an extract of inula britannica has been isolated that has use as a cancer preventative agent due to its activity to induce apoptosis in cancer cells . the extract contains several sesquiterpene compounds , including but not limited to the sesquiterpene lactones known as britannilactone , 1 - o - acetylbritannilactone ( oabl ), and 1 , 6 - o , o - diacetylbritannilactone ( oodabl ). the extract and the chemicals isolated therefrom can be used as a pharmaceutical for cancer treatment and / or prevention as well as a medical food , or nutraceutical , and a dietary supplement . the flowers ( approximately 10 kg ) of inula britannica var . chinensis were extracted three times with 95 % ethanol . the chloroform - soluble fraction of the ethanol extract ( 500 g ) was chromatographed on a silica gel column eluting with a gradient of chloroform - methanol . from the fraction collected with chloroform - methanol ( 20 : 1 ), 1 , 6 - o , o - diacetylbritannilactone ( 52 g ) was obtained . from the fraction collected with chloroform - methanol ( 10 : 1 ), 1 - o - acetylbritannilactone ( 10 . 5 g ) was obtained . experiments were performed to determine the activity of two of the sesquiterpene compounds isolated from inula britannica , oabl and oodabl , as cancer preventative agents . using a western blot technique , the ability of these compounds to phosphorylate bcl - 2 in cancer cells was examined . using the breast cancer cell line mcf - 7 , it was found that oabl induced bcl - 2 phosphorylation , with effective doses of 10 and 20 μm . oodabl induced bcl - 2 phosphorylation at lower effective doses , 2 . 5 and 5 μm . these results were compared to the ability of a known chemotherapeutic paclitaxel , which induced bcl - 2 phosphorylation at a dose of 100 μm . these data indicate that the sesquiterpenes are more potent than paclitaxel at inducing bcl - 2 phosphorylation in mcf - 7 cells . in two ovarian cancer cell lines , ovcar and pa - 1 , similar results were seen . in ovcar cells , oodabl induced bcl - 2 phosphorylation at a dose of 5 μm , while oabl induced bcl - 2 phosphorylation at a dose of 10 μm . in pa - 1 cells , oodabl induced bcl - 2 phosphorylation at a dose of 5 μm , while oabl induced bcl - 2 phosphorylation at a dose of 10 μm . in a prostate cancer cell line , these compounds were also able to induce bcl - 2 phosphorylation . oodabl induced bcl - 2 phosphorylation at a dose of 5 μm while oabl induced bcl - 2 phosphorylation at a dose of 10 μm . using cleavage of parp as an indicator of apoptosis , the compositions of the present invention were tested in pa - 1 ovary cells . parp is a 116 kd nuclear poly ( adp - ribose ) polymerase that appears to be involved in dna - repair , predominantly in response to environmental stress ( satoh , m . s . and t . lindahl . 1992 . nature 356 : 356 - 358 ). parp is important for cells to maintain their viability . cleavage of parp facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis ( oliver , f . j . et al . 1998 . j . biol . chem . 273 : 33533 - 33539 ). this protein can be cleaved by many ice caspases to form a 85 kd protein in cells undergoing apoptosis . pa - 1 cells were treated with oodabl for 24 hours . cells were lysed and parp cleavage was measured by western blot using a monoclonal antibody to parp ( pharmingen , inc ., san diego , calif .). oabl induced parp cleavage at doses of 10 and 20 μm , while oodabl induced parp cleavage at a dose of 5 μm . the effect of oodabl on cell cycle was analyzed by flow cytometery using breast cancer cell lin t47d . oodabl arrested cells at the g2 / m phase at a 20 μm concentration as compared to control cells . the effect of oodabl on microtubules was examined by indirect immunofluorescence of mcf - 7 cells using an antibody to tubulin after 12 hours treatment with either a vehicle control , 10 μm paclitaxel ( positive control ), or 20 μm oodabl . the results showed , that oodabl polymerized microtubules like paclitaxel . in a tunel assay , the effect of oodabl on late apoptosis was examined . hl - 60 cells lines were subjected to flow cytometry analysis using apo - brdu . apoptosis was detected by incorporation of br - dutp using a fluorescein labeled anti - brdu monoclonal antibody after treatment with a vehicle control , 1 μm camptothecin or 20 μm oodabl for 12 hours . oodabl was shown to induce apoptosis , as did the positive control camptothecin . cell viability was then assessed in a microculture tetrazolium / formazan assay ( mtt ; scudiero , d . a . et al . 1988 . cancer res . 48 : 4827 - 4833 ) using a variety of cell lines . absorbance was measured at 550 nm and cell viability was expressed as the percentage of drug treated cells relative to that of controls . the ic 50 was then defined as the concentration of drug that produced a 50 % decrease in cell viability relative to controls . oodabl was first tested in a variety of cell lines . results in mcf - 7 cells treated with various concentrations of oodabl ( 1 . 25 , 12 . 5 , 25 . 50 and 100 μm oodabl ) showed that cell viability decreased with treatment in a dose - dependent manner . the ic 50 was less than 12 . 5 μm . in pa - 1 cells treated with various concentrations of oodabl ( 1 . 953 , 3 . 9 , 7 . 815 , 15 . 625 , 31 . 25 , and 62 . 5 μm oodabl ), cell viability was decreased in a dose - dependent manner with an ic 50 of less than 7 . 815 μm . in du - 145 cells treated with various concentrations of oodabl ( 3 . 4 , 7 . 86 , 15 . 6 , 31 . 5 , 62 . 5 , and 125 μm oodabl ), cell viability was decreased in a dose - dependent manner with an ic 50 of less than 15 . 6 μm . in nci - h - 460 cell treated with various concentrations of oodabl ( 3 . 9 , 7 . 81 , 1 { grave over ( )} 5 . 62 , 31 . 25 , 62 . 5 and 125 μm oodabl ), cell viability was decreased in a dose - dependent manner with an ic 50 of between 31 . 25 and 62 . 5 μm . in nih 3t3 ( normal mouse fibroblasts ) cells treated with various concentrations of oodabl ( 1 , 10 , 20 and 50 μm oodabl ), cell viability was decreased in a dose - dependent manner with an ic 50 of 50 μm . oabl was then tested in some of these same cell lines . in mcf - 7 cells , oabl was tested at doses of 0 . 3 nm , 3 nm , 30 nm , 300 nm , 3 μm , and 30 μm . results showed that oabl decreased cell viability with an ic 50 of around 200 μm . in pa - 1 cells , oabl ( 1 . 953 , 3 . 9 , 7 . 815 , 15 . 62 , 31 . 25 and 62 . 5 μm ) decreased cell viability with an ic 50 of about 2 μm . in du - 145 cells , oabl ( 4 . 68 , 9 . 37 , 18 . 75 , 37 . 5 , 75 and 100 μm ) decreased cell viability . cell cytotoxicity was also assessed by a clonogenic assay . mcf - 7 breast cells were treated with various concentrations of oodabl ( 625 nm , 1 . 25 , 2 . 5 , 5 and 10 μm ) for 15 days and cells were then stained with methylene blue and colonies counted . the ic 50 was in the range of 2 . 5 to 5 μm oodabl . pc - 3 prostate cells were treated with various concentrations of oodabl ( 20 and 200 nm , and 2 and 20 μm ) for 15 days and cells were then stained with methylene blue and colonies counted . the ic 50 was in the range of 200 nm oodabl . rko cells were treated with various concentrations of oodabl ( 20 and 200 nm , and 2 and 20 μm ) for 15 days and cells were then stained with methylene blue and colonies counted . the ic 50 was in the range of 20 μm oodabl . baby rat kidney cells were transformed with e1a and transfected with the bcl - 2 gene to form brk - 4b - bcl - 2 cells . these cells were treated with various concentrations of oodabl ( 20 and 200 nm , and 2 , 10 and 20 μm ) for 15 days and cells were then stained with methylene blue and colonies counted . the ic 50 was in the range of 200 nm oodabl . baby rat kidney cells were transformed with e1a and transfected with bcl - 2 gene in which phosphorylation sites were mutated to form phosphomutant brk - 4b - bcl - 2 cells . these cells were treated with various concentrations of oodabl ( 20 and 200 nm , and 2 and 20 μm ) for 15 days and cells were then stained with methylene blue and colonies counted . the ic 50 was in the range of 2 μm . the level of bcl - 2 phosphorylation was then assessed in the non - mutated and mutated brk - 4b - bcl - 2 cells using a western blot assay . cells were initially treated for 12 hours with the test compound , oodabl ( at concentrations of 10 , 20 , 30 , 40 , or 60 μm ). taxol was used as a positive control at a concentration of 5 μm . cells were then lysed in ice cold radio - immune precipitation buffer with inhibitors . equivalent amounts of proteins were electrophoresed by 12 % dodecyl sulfate - polyacrylamide gel electrophoresis and transferred to nitrocellulose . bcl - 2 and phosphorylated bcl - 2 proteins were detected using a monoclonal bcl - 2 primary antibody and a secondary goat anti - mouse horseradish peroxidase conjugated antibody followed by enhanced chemiluminescence detection . the results showed that there was a dose - dependent increase in bcl - 2 phosphorylation with oodabl in the non - mutated cells . taxol also produced an increased in protein phosphorylation . in the mutated rat kidney cells , there was no phosphorylation evident with either taxol or oodabl . these data demonstrate that the extract of inula britannica has use as a cancer preventative and treatment agent due to its activity to induce apoptosis and cell cytotoxicity in cancer cells . the extract and the chemicals isolated therefrom , oabl and oodabl , can be used as a pharmaceutical for cancer treatment and / or prevention as well as a medical food , or nutraceutical , and a dietary supplement . the data presented support the development of either foods for animal consumption , where animals include humans , or as dietary supplements for animals including humans . these foods and supplements are referred to by those of skill in the art as “ nutraceuticals ”. compositions of the present invention would be useful as nutraceuticals for prevention or treatment of cancer . one of skill would be able to use the results of experiments in cells and animals to determine an effective amount to be administered in humans . an effective amount would be an amount that induces apoptosis or inhibits tumor growth either in vitro or in vivo in animals . for example , human test doses can be extrapolated from effective doses in cell studies , such as ic 50 values , or from effective doses in vivo by extrapolating on a body weight or surface area basis . such extrapolations are routine in the art . further , one of skill would know how to formulate or prepare diets or dietary supplements containing the analogs . in the case of animal diets , the analogs could be added in concentrations up to 5 % by weight and mixed according to methods routine in the art . dietary supplements for animals or humans could be prepared in a variety of forms that would include but not be limited to liquid , powder , or solid pill forms . pill forms for the supplements would be prepared by methods routine in the art of dosage formulation and could include but not be limited to production of gel capsules , time - release capsules , or solid pills formulated with excipients and binders . again , one of skill in the art would know how to formulate the extracts or compounds isolated therefrom based on its chemical nature and the desired effect . the extract and / or the compounds isolated therefrom could also be administered topically in liquid or creme of lotion forms or by injection . injectable forms would be prepared by solubilizing in a pharmaceutically acceptable vehicle .
compositions and methods for preventing and treating cancer are provided that comprise extracts of inula britannica or compounds isolated therefrom . the compounds of 1 - o - acetylbritannilactone or 1 , 6 - o , o - diacetylbritannilactone are isolated and used for inducing apoptosis in cells and for treating cancer in an animal . induction of apoptosis comprises contacting cells with a composition comprising the compounds in an amount sufficient to induce bcl - 2 protein phosphorylation . further , treatment of cancer in an animal comprises administering to an animal a composition comprising the compounds in an amount sufficient to induce bcl - 2 protein phosphorylation to treat the cancer .