Patent Application: US-95480507-A

Abstract:
this invention relates to the preparation and use of anti - cancer compounds / formulation containing cucurbitacins . said formulation comprises active ingredients , particularly cucurbitacin b and cucurbitacin d , with the efficacy of anti - proliferation and inducing cellular apoptosis . said formulation owns the anticancer activity . this invention also provides a method of isolating and purifying the active ingredients in lab - scale and in industrial - scale .

Description:
as used herein , the term “ extract ( s )” denotes all possible extracts from cucurbitaceae family , for example , but not limited to , trichosanthes , cucurbita pepo , cucumis sativus and citrullus ecirrhosus , which are obtained during the sample preparation process regardless of solvent and conditions . as used herein , the term “ ingredient ( s )” denotes all possible products that are obtained during the sample purification process and contains lead compounds from herbs cucurbitaceaes . as used herein , the term “ compound ( s )” denotes all possible lead compounds , either isolated from natural products or chemically synthesized , responsible for the biomedical activity motioned in this patent . as used herein , the term “ cucurbitacins ” denotes all the chemical analogs of cucurbitacin , for example , but not limited to , cucurbitacin a , cucurbitacin b , cucurbitacin c , cucurbitacin d , cucurbitacin e , cucurbitacin f , cucurbitacin h , cucurbitacin i , cucurbitacin j , cucurbitacin l , cucurbitacin 0 , cucurbitacin p , cucurbitacin q and cucurbitacin s as shown in fig1 . as used herein , the term “ formulation ( s )” denotes all possible formulations that consist either single or combined ingredients of the traditional herbal medicine cucurbitaceae , and / or extracts derived thereof , and / or ingredients isolated thereof , and / or compound ( s ) purified thereof . as used herein , the term “ trichosanthes ” denotes any species of the trichosanthes genus . examples of such plant include , but are not limited to , trichosanthes kirilowii maxim , trichosanthes rosthornii harms , trichosanthes japonica regel . as used herein , the terms “ cucurbitaceae ” and “ trichosanthes ” also denote any constituent of the herbal plant . examples of such constituents include , but are not limited to , root , stem , leaf , flower , fruit and seed . as used herein , the term “ l ” means liters , “ ml ” means milliliters , “ μl ” means microliter , “ g ” means grams , “ mg ” means milligram , “ ng ” means nanograms , and all temperatures are in degrees celsius . as used herein , the term “ cancer ” denotes any type of malignant disease characterized by neoplastic , tumorigenic or malignant cell growth . examples of thalassemia include , but are not limited to , prostate cancer , breast cancer , lung cancer , liver cancer , colon cancer , gastric cancer , and leukemia . as used herein , the term “ non - polar ” means any organic solvent with a polarity index ( snyder 1978 ) of not greater than 2 . 0 , and preferably not greater than 1 . 6 . examples of such non - polar solvents include , but are not limited to , hexane , petroleum ether , carbon tetrachloride , and mixtures of any solvents with the specified polarity index . as used herein , the term “ polar ” means any organic solvent with a polarity index ( snyder 1978 ) of greater than 2 . 0 , and preferably greater than 4 . 0 , and generally easily miscible with water . examples of such moderately polar solvent include , but are not limited to , methanol , ethanol , acetonitrile , and mixtures of any solvents with the specified polarity index . cucurbitacins are group of complex compounds found in plants of cucurbitaceae family . they are responsible for the bitter taste in the eggplant or cucumber . the common cucurbitacins identified including cucurbitacins a , cucurbitacins b , cucurbitacins d , cucurbitacins e , cucurbitacins i and cucurbitacins q . cucurbitaceaes that are rich in cucurbitacins , include trichosanthe , cucurbita pepo , cucumis sativus and citrullus ecirrhosus . other herb like picrorhiza kurroa from the scrophulariaceae family ( stuppner and wagner , 1989 ) or iberis umbellate from the cruciferae family ( dinan , 1997 ) are also rich in cucurbitacins . cucurbitacins can be distributed among the whole plant , but usually more abundant in the seeds and fruits of the plant . some members of cucurbitaceae which are rich in cucurbitacins have been used as traditional herbal medicines for a long history . for example , trichosanthes has been widely prescribed by herbal practitioners for thousands of years in china . the earliest record of trichosanthes application was recorded by the ancient emperors huangdi and shennong 5000 years ago in “ shennong ben cao jing ”, which is the herbal medicine handbook marking the classical pharmacopoeia of the heavenly husbandman . this invention provides a method for extracting , and purifying cucurbitacins from cucurbitaceaes . accordingly , a first aspect of this invention provides a method for extracting fractions that contains cucurbitacins from the plant tissues of cucurbitaceaes , for example , but not limited to , trichosanthes . either water or organic solvents , preferably their mixture , can be used to prepare the extract . a second aspect of the invention provides a method for isolating active ingredients from herb cucurbitaceaes , for example , but not limited to , trichosanthes . fractionally isolated ingredients can be prepared according to different purification procedures . examples of such procedures include , but are not limited to , rotary evaporation , organic solvents extraction , centrifugation , solid phase extraction , chromatography and etc . referring to fig2 , the original plant materials may be sliced , dried , or physically disintegrated prior to processing . the extraction of herbal plant cucurbitaceaes , for example , but not limited to trichosanthes , may be obtained by any method known in the art , but preferably obtained by soaking the dried plant tissues in water or polar organic solvents or their mixture at any ratio . such mixture should be enclosed and incubated at a certain temperature , which is usually , but not limited to , ranges between the room temperature and boiling temperature of the solvent . resulting extract contains biological active ingredients and compounds in liquid phase . the liquid phase is isolated from the remaining insoluble materials by any means known in the art , but preferably by filtrating through medical gauze . remaining insoluble materials may be further removed by centrifugation . the resulting liquid ( fraction a ) is typically clear and additional filtration will be performed if necessary . the previous obtained fraction a can be optionally further concentrated into a viscous liquid phase by any means known in the art , preferably by rotary evaporation . fraction a can also be optionally extracted with a non - polar solvent to remove those essentially produced contaminants as pigments , lipids , fatty acids and waxes from aqueous phase . further purified ingredients can be obtained if fraction a is processed by subsequent separation methods . examples of such methods include , but not limited to , liquid - liquid extraction , solid phase extraction ( spe ), super filtration , super critical extraction and etc . for liquid - liquid extraction , a polar organic solvent is always provided to extract a mixture of partially purified ingredients . for spe , the column is generally eluted by a first polar organic solvent to remove the irrelative ingredients , and then eluted by a second polar organic solvent , usually with less polarity index , to wash out ingredient comprising the active compounds . finally the second elution solvent is collected ( fraction c ). this fraction c is then further concentrated by rotary evaporation and filtrated through 0 . 22 μm filter ( fraction d ). the cucurbitacins in fraction d can be isolated by further separation methods . examples of such methods include , but not limited to , thin layer chromatography ( tlc ), gas chromatography ( gc ), liquid chromatography ( lc ), and high - performance liquid chromatography ( hplc ), of which hplc is preferred . different columns can be adopted during hplc purification . examples of such columns include , but not limited to , normal phase columns , reverse phase columns , ion - exchange columns , and size - exclusion columns , of which c 18 reverse phase columns are preferred . cucurbitacins are a group of highly structurally diverse triterpenes , with a rich variety of side chain derivatives and different pharmacological activities . although the cytotoxicity of cucurbitacin b and d was known before 1800 ad , very little is known about the mechanism of the effect of cucurbitacin b and d at the cellular and molecular level , which accounts for the relatively slow advance in cucurbitacin based anti - cancer drug discovery . with more structurally diverse cucurbitacin - related compounds being isolated and characterized from natural sources , coupled with the advance of molecular pharmacology of cancer and inflammatory diseases , which allows activities to be assayed rapidly and molecular mechanisms deciphered , it can be anticipated that cucurbitacins could be used as templates for anti - inflammatory and anti - cancer drug discovery . one kilogram of cucurbitacin - containing plant , trichosanthes , is crushed into small pieces and oven dried . deionized water or polar organic solvent or their mixture , 30 - 60 % ethanol is preferred , is added into the trichosanthes for extraction in a 5 l bottle ( ratio approximately : 1 kg herb : 4 l extraction solvent ). the mixture is mixed well and incubated in a 60 ° c . ultrasonicator over night with sonication occasionally . then the insoluble substance is removed by passing the mixture through a cheese cloth . then the sedimentation is spun down and clear fitrate is collected . the extract from section a is further purified by solid phase extraction method using c 18 column . the extract is firstly loaded into the absorbent and the cucurbitacins are eluted by organic solvent ( ethanol is preferred ). the cucurbitacin - containing eluent is collected in sample collection tube . the eluent is then rotary evaporated to a small volume . an organic solvent ( ethanol is preferred ) is added into the eluent until a clear solution obtained . the herbal extract from section b is then purified by hplc technique using c 18 column . it is firstly purified by a waters © atlantis d c 18 column using acetonitrile and water as mobile phase . the fraction containing cucurbitacin b and cucurbitacin d is collected . the fraction containing cucurbitacin b from section c is then purified by waters © symmetry prep c 18 column using methanol and water as mobile phase and the fraction containing cucurbitacin b is collected . the collected fraction is then purified again by waters © symmetry prep c 18 column using acetonitrile and water as mobile phase to obtain pure cucurbitacin b . the fraction containing cucurbitacin d from section c is then purified by waters © symmetry prep c 18 column using methanol and water as mobile phase and the fraction containing cucurbitacin d is collected . the collected fraction is then purified again by waters © symmetry prep c 18 column using acetonitrile and water as mobile phase and the fraction containing cucurbitacin d is collected . the collected fraction is finally purified by a waters © atlantis d c 18 column using methanol and water as mobile phase to obtain pure cucurbitacin d . twenty kilograms of cucurbitacin - containing plant , trichosanthes , are crushed into small pieces and oven dried . deionized water or polar organic solvent or their mixture , 30 - 70 % ethanol is preferred , is added into the trichosanthes for extraction in a 100 l reaction tank ( ratio approximately : 1 kg herb : 4 l extraction solvent ). the mixture is mixed well and incubated in a 60 ˜ with constant stirring . the insoluble substance is removed by passing the mixture through a metallic mesh . then the extract is allowed to settle at room temperature for overnight and the upper clear solution is obtained . the extract is subjected to pass through resins , for example , dm11 , and cucurbitacins adhered on the resins are eluted by organic solvent ( ethanol is preferred ). the eluent is concentrated and adjust to ethanol content below or equal to 40 %. it is then purified by solid phase extraction method using c 18 column . the extract is loaded into the absorbent and cucurbitacins are eluted by organic solvent ( ethanol is preferred ). the cucurbitacin - containing eluent is collected in sample collection vessel . the eluent is then rotary evaporated to a small volume . an organic solvent ( ethanol is preferred ) is added into the eluent until a clear solution obtained . the herbs extract from section b is then purified by preparative hplc technique using c 18 columns . it is firstly purified by a waters © atlantis prep d c 18 column using ethanol and water as mobile phase . the fraction containing cucurbitacin b and cucurbitacin d is collected . the fraction containing cucurbitacin b from section c is then purified by waters © symmetry prep c 18 column using methanol and water as mobile phase and the fraction containing cucurbitacin b is collected . the collected fraction is then purified again by waters © symmetry prep c 18 column using acetonitrile and water as mobile phase to obtain pure cucurbitacin b . the fraction containing cucurbitacin d from section c is then purified by waters © symmetry prep c 18 column using methanol and water as mobile phase and the fraction containing cucurbitacin d is collected . the collected fraction is finally purified by a waters © atlantis d c 18 column using methanol and water as mobile phase to obtain pure cucurbitacin d . cucurbitacin b and cucurbitacin d produced cytostatic effect on human cancer cell lines human cancer cell lines from the 59 - ncl cancer cell line panel including leukemia , melanoma and cancers of breast , brain , colon , lung , ovary , prostate and kidney , were purchased from the national institute of cancer ( usa ). the growth inhibition ( gi 50 ) of these cell lines treated with cucurbitacin b and cucurbitacin d in various concentrations was investigated . 59 human cancer cell lines were maintained in rpmi 1640 ( invitrogen life technologies , calif ., usa ) supplemented with 5 % fetal bovine serum , 2 mm l - glutamine and 1 % penicillin / streptomycin ( invitrogen life technologies ) at 37 ° c . with 5 % co 2 . all chemicals were purchased from sigma ( st . louis , mo ., usa ) unless specified otherwise . sulforhodamine b ( srb ) assay was applied to determine the cytostatic effect of cucurbitacin b and cucurbitacin d . srb is a dye which binds to cellular proteins and will be dissolved in base . the biomass of total protein can be measured at 520 nm using a plate reader . cells were inoculated into 96 - well microtiter plates including “ time zero ” ( tz ) plates in 100 μl at cell concentrations from 5000 to 40 , 000 cells per well according to nci guideline . the cells were incubated at 37 ° c . with 5 % co 2 for 24 hours . cucurbitacin b and cucurbitacin d were added to the cells of final concentrations ranging from 0 . 488 to 4000 nm for 48 hours at 37 ° c . with 5 % co 2 . cold trichloroacetic acid ( tca ) was added at final concentration of 10 % ( w / v ) to adherent cells and 16 % w / v to suspension cells for cell fixation for at least 60 minutes at 4 ° c . the supernatant was discarded and the plates were washed in tap water for 5 times and air dried . 0 . 4 % ( w / v ) srb solution was added to stain the cells for 10 minutes at room temperature . the plates were then washed with 1 % acetic acid ( merck , darmstadt , germany ) for 5 times and air dried . bound srb was solubilized with 100 - 200 ul per well of 10 mm trizma base and absorbance was measured at a wavelength of 520 nm . the percentage of growth inhibition was calculated as follows : the growth inhibition of 50 % ( gi 50 ) was obtained from the dose response curve of percentage of inhibition against dosage . both cucurbitacin b and cucurbitacin d inhibited the growth of 59 cancer cell lines dose - dependently . different cell lines response differently to the two compounds . for cucurbitacin b , gi 50 varies from 5 . 8 to 164 nm , which is much lower than those treated with cucurbitacin d . prostate cancer is the most sensitive type of cancer when treated with cucurbitacin b ( mean gi 50 = 17 nm ). for cucurbitacin d , the gi 50 varies from 14 to 354 nm while melanoma is the most sensitive cancer type ( mean gi 50 = 60 nm ). ( fig3 and 4 ) cucurbitacin b and cucurbitacin d produced cell cycle arrest on human cancer cell lines mutation causes the cancer cells to proliferate unrestrictedly . it may result from abnormal cell cycle control . human cancer cell lines from the 59 - ncl cancer cell line panel including leukemia , melanoma and cancers of breast , brain , colon , lung , ovary , prostate and kidney were purchased from the national institute of cancer . cell lines which possess the highest or lowest sensitivity ( according to gi 50 ) in response to cucurbitacin b and cucurbitacin d were selected ( fig5 ). they were treated with cucurbitacin b and cucurbitacin d in three different concentrations according to the gi 50 to elucidate the ability to cause any changes in cell cycle . cancer cell lines were maintained in rpmi 1640 supplemented with 5 % fetal bovine serum , 2 mm l - glutamine and 1 % penicillin / streptomycin at 37 ° c . with 5 % co 2 . all chemicals were purchased from sigma unless specified otherwise . cells were inoculated into tissue culture flask at cell concentrations from 50000 to 400 , 000 cells / ml according to the ncl guideline . the cells were incubated at 37 ° c . with 5 % co 2 for 24 hours . cucurbitacin b and cucurbitacin d were added to the cells of final concentrations ranging from 6 to 350 nm ( gi 50 , ½ gi 50 and ¼ gi 51 ) of particular cell line ) for 48 hours at 37 ° c . with 5 % co 2 . cells were then harvested and fixed in 80 % cold ethanol for 30 minutes at − 20 ° c . the ethanol was removed by centrifugation . 500 μl of pi / rnase solution ( 10 μg / ml propidium iodine and 300 μg / ml rnase ) ( becton dickinson , calif ., usa ) was added to stain the cells which were incubated at room temperature for 15 min and filtered with 53 μm nylon mesh . fifteen thousands cell cycle events were collected by the facs caliber ( becton dickinson ) and the cell cycle distribution was analyzed by the modfit ™ lt software ( becton dickinson ). less g 2 / m arrest in the cell lines treated with cucurbitacin b ( 6 out of 18 ) were observed ( fig6 ) while half of cell lines ( 9 out of 18 ) treated with cucurbitacin d were arrested in g 2 / m ( fig7 ). during the 48 - hour treatment , endoreduplication ( 8 n ) was observed in most cell lines . a leukemia cell line , hl60 ( tb ) ( fig8 ), and a cns cell line , sf - 295 ( fig9 ), both displayed g 2 / m arrest and endoreduplication with cucurbitacin b and cucurbitacin d treatments , respectively . cucurbitacin b and cucurbitacin d induced apoptosis in human cancer cell lines apoptosis is the important mechanism for cell death in normal cells . in cancer cells , this mechanism fails and cells proliferate without control . human cancer cell lines from the 59 - nci cancer cell line panel including leukemia , melanoma and cancers of breast , brain , colon , lung , ovary , prostate and kidney were purchased from the national institute of cancer . cell lines which possess the highest or lowest sensitivity ( according to gi 50 ) in response to cucurbitacin b and cucurbitacin d were selected ( fig5 ). they were treated with cucurbitacin b and cucurbitacin d at three different concentrations to elucidate their ability to induce any changes in cell cycle . cancer cell lines were maintained in rpmi 1640 supplemented with 5 % fetal bovine serum , 2 mm l - glutamine and 1 % penicillin / streptomycin at 37 ° c . with 5 % co 2 . all chemicals were purchased from sigma unless specified otherwise . cells were inoculated into tissue culture flask at cell concentrations from 50000 to 400 , 000 cells / ml according to the ncl guideline . the cells were incubated at 37 ° c . with 5 % co 2 for 24 hours . cucurbitacin b and cucurbitacin d were added to the cells of final concentrations ranging from 6 to 350 nm ( gi 50 , ½ gi 50 and ¼ gi 50 of particular cell line ) for 48 hours at 37 ° c . with 5 % co 2 . cells ( 3 × 10 ) were then harvested and stained with 5 μl annexin v - fitc and 10 μl propidium iodine ( becton dickinson , calif ., usa ) for 15 minutes at room temperature . ten thousands events were collected by the facscaliber and the percentage of different cell populations were analyzed by the cellquest ™ software ( becton dickinson ). our results indicated that cucurbitacin b induced apoptosis in 9 out of 18 cell lines ( fig1 and 11 ) while cucurbitacin d induced apoptosis in 11 out of 18 cell lines ( fig1 and 13 ). these 2 compounds induced apoptosis in a dose - dependent manner . cucurbitacin b and cucurbitacin d induced apoptosis by the activation of parp cell signaling pathway — cell cycle and apoptosis cucurbitacin b and cucurbitacin d induced apoptosis in human leukemia cell lines , hl 60 and regulated cell cycle via mitogen - activated - protein kinase ( mapk ) signaling pathway . control cells , as well as cells treated with cucurbitacin b or cucurbitacin d , were harvested and collected by centrifugation . whole cell extracts were then prepared by lysing the cells using 4 % sodium dodecyl sulfate ( sds ) gel sample buffer . cell extracts were boiled for 10 min and chilled on ice , subjected to 12 % sds - polyacrylamide gel electrophoresis , and transferred to a pvdf membrane . each membrane was cut into to two pieces with one piece incubated at 4 ˜ overnight with antibodies against cell cycle signaling proteins , such as erk , phosphorlated - erk , p38 , phosphorlated - p38 , cyclin e , retinoblastoma , phosphalated - retinoblastoma and c - myc , and apoptotic protein ( parp ). β - actin was used as a control for protein loading . all antibodies were obtained from cell signaling technologies ( usa ). then membranes were incubated at 37 ˜ for 1 h with secondary antibody conjugated with peroxidase , and the signal was detected using chemiluminescence detection reagent . the relative protein level was calculated as the ratio of the optical density of the protein of interest to that of β - actin . apoptosis was found upon cucurbitacin b or cucurbitacin 1 ) incubation . it is demonstrated by the cleavage of parp , an inducer of apoptosis , when induced with cucurbitacin b or cucurbitacin d treatment ( fig1 ). parp , a polypeptide of about 118 kda , will cleave into two fragments of 89 kd and 24 kd when activated and results in the consequence of dna breakage during apoptosis . mapk signaling pathway is a downstream signaling cascade that regulates both cell cycle progression and arrest . it includes four families : the extracellular signal - regulated kinases ( erks ), the c - jun nh 3 - terminal kinases / stress activated protein kinase , the p38 mapks , and the erk5 or big mapks ( jones et al ., 2005 ). as shown in fig1 , our results demonstrated that cell cycle arrest was induced by cucurbitacin b or cucurbitacin d treatment as a consequence of erk activation , followed by cyclin e down - regulation , inhibition of retinoblastoma &# 39 ; s phosphorylation , and ultimately down - regulation of c - myc . c - myc is an onco - protein which is found to be amplified in many types of tumor , including breast , cervical and colon cancers , as well as in squamous cell carcinomas of the head and neck , myeloma , non - hodgkin &# 39 ; s lymphoma , gastric adenocarcinomas and ovarian cancer ( pelengaris et al ., 2003 ). fig1 illustrated that the signaling proteins were regulated in a dose dependent manner with statistical significance . ahmad , a ., khan , k . a ., ahmad , v . u ., qazi , s ., 1986 antibacterial activity of juliflorine isolated from prosopis juliflora . planta med 4 : 285 - 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