Establishment Of Suspension Cell Lines And Stable Isotopic Tracer Studies Of THSG Synthesis Pathway Of Polygonum Multilforum Thunb.

THSG （2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside） is the main active ingredient ofPolygonum multiflorum Thunb., which posses anti-inflammatory, antioxidant, regulating lipid,anti-tumor, calming the nerves, prevention and treatment of Alzheimer’s disease. At presentthe study focus on the extraction and pharmacological effect of THSG, yet THSGbiosynthetic pathway has not been reported. We are committed to the biosynthesis pathway ofTHSG. First, calli were induced, then suspension cell line was established. Normal precursorswere added and stable isotope precursor was used to tracer cells of Polygonum multiflorumThunb. THSG was speculated by HPLC-MS/MS, on the basis of results THSG biosyntheticpathway was deducted. The main results of this article are as follows:（1） Induction medium of Polygonum multiflorum Thunb. was obtained by orthogonalexperiments which contained1mg·L^-12,4-D and0.4mg·L^-1NAA in MS medium. In thismedium, calli can be induced in15days, and induction rate of calli was96.7%. The inductioneffect of root, stems and leaves of Polygonum multiflorum Thunb. was evaluated and THSGcontent was examined, and found that root, stems and leaves of Polygonum multiflorumThunb. can be induced. The highest content of THSG in root calli was1.5mg·g^-1, so rootcalli were used to transfer and culture. Calli were fine and close which can’t be suspended.Calli were transferred in liquid MS medium for4days, then transferred to solid MS mediumcontaining different plant growth hormone, at last loose and friable calli were acquired, andthe growth hormone were0.2mg·L^-16-BAand0.5mg·L^-1NAA.（2） The growth effect was compared in MS, B5and N6medium, and MS medium is the bestmedium. The cell DW （dry weight） and THSG content in this medium was respectively6.35g·L^-1and42.23mg·L^-1, so MS medium was selected as suspension medium.（3） Suspension cells were spread on the plate, and cultured for30days. Fast-growing smallcell clusters were selected and transferred on the plate, and then transferred once every15days for six times. THSG content of different small cell cluster was examined and ultimatelya high content cell line of THSG was acquired, and DW and THSG content of this high yieldcell line of Polygonum multiflorum Thunb. was7.46g·L^-1and56.23mg·L^-1respectively, yetDW and THSG content of the original cell line was respectively6.55g·L^-1and43.39mg·L^-1. DW and THSG content of this high content cell line of Polygonum multiflorum Thunb.respectively increased by13.89%and29.59%than the original cell line. By optimization offermentation conditions, the best culture conditions were100rpm,26℃and dark culture for16days.（4） In order to screen precursors of THSG biosynthetic pathway and feeding studies,Different concentration of phenylalanine was added in suspension medium of Polygonummultiflorum Thunb. in the10thday,105.15mg·L^-1of THSG was acquired which increased by90.38%in60mg·L^-1phenylalanine than the control group (55.23mg·L^-1). Differentconcentrations of cinnamic acid were added in suspension cell medium. THSG can can reachthe maximum content in40mg·L^-1cinnamic acid:123.03mg·L^-1which increased by122.76%than the control group (55.23mg·L^-1). Different concentration of sodium acetatewas added in suspension medium.20mg·L^-1sodium acetate was added in suspensionmedium. The maximum production of THSG can be reached:89.08mg·L^-1than the controlgroup (55.23mg·L^-1) which increased by61.29%. Mixed precursors of differentconcentration of phenylalanine, cinnamic acid and sodium acetate were added in liquid MSmedium, and the maximum content of THSG acquired in40mg·L^-1cinnamic acid and20mg·L^-1sodium acetate was162.65mg·L^-1than the control group (55.23mg·L^-1) whichincreased by194.50%, so mixed precursor had best effect.（5） U^-13C9phenylalanine was added in liquid MS medium of Polygonum multiflorum Thunb.Then THSG was detected by HPLC-MS/MS,¹³C phenylalanine can be combined into THSG,which confirmed phenylalanine was precursor of THSG. U^-13C9cinnamic acid was added inliquid MS medium. Then THSG was detected by HPLC-MS/MS,¹³C cinnamic acid can becombined into THSG, which confirmed that cinnamic acid was a precursor of THSG, and thetracer experiment proved hypothetical THSG biosynthetic pathway.Based on the above findings, we proved THSG biosynthetic pathway: phenylalanine asprecursor under phenylalanine ammonia lyase, then synthesize cinnamic acid, after a fewsteps biosynthetic reaction further synthesize THSG.