Research On Cell Cultures And Formation Of Secondary Metabolite Of Cistanche Deserticola Y.c.ma

We induced Callus from the seeds of Cistanche deserticola Y.C.Ma using plant cell culture techniques. The subculture of Callus and cell suspension culture methods were established. The effects of several precursors of phenylethanoid glycosides(PeGs) and fungal elicitors on the Dry Weight (DW) of cell suspension culture and the content of PeGs and echinacoside (Echin) were systematically studied.Under current conditions, the quality control and in-vitro antioxidant activity of the culture was initially investigated.The optimal culture conditions of the callus were as follows: MS+YE 800 mg/L+NAA 1.0 mg/L+6-BA 1.5 mg/L+2,4-D 0.5 mg/L+Sucrose 30 g/L+Agar 6.5 g/L; light period: 12 h/d; light intensity: 58.4μmol·m^-2·s^-1; culture temperature: 26±1℃; pH=5.8. The results showed that light yellow callus was high-yield cell line for the production of secondary metabolites. The maximum Dry Weight of callus reached 12.720 gDW/L on the 24^th day, while the highest contents of PeGs and Echin reached 11.467 and 6.041 mg/g respectively on the 20^th d.Conditions of cell suspension culture were as follows: MS+YE 800 mg/L+NAA 2.0 mg/L+6-BA 0.5 mg/L+2,4-D 0.2 mg/L+Sucrose 30 g/L; light period: 12 h/d; light intensity: 58.4μmol·m^-2·s^-1; culture temperature: 26±1℃; rotation speed: 110 rpm; inoculation concentration: 3.2 g DW/L; pH=5.8. The results showed that the maximum of Dry Weight, the contents of PeGs and Echin reached 8.108 gDW/L, 12.105 and 5.980 mg/g respectively on the 20^th d.The results showed that the inoculation of fungal elicitors had marked effects on the cell dry weight, the contents of PeGs and Echin among the six fungal elicitors in the cell suspension culture of C.deserticola .Cladosporium fulvum(C. fulvum) played an important role.We added 80 mg/L C.fulvum on the 16^th day,and the cell Dry Weight,the contents of PeGs and Echin reached the maximum 14.055 gDW/L,18.980 and 7.230 mg/g respectively after 6 days.We also investigated the effects of precursors feeding on the cell Dry Weight and the accumulation of secondary metabolites in the cell suspension culture of C.deserticola. The results showed that the contents of PeGs and Echin were greatly enhanced by feeding L-Phenylalanine and L-Tyrosine together, and the contents were higher than feeding them individually. When the combination of 0.330 g/L L-Phenylalanine and 0.362 g/L L-Tyrosine were added to the medium, the cell Dry Weight and the contents of PeGs and Echin of suspension cell culture reached the maximum 12.320 gDW/L, 48.500 and 20.180 mg/g respectively on the 20^th day.The results showed that the combination of precursor feeding and elicitation could enhanceed the production of secondary metabolites in the cell suspension culture of C. deserticola. When the combination of 0.330 g/L L-phenylalanine and 0.362 g/L L-Tyrosine were added on 0^th day followed by the addition of 80 mg/L C.fulvum elicitor on the 16^th day, the cell dry weight and the contents of PeGs and Echin reached the highest value , 14.691 gDW/L, 50.547 and 23.860 mg/g on the 22^th day,which were respectively 1.812, 4.176 and 3.990 times to the values without precursors and elicitors,the contents of PeGs and Echin were respectively 6.230 ,7.187 times to the values of wild C.deserticola.The comparative study of in-vitro antioxidant activity between suspension culture cells and wild materials of C. deserticola was carried out for the first time in this study by inhibiting the production of malondialdehyde(MAD), which is lipid peroxidation product of hepatic cytomicrosome. The detection of MAD levels indicated that the antioxidant activity of cell suspension culture showed the dose-dependent pattern. When the concentration of PeGs from cells suspension culture and wild material of C. deserticola were 0.04 mg/mL and 0.2 mg/mL respectively, they both had conspicuous antioxidant activity and could inhibit the production of MDA.