Patent Abstract:
method and pharmaceutical composition for treating anemia related symptoms . the method and formulation involve the use of one or more cucurbitacin analogs as active ingredients , for example , cucurbitacin d , which are capable of increasing hemoglobin expression , reactivating fetal or adult hemoglobin and inducing γ - globin .

Detailed Description:
for the purpose of practicing the present invention , there are rich natural resources to obtain cucurbitacin analogs . cucurbitacins belong to a group of complex compounds found in plants of the cucurbitaceae family . the bitter taste in the eggplant or cucumber , members of the cucurbitaceae family , are due to the presence of cucurbitacins . the cucurbitacin analogs commonly found in nature resources are cucurbitacins a , cucurbitacins b , cucurbitacins d , cucurbitacins e , cucurbitacins i and cucurbitacins q . cucurbitaceaes species that are rich in cucurbitacins , include trichosanthe , cucurbita pepo , cucumis sativus and citrullus ecirrhosus . other herbs such as picrorhiza kurroa of the scrophulariaceae family ( stuppner and wagner , 1989 ) and iberis umbellate of the brassicaceae family ( dinan , 1997 ) are also found rich in cucurbitacins . cucurbitacins can be found in all parts of the plant , but usually are more concentrated in the seeds and fruits . some members of cucurbitaceae with high levels of cucurbitacins have been used as traditional herbal medicines for a long time . for example , trichosanthes has been widely prescribed by herbal medical practitioners for thousands of years in china . therefore , these herbal plants provide rich resources for obtaining various cucurbitacin analogs in practicing the present invention . on the other hand , synthetic effort can be made to provide alternative routes of obtaining pharmaceutically useful cucurbitacin analogs , which may be carried out on a larger scale and more economically . the following lists the chemical structures , formula and mass of cucurbitacin analogs , including cucurbitacin a , b , c , d , e , f , h , i , j , l , o , p , q and s : cucurbitacin a formula c 32 h 46 o 9 mass 574 . 314 cucurbitacin b formula c 32 h 46 o 8 mass 558 . 3192 cucurbitacin c formula c 32 h 48 o 8 mass 560 . 3348 cucurbitacin d formula c 30 h 44 o 7 mass 516 . 3087 cucurbitacin e formula c 32 h 44 o 8 mass 556 . 3035 cucurbitacin f formula c 30 h 46 o 7 mass 518 . 3243 cucurbitacin h formula c 30 h 46 o 8 mass 534 . 3192 cucurbitacin i formula c 30 h 42 o 7 mass 514 . 293 cucurbitacin j formula c 30 h 44 o 8 mass 532 . 3036 cucurbitacin l formula c 30 h 44 o 7 mass 516 . 3087 cucurbitacin o formula c 30 h 46 o 7 mass 518 . 3243 cucurbitacin p formula c 30 h 48 o 7 mass 520 . 3399 cucurbitacin q formula c 32 h 48 o 8 mass 560 . 3348 cucurbitacin s formula c 30 h 42 o 6 mass 498 . 298 for the purpose of understanding the present invention and construing the scope of the appended patent claims , “ cucurbitacin analog ” means any compound having a backbone structure shared by the above listed examples . it is contemplated that a cucurbitacin analog shares the similar properties and pharmaceutical applications as cucurbitacin d in practicing the present invention . cucurbitacin d is an exemplified embodiment disclosed herein . it is also contemplated that a cucurbitacin analog , such as cucurbitacin d , may be obtained through total synthesis or semi - synthesis . it is further contemplated , as a person with ordinary skill in the art would understand , that a cucurbitacin analog , such as cucurbitacin d , may be made in various possible racemic , enantiomeric or diastereoisomeric isomer forms , may form salts with mineral and organic acids , and may also form derivatives such as n - oxides , prodrugs , bioisosteres . “ prodrug ” means an inactive form of the compound due to the attachment of one or more specialized protective groups used in a transient manner to alter or to eliminate undesirable properties in the parent molecule , which is metabolized or converted into the active compound inside the body ( in vivo ) once administered . “ bioisostere ” means a compound resulting from the exchange of an atom or of a group of atoms with another , broadly similar , atom or group of atoms . the objective of a bioisosteric replacement is to create a new compound with similar biological properties to the parent compound . the bioisosteric replacement may be physicochemically or topologically based . making suitable prodrugs , bioisosteres , n - oxides , pharmaceutically acceptable salts or various isomers from a known compound ( such as those disclosed in this specification ) are within the ordinary skill of the art . therefore , the present invention contemplates all suitable isomer forms , salts and derivatives of the above disclosed cucurbitacin analogs , which are all within the meaning of “ cucurbitacin analog ” for the purpose of construing the claims . the extraction of herbal plant cucurbitaceaes may be performed according to any conventional methods known in the art . the following flow - chart presents an exemplary extracting process , which can be employed to make extracts from a plant of the cucurbitaceaes family , for example , trichosanthe . according to the flow - chart , the original plant materials may be sliced , dried , or physically disintegrated prior to processing . then the extraction is preferably conducted 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 , 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 b ). this fraction b is then further concentrated by rotary evaporation and filtrated through 0 . 22 μm filter ( fraction c ). single compound responsible for the biological activity can be isolated from fraction c 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 c18 reverse phase columns are preferred . in one embodiment of the invention , the active compound is purified by reverse phase c18 hplc , using a gradient elution protocol , from 0 % to 60 % methanol . the resulting product will comprise active compound in essentially pure form . however , the purity can be improved if the hplc purification process is repeated . fig1 provides an example of the pure active compound ( referred to as “ lead compound ”) derived from hplc separation , the purity plot ( from waters ® millenmium32 software ) of which is indicated in fig2 . fig3 provides an example of the ultraviolet / visible spectrum of the pure active compound derived from the photodiode array ( pda ) detector from hplc separation module ( waters ® alliance ® 2695 / pda 996 ). to get the powder form of the compound , the collected solution should be rotary evaporated followed by frozen and lyophilized . this active compound is identified to be cucurbitacin d . cucurbitacins in a deacetylation form , a form which is naturally occurring in very trace amount or even not occurring , may be more biologically active than the parent compounds . deacetylation of acetyl group ( s ) to produce a corresponding deacetylated cucurbitacin analogue ( which is also referred to as “ deacetylated entity ”) can be effected by various chemical reactions . examples of such reactions include , but not limited to , saponifaction by potassium carbonate , sodium methoxide and magnesium methoxide in methanol , as well as reduction by lithium aluminum hydride and other reducing agents , of which potassium carbonate in methanol is preferred . a range of cucurbitacins can be subject to this deacetylation reaction . below is an example , showing cucurbitacin b undergoing deacetylation to afford cucurbitacin d : one kilogram of cucurbitacin - containing plant , trichosanthes , is crushed into small pieces and oven dried . 40 % ethanol 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 , i . e ., the clear filtrate from step a , is further purified by solid phase extraction method using c18 column . the extract is firstly loaded into the absorbent c18 and the cucurbitacins are eluted by ethanol . the cucurbitacin - containing eluent is collected in sample collection tube . the eluent is then rotary evaporated to a small volume . ethanol is added into the eluent until a clear solution obtained . the herbal extract from step b is then purified by hplc technique using c 18 column . it is firstly purified by a waters © atlantis d c 18 column ( 10 mm × 150 mm ) using 60 % acetonitrile and 40 % water as mobile phase . the fraction containing cucurbitacin b and cucurbitacin d is collected . the fraction containing cucurbitacin b from step c is then purified by waters © symmetry prep c 18 column ( 7 . 8 mm × 150 mm ) using 60 % methanol and 40 % 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 ( 7 . 8 mm × 150 mm ) using 40 % acetonitrile and 60 % water as mobile phase to obtain pure cucurbitacin b . the fraction containing cucurbitacin d from step c is then purified by waters © symmetry prep c 18 column ( 7 . 8 mm × 150 mm ) using 55 % methanol and 45 % 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 ( 7 . 8 mm × 150 mm ) using 28 % acetonitrile and 72 % water as mobile phase and the fraction containing cucurbitacin d is collected . the collected faction is finally purified by a waters © atlantis d c 18 column ( 10 mm × 150 mm ) using 60 % methanol and 40 % water as mobile phase to obtain pure cucurbitacin d . twenty kilograms of cucurbitacin - containing plant , trichosanthes , are crushed into small pieces and oven dried . 40 % ethanol 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 ° c . 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 passed through a large column packed with dm11 , an absorbent , and cucurbitacins adhered on the resins are eluted by ethanol . the eluent is concentrated and adjust to ethanol content below or equal to 40 %. it is then purified by solid phase extraction method using c18 column . the extract is loaded into the absorbent ( dm11 ) and cucurbitacins are eluted by ethanol . the cucurbitacin - containing elutent is collected in a sample collection vessel . the eluent is then rotary evaporated to a smaller volume . ethanol is added into the eluent until a clear solution obtained . the herbs extract from section b is then purified by preparative hplc technique using c18 columns . it is firstly purified by a waters © xterra rp 18 column ( 19 mm × 150 mm ) using ethanol and water as mobile phase running in gradient , where ethanol content from 35 % to 50 %. the fraction containing cucurbitacin b and cucurbitacin d is collected . the fraction containing cucurbitacin b from step c is then purified by waters © symmetry prep c 18 column ( 19 mm × 150 mm ) using 45 % ethanol and 55 % water as mobile phase and the fraction containing cucurbitacin b is collected . the collected fraction is then purified again by waters © sunfire prep obd c 18 column ( 19 mm × 150 mm ) using 40 % acetonitrile and 60 % water as mobile phase to obtain pure cucurbitacin b . the fraction containing cucurbitacin d from step c is then purified by waters © symmetry prep c 18 column ( 19 mm × 150 mm ) using 40 % ethanol and 60 % water as mobile phase and the faction containing cucurbitacin d is collected . the collected fraction is then purified again by waters © sunfire prep c 18 column ( 19 mm × 150 mmn ) using 35 % acetonitrile and 65 % water as mobile phase and the fraction containing cucurbitacin d is collected . the collected fraction is finally purified by a waters © xbridge prep c 18 column ( 10 × 150 mm ) 30 % acetonitrile and 70 % water as mobile phase to obtain pure cucurbitacin d . 15 mg of cucurbitacin b from example 1 ( d1 ) or example 2 ( d1 ) is added to a mixture containing excess amount of potassium carbonate ( aldrich ) in dry methanol . the mixture is stirred under nitrogen or argon at room temperature for 3 hours and is quenched with excess amount of saturated ammonium chloride ( aldrich ). the aqueous mixture is then extracted with ethyl acetate twice . the salt in the combined organic extract is removed by passing through a short pad of silica gel ( merck ) and eluted with ethyl acetate . the solvent is removed by rotary evaporation and the resultant crude oil is suspended in methanol for separation according to the method in example 1 ( d1 ) or example 2 ( d1 ). extracts , a , b and c were obtained by a procedure detailed in the following : ingredients , a , b and c were further isolated from extract b of example 4 by a procedure detailed in the following : for the purpose of construing the claims , the term “ extract ” includes , but is not limited to , extract a , extract b , extract c , ingredient a , ingredient b and ingredient c . the present invention completes any form of extracts made according to any methods known to people ordinarily skilled in the art . the k562 cell line is considered to be a multipotent hematopoietic stem cell because it has multiple - lineage markers . the cells could be induced to erythrocytic , monocytic , granulocytic and megakariocytic differentiation using various materials . since its discovery , k562 cell line has been extensively used as a model in studies of erytlroid differentiation and regulation of globin gene expression . k562 cells ( atcc ) were cultured in rpmi 1640 ( gibco ) supplemented with 10 % fetal calf serum ( fcs , gibco ) and 1 % psn ( gibco ). the cultures were maintained under a humidified atmosphere with 95 % air / 5 % co 2 at 37 ° c . the hemoglobin positive k562 cells can be identified by 3 , 3 ′, 5 , 5 ′- tetramethylbenzidine ( 1 mb , sigma ) staining . in brief , 10 μl cell suspension was pipetted out and mixed with 10 μl tmb working solution . five minutes later , cells were scored as positive ( blue ) or negative ( pale yellow ) at 200 × microscope . before the assay , k562 cells were scored first by tmb staining to ensure there was no self differentiation ( tmb positive cell percentage less than 1 . 0 %). 100 μl / well complete rpmi 1640 was added into a 96 - well plate , and 180 μl was added in the first well . 20 μl trichosanthes extract ( or compounds to be tested ) was added into the first well immediately . then 100 μl of the mixture from the previous well ( the first well ) was transferred to the next well ( the second well ). in the same fashion , 100 μl was transferred from the second to the third . this dilution process is continued until the last well so that each well on the plate contains half of the trichosanthes extract amount as contained in the previous well but twice as mush as the next well . 100 μl k562 cell suspension with the density of 4 × 10 4 cells / ml was then added to each well of the 96 - well plate . the final cell suspension was mixed well and cultured in the 37 ° c . co 2 incubator for six days . then the hemoglobin positive cells were recorded by tmb staining . in addition to various trichosanthes extracts ( extracts a , b , c ; ingredients a , b , c ), purified cucurbitacin d and some previously reported compounds with potential activity on hemoglobin induction were also tested in the same fashion as above described . the results are presented in table 1 and 2 , which demonstrated that cucurbitacin d isolated from trichosanthes , as well as various trichosanthes extracts ( extacts a , b , and c ) and ingredients ( ingredients a , b and c ), can significantly induce erythrocytic differentiation and hemoglobin expression . extacts a , b , and c were prepared according to example 4 and ingredients a , b and c were prepared according to example 5 . preparation of lead compound was described previously and shown in fig1 . dose - dependent effect of cucurbitacin d was studied on k562 cells which were cultured and assayed as described the previous section . briefly , k562 cell suspension was mixed with serial two - fold diluted medium containing cucurbitacin d and cultured for six days . then the hemoglobin positive cells were recorded by tmb staining . similarly , k562 cells treated by a serial of two - fold diluted hydroxyurea or cucurbitacin d were cultured for 6 consecutive days . the concentration of cucurbitacin d used was serial diluted for 12 times , ranged from 100 ng / ml to 48 . 8 pg / ml , while hydroxyurea was from 50 . 0 μg / ml to 24 . 4 ng / ml . as shown in fig4 , the effect was dose - related and no effect was detected at a dosage lower than 0 . 1 ng / ml . however , a reduced effect was observed at the high dosage , which could be due to the cytotoxic effect of cucurbitacin d . this cytotoxic effect may be useful in antitumor applications . the dose - response curve provided the direct evidence that the hemoglobin inducing activity is attributed to cucurbitacin d . the results in fig4 are normalized and indicated as mean ± sd . ( sd = standard deviation ). the mrna of α - and γ - globin in cucurbitacin d and hydroxyurea treated k562 cells were analyzed by rt - pcr to investigate the globin gene expression at molecular level . the procedures for isolating total rna were described as follows . the frozen cell lysate was thawed by incubating in a 70 ° c . water bath for 10 minutes with constant vortexing to shear the dna . the thawed cell lysate was then chilled on ice immediately . the gt lysate was homogenized by drawing it into a sterile hypodermic syringe and expelling it through a 23 - gauge needle for several times . approximately 2 ml of the gt lysate was overlayered on 1 ml 5 . 7 m cesium chloride cushion in an ultra - centrifuge polyallomer tube . to avoid cracldng , the tubes were topped up with gt solution . the centrifuge tubes were balanced by adding in gt solution ( with a deviation less than 0 . 01 g ). it was then ultra - centrifuged at 32000 rpm for 18 hours at 18 ° c . after centrifugation , the overlaying supernatant was removed by aspiration . the tubes were inverted quickly and drained for a while to remove excess supernatant and to allow the rna pellet to dry . afterwards , the bottom 0 . 5 cm of the tube containing the clear rna pellet was cut off with a new sterile scalpel blade . the rna pellet was then rinsed out and resuspended very carefully to a new eppendorf tube with a total of 400 μl ddh 2 o . an aliquot of 1 ml absolute ethanol and 45 μl of 3m sodium acetate ( ph 4 . 8 ) were added . the rna was pelleted by centrifugating at top speed for 30 minutes . the rna pellet was washed with 70 % ethanol to remove any residual salts . the vacuum dried rna pellets were resuspended with ddh 2 o to the final concentration of 0 . 1 μ / μl and followed by rt - pcr . an aliquot of 10 μl diluted rna solution was added to a new eppendorf tube and incubated in 65 ° c . water bath for 10 min to denature rna . the rna solution was chilled on ice for 2 min , followed by vortex and spin down . the following reaction agents were added respectively as following : m - mlv rt buffer ( 5 ×, 4 μl ), dtt ( 2 μl ), rnasin ( 40 u / μl , 1 μl ), oligo dt ( 0 . 1 μg / μl , 1 μl ), dntp ( 10 mm , 1 μl ), m - mlv reverse transcriptase ( 1 μl ), rna ( 0 . 1 μg / μl , 10 μl ). the mixture was incubated at 37 ° c . for 1 hour . the reverse transcripted product was dilute by 80 μl h 2 o to the final rna concentration of 0 . 01 μg / μl . the standard pcr procedure was followed using 10 μl of diluted reverse transcripted product as template dna each time . the pcr conditions for different primers were shown in the following table 3 . k562 cells treated with cucurbitacin d ( 12 . 5 ng / ml ) and hydroxyurea ( 25 . 0 μg / ml ) were cultured for six days . total mrna extracted was followed by rt - pcr analysis . as illustrated in fig5 , α - and γ - globin mrna were increased in both hydroxyurea - treated and cucurbitacin d - treated cells , but more significantly in the latter . the results indicate that the fetal hemoglobin inducting activity of hydroxyurea and cucurbitacin d are likely to be caused by an increase in transcription of globin genes . the number in fig5 represents as follows : lane 1 : ikb plus dna marker ; lane 2 : control cells , gapdh gene ; lane 3 : hydroxyurea - treated cells , gapdh gene ; lane 4 : cucurbitacin d - treated cells , gapdh gene ; lane 5 : control cells , α - globin gene ; lane 6 : hydroxyurea - treated cells , α - globin gene ; lane 7 : cucurbitacin d - treated cells , α - globin gene ; lane 8 : control cells , γ - globin gene ; lane 9 : hydroxyurea - treated cells , γ - globin gene ; lane 10 : cucurbitacin d - treated cells , γ - globin gene . k562 cells were cultured with hydroxyurea ( hu , 25 mg / ml ) and cucrbitacin d ( 12 . 5 ng / ml ) for 6 days . then the cells were conjugated with pe labeled mouse anti human fetal hemoglobin monoclonal antibodies ( becton dickinson ) followed by facs analysis . in brief , 1 × 10 6 cells were collected in a 15 ml falcon tube and centrifuged in 150 rpm for 5 min . the cell pallet was washed with pbs . then cells were fixed by gently mixing with 1 ml pbs with 4 % formaldehyde ( 37 - 40 %, merck ) for 1 hour at room temperature . the fixed cells were washed once with pbs and then were resuspended in 100 μl 0 . 01 % triton x - 100 ( merck ) in pbs / 0 . 1 % bsa ( sigma ). 20 μl monoclonal antibody was added in , followed by mixing thoroughly and incubating for 30 min . in darkness at room temperature , with frequent gentle shaking . finally the cells were washed with 1 × pbs with 0 . 1 % sodium azide once and kept in 4 ° c . until analysis . as shown in fig6 from fsc and ssc , k562 cells treated by cucurbitacin d showed no obvious cell morphology changes , comparing with negative control and untreated cells . however , there existed obvious morphology changes in hu - treated cells . this could be due to the cytotoxicity of hydroxyurea in such high dosage . expression of fetal hemoglobin was induced in both compound treated cells , but more significantly in the cucurbitacin d treated cells . the results also provide evidence that cucurbitacin d can positively induce not only adult hemoglobin , but also fetal hemoglobin expression . in fig6 , i is negative control ; ii is untreated ; iii is treated with 25 mg / ml hydroxyurea ; and iv is treated with 12 . 5 ng / ml cucurbitacin d . ( e ) hemoglobin expression on peripheral blood monocytes ( pbmc ) derived human erythroid progenitor cells treated by different compounds peripheral blood was freshly phlebotomized and kept in heparinized tubes . pbmc were immediately isolated as described below . freshly drawn human peripheral blood or bone marrow or mobilized peripheral blood , not older than 8 hours , was treated with an anti - coagulant ( e . g . heparin , edta , citrate , acid citrate dextrose anticoagulant ( acd - a ) or citrate phosphate dextrose ( cpd )). the cells were diluted with 2 - 4 volumes of pbs . 35 ml of diluted cell suspension was carefully layered over 15 ml ficoll paque ® ( 1 . 077 density ) in a 50 ml conical tube and centrifuged at 400 × g for 30 - 40 minutes at 20 ° c . in a swinging - bucket rotor ( without brake ). the upper layer was aspirated , leaving the mononuclear cell layer undisturbed at the interphase . the interphase cells ( lymphocytes , monocytes , and thrombocytes ) were carefully transferred to a new 50 ml conical tube . the conical tube was filled with pbs , mixed and centrifuged at 300 × g for 10 minutes at 20 ° c . the supernatant was carefully removed completely . for removal of platelets , the cell pellet was resuspended in 50 ml of buffer and centrifuge at 200 × g for 10 - 15 minutes at 20 ° c . the supernatant was carefully removed completely . by repeating this last washing step , most of the platelets remained in the supernatant upon centrifuigation at 200 × g . alternatively , the cells suspended in pbs or medium were layer on nycoprep ™ ( 1 . 063 density ) and centrifuge for 15 minutes at 350 × g . the mononuclear cells precipitated and the platelets remained in the supernatant . the cell pellet was resuspended in an appropriate buffer . the cells were counted and preceded to two phase culture system . epo - independent phase i : isolated peripheral blood ( pb ) cultured at a density of 5 × 10 6 cells / ml in rpmi 1640 medium supplemented with 10 % fcs and 1 % psn and 10 % human bladder carcinoma cell line 5637 conditioned medium ( cm5637 ). the cm5637 were prepared as following : human bladder carcinoma cells 5637 were cultured for 10 days . the medium was collected and centrifuged at 300 × g for 10 minutes . the supernatant was filter sterilized and stored at 4 ° c . until use . the cultures were maintained under a humidified atmosphere with 95 % air / 5 % co 2 at 37 ° c . for 5 days . epodependent phase ii : the nonadherent cells were harvested and washed by pbs after 5 days culture in phase i . the nonadherent cells were cultured in freshly prepared medium for 4 days . the fresh medium composed of 30 % fcs , 1 % bsa , 1 × 10 − 5 mol / l β - mercaptoethanol , 1 . 5 mmol / l glutamine , 1 × 10 − 6 mol / l dexamethasone , and 1 u / ml rhepo in rpmi 1640 . after cultured for 4 days in phase ii , the lymphocytes were removed as described . the cells were spun down , harvested and resuspended in pbs . the medium was saved . the cells in pbs were carefully layered on a percoll ® solution ( 1 . 0585 density ) in a 50 ml conical tube and centrifuged at 1 , 000 × g for 20 minutes at room temperature . the upper layer solution , which containing the proerythroblasts , was collected and transferred to a new 50 ml conical tube . the conical tube was filled with pbs , mixed and centrifuged at 300 × g for 10 minutes at 20 ° c . the supernatant was carefully removed completely . the cells were resuspended in the saved medium supplemented with hydroxyurea ( 25 . 0 μg / ml ) or cucurbitacin d ( 12 . 5 ng / ml ) and cultured under a humidified atmosphere with 95 % air / 5 % co 2 at 37 ° c . for 7 days before further analysis . human pbmc / bm / pbsc derived progenitor cells were cultured in a density of 5 × 10 6 cells / ml in rpmi1640 medium supplemented with 30 % fetal calf serum ( fcs ), 1 % psn , 10 % cm5637 , and 1 u / ml rhepo . for assay groups , hydroxyurea ( 25 . 0 μg / ml ) or cucurbitacin d ( 12 . 5 ng / ml ) was also added in the medium . the cultures were maintained under a humidified atmosphere with 95 % air / 5 % co 2 at 37 ° c . seven days after the addition of hydroxyurea and cucurbitacin d , the cells were assayed by tmb staining as described in example 5 and the number of hemoglobin positive cells were scored . the result indicates both hu and cucurbitacin d were able to induce hemoglobin expression in normal erythroid progenitor cells . the activity of cucurbitacin d appeared to be better than hydroxyurea ( fig7 ). with the newly available anti - fetal hemoglobin monoclonal antibodies , it is convenient to detect the fetal hemoglobin containing cells ( f cells ) by confocal microscopy . fetal hemoglobin belongs to intracellular antigens which need to be detected by membrane permeablizing techniques . the fetal hemoglobin expression of human bm mononuclear cells and k562 cells treated with cucurbitacin d and hydroxyurea were analyzed by immunofluorescence confocal microscope . the bm cells were collected from a healthy donor . the mononuclear cells were immediately isolated from the bm cells as method 5 . 3 . 1 described . the cells were subsequently cultured as described in example 8 . on the other hand , k562 cells treated with cucurbitacin d ( 12 . 5 ng / ml ) and hydroxyurea ( 25 . 0 μg / ml ) were cultured for six days . the fetal hemoglobin expression in both studies was analyzed by immunofluorescence confocal microscope as follows . bm mononuclear cells / k562 cells treated with different compounds , at the initial density of 2 × 10 4 cells / ml , were cultured in 10 ml cell culture flask for 6 days . an aliquot of 1 × 10 6 cells were collected in a 15 ml falcon tube and spun down at 150 × g for 5 min . the cell pellet was washed once with 1 × pbs , and then the cells were resuspended in 50 μl pbs . an aliquot of 20 μl monoclonal antibody solution was added into the cell suspension . the mixture was incubated for 30 min in dark at 4 ° c . the cells were washed twice with 1 × pbs with 0 . 1 % sodium azide . the cells were resuspended in 0 . 2 ml 4 % formaldehyde in pbs and kept in dark at 4 ° c . before confocal microscopic analysis , the cells were mixed thoroughly by vortex . an aliquot of 20 μl cell suspension was transferred on a glass slide , with a cover slip carefully covered on the top . the result indicates cucurbitacin d could induce a higher level expression of fetal hemoglobin than hydroxyurea in bm mononuclear cells ( fig8 ) and in k562 cells ( fig9 ). in fig8 , from top row to bottom row , the images are untreated , hu - treated and cucurbitacin d - treated cells , stained by anti - fetal hemoglobin monoclonal antibodies , respectively . the left column is transmission images while the right column is immunofluorescence confocal images . once the chemical compound having a desired medical effect is identified in an herb and substantially pure preparations of the compound are obtained either by isolating the compound from natural resources such as plants or by chemical synthesis , various pharmaceutical compositions or formulations can be fabricated from partially purified extract or substantially pure compound using existing processes or future developed processes in the industry . specific processes of making pharmaceutical formulations and dosage forms ( including , but not limited to , tablet , capsule , injection , syrup ) from chemical compounds are not part of the invention and people of ordinary skill in the art of the pharmaceutical industry are capable of applying one or more processes established in the industry to the practice of the present invention . alternatively , people of ordinary skill in the art may modify the existing conventional processes to better suit the compounds of the present invention . the following information is provided for easy reference . a “ pharmaceutically acceptable carrier ” is determined in part by the particular composition being administered and in part by the particular method used to administer the composition . a wide variety of conventional carrier may be suitable for pharmaceutical compositions of the present invention and can be selected by people with ordinary skill in the art . the dose administered to a subject , in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the subject over time . this effective dosage is referred to as “ pharmaceutically effective amount .” the effective amount or dosage of an active ingredient can be determined by people skilled in the art . while there have been described and pointed out fundamental novel features of the invention as applied to a preferred embodiment thereof , it will be understood that various omissions and substitutions and changes , in the form and details of the embodiments illustrated , may be made by those skilled in the art without departing from the spirit of the invention . the invention is not limited by the embodiments described above which are presented as examples only but can be modified in various ways within the scope of protection defined by the appended patent claims . arisawa , m ., pezzuto , j . m ., kinghorn , a . d ., cordell , g . a ., farnsworth , n . r ., 1984 plant anticancer agents . xxx : cucurbitacins from ipomopsis aggregata ( polemoniaceae ). j pharm sci . 73 ( 3 ): 411 - 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