text: the invention utilizes rapid and inexpensive bench - top bioassays for initial evaluation of the tumor suppressing properties of a plant extract by treating tissue that has been exposed to a known tumor - inducing agent with the extract and determining the level of suppression of tumor formation and / or growth . in a parallel assay , the effect of the extract on mutagenicity is estimated . mutagenicity is known to be closely associated with carcinogenicity . tumor suppression is evaluated following the well - known agrobacterium tumor induction assay procedure . the assay depends on the unique property of agrobacterium tumefaciens of inserting a portion of its tumor - inducing ( ti ) plasmid into the chromosomal dna of a wounded plant , causing tumor - like growths on the plant , a condition commonly known to those skilled in the art as crown gall disease . antitumor potential is measured by tumor suppression on aseptic potato discs treated with the plant extract . see a . b . galsky , et al . j . nat . cancer inst . 1981 . 67 : 689 - 692 , incorporated herein by reference , in its entirety . antitumor activity detected by the agrobacterium assay is correlated with suppression of tumors in animals ; thus , plant extracts that suppress agrobacterium - induced tumor formation have significant potential as human anticarcinogens . mutagenicity is evaluated following the ames salmonella mutagenic assay , a bacterial reverse mutation test using a histidine - dependent autotrophic mutant of salmonella typhimurium lacking both normal dna repair mechanisms and the ability to grow on a histidine - free medium . see , d . r . maron and b . ames . mutation research . 1980 . 113 : 173 - 215 , incorporated herein by reference in its entirety . as one skilled in the art understands , the test assumes that dna - altering chemicals cause mutations and that mutagenicity is closely associated with carcinogenicity . induced mutations are estimated by the number of bacteria capable of growing on a histidine - free medium . all extracts were made from vegetative material ( leaves and rhizomes ) of shortia galacifolia t .& amp ; g ., diapensiaceae , commonly recognized as “ oconee bell .” materials were collected in the winter ( dormant season ) from native populations in oconee county , s . c ., and from transplanted populations at the south carolina botanical garden , clemson , s . c . species identification was verified professionally by the clemson university herbarium ( clemson university , herbarium identification # 63415 , clemson , s . c .). plant samples were washed for 5 minutes under running tap water and dried at room temperature ( approximately 20 c or 68 f ) overnight . materials were divided into one of three categories : leaf material , new growth rhizomes , and mature rhizomes and dried in an oven at 40 c ( 104 f ) for 48 hours . approximately 5 g of dried sample were ground to a fine powder . crude extracts of plant material were prepared by mixing 1 - g aliquots of the powdered material with 8 ml of 80 % methanol and agitating on a lateral shaker for 8 hours at room temperature ( approximately 20 c , 68 f ). samples are then centrifuged at 1800 × g for 20 minutes and the supernatant saved . the residual pellet from this centrifugation was extracted following the preceding procedure . the supernatants from the two centrifugations were combined and evaporated to dryness at 40 ° c . ( approximately 104 f ) under a gentle flow of filtered air . samples were reconstituted to a 1 g / ml concentration with 100 % ethanol or with dimethyl sulfoxide ( dmso ). the reconstituted samples are filtered with a 0 . 45 μm membrane disk filter ( versapor acrodisc ®, v45w500 , pall gelman , ann arbor , mich .) to remove particulate matter and finally were stored at 4 c ( approximately 39 f ) in the dark until used . the preceding extraction was modified using 100 % hexane or 80 % methanol for extractions and dilute dmso for testing . antitumor effects of extracts were tested using the potato tumor induction method of mclaughlin &# 39 ; s laboratories . 1 a . tumefaciens was cultured in difco yeast extract medium for a period of 48 - 72 hours at 28 ° f . ( approximately 118 f ) for use in the assay . test tissue was prepared from organically grown , red - skinned potatoes ( solanium tuberosum l ., solanaceae ) as follows : tubers were washed under running tap water and surface sterilized with a 0 . 6 % sodium hypochlorite solution for 20 minutes . a thin slice was cut from each side of the tuber to produce flat , peeled surfaces . the tubers next were sterilized by immersion in a 1 . 2 % sodium hypochlorite solution for 15 minutes . cores of tissue were aseptically cut from each tuber using a # 9 ( 11 mm ) cork borer . the cylinders of tissue were rinsed twice in distilled water adjusted to ph 40 , and pieces approximately 2 cm in length are cut from each end of this cylinder and discarded . the remainder of the cylinder is cut into 0 . 5 cm discs . potato discs were rinsed in sterile water and transferred to a well - culture plate ; a 24 - well culture plate ( corning costar ®, model 3524 , cambridge , mass .) is appropriate , but not a limitation . the well - culture plate was prepared with 1 . 5 ml water agar ( 1 . 5 %) per well . an a . tumefaciens suspension was prepared by adding 7 - 10 loops of cultured bacteria to 15 ml phosphate buffered saline ( pbs , ph7 . 2 ) until obtaining an absorbance value of 0 . 96 ± 0 . 2 at 600 nm , which corresponds to approximately 1 × 10 9 colony forming units . the assay included three controls . camptothecin ( cat . no . c9911 ; sigma chemical ; saint louis , mo . ), a known anti - mitotic , anti - neoplastic agent was added to discs inoculated with a . tumefaciens as positive inhibitory control . plant extracts were diluted with 50 % ethanol ; thus , a . tumefaciens inoculated disks treated with a 50 % ethanol solution were a positive control for tumor inhibition by 50 % ethanol . a solvent control using 50 % ethanol added to discs not inoculated with a . tumefaciens tested for solvent induced tumor formations . all reagents controls and plant extracts were filter - sterilized ( 0 . 2 um ht tuffryn ® acrodisc ®, syringe filter , pall gelman , model 4192 , ann arbor , mich .). the positive inhibitory control contained 600 μl camptothecin stock ( 100 μg / ml )+ 150 μl sterile distilled water + 750 μl a . tumefaciens stock ; the positive control had control had 600 μl 50 % ethanol , 150 μl sterile distilled water + 750 μl a . tumefaciens stock ; for the solvent control , pbs replaced the a . tumefaciens stock ; test samples contained 600 μl dilute plant extract , 150 μl sterile distilled water + 750 μl a . tumefaciens , potato discs on water agar were overlaid with 50 μl of these solutions . two culture plates with 12 discs per plate were used for each combination , for a total of 24 replicates per combination . plates were incubated in the dark at room temperature ( 20 c or 68 f ) for 14 days . discs were stained with lugof &# 39 ; s potassium iodide reagent to facilitate tumor detection . see , n . r . ferrigni , et al . 1982 . 45 : 679 - 686 . to facilitate tumor detection . tumors on each stained potato disc were counted with the aid of a dissecting microscope . total number of tumors on control discs was compared with the total on extract treated discs and percent tumor inhibition calculated , table 1 . the shortia extract ( 1 g / ml ) was diluted in 50 % ethanol to obtain four concentrations thereby evaluating the effects of extract concentration on tumor suppression , table 1 . dilutions were 10 mg / ml ( 1 : 100 ), 1 mg ( ml ( 1 : 1000 ), 0 . 1 mg / ml ( 1 : 10 , 000 ), and 0 . 10 mg / ml ( 1 : 100 , 000 ). as previously described , comparisons included three controls and the dilutions in two culture dishes of 12 discs , each for a total of 24 replications . the entire assay was repeated three times . overall , an extract from vegetative material of plants of shortia galacifolia t .& amp ; g . suppressed tumor formation in discs of potatoes induced by a . tumefaciens . appropriate controls confirm that the detected tumor suppression is not an artifact , nor is it a product of tissue preparation protocol or a response to any solvent or combinations of solvents . the assay results illustrating the effects of concentrations of a plant extract derived from different tissues of shortia galacifolia are summarized in table 1 in which ‘ cont ’ is the positive control in which only the tumor inducing agent , a . tumefaciens was added , and no tumor suppression observed , as would be anticipated . ( the positive , camptothecin control resulted in effectively complete ( 98 . 2 %) tumor inhibition resulting from effects on the tumor inducing agent , a . tumefaciens , and as anticipated , the 50 % ethanol control , absent the inducing agent had no effect .) ‘ lf ’ in table 1 indicates leaf tissue as a source of the extract , ‘ nrh ’ new rhizome tissue , and ‘ mrh ’ mature rhizome tissue as the source of the extract . dilutions are 50 % ethanol dilutions of each extract source . the % tumor inhibition was calculated as from table 1 , clearly the extract suppressed tumor formation compared with the positive control , cont . overall extract from mature rhizomes suppressed tumors more than extracts from either leaves or new rhizomes , which were essentially comparable , 81 . 8 % suppression vs 69 . 9 and 70 . 0 %. generally higher concentrating ( lower dilution ) of the extract produced greater inhibition . although statistically significant , average concentration differences appeared to be of lesser practical significance because of the potential effects of a tissue × concentration interaction that did not alter the fundamental finding of effectiveness of the extract in tumor suppression . in independent , parallel studies , rhizome extracts were made with both hexane and 80 % methanol . in these studies , dilution effects were more pronounced , table 2 . the solvents hexane and methanol ( 80 %) were equally effective at the highest concentration ( 10 mg / ml ), 63 . 4 % and 63 . 8 % inhibition , respectively , and effectiveness decreased with both reagents more than with ethanol , table 1 . from tables 1 and 2 , it is clear that an extract from vegetative parts of plants of shortia galacifolia suppress tumors induced by a . tumefaciens in potatoes . because of the biological similarities between tumor growth in plant tissues , such as tubers , and in certain animal tissues , as one skilled in the art recognizes , parallel tumor suppression by such extracts on animal tissue is anticipated by the invention . antimutagenic effects of extracts were assayed by the ames salmonella / microsome mutagenic test with modifications . see d . r . maron and b . ames . mutation research . 1980 . 113 : 173 - 215 and k . mortelmans and e . zeiger . mutation research . 2000 . 455 : 229 - 260 both of which are herein incorporated by reference in their entirety . extracted plant samples were diluted 1 : 1000 with dmso . master plates were prepared from stock cultures of salmonella typhimurium ( strain ta 100 , xenometrix , san diego , calif .). colonies chosen to generate overnight cultures were grown in 5 ml nutrient broth # 2 ( cat no . cm 0067 ; oxid products ; basingstone , hampshire , uk ) at ° c . to a density of 1 - 2 × 10 9 per ml ( absorbance of 0 . 96 ± 0 . 2 at 600 nm ). cells were diluted 1 : 10 prior to use in tests . the assay consisted of combining either controls or the test compound and the salmonella tester strain the mutagen , s9 liver microsomes in a soft top agar poured onto a minimal agar plate lacking histidine . top agar ( 0 . 6 % noble agar and 0 . 5 % sodium chloride ) was melted and 10 ml mm / l - histidine . hcl / 0 . 5 mm biotin was added to 100 ml agar . twenty ml of human s9 mix ( moltox ™; product 14 - 102 ; boone , n . c .) { 8 mm mgcl 2 , 33 mm kcl , 5 mm glucose - phosphate , 4 mm nicotinamide adenine dinucleotide phosphate ( nadp ), 100 mm sodium phosphate , ph 7 . 4 and 0 . 04 ml s9 } were prepared immediately before assay . the s9 mix was divided into four 4 ml portions each of which had one of the following added : 10 . 4 μl dmso , 10 . 4 μl leaf extract , 10 . 4 μl new growth rhizome extract , or 10 . 4 μl nature rhizome extract . test compounds with the s9 mix were incubated for 30 minutes at 37 ° c ., filter sterilized ( 0 . 2 μm acrodisc ® v45w500 ; pall galman , ann arbor , mich . ), and divided into two 1 . 5 ml duplicate aliquots . one aliquot of each duplicate set was treated with 6 μl ( 1 mg / ml ) of the mutagen 2 - aminianthuracene ( 2 - aa ) [ cas no . 613 - 13 - 8 ], and the second had 6 μl dmso added and served as a control for each treatment . all aliquots were incubated 30 min at 37 ° c . each aliquot was divided into three 500 μl portions in plastic culture tubes , and 100 μl salmonella typhimurium dilution and 2 ml molten top agar was added to each tube . each aliquot was vortexed briefly and immediately poured on petri dishes containing the minimal glucose agar ( mga ) which were allowed to solidify , then inverted and incubated at 37 ° c . in the dark for 48 hours , after which revertant colonies on control and test plates were counted . in a separate study , uv - c ( 254 nm ) radiation was used as the mutagen or positive control for bacterial mutation . cells of s . typhimurium were irradiated with a uv - c lamp at a dose rate of approximately 1 j / m2 . mutated ( exposed ) cells were added immediately to 100 μl sterile water and either 100 μl plant extracts ( equivalent to 100 μg ) or to dmso alone . controls consisted of an identical set of tubes with water and extracts , but with bacterial cells not exposed to radiation . molten agar was added to all tubes , poured on to petri dishes with mga and incubated at 37 ° c . for 48 hours . tumor growth and development are known to be associated with increased rates or incidents of mutation . thus , suppression of mutation may be an acceptable measure of tumor inhibition . the mutagen 2 - aa dramatically increased mutation rate based on revertant colonies formed in the presence of 2 - aa , table 3 , in which the number of colonies formed ( 991 . 6 ) is approximately 20 - fold greater than the control ( 48 . 2 ). the inhibition effect of the leaf extract is apparent on mutation rate is apparent in comparisons of the colonies formed with 2 - aa control c 991 . 6 versus 2 - aa in combination of leaf , new growth rhizome , and mature rhizome 303 . 3 , 2029 , and 254 . 0 , respectively and the corresponding percent inhibition , 73 . 0 , 82 . 5 , and 78 . 2 , respectively , table 3 . from table 3 , the inhibition effects of the shortia extract are obvious and in agreement with tumor suppression result summarized in table 1 and 2 . the preceding methods and examples are presented by way of illustration , not limitation . the invention anticipates different methods of isolation of extracts as well as different tissues and dilutions of the extracts . thus , the broadest interpretation should be given to the appended claims .