Patent Application: US-19434502-A

Abstract:
the present invention provides medicinally active extracts and fractions , and a method for preparing the same by extracting and fractioning constituents from the tissue of plant components of the anoectochilus family . these active extracts and fractions are useful for preventing or inhibiting tumor growth .

Description:
we adapted the anti - tumor drug - screening strategy developed at the nci ( monks et al ., 1991 ) with some modifications , primarily aimed at evaluating specific cytotoxicity on tumor cells by a . formosanus plant extracts . we employed a defined and reproducible “ bioactivity - guided fractionation strategy and protocol ” to evaluate the potential anti - tumor cell effects of the a . formosanus total plant extracts and the bio - organically derived fractions . the mechanism - specific cytotoxic activity on tumor cells obtained from specific plant extracts of a . formosanus is apparently involved in the induction of apoptosis , as evaluated using flow cytometry , western - blot analysis , and caspase ( s ) activity assays . these findings are very similar to various published studies demonstrating the apototic effects induced by specific anti - cancer drugs . see for instance the published studies of whllie et al ., 1980 and jiang et al ., 1996 ). b 16 , mcf - 7 , and hepg2 cells were grown in atcc - recommended culture media , containing 10 % fetal bovine serum , 100 u / ml penicillin , and 100 μg / ml streptomycin as supplements . rpmi 1640 ( gibco brl , usa ) was used to grow b16 and mcf - 7 cells , and minimum essential medium ( mem , gibco brl , usa ) for hepg2 cells . the effect of a . formosanus plant extracts and the derived fractions on growth of target tumor cells was mainly evaluated by using mtt colorimetric assay as described elsewhere ( mosmann , 1983 ) with minor modifications . the tetrazolium ring of mtt ( which confers a yellow color ) is cleaved by mitochondrial dehydrogenase in living cells , and this produces the brown colored formazan precipitates . the relative absorbance changes at 570 nm are then measured . for mtt assays , target tumor cells ( 5 × 10 3 to 1 × 10 4 ) were incubated with or without plant extracts in 96 well titer plates for 1 to 3 days . after incubation , adherent cells were washed once with fresh medium , and mtt dye ( 0 . 5 mg / ml in pbs ) was then added into the corresponding cell culture medium as described above , and reactions allowed to be carried out at 37 ° c . for 4 hours . the insoluble reaction product was then collected by centrifugation , dissolved in 100 μl of dmso , and incubated at 37 ° c . for 2 hours . all assays were performed in quadruplicate cell sets . the cell proliferation - inhibition effect was expressed as the relative absorption at 570 nm using a microplate reader ( labsystems multiskan ms , finland ). the percentage of cell survival after treatment with plant extracts was calculated using the following formula : viable cell number (%) od 570 ( treated cell culture )/ od 570 ( control ( un - treated ) cell culture )× 100 %. for certain test cells , a 3 h - thymidine incorporation assay was also employed to determine the anti - cell proliferation activity in plant extracts of a . formosanus , by following the stringent experimental protocol , as described by seufferlein and rozengurt ( 1995 ). the apoalert annexin v - egfp method ( clontech , palo alto , calif .) was used to assess for apoptosis in test cells ( zhang et al ., 1997 ). mcf - 7 tumor cells were treated for 4 hours with ea fraction ( 1 mg / ml ) or the vehicle ( 0 . 1 % dmso ), washed with fixing solution , and stained with annexin v - egfp and / or propidium iodide for 15 min in dark . cells were viewed using a nikon eclipse e800 inverted fluorescentscope . test tumor cells ( 1 × 10 6 cells ), with or without treatment using the evaluating plant extracts or the derived phytochemicals , were harvested and fixed with 1 ml ice - cold , 70 % ethanol at 4 ° c . for 2 hr . cellular dna was stained for 30 minutes in the dark with pi staining solution containing 50 μg / ml propidium iodide ( pi ) and 100 μg / ml rnase a in pbs buffer . the percentage of cells with a sub - g 1 dna content over total dna was taken as a measure of the apoptotic cell population using a coulter epics xl flow cytometer ( beckman coulter , usa ). data were generated from samples with at least 10 , 000 cells per assay and analyzed with the dna multicycle program ( beckman coulter , usa ). 3 . induction of fasl expression on mcf7 cells and flow cytometry analysis of fasl expression aliquots of 1 × 10 6 / ml cells were cultured for overnight in six - well flat - bottom plates in rpmi 1640 medium supplemented with 10 % heat inactivated fetal bovine serum . ea plant extract ( 1 mg / ml ) or 0 . 4 % dmso was then added to growth media and incubated for 5 . 8 or 16 hours . after treatment , cells were harvested using 0 . 5 ml / well trypsin / edta and washed twice in phosphate buffered saline ( pbs ), ph 7 . 2 . aliquots of 5 × 10 5 cells in a final volume of 100 μl each pbs were incubated for 30 min at 20 ° c . with 1 μg mouse anti - human fasl igg1 , clone nok - 1 ( pharmingen , usa ), washed once , and resuspended in 100 μl pbs . a mouse igg1 , clone mopc - 21 ( pharmingen , usa ) was used as the isotype control . for secondary staining , cells were incubated for 30 min at 20 ° c . in the dark with 1 μg fitc - conjugated rate anti - mouse igg , clone lo - mg1 – 15 ( biosource , california , usa ), and washed twice in pbs prior to analysis by a coulter epics xl flow cytometer ( beckman coulter , usa ) using the expo xl 4 cytometer software . test cells ( 2 × 10 6 ) were washed with pbs once and lysed in 100 μl lysis buffer ( ph 7 . 0 , 20 mm pipes , 10 mm nacl , 1 mm edta , 0 . 1 % chaps , 10 % sucrose , 10 mm dtt , 5 mm hepes , 0 . 05 % ( v / v ) triton x - 100 , 1 mm mgcl 2 , 2 . 5 mm egta , and 0 . 438 % ( w / v ) β - glycerol - phosphate ) and 2 μl protease inhibitor cocktail set iii ( calbiochem co ., usa ) for 20 minutes on ice , and vortex - mixed every 5 minutes . pipes , chaps and hepes chemicals were purchased from sigma chemical co . ( mo , usa ). after centrifugation , protein concentration in the supernatant was determined as described by bradford ( 1976 ), using bovine serum albumin as a standard . aliquots of 40 μg of protein were separated on a 5 – 20 % gradient mini - sds - polyacrylamide gel ( laemmli , 1970 ), and then transferred onto a pvdf membrane ( millipore ). pvdf filters were blocked for non - specific binding with 3 % non - fat dry milk in tbs buffer ( 10 mm tris , ph 7 . 5 and 100 mm nacl ), and probed with specific primary antibodies in blocking solution at 4 ° c . overnight with agitation . rabbit anti - actin ( oncogene research products ), mouse anti - parp ( related products ), mouse anti - cytochrome c ( pharmingen ), rabbit anti - nf - κb ( oncogene research products ), mouse anti - porcaspases - 2 , - 7 , and - 9 and anti - porcaspase - 8 ( bd research products ) were used as primary antibodies . after washing with tbst buffer ( 0 . 1 % tween 20 in tbs buffer ), blotting membrane was treated with alkaline phosphatase - conjugated secondary antibodies at room temperature for 3 h . the reacted protein bands were visualized by colorimetric method or by an enhanced chemi - luminescence system ( ecl , amersham pharmacia biotech co ., uk ) for image analysis on kodak biomax ms films ( eastman kodak company , usa ). the animal tumor system employed was the mouse strain c57bl / 6j , or often called c57 or b6 mouse system harbored with murine b - 16 melanoma cells . possible in vivo effects of anoectochilus formosanus plant extract on tumor growth were evaluated in vivo using a moderate tumor cell load of b16 melanoma cells inoculated and grown in 2 – 3 month old test mice . five groups of mice , each with 7 mice per test group , were employed . a total of 1 × 10 5 b - 16 tumor cells suspended in 50 μl of phosphate buffered saline ( pbs ) was implanted into the dermal tissue ( intra - dermally ) of the right abdominal area of each mouse on day 0 . three sets of mice were tested for the ah - sup treatment , by intra - peritoneal ( i . p .) or intra - tumoral ( i . t .) injections of test plant extracts , or by the force - feeding ( f . f .) mode for testing anti - tumor effects . specifically , from day 2 on , test mice in group 1 received pbs only via i . p . injection ( control 1 ), and group 2 mice were treated with pbs only via force - feeding and i . p . injection concomitantly ( control 2 ). groups 3 , 4 and 5 mice were treated with ah - sup solution by i . t . injection , i . p . injection , and force - feeding by mouth , respectively . the injection dosage received by each test mouse was 1 mg of ah - sup in 100 μl pbs per day , while the force - fed mice took 2 mg in 200 μl per day orally . both test groups were treated for 7 consecutive days after inoculation of tumor cells . starting on day 8 , the frequency of the treatment was changed to a slower pace , by treating mice every two days until day 21 . in the case of force - feeding , the relative herb dosage tested in mice was equivalent to the ingestion or uptake of a 50 kg person drinking two servings of instant herb tea powders ( 2 tea bags ) made from about 30 g of dried a . formosanus plant . 6 . metabolite profiling and the index or composition compound analysis of ah - sup and ea fractions of anoectochilus formosanus analytical high performance liquid chromatographic ( hplc ) analyses were performed using a waters hplc system equipped with a waters 600 controller , waters delta 600 pump , and 2487 duel λ absorbance detector . a 5 μm c - 18 column ( 250 × 10 nm , merck , germany ) was employed for the analysis of ah - sup fraction with two solvent systems , methanol - water ( 90 : 10 , v / v ) ( a ) and methanol ( b ). the elution conditions were performed as follows : 0 to 5 minutes , 80 % a to b ( isocratic ); 5 to 30 minutes , 80 – 0 % a to b ( linear gradient ); 30 to 60 minutes , 100 % b ( isocratic ). the detector wavelength was set at 254 nm . a 5 μm si - 60 column ( 250 × 10 nm merck , germany ) was employed for the fa active fraction with two solvent systems , n - haxane ( a ) and ethyl acetate ( b ). the elution gradient profile was performed as follows : 0 – 5 min , 70 % a to b ( isocratic ); 5 – 15 min , 70 – 30 % a to b ( linear gradient ); 15 – 20 min , 30 % a to b ( isocratic ); 20 – 30 min 30 %– 0 % a to b , the flow rate was 5 ml / min , and the detector wavelength was set at 280 nm . the structures of compounds 1 to 8 in the all - sup fraction and compounds 1 to 6 in the ea fraction were then elucidated using various spectroscopic analyses . uv spectra of test compounds were recorded with a jasco v - 550 spectrometer , and ir spectra obtained from a bio - rad fts - 40 spectrophotometer . electron - impact mass spectrometry ( eims ) and high resolution electron - impact mass spectrometry ( hreims ) data were collected with a finnigan mat - 958 mass spectrometer , and the nmr spectra recorded with bruker avance 500 and 300 mhz ft - nmr spectrometers , at 500 mhz ( 1 h ) and 75 mhz ( 13 c ). quantification of the content of index compound in ea fraction was done by a hplc analysis using a known concentration of ea fraction . individual peak area corresponding to index compound in the hplc profile ( metabolite profile ) of the ea fraction was determine at the observed maximal absorbance of od 280 . the standard calibration curves ( peak areas vs . concentrations ) of the compounds , ranging from 0 . 05 to 1 mg / ml , revealed good linearity and r 2 values (& gt ; 0 . 98 ). quantification of the content of index compound in ea fraction was done by a hplc analysis using a known concentration of ea fraction , and the peak area of specific compound was then determined and referred to their contents in the extract based on the standard calibration curve . fresh plants of a . formosanus were purchased from a reputable anoectochilus cultural station and the plants &# 39 ; identity and authenticity were validated by the specific morphological and anatomical features of the flowers ( lin et al ., 1978 ). fresh ( 1000 g ) or dried ( 100 g ) whole plants of a . formosanus , including leaf , stem , and root tissues , were homogenized by grinding with a mortar and pestle , and extracted by a slow and steady boiling ( about 100 ° c .) process for one hour with three volumes of distilled water . the extraction procedure described above was then repeated once . the supernatants from this two time extractions were collected by centrifugation at 24 , 000 × g for 20 minutes at 25 ° c ., and then lyophilized to dryness using a lyophilizer ( labconco , usa ). this preparation gave rise to the total af - hot crude extract ( ca . 49 g in dry weight ). this af - hot extract was dissolved in phosphate buffer saline ( pbs ) and employed for anti - cell proliferation activity on target tumor cells . a defined “ bioactivity - guided fractionation strategy and protocol ” was employed for further fractionation of the af - hot of a . formosanus , as described in fig1 and discussed above . various sub - fractions were obtained from the af - hot crude extract using a stepwise ethanol fractionation procedure . ah - i , ii , and iii fractions were the ethanol - precipitated materials derived from the af - hot samples that were sequentially extracted organically with 50 %, 75 %, and 87 . 5 % ( v / v ) ethanol , respectively , and the ah - sup was the remaining soluble form / fraction of the af - hot sample treated with 87 . 5 % ethanol . in summary , equal volume of anhydrous ethanol was slowly added into the af - hot crude extract with stirring and allowed to stand at room temperature for 30 minutes . the ah - i fraction (˜ 17 . 6 % in dry weight ) was collected from the pellet by centrifugation at 24 , 000 × g for 20 min at 4 ° c ., and the supernatant was then further added with anhydrous ethanol to reach a final ethanol concentration of 75 % ( v / v ) by using the same procedure as described in preparation of ah - i fraction . in this step , ah - ii fraction (˜ 4 . 4 % in dry weight of the original plant materials ) was obtained . the ah - iii (˜ 1 . 7 % in dry weight ) and ah - sup (˜ 23 % in dry weight ) fractions were obtained from the 87 . 5 % ( v / v ) ethanol precipitates and the soluble fraction , respectively . a further fractionation procedure on the all - sup ( 19 . 3 g ) was then subjected to differential solvent partitioning using ethyl acetate ( ea ) and followed by butanol ( buoh ) to yield the ea ( about 1 . 2 g ), buoh ( about 3 . 4 g ), and water ( 14 . 4 g ) subfractions , respectively ( fig1 ). in these organic solvent fractionation steps , ethyl acetate was mixed vigorously with af - sup at an equal volume ( 1 : 1 ) and allowed to stand at room temperature until a separated and clear organic ea and aqueous layers were obtained . the ea fraction was then collected and the aqueous layer was extracted again with ethyl acetate . the two ea fractions were pooled and evaporated to dryness using a rotavaper . the aqueous layer was then further extracted with butanol using the same procedures as was performed for the ethyl acetate extraction , yielding the buoh and water subfractions . the af - hot , ah - i , ah - ii , ah - ii and ah - sup fractions thus obtained were dissolved in phosphate buffered saline ( pbs ), and the ea , buoh and water fractions were dissolved in dimethyl sulfuric oxide ( dmso ) solvent for subsequent use in the following experiments . mtt ( tetrazolium ) assay ( mosmamn , 1983 ), a calorimetric assay for measuring various dehydrogenase activity in mitochondria of living cells , and 3 h - thymidine incorporation assay , for measuring dna synthesis in viable cells , were employed for evaluation of anti - tumor cell activity . three typical tumor cell lines , i . e ., human mammary tumor ( mcf - 7 ), mouse melanoma ( b16 ), and human hepatoma ( hepg2 ), were used in the same or parallel tests . fig2 shows the cytotoxic effect of af - hot on the mcf - 7 , hepg2 , and b16 tumor cells in the presence or absence of fetal bovine serum in growth medium of test tumor cell cultures . a very similar level of anti - tumor cell effect was observed for total plant extract af - hot regardless of whether the culture medium was supplemented with or without fetal bovine serum . this is a useful and important baseline information , demonstrating that the potential interference between fetal bovine serum components and test plant extracts apparently does not play a role in the anti - tumor cell proliferation activity as conferred by plant extracts of a . formosanus . the total crude extract ( ah - hot ) prepared from two different sets of starting plant materials were observed to confer similar or comparable trends of cytotoxic effects on test tumor cells ( data not shown ). based on mtt assays , the ah - sup sample showed the most significant cytotoxic or anti - proliferation effect on mcf - 7 , b16 , and hepg2 cells , as compared to those conferred individually by the ah - i , ah - ii , and ah - iii fractions derived from the af - hot total plant extract . less than 45 to 55 % of tested cells were detected as viable for mcf - 7 , hepg2 , and b16 tumor cells when they were treated with ah - sup a dosage of 1 . 5 mg / ml for 48 hours . whereas , more than 80 % viable tumor cells were observed when all three tumor cell lines were treated with the same dose of ah - i , ah - ii , or ah - iii fractions . a similar trend of cytotoxic effects of af - hot and ah - sup was also observed when the test tumor cells were evaluated by 3 h - thymidine incorporation assays . the relative effects on cytotoxicities ( or anti - tumor cell activity ) of the total crude extract ( ah - hot ), ah - sup , ea , buoh , and water fractions were further characterized and compared using mtt assays . as shown in fig3 , the ea fraction conferred the highest levels of cytotoxic effect on all tested cell lines , as compared to the af - hot or other organic subfractions of a . formosanus extracts . table 1 summarizes the effective dosage for 50 % inhibition on cell proliferation ( ed 50 ) of four different bio - organic subfractions ( ah - sup , ea , buoh , and water ) on mcf - 7 , b16 , and hepg2 cells . the ed 50 of the ea fraction for mcf - 7 human mammalian adenocarcinoma cells was detected at 0 . 08 mg / ml , and this is 10 - fold less than that obtained for ah - sup subfraction . formosanus plant on three targeted human or mouse tumor cells ed 50 the effective dosage ( ed ) for inhibiting 50 % of cell proliferation . light and fluorescent microscopic analyses of morphology and culture behavior of test tumor cells light microscopy analysis showed that the morphology of mcf - 7 cells in culture had significantly altered after the treatment with plant extracts of a . formosanus . at a dose of 1 mg / ml , ea fraction exhibited the most significant effects on the morphological change of mcf - 7 cells , as compared to that of ah - sup and af - hot treatment ( fig4 ). similar results were also observed for the hepg2 and b 16 tumor cells ( data not shown ). these observations are in good consistence with the observed ed 50 values of these extract fractions as was represented in table i . to further verify if the treated mcf - 7 cells were progressing toward apoptosis , fluorescent microscopy analyses were performed using annexin v - gfp and propidium iodide staining . fig5 shows that an early redistribution of plasma membrane phosphatidylserine was readily detected in mcf - 7 cells after treatment with ea fraction for four hours . this result shows that ea fraction has effectively induced apoptosis on mcf - 7 cells . fig6 shows the flow cytometric profiling of the change of dna content and cell cycle behaviors of mcf - 7 cells , as a function of time by treatment with the ea fraction . approximately a level of 48 % of apoptotic dna level ( as determined from the sub - g1 peak ) was obtained for mcf - 7 cells post treatment with ea fraction ( 0 . 25 to 1 mg / ml ) for 48 hours , and this apoptotic dna level was increased to 71 % at the 72 hours post treatment . under the same experimental conditions , similar dna profiles were detected in hepg2 cells compared to that of mcf - 7 cells , whereas approximately 72 % apoptotic dna level was already detected in b16 cells at the 48 hours treatment . ah - sup fraction ( 6 mg / ml ) of a . formosanus also exhibited a similar effect on the dna content and cell cycle behaviors of mcf - 7 and b16 cells ( data not shown ). these results show that the cytotoxic effect of a . formosanus on test tumor cells is likely mediated via the process of programmed cell death or apoptosis ( williams , 1991 ). it is known that expression of fas ligand ( fasl ) can mediate apoptosis by binding to its cognate receptor fas ( naujokat et al ., 1999 ). in this study , flow cytometry analyses using a specific anti - human fasl igg1 conjugated with fluorophore ( fitc ) was employed to evaluate whether the observed apoptosis of mcf - 7 induced by ea extract of a . formosanus was triggered by the expression of fasl . as shown in fig7 , the expression of fasl protein was increased drastically from 10 % to 76 %, relative to the non - treated control mcf - 7 cells , when the tumor cells were treated with the ea fraction from 5 hours to 16 hours in culture ( fig7 a & amp ; b ). several single compound drugs exhibiting anti - tumor activities , including taxol and etoposide , have been demonstrated to cause mitochondrial damage and content release , resulting in cytosolic accumulation of cytochrome c ( fang et al ., 1998 ). we have evaluated the possible effects of a . formosanus plant extracts on release of mitochondria cytochrome c into cytosol of test tumor cells . fig8 shows that a significant increase of the cytosolic cytochrome c levels in mcf - 7 cells was observed when treated with ea ( or ah - sup ) fraction of a . formosanus plant extracts . caspase proteases have been shown to drive apoptotic signaling and execution by cleaving critical cellular proteins specifically and solely after the aspartate residues ( wolf and green , 1999 ). caspases exist as latent zymogens , however , once activated by apoptotic signals , the pro - caspases are proteolytically cleavaged and begin to take action . the activated apoptotic initiators ( caspases - 8 and - 9 ) in turn can activate the executioner caspases ( caspases - 3 , - 6 , and - 7 ). in our study , pro - caspase - 8 (˜ 55 kda ) was found proteolytic cleavaged in mcf - 7 cells after treatment with ea extract within approximately 16 hours ( fig8 a ). for the case of caspase - 7 precursor protein , ea extract was detected to induce the proteolytic processing of caspase - 7 , starting at 24 to 32 hours post - treatment . caspase - 9 , known to involve in the early activation cascade was also activated in ea - treated mcf - 7 cells . treatment of mcf - 7 cells with ea extract have also resulted in a time - dependent proteolytic cleavage of poly ( adp - ribose ) polymerase ( parp ), another hallmark of apoptosis , with an accumulation of the 28 kda and the concomitant disappearance of the full - size 116 kda molecule ( fig8 b ). it has been previously reported that nf - κb activation can result in protection against tnf - α - induced apoptosis in hela cells , a cervical - cancer cell line , in vitro ( bursch et al ., 1992 ). the rel a subunit ( p65 ) of nf - κb is known to be directly involved in the inhibition of apoptosis ( liu et al ., 1996 ). in this study , after ea treatment there was detectable effect on the suppression of rel a protein level in mcf - 7 cells ( fig8 a ). expression of the rel a subunit in mouse b16 melanoma was significantly suppressed as a function of the treatment time period with ah - sup ( 6 mg / ml ) or ea fraction ( 0 . 25 – 1 . 0 mg / ml ) of a . formosanus ( data not shown ). a flux of caspase 3 activity , an increase from 4 to 24 hours and then a decrease from 24 to 48 hours , as a function of treatment time by ah - sup in b16 melanoma cells was observed ( data not shown ). these results indicate that specific apoptosis mechanisms are involved in the observed killing of targeted tumor cell lines , instead of general , non - specific chemical toxification or killing . on the basis the results of flow cytometry , western - blot analysis , and caspase ( s ) activity assays , we proposed a possible mechanism for the apoptosis in mcf - 7 tumor cells induced by the plant extract of a . formosanus ( fig1 ). in vivo inhibition of b16 tumor growth in c57bl / 6j mouse model suppression or retardation of in vivo tumor growth by a . formosanus plant extract in the cs7bi / 6j mouse tumor model was also demonstrated as another aspect of this invention . three commonly used drug - administering methods , including intra - tumoral injection ( group 3 ), intraperitoneal injection ( group 4 ) and force - feeding via oral administration ( group 5 ), were performed in parallel in this study . fig9 summarizes the experimental results . in all three cases , test mice treated with the ah - sup fraction showed a significant delay of tumor growth , as observed at the early stage for in vivo tumor growth ( between 6 to 10 days post treatment ) ( fig9 ). the highest inhibitory effect on tumor growth was observed between 8 and 16 days post - tumor cell implantation . on day 17 , all of the experimental test groups , including the i . t . injection , i . p . injection , and the force - feeding groups , have maintained with a set between 4 to 7 mice per group , with tumor size measured below 12 mm in diameter . in contrast , only 3 and 2 mice were with tumor size below 12 mm in the non - ah - sup treated , control 1 and control 2 groups , respectively ; and 3 mice in control 2 groups were scarified due to overburden of tumor load ( fig9 ). on day 21 , all surviving mice in both control groups had effectively grown tumors to more than the critical size limit of 15 mm tumor in diameter , legally allegeable for sacrificing as test mice according to animal room regulation rules . in contrast , at the same experimental stage of day 21 post - treatment , there were still 4 mice in group 3 , two mice in group 4 , and one mouse in group 5 that still have tumors exhibiting a size of smaller than 15 mm in diameter ( data not shown ). these results suggest that all of the three tested administration methods for delivery of herbal extracts can provide a significant level of anti - tumor effects , with the most remarkable results obtained from the group 3 mice that were i . p . injected with ah - sup fraction of a . formosanus . specifically , an inhibition of more than 70 % tumor growth in test mice was obtained when the animals were intra - tumorally administered with a plant extract of a . formosanus . results from these experiments indicate that in vivo growth of b16 melanoma tumor in c57bl / 6j mice can be effectively and readily inhibited by administration of the partially purified plant extracts of a . formosanus under the experimental conditions of this invention . analytical high performance liquid chromatographic ( hplc ) analyses were performed using a waters hplc system equipped with a waters 600 controller , waters delta 600 pump , and 2487 duel λ absorbance detector . the three chromatograms of ah - sup plant extract from anoectochilus formosanus were obtained from a c - 18 reverse phase hplc system . the index compounds are herein to represent the defined chemical composition and / or characteristics of a mixture of relatively crude plant extract substances or phytochemicals . for the case of ah - sup , the eight candidate index compounds identified by using ir , mass and nmr spectrometric analyses are : compounds 1 : nicotinic amide ; 2 : adenosine ; 3 : cytosine ; 4 : isorhamnetin 3 , 4 ′- o - β - d - diglucopyranoside ; 5 : isorhamnetin 3 - o - β - d - glucopyranoside ; 6 : caffeic acid ; 7 : ( 6r , 9s )- hydroxy - megastima - 4 , 7 - diene - 3 - one - 9 - o - β - dglucopyranoside ; and 8 : kinsenone ( fig1 a ). according to the results of ms and nmr analyses and by comparing with the spectral data with literatures ( wang et al , 2002 ; ali et al ., 1997 ), six compounds isolated from ea of a . formosanus were identified as long chain aliphatic acid ( 1 ; retention time ( rt )= 6 . 0 min ). α - amyrin trans - p - hydroxy cinamate ( 2 ; rt = 8 . 9 min ), α - amyrin cis - p - hydroxy cinamate ( 3 ; rt = 9 . 7 min ), isorhamnetin ( 4 ; rt = 13 . 5 min ), kinsenone ( 5 ; rt = 14 . 6 min ), p - hydroxybenzyl alcohol ( 6 ; rt = 18 . 0 min ) ( fig1 b ). quantitative determination of the content of candidate index compounds 2 ( adenosine ) and 5 ( isorhamnetin 3 - o - β - d - glucopyranoside ) in ah - sup fraction and candidate index compounds 2 ( α - amyrin trans - p - hydroxy cinamate ) and 4 ( isorhamnetin ) in ea fraction were characterized by analytical hplc . individual peak areas of the maximal uv absorbance corresponding to the candidate index compounds in the hplc profile of ah - sup and ea fraction were monitored and determined . calibration curves ( peak area vs . concentrations ) of the α - amyrin trans - p - hydroxy cinamate and isorhamnetin standards , ranging from 0 . 05 to 1 mg / ml , revealed a good linearity and r 2 values (& gt ; 0 . 98 ) ( data not shown ). quantification of the candidate index compounds in ah - sup and ea fraction was performed based on the known concentration of the specific fractions used for hplc analysis , and the calculated amounts of peak areas of the candidate compounds corresponded well with the compounds revealed by our hplc profiling analysis . each gram of ah - sup fraction of a . formosanus contains 0 . 72 mg ( 0 . 072 %) and 3 . 4 mg ( 0 . 34 %) of adenosine and isorhamnetin 3 - o - β - d - glucopyranoside , respectively . ea extract of a . formosanus was found to contain 4 . 9 mg ( 0 . 49 %) and 52 . 3 mg ( 5 . 23 %) of α - amyrin trans - p - hydroxy cinamate and isorhamnetin , respectively in one gram of the extract . the spectral data of adenosine and isorhamnetin 3 - o - β - d - glucopyranoside , α - amyrin trans - p - hydroxy cinamate , and isorhamnetin are summarized as follows : colorless needle crystal ; mp 234 – 235 ° c . ; eims for c 10 h 13 n 5 o 4 found 274 . 2 ; 1 hnmr ( in d 2 o ): σ ( ppm ) 8 . 18 ( 1h , s ), 8 . 08 ( 1h , s ), 5 . 91 ( 1h , d , j = 6 . 0 hz ), 4 . 28 ( 1h , dd , j = 5 . 2 , 3 . 0 hz ), 4 . 15 ( 1h , dd , j = 5 . 2 , 3 . 0 hz ), 3 . 78 ( 1h , d , j = 1 . 5 hz ), 3 . 69 ( 1h , d , j = 1 . 5 hz ). yellow amorphous ; mp 162 ° c . ; eims for c 21 h 20 o 12 found 464 . 38 ; 1 hnmr ( in d 2 o ): σ ( ppm ) 7 . 96 ( 1h , d , j = 2 . 0 hz ), 7 . 56 ( 1h , dd , j = 8 . 0 , 2 . 1 hz ), 7 . 04 ( 1h , d , j = 7 . 8 hz ), 6 . 39 ( 1h , d , j = 2 . 0 hz ), 6 . 20 ( 1h , d , j = 2 . 0 hz ), 5 . 39 ( 1h , d , j = 7 . 0 ), 3 . 72 ( 1h , dd , j = 11 . 5 , 2 . 0 hz ), 3 . 54 ( 1h , m ), 3 . 44 ( 2h , m ), 3 . 23 ( 1h , m ). white solid ; mp 105 – 106 ° c . ; eims for c 29 h 56 o 3 found 572 . 43 ; 1 hnmr ( cdcl 3 ): σ ( ppm ) 7 . 62 ( d , j = 8 . 0 ), 7 . 45 ( d , j = 8 . 0 ), 6 . 85 ( d , j = 18 . 2 ), 6 . 32 ( d , j = 18 . 2 ), 5 . 22 ( m ), 4 . 70 ( m ), 1 . 17 ( s ), 1 . 01 ( s ), 1 . 00 ( s ), 0 . 96 ( s ), 0 . 90 ( s ), 0 . 89 ( s ), 0 . 86 ( s ). yellow amorphous ; eims for c 16 h 12 o 7 found 312 . 6 ; 1 hnmr ( cdcl 3 ): σ ( ppm ) 7 . 96 ( d , j = 2 . 0 ), 7 . 56 ( dd , j = 8 . 0 , 2 . 1 ), 7 . 04 ( d , j = 7 . 8 ), 6 . 39 ( d , j = 2 . 0 ), 6 . 20 ( d , j = 2 . 0 ). ahinad , n ., feyes , d ., nieminen , a ., agarwal , r ., and mukhtar h . 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