Study On Analysis Of Active Ingredients Of Flavonids In Anoectochilus Roxburghii And Nucleosides In Goodyera Matsumurana By LC-MS And Fingerpring In Goodyera Matsumurana

LC-MS technology is one of the important means of quantitative and qualitative analysis of the traditional Chinese medicine chemical composition. In this thesis, the four kinds of flavonids in Anoectochilus roxburghii(A roxburghii.) and the six kinds of nucleoside in Goodyera matsumurana were analyzed by liquid chromatography-mass spectrometry(LC-MS) method. Based on the systemic chemical research of Goodyera matsumurana,the HPLC fingerprint of it was established, to provide the basis for Goodyera matsumurana overall quality control,and by using the established fingerprint,to distinguish the same families different species of Anoectochilus roxburghii and Goodyera matsumurana.The thesis contains three chapters.Chapter one: Analysis on the four kinds of flavonids substances from Anoectochilus roxburghi by LC-MS.To extract the four kinds of flavonids(isorhamnetin-3-O-neohesperidoside,isoquercitrin, quercetin and isorhamnetin) from Anoectochilus roxburghii(A roxburghii.) by Ultrasonic-Assisted Extraction,and to identify and determine the four kinds of flavonids by HPLC-ESI/MS. Chromatographic condition: Agilent TC-C18 column(4.6 mm×250 mm,5 μm).The mobile phase was composed of solvent A(0.8% acetic acid aqueous solution and 0.2% ammonia)and solvent B(methanol). Linear gradient program was performed as follows: 0-10 min,linear gradient 50%-58% B; 10.01-30 min, isocratic elution 65% B. The flow rate was 1.0 ml/min. The injection volume was 20μl. The column temperature and wavelength were set at 30°C and 368 nm, respectively.Mass Spectrum conditions:electrospray ion source(ESI),positive ion monitoring. The content of the four kinds of flavonoids isorhamnetin-3-O-neohesperidoside,isoquercitrin,quercetin and isorhamnetin in different origin of A roxburghii from Fujian and Guangxi were 0.0940mg/g and 0.0890mg/g, 0.0530mg/g and 0.0670mg/g, 0.0390mg/g and 0.0420mg/g, 0.0700mg/g and 0.0610mg/g, respectively.The method has fast and convenient characteristics. There is a difference in the content of four kind of flavonoids in different origin A. roxburghii. This is the first report that isorhamnetin-3-O-neohesperidosid coexist in A roxburghii.Chapter two: Analysis on the six kinds of nucleosides substances from Goodyera matsumurana by LC-MS.To extract the six kinds of nucleoside(cytidine,uridine,adenine, inosine, guanosine and adenosine) from Goodyera matsumurana by Ultrasonic-Assisted Extraction,and to identify and determine the six kinds of nucleoside by HPLC-ESI/MS.The chromatographic separations were performed with an Agilent TC-C18column(4.6 mm×250 mm i.d., 5 μm). The mobile phase was composed of solvent A(0.1mol/L ammonium acetate) and solvent B(methanol) in linear gradient mode, The flow rate was 1.0ml/min. The column temperature and UV wavelength were set at 30°C and 260 nm, respectively. The injection volume was 20 μl. Mass Spectrum conditions: electrospray ion source(ESI), positive ion monitoring.Cytidine, uridine, adenine, inosine, guanosine and adenosine in Goodyera matsumurana were identified.The linearity was good within the concentration range of 1.25-80μg/ml(r≥0.9994) for the six kinds of nucleoside.The average recovery of them were 92.7%-96.8% with an RSD≤1.6%. The content of the six kinds of nucleoside(cytidine,uridine,adenine, inosine, guanosine and adenosine) from Goodyera matsumurana were 0.106mg/g,0.339mg/g,0.0699 mg/g,0.0820mg/g,0.0308mg/g,0.107mg/g,respectively. The proposed method is simple, accurate and rapid. So far there is no report to indicate that Goodyera matsumurana contains these six important bioactive nucleoside compounds. It provides a scientific basis for further study clinical test and pharmacology of Goodyera matsumurana.Chapter three: Study on HPLC fingerprint of Goodyera matsumurana.Based on the systemic chemical research of Goodyera matsumurana. the HPLC fingerprint of it was established. By optimizing extraction conditions, the mobile phase systems, the detection methods of wavelength,established the HPLC fingerprint of Goodyera matsumurana in the best condition,ensure the medicinal materials HPLC fingerprint has specificity, characteristic and reproducibility.