| Puerarin, geniposide, baicalin and naringin are the bases of the clinical commonly used Chinese herbal medicine, has been widely used in modern pharmaceutical preparations, the pharmacological activities and targets is a research hotspot in the field of traditional Chinese medicine. Clinical studies have demonstrated that puerarin has improve immunity, enhance myocardial contraction force, protect myocardial cells, reduce blood pressure, antiplatelet aggregation, fall blood sugar, fall hematic fat action such as; Geniposide has antipyretic, analgesic, sedative, cholagogue function, protect liver, anti-microbial, antithrombotic, anti-inflammatory, treatment of soft tissue injury, induced inhibition; Baicalin has bacteriostatic, diuresis, anti-inflammatory, resist allergy, spasmolysis, cholagogue, anticancer etc; Naringin has cough phlegm, anti-inflammation, antivirus, antitumor, resistance mutation, allergy, fight ulcer, analgesia, blood pressure, reduce the formation of blood clots, improve local microcirculation and nutrition, and so on.Puerarin, geniposide, baicalin, naringin four substances belong to the traditional Chinese medicine (TCM) of small molecules, traditional Chinese medicine analysis of small molecules with spectrophotometer, thin layer chromatography, capillary electrophoresis, coulomb titration method, high performance liquid chromatography (HPLC) method, the above four kinds of material analysis mainly USES the HPLC method, although this method is accurate and reliable, but when used in the detection of a large number of samples, take longer, especially in the study of the metabolism inside body and organization distribution, need to trace biological samples such as the hippocampus of mice, cerebrospinal fluid prior to more complex processing procedure, to remove the macromolecular material such as protein, making it difficult to obtain satisfactory results, and even cannot be determined.Immune analysis method is a kind of to the specificity of the antigen and antibody, reversible reaction based on the analysis method, this method before the process is very simple and even does not require any prior to processing, can be measured directly, and strong specificity, high sensitivity, convenient and quick, the analysis of low cost, safe and reliable, to trace biological samples has obvious advantages.Objective:to establish puerarin, geniposide, baicalin, HPLC method for the determination of naringin content and indirect competitive ELISA method, by comparing the HPLC method and the indirect competitive ELISA method determination of puerarin in related drugs, geniposide, baicalin, naringin content, confirm that the indirect competitive ELISA method in content detection reliability, in the process to be used for future research in biological samples of puerarin, geniposide, baicalin, naringin content analysis.Methods:puerarin, geniposide, baicalin, HPLC method for the determination of naringin content, including chromatographic conditions for the chromatographic column:puerarin Agilent ZORBAX SB-C18(4.6x150mm, including5m), mobile phase:methanol:water=5(V/V) captured, detection wavelength:250nm, the column temperature:25℃, the flow rate:1.0ml/min, sample quantity:10(including1; Geniposide chromatographic conditions for the chromatographic column:Agilent ZORBAX SB-C18(4.6x250mm, including5m), mobile phase:acetonitrile:water=5(V/V) captured, detection wavelength:238nm, the column temperature:25℃, the flow rate:1.0ml/min, sample quantity:10(including1. Baicalin chromatographic conditions for the chromatographic column:Agilent ZORBAX SB-C18(4.6x250mm, including5m), mobile phase:acetonitrile:=0were1%phosphoric acid (V/V), detection wavelength:276nm, the column temperature:25℃, the flow rate:1.0ml/min, sample quantity:10(including1. Naringin chromatographic conditions for the chromatographic column:Agilent ZORBAX SB-C18(4.6x150mm, including5m), mobile phase:acetonitrile:0.05%phosphoric acid=0(V/V), were detected wavelength:250nm, the column temperature:25℃, the flow rate:1.0ml/min, sample quantity:10(including1. Methodology for each method to investigation, the standard curve, detection range, recovery rate, precision, reproducibility, stability, and to determine the method is reliable.Establish puerarin, geniposide, baicalin, indirect competitive ELISA method for the determination of naringin content, and the method of linearity, detection range, between difference, the difference between hole and recovery.Respectively with the indirect competitive ELISA method and HPLC method to detect puerarin, geniposide, baicalin, naringin into drugs, determination of content of the two methods are compared, the results of the observation results of correlation.Results:HPLC method and the indirect competitive ELISA method methodology inspection results are good, HPLC method four substances R2value is above0.999, average recovery is above95%, precision, reproducibility, stability, RSD value is less than3%; Indirect competitive ELISA method four substances R2value is above0.9, the average recovery is above95%, between difference, the difference between hole in ELISA method acceptable range.Conclusion:the HPLC method and the indirect competitive ELISA method for the determination results are consistent, within the scope of the test show good correlation, the indirect competitive ELISA method can be used for containing the puerarin consociation sibeline, geniposide, baicalin, the content of naringin medicine or compound analysis. Indirect competitive ELISA method is credible, the detection of small molecules content for traditional Chinese medicine provides a convenient, in the process of metabolism, etc for the future study of trace puerarin in biological samples, geniposide, baicalin, naringin content analysis laid a solid foundation. |
PDF Full Text Request