Inducement Of Aspergillus Nigertoontriterpenoid Biosynthesis In Suspended Cultured Cyclocarya Paliurus Cells And Its Mechanism

Cyclocarya paliurus which survived in the ice age most distribute in the provinces along the Yangtze river basin in China.A large number of studies showed that its leave contains many function component such as triterpenes,flavonoids and polysaccharide,and have the physiological and pharmacological function of antidiabete,lowering blood lipid,hepatoprotective.In this paper,the plant cell culture technology was used to establish a stable Cyclocarya paliurus suspension culture,a triterpenoid fingerprint of the cell was drawn,the concentration,the adding time and the duration time of exogenous Aspergillus Niger elicitor were optimized and the relationship of triterpenoid biosynthesis with NO,H2O2,JA,SA and MARK signal pathway was studied.The main results were as follows:A stable Cyclocarya paliurus suspension culture was established,HPLC testing conditions and triterpenoid extracting conditions were optimized,the methodology was discussed to draw the triterpenoid fingerprint of Cyclocarya paliurus cell.The optimized HPLC condition was as the following: the mobile phase consisted of water containing0.02%(v/v)glacial acetic acid(A)and methanol containing 0.02%(v/v)glacial acetic acid(B)and the gradient program was as follows: 0.0–10.0 min,linear gradient 80.0–82.5% B;10.0–30.0min,linear gradient 82.5–90.0% B;30.0–40.0 min,isocratic 90.0% B.The peaks were recorded by a DAD detector at 210 nm.The optimal extraction condition was: 60℃,1/50(m/v,g/ml),60 min.The precision,stability,repeatability and recovery of this method are all good,the method was reliable to analyze.Ten peaks in the HPLC chromatogram were assigned as “characteristic peaks” 5 peaks were determined to maslinic acid,corosolic acid,betulinic acid,oleanolic acid and ursolic acid,the remaining five was not yet identified.Triterpenoid biosynthesis of the cells was improved after being induced by exogenous Aspergillus Niger elicitor.The optimized conditions for inducing was: the elicitor’s adding time,concentration and duration time were respectively 4-day-later after cell inoculation,200 μg/ml and 3 days.The content and yield of triterpenoids both peaked in the optimal condition,which were 10.79 times and 10.21 times higher than that of the control,respectively were 49.52 μg/mg DW(Dry weight)and 30.42 mg/40 ml.The relationship of triterpenoid biosynthesis with NO,H2O2,JA,SA was analyzed through adding the corresponding quenching agent and inhibitors.Test results proved that NO,H2O2,JA except SA were all related to triterpenoid biosynthesis of the inducing cell.In addition,some improvement of triterpenoid biosynthesis was caused by the JA promoted by NO and H2O2,and there were still JA from other ways in the cell to promote the biosynthesis of triterpenoid.The influence of Phospho-JNK、Phospho-p38 and Phospho-ERK which belong to MAPK on the biosynthesis of triterpenoid and the growth of the cell was studied.The results showed that Phospho-JNK signal pathway was the most influential factor,once this pathway was blocked,the cell biomass would be greatly reduced and most of the triterpenoid biosynthesis in inducing cell would be blocked,too.Above all,the triterpenoid fingerprint of Cyclocarya paliurus cell was reliable to be used to analyze the cell triterpenoid.Exogenous Aspergillus Niger inducer could effectively promote the biosynthesis of triterpenoid in Cyclocarya paliurus suspension cultured cell.In the inducing cell,some promotion of triterpenoid biosynthesis was caused by the JA promoted by NO and H2O2.In the MAPK,the Phospho-JNK signal pathway greatly influenced the cell growth and the biosynthesis of triterpenoid in the inducing cell.