| 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. |
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