surprisingly and in accordance with the present invention , there is provided in vivo evidence supporting the usefulness of the present antitumor composition . normal human fibroblasts were purchased from biopredic international ( rennes , france ). this frozen culture was obtained from a 36 - years old female abdominal surgical waste and the cells used in this work was from the 7 th to the 12 th passage of the culture . m4beu and m3dau , human melanoma cell lines , was established in the laboratory of dr . j . f . doré ( institut national de la santé et de la recherche médicale - inserm , unit 218 , lyon , france ) from metastatic biopsy specimens ( thomasset , n . et al . ; british journal of cancer , 46 , 58 - 66 ( 1982 )). murine colon carcinoma cell line ct - 26 was obtained from dr i . j . fidler ( university of texas m . d . anderson cancer center , houston ). breast cancer adenocarcinoma mcf - 7 and mda - mb - 231 , prostatic adenocarcinoma pc - 3 , ovary adenocarcinoma sk - ov - 3 , lung carcinoma a - 549 , colon adenocarcinoma dld - 1 , human cell lines and melanoma 1b16 and fibroblast l - 929 murine cell lines were purchased from american type of culture collection ( atcc ) or the european collection of cell cultures ( ecacc ; salisbury , united - kingdom ). stock cell cultures were maintained as monolayers in 75 - cm 2 culture flasks in glutamax ™ eagle &# 39 ; s minimum essential medium with earle &# 39 ; s salts ( gibco - brl , paisley , scotland ) supplemented with 10 % foetal calf serum ( sigma ), and 5 ml of a 100 × solution of vitamins ( gibco ), 5 ml of 100 mm sodium pyruvate ( gibco ), 5 ml of 100 × non - essential amino acids ( gibco ) and 2 mg of gentamicin base ( gibco ). cells were grown in a humidified 37 ° c . incubator containing 5 % co 2 . cells were plated at a density of 5 × 10 3 cells per well in 96 - well microplates ( nunclon ™, nunc ) in 100 μl of culture medium and were allowed to adhere for 16 h before treatment . then , 100 μl of culture medium containing essential oil of pinus divaricata , essential oil of thuya occidentalis , essential oil of abies balsamea , α - humulene ( sigma - aldrich ); isocaryophyllene ( sigma - aldrich ) were added and incubated at 37 ° c . for 48 h . all compounds were dissolved in ethanol and the final concentration of ethanol in the culture medium was maintained at 0 . 5 % ( v / v ). the effect of essential oils and compounds of oil on the proliferation of tumour cells was assessed using resazurin reduction test . resazurin was recently identified as the alamar blue dye ( o &# 39 ; brien , j . et al . ; european journal of biochemistry , 267 , 5421 - 6 , ( 2000 )). the resazurin reduction test ( rrt ) was carried out according to the protocol described here . briefly , plates were rinsed by 200 μl pbs ( 37 ° c ., gibco ) at 37 ° c . using an automatic microplate washer ( cell wash ™, labsystems , helsinki , finland ) and emptied by overturning on absorbent toweling . then , 150 μl of a 25 μg / ml solution of resazurin in rpmi 1640 without phenol red was added in each well using an automatic microvolume dispenser ( multidrop 384 ™ labsystems ). the plates were incubated 1 h at 37 ° c . in an humidified atmosphere with 5 % of co 2 for fluorescence development by living cells . fluorescence was then measured on the automated 96 - well plate reader fluoroskan ascent fl ™ ( labsystems ) using an excitation wavelength of 530 nm and an emission one of 590 nm . the fluorescence is proportional to the number of living cells in the well and ic 50 ( drug concentration required to inhibit cell proliferation by 50 %) was calculated from the curve of concentration - dependent survival percentage , defined as the fluorescence in experimental wells as a percentage of that in control wells , with blank values subtracted . b612f1 male mice , 18 - 20 g , were obtained from iffa credo ( lyon , france ). murine leukemia cell line , l1210 ( 1 × 10 5 ), were implanted intraperitoneally on day 0 . the treatment were administered intraperitoneally on a day - 1 , - 5 and - 9 schedule and was delivered as one injection of 0 . 2 ml / 25 g mouse body weight . each experimental group contained six animals . each agent including essential pine oil , β - caryophyllene ( fig1 b ), α - humulene ( fig1 a ) and isocaryophyllene ( γ - caryophyllene , fig1 c ) were administered at 1000 mg / kg . the essentials oil of jack pine ( pinus banksiana or pinus divaricata ), eastern white cedar ( thuya occidentalis ), balsam fir ( abies balsamea ), were evaluated for their cytotoxic activity against various tumor cell lines ( table 1 ). the cell growth inhibition were measured by the fluorescence induced by the metabolic transformation of resazurin in resorufin . the fluorescence is proportional to living cells and allow to evaluate the concentration inhibiting 50 % of cell growth ( ic 50 ). several essential oils have demonstrated a cytotoxic activity against all tumor cell lines tested including : breast adenocarcinoma mcf - 7 and mda - mb - 231 , prostatic adenocarcinoma pc - 3 , ovary adenocarcinoma sk - ov - 3 , lung carcinoma a - 549 , colon adenocarcinoma dld - 1 and melanoma m4beu and m3dau . cytotoxic activity of sesquiterpene oil constituent was evaluated against various tumor cell lines in order to identify bioactive compounds . a sesquiterpene of the humulane family , α - humulene ( also known as α - caryophyllene , 2 , 6 , 6 , 9 - tetramethyl - 1 , 4 , 8 - cycloundecatriene ) ( fig2 ), is a compound of essential oil demonstrating a significant activity against tumor cells ( table 2 ). indeed , α - humulene is active (& lt ; 100 μmol ) against breast adenocarcinoma mcf - 7 , prostatic adenocarcinoma pc - 3 , lung carcinoma a - 549 , colon adenocarcinoma dld - 1 and melanoma m4beu ( table 2 ). cytotoxic activity of a sesquiterpene related to α - humulene ( fig3 ), isocaryophyllene , have been evaluated . isomer cis of β - caryophyllene or isocaryophyllene ( also known as γ - caryophyllene , z - caryophyllene , cis - 4 , 11 , 11 - trimethyl - 8 - methylenebicyclo [ 7 . 2 . 0 ] undec - 4 - ene ) demonstrated as α - humulene , a cytotoxic activity (& lt ; 100 μmol , table 2 ) against all tumor cells tested . moreover , isocaryophyllene shown to be slightly less active against fibroblastic normal cell in comparison to tumor cells (& gt ; 100 μmol , table 2 ). antitumor activity of essential oil of pine on murine l1210 tumor cells implanted in b6d2f1 mice was evaluated . in fig2 , the results show that the essential oil of pine increase the survival of mice . moreover , the antitumor activity of sesquiterpene including β - caryophyllene in combination with humulene and β - caryophyllene in combination with isocaryophyllene was evaluated in vivo . the results appearing in fig3 shows that β - caryophyllene with humulene as well as β - caryophyllene with isocaryophyllene resulted in an increased life span . while the invention has been described in connection with specific embodiments thereof , it will be understood that it is capable of further modifications and this application is intended to cover any variations , uses , or adaptations of the invention following , in general , the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth , and as follows in the scope of the appended claims .
the present invention relates to novel antitumor compositions , the use of sesquiterpene derivatives as antitumor agents and to methods of treatment thereof . more precisely , the present invention relates to an antitumor composition comprising a therapeutically effective quantity of at least one sesquiterpene derivative in association with a pharmaceutically acceptable carrier .