| Stellera chamaejasme L.is a perennial herbaceous plant,also known as red stigma,broken grass,etc.,The dry root is its medicinal site,its bitter taste,flat,Xin,toxic,and there are by expectorant water,expectorant insecticidal effect.Modern pharmacological studies have shown that flavonoids and coumarin compounds in Stellera chamaejasme have got good biological activity and high content.However,there is no quantitative method for the simultaneous determination of flavonoids and coumarins in the current quality control of Stellera chamaejasme.Stellera chrysanthemum is toxical,but in the medication with Euphorbiaceae Lepidoptera and Euphorbia halberdia confused,in order to medication accuracy,should establish a comprehensive quality control methods.In the existing research on the fingerprints of Stellera chamaejasme,the origin of the selection is single and can not be well represented.Stellera chamaejasme L.was used in the treatment of tumor.Pharmacological studies have shown that its alcohol extract,water extract and total flavonoids all have a wide range of anti-tumor activity,but the specific role of the effective ingredients is not clear.In this study,the key components of flavonoids of Stellera chamaejasme L.antitumor were studied,and the purified flavonoid of Stellera chamaejasme L.was extracted and its fingerprint was established.The total flavonoids were applied to lung cancer A549 cells and the IC50 was calculated.The relationship between the total flavonoid fingerprint and IC50 was established by gray correlation analysis and multiple linear regression method,and the key components of anti-tumor were determined.To determine its anti-tumor material basis for the development of its anti-tumor effect of quality control methods to lay the foundation.1 Umbelliferone and Chamechromone in Stellera chamaejasme L.were determined by UPLCThe UPLC analysis was performed on Waters BEH C18 column(2.1mm×100mm,1.7 μm),using acetonitrile(A)and a 0.1% phosphoric acid solution(w/v,B)as the mobile phase at a flow rate of 0.3 mL/min.The wavelength 327 nm was chose for the umbelliferone while 297 nm was set for chamechromone.Regression analyses revealed a good linear relationship between peak and concentration(r1 = 0.9999,and r2 = 0.9998).Accuracy and precision were also within the required limits.Average recoveries were 99.20% and 99.63%,as well as RSD were 1.81%,1.65%,respectively.Content range of umbelliferone in Stellera chamaejasme L.was 0 mg·g-1-0.602mg·g-1,the content range of chamechromone in Stellera chamaejasme L.was 6.08mg·g-1-33.37mg·g-1.The method was simple,accurate and reliable,and can be used for quality control of traditional Chinese medicine of Stellera chamaejasme L.2 Establishment the fingerprint of Stellera chamaejasme L.by HPLCThe HPLC analysis was performed on Hypersil GOLDaQ-C18(250mm × 4.6mm,5μm);The mobile phase was consisted of acetonitrile(A)and 0.1% phosphate aqueous solution(B)in gradient elution: 0 ~ 7min,90% ~ 80% 17 ~ 32 min,65% ~ 60% B;32 ~ 42 min,60% ~ 50% B;42 ~ 45 min,50% ~ 40% B;45 ~ 55 min,40%.The determined wavelength was 290 nm.The flow rate was 1.0mL· min-1 and sample injection volume was 10μL.The column temperature was 25℃.Methodological verification shows that it has good precision and high specificity.All common peaks were determined by similarity evaluation and cluster analysis method.The similarity was above 0.9 between different producing areas,which indicated that the chemical composition of different producing areas was basically the same.3 Establishment the fingerprint of Stellera chamaejasme L.by UPLCThe UPLC analysis was performed on Waters BEH C18 column(2.1 × 100 mm,1.7 μm).The mobile phase was consisted of acetonitrile(A)and 0.1% phosphate aqueous solution(B)in gradient elution: 0 ~ 2min,90% ~ 80% B;Min,80% ~ 70% B;3.5 ~ 6.5min,70% ~ 60% B;6.5 ~ 8min,60% ~ 50% B;8 ~ 10 min,50% ~ 40% B;10 ~ 15 min,40%;The determined wavelength was 290 nm.The flow rate was 0.3 mL· min-1 and sample injection volume was 2 μL.The column temperature was 25℃.Methodological verification showed that it has good precision and high specificity.Using the similarity evaluation and clustering analysis,the quality of the medicinal materials was evaluated comprehensively,and 16 common peaks were determined.The similarity between the different producing areas was above 0.9,which was in accordance with the fingerprints established by the HPLC method,and the UPLC had better separated effection.4 Establishment the fingerprint of flavonoids of Stellera chamaejasme L.by UPLCThe HPLC analysis was performed on Hypersil GOLDaQ-C18(250mm × 4.6mm,5μm);The mobile phase was consisted of acetonitrile(A)and 0.1% phosphate aqueous solution(B)in gradient elution: 0-10 min,80% ~ 65% 25 min,50% B;The determined wavelength was 290 nm.The flow rate was 1.0mL· min-1 and sample injection volume was 10μL.The temperature of column was 25℃.Methodological verification showed that the precision is good and the specificity is high.Eleven common peaks were determined using the similarity evaluation system.5 The Inhibitory effection of flavonoids in Stellera chamaejasme L.on lung cancer A549 cellsMTT assay was used to reveal the inhibitory effect of flavonoids of Stellera chamaejasme L.on proliferation of A549 cells.The result showed that maximum inhibition rate was 81.76% at 48 h.The inhibition rates of flavonoids in Stellera chamaejasme L.were different because of the producing areas.6 The anti-tumor effection of flavonoids in Stellera chamaejasme L.The gray correlation sequencing between the 11 common peaks of the SCTF fingerprint and the anti-tumor IC50 was P1,P7,P6,P2,P11,P10,P5,P8,P9,P4,P3,which P2 was umbelliferone and P7 was chamechromone.The multivariate linear regression analysis of 11 common peaks and anti-tumor IC50 showed that P1,P4,P5,P7,P9,P10,P11 and antitumor effects were related,fingerprint–efficacy equation: Y =-0.591P1-2.044P4 + 0.442P5 + 0.372P7 + 2.969P9-0.996P10 + 0.560P11;P1,P4 and P10 were negatively correlated with the effect of A549 cells,while P5,P7,P9 and P11 were positively correlated with the effect of A549 cells.In this study,UPLC method was used to establish the content of umbelliferone and chamechromone for the first time.The fingerprints between HPLC and UPLC were different,and the study of medicinal materials of source is more extensive than the previous study.The method of fingerprint–efficacy was used to determine the anti-tumor effect of Stellera chamaejasme L.,which laid the foundation for the establishment of more targeted quality control method. |
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