| Terminalia chebular Retz.is a dry mature fruit of Terminalia chebular Retz.and its variants,Terminalia chebula Retz.var.tomentella Kurt.,which is widely used in Tibetan medicine and Mongolian medicine.It is rich in chemical components,including tannins,phenols,phenylpropanoids,flavonoids and triterpenes,among which hydrolysable tannins are the most abundant.Modern pharmacology research has shown that Terminalia chebular Retz.have many pharmacological effects such as anti-oxidation,antibacterial,blood lipid regulating,anti-virus,anti-tumor,liver protection,anti-inflammatory,antitussive,analgesic,detoxification,hypoglycemic,and promotion of wound healing.The 2015 edition of the Chinese Pharmacopoeia does not regulate the quality control of any component of Terminalia chebular Retz.,and there are few reports on the method of quality control of Terminalia chebular Retz.Tannin is the main component of Terminalia chebular Retz.In recent years,research on tannins has become increasingly widespread.As the main component of Terminalia chebular Retz,more in-depth research is needed to provide data support for the improvement of Terminalia chebular Retz.This thesis establishes the extraction and purification technology of tannins fraction of Terminalia chebular Retz.,studies the quality control methods of Terminalia chebular Retz and it’s tannins fraction,and provides scientific basis for the development and utilization of Terminalia chebular Retz.The specific study is as follows:1 Study on the the extraction and purification technology of the tannins fraction from Terminalia chebular Retz.The extraction technology of tannins fraction of Terminalia chebular Retz.has been establish.The effects of different particle size on the ratio of extraction were investigated.Methods Using L9(34)orthogonal design to determine the content of tannins,gallic acid,punicalagin,methyl gallate,corilagin,1,2,3,4,6-O Pentagalloylglucose and ellagic acid as the index of evaluation,research the extraction solvent,solvent consumption,extraction time,extraction times on the ratio of extraction.According to the results of SAS analysis,the optimal extraction process of Terminalia chebular Retz.have been establish:use 10 times dosage 60%ethanol,to extract the medical material 3 time,9 hours every time.The results show that the extraction technology is reasonable and stable.The Purification technology of tannin from Terminalia chebular Retz.by macroporous resin has been establised.Determine the content and metastatic rate of tannins,gallic acid,punicalagin,methyl gallate,corilagin,1,2,3,4,6-O Pentagalloylglucose and ellagic acid as the index of evaluation to optimization the purification process of macroporous resin.The extraction concentrated to 0.2g·mL-1 and centrifuged at 4000 r·min-1 for 30 min.According to 1:5 the volume ratio of the raw material and the macroporous resin,supernatant liquid absorbed by HPD-400 macroporous resin(Ratio of diameter to height is 1:7).The absorption speed was 1mL·min-1.After the adsorption,2BV 10%ethanol was used to remove the impurity,the impurity removal speed was 0.5mL·min-1,then elute with 5BV 70%ethanol,the elution rate was 2mL·min-1.Enrichment Process is verified by 3 batches tannins extracted from Terminalia chebular Retz.The content of tannins is above 60%and the transfer rate is above 65%,which shows that the method is reproducible and stable.2 Study of methods to control the quality of Terminalia chebular Retz.and its tannins fraction.Establishment the method of determination of tannins in tannins constituent and Terminalia chebular Retz.by spectrophotometry.Linear regression examination is used for this study,and found that GA has a good linear relationship in the range of 1.08～10.08μg·mL-1.The linear regression equation is Y=102.1469X+0.0087,r=0.9982(n=6).The methodological study showed that this method had a good repeatability and reproducibility.For the first time to establish a HPLC-DAD method to determine the contents of corilagin,punicalagin A,methyl gallate,punicalagin B,1,2,3,4,6-O pentagalloylglucose ellagic acid and gallic acid,in Terminalia chebular Retz.The linear regression equation of corilagin,punicalagin A,methyl gallate,punicalagin B,1,2,3,4,6-O pentagalloylglucose ellagic acid and gallic acid are Y=2597250.9110 X+26.6861,r=0.9998.Y=2974518.9674 X+4.7036,r=0.9997;Y=2469081.4701 X-23.5474,r=0.9995;Y=2260093.964 9 X+5.4562,r=0.9993;Y=1701356.1444 X+6.3150,r=0.9998;Y=2682438.5283 X+8.1821,r=0.9998;Y=9324243.5299 X+109.4421,r=0.9994.A good linearity between peak area and the concentration was found in the range of 0.186～3.720 μg,0.046～0.552 μg,0.029～0.192μg,0.182～2.730 μg,0.069～1.388 μg,0.324～6.480 μg,0.167～1.672 μg for gallic acid,punicalagin A,methyl gallate,punicalagin B,corilagin,1,2,3,4,6-O pentagalloylglucose and ellagic acid respectively.The method ology study show that the method of HPLC to determine the contents of gallic acid,punicalagin A,methyl gallate,punicalagin B,corilagin,1,2,3,4,6-O pentagalloylglucose and ellagic acid in Terminalia billerica(Gaertn.)Roxb.was precise,valid and satisfactory,it can be a method for quality control of Terminalia chebular RetzFor the first time to establish a high performance liquid chromatography(HPLC)method to determine the contents of gallic acid,punicalagin A,methyl gallate,punicalagin B,corilagin,1,2,3,4,6-O pentagalloylglucose and ellagic acid in Tannin fraction.The method ology study shows that the method of HPLC to determine the contents of gallic acid,punicalagin A,methyl gallate,punicalagin B,corilagin,1,2,3,4,6-O pentagalloylglucose and ellagic acid in Tannin fraction was precise,valid and satisfactory,it can be a method for quality control of Tannin fraction.To identify chemical constituents and analysis of their fragmentation pathways in the extract found in Terminalia chebular Retz.The extract of Terminalia chebular Retz.were analyzed using high performance liquid chromatography coupled with high-resolution mass spectrometry,chromatographic separation was achieved on C18 reverse phase column(4.6 mm×250 mm,5μm)with the mobile phase of Methanol-water(0.2%acetic acid)and step gradient Elution,and the flow rate was 1.0 mL/min.The ESI-MS analysis was operated at the range of m/z 100-2000 in negative ionization mode.The results indicated that 62 compounds from Terminalia chebular Retz.are identified,including acids,hydrolysable tannins,triterpenoids,by the accurate mass of molecular and product ions provided by LIC-Orbitrap mass spectrometer,the element compositions analysis,standards and literatures.14 of these compounds are found from this specie for the first time.It is an effective method to provide chemical information concerning the constituents in herbal medicines,which could be helpful for the quality control and further phytochemical studies of Terminalia chebular Retz. |
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