Study On The Anti-tumor Activity, Chemical Composition And Content Determination Of The Uyghur Medicine Anaqi Roo

Background:The root of Anacyclus pyrethrum（L）DC（family of Asteraceae）,A.pyrethrum,is representative of Traditional Uygur Medicine（TUM）.With function of removing phlegm and evacuating stagnation to alleviate pain,it was widely used in clinical treatment,including vitiligo,tumor and rheumatoid arthritis.Recently,domestic academic research of A.pyrethrum mainly focused on clinical applications,but few on the material basis and pharmacological activity.From preliminary drug screening,we found that the root of A.pyrethrum had the effect of inhibiting the proliferation of tumor cells.It has been reported that the acute toxicity studies of the root of A.pyrethrum have shown that high,medium and low doses of extract have no toxic effects on mice.Thus,the root of A.pyrethrum is a potentially low toxicity,anti-tumor drug.Objective:Confirm the main effective part of the root of A.pyrethrum through comparing the anti-cancer activity of the different extraction parts.Index components that systematic separated from the root of A.pyrethrum were chose for quantitative analysis,in order to lay a foundation for future study of the root of A.pyrethrum.Method:Part 1:Anti-proliferation and pro-apoptosis effect of dichloromethane fraction from the root of A.pyrethrum to human liver cell line HepG2:The CCK-8 assay was used to study the antitumor effect of the three extracts（dichloromethane fraction,ethyl acetate fraction and n-butanol fraction）from the root of A.pyrethrum and the main bioactive part was determined through growth inhibition ratio comparison Hoechst staining and flow cytometry were used to detect the effect of active site on the apoptosis of tumor cells.Part 2:The extraction and separation of the chemical constituents from the root of A.pyrethrumMethods of silica gel,C¹⁸,Sephadex LH-20 chromatographic were applied to make a systemic chemical investigation of the root of A.pyrethrum.The structures of the separated compounds were identified by High Performance Liguid chromatography（HPLC）,mass spectrometry（MS）and nuclear magnetic resonance（NMR）.Part 3:Rapid identification of N-alkylamides in the root of A.pyrethrumFragmentation pathways and patterns of N-alkylamides were deduced and summarized by HPLC-ESI-MS/MS,and UPLC/Q-TOF-MS was used to make a further confirm.The regular mass spectrometric pattern of N-alkylamides combined with UPLC/Q-TOF-MS method was established to the rapid identification of N-alkylamides in the root of A.pyrethrum.Part 4:Quantitative analysis of the root of A.pyrethrum by HPLCAn HPLC method was used for simultaneous determination of 5 N-alkylamides that separated from the root of A.pyrethrum for the first time,including Deca-2E,4E-dienoicacid 4-hydroxyphenylethylamide,Deca-2E,4E-dienoicacid isobutylamide,Dodeca-E,4Edienoicacid 4-hydroxyphenylthylamide,Tetradeca-2E,4E,8E-trienoic acid 4-hydroxyphenyl-ethylamide,Tetradeca-2E,4E-dienoic acid 4-hydroxyphenylethylamide.The analyses was conducted on a type of LC-20A HPLC and performed on Agilent Extend C¹⁸ column（4.6 mm×250mm,5μm）with mobile phase of methanol（B）and water（A）（0-12min:30%A,12-14min:30%A～20%A,14-40min:20%A）.The flow rate was 0.8 mL·min-1,the detection wavelength was 241 nm,and the column temperature was 25℃.An HPLC method was used for determination of chlorogenic acid in the root of A.pyrethrum for the first time.HPLC analysis was actualized on an Agilent TC-C¹⁸（5 μm,4.6 mm×150 mm）column with mobile phase of acetonitrile-0.4%phosphoric acid solution（13:87,V/V）.The flow rate was 0.8 mL·min-1.Detection wavelength was 327 nm and column temperature was 40℃.Results:Part 1:CCK-8 assay showed that dichloromethane fraction exhibited the highest degree of cytotoxicity than others.The proliferation of HepG2 was inhibited by the dichloromethane fraction in a dose-dependent manner with an IC50 of 78.94μg/mL.Flow cytometry and Hoechst staining showed dichloromethane fraction can increase the apoptosis ratio of HepG2 in a dose dependent manner.Part 2:12 monomeric components were obtained from A.pyrethrum,including 7 N-alkylamides,1 coumarin,1 steroidal saponin and 3 flavonoid glycosides.They are identified as Deca-2E,4E-dienoicacid isobutylamide,Deca-2E,4E-dienoic acid 4-hydroxyphenylethylamide,Dodeca-2E,4E-dienoic acid 4-hydroxyphenylethylamide,Undeca-2E,4E-diene-8,10-diynoic acid phenylethylamide,Tetradeca-2E,4E-dienoic acid 4-hydroxyphenylethylamide,Tetradeca-2E,4E,8E-trienoic acid 4-hydroxyphenyl-ethylamide,Tetradeca-2E,4E-diene-8,10-diynoic acid isobutylamide,Isoscopoletin,Daucosterol,Quercetin-7-O-β-D-glucopyranoside,Isorhamnetin-7-O-β-D-glucopy-ranoside,Kaempferol-7-O-β-D-glucopyranoside.Among them,except for Deca-2E,4E-dienoic acid 4-hydroxyphenylethylamide,Dodeca-2E,4E-dienoic acid 4-hydroxy-phenylethylamide and Tetradeca-2E,4E-diene-8,10-diynoic acid isobutylamide,other compounds were isolated from this herbs for the first time.Tetradeca-2E,4E,8E-trienoic acid 4-hydroxyphenylethylamide was identified as a novel compound.Part 3:N-alkylamides presented the similar fragmentation pathway.5 N-alkylamides are prone to occur[M+H]⁺ ions in the positive ion mode.α-cracking in N-position would occur further that presented as losing amine,CO and H2O to form the main fragments.Based on the regular mass spectrometric pattern of N-alkylamides with UPLC/Q-TOF-MS,20 N-alkylamides were identified in the positive ionization mode.5 of them,Undeca-2E,4E-diene-8,10-diynoic acid 4-hydroxyphenylethylamide,Dodeca-2E,4E,nE-trienoic acid 4-hydroxyphenylethylamide,Tetradeca-2E,4E,nE-trienoic-8,10-diynoic acid IB A,Tetradeca-2E-diny-8,10-diynoic acid IB A,Tetradeca-2,4E,nE-trienoic acid IB A were identified as novel compounds.（2E,7Z）-N-isobutyl-2,7-tridecadiene-10,12-diynamide and（2E,6Z,8E）-N-isobutyl-2,6,8-decatrien amide were identified for the first time in this herb.Part 4:An HPLC method was established to determine 5N-alkylamides in the root of A.pyrethrum simultaneously for the first time through investigating the extraction time of extracts,detecting wavelength of HPLC and proportion of mobile phase.The results showed that Deca-2E,4E-dienoicacid 4-hydroxyphenylethylamide,Deca-2E,4E-dienoicacid isobutylamide,Dodeca-2E,4E-dienoicacid 4-hydroxyphenyl-ethylamide,Tetradeca-2E,4E,8E-trienoic acid 4-hydroxyphenylethylamide,Tetradeca-2E,4E-dienoic acid 4-hydroxyphenyl-ethylamide have good average recovery rates in the linear range of their linear relationship with the regression equation of Y=83176X-4896.8,r=0.9999;Y=205274X+143461,r=0.9999;Y=62833X-2853.5,r=0.9998;Y=9436.1X-1749,r=0.9999;Y=16982X-4020.1,r=0.9999,respectively.With excellent linearity,this method was quickly,simple and precision.It can be employed for the quality evaluation of N-alkylamides components in the root of A.pyrethrum.Besides,An HPLC method was established for determination of chlorogenic acid in the root of A.pyrethrum for the first time.To all,we established method of determination of content of the root of A.pyrethrum and further provide some references for the quality control of it.Conclusion:The dichloromethane fraction of the root of A.pyrethrum can significantly inhibit the proliferation of HepG2 and increase cell apoptosis.Through the systematic separation and analysis of the chemical constituents of dichloromethane and ethyl acetate fraction of the root of A.pyrethrum,a method for rapid identification of N-alkylamides in the root of A.pyrethrum is established on the one hand;through the selection of indicators,a method for the content determination in the root of A.pyrethrum which can fill the blank of root of A.pyrethrum medicine quality standard was established on the other hand.