Comparative Analysis Of Flos Caryophyllata And Fructus Caryophylli By High-speed Counter-current Chromatography

Eugenia caryophylla Thunb., a commonly used herb, is documented in the Traditional Chineses Medicine (TCM) collection edition 2005. Flos Caryophyllate is the dry flower buds of Eugenia caryophylla Thunb., while Fructus Caryophylli is the mature fruits of Eugenia caryophylla Thunb.. It has been clearly known that effective components of these herbs are their secondary metabolites. The present paper comparatively analyzed chemical components of Flos Caryophyllata and Fructus Caryophylli by high-speed counter-current chromatography technique.This dissertation consists of three sections:The first part is the introduction section. In this section, an extensive review including chemical components, pharmaceutical effect, purification methods of Flos Caryophyllate and Fructus Caryophylli was included. A brief review about the latest progress of HSCCC was also included in this section.The second section focuses on Comparative analysis of Flos Caryophyllata and Fructus Caryophylli by high-speed counter-current chromatography. The herbs were first extracted by 95% ethanol continous reflux. The crude extracts were further extracted by petroleum ether and ethyl acetate sequentially, followed by macroporous resin fractionation. The fractions were then separated by HSCCC in a stepwise elution technology respectively. The two-phase solvent system composed of hexane: ethyl acetate: methanol: water (5:8:6:13, v/v/v/v, A solvent system) and hexane: ethyl acetate: methanol: water (5:8:9:10, v/v/v/v, B solvent system) that were optimized and determined according to their physical parameters. The separation was initially done by filling the column with upper phase of A solvent system as the stationary phase followed by elution with lower phase of A solvent system for 210 min. Afterwards it eluted with lower phase of B solvent system for another 340 min. Rotation speed of the chromatography was 880 r/min and the flow rate maintains 1.2 mL/min. The sample volume was 70 mg. Nine peaks were identified among which 6 peaks were consistent from both samples. The average of relative standard deviation (RSD) from the 6 peaks'HSCCC retention times was 2.260%, which is much lower than the national standared criteria 3%. Overlay ratios between two HSCCC chromatograms were around 66.67%, which manifest efficiency of HSCCC method in the test. In addition, five compounds including ellagic acid, 5,7-dimethoxy-2-methyl-chromen-4-one, 5,7-dimethoxy- 2,6-dimethyl- chromen -4-one, 3,5,3'4'-tetrahydroxy-7-methoxyflavone and quercetinpurified from the two herbs, among which quercetin, ellagic acid and 3,5,3'4'-tetrahydroxy-7-methoxyflavone are known compounds isolated from Eugenia caryophylla Thunb.. To be mentioned here, 5,7-dimethoxy- 2,6-dimethyl-chromen-4-one is a compound that firstly identified by NMR information so far.Based on the second section, compounds from Fructus Caryophylli was systematically isolated by using HPLC method. Three compounds including gallic acid, bancroftione and recrystallized 5,7,3',4',5'-pentamethoxyflavanone were purified. Among which 5,7,3',4',5'-pentamethoxyflavanone and was firstly identified from Fructus Caryophylli.