CN102232942A - Antrodia camphorata cyclohexenone compound for inhibiting growth of lymphoma tumor cells - Google Patents

Abstract

The invention relates to a new application of a compound, which 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-methyl-5 (3, 7, 11-trimethyl-dodeca-2, 6, 10-trienyl) -cyclohexenone) obtained by separating and purifying antrodia camphorata extract.

Description

Antrodia antrodia cyclohexenone compound for inhibiting the growth of lymphoma 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 Antrodia cinnamomea to inhibit the growth of lymphoma tumor cells. the

Background technique

Lymphoma is characterized by the destruction of normal lymphocytes and their replacement by immature lymphocytes. Lymphoma is a malignant tumor that is prone to occur in the lymphatic system in the body, including lymphocytes, lymphatic vessels, and lymph glands; on the other hand, lymphocyte regulatory organs responsible for the production, storage, and division of lymphocytes include bone marrow, spleen, and lymphocytes. Thymus lesions can also induce lymphoma. Clinically, lymphoma is often divided into Hodgkin's lymphoma (Hodgkin's disease) and non-Hodgkin's lymphoma (Non-Hodgkin's lymphoma) according to the different pathological types of tumors. Jerkin's lymphoma is the most common. the

Possible causes of lymphoma include a lack of immune system, viral infection, use of radiation or drugs, and genetics. Studies have pointed out that the incidence rate of lymphoma in all age groups is very average, and the proportion of men and women is about equal. Lymphoma is the most common early symptom of lymph gland enlargement, especially in the neck, armpit or groin Lymph nodes, and often painless clusters of lymph nodes, some patients will have systemic symptoms, such as fever, night sweats, weight loss, long-term fatigue or weakness, itchy skin rash and other symptoms; in addition, About one-third of the initial lesion of lymphoma is not in the lymph glands, but in internal organs, such as the stomach, large and small intestine, and the mediastinal cavity of the lungs. Symptoms include stomach pain, gastric bleeding, intestinal obstruction, or lung breathing Difficulty etc. the

The treatment of lymphoma varies according to the pathological type and clinical stage of the cancer. The main treatment methods include radiation therapy, chemotherapy, bone marrow transplantation or peripheral blood stem cell transplantation, etc. Generally speaking, the malignant degree is low and it belongs to the early local lymphoma Radiation therapy is suitable for patients with high malignancy, and patients with high malignancy and middle-advanced stage, because their condition deteriorates quickly and spreads easily throughout the body, chemotherapy is the main treatment. However, whether it is radiation therapy or chemotherapy, it often leads to many Side effects or uncomfortable symptoms. Therefore, if an effective natural treatment substance without side effects can be found, it will definitely improve the efficacy of lymphoma treatment and avoid other uncomfortable symptoms. 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, porous perennial fungi 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, 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-methylenedioxy-6, 6'-dimethyl biphenyl) (Chiang et al., 1995), with ergostane (ergostane) as the skeleton New triterpenoids antcinA, 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 E (Chen and Yang, 1995; Yang 1996), a new compound with lanostane as the backbone 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 Cinnamomea inhibiting cancer, and the application of Antrodia Cinnamomea to cancer such as lymphatic 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;

Wherein, X is oxygen (O) or sulfur (S), Y is oxygen or sulfur; R1 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 aforementioned compound, the present invention applies it to inhibiting the growth of tumor cells, so that it can be further used in the pharmaceutical composition for treating cancer to enhance the therapeutic effect of cancer. The scope of application of the compound in the present invention includes inhibiting the growth of lymphoma 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 lymphoma 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 manufactured according to the preparation method of general conventional pharmacy, and formula (1) or/and formula (2) active ingredient dosage is mixed with more than one kind of carrier, prepares the required dosage form, this 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 should be defined by the appended 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 fractionated liquid with tumor suppressing effect, and the components that may produce tumor suppressing effect were further tested for the inhibitory effect on lymphoma tumor cells. Finally, it is found that the compound of formula (1)/formula (2) in the present invention has the effect of inhibiting the growth of lymphoma 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 the growth of tumor cells, in the present invention, the cell survival rate of lymphoma tumor cells is carried out according to the anti-tumor drug screening mode of the National Cancer Institute (National Cancer Institute, NCI) with MTT analysis method in the present invention. test. It was confirmed by these tests that 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodecatriene)-2 - Cyclohexenone for lymphoma tumor cells: the U937 cell line can reduce its survival rate, and at the same time reduce the concentration required for half-inhibition rate of growth (i.e. IC ₅₀ value), so 4-hydroxyl-2 , 3-dimethoxy-6-methyl-5(3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone, applied to lymphoma tumor Cell growth inhibition, and further can be used in the treatment of lymphoma. Hereby, the aforementioned embodiment 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 alcohol It is preferably ethanol, stirred and extracted at 20-25°C for at least 1 hour, then filtered with filter paper and a 0.45 μm filter membrane, and the filtrate was collected to obtain the Antrodia camphorata extract. the

The Antrodia camphorata extract collected above was analyzed by using a high performance liquid chromatography (High Performance Liquid chromatography) with an RP18 chromatography tube (column), and methanol (A) and 0.1% to 0.5% acetic acid aqueous solution (B) ) as the 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, B ratio 10% to 90%; 35 to 40 minutes, the B ratio is 10% to 95%), eluting at a speed of 1ml per minute, and simultaneously analyzing with a UV-visible 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:

Anti-lymphoma tumor cell activity test in vitro

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 lymphoma tumor cell U937 to test the viability of tumor cells. Among them, the test of cell viability can be analyzed by conventional MTT assay, and the lymphoma tumor cell U937 is a human histiocytic lymphoma 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 tetrazolium reductase) is reduced to formazan which is water-insoluble and blue-purple in color, so the survival rate of cells can be judged and calculated by whether formazan is formed or not. the

First, human lymphoma cell U937 was cultured in RPMI1640 medium containing 10% fetal bovine serum, which also contained 10 U/ml of penicillin and 100 μg/ml of streptomycin (Streptomycin), and cultured in 5% CO ₂ , 37°C environment 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-dodecatriene)-2-cyclohexenone was used as the test group, and cultured at 37° C. and 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 lymphoma tumor cells

It can be seen from Table 1 that through 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 U937 human lymphoma tumor cells is 9.71 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 lymphoma 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 lymphoma tumor cells. On the other hand, because the cyclohexenone compound of Antrodia antrodia is a naturally extracted substance, when it is used to inhibit lymphoma, 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 chemotherapeutic drugs and reduce the side effects caused by these chemotherapeutic agents; in addition, it can also be prepared into a pharmaceutical composition for the treatment of lymphoma, wherein, the pharmaceutical composition contains effective doses of Antrodia antrodia cyclohexenone In addition to the compound, a pharmaceutically acceptable carrier may also be included. 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 manufactured according to the preparation method of general known pharmacy, 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 lymphoma tumor disease. the

Claims (16)

1. A compound with the following structural formula is utilized to prepare the application of the medicine for inhibiting lymphoma tumor cell growth:

Wherein, 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 according to 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 according to claim 2, wherein the compound is isolated from the extract of Antrodia camphorata. 4. The application according to claim 3, wherein the compound is separated and prepared from the water extract of Antrodia camphorata. 5. The application according to claim 3, wherein the compound is separated and prepared by 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 use according to claim 6, wherein the alcohols are ethanol. 8. The application according to claim 1, wherein the lymphoma tumor cells are U937 cell lines. 9. A pharmaceutical composition for inhibiting the growth of lymphoma tumor cells, comprising an effective dose of the compound according to claim 1 and a pharmaceutically acceptable carrier. 10. The pharmaceutical composition according to 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 according to claim 10, wherein the compound is isolated from the extract of Antrodia camphorata. 12. The pharmaceutical composition according to claim 11, wherein the compound is isolated from the water extract of Antrodia camphorata. 13. The pharmaceutical composition according to claim 11, wherein the compound is isolated from the organic solvent extract of Antrodia camphorata. 14. The pharmaceutical composition according to claim 13, wherein the organic solvent is selected from the group consisting of esters, alcohols, alkanes and haloalkanes. 15. The pharmaceutical composition according to claim 14, wherein the alcohol is ethanol. 16. The pharmaceutical composition according to claim 9, wherein the lymphoma tumor cell is U937 cell line.