| Camptothecin(CPT)and 10-hydroxycamptothecin(HCPT)are two main alkaloids in Camptotheca acuminata and they can be used as the materials for synthezing anti-tumour drugs.Because the CPT and HCPT have to be obtained from C.acuminata,so it is very important to study CPT and HCPT distribution and change trend in Camptotheca acuminate.Split ring loganic synthase(SLS)and tryptophan decarboxylase(TDC)are two key enzymes in biosynthesis of CPT derivatives.In this paper,C.acuminata seeds and seedlings were used as the research materials,on the basis of optimizing the CPT,HCPT determination method,and SLS,TDC activity analysis,CPT and HCPT content changes were discussed in C.acuminata germinating and seedling stages.In addition,the correlation of CPT,HCPT content changes and SLS,TDC activities were analyzed.1.Study and establish the analysis method of HCPT and CPT by HPLC and LC-MS/MS.(1)Sample preparation method for analysis the contents of HCPT and CPT in C.acuminata was established.Materials were dried at 80?,crushed and then ultrasonic extraction for 60min.The extraction conditions as follows:55%ethanol as extraction solvent,25:1(mL:g)of liquid-solid ratio,40kHz of ultrasonic frequencies,40? of extraction temperature.(2)The determination method of HCPT and CPT by HPLC were established.Chromatographic column:HiQ SiL C18 column(4.6mm?×250mm,5?m);mobile phase:acetonitrile-water(3:7/V:V);flow rate:1.0mL/min;detection wavelength:254 nm(CPT)and 366 nm(10-HCPT);column temperature:25?;injection volume:10 u L.(3)The determination method of HCPT and CPT by LC-MS/MS were established.Chromatographic conditions:mobile phase containing 0.2%formic acid in water-methanol(1:1,v/v);column was Symmetry Shield TM RP18 column(3.9 mm(?×150 mm,5 ?m);flow rate of 1.0 mL/min(split ratio 7:3).MS conditions:electrospray ionization source in positive ion mode;ion source temperature of 300?;,ionizing voltage 5500V;detection method:select multiple reaction monitoring(MRM),for quantitative analysis of ion reactions m/z 365.3/321.1(HCPT)and m/z 349.3/305.3(CPT).2 The assay methods of SLS and TDC activities were established.After the enzyme solution was added to loganic,the amount of split ring loganic in the unit of time was used to express SLS activity with?mol·g-1(FW)·h-1,in which split ring loganic was quantified by inner standard method.HPLC conditions:HiQ SiL C18 column(4.6mm?×250mm,5?m);acetonitrile-water(within 40 minutes,the volume percentage of acetonitrile from 15%to 35%)of mobile phase;0.5mL/min of flow rate;240 nm of detection wavelength;25? of column temperature;10 ?L of injection volume.7-hydroxy-6-methoxycoumarin was used as internal standard.After the tryptophan was added to the preparation enzyme solution,the amount of serotonin in the unit of time was used to express TDC activity with?mol·g-1(FW)·h-1,in which tryptophan was quantified by inner standard method.HPLC conditions:HiQ SiL C18 column(4.6mm?×250mm,5 ?m);methanol-10 mM KH2P04 solution(V:V/7:3,containing 7 mM SDS)of mobile phase;1.0 mL/min of flow rate;280 nm of detection wavelength;25?of column temperature;10 ? L of injection volume.5-methyl tryptamine was used as internal standard.3.The correlation of the main alkaloid content and enzyme activity in C.acuminata germinating and seedlings were analyzed.In the process of seed germination,HCPT content and SLS activity had a significant positive correlation;CPT content and TDC activity was positively correlated;HCPT content,TDC activity and CPT content,SLS activity was negatively correlation.In the process of seedlings growth,the contents of CPT,HCPT in leaves and TDC activity was negatively correlated;the contents of CPT,HCPT in leaves and SLS activity was negatively correlated.However,the contents of CPT,HCPT in roots and stems and the SLS,TDC activities were both positively correlated.These research results obtained in the work will offer some data for studying CPT compounds metablization and developing C.acuminata resources. |
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