HPLC analysis of Echinacea & Hypericum perforatum extracts, fractionation of Hypericum perforatum extract and alkamide stability in Echinacea purpurea extracts
Six Echinacea phenolic compounds, caffeic acid, chlorogenic acid, caftaric acid, echinacoside, cynarin, and cichoric acid, two synthesized Echinacea alkamides, alkamide 2 and alkamide 10, five Hypericum flavonoids, rutin, hyperoside, isoquercitrin, quercitrin, and quercetin, were available to determine their extinction coefficients using the spectrophotometer. The Soxhlet extraction protocol was proved to be more efficient than the shaking extraction protocol to make both Echinacea and Hypericum extracts. The E. purpurea extracts made by same amount and same source of E. purpurea roots showed that the Soxhlet extract contained more phenolic compounds and alkamides than the Shaking extract. Two HPLC analyses, Echinacea phenolic gradient and alkamide gradient, were developed to quantify and qualify the Echinacea extracts. Hypericum flavonoid and hypericin HPLC gradients were established to quantitatively determine the flavonoids and pseudohypericin and hypericin in the Hypericum extracts. Three fractions of Hypericum extracts, flavonoids fraction, pseudohypericin fraction, and hypericin fraction were provided for cytotoxicity testing. The degradation of several major alkamides in E. purpurea extracts were monitored in four different storage conditions, phenolic-poor and phenolic-rich dry E. purpurea extracts, and phenolic-poor and phenolic-rich DMSO E. purpurea extracts at three high accelerated temperatures (70, 80, and 90 °C). The degradation of alkamides followed the first order reaction kinetics. The phenolic acids acted as antioxidant for the oxidation of the alkamides in dry E. purpurea extracts, while the phenolic acids took the opposite activity for the oxidation of alkamides in DMSO E. purpurea extracts. The predominant alkamides in dry and DMSO E. purpurea extracts followed this degradation order: alkamide 1, alkamide 2, alkamide 6, alkamide 8 and alkamide 9 > alkamide 3, alkamide 5 and alkamide 7. The energy activation of all alkamide degradation was about 100 kJ/mol in dry either phenolic-rich and phenolic-poor E. purpurea extracts, while about 60 kJ/mol energy activation was for all alkamides in phenolic-rich DMSO E. purpurea extracts. The prediction of the degradation rate constants and half-lives for the alkamides were calculated by Arrhenius equation. The predominant alkamides were proven to be very stable in E. purpurea extracts during the storage at room temperature or lower temperatures.