| Objective:The number of deaths from cardiovascular and cerebrovascular diseases in many years is the highest in chronic non-communicable diseases worldwide..Huangyangning tablets are mainly composed of Cyclovirobuxine D and related excipients.They have been used clinically for the treatment of cardiovascular diseases for many years..Cyclovirobuxine D is a ring of Alternane steroidal alkaloids from the boxwood of the boxwood of the genus Phyllanthus and its genus.In the"Compendium of Materia Medica",boxwood was collected as medicinal,and it was used by the people to control cardiovascular diseases.Cyclovirobuxine D as its active ingredient was included in the 2000 edition of the Chinese Pharmacopoeia and was used up to now.At present,cyclovirobuxine D is mainly found in the boxwood of the boxwood of the genus Phyllanthus,and its content is relatively low.The content of the carotenoid in the thickest stem of the lobular boxwood is 0.3%,and the extraction yield is relatively low.Since the structure of cyclovirobuxine D does not contain a conjugated group,it cannot be directly detected by the ultraviolet detector commonly used in high performance liquid chromatography.In summary,this research studied the preparation process of Cyclovirobuxine D,explored the detection method of Cyclovirobuxine D,and improved the extraction process of Cyclovirobuxine D.I hope to reduce the production cost of Cyclovirobuxine D.Methods:1.Study on HPLC method of cyclovirobuxine DThe content of cyclovirobuxine D detected by non-aqueous titration was adopted by the 2015Chinese Pharmacopoeia.This method uses conventional alkaloid detection method,glacial acetic acid dissolution,perchloric acid titration,crystal violet indication.On the one hand,the method has low sensitivity and titration end point.It was judged that there was a human operation error,and on the other hand,the content of the total alkaloid was detected.The literature reports a direct ultraviolet absorption measurement method,but since the cyclovirobuxine D belongs to the terminal ultraviolet absorption,the signal interference is large and it is difficult to be detected by the detector.Combined with the structural characteristics of cyclovirobuxine D,the content of secondary amine groups was used to determine the content by derivatization with phenyl isocyanate.2.Exploration of the preparation technology of cyclovirobuxine DComparing the yields of two conventional alkaloid extraction methods,such as acid alcohol extraction and alkalization extraction method,it is determined that the most suitable alkalization conditions are selected by alkalization extraction method and orthogonal optimization experiment,and the volume of extraction solution,extraction time,The effect of the number of extractions on the yield of baicalin,and the extraction and separation process of cyclovirobuxine D was established.Results:1.A method for the determination of Cyclovirobuxine D by high performance liquid chromatography was established.The following chromatographic conditions were as follows:Agilent 1260 high performance liquid chromatography,Elite C18 reverse high performance liquid chromatography column,81%methanol and 19%water as mobile phase,column temperature 30°C,flow rate 1 mlˇmin-1.The reagent is phenyl isocyanate,which is dissolved in chloroform dried with anhydrous sodium sulfate.After phenyl isocyanate is used as a derivatization reagent,the chloroform dried by anhydrous sodium sulfate is dissolved,and after derivatization reaction of cyclovirobuxine D,N-N?substituted urea is formed,and the detection wavelength is 242 nm.The linear relationship between the amount of substance D and the peak area of the cyclovirobuxine in the range of 40 ng to 400 ng was good,and the 12-hour stability RSD was less than2%.This method can be used for the analysis of the content of cyclovirobuxine D.2.Explored a new extraction path for the extraction of D.The following is the extraction separation method:the boxwood powder was ammoniated with 1-fold absolute ethanol and concentrated ammonia water,and then extracted with 5-fold cyclohexane after1 hour of ammoniation,and the mixture is triturated by steaming to obtain a paste.Methane transfer,chloroform transfer solution and sodium dihydrogen phosphate buffer solution are mixed,so that cyclovirobuxine D and sodium dihydrogen phosphate are dissolved in water,monohydrogen phosphate is transferred from the organic layer to the water layer,and the organic layer is discarded.Adding concentrated ammonia water to the water layer to reduce it,Cyclovirobuxine D precipitates in the form of precipitation,and then excessive purified water washes the phosphate adhering to its surface.After drying,the yield and content of Cyclovirobuxine D are calculated and determined.Conclusion:1.Under the established chromatographic conditions,the concentration of D-derivatives of cyclovirobuxine has a good linear relationship with the corresponding peak area within the detection range.The theoretical plate number,resolution,tailing factor,instrument precision,method recovery rate,and stability test all meet the testing requirements.2.The preparation technology of Cyclovirobuxine D was determined by orthogonal experiment.3.The yield and purity of Cyclovirobuxine D need to be further optimized. |
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