【作者】 周小菲；

【导师】 高增平；

【作者基本信息】 北京中医药大学 ， 中药化学， 2012， 硕士

【摘要】 本论文分为三个部分第一部分文献综述本论文通过系统查阅2012年以前国内外的相关文献,对间苯三酚类成分的植物来源、结构类型、药理活性、合成途径以及绵马贯众的化学成分、药理活性、质量研究等进行了较深入的总结分析。第二部分实验研究1.东北贯众素ABBA对照品的制备先后尝试了加压薄层色谱法、重结晶法、高速逆流色谱法、反相硅胶柱分离法,减压正相硅胶柱分离法、正相硅胶柱分离法以及薄层色谱制备法等多种方法分离制备东北贯众素ABBA,最后,通过薄层色谱制备的方法制备出了东北贯众素ABBA对照品,并首次从绵马贯众中分离得到东北贯众素ABBP。2.薄层色谱鉴别研究改进了现行版药典记载的绵马贯众项下的薄层鉴别方法,对薄层板、活化条件、展开剂、预饱和时间、显色温度和时间进行了选择,并通过正交试验考察了相对湿度、温度和预饱和时间对薄层分离效果的综合影响。最后,确定改进后的薄层条件为：将枸橼酸-磷酸氢二钠缓冲液(pH7)浸润后的GF254预制板于110℃活化30min,以正已烷-氯仿-甲醇(10：20：1)为展开剂,控制相对湿度为50%,温度为25℃,薄层板置展开缸中预饱和1h,展开,取出,立即喷以0.3%的坚牢蓝BB盐稀乙醇溶液,用吹风机热风吹至斑点清晰。实验证明,改进后的TLC鉴别方法分离效果良好,且减少了实验中制板和环境温度、湿度等不稳定因素的影响,简化了实验操作步骤,大大缩短了实验时间,提高了实验效率。建立了绵马贯众的生物自显影TLC鉴别方法,使其质量评价与其抗氧化活性直接相关联,从而更好地控制绵马贯众的质量。3.检查研究对绵马贯众进行了水分、总灰分以及酸不溶性灰分的测定,为建立绵马贯众质量标准提供了参考,建议绵马贯众炭的水分限度不超过7%,总灰分含量不超过8%,酸不溶性灰分含量不超过0.6%；绵马贯众饮片的酸不溶性灰分含量不超过1%。4.总间苯三酚的含量测定利用比色法测定了12批绵马贯众药材、12批绵马贯众饮片以及3批绵马贯众炭中总间苯三酚的含量,以东北贯众素ABBA为对照品,建立回归方程y=19.1x-0.084,r=0.9998,结果显示,对照品的浓度在12.12μg/ml～48.48μg／ml范围内与吸收度值呈良好的线性关系。由测定结果可知,购自不同地区的绵马贯众药材、饮片所含总间苯三酚的量虽然存在一定差异,但绝大部分具有较好的一致性,仅各有一批药材和饮片的总间苯三酚含量极低,提示这2批样品可能为伪品。3批绵马贯众炭的含量测定结果显示,其总间苯三酚含量在药材含量的十分之一以下,说明炮制过程中大部分间苯三酚类成分已经遭到破坏。5.指纹图谱研究首次建立了绵马贯众的HPLC指纹图谱测定方法,精密度、重复性和稳定性均符合要求。测定了12批绵马贯众药材、12批绵马贯众饮片以及3批绵马贯众炭的指纹图谱,通过相似度软件分析,11批绵马贯众药材的相似度在0.908和0.987之间,均高于0.900,有l批样品相似度仅为0.182；11批绵马贯众饮片的相似度在0.936和0.992之间,均高于0.900,有1批样品相似度仅为0.183；3批绵马贯众炭的相似度分别为0.869,0.661,0.893。其中,绵马贯众药材和饮片各有一批样品与对照指纹图谱差异显著,证明本文所建立的绵马贯众HPLC指纹图谱检测方法可有效鉴别绵马贯众药材和饮片第三部分总结与讨论起草绵马贯众药材及饮片质量标准草案,并对本论文进行归纳总结,得出创新点：1.改进绵马贯众TLC鉴别方法,极大简化了实验步骤,缩短了实验时间,提高了实验效率。2.首次将TLC-生物自显影技术用于绵马贯众的鉴别,将其抗氧化活性检测与TLC鉴别完美结合。3.首次建立了绵马贯众的指纹图谱测定方法。更多还原

【Abstract】 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.更多还原