| AimIn the past hundred years,the population has increased sharply,the wild medicinal resources are short,and the cultivated medicinal materials have become the main source of commodities.The quality of medicinal materials is closely related to the environment,and the cultivation of medicinal materials also leads to the decline of the quality of medicinal materials.Improving the quality of cultivated medicinal materials is the key and difficult point in the research of traditional Chinese medicine resources,so it is urgent to find an effective method.Scutellaria baicalensis was studied in the early stage by our team.Reactive oxygen species were used to reproduce the stress conditions and regulate the physiological metabolism of plants and the fresh roots of S.baicalensis to increase the content of secondary metabolites.Plant cells have totipotent and complete metabolic units,the mechanism of ROS regulating secondary metabolism in S.baicalensis suspension cells can be clarified more clearly.Cell suspension culture is not affected by the season,which can realize the production of secondary metabolites of medicinal in factory and provide a new way for the development of traditional Chinese medicine resources.MethodsTissue culture technique to induce suspension of S.baicalensis,using PEG-6000 to simulate drought,exogenous H2O2 and O2-.carrier Na2S2O4 mimic ecological factors,effects of these treatments on ROS content and secondary metabolism of S.baicalensis suspension cells were studied,and the ROS content,antioxidant enzyme activity,gene expression and activity of flavonoids synthesis related enzymes,and the significance and correlation between flavonoids secondary metabolites in S.baicalensis cells.To elucidate the mechanism of metabolic changes and the formation mechanism of S.baicalensis combined with UPLC-Triple-TOF-MS/MS.The enzyme activity and substance content were determined by spectrophotometry and HPLC.Results1.Optimal conditions for induction and optimization of S.baicalensis suspension cells?1?Optimal conditions for inducing S.baicalensis callus:The stem segment of S.baicalensis seedling was explant,MS adding 2,4-D 1.0 mg/L+6-BA 1.0 mg/L,callus induction rate of S.baicalensis was 88.00%,which was light yellow,loose and granular,secondary cultureat 21 d.?2?Optimal conditions for inducing S.baicalensissuspension cells:The callus of S.baicalensis was induced,MS liquid medium adding 1.0 mg/L6-BA+0.2 mg/L2,4-D,the inoculation amount was3.0 g/bottles,sucrose 3%,pH 5.8,25 1?,120 r/min suspension culture,secondary cultureat 12 d.2.Effects of PEG on the metabolism of S.baicalensis suspension cells?1?ROS content:The content of O2-.in PEG treatment group were significantly higher than those of the control group and were proportional to the PEG concentration.There was no significant difference of H2O2 content between 4%PEG treatment group and the control group,and the H2O2content of 8%and 16%PEG treatment group was lower than that of the control group.?2?The antioxidant enzyme system effect:The activity of SOD in PEG treatment groups were higher than that in control group within 48 h before culture.The activity CAT in PEG treatment groups were higher than that in control group.The POD activity in 4%and 8%PEG treatment groups were significantly higher than that in control group.The activity of APX in PEG treatment group was3.35 times higher than that in control group at 60 h.?3?Secondary metabolic effects:The activity of PAL in 4%,8%,16%PEG treatment group increased by 11.60%,23.93%,14.92%,respectively,within 12-24 h,which promoted the synthesis and accumulation of flavonoids.The contents of baicalin,wogonoside,baicalein,and wogonin being increased by 17.51%%,2.71%,93.00%,and 64.29%for 4%PEG at 24 h respectively,most notably from the aglucons with high activities.?4?Significance and correlation analysis:The contents of wogonin and O2-.,baicalin and baicalein were significantly positively correlated in 4%PEG treatment group.The contents of H2O2was significantly positively correlated with contents of O2-.and baicalein,and the contents of O2-.was significantly negative correlation with baicalein,wogonin,and POD activity in 8%PEG treatment group.The CAT activity was significantly positively correlated with PAL activity and baicalin content in 16%PEG treatment group.The correlation indicates that ROS,antioxidant enzymes and flavonoids are in dynamic equilibrium,antioxidant enzymes and flavonoids synergistically eliminate excess ROS.3.Effects of exogenous H2O2 on metabolism of S.baicalensis suspension cells?1?ROS content:The contents of O2-.was significantly higher than that in control group except24-48 h in S.baicalensis suspension cells by exogenous H2O2 treatment.The contents of H2O2 was significantly higher than that in the control group,and the H2O2 content was proportional to the exogenous H2O2 concentration in 0-48 h.?2?The antioxidant enzyme system effect:The activity SOD,CAT,POD,APX of anti-oxidative enzyme were significantly improved in the treatment of S.baicalensis suspension cells with medium and low concentration of exogenous H2O2.?3?Secondary metabolic effects:The PAL activity increased 6.08 times,2.54 times and 12.23times?P<0.05?in10?mol/L,0.1?mol/L,0.001?mol/Lexogenous H2O2 treatment group,which promoted the synthesis and accumulation of flavonoids.The baicalin,wogonoside,baicalein and wogonin were increased by 2.64%%,22.41%,43.19%,115.65%for 0.1?mol/L exogenous H2O2 at 72 h.?4?Significance and correlation analysis:The contents of H2O2 and baicalin.,baicalein content and APX activity were significantly negative correlated,SOD activity and wogonoside content was significantly positively correlated in 10?mol/L exogenous H2O2 treatment group.The SOD activity and O2-.content,the CAT and PAL activity,.baicalin and wogonoside content,baicalein and wogonin content were significantly positively correlated in 0.1?mol/L exogenous H2O2 treatment group.The SOD and CAT activity,the SOD activity and wogonoside content,baicalein and wogonin content were significantly positively correlated in 0.001?mol/L exogenous H2O2 treatment group.The correlation indicates that ROS,antioxidant enzymes and flavonoids are in dynamic equilibrium,antioxidant enzymes and flavonoids synergistically eliminate excess ROS.4.Effects of Na2S2O4 on metabolism of S.baicalensis suspension cells?1?ROS content:The contents of O2-.was significantly higher than that in the control group,and the O2-.content was proportional to the Na2S2O4 concentration.The contents ofthe H2O2 increased significantly in low concentration Na2S2O4 treatment group.?2?The antioxidant enzyme system effect:The activity of SOD,CAT,APX were higher inNa2S2O4 treatment group than that in control group.The activity of POD was significantly higher in10?mol/L,0.1?mol/L Na2S2O4 treatment group than that in control group.?3?Secondary metabolic effects:The PAL activity increased by 22.60%,24.89%and 26.66%at12 h,12 h,48 h in 10?mol/L,0.1?mol/L,0.001?mol/L Na2S2O4 treatment group,respectively,which promoted the synthesis and accumulation of flavonoids.The baicalin,baicalein,and wogonin contents were increased by 22.54%,33.76%,and 97.75%in 0.001?mol/L Na2S2O4 at 24 h,respectively.?4?Significance and correlation analysis:The SOD and PAL activity,the SOD activity and baicalin content,.the POD and APX activity,the POD and PAL activity,the APX and PAL activity were significantly positively correlated in 10?mol/L Na2S2O4 treatment group.The POD and PAL activity,the POD activity and baicalin content were significantly positively correlated in 0.1?mol/L Na2S2O4 treatment group.The POD activity and wogonoside content significantly negative correlation in 0.001?mol/L Na2S2O4 treatment group.The O2-.and baicalin content,the O2-.and wogonoside content,the H2O2content and PAL activity,the H2O2andbaicalin content,PAL and SOD activity,PAL activity and baicalin content,CAT and APX activitywere significantly positively correlated in 0.001?mol/L Na2S2O4 treatment group.The correlation showed that PAL activity was strongly correlated with flavonoids,ROS can enhance PAL activity,indicating that PAL promoted the synthesis and accumulation of flavonoids.At the same time,it showed that antioxidant enzymes and flavonoids could eliminate excess ROS.5.Metabolomics research on PEG simulated drought in S.baicalensis suspension cellsSpectrum of S.baicalensis suspension cells was established by UPLC-Triple-TOF-MS/MS analysis technique,a total of 21 chemical constituents were identified.A cytochemical profile was established of S.baicalensis suspension cells by PEG simulated drought and normal culture.Nine different components were identified,which were shikimic acid,citric acid,chrysin,3,5,7,2',6'-Pentahydroxy flavanone,eriodictyol,baicalin,wogonoside,baicalein,wogonin.The chemical structure showed that most of them were free phenolic hydroxyl or methoxy flavonoids,with high antioxidant activity.The content of citric acid decreased and the content of shikimic acid increased by PEG simulated drought,indicating that secondary metabolism was enhanced.At ROS excess,baicalein,wogonin,3,5,7,2',6'-Pentahydroxy flavanone,eriodictyol and other antioxidant glycosides and free phenolic hydroxyl or methoxyflavone content were higher.As ROS content decreases,these glycosides are converted into corresponding glycosides with low antioxidant activity and stored,and the free phenolic hydroxyl or methoxy flavonoids content decreases.By biosynthesis and transformation between these flavonoids,similar to complex buffer solutions,maintaining the homeostasis ROS plants,which clarified the mechanism of quality formation of medicinal materials and explained the diversity,complexity and ecological significance of medicinal plants.ConclusionsPEG-6000 simulated drought,exogenous H2O2 and O2-.carrier Na2S2O4 mimic ecological factors,after treatment of S.baicalensis suspension cells,O2-.and H2O2 content significantly increased,activated the antioxidant enzyme defense system,increased varying degrees the activity of SOD,CAT,POD,APX enhanced the secondary metabolites synthesis and accumulation of S.baicalensis cells,and the antioxidant enzyme system and secondary metabolites synergistically eliminated excess ROS.Appropriate ROS reappearance of stress conditions to treat with S.baicalensis suspension cells,can achieve the purpose of regulating cell metabolism and improving secondary metabolites. |
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