蜘蛛香环烯醚萜有效部位的纯化工艺研究

【作者】 李萍；

【导师】 王宝华； 闫兴丽；

【作者基本信息】 北京中医药大学 ， 中药制药学， 2016， 硕士

【摘要】 前期实验研究表明蜘蛛香环烯醚萜类成分对肠易激综合征有很好的治疗效果,为能够将蜘蛛香环烯醚萜有效部位作为五类新药应用开发,本文对蜘蛛香环烯醚萜有效部位的纯化工艺进行研究,分别利用大孔吸附树脂、萃取、硅胶柱层析、聚酰胺柱层析、酸水解等多种纯化方法对其进行纯化,干膏中环烯醚萜有效部位的含量以及ZXX(环戊烷-吡喃-7-甲醛,4-乙氧基甲基)和ZXY(缬草醛)的含量得到了很大程度的提高,为后续工艺研究提供原料,中试经过大孔吸附树脂、硅胶柱层析、SephadexLH-20以及重结晶等方法进行分离纯化,得到纯度大于98%的环烯醚萜类单体化合物作为对照品,为进一步监测纯化工艺流程、药理研究、临床应用提供物质基础。1.目的：分离制备蜘蛛香环烯醚萜类成分对照品ZXX和ZXY,对蜘蛛香环烯醚萜有效部位以及指标性成分ZXY和ZXX的含量进行研究,完善其质量评价体系；探索优化蜘蛛香环烯醚萜有效部位的纯化工艺,以期有效部位的纯度达到50%以上。2.方法：(1)使用乙醇回流法提取蜘蛛香药材并采用大孔吸附树脂、硅胶柱色谱、凝胶柱色谱以及重结晶相结合的方法制备蜘蛛香环烯醚萜类成分对照品ZXX和ZXY；采用核磁共振谱鉴定对照品ZXX和ZXY,利用薄层色谱法(TLC)及高效液相色谱法(HPLC)进行纯度检测；同时考察对照品的稳定性。(2)采用紫外分光光度法,建立蜘蛛香环烯醚萜有效部位的含量测定方法。(3)采用高效液相色谱法,建立同时测定蜘蛛香中两个指标性成分ZXY和ZXX的含量测定方法。(4)采用多种纯化方法(高速离心除杂、大孔吸附树脂、萃取、硅胶柱层析、聚酰胺柱层析、酸水解等)对蜘蛛香环烯醚萜有效部位进行富集,进而得到较优化的纯化工艺,使蜘蛛香环烯醚萜有效部位的纯度得到较大幅度的提高,并使两个环烯醚萜类成分ZXX和ZXY所占有效部位的比例增大。3.结果：(1)使用乙醇回流法提取蜘蛛香药材并采用硅胶柱色谱和凝胶柱色谱分离相结合的方法制备了蜘蛛香环烯醚萜类成分对照品ZXX和ZXY；其理化检识、薄层色谱分析及高效液相色谱法分析检测结果表明,所制得的两个对照品均为单一稳定化合物,符合中药质量标准用化学对照品的技术要求,可用于中药质量标准的分析检验。(2)建立了紫外分光光度法测定蜘蛛香环烯醚萜有效部位含量的方法,线性回归方程：Y=0.0472x+0.0287,R2=0.9996,线性范围为2.088-14.616μg/ml;方法学考察表明,所建立的含量测定方法结果准确,操作简便,可用于蜘蛛香药材环烯醚萜有效部位的含量测定。(3)采用高效液相色谱法建立了同时测定蜘蛛香药材2个指标性成分ZXY和ZXX含量的方法,ZXY的线性回归方程：Y=4793006.518x+5375.242,R2=0.999,ZXY进样量在0.03744～0.2246μg范围内线性关系良好,方法学考察均符合要求；ZXX线性关系回归方程：Y=5247320.055x-185.80,R2=0.999,ZXX进样量在0.0416--0.2496gg范围内线性关系良好,方法学考察均符合要求,所建立的含量测定方法操作简单、结果可靠,可用于蜘蛛香药材的质量控制。(4)采用高速离心的方法对蜘蛛香乙醇提取液进行除杂情况研究,实验考察了在3000、4500、6000、7500r/min四个不同转速条件下以及在10、20、30、60min不同时间条件下的离心情况,结果表明蜘蛛香环烯醚萜有效部位以及ZXX和ZXY的转移率、损失率与离心转速和时间均无显著性联系,考虑到节约能源与保护仪器,最终确定离心条件选择3000r/min,离心10min。(5)对蜘蛛香大孔树脂洗脱物再纯化的工艺进行研究,大孔树脂条件定为：树脂柱径高比为(1：6),上样流速为2BV/h,吸附平衡后用2BV的30%乙醇溶液除杂,5BV的90%乙醇溶液洗脱,洗脱流速为4BV/h,收集洗脱液；再次上大孔树脂,收集洗脱液。干膏中蜘蛛香环烯醚萜有效部位的含量为75.23%,比原药材提高了14.84倍；干膏中ZXY的含量从0.0303mg/g生药提高到了0.5032mg/g干膏,提高了16.61倍；干膏中ZXX的含量从0.0914mg/g生药提高到了1.5423mg/g干膏,提高了16.87倍。而蜘蛛香大孔吸附树脂洗脱液再萃取工艺,得到的干膏中环烯醚萜有效部位的含量达到了78.39%,ZXY和ZXX含量为0.4642mg/g干膏及0.7397mg/g干膏,比原药材的含量各提高了15.32倍及8.09倍,大孔树脂洗脱物再洗脱以及大孔树脂洗脱物再萃取的工艺,均能将蜘蛛香环烯醚萜成分富集,是较好的纯化方法。(6)对萃取纯化蜘蛛香环烯醚萜有效部位的工艺进行研究,通过比较石油醚、乙酸乙酯、正丁醇和环己烷等萃取剂的萃取效果,得出乙酸乙酯萃取效果最好,同时选用L9(34)正交设计表,3因素为：萃取液浓度(A)、萃取倍量(B)、萃取次数(C),每因素设3水平,最终萃取条件定为：萃取药液浓度为O.1g生药/ml,1倍量乙酸乙酯,萃取3次。验证实验结果可知：干膏中环烯醚萜有效部位的含量达到54.19%,干膏中环烯醚萜类成分ZXY的含量为0.3315mg/g,提高了10.94倍,ZXX的含量为0.9821mg/g,提高了10.75倍。(7)对硅胶柱层析纯化蜘蛛香环烯醚萜有效部位的工艺进行研究,对径高比进行考察,同时从节约试剂保护环境以及考虑分离效率的综合考虑,选择径高比(1：2)的硅胶柱进行蜘蛛香环烯醚萜有效部位的富集纯化；同时对洗脱剂进行考察,最终得到石油醚-乙酸乙酯(4：1)可将蜘蛛香环烯醚萜类成分ZXY、ZXX尽可能多的洗脱下来,可利用洗脱剂石油醚-乙酸乙酯(4：1)来富集ZXY、ZXX。(8)聚酰胺柱层析纯化蜘蛛香环烯醚萜有效部位的工艺研究,通过比较不同洗脱剂(水、10%乙醇、20%乙醇、30%乙醇、40%乙醇、90%乙醇)的洗脱效果得出洗脱溶剂水以及30%乙醇具有较好的洗脱效果,最终选择的洗脱工艺：用11BV的水洗脱,再用5BV的30%的乙醇洗脱。聚酰胺柱层析可将环烯醚萜有效部位的纯度提高到12.99%,能把黄酮类成分除去,增大了其纯度,本实验可作为纯化工艺的参考。(9)酸水解纯化蜘蛛香环烯醚萜有效部位的工艺研究,实验过程中分别采用硫酸和盐酸进行酸水解,虽然硫酸的水解效果较盐酸好,但考虑到硫酸根不易去除,最终选择用盐酸酸水解。盐酸酸水解后,总环烯醚萜类成分的含量为18.25%,ZXY的含量为2.1504mg/g,ZXX的含量为9.5919mg/g。水解之后总环烯醚萜类成分的含量是未水解的3.53倍；水解后ZXY的含量是未水解的70.97倍；水解后ZXX的含量是未水解的104.94倍,酸水解能显著提高环烯醚萜有效部位以及ZXY和ZXX的含量。4.结论：(1)制备的两个蜘蛛香对照品均符合中药质量标准分析检验用对照品的要求；所建立的质量评价方法专属性强、重复性良好,可用于蜘蛛香药材的质量控制。(2)优选的蜘蛛香环烯醚萜有效部位纯化工艺,包括大孔吸附树脂、萃取、硅胶柱层析、聚酰胺柱层析、酸解等,可以显著提高环烯醚萜有效部位以及ZXX(环戊烷-吡喃-7-甲醛,4-乙氧基甲基)和ZXY(缬草醛)的含量,为蜘蛛香药材的进一步开发利用奠定基础。更多还原

【Abstract】 Based on preliminary experimental research, it shows that Valeriana jatamansi Jones iridoid class composition have a good therapeutic effect on irritable bowel syndrome(IBS). In order to help make the effective parts of Valeriana jatamansi Jones iridoid to meet the requirements of the V class of new Chinese drugs, this paper focus on purification process of these effective parts. The purification process including using macroporous resin, extraction, silica gel, gel, polyamide and acid hydrolysis method. These purification methods make the content of iridoid effective parts, ZXX(cyclopentane-formaldehyde-7-4-ethoxymethyl) and ZXY (valerian aldehyde) greatly improved. As the result, it can provide more raw materials for the process of follow-up study. Pilot test using macroporous resin and silica gel column chromatography to make ridoid compound monomer purity greater than 98%, which provide the material basis for the further testing of purification process, pharmacological research and clinical applications.Objectives:Isolating and preparing the reference ZXX and ZXY of Valeriana jatamansi Jones. Researching the content of Valeriana jatamansi Jones iridoid effective parts and the major iridoid class composition ZXY and ZXX and improving its quality evaluation system. Exploring the optimization purification process of Valeriana jatamansi Jones iridoid effective parts, in order to make the purity of the effective part of more than 50%.Methods:(1) Using the method of ethanol reflux to extract Valeriana jatamansi Jones and applying combination of macroporous resin, silica gel column chromatography, gel column chromatography and recrystallization method to separate reference substance ZXX and ZXY from Valeriana jatamansi Jones. Using nuclear magnetic resonance spectrum to identify ZXX and ZXY, and using TLC and HPLC method to detect the purity and to survey the stability of the reference substance.(2) To establish a method for the determination of the total iridoid compounds from Valeriana jatamansi Jones by UV-VIS spectrophotometry.(3) To establish a method for the determination of the compounds of ZXX and ZXY from Valeriana jatamansi Jones by HPLC.(4) Using a variety of purification methods (high-speed centrifugal, macroporous resin, extraction, normal phase silica gel column chromatography, polyamide column chromatography, acid hydrolysis method) to enrich the iridoid effective part of Valeriana jatamansi Jones. In this way, we could get the more optimized purification method, and increase the determination of total iridoid compounds from Valeriana jatamansi Jones and the percentage of ZXX and ZXY.Results:(1) Using the method of ethanol reflux to extract Valeriana jatamansi Jones and the combination methods of silica gel column chromatography and gel column chromatography to separate Valeriana jatamansi Jones reference substance ZXX and ZXY. The detection result using spectroscopic methods, TLC, HPLC shows that the prepared two reference compounds are single, stable, in line with the technical requirements of the quality standards of Chinese medicine chemical reference substance. It can be used in Chinese medicine quality standards for analytical testing.(2) Established the content determination method of the total iridoid compounds using UV spectrophotometry. The linearity of iridoids were in good linearity within the ranges of 2.088-14.616μg·μl-1. The method is accurate, simple and could be used for the quality control of Valeriana jatamansi Jones.(3) Established the content determination method of baldrinal and 11-ethoxyviburtinal. The regression eqution were Y=4793006.518x+5375.242 (R2=0.999), Y=5247320.055x-185.80 (R2=0.999). The two compostions were in good linearity within the ranges of 37.44-224.64μg,41.6-249.6μg separately. The method is accurate, simple, rapid, and could be used for the quality control of Valeriana jatamansi Jones.(4) Studied the Valeriana jatamansi Jones ethanol extract impurity using high-speed centrifugation method. The experiences research the centrifugal results under different centrifugation speed of 3000、4500、6000、7500r/min and different lengths of 10、20、 30、60 minutes. The results show that the transfer rate, loss rate of Valeriana jatamansi Jones iridoid effective parts and ZXX, ZXY has no significant contact with centrifugal speed and time. Taking into account the energy conservation and equipment protection, the centrifugal conditions were set to 3000r/min, 10min.(5) Study on the repurify process of the macroporous resin elution. The macroporous resin condition were:resin column diameter to height ratio of 1:6, the sample flow rate 2BV/h, with 2BV 30% ethanol solution remove impurities after adsorption equilibrium,5BV 90% ethanol elution flow rated 4BV/h, collecting the eluent; upper macroporous resin again, collecting the eluent. The purity of jatamansi iridoid effective parts is 75.23%,14.84 times of the medicine materials; jatamansi original herbs ZXY content from 0.0303mg/g increased to 0.5030mg/g, a 16.60-fold increase, with the ZXX content from 0.0914mg/g increased to 1.5417mg/g, a 16.87-fold increase. As to the reextraction process of macroporous adsorption resin elution liquid, in the meanwhile, the resulting transfer iridoid effective part was 42.11%. The content of ZXY and ZXX were 0.4642mg/g, 0.7397mg/g, separately, which is 15.32 times,8.09 times than the purity of the original ingredients. Therefore, both of the upper two process can enrich the components of jatamansi iridoid.(6) Study on the extraction purification process of jatamansi iridoid effective parts. Comparing with the extraction rate of petroleum ether, ethyl acetate, n-butanol and cyclohexane, ethyl acetate obtained the best results. L9 (34) orthogonal design table was selected, three factors were:extract concentration (A), extraction times of amount(B), extraction times (C), with 3 levels for each factor. The final extraction conditions were as follows:extraction solution concentration was O.lg raw medicine/ml,1-fold amount of ethyl acetate, and extract 3 times. The verified experimental result shows that the purity iridoid effective parts reached 54.19%, the amount of iridoid Constituents ZXY is 0.3315mg/g,10.94 times than the medicine materials, and the amount of ZXX was 0.9821mg/g,10.75times than the medicine materials.(7) Study on the silica gel column chromatography process of jatamansi iridoid effective parts. Comparing different high ratio of diameter, at the same time considering to protect the environment and to save agents from the separation efficiency, we choose a high-diameter ratio of 1:2 silica gel column to enrich and purify the effective part of jatamansi iridoids. At the same time to inspect the eluent, the result shows that the ratio of petroleum ether: ethyl acetate was 4:1 can elute more ZXY and ZXX. So this paper choose this ratio to enrich ZXY and ZXX.(8) Study on the polyamide column chromatography purification process of jatamansi iridoid effective. Comparing with the elution effect of different eluant (water,10% ethanol,20% ethanol,30% ethanol,40% ethanol,90% ethanol), the result indicated that water and 30% ethanol elution has the best effect. So the finally elute process is eluted with 11BV water first and then 5BV 30% ethanol. Polyamide column chromatography can increased the purity of iridoid effective part to 12.99%, which can be used as a reference for the purification process.(9) The process results of acid hydrolysis of purified Valeriana jatamansi Jones iridoid effective part of show that, between two kinds of acid, sulfuric acid and hydrochloric acid, sulfuric acid has better effect on hydrolysis. However, sulfate was difficult to remove. As the result, hydrochloric acid was the best choose. After hydrochloric acid hydrolysis, the content of total iridoid class component was 18.25%, the content of ZXY is 2.1504mg/g, and the content of ZXX was 9.5919mg/g. Comparing the not hydrolyzed method with the hydrochloric acid hydrolysis, the content of the total content of iridoid class ingredients of the latter is 3.53 times of the fommer, and the content of ZXY of the latter is 70.97 times of the fommer,as well as the content of ZXX of the latter is 104.94 times of the fommer. Acid hydrolysis significantly increase the content of iridoid effective parts, ZXY and ZXX.Results:(1) Two reference preparation of Valeriana jatamansi Jones are in line with traditional Chinese medicine quality standards with reference analytical testing requirements. The established quality evaluation method has strong specificity, good repeatability and can be used to measure Valeriana jatamansi Jones quality control.(2) The optimized purification process of jatamansi iridoid effective part, including macroporous resin, extraction, silica gel, polyamide, acid hydrolysis, can significantly improve the contents of iridoid effective parts and ZXX (cyclopentane-pyran-7-formaldehyde,4-ethoxy methyl) and ZXY (baldrinal), which lay the foundation of the further development and utilization of Valeriana jatamansi Jones.更多还原