Patent Abstract:
use and application of onjisaponin b derived and isolated from radix polygalae as novel autophagy enhancer are provided . a method of preventing , treating and / or delaying the onset of neurodegenerative diseases comprising administering an effective amount of onjisaponin b is also provided .

Detailed Description:
as used herein and in the claims , “ comprising ” means including the following elements but not excluding others . in this study , novel autophagy enhancers with neuroprotective effects by evaluating their efficacy in enhancing the clearance of mutant huntingtin and α - synuclein , and in increasing of cells viability , are identified . through screening of their library of natural product extracts from the chinese medicinal herbs , inventors have demonstrated that both ethanol extract of radix polygalae and its single component , onjisaponin b , are able to induce autophagy with potential therapeutic application in neurodegeneration . the formula , molecular weight and accurate mass of onjisaponin b are summarized in table 1 below . the reference cited throughout this application is identified in square bracket as “[ xx ]” with xx referring to the number of the corresponding reference on the “ references ” list . the following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention . they should not be considered as limiting the scope of the invention , but merely as being illustrative and representative thereof . this example describes in vitro cytotoxicity of onjisaponin b in a rat adrenal pheochromocytoma cells ( pc12 ). cell culture and cytotoxicity assay : the test compound of onjisaponin b was dissolved in dmso at a final concentration of 100 mmol / l and stored at − 20 ° c . cytotoxicity was assessed using the 3 -( 4 , 5 - dimethylthiazol - 2 - yl ) - 2 , 5 - diphenyltetrazolium bromide assay as described previously [ 28 ]. pc - 12 , 4000 cells were seeded on 96 - well plates per well . after overnight pre - incubation , the cells were exposed to different concentrations of onjisaponin b ( 0 . 039 - 100 μmol / l ) for 2 days . subsequently , 10 μl of mtt reagents was added to each well and incubated at 37 ° c . for 4 hours followed by the addition of 100 μl solubilization buffer ( 10 % sds in 0 . 01 mol / l hcl ) and overnight incubation . absorbance at 585 nm was determined from each well the next day . for cell viability assay measured by crystal violet staining , pc - 12 cells were incubated in 35 mm disc followed by the addition of onjisaponin b at the indicated concentrations for 24 hours . the cells were then incubated with crystal violet for 10 minutes followed by a ddh 2 o wash . the stained cells image was captured by ccd digital camera spot rt3 ™ under the nikon eclipse 80i microscope with 4 × magnification . cell viability was quantified by dissolving the stained cells in 10 % acetic acid ( 200 μl / well ). the colorimetric reading of the solute mixture was then determined by spectrophotometer at od 560 nm the percentage of cell viability was calculated using the following formula : cell viability (%)= cells number treated / cells number dmso control × 100 . data was obtained from three independent experiments . results : there was no toxicity observed overtly in pc - 12 cells treated with onjisaponin b for 48 hours as revealed by mtt assay as shown in fig2 a . besides , no significant morphological damage was found in pc12 cells treated with onjisaponin b for 24 hours as revealed by the crystal violet assay as shown in fig2 b . conclusion : the result shows that onjisaponin b is non - toxic in rat adrenal pheochromocytoma cells ( pc - 12 ). this example describes an in vitro study to demonstrate the autophagic effect of onjisaponin b . quantification of autophagy gfp - lc3 puncta . gfp - lc3 puncta formation was quantified as described previously [ 2 ]. in brief , cells grown on coverslips in a 6 - well plate were fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides were mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif .) and examined by fluorescence microscopy . the number of gfp - positive cells with gfp - lc3 puncta formation was examined under the nikon eclipse 80i microscope . representative images were captured with ccd digital camera spot rt3 ™ ( diagnostic instruments , inc ., melville , n . y .). to quantify autophagy , the percentage of cells with punctate gfp - lc3 fluorescence was calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields was scored . detection of autophagic marker protein lc3 conversion . after drug treatments with or without autophagy inhibitor ( 10 g / ml e64d and pepstain a ), cells were harvested and lysed in ripa buffer ( cell signaling technologies inc ., beverly , mass .). the cell lysates were then resolved by sds - page . after electrophoresis , the proteins from sds - page were transferred to nitrocellulose membrane which was then blocked with 5 % non - fat dried milk for 60 minutes . the membrane was then incubated with lc3 primary antibodies ( 1 : 1000 ) in tbst overnight at 4 ° c . after that , the membrane was further incubated with hrp - conjugated secondary antibodies for 60 minutes . finally , protein bands were visualized by using the ecl western blotting detection reagents ( invitrogen , paisley , scotland , uk ). results : the positive control drug , rapamycin , demonstrated a markedly increase in gfp - lc3 puncta formation . while the other active components of radix polygalae including polygalacic acid and senegenin showed no autophagy activity in cells , onjisaponin b increased the formation of gfp - lc3 puncta formation in a dose dependent manner as revealed by fluorescent microscopy as shown in fig3 a . in addition , western blot analysis of autophagic marker lc3 also demonstrated the increase of lc3 - ii conversion upon onjisaponin b treatment as shown in fig3 b . furthermore , onjisaponin b increased the rate of lc3 - ii formation in the presence of protease inhibitors as shown in fig3 c , suggesting that onjisaponin b induces autophagic flux , rather than promotes the blockage of fusion between autophagosome and lysosome . conclusion : these data proved that onjisaponin b has the autophagic effect and suggested that onjisaponin b is a novel autophagy enhancer in radix polygalae . this example describes an in vitro study to demonstrate the autophagic effect of onjisaponin b is dependent on the presence of autophagy - related gene 7 ( atg7 ). quantification of autophagy gfp - lc3 puncta in atg7 wild type and deficient mefs . gfp - lc3 puncta formation was quantified as described previously [ 2 ]. in brief , both atg7 wild - type and deficient mouse embryonic fibroblasts ( mefs ) grown on coverslips in a 6 - well plate were treated with indicated concentrations of onjisaponin b . the cells were then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides were mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif .) and examined by fluorescence microscopy . the number of gfp - positive cells with gfp - lc3 puncta formation was examined under the nikon eclipse 80i microscope . representative images were captured with ccd digital camera spot rt3 ™ ( diagnostic instruments , inc ., melville , n . y .). to quantify for autophagy , the percentage of cells with punctate gfp - lc3 fluorescence was calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields was scored . results : onjisaponin b was found to induce gfp - lc3 puncta formation in wild type atg7 cells but not in atg7 - knockout mouse embryonic fibroblasts as shown in fig4 . conclusion : the results show that onjisaponin b works as a novel autophagy enhancer which depends on autophagy related gene , atg7 , for the induction of autophagy . this example describes an in vitro study to demonstrate the molecular mechanism of onjisaponin b in autophagy induction . detection of mtor signaling marker proteins . pc12 cells treated with indicated time and concentrations of onjisaponin b were harvested and lysed in ripa buffer ( cell signaling ). the cell lysates were then resolved by sds - page . after electrophoresis , the proteins from sds - page were transferred to nitrocellulose membrane which was then blocked with 5 % non - fat dried milk for 60 minutes . the membrane was then incubated with p - p70s6k , p70s6k , p - ampk , ampk primary antibodies ( 1 : 1000 ) in tbst overnight at 4 ° c . respectively . after that , the membrane was further incubated with hrp - conjugated secondary antibodies for 60 minutes . finally , protein bands were visualized by using the ecl western blotting detection reagents ( invitrogen ). quantification of onjisaponin b - mediated autophagy in the presence of specific inhibitors . gfp - lc3 puncta formation was quantified as described previously [ 2 ]. in brief , pc12 cells expressing gfp - lc3 were treated with onjisaponin b ( onji , 25 μm ) in the presence of ampk inhibitor ( compound c ) ( cc , 5 μm ) or 3 - methyladenine ( 3 - ma , 5 mm ) for 12 hours . the cells were then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides were mounted with fluorsave ™ mounting media ( calbiochem ) and examined by fluorescence microscopy . to quantify for autophagy , the percentage of cells with punctate gfp - lc3 fluorescence was calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields was scored . results : onjisaponin b was found to activate the phosphorylation of ampk in a time dependent manner ( as observed from columns 2 - 4 of fig5 a that show the results of the study in which protein bands of 25 μm onjisaponin b were visualized at the 8 th , 16 th and 24 th hour ) and dose dependent manner ( as observed from columns 5 - 8 of fig5 a that show the results of the study in which protein bands of onjisaponin b at concentrations of 3 μm , 6 . 25 μm , 12 . 5 μm and 25 μm were visualized at the 24 th hour ), and this activation was also accompanied by a concomitant reduction in its downstream p70s6k phosphorylation . in addition , there was a significant reduction in onjisaponin b - induced gfp - lc3 puncta formation in pc12 cells treated with the presence of ampk inhibitor ( compound c ) or the autophagy inhibitor ( 3 - methyladenine , 3 - ma ), which is a specific inhibitor of the class iii pi3k responsible for autophagy induction as shown in fig . 5 b . conclusion : the results show that onjisaponin b activates autophagy through an ampk - mtor signaling pathway . this example describes an in vitro study to demonstrate the clearance of mutant huntingtin and mutant a53t α - synuclein by onjisaponin b . removal of mutant huntingtin and mutant α - synuclein . pc 12 cells were transfected transiently with egfp - hdq74 plasmids for 24 hours using lipofectamine plus ltx reagent ( invitrogen ) according to the manufacturer &# 39 ; s protocol . the transfected cells were then treated with onjisaponin b for 24 hours . the removal of mutant huntingtin , egfp - hdq74 , was then quantitated by immunoblotting with antibody against egfp . to measure the clearance of α - synuclein in cellular model , the overexpression of mutant a53t α - synuclein was first induced by the addition of doxycycline ( 1 μg / ml ) ( sigma ) for 24 hours [ 11 ], by using the doxycycline inducible pc 12 cell line transfected with a53t α - synuclein plasmid . the expression of mutant a53t α - synuclein was then switched off by removing doxycycline from medium . cells were then incubated with onjisaponin b for a further 24 hours . the clearance of mutant α - synuclein was then measured by immunoblotting with antibody against myc tag . results : both ethanol extract of radix polygalae and onjisaponin b enhanced the clearance of overexpressed egfp - tagged mutant huntingtin with 74 cag repeats as measured by immunoblotting against egfp antibody as shown in fig6 a . on the other hand , the expression of a53t α - synuclein can be switched on by adding the chemical agent , doxycycline . upon the induction by doxycycline , whether specific compounds enhance the clearance of mutant proteins [ 11 , 29 , 30 ] can be evaluated . as shown in fig6 b , both radix polygalae ethanol extract and onjisaponin b accelerate the clearance of myc - tagged mutant a53t α - synuclein , whereas cells without onjisaponin b or ethanol extract incubation showed no removal of mutant protein after doxycycline induction . the fold change in both fig6 a and 6 b indicated the fold change of egfp - hdq 74 level . conclusion : the results show that onjisaponin b works as a useful neuroprotective agent through accelerating the clearance of mutant huntingtin and α - synuclein in vitro . this example describes an in vitro study to demonstrate that the clearance of mutant huntingtin by onjisaponin b requires autophagy induction . quantification of mutant huntingtin aggregates in pc12 , atg7 wild type and deficient mefs . in brief , both egfp - hdq74 transfected pc12 , atg7 wild - type and deficient mouse embryonic fibroblasts ( mefs ) grown on coverslips in a 6 - well plate were treated with indicated concentrations of onjisaponin b . the cells were then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides were mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif .) and examined by fluorescence microscopy . the number of cells with gfp - aggregates formation was examined under the nikon eclipse 80i microscope . representative images were captured with ccd digital camera spot rt3 ™ ( diagnostic instruments , inc ., melville , n . y .). to quantify the clearance of mutant huntingtin , the percentage of cells with gfp - aggregates was calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields was scored . detection of autophagic marker protein lc3 conversion and htt 74 mutant aggregates . after drug treatments for atg7 wild type and deficient mefs , cells were harvested and lysed in ripa buffer ( cell signaling technologies inc ., beverly , mass .). the cell lysates were then resolved by sds - page . after electrophoresis , the proteins from sds - page were transferred to nitrocellulose membrane which were then blocked with 5 % non - fat dried milk for 60 minutes . the membrane was then incubated with lc3 primary antibodies ( 1 : 1000 ) in tbst overnight at 4 ° c . after that , the membrane was further incubated with hrp - conjugated secondary antibodies for 60 minutes . finally , protein bands were visualized by using the ecl western blotting detection reagents ( invitrogen , paisley , scotland , uk ). results : immunocytochemistry analysis further confirmed that onjisaponin b enhanced the clearance of inclusions formed by egfp - hdq74 as shown in fig7 a . to further confirm that the protective effect of onjisaponin b was due to an atg7 dependent autophagic effect , wild type atg7 and atg7 - knockout mouse embryonic fibroblasts were transfected with egfp - hdq74 for fluorescent inclusions formation . results showed that onjisaponin b enhanced the clearance of egfp - hdq74 inclusions as illustrated in fig7 b ; further , as shown in fig7 c , the rate of lc3ii formation in wild type atg7 cells but not in atg7 - knockout cells suggested the compound - mediated neuroprotective effect was autophagy dependent . conclusion : the enhanced clearance of mutant huntingtin by onjisaponin b requires the induction of autophagy in cells . this example describes an in vitro study to demonstrate the reduced oligomerization of α - synuclein mediated by onjisaponin b . bimolecular fluorescence complementation ( bifc ) assay . in brief , hela cells transfected with both gfp - n terminal - α - synuclein ( gns ) and α - synuclein - gfp - c terminal ( sgc ) plasmids were incubated at 37 ° c . for 4 hours . then , the transfected cells were incubated with different concentrations of radix polygalae ethanol extract or onjisaponin b for a further 24 hours at 30 ° c . [ 31 ]. fluorescent signals upon complete gfp fluorophore reconstitution in cells were then detected by flow analysis . results : by using the bimolecular fluorescence complementation ( bifc ) assay , the oligomerization of α - synuclein in living cells [ 31 ] can be directly quantified . in brief , α - synuclein proteins fused with two different non - fluorescent gfp terminal fragments , ( i ) gfp - n terminal - α - synuclein ( gns ) or ( ii ) α - synuclein - gfp - c terminal ( sgc ) respectively were used [ 31 , 32 ]. upon oligomerization of α - synuclein , the two non - fluorescent fractions of gfp will therefore reconstitute the complete gfp fluorophore and transmit gfp fluorescent signal within cells . this signal in turn can be quantified by flow cytometry analysis . as measured by flow analysis as shown in fig8 , both radix polygalae crude extract and onjisaponin b inhibit the oligomerization of α - synuclein in hela cells transfected with gns and sgc . conclusion : from the result , the decrease of gfp fluorescent signal suggested that both radix polygalae ethanol extract and its active component onjisaponin b may play a protective role in parkinson &# 39 ; s disease through inhibiting α - synuclein oligomerization , which is a crucial step for α - synuclein aggregation . taken together , these novel findings provide evidence to further support the neuroprotective function of radix polygalae ethanol extract and onjisaponin b in cellular model . this example describes an in vitro study to demonstrate that onjisaponin b reduces toxicity in pc - 12 cells expressing either mutant huntingtin or mutant a53t α - synuclein . cell cytotoxicity assay : cytotoxicity was assessed using the 344 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide assay as described previously [ 28 ]. pc - 12 cells were transfected with or without mutant huntingtin egfp - hdq74 for 24 h . the pc - 12 cells were then seeded on 96 - well plates with 4000 cells per well and then exposed to the indicated concentrations of onjisaponin b ( 25 μmol / l ) for 2 days . subsequently , 10 μl of mtt reagents was added to each well and incubated at 37 ° c . for 4 hours , followed by the addition of 100 μl solubilization buffer ( 10 % sds in 0 . 01 mol / l hcl ) and overnight incubation . absorbance at 585 nm was determined from each well on the next day . the percentage of cell viability was calculated using the following formula : cell viability (%)= cells number treated / cells number dmso control × 100 . data was obtained from three independent experiments . flow cytometry analysis : doxycycline - inducible pc - 12 cell lines transfected with mutant a53t α - synuclein were induced with doxycycline ( 1 μg / ml ) for 24 h and the expression of transgene was then switched off by the removal of doxycycline . cells were then treated with onjisaponin b ( 50 μm ) for a further 24 h after doxycycline removal . the control group ( ctrl ) contained cells without the addition of doxycycline , while the dox group contained cells with the removal of doxycycline ( 1 μg / ml ) after 24 h of induction but without treatment of onjisaponin b . cell death was then assessed by flow cytometry using annexin v staining kit and propidium iodide staining . data from the bar chart represented the means ± s . d . of three independent experiments . ** p & lt ; 0 . 01 ; * p & lt ; 0 . 05 . flow cytometry was carried out using a facscalibur flow cytometer . data acquisition and analysis were performed with cellquest . results : with the result that onjisaponin b treatment enhanced the clearance of mutant aggregate - prone proteins , onjisaponin b was studied to see if it plays a protective role in reducing cell death induced by mutant huntingtin or mutant a53t α - synuclein expression . to study the toxicity of mutant huntingtin or mutant a53t α - synuclein in cells , pc - 12 cells transfected transiently with egfp - hdq 74 or myc - tagged mutant a53t α - synuclein were used to examine the effect of onjisaponin b on cell viability . as shown in fig9 a and 9 b , while transient expression of mutant huntingtin or mutant a53t α - synuclein lead to a decrease in cell viability , the addition of onjisaponin b reduced toxicity in pc - 12 cells expressing either mutant huntingtin ( as shown in fig9 a ) or mutant a53t α - synuclein ( as shown in fig9 b ), respectively . conclusion : in consistent with the results from the above examples that onjisaponin b increases the clearance of mutant huntingtin and mutant a53t α - synuclein and reduces oligomerization of α - synuclein , the result of this example further demonstrated the potential therapeutic role of onjisaponin b working as a neuroprotective agent , through lowering of mutant huntingtin and α - synuclein toxicity in cells . in summary , throughout these studies , onjisaponin b is proven to show no in vitro toxicity and possess autophagic effect , which is further demonstrated to be atg7 - dependent . also , the activation of autophagy by onjisaponin b is demonstrated to be through an ampk - mtor signaling pathway . onjisaponin b is further shown to be a useful neuroprotective agent through accelerating the clearance of mutant huntingtin and α - synuclein in vitro , in which such clearance requires the induction of autophagy in cells . in addition , onjisaponin b is proven to inhibit the oligomerization of α - synuclein and reduce toxicity in pc - 12 cells expressing either mutant huntingtin or mutant a53t α - synuclein . taken together , these findings provide evidence to further support the neuroprotective function of onjisaponin b and its medical application in treating neurodegenerative disease . the exemplary embodiments of the present invention are thus fully described . although the description referred to particular embodiments , it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details . hence this invention should not be construed as limited to the embodiments set forth herein . 1 . levine b , kroemer g . autophagy in the pathogenesis of disease . cell 2008 jan . 11 ; 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