Study On Mechanism Of Low Temperature Enhancing Hypericin Accumulation In Hypericum Perforatum

Hypericum perforatum L.(family Hypericaceae)also known as St.John's Wort,is a traditional Chinese herbal medicine which has been used worldwide for antibacterial,anti-inflammatory,arrest bleeding and detumescence.Aerial parts with pharmacological activity including anti-cancer,anti-viral and anti-oxidant,largely relying on its chemical constituents such as naphthodianthrones,flavonoids and volatile oil;of these,Hyp has a significant effect of mild to moderate depression,makes H.perforatum become one of the globally focused and researched herds.At present,Hyp was mainly obtained from wild that lead to the plant being endangered and could not meet the market demand.Therefore,modern biotechnology is needed to increase the planting area and regulate the accumulation of major bioactive compounds.In this study,the system of tissue culture and hardening for H.perforatum was established with taking mature seeds as original explants;and further studied the effects of temperatures on plant growth,major bioactive compounds accumulation and key genes expression.The results are as follows:1)The seeds germination rate reached 51% based on the medium MS+4 g/L agar+20 g/L sucrose + light(200 lx)24 h/d;the number of stem internode reached 11 with supplemental 1.0 mg/L 6-BA+0.5 mg/L NAA+1.0 g/L AC;the root number and length respectively reached 4 and 8 cm with supplemental 2.0 mg/L IBA+1.0 g/L AC;and the seedling survival rate was 85% under the vermiculite: peat=(1:2).2)The growth characteristics,contents of major bioactive compounds,anti-oxidant capacity and key genes expression levels of the plants at different temperatures(15,22 and 30?)were measured and analyzed.Results showed that aerial parts biomass was greater at 15? with 1.3 and 1.6-fold increase compared to at 22 and 30?,chlorophyll content,stem number and leaf area has also enhanced at low temperature.Hyp content was greater 1.9 and 5.6-fold compared to 22 and 30? on a per plant basis,and the contents of other bioactive compounds(flavonoids and phenolics)as well as antioxidant capacity(DPPH scavenging activity and FRAP value)also significantly increase with the temperatures decrease.The expressions of eight genes(psbA,psbB,psbC,psbD,ycf3,ycf4,ycf5 and matK)related to photosynthesis and two genes(PKS and Hyp-1)involved in Hyp biosynthesis were also up-regulated 0.7 to 3.8-fold at 15? compared with 22?.3)The ethanol extract from aerial parts at different temperatures was analyzed by GC-MS,81 kinds of components were identified in the extracts;among them,37,39 and 43 kinds of compounds were identified at 15,22 and 30?,respectively;8 compounds were found in three temperatures;and O-(4-ethylbenzoyl)-1,2-Benzenediol(3.18%)?Triethyl phosphate(2.59%)?3-Ethyl-5-(2-ethylbutyl)-Octadecane(2.20%)were higher at low temperature.