| The object of my study is Rhizoma Pardis,known as Paris polyphylla var.chinensis?Franch.?Hara which is one of wild Chinese herbal medicine from Qin-Ba Mountain area.The samples of saponins were prepared by reflux extraction of ethanol,separation and purification.The flow injection chemiluminescence?CL-FIA?analysis technique was used to build a new analysis method to analyze and detect the saponins and Polyphyllin I monomer.The sensitivity and feasibility of CL-FIA were investigated by means of conditional and parameter optimization,methodological verification and interference test,combined with the comparison results of HPLC and UV analysis.The main research contents and experimental results are as follows.?1?The extraction technique of the Paris saponins was optimized by response surfacere method based on the single factor experiment of the saponin extraction,and the saponins were purified by macroporous adsorption resin.Under the optimal conditions,the total contents of saponins in the samples detected by HPLC or UV respectively were21.938 mg·g-1 and 22.192 mg·g-1.In addition,on the basis of extraction and purification of Paris saponins,the monomer components of the reconstituted saponin I were separated by silica gel column chromatography and analysised by HPLC showed that the content of saponin I was between 2.068 mg·g-1 and 2.098 mg·g-1 and the purity was 95.40%.The results showed that the method of purification of saponins and the separation process of saponin I monomer are feasible,which laid a foundation for our later in-depth study on CL-FIA analysis of Saponins.Besides,to determine seven kinds of steroidal saponins in 15 batches of Rhizoma Paridis samples from different sources by HPLC,and HPLC fingerprints were established.The results showed the obvious difference of saponins content of Rhizoma Paridis plants from different producing areas and variety classes.?2?The study showed that the oxidation of Luminol by H2O2 produces a weak intensity of chemiluminescence,and the Cu2+ can increased the chemiluminescence intensity,while the Polyphyllin can decreased the chemiluminescence intensity of Luminol-H2O2 system.The Polyphyllin restrain the chemiluminescence intensity of KMnO4-Luminol system obviously in an acidic medium.Based on Luminol-H2O2 and KMnO4-Luminol chemiluminescence systems,a method was established for CL-FIAanalysis Rhizoma Paridis saponin.The results showed that the linear range of Luminol-H2O2 system testing for 3 kinds of standards respectively were 0.305 ?g·mL-1 to3.050 ?g·mL-1,0.308 ?g·mL-1 to 3.080 ?g·mL-1 and 0.311 ?g·mL-1 to 3.110 ?g·mL-1,the detection limit respectively were 0.202,0.224 and 0.215 ?g·mL-1,the total contents of saponins in the samples was 21.003 mg·g-1,RSD?1.68%.To KMnO4-Luminol system,the results showed that the linear range testing for 3 kinds of standards respectively were 0.140?g·mL-1 to 2.330 ?g·mL-1,0.142 ?g·mL-1 to 2.353 ?g·m L-1 and 0.144 ?g·mL-1 to 2.376?g·mL-1,the detection limit were 0.102,0.135 and 0.125?g·m L-1 respectively,the total contents of saponins in the samples was 21.248 mg·g-1,RSD?1.78%.The results showed that instrument precision were good?RSD?2.98%,n=11?for Luminol-H2O2 system and KMnO4-Luminol system.The determination result of Polyphyllin from CL-FIA was similar to the results from UV and HPLC.The KMnO4-Luminol-H+ system was showing the wide linear detection range,low detection limit and high precision,by compared the accuracy with two systems.?3?Two kinds of chemiluminescence systems were used to determine the CL-FIA method of Polyphyllin I monomer by studying the factors such as ligands,reaction medium type,medium concentration and instrument parameters of chemiluminescence system.The feasibility of the CL-FIA detection method was established.Under the different media conditions,the Polyphyllin I can enhance the chemiluminescence intensity of Ce???-Rhodamine 6G system,while restrain the chemiluminescence intensity of Luminol-NaIO4 system.Under the optimized reaction conditions,the linear ranges of the two systems were 1.275 ?g·mL-1 to 4.250 ?g·mL-1 and 0.855 ?g·mL-1 to 4.250 ?g·mL-1respectively.The detection limits were 0.793 ?g·mL-1and 0.697 ?g·mL-1.Based on the the two systems detecting Polyphyllin I in samples,its content were 1.938 mg·g-1 and 1.956mg·g-1respectively.The results of the comparative analysis showed that the detection value of Luminol-NaIO4 system was relatively satisfactory in two chemiluminescence detection systems.However,the accuracy of CL-FIA was lower than HPLC.The results showed that there were many influencing factors in CL-FIA,and the effect of luminescent agent and oxidant was significant.The test results of the sample can be affected by different chemiluminescence system,different system setting parameters,different component concentrations and other aspects.Therefore,the selection of suitable chemiluminescence system for the determination of different medicinal materials anddifferent active ingredients of different medicinal materials is the key to the construction of CL-FIA method.Secondly,its accuracy and sensitivity are closely related to the settings and optimizing of the type,concentration and instrument parameters of the reaction medium.Through the longitudinal and horizontal comparative analysis,the results showed that the method established by flow injection-chemiluminescence for Polyphyllins analysis in this experiment was less accurate than UV or HPLC.However,the CL-FIA method showed the wider linear range,lower detection limit,faster analysis speed and lower experimental cost.Therefore,the method can be used to quality analysis of Paris polyphylla and detecting polyphyllins practically in Paris polyphylla. |
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