| Objectives:The extraction,purification and pilot scale-up of the total saponins from the above ground parts of Paris polyphylla var.yunnanensis were studied.The quality control of the total saponin extract was studied to elaborate its material basis,physical and chemical properties,identification,inspection,characteristic map and content determination.It provides a scientific basis for the further development and utilization of the total saponins of Paris polyphylla.Methods:Process research:the extraction methods of total saponins from the above ground parts of Paris polyphylla var.yunnanensis were selected by conventional methods,the single factor of the process was investigated,and the orthogonal experimental design was used to optimize the extraction process;the extraction process was enlarged to investigate the stability and applicability of the small-scale test process,and the samples were also prepared for the next purification process experiment;the purification process of macroporous adsorption resin was studied,and the tree was studied In order to verify the stability and applicability of the technology,a pilot test was carried out on the extraction and purification process to prepare a pilot sample for the quality study of the total saponins in the above ground part of Paris polyphylla var.yunnanensis;Study on chemical components:separation and structure identification of the small polar parts of the total saponins in the aboveground part of Paris polyphylla var.yunnanensis were carried out by means of silica gel column chromatography,medium pressure column chromatography and compound structure analysis;Quality research:according to the relevant technical requirements of Chinese Pharmacopoeia(2015 Edition),the quality items of the total saponins in the above ground part of Paris polyphylla var.yunnanensis were investigated in terms of properties,TLC identification,inspection,characteristic map and content determination.Results:1.Process researchthrough orthogonal optimization,small-scale test and pilot scale-up test,the preparation process of the total saponins of the above ground part of Paris polyphylla var.yunnanensis was finally determined as:60%ethanol reflux extraction for three times(14 times of the solvent),combined with the extraction solution,cooling,centrifugation,HPD-100 macroporous resin purification on the concentrated extraction solution,40%ethanol elution for 2-2.5bv,85%ethanol elution for 2.5-3bv,collection for 85%eluent,concentration and drying Dryness is what you get.The average content of total saponins was 22.82%and the average transfer rate was 89.33%;2.Study on chemical composition20 known compounds were isolated and identified from the small polar parts of total saponins by silica gel column chromatography and medium pressure column chromatography,mainly including fatty acids,lipids(compounds 1,10,11),biphenyls(compounds 17),flavonoids,glycosides(compounds 2,36),enols,acids(compounds 10,11,12),phytosterols(compounds 14),spirostanes(compounds 6,7)Steroidal saponins(compounds 19,20)and other compounds,including compounds 2,3,5-7,10,11,13,15-17 and other 11 compounds were isolated from this genus for the first time.3.Quality research(1)Thin layer identification:the silica gel thin layer identification method for the total saponins of Paris polyphylla was established with Paris polyphylla saponins ?,?,h,PA,VI and VII as the reference materials.The method was verified by several batches of small samples and three batches of medium samples.Dichloromethane methanol ethyl acetate water(2:2:4:1,lower layer)was used as the developing agent,which was developed once,taken out,dried in the air,soaked in 10%sulfuric acid ethanol solution,and added at 105? Heat until the spots are clear.(2)Water content:according to the test of water content determination method(the second method of general rule 0832 in the fourth part of Chinese Pharmacopoeia,2015 Edition),the water content of total saponins in the above ground parts of three batches of Paris were 5.48%,4.68%and 3.50%,respectively.(3)Residue on Ignition:according to the test method of residue on ignition(general principles of the fourth part of Chinese Pharmacopoeia,2015 edition,0841),the residue on ignition of three batches of total saponins in the aboveground part of Paris were 1.31%,0.86%and 0.72%,respectively.(4)Heavy metals and harmful elements:according to the test method of determination of lead,cadmium,arsenic,mercury and copper(general rule 2321,Chinese Pharmacopoeia,2015 Edition),the content of heavy metals in the total saponins of the above ground parts of three batches of Paris all meet the limit requirements.(5)Pesticide residue:according to the test of the method for the determination of pesticide residue(item 2,method 1,general principles 2341,Chinese Pharmacopoeia,2015 Edition),the residue of organochlorine pesticide meets the limit requirements of"general principles 2341,pesticide residue determination method,Chinese Pharmacopoeia,2015 edition"?(6)Resin residual solvent residual:the amount is up to the requirements.(7)HPLC method was used to establish the HPLC characteristic map of total saponins in the above ground part of Paris polyphylla var.yunnanensis.The similarity between the chromatograms of six batches of tested samples and the control characteristic chromatograms was greater than 0.99.(8)Content determination:refer to the content determination of Paris ?,?,?and ? in the first part of Chinese Pharmacopoeia(2015 Edition),increase the determination of Paris PA,carry out preliminary methodological investigation on the method,the theoretical plate number and resolution of the detected component chromatographic peak meet the requirements,with good accuracy.Conclusions:1.Through the study of extraction and purification process,the preparation process of the total saponins of the above ground part of Paris polyphylla var.yunnanensis was determined.The process was proved to be stable and feasible by many small and medium-sized tests and can be used in industrial production.2.Through the study of the main chemical components of the total saponins,the main chemical components of the total saponins of Paris polyphylla were basically clarified3.The quality control method of the total saponins in the above ground part of Paris polyphylla var.yunnanensis was established through the preliminary study on the quality of identification,characteristic map,content and characteristic map,residue of ignition and heavy metal detection. |
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