Abstract:  

A reversed-phase HPLC method with photodiode array detection was established for the simultaneous analysis of hypericin and hyperforin in the methanolic extracts of three widely growing Hypericum species of Jordanian origin, namely: Hypericum empetrifolium Willd., Hypericum sinaicum Hochst. & Steud. ex Boiss, and Hypericum triquetrifolium Turra. The samples were extracted with methanol under reflux at 50 ?C. The components were separated by RP-C18 chromatography column using gradient mobile phase consisting of 20 mM ammonium acetate?acetonitrile over 45-min with 1 mL/min flow rate, wavelength range 250?600 nm (at 287 nm for hyperforin and 590 nm for hypericin). Quantification of hypericin and hyperforin was performed using external reference standards. The standard curves were linear over the concentration ranges, 10?100 ppm for both hypericin and hyperforin. Wide-ranging differences were observed in hypericin and hyperforin content among the investigated Hypericum species. Their hypericin content varied from 0.002% to 0.020%, while their hyperforin content varied from 0.013% to 0.989%. H. sinaicum showed the highest hyperforin content of 0.989% (w/w); while H. triquetrifolium and H. sinaicum showed the highest hypericin content of ?0.020% (w/w). This method can be utilized as an invaluable tool for standardization and quality evaluation of St. John's wort herbal preparations simultaneously against hypericin and hyperforin, which are the generally accepted marker compounds.