| Chloranthus japonicus Sieb. which belongs to the genus Chloranthus in chloranthaceae family is perennial herbaceous plants. It was produced in Heilongjiang, Liaoning, Jilin, shanxi, Gansu province of China. The whole plants and their roots have been traditionally used for hundreds of years to treat boils, dermatopathia, snake bite, and widely used in folk. The rencent years’researches are focus on biological, chemical constituents were isolated and identified,but only few studies on quality control.The research on the basis of the former and our task group studies, in order to find a more reasonable method of quality control, we attempt to establish its melhod for HPLC fingerprint analysis.The sample preparation methods were screened and the chromatographic conditions including the mobile phase, detection wave. This method was used to built the fingerprint of13batches Chloranthus japonicus Sieb. to reveal the differences in chemical composition. In this study we also use LC-MS hyphenated techniques to identify the chemical constituents. In order to provide reference of the Chloranthus japonicus Sieb. reasonably and timely recovery we detection the content of acid and coumarin substance in different parts and different harvest seasons.The results are as follows:(1)The paper establishes a method for HPLC fingerprint analysis. The sample preparation methods were screened and the chromatographic conditions were optimized in this research. The Chloranthus japonicus Sieb. preparation method is:the powdered sample was accurately weighted (1.0g) and extracted with5mL methanol under ultrasonic for30min two times, merginged extracted liquid, filter. Phenomenex C18(250mm×4.6mm,5μm) column was applied, with mixtures of water and methanol as mobile phase in a gradient mode.(B; Omin,15%;5min,25%;20min,30%;45min,40%;95min,60%).The flow rate was1.0mL/min, the temperature of column was25℃, the wavelength of detection was323nm, the recording time was95min, the injection volume was10uL(2)HPLC was used to establish fingerprint chromatograph of13batches of Chloranthus japonicus Sieb.in Heilongjiang, Liaoning, Jilin and shanxi provinces. As rosmarinic acid to a mark peak,12common peaks were confirmed. The relative retention time were more stable,the relative peak area had great differences, indicating that there were some difference between different origin Chloranthus japonicus Sieb..We used the software named "chromatographic fingerprints of TCMs similarity evaluation system2004A Edition" to analyze and evaluate the obtained chromatogram, the result was that the Chloranthus japonicus Sieb.’s fingeprint exist some differences in different regions. Cluster analysis was used to cluster the similar sample. The13batch samples were divided into2clusters through the software at last. The method was simple, stable, reliable, and reproducible, it can provide a reference for the development of Chloranthus japonicus Sieb. quality standars.(3)LC-MS was used to preliminary characterize and determine the content of fumaric acid, isofraxidin, chlorogenic acid, rosmarinic acid content. The results display that isofraxidin was mainly exist in the root of Chloranthus japonicus Sieb. and the content was higher than0.02%(the Chinese Pharmacopoeia Edition Regulation the Sarcandra glabra Nakai). The fumaric acid was mainly exist in the stems and leaves, the result was display that the content of it in April and May was higher than that in June and July. The chlorogenic acid was mainly exist in the leaves, and the content of it in April was the higher than other months. The rosmarinic acid was mainly exist in the leaves, and the content of it in June was higher than other months. The content of rosmarinic acid in Chloranthus japonicus Sieb. is higher than0.02%(the Chinese Pharmacopoeia Edition Regulation the Sarcandra glabra Nakai). This results provide basis for harvesting time and reasonable use of Chloranthus japonicus Sieb. |
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