Study On Separation And Purification Of Hyperforin

Hypericum perforatum Linn,a plant of Garcinia family, is one of Chinesetraditional medicines. It is also named St John's wort in Europe and America. Ithas been used to treat depression for hundred years. Recently pharmacologicaland clinical studies in St.John's wort indicated hyperforin is the 'true'activecomponent with the antidepression properties. In this paper, the analyticalmethods and preparation of hyperforin were studied. The main contents of thispaper were as follows: 1) A novel high performance thin layer chromatographic (HPTLC) and thinlayer chromatographic (TLC) methods to qualitative analyse hyperforin weredeveloped novelly in China. The optimal conditions of TLC were as follows:silica gel GF254 as stationary phase, n-hexane-ethyl acetate (5:1, v/v) as mobilephase. The Rf value of hyperforin was 0.41. 2) A new method of the reverse phase high performance liquidchromatography (HPLC) determination of hyperforin was developed. Theoptimal HPLC conditions were as follows: Alltima C18 column as the stationaryphase, gradient elution, A solvent: 80%methanol+1%acetic acid,B solvent:methanol, 0min～10min,0%B～100%B, 10min～40min,100%B, 1mL/min offlow rate, 30℃ of column temperature, UV detector (270nm). Under theseconditions, a baseline separation of hyperforin was obtained and the retentiontime of hyperforin is 14.0 minutes. The calibration equation is M=1.3238A-0.013921. The related coefficient r is 0.9993, the linear range is 0.5μg to 10μg, when the signal-to-noise ratio was 3:1, the LOD was 7.34×10-7μg. The RSDis 1.14%. The average recovery is 98.34 %. 3) Extraction of hyperforin from Hypericum perforatum were studied. Theextraction was intensified by ultrasonic method. Then the crude extracts ofhyperforin was obtained. The separation of hyperforin from crude extracts bypreparative thin layer chromatography (PTLC) was studied. The optimal IIIconditions of PTLC were as follows: silica gel GF254 as stationary phase;n-hexane-ethyl acetate (4.5:1, v/v) as mobile phase. The purity of hyperforinwas 76.64%. The separation of hyperforin from crude extracts with silica columnchromatography was studied. The optimal conditions of silica columnchromatography were as follows: column chromatography silica gel as stationaryphase, n-hexane-ethyl acetate (6:1, v/v) as mobile phase, fractively collection,combining the same constituents. The purity of separated hyperforin was61.14%. 4)The separation of hyperforin from crude extracts with dry columnchromatography was studied firstly. The optimal conditions of dry columnchromatography were as follows: silica gel GF254 as stationary phase, n-hexane-ethyl acetate (4.5:1, v/v) as the mobile phase, the purity of hyperforin was72.13%. 5)By combination of PTLC and preparative high performance liquidchromatography (PHPLC), a high-purity hyperforin sample was obtained. Thepurity of sample was higher than 98%. 6)The product was identified as hyperforin by ESI-MS. The ESI+-MSand ESI--MS spectrum of the product was in accord with literatures. TheESI-MS was elucidated and the fragmentation pathway of hyperforin wassuggested.