Study On Accumulation And Extraction Of Physiological Active Substances In Adventitious Roots Of Co-cultured Echinacea

In this study,adventitious roots from the co-cultivation of Echinacea purpurea Moench and Echinacea pallida Moench were used as experimental materials to conduct liquid suspension culture in a bioreactor and the conductivity and pH in the medium were determined.The dynamic changes of adventitious root growth and the accumulation of physiologically active substances were investigated,including the dry weight of adventitious roots,polyphenol content,etc.,and the growth kinetics model of adventitious roots was established by co-cultivation;methyl jasmonate（MeJA）and salicylic acid（SA）were used.As an inducer,promote the accumulation of target substances in adventitious roots;optimize the extraction process of adventitious root active substances by analyzing the correlation of adventitious root drying temperature,time and solvent,and secondary metabolite content.Research and development of co-culture technology of Echinacea adventitious root bioreactor to provide a new method for the production of total phenols and caffeic acid derivatives and other physiologically active substances,provide new ideas for the rational development and utilization of Echinacea,can effectively protect wild Echinacea resources,and promote Echinacea the modernization of traditional Chinese medicine.The main findings are as follows:（1）Establishment of Dynamic Model for the Growth of Adventitious Roots in Co-cultured EchinaceaThe adventitious roots of Echinacea were co-cultured with Pa+Pu.After 0¹0 days of inoculation,the biomass increased slowly and the content of secondary metabolites even decreased.This time was the growth lag period;the biomass increased rapidly from 10 to 35days.The content of secondary metabolites also increased significantly.When cultured for 35days,the biomass of the same generation product could reach the peak value.The dry weight of harvested adventitious roots was 37.34 g,and the content of total phenols in adventitious roots were 60.45 mg/g DW,this period was logarithmic growth phase;35 to 50 days,biomass same secondary metabolite the contents of these substances remain stable without any significant decrease.This time is the stationary phase.The results of dynamic research on adventitious root growth and material synthesis showed that the content of caffeic acid derivatives in co-cultivated adventitious roots was the same as biomass,and the total amount of caffeic acid derivatives remained unchanged or even slightly decreased during the lag phase.Starting from the logarithmic phase,the total content of caffeic acid derivatives increased rapidly and reached a maximum of 44.16 mg/g DW on the 35th day.The pH value increased with the increase of culture time.The change trend of the chelation force of EC and Fe²⁺was consistent,and both decreased with the increase of culture time.The trends of DPPH and ABTS were similar,falling before the 5th day of culture,followed by a sudden increase,and finally remained unchanged.Reducing power increased first and decreased with the increase of culture time.（2）Effect of Inducer on Physiologically Active Substances in Adventitious Root System of Co-cultured EchinaceaBy adding MeJA and SA,the co-culture of adventitious root polyphenols with Echinacea purpurea and Echinacea purpurea was studied.It was found that MeJA was more effective than SA.Therefore,MeJA was chosen as an inducer in subsequent experiments.When the adventitious roots of Echinacea purpurea and Echinacea purpurea were co-cultured,adding a certain concentration of MeJA could significantly increase the content and yield of secondary metabolites.The content of adventitious root total phenol was 81.28 mg/g DW（increased30.32%）.When the concentration of MeJA inducer was 25μM,the total phenol content and yield of adventitious roots were significantly higher than that of other treatments,and the total content of caffeic acid and PAL activity in adventitious roots were significantly increased.The total phenol content was 80.86 mg/g DW.The activity of PAL was highest at 24 h after adding MeJA to 24.03 U/mg;then the activity of PAL gradually decreased to 12.47 U/mg;meanwhile,the activities of POD and SOD were the highest.（3）Optimization of Extracting Process of Physiologically Active Substance from Adventitious Roots of EchinaceaResponse surface methodology was used to optimize the extraction of polyphenols from the adventitious roots of co-cultured Echinacea.The PB test was used to determine the five factors that had a significant effect on the time,ratio of liquid to material,temperature,ethanol concentration,and number of extractions.The CCD method was used to optimize the optimal extraction conditions and to study the interactions between various factors.The results showed that the interaction of temperature*time,temperature*ethanol concentration and temperature*liquid-to-liquid ratio had a significant effect on the extraction rate of adventitious roots.The best extraction conditions were calculated by the CCD method.The optimal extraction temperature was 68.33°C,the optimum extraction time was 2.43 h,the optimal ethanol concentration was 56.56%,and the optimal liquid-to-feed ratio was 91.49:1.The estimated lower polyphenol content was 63.37 mg/g DW and the verification result was65.84 mg/g DW.