Determination Of Three Active Ingredients In Rhizoma Of Anemone Altaica By CL-FIA

The traditional Chinese medicine Anemone Rhizoma?Jiujie Changpu?,is the dry rhizoma of Anemone altaica Fisch.ex C.A.Mey.It is mainly distributed in northwest China and central China.It is used clinically to treat neurasthenia,epilepsy and rheumatic joint pain.In this study,A.altaica rhizomes distributed in Shaanxi area were used as test materials,flow-injection-chemiluminescence?CL-FIA?technology was used to establish the analytical methods for the determination of three active components of A.altaica,including anemonin,oleanolic acid and esculin sesquihydrate.Through determination and comparison of parallel samples by HPLC,the feasibility analysis of the established analysis system was carried out;the main research contents and results were as following.Based on the single factor experiment,the extraction and separation process of anemonin in A.altaica was optimized by orthogonal test.The NaOH-Luminol-NaIO₄system,H₂O₂-Methylene blue-FeSO₄ system and KMnO₄-Luminol-H₂SO₄ system for detecting anemonin were constructed based on CL-FIA.It was found that the detection limits of the three chemiluminescent systems were 0.0086?g·mL^-1,0.0051?g·mL^-1 and0.0048?g·mL^-1,respectively;the yields were 0.098 mg·g^-1 and 0.097 mg·g^-1 and 0.095mg·g^-1 respectively.Comparing with the results of HPLC analysis,the three chemiluminescence detection systems had higher precision and sensitivity,lower detection limit,and can be used for the analysis and detection of anemonin.Based on the single factor experiment,the orthogonal design was used to optimize the extraction and separation process of oleanolic acid from the A.altaica.The KMnO₄-Luminol-H₂SO₄ chemiluminescence system was selected and the sample were successfully determined,the yield was 0.77 mg·g^-11 and the detection limit was 0.0486?g·mL^-1.The experimental study showed that in the established chemiluminescence system,oleanolic acid had a good linear range in the acid medium,while in the alkaline medium the system light emission was irregular,and the numerical value was unstable,which was not suitable for the detection of oleanolic acid.Compared with HPLC results,CL-FIA analysis showed a good result for oleanolic acid determination.The content of esculetin compounds in A.altaica was relatively low.CL-FIA technology was used to test the determination of esculin in two chemiluminescence systems:NaIO₄-Luminol-NaOH and KMnO₄-Luminol-H₂SO₄.The detection limits were 0.0467?g·mL^-1 and 0.0387?g·mL^-1,respectively.The results showed that there was no difference in the aniline yield between the two systems,which was 0.071 mg·g^-1.Compared with HPLC,the esculin yield was 0.079 mg·g^-1,the comparison showed that the content of esculin was low in A.altaica,and there was no significant difference between the two methods;in addition,the chemiluminescence system constructed by acidic media was more stable than the alkaline luminescence value.The comparison between the study and the references showed that the contents of the three active ingredients of anemonin,oleanolic acid,and esculin were low in A.altaica;the flow injection-chemiluminescence analysis established in this study all got satisfactory results.Different choices of the chemiluminescent agents,oxidants and chemical reaction conditions and system parameters all affected the CL-FIA results in analyzing different active ingredients in different plants resources.In addition,its sensitivity and selectivity were limited in the analysis of the mixture.Therefore,the selection of chemiluminescence system and the optimization of reaction conditions were crucial for the establishment of the method.The CL-FIA system constructed in this test had the characteristics of low cost and high sensitivity for the detection of A.altaica,which providing a new approach for quality analysis of this medicinal material or analysis and detection of such components,and it was applicable.