Isolation And Identification Of The Chemical Constituents Of Terminalia Chebula Fruits And Comparison Of The Effects Of Two Fractionation Methods

This research was carried out to investigate the chemical constituents of the fruits of Terminalia chebula,their anti-oxidant activities and the effects of two fractionation methods.As the results,21 chemical constituents were isolated through various chromatographic methods and identified by analysis of their MS and NMR(1H?13C?HMBC?1H-1HCOSY)spectra data as corilagin(1),chebulagic acid(2),ellagic acid(3),gallic acid(4),chebulic acid(5),shikimic acid(6),13-methylchebulate(7),11-methylchebulate(8),5-O-galloylshikimic acid(9),11,12-dimethylchebulate(10),chebumeinin B(11),punicalagin(12),1,4-di-O-galloyl-?-D-Glc(13),terflavin A(14),tellimagrandin(15),casuarinin(16),pentagalloyl glucose(17),chebulinic acid(18),1,3,6-tri-O-galloyl-?-D-glcose(19),1-O-galloyl-6-O-cinnamoylglucose(20),and chebuloside ?(21).Bio-assay experiments revealed that most of the constituents(1-5,7-20)showed strong DPPH radical scavenging activity,which should be caused by the phenolic hydroxyl groups in their structures.The mono-methyl ester of chebulic acid,13-methylchebulate and 11-methylchebulate(ECso values:9.8 and 15.1?g/mL,respectively),were less active than chebulic acid(ECso=4.6 ?g/mL).The dimethyl ester compound,11,12-dimethylchebulate(EC50=21.5 ?g/mL),was the least active among chebulic acid and its esters.These results suggested that carboxyl groups are more important than ester groups in the structures of chebulic acid and its derivatives for their DPPH radical scavenging activity.In the experimental study of hydroxyl radical scavenging activity,all the tested compounds(1-20)showed good activity with chebulic acid being most potent.Similar to that on DPPH,down trend hydroxyl radical scavenging activity were found among chebulic acid,13-methylchebulate,11-methylchebulate,and 11,12-dimethylchebulate.An LC-MS method was established to quantify the 21 compounds in fractions obtained from macroporous resin and solvent partition to compare the fractionation effects of the two methods.The results showed that the water,water-methanol 1:1,and methanol eluted parts from the macroporous resin column contained similar chemical constituents respective to the water,n-butanol,and ethylacetate soluble parts of the solvent partition method.Most of the above mentioned Terminalia constituents were found in the water eluted part of the macroporous resin,and the water phase of the solvent partition method.Meanwhile,each of the methods has their own advantages for enriching some compounds.Macroporous resin could enrich more than 85%of chebulic acid(5),shikimic acid(6),and 13-methylchebulate(7)to the water eluted part.On the other hand,solvent partition could enrich more than 90%of 1-O-galloyl-6-O-cinnamoylglucose(20)to the ethylacetate phase.