Quality Control Method Of The Rhizome Of Dryopteris Crassirhizoma

Part one:Literature reviewThe documents about the present research situation in Chinese medicine Guan Zhong were summed up and analyzed. According to studying on the related literature domestic and overseas until2012, a review on the research progress of phloroglucinol compounds’ plant origin, structure classification, pharmacological activities, synthetic routes and Dryopteris crassirhizoma’s chemical constituents, pharmacological activities and quality control research was presented.Part two:Experimentation1. Preparation of reference substance of Dryocrassine ABBATo prepare Dryocrassine ABBA, many methods has been tried, including overpressured-layer chromatography(OPLC), recrystallization, High speed counter-current chromatography (HSCCC), reverse phase silica gel column chromatography, vacuum silica gel column chromatography, silica gel column chromatography and preparative thin-layer chromatography. Finally, reference substance of Dryocrassine ABBA was gotten by preparative thin-layer chromatography, and Dryocrassine ABBP was isolated from Dryopteris crassirhizoma for the first time.2. TLC identification method of Dryopteris crassirhizomaStudying on the different type of plates, activated conditions, developing solvents, monitor conditions to improve the TLC identification method of Dryopteris crassirhizoma recording in Chinese pharmacopoeia2010edition. Further, using orthogonal experiment method to investigate the influence of temperature, relative humidity, saturating time on the TLC results. When the relative humidity was50%, the temperature was at25℃, infiltrate silica plate in pH7citric acid-disodium hydrogen phosphate buffer solution,110℃activated for30min, the developing solvent was n-hexane-chloroform-methanol(10:20:1), saturated for1h, the TLC separation for Dryopteris crassirhizoma was satisfied and with good repeatability. It has been proved by experiments that the improved method has better separation effect and it can reduce influence of environmental temperature, humidity and other factors, and remarkably save the time so that it can improve the experimental efficiency.Establishing the TLC-bioautography identification method of Dryopteris crassirhizoma, associate its antioxidative activity with quality evaluation to control its quality well.3. Research on content determination of moisture,total ash,acid-insoluble ash in Dryopteris crassirhizoma. According to the results, we suggest that the moisture content, total ash content and acid-insoluble ash content of Dryopteris crassirhizoma charcoal should be controlled no more than7%,8%, and0.6%; the acid-insoluble ash content of Dryopteris crassirhizoma pieces should be controlled no more than1%.4. Content of total dryocrassins was evaluated by colorimetryEvaluating content of total dryocrassins in12batches of Dryopteris crassirhizoma materials,12batches of Dryopteris crassirhizoma pieces, and3batches of Dryopteris crassirhizoma charcoal by colorimetry. Regression equations of Dryocrassine ABBA were y=19.1x-0.084, r=0.9998, which showed that the calibration curve was linear within the range of12.12μg/ml～48.48μg/ml for Dryocrassine ABBA. The result shows that, there were some differences among Dryopteris crassirhizoma materials and pieces, but most of them have good consistency. In addition, there were2samples’total dryocrassins content were extremely low which respectively belong to Dryopteris crassirhizoma materials and pieces, it seems like that they were counterfeits. According to the results of Dryopteris crassirhizoma charcoal, we can find that their total dryocrassins were lower than1/10of Dryopteris crassirhizoma materials and pieces, implying that most of dryocrassins in them have been destoried during processing.5. Study of HPLC fingerprint spectrumEstablishing the determination method of HPLC fingerprint spectrum related to Dryopteris crassirhizoma for the first time. And evaluating fingerprint spectrums of12batches of Dryopteris crassirhizoma materials,12batches of Dryopteris crassirhizoma pieces, and3batches of Dryopteris crassirhizoma charcoal. Then, analyzing the fingerprint spectrums by similarity calculation software, the results showed that, the similarity of11batches of Dryopteris crassirhizoma materials were ranged from0.908to0.987, there was1sample’s similarity just was0.182; the similarity of11batches of Dryopteris crassirhizoma pieces were ranged from0.936to0.992, there also was1sample’s similarity just was0.183; and the similarity of3batches of Dryopteris crassirhizoma charcoal were0.869,0.661,0.893respectively. From the result above, we can get that the method was steady and reliable.Part three:Summary and discussionTo draft the quality standard draft of Dryopteris crassirhizoma materials and pieces, and summarize the paper and carry out the Innovations as below:1. Improved the TLC identification method recording in Chinese pharmacopoeia2010edition so that we can simplify experimental procedure, save experimental time and improve experimental efficiency.2. First time to establish the TLC-bioautography identification method of Dryopteris crassirhizoma, associate its antioxidative activity with quality evaluation to control its quality well.3. To establish the HPLC fingerprint spectrum of Dryopteris crassirhizoma for the first time.