zhankuic acid B and zhankuic acid C after extracting the body of A. camphorata with ethanol (Chen, CH, and Yang, SW 1995. New steroid acids from Antrodia cinnamomea, -a Fungus parasitic on Cinnamomum micranthum. J. Nat. Prod. 58: 1655- 1661 ). In addition, in 1995, Chiang et al. also found three other new triterpenoids 4 conjugates, sesquiterpene lactone and two diacids, from the fruiting body extract. This is anrocin, 4,7-dimethoxy-5-mercapto-1,3-benzodioxole (4,7-dimethoxy- 5-methy-l, 3-benzodioxole) and 2,2', 5,5'-tetradecyloxy-3,4,3',4'-bis-indenylenedioxy-6,6'-dimercaptobiphenyl (2,2',5,5 ^, -teramethoxy -3,4,3 ^, ,4 ^, -bi-methylenedioxy-6,6'- dimethylbiphenyl ) (Chiang, H. C, Wu, DP, Cherng, IW, and Ueng, CH 1995. A sesquiterpene lactone, phenyl and biphenyl Compound from Antrodia cinnamomea. Phytochemistry. 39:613-616). In 1996, Cherng et al. found four new triterpenoids in the same way: antcin E, antcin F, methyl antcinate G, methyl antcinate H ( Cherng, IH, Wu, DP, and Chiang, HC 1996. Triteroenoids from Antrodia cinnamomea. Phytochemistry. 41 :263-267 ); and Yang et al. found two new compounds, zhankuic acid D, zhankuic acid E, and three lanostanes, which are based on ergostere. New compounds of the skeleton: 15 α-acetyl-dehydrosulphurenic acid, dehydroeburicoic acid and dehydrasulphurenic acid (dehydrasulphurenic acid) Yang, SW, Shen, Y. C, and Chen, CH 1996. Steroids and triterpenoids of Antrodia cinnamomea- a fungus parasitic on Cinnamomum micranthum. Phytochemistry. 41 : 1389-1392 ). Although it is known from many experiments, the extract of Antrodia camphorata has cancer inhibition. Efficacy (such as the aforementioned Chen (1995)), but the study of which active ingredients can achieve the effect of inhibiting tumor cells, is still in the experimental stage, there is no specific active ingredient published, so if the extract can be further purified Analysis, to find out its really effective anti-cancer ingredients, will greatly help the treatment of human cancer. Summary of the invention

In order to clarify what component of the extract of Antrodia camphorata has anti-cancer effect, the present invention separates and purifies a novel compound having the structural formula of formula (1) from the extract of Antrodia camphorata:

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

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

a compound of formula (2) having the chemical name 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-trimethyl-2,6,10-dodeca carbon) Trienyl-2-cyclohexenone (4-hydroxy-2,3-dimethoxy-6-methy-5-(3,7,11-trimethyl-dodeca-2,6, 10-trienyl)-cyclohex-2 -enone ) , the molecular formula is C ₂₄ H ₃₈ 0 ₄ , the appearance is light yellow powder, and the molecular weight is 390.

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

By the above-mentioned compound, the present invention is applied to inhibit the growth of tumor cells, and can be further applied to a pharmaceutical composition for treating cancer, thereby enhancing the therapeutic effect of cancer. The application range of the compound of the present invention includes inhibiting the growth of cells such as breast cancer tumor cells, liver cancer tumor cells and prostate cancer tumor cells, so that the tumor cells cannot grow rapidly, thereby inhibiting tumor proliferation and delaying tumor deterioration. Therefore, it can be further utilized for the treatment of cancers such as breast cancer, liver cancer and prostate cancer.

On the other hand, in the present invention, the compound of the formula (1) or/and the formula (2) can also be used for the treatment of components of pharmaceutical compositions such as breast cancer, liver cancer and prostate cancer, thereby suppressing the growth of the tumor cells. . The aforementioned pharmaceutical composition may further comprise a pharmaceutically acceptable carrier in addition to an effective amount of the compound of the formula (1) or / and the formula (2). The carrier may be an excipient (such as water), a filler (such as sucrose or starch), a binder (such as a cellulose derivative), a diluent, a disintegrant, an absorption enhancer or a sweetener, but is not limited thereto. . The pharmaceutical composition of the present invention can be produced according to a generally known pharmaceutical preparation method, and a dosage form of the active ingredient of the formula (1) or/(2) is mixed with one or more carriers to prepare a desired dosage form, and the dosage form is prepared. These may include lozenges, powders, granules, capsules or other liquid preparations, but are not limited thereto.

In addition, since the compound of the present invention has antioxidant activity at the same time, it can also be added to health foods, foods, medicines, cosmetics, and its antioxidant capacity can be used to prevent cardiovascular diseases or to avoid cell mutation equivalents. To help the health of the human body.

The embodiments of the present invention are further described in the following description, which is set forth to illustrate the invention and not to limit the scope of the present invention. In the scope of the invention, the scope of the invention should be construed as limited by the appended claims. detailed description

First, Antrodia camphorata mycelium, fruiting body or a mixture of the two is taken, and extracted by water or an organic solvent by a known extraction method to obtain an aqueous extract of Antrodia camphorata or an organic solvent extract. Wherein, the organic solvent may include an alcohol (such as methanol, ethanol or propanol), an ester (such as ethyl acetate), an alkane (such as hexane) or a halogen (such as methyl chloride, ethyl chloride), But it is not limited to this. Among them, preferred are alcohols, and more preferably ethanol.

The extracted aqueous extract of Antrodia camphorata or the organic solvent extract can be further separated and purified by high performance liquid chromatography, and then each of the fractions is tested for its anticancer effect. Finally, the components of the cancer-suppressing effect were analyzed for component analysis, and the components that may have a tumor suppressing effect were further tested for inhibition of different cancer tumor cells. Finally, it was found that the compound of the formula (1) / formula (2) in the present invention has an effect of inhibiting the growth of tumor cells of different cancers, and the compound has not been found in Antrodia camphorata compared with the known literature, and thus is novel. Compound.

For convenience of description of the present invention, 4-hydroxy-2,3-didecyloxy-6-mercapto--5 of the formula (2) will be exemplified below.

(3,7,11-tridecyl-2,6,10-dodecatriene)-2-cyclohexenone compound will be described. Further, in order to confirm 4-hydroxy-2,3-dimethoxy-6-mercapto-5(3,7,11-tridecyl-2,6,10-dodecatriene)-2-ring The inhibitory effect of the hexenone compound on tumor cell growth, the present invention is based on the MTT assay, according to the National Cancer Institute (NCI) anti-tumor drug screening mode, including breast cancer, liver cancer and prostate. Tumor cells such as cancer are tested for cell viability. As confirmed by these tests, 4-hydroxy-2,3-dimethoxy-6-mercapto-5(3,7,11-tridecyl-2,6,10-dodecatriene)-2- Cyclohexenone for breast cancer tumor cells (including MCF-7 and MDA-MB-231), liver cancer tumor cells

(including Hep 3B and Hep G2) and prostate cancer cells (including LNCaP and DU-145) can reduce their survival rate, and simultaneously reduce the concentration required for growth half inhibition (ie IC _{5 )} . Value), therefore by 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-dodecatriene) - 2-cyclohexenone is used for the growth inhibition of tumor cells including breast cancer, liver cancer, and prostate cancer, and is further applicable to the treatment of cancers such as breast cancer, liver cancer, and prostate cancer. The foregoing embodiment is described in detail: 3⁄4 under:

Example 1: 4-Hydroxy-2,3-dimethoxyoxy-6-mercapto-5 (3,7,11-trimethyl-2,6,10-dodecatriene)-2-ring Separation of hexenone

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

The collected Antrodia camphorata extract was analyzed by high performance liquid chromatography using a column of RP18, and decyl alcohol (A) and 0·1%~0· 5% aqueous acetic acid solution (B) as mobile phase (the ratio of the solution is: 0-10 Minute, B ratio is 95% ~ 20%; 10~20 minutes, B ratio is 20%~10%; 20-35 minutes, B ratio is 10%~10%; 35-40 minutes, B ratio is 10%~ 95%), eluted at a rate of 1 ml per minute, and analyzed by an ultraviolet-visible full-wavelength detector.

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

1.51, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 111, 2.25, 3.68, 4.05, 5.07 and 5.14. ¹³ C-NMR (CDC13) 5 (ppm): 12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 39.71, 39.81, 4.027, 43.34, 59.22, 60.59, 120.97, 123.84, 124.30, 131.32, 135.35, 135.92, 138.05, 160.45 and 197.12.

4-Hydroxy-2,3-dimethoxy-6-mercapto-5(3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexene After comparison of the ketone with the chemical formula database, the same compound structure as described above was not found, and therefore, the compound is a novel and unprecedented compound.

Example 2: Activity test of anti-breast cancer tumor cells in vitro

To further test the inhibitory effect of the compounds found in Example 1 on tumor cells, this example will be based on the National Cancer Institute (NCI) anti-tumor drug screening mode, first isolated in Example 1. 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone compound Tumor cell viability was tested by adding MCF-7 and MDA-MB-231 human tumor cell culture medium. Cell viability assays can be performed using known MTT assays, while MCF-7 and MDA-MB-231 are both human breast cancer tumor lines.

MTT assay is a commonly used analytical method for analyzing cell proliferation, percent of viable cells, and cytotoxicity. Wherein, MTT (3- [4,5- dimethylthiazol- 2-yl] 2,5-diphenyltetrazolium bromide) is a yellow dye, which can be absorbed by the living cells and granulocytes number of glands Perot ^p sat reduction tetra The enzyme (succinate tetrazolium reductase) is reduced to insoluble and blue-violet formazan, so the survival rate of the cells can be judged and calculated by the formation of formazan.

First, human breast cancer cells MCF-7 and MDA-MB-231 were separately cultured in fetal bovine serum. Incubate for 24 hours in the culture solution. The proliferated cells were washed once with PBS, and the cells were treated with 1 time trypsin-EDTA, followed by centrifugation at 1,200 rpm for 5 minutes, the cells were pelleted and the supernatant was discarded. Then, 10 ml of the new culture solution was added, and the cells were resuspended by shaking slightly, and the cells were placed in a 96-well microplate. During the test, 30, 10, 3, 1, 0.3, 0.1 and 0.03 g/ml ethanol extract of Antrodia camphorata (control group, total extract without purification) and 4-hydroxy-2 were added to each well. 3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-dodecatriene)-2-cyclohexanone (test group), at 37 Incubate for 48 hours at °C, 5 % C0 ₂ . Thereafter, 2.5 mg/ml of MTT was added to each well in the dark, and after 4 hours of reaction, the reaction was stopped by adding lysisμΙ lysis buffer to each well. Finally, the absorbance was measured by an enzyme immunoassay at an absorption wavelength of 570 nm to calculate the cell survival rate, and the concentration required for the growth half inhibition rate (i.e., IC ₅ value) was calculated. The results are shown in Table 1.

Table 1: Test results of breast cancer tumor cell survival in vitro Test sample IC ₅₀ ( g / ml )

Control group (added to Antrodia camphorata extract)

MCF-7 1 1.132

MDA-MB-231 25.812

Test horse full group (join 2)

MCF-7 0.852

MDA-MB-231 1.03 1

As can be seen from Table 1, by 4-hydroxy-2,3-dimethoxy-6-mercapto-5(3,7,11-tridecyl-2,6,10-dodecatriene) -2-cyclohexenone of action, which for the MCF-7 human breast cancer tumor cells IC ₅₀ value of 0.852 g / ml, for MDA-MB-231 human breast tumor cells IC _5. The value is 1.031 μ^Ώύ compared to the IC ₅ measured by the extract of the Antrodia camphorata. The value is much lower, so it can be confirmed that 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10- in the extract of Antrodia camphorata) Decadotriene)-2-cyclohexenone can indeed be utilized for the inhibition of breast cancer tumor cell growth.

Example 3: Activity test for adjuvant treatment of breast cancer tumor cells in vitro

This test was also tested according to the National Cancer Institute's in vitro screening model. First, human breast cancer cells MCF-7 and MDA-MB-231 were cultured for 24 hours in culture medium containing fetal bovine serum, and the proliferated cells were washed once with PBS and doubled with trypsin-EDTA. The cells were treated, followed by centrifugation at 1,200 rpm for 5 minutes, the cells were pelleted and the supernatant was discarded. Then, 10 ml of the new medium was added, and the cells were suspended by gentle shaking. Before the test, the cells were treated with 0.0017 g/ml paclitaxel (Taxol) for 72 hours, and then the cells were placed in a 96-well microplate, and then O g/ml was added to each well (control group), 30, 10, 3, 1, 0.3, 0.1 and 0.03 g/ml 4-hydroxy-2,3-didecyloxy-6-mercapto-5 (3,7,11-tridecyl-2) isolated in Example 1. , 6,10-dodecatriene)-2-cyclohexenone (test group), cultured at 37 ° C, 5 % C0 ₂ for 48 hours. Thereafter, 2.5 mg/ml of MTT was added to each well in the dark, and after 4 hours of reaction, the reaction was stopped by adding lysisμΙ lysis buffer to each well. Finally, the absorbance was measured by an enzyme immunoassay at an absorption wavelength of 570 nm to calculate the cell survival rate, and the concentration required for growth half inhibition (ie, IC ₅ value) was calculated. The results are shown in Table 2.

Table 2: Test results of inhibition of breast cancer tumor cells after adjuvant treatment with paclitaxel in vitro Test sample Results Control group Cell viability (%)

MCF-7 (0.0017 g/ml Taxol) 65 ±1

MDA-MB-23 1 0.0017 g/ml Taxol) 76±3

Test group IC ₅₀ ( g/ml)

MCF-7 (0.0017 g/ml Taxol+ type 2) 0.009

MDA-MB-23 1 0.0017 g/ml Taxol+ type 2) 0.01 1

As can be seen from Table 2, 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-10) through the synergistic effect of paclitaxel two cyclododecatriene) -2-cyclohexenone IC ₅ to the MCF-7 human breast tumor cells. Value reduced to 0.009 g / ml, for MDA-MB-231 human breast tumor cells IC _5. The value was also lowered to about 0.011 g/ml, so that 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2) in the extract of Antrodia camphorata was confirmed. 6,10-dodecatriene)-2-cyclohexenone can indeed be used for the inhibition of growth of breast cancer tumor cells, and has a better inhibitory effect under the synergistic effect of paclitaxel.

Example 4: Activity test of anti-hepatocarcinoma tumor cells in vitro

This test was also carried out according to the National Cancer Institute anti-tumor drug screening mode, and 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11) isolated in Example 1. - triterpene-2,6,10-dode carbon The triene)-2-cyclohexenone compound was cultured by adding Hep 3B and Hep G2 human liver cancer tumor cell culture medium, thereby performing tumor cell viability test.

First, human hepatoma cells Hep 3B and Hep G2 were cultured for 24 hours in a culture medium containing fetal bovine serum, respectively. The proliferated cells were washed once with PBS, and the cells were treated with 1-fold trypsin-EDTA, followed by centrifugation at 1,200 rpm for 5 minutes, the cells were pelleted and the supernatant was discarded. Thereafter, 10 ml of the new culture solution was added, the cells were resuspended by gentle shaking, and the cells were placed in a 96-well microplate. During the test, 30, 10, 3, 1, 0.3, 0.1 and 0.03 g/ml of Antrodia camphorata ethanol extract (control group, total extract without purification) and 30, 10, 3 were added to each well. 1, 1, 0.3, 0.1 and 0.03 g/ml of 4-hydroxy-2,3-dimethoxy-6-mercapto-5(3,7,11-trimethyl-2,6,10-12 Carbotriene)-2-cyclohexenone (test group), cultured at 37 ° C, 5 % C0 ₂ for 48 hours. Thereafter, 2.5 mg/ml of MTT was added to each well in the dark, and after 4 hours of reaction, the reaction was stopped by adding lysisμΙ lysis buffer to each well. Finally, the absorbance was measured by an enzyme immunoassay at an absorption wavelength of 570 nm to calculate the survival rate of the cells, and the IC ₅ was calculated. Value, the results are shown in Table 3.

Table 3: Test results of inhibition of liver cancer tumor cells in vitro

Test sample IC ₅ . ( g/ml ) Control group (added to Antrodia camphorata extract}_

Hep 3B 5.121

Hep G2 18.631

Test horse full group (join 2)

Hep 3B 0.005

Hep G2 1.679

As can be seen from Table 3, by 4-hydroxy-2,3-dimethoxy-6-mercapto-5(3,7,11-tridecyl-2,6,10-dodecatriene) -2- cyclohexenone action, for which the value of IC Hep 3B ₅₀ human hepatoma tumor cells is reduced to 0.005 g / ml, for human hepatocarcinoma Hep G2 cells IC _5. The value was reduced to 1.679 g/ml compared to the IC ₅ measured for the extract of the Antrodia camphorata. The value is much lower, so 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10- in the extract of Antrodia camphorata can be confirmed. Decadotriene)-2-cyclohexenone can indeed be utilized for the inhibition of growth of liver cancer tumor cells. Example 5: Activity test for adjuvant treatment of liver cancer tumor cells in vitro

This test was also tested according to the National Cancer Institute's in vitro screening model. First, human hepatoma cells Hep 3B and Hep G2 were cultured in culture medium containing fetal bovine serum for 24 hours, and then the proliferated cells were washed once with PBS, and the cells were treated with 1 time trypsin-EDTA, followed by treatment. After centrifugation at 1,200 rpm for 5 minutes, the cells were pelleted and the supernatant was discarded. Then add 10 ml of new medium and shake gently to resuspend the cells. Before the test, 0.0043 g/ml of lovastatin was added to the Hep 3B cell line, and 0.0017 g/ml of taxol was added to the Hep G2 cell line. The cells were treated for 72 hours, and the cells were placed in a 96-well microplate. Then, O g/ml (control group), 30, 10, 3, 1, 0.3, 0.1 and 0.03 g/ml of 4-hydroxy-2,3-di isolated in Example 1 were added to each well.曱oxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-dodecatriene)-2-cyclohexenone (test group), at 37 ° C, Incubate for 48 hours at 5 % C0 ₂ . Thereafter, 2.5 mg/ml of MTT was added to each well in the dark, and after 4 hours of reaction, the reaction was stopped by adding lysisμΙ lysis buffer to each well. Finally, the absorbance was measured by an enzyme immunoassay at an absorption wavelength of 570 nm to calculate the survival rate of the cells, and the IC ₅ was calculated. Value, the results are shown in Table 4.

Table 4: Test results of inhibition of hepatocarcinoma cells after adjuvant treatment with paclitaxel in vitro Test sample Results Control group Cell viability (%)

Hep 3B (0.0043 g/ml Lovastatin) 61 +3

Hep G2 (0.0017 g/ml Taxol) 81 +2 test group IC ₅ o ( g/ml )

Hep 3B (0.0043 g/ml Lovastatin + Formula 2) 0.002

Hep G2 (0.0017 g/ml Taxol + Formula 2) 0.008 As can be seen from Table 4, through the synergistic action of lovastatin and paclitaxel, 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3, 7,11-trimercapto-2,6,10-dodecatriene)-2-cyclohexenone for Hep 3B human hepatoma tumor cells IC ₅ . The value was reduced to 0.002 g/ml, and the IC ₅₀ value of Hep G2 human liver cancer tumor cells was also reduced to about 0.008 g/ml, so that 4-hydroxy-2,3-dimethoxy-6 in the extract of Antrodia camphorata was confirmed. - mercapto-5 (3,7,11-tridecyl-2,6,10-dodecatriene)-2-cyclohexenone can indeed be used in the liver The inhibition of the growth of cancer tumor cells, and the synergistic effect of paclitaxel, has a better inhibitory effect. Example 6: Activity test of in vitro anti-prostate cancer tumor cells

This test was also carried out according to the National Cancer Institute anti-tumor drug screening mode, and 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11) isolated in Example 1. -Tridecyl-2,6,10-dodecatriene)-2-cyclohexenone compound, which is cultured by adding LNCaP and DU-145 human prostate cancer tumor cell culture medium, thereby performing tumor cell storage Activity test.

Human prostate cancer cells LNCaP and DU-145 were first cultured in culture medium containing fetal bovine serum for 24 hours. The proliferated cells were washed once with PBS, and the cells were treated with 1 time trypsin-EDTA, followed by centrifugation at 1,200 rpm for 5 minutes, the cells were pelleted and the supernatant was discarded. Then, 10 ml of the new culture solution was added, and the cells were resuspended by gentle shaking, and the cells were placed in a 96-well plate. During the test, 30, 10, 3, 1 and 0.3 g/ml ethanol extract of Antrodia camphorata (control group, total extract without purification) and 30, 10, 3, 1 and 0.3 g were added to each well. /ml 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-dodecatriene) isolated from Example 1. -2-Cyclohexenone (test group), cultured at 37 ° C, 5 % C0 ₂ for 48 hours. Thereafter, 2.5 mg/ml of MTT was added to each well in the dark, and after 4 hours of reaction, the reaction was stopped by adding lysisμΙ lysis buffer to each well. Finally, the absorbance was measured by an enzyme immunoassay at an absorption wavelength of 570 nm to calculate the survival rate of the cells, and the IC ₅ was calculated. Value, the results are shown in Table 5.

Table 5: Test results of in vitro inhibition of prostate cancer tumor cells

Test sample IC ₅ . ( g/ml ) control group (added to Antrodia camphorata extract)

LNCaP 1 1.491

DU- 145 41.392

Test horse full group (join 2)

LNCaP 2.378

DU- 145 1.812

As can be seen from Table 5, by 4-hydroxy-2,3-dimethoxy-6-mercapto-5(3,7,11-tridecyl-2,6,10-dodecatriene) -2- cyclohexenone action, for which the value of IC LNCaP human prostate cancer ₅₀ tumor cells is reduced to 2.378 g / ml, for the DU-145 human prostate tumor cells IC _5. The value is reduced to 1.812 g/ml, compared to the IC ₅ measured by the extract of Antrodia camphorata. The value is much lower, so it can be confirmed that 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10- in the extract of Antrodia camphorata) Decadotriene)-2-cyclohexenone is indeed able to utilize the inhibition of growth of prostate cancer tumor cells.

Example 7: Activity test for adjuvant therapy of prostate cancer cells in vitro

This test was also tested according to the National Cancer Institute's in vitro screening model. First, human prostate cancer cells LNCaP and DU-145 were cultured in culture medium containing fetal bovine serum for 24 hours, and then the proliferated cells were washed once with PBS and treated with 1 time trypsin-EDTA. The cells were then centrifuged at 1,200 rpm for 5 minutes, the cells were pelleted and the supernatant discarded. Then, 10 ml of the new medium was added, and the cells were suspended by gentle shaking. Before the test, 0.0017 g/ml paclitaxel was added to the LNCaP cell line test, and 0.0043 g/ml paclitaxel was added to the DU-145 cell line for 72 hours, and then the cells were placed in a 96-well microplate, respectively. O g/ml (control), 30, 10, 3, 1, 0.3, 0.1 and 0.03 g/ml of 4-hydroxy-2,3-dimethoxy group isolated in Example 1 were added to each well. -6-mercapto-5 (3,7,11-tridecyl-2,6,10-dodecatriene)-2-cyclohexenone (test group), at 37 ° C, 5 % C0 Incubate for 48 hours under ₂ hours. Thereafter, 2.5 mg/ml of MTT was added to each well in the dark, and after 4 hours of reaction, the reaction was stopped by adding lysisμΙ lysis buffer to each well. Finally, the absorbance was measured by an enzyme immunoassay at an absorption wavelength of 570 nm to calculate the survival rate of the cells, and the IC ₅ was calculated. Value, the results are shown in Table 6.

Table 6: Test results of in vitro inhibition of prostate cancer tumor cells after paclitaxel adjuvant therapy

Test sample Results Control cell survival rate (%)

LNCaP (0.0017 g/ml Taxol) 56±3

DU- 145 (0.0043 g/ml Taxol) 70±2

Test group IC ₅ o ( g/ml )

LNCaP (0.0017 g/ml Taxol+ type 2) 0.961

DU- 145 (0.0043 g/ml Taxol+Form 2) 0.515 As can be seen from Table 6, through the synergistic effect of paclitaxel, 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7, 11- three Yue dodecene three women 2,6,10-yl) -2-cyclohexenone for the human LNCaP prostate tumor cells IC _5. Value reduced to 0.961 g / ml, for DU-145 ₅₀ IC values of human prostate cancer cells is also reduced to about 0.515 g / ml, compared to the mixture was extracted Antrodia measured IC _5. The value is much lower, because This confirms 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-dodecatriene) in Antrodia camphorata extract. -2-cyclohexenone can indeed be used for the inhibition of growth of prostate cancer tumor cells, and has a better inhibitory effect under the synergistic effect of paclitaxel.

Example 8: Test for antioxidant activity in vitro

The general antioxidant activity test is to use human low density lipoprotein (LDL), add copper ions (Cu ²⁺ ) to the sample to be tested for oxidation reaction, and detect the reaction result of diene on LDL. The water-soluble analog Trolox of vitamin E was used as a control (the potency value was set to a standard value of 1.0 when the concentration of Trolox was 2 μΜ), and the antioxidant activity of the sample to be tested was calculated.

First, prepare secondary pure water (control group), and prepare 5 mM sodium phosphate buffer (SPB), ΙμΜ with 2 μΜ of Trolox (control group), and 4 (^g/ml isolated from Example 1). 4-hydroxy-2,3-dimethoxy-6-mercapto-5 (3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone ( Test group). After measuring the low-density cholesterol concentration (LDL-C) by enzyme method, the LDL was diluted to 0.10-0.25 mg/ml with 5 mM SPB. 96-well quartz microplate was taken, and ΙΟΟμΙ was added first. LDL, respectively, adding the aforementioned predetermined concentration of Trolox and the compound isolated in Example 1, and then separately adding a CuS0 ₄ solution to initiate the oxidation reaction (the concentration of copper (II) per 250 μl hole is 5.0 μΜ), and finally The microplate is placed in an enzyme immunoassay analyzer (ELISA reader) for detection. The enzyme immunoassay microplate analyzer has a detection wavelength of 232 nm, a temperature of 37 ° C, a monitoring time of 12 hours, and a sampling interval of 15 minutes. The results are shown in Table 7.

Table 7: Test results of antioxidant activity in vitro

Test sample Tlag (minute) ATlag (minute) Effective value

3⁄40 (control group, Tlag ₀ ) 185

1 μΜ Trolox (control) 266 81 0.48

2 μΜ Trolox 344 159 1.00

40 g/ml Formula 2 439 208 1.30 Note 1: Tlag (point) refers to the intersection of the lag phase and the propagation phase at the absorption wavelength of 234 nm; ATlag (minute) means each The difference between the Tlag time and the TlagO time of the test sample.

Note 2: When the potency value is >0.5, it indicates that it has antioxidant effect.

As can be seen from Table 7, 4-hydroxy-2,3-didecyloxy-6-mercapto-5 (3,7,11-tridecyl-2,6,10-ten Dicarbotriene)-2-cyclohexenone has a potency value of 1.30, which is much higher than the standard value of 0.5 with antioxidant capacity, that is, it has considerable antioxidant capacity and can therefore be used as a health food. The ingredients in foods, medicines, cosmetics, and cosmetics can be used to prevent cardiovascular diseases or to avoid cell mutation equivalents, which will greatly contribute to the human health of the application.

Claims

Claim

1. A compound comprising the following structural formula:

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

2. The compound according to claim 1, wherein the compound is isolated from burdock

The compound according to claim 2, wherein the compound is isolated from an organic solvent extract of Antrodia camphorata.

The compound according to claim 3, wherein the organic solvent is a group selected from the group consisting of esters, alcohols, alkanes or alkanes.

5. The compound of claim 4, wherein the alcohol is ethanol.

The compound according to claim 2, wherein the compound is isolated from an aqueous extract of Antrodia camphorata.

The compound according to claim 1, wherein the compound is 4-hydroxy-2,3-dimethoxy-6-methyl-5 (3,7,11-trimethyl-2) ,6,10-dodecatriene)-2-cyclohexenone

(4-hydroxy-2,3-dimethoxy-6-methy-5(3,7,ll-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone).

8. Use of a compound according to claim 1 or 7 for inhibiting the growth of breast cancer tumor cells.

9. Use of the compound according to claim 8 for inhibiting growth of breast cancer tumor cells, characterized in that the compound is isolated from Antrodia camphorata.

10. The use of the compound for inhibiting the growth of breast cancer tumor cells according to claim 9. Use, characterized in that the compound is isolated from an organic solvent extract of Antrodia camphorata.

The use of the compound according to claim 10 for inhibiting the growth of breast cancer tumor cells, characterized in that the organic solvent is selected from the group consisting of esters, alcohols, alkanes or alkanes.

12. Use of the compound according to claim 11 for inhibiting growth of breast cancer tumor cells, characterized in that the organic solvent is ethanol.

13. Use of the compound according to claim 9 for inhibiting growth of breast cancer tumor cells, characterized in that the compound is isolated from an aqueous extract of Antrodia camphorata.

The use of the compound for inhibiting the growth of breast cancer tumor cells according to claim 8, wherein the breast cancer tumor cells are MCF-7 or MDA-MB-231 cell lines.

15. Use of a compound according to claim 1 or 7 for inhibiting the growth of liver cancer tumor cells.

16. Use of the compound according to claim 15 for inhibiting growth of liver cancer tumor cells, characterized in that the compound is isolated from Antrodia camphorata.

17. Use of the compound according to claim 16 for inhibiting the growth of liver cancer tumor cells, characterized in that the compound is isolated from an organic solvent extract of Antrodia camphorata.

The use of the compound for inhibiting the growth of liver cancer tumor cells according to claim 17, wherein the organic solvent is selected from the group consisting of esters, alcohols, alkanes or alkanes.

19. Use of the compound according to claim 18 for inhibiting growth of liver cancer tumor cells, characterized in that the organic solvent is ethanol.

20. Use of the compound according to claim 16 for inhibiting the growth of liver cancer tumor cells, characterized in that the compound is isolated from an aqueous extract of Antrodia camphorata.

The use of the compound for inhibiting the growth of liver cancer tumor cells according to claim 15, wherein the liver cancer tumor cells are Hep 3B or Hep G2 cell lines.

22. Use of a compound according to claim 1 or 7 for inhibiting the growth of a fine cell of a prostate cancer tumor.

23. Use of the compound according to claim 22 for inhibiting the growth of prostate cancer tumor cells, characterized in that the compound is isolated from Antrodia camphorata.

24. The use of the compound according to claim 23 for inhibiting the growth of prostate cancer tumor cells, characterized in that the compound is isolated from an organic solvent extract of Antrodia camphorata.

The use of the compound according to claim 24 for inhibiting the growth of prostate cancer tumor cells, characterized in that the organic solvent is selected from the group consisting of esters, alcohols, alkanes or alkanes.

26. Use of the compound according to claim 25 for inhibiting growth of prostate cancer tumor cells, characterized in that the organic solvent is ethanol.

27. Use of the compound according to claim 23 for inhibiting the growth of prostate cancer tumor cells, characterized in that the compound is isolated from an aqueous extract of Antrodia camphorata.

28. The use of the compound according to claim 22 for inhibiting the growth of prostate cancer tumor cells, characterized in that the prostate cancer tumor cells are LNCaP or DU145 cell lines.

The compound according to claim 1 or 7, wherein the compound has antioxidant activity.

30. A pharmaceutical composition for inhibiting growth of a tumor cell, comprising an effective amount of the compound of claim 1 and a pharmaceutically acceptable carrier, wherein the tumor cell is selected from the group consisting of breast cancer, liver cancer or prostate Cancerous tumor cells.

31. A pharmaceutical composition for inhibiting growth of a tumor cell, comprising: an effective amount of the compound of claim 7 and a pharmaceutically acceptable carrier, wherein the tumor cell is selected from the group consisting of breast cancer, liver cancer Or tumor cells that prostate cancer.