Patent Publication Number: US-2011059122-A1

Title: Inhibition of the Survival of Oral Cavity Cancer by Cyclohexenone Compounds from Antrodia Camphorata

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a new application for inhibiting cancer cell survival, in particular to the application for inhibiting the survival of oral cavity cancer cells by a compound isolated and purified from  Antrodia camphorata.    
     2. The Prior Arts 
     Oral cavity is an important gateway of the human body to proceed with basic functions of breath, food taking, and linguistic expression in our daily life. Oral cavity cancer is an oral malignancy arose in the oral cavity area such as lip, buccal mucosa, lower gingival, upper gingival, retromolar trigone, bottom of oral cavity, hard palate, or the front of tongue. Oral cavity cancer is a common cancer disease in Taiwan. The possible risk factors of causing oral cancer include chewing betel nuts, frequently eating acrid food, smoking and alcohol drinking. The incidence and mortality of oral cavity cancer are increasing yearly and the prevalent age has decreased to 40-50 years old, which is younger than before and in which 90 percent of the patients are male. The major cause of oral cavity cancer is the deterioration of squamous cell and a small part of oral cavity cancer is adenocarcinoma, which is caused by the deterioration of small saliva glands. Buccal squamous cell carcinoma and tongue cancer are the majority of carcinogenic lesions of oral cavity cancer patients in Taiwan area. 
     The symptoms of early-stage oral cavity cancer include the existence of blood in saliva or phlegm and the formation of white or red spots without pain. With the advanced development of cancer, the symptoms further includes the existence of pain, hardly healed ulcer or protrudent ulcerated lump, and chronic wounds lasted for six months. Patients with early-stage oral cavity cancer usually don&#39;t feel pain and sometimes they still feel painless even though the tumor is formed. Therefore, oral cavity cancers are easy to be ignored, and generally are not found until symptoms are obvious. The disturbances to patients&#39; life quality caused by oral cavity cancer mainly include appearance, eating and pain; further include the problems of neck stiffness, hard to open mouth, difficult to speak, few secretion of saliva, damaged tooth and bad appetite, and thus resulting in serious adjustment difficulties of patients in physiology, psychology and society. 
     Generally, the treatment of oral cavity cancer is mainly based on surgical excision. Since the lesion parts to be resected may be face or neck skin that will cause negative effects on appearances of patients after surgery, age, toleration, and willingness of patients to accept surgery should also be seriously considered. With regard to the tumors that are not suitable to or cannot be excised by surgery, radiotherapy in combination with chemotherapy may be considered. However, the treatments of surgery, radiotherapy, chemotherapy, or combination therapy usually lead to some sequelae such as tightly closed teeth, facial deformity, inarticulacy, and dysphagia, which worsen patients&#39; life quality. Besides, radiotherapy or chemotherapy also causes many adverse side effects or complications, and the overall 5-year survival rates of patients resulted is less than 20 percent. Thus, the research and development of a therapeutic substance which can effectively treat oral cavity cancer with no above-mentioned sequelae or side effects is urgently desired. 
       Antrodia camphorata  is also known as various names such as Chang-Chih,  Ganoderma comphoratum, Antrodia camphorata, Taiwanofungus camphorata , and Camphor Mushroom . . . etc., a genus of Basidiomycoya, Homobasidiomycetes, Aphyllophorales, Polyporaceae, and Antrodia in Fungi, and also a perennial mushroom. It is a Taiwan endemic species of fungi and received its name because it only grows on the inner wall of the hollow material from Taiwan&#39;s endemic Lauraceae tree species,  Cinnamomum kanehirai . The price of  Antrodia camphorata  is very high due to the extremely slow growth rate of natural  Antrodia camphorata.    
     The fruiting bodies of  Antrodia camphorata  are perennial, sessile, hard and woody, which exhale strong smell of sassafras (camphor aroma). The appearances are various with plate-like, bell-like, hoof-like, or tower-like shapes. They are reddish in color and flat when young, attached to the surface of wood. Then the brims of the front end become reversely curled tilting and extending to the surroundings. At the same time, the color turns to be faded red-brown or cream yellow brown, with ostioles all over. This region is of very high medical value. 
     In traditional Taiwanese medicine, the curative effects of  Antrodia camphorata  include removing rheumatism, smoothing vitality, nourishing blood, eliminating bruises, benefiting spleen and stomach, lessening accumulation, detoxification, subsiding swelling, sedation and relieving pain, and is used as a great antidote for detoxifying food poisoning, diarrhea, vomiting and pesticide poisoning. Furthermore, it has adjuvant therapeutic effects on liver and stomach dysfunction and the diseases of blood circulation.  Antrodia camphorata , like general edible and medicinal mushrooms, is rich in numerous nutrients including polysaccharides (such as β-glucosan), triterpenoids, superoxide dismutase (SOD), adenosine, proteins (immunoglobulins), vitamins (such as vitamin B, nicotinic acid), trace elements (such as calcium, phosphorus and germanium and so on), nucleic acid, agglutinin, amino acids, steroids, lignins and stabilizers for blood pressure (such as antrodia acid) and so on. These physiologically active ingredients are believed to exhibit effects such as: anti-tumor activities, increasing immuno-modulating activities, anti-allergy, anti-bacteria, anti-hypertension, decreasing blood sugar, decreasing cholesterol, etc. 
     Triterpenoids are the most studied components among the numerous compositions of  Antrodia camphorata . Triterpenoids are the summary terms for natural compounds, which contain 30 carbon atoms with the pent- or hex-acyclic structures. The bitter taste of  Antrodia camphorata  is from the component of triterpenoids. Three novel ergostane-type triterpenoids (antcin A, antcin B, antcin C) were isolated by Cherng et al. from the fruiting bodies of  Antrodia camphorata  (Cherng, I. H., and Chiang, H. C. 1995. Three new triterpenoids from  Antrodia cinnamomea . J. Nat. Prod. 58:365-371). Three new compounds zhankuic acid A, zhankuic acid B and zhankuic acid were extracted from the fruiting bodies of  Antrodia camphorata  with ethanol by Chen et al. (Chen, C. H., and Yang, S. W. 1995. New steroid acids from  Antrodia cinnamomea , —a fungus parasitic on Cinnamomum micranthum. J. Nat. Prod. 58:1655-1661). In addition, Cherng et al. also found three other new triterpenoids from the fruiting bodies of  Antrodia camphorata , which are sesquiterpene lactone and 2 biphenyl derived compounds, 4,7-dimethoxy-5-methyl-1,3-benzodioxole and 2,2′,5, 5′-teramethoxy-3,4,3′,4′-bi-methylenedioxy-6,6′-dimethylbiphenyl (Chiang, H. C., Wu, D. P., Cherng, I. W., and Ueng, C. H. 1995. A sesquiterpene lactone, phenyl and biphenyl compounds from  Antrodia cinnamomea . Phytochemistry. 39:613-616). In 1996, four novel ergostane-type triterpenoids (antcins E and F and methyl antcinates G and H) were isolated by Cherng et al. with the same analytic methods (Cherng, I. H., Wu, D. P., and Chiang, H. C. 1996. Triteroenoids from  Antrodia cinnamomea . Phytochemistry. 41:263-267). And two ergostane related steroids, zhankuic acids D and E together with three lanosta related triterpenes, 15 alpha-acetyl-dehydrosulphurenic acid, dehydroeburicoic acid, and dehydrosulphurenic acid were isolated by Yang et al. (Yang, S. W., Shen, Y. C., and Chen, C. H.1996. Steroids and triterpenoids of  Antrodia cinnamomea —a fungus parasitic on  Cinnamomum micranthum . Phytochemistry. 41:1389-1392). 
     Although  Antrodia camphorata  extracts were reported to have the above mentioned effects from the previously published experimental results, and the several compounds were analyzed and identified successfully, further works are needed to identify the effective compounds to inhibit cancer growth and thus to contribute beneficial effects on cancer therapy such as the treatment and prevention of oral cavity cancer. 
     SUMMARY OF THE INVENTION 
     In order to identify the anti-cancer compounds from the extracts of  Antrodia camphorata , the compound of the formula (1) was isolated and purified in the present invention, 
     
       
         
         
             
             
         
       
     
     wherein X and Y can be oxygen, nitrogen or sulfur, R 1 , R 2  and R 3  are each a hydrogen atom, methyl or (CH 2 ) m —CH 3  and m=1-12; n=1-12. 
     A preferred compound of the general formula (1) is 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone as shown in formula (2), with molecular formula of C 24 H 38 O 4 , appearance of pale yellow powder and molecular weight of 390. 
     
       
         
         
             
             
         
       
     
     Cyclohexenone compounds having the structures of formula (1) and formula (2) are purified from aqueous extraction or organic solvent extraction of  Antrodia camphorata . The organic solvents used include, but not limited to, alcohols such as methanol, ethanol or propanol, esters such as ethyl acetate, alkanes such as hexane, or halogenated alkanes such as chloromethane, chloroethane. Among them, alcohol is preferred, and ethanol is particularly preferred. 
     Cyclohexenone compounds of the present invention are applied in inhibiting the survival of cancer cells, which can further be used as a pharmaceutical composition for treating cancer and to enhance the cancer therapeutic effects. The compounds of the invention can be applied in inhibiting the survival of oral cavity cancer cells, which result in delaying the growth of the cancer cells and suppressing proliferation of the cancer cells, and further inhibiting cancer deterioration. The preferred compound is 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone of the formula (2). 
     On the other hand, the compounds of formula (1) and/or formula (2) in the present invention can be incorporated into pharmaceutical compositions or medicaments for treating oral cavity cancer to inhibit the survival of cancer cells. The pharmaceutical compositions include not only the compounds of formula (1) and/or formula (2), but also the pharmaceutically accepted carriers. Examples of such carriers include, 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 or medicament can be manufactured through mixing the compounds of formula (1) and/or formula (2) with at least one of the carriers by means of conventional methods known in the pharmaceutically technical field, which can be formulated in the form of, but are not limited to, powder, tablets, capsules, pellets, granules or other liquid formulation. 
     The present invention is further explained in the following embodiment illustration and examples. Those examples below should not, however, be considered to limit the scope of the invention, it is contemplated that modifications will readily occur to those skilled in the art, which modifications will be within the spirit of the invention and the scope of the appended claims. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The aqueous or organic solvent extracts of  Antrodia camphorata  were subjected to high-performance liquid chromatography (HPLC) for isolation and purification. Each fraction was recovered and applied to anti-cancer assay. The potent fractions with anti-cancer effects were analyzed for the composition and further assayed against oral cavity cancer cells. The above approach then led to the identification of compounds of formula (1) and formula (2) in inhibiting the survival of oral cavity cancer cells. 
     The compound 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone of the formula (2) is explained below as an example for the present invention. The anti-cancer effects of 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the anti-cancer drug screening model of National Cancer Institute (NCI) to analyze survival rates on oral cavity cancer cell line SCC-25. These assays have proved that cyclohexenone compounds from  Antrodia camphorata  decreased the survival rates of oral cavity cancer cell line SCC-25, and simultaneously showed low half inhibition concentration (IC 50 ) value. Therefore, cyclohexenone compounds from  Antrodia camphorata  can be used for inhibiting the survival of oral cavity cancer cells and further be applied for the treatment of oral cavity cancer. The details of the examples are described as follows: 
     Example 1 
     Isolation of 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone 
     One hundred grams of mycelia, fruiting bodies or mixture of both from  Antrodia camphorata  were placed into a flask. A proper amount of water and alcohol (70-100% ethanol solution) was added into the flask and were stirred at 20-25° C. for at least 1 hour. The solution was filtered through both a filter paper and a 0.45 μm membrane, and then collected as the extract. 
     The extract of  Antrodia camphorata  was subjected to High Performance Liquid chromatography (HPLC) analysis. The separation was performed on a RP18 column using a mobile phase consisted of methanol (A) and 0.1-0.5% acetic acid (B), with the gradient conditions: the ratio of (B) from 95% to 20% 0-10 minutes, from 20% to 10% 10-20 minutes, kept 10% 20-35 minutes, and increased from 10% to 95% 35-40 minutes at the flow rate of 1 ml/min. The column effluent was monitored with a UV-visible detector. 
     The fractions collected during 25-30 min were concentrated to yield 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone, a product of pale yellow powder. The analysis of 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone showed the molecular formula of C 24 H 38 O 4 , molecular weight of 390, and melting point of 48° C.˜52° C. Investigation of NMR spectra showed that  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, 124.30, 131.05, 135.35, 135.92, 138.05, 160.45, and 197.12. 
     Example 2 
     In Vitro Survival Assay for Anti-Oral Cavity Cancer Effects 
     Inhibiting effects of oral cavity cancer cells by cyclohexenone compounds of  Antrodia camphorata  from example 1 were assessed according to the anticancer-drug screening model of National Cancer Institute (NCI). The compound 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone from example 1 was added into the culture media of oral cavity cancer cell line SCC-25 to determine the survival rates. Survival of cell was analyzed using MTT assay. SCC-25 cell line was a human squamous cell carcinoma cell line. 
     MTT assay is commonly used to analyze cell proliferation, survival rate of viable cells and cytotoxicity. MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a yellow dye which can be converted to water-insoluble purple formazan on the reductive cleavage of its tetrazolium ring by the succinate tetrazolium reductase in mitochondria of cells. The amount of formazan produced is used to detect the number of viable cells and calculate the survival rates. 
     The SCC-25 cells were cultivated in DMEM/F12(1:1) medium supplemented with 10% fetal bovine serum and 400 ng/ml of hydrocortisone at 37° C., 5% CO 2  for 24 hours. Proliferated cells were washed once with PBS, treated with 1× trypsin-EDTA, and centrifuged at 1200 rpm for 5 min. The supernatant was removed and the cell pellet was resuspended in 10 ml of fresh medium by gently shaking. Cells were seeded onto 96-well plates. Cells treated with the crude extracts of  Antrodia camphorata  (total ethanol extracts, not purified) were designed as the control group; and cells treated with 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone were designed as the experiment group. Both substrates were added in the concentration of 30, 10, 3, 1, 0.3, 0.1 and 0.03 μg/ml respectively. Cells were cultivated at 37° C., 5% CO 2  for 48 hours. Afterward, 2.5 mg/ml of MTT solution was added to each well and incubated in the dark for 4 hours, followed by the addition of 100 μl of lysis buffer to stop the reaction. The absorbances were measured at 570 nm with an ELISA Reader to determine the survival rates. The half inhibition concentration (IC 50 ) value was also calculated and listed in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Results of in vitro survival assay for 
               
               
                 inhibition of oral cavity cancer cells 
               
            
           
           
               
               
               
            
               
                   
                 Sample 
                 IC 50  (μg/ml) 
               
               
                   
                   
               
               
                   
                 Experiment group (formula 2) 
                 1.05 
               
               
                   
                 SCC-25 
               
               
                   
                   
               
            
           
         
       
     
     Refers to the result of table 1, the IC 50  value of 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone toward SCC-25 was 1.05 μg/ml, which was significantly lower than those of total extracts from  Antrodia camphorata  (data not shown). Therefore, 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone from  Antrodia camphorata  can be utilized to inhibit the survival of oral cavity cancer cells. 
     In summary, the compound 4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone isolated from Antrodia camphorata according to the present invention can be used to effectively inhibit the survival of human oral cavity cancer cells. The cyclohexenone compounds from  Antrodia camphorata  won&#39;t induce uncomfortable side effects, toxicity or complications when being applied for treating oral cavity cancer. Moreover, these compounds of the invention can also be used concurrently with chemotherapy drugs when treating oral cavity cancer in order to reduce the using amount of chemotherapy drugs as well as decreasing the side effects resulted from chemotherapy drugs. In addition, it can be incorporated into pharmaceutical compositions. The pharmaceutical compositions include not only effective amount (or active dose) of the cyclohexenone compounds from  Antrodia camphorata  of the present invention, but also the pharmaceutically accepted carriers. Examples of such carriers include, 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 composition of the present invention can be manufactured through mixing the compound of cyclohexenone from  Antrodia camphorata  with at least one of the carriers by means of conventional methods known in the pharmaceutically technical field, and can be formulated in the forms of powder, tablets, capsules, pellets, granules or other liquid formulation, but are not limited to. The purpose for treating oral cavity cancer can then be accomplished.