Production Of Flavonoids By Cell Suspension Culture Of Hypericum Perforatum

Hypericum perforatum, commonly known as St. John’s wort, is a perennial herb native to Europe and also a traditional medicinal plant which has been utilized in Chinese folk medicine for a range of purposes. The medicinal applications of H. perforatum, including skin wounds, eczema, burns, diseases of the alimentary tract and psychological disorders, have been related to the flavonoids of the plant, which comprise the major group of biologically active metabolites in H. perforatum. The extracts of H. perforatum currently used in foods and pharmaceutics, which are mostly composed of flavonoids, are mainly obtained from the top aerial parts collected in the flowering stage. However, the quality of the flavonoids-rich extracts derived from field-grown plants may be affected by many environmental factors as well as biological processes. Furthermore, field cultivation of H. perforatum requires a long growth period and plant management, which is a slow and laborious process. Therefore, an alternative method for more efficient and controllable production of flavonoids from H. perforatum is urgently required.In this work, stem from H. perforatum was uesd to achieve callus. After several subculture,cell suspension culture was built in MS medium(1mg/L2,4-D+0.2mg/L6-BA) under12h/d light,25±2℃and100rpm.Spectrophotometry and high performance liquid chromatography (HPLC) were used to detect flavonoids, results showed that H. perforatum suspension cells could produce flavonoids, which mainly contained hyperoside and quercetin.Effect of precursor and elicitor on H. perforatum cell growth and flavonoids production were investigated in order to improve the flavonoids production. When phenylalanine concentration was50mg/L, flavonoids production achieved the highest of161.26±28.47mg/L. During the screen of elicitors, results showed the most outstanding effect of MeJA on cell growth and flavonoids production. The optimal elicitation strategy was to add100μM Methyl jasmonate on day15, under this circumstance biomass achieved5.62±0.45g/L DW, and the flavonoids production of MeJA elicited cells was280.40±38.99mg/L, which was2.7times of control. Meanwhile, the effect of light, sucrose concentration, aggregate size and inoculum density on cell growth and flavonoids yields were investigated. The best strategy was to screen for cell aggregate of300μm and cultivated under12h/d light,with25g/Lsucrose concentration and0.6g/30mL FW inoculum density. Under above conditions, cell mass was10.95±1.22g/L DW after20d cultivation and flavonoids production achieved365.18±17.20mg/L.The activities of key enzymes (CAT, PPO and PAL) related to flavonoids biosynthesis and cell growth in plants were investigated, in order to illustrate the mechanism of promoting H. perforatum cell growth and flavonoids accumulation in enzyme aspect. Light and MeJA could decrease CAT activity,and also increased the activity of PPO and PAL, therefore induced the defense response and the accumulation of secondary metabolites,which was consistent with the increase of cell mass and flavonoids production that we observed.Enzyme activity in suspension cells of different aggregate size was different, which resulted in the final difference of biomass and flavonoids production.