| The leaves of Aralia elata belong to dried leaves of Aralia elata(Aralia elata(Miq.)Seem.),which have the function of clearing heat,promoting diueresis,benefiting Qi and tonifying kidney.Currently,Aralia elata leaves capsules,which take the extract of total saponins of the Aralia elata leaves as the material,have become the hospital preparations of the Second Affiliated Hospital,Helongjiang University of Chinese Medicine.They are mainly used adjuvant therapy for cancer patients.Our research group has found that the triterpene saponins in Aralia elata leaves are the pharmacodynamic material basis to fight against tumor in the early research.To further explain the function mechanism and pharmacokinetics characteristics of antitumor active fraction(extract of total saponins)extracted from Aralia elata leaves,the pharmacokinetic characteristics of 6 kinds of triterpene saponins components were studied after given the total saponins of Aralia elata leaves through overall animal research by adapting the technology of LC-MS in this research,and the mutual function risk of metabolic drug interactions was evaluated basically.In the meanwhile,this research uses the metabonomics method to research the differences of the metabolites from the 3 species cancer cells after given the total saponins of Aralia elata leaves.By analyzing and verifying the biological significance of the difference matters of the metabolites,this research clarifies the anti-tumor function mechanism of the effective parts of the Aralia elata leaves.The research contents mainly include following parts.1.Determination of Aralia elata leaves and its effective anti-tumor partsA HPLC-ELSD method was developed for simultaneous quantitative determination of ten triterpenoid saponins in Aralia elata leaves and its antitumor active fraction.The analysis was carried out on a Diamonsil C18 column(4.6 mm×250 mm,5?m,id)using gradient elution with the mobile phase consisting of water and acetonitrile.The flow rate was 0.8 mL/min.The detector is an ELSD,the carrier gas flow rate is 3 L/min,and the drift tube temperature is 110?.There are altogether 53 batches samples from 11 different production areas,and four kinds of triterpene saponins in these samples were tested by the above-mentioned method.The results showed that the samples in May have comparatively less triterpenoid saponins,while triterpenoid saponins show more in samples of July and August;BUT wide differences appear among samples of June and September,four kinds of triterpenoid saponins content was relatively high in late June and early September.Therefore,according to the result,the optimal harvest time for the Aralia elata leaves is July and August,but the harvest time of different areas had better postpone or bring it forward in accordance with their own regional condition.The efficient,sensitive and reliable analysis method was established to test four kinds of triterpene saponins of Aralia elata leaves and its antitumor active fraction.It can be used for the quality control of Aralia elata leaves and its antitumor active fraction.2.The pharmacokinetic study of the anti-tumor part of Aralia elata leavesA LC-MS/MS method was developed for simultaneous quantitative determination of six triterpenoid saponins in rat plasma.The analysis was performed on an ACQUITYUPLC HSS T3(100mm×2.lmm,1.7?m,id)using gradient elution with the mobile phase consisting of water(0.1%formic acid)and acetonitrile(0.1%formic acid).The flow rate was 0.3 mL/min.The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring(MRM)mode via electrospray ionization(ESI)source.This method was applied to measuring the concentration of 6 kinds of triterpenoid saponins in rat's plasma after the rat took the medicine,then the drug concentration-time curve were plotted,the main pharmacokinetic parameters were calculated.The result indicated these 6 kinds of Aralia elata saponins would not accumulate in rat's body easily because they were quickly absorbed and eliminated.Besides,since the 6 kinds of Aralia elata saponins have the similar chemical structure as well as similar drug concentration-time curve in blood,and they tend to have similar oral absorption and elimination features.3.The influence of the anti-tumor part of Aralia elata leaves&saponins to P450 enzyme.A LC-MS/MS method was developed for simultaneous quantitative determination of six metabolites of probe substrate.The analysis was performed on an ACQUITY UPLC HSS T3(100mm×2.1mm,1.7?m,id)using gradient elution with the mobile phase consisting of water(0.1%formic acid)and acetonitrile(0.1%formic acid).The flow rate was 0.3 mL/min.The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring(MRM)mode via electrospray ionization(ESI)source.This method was applied to assess the influence of the anti-tumor part of Aralia elata leaves&saponins to enzymes in human liver microsomes.The result showed the anti-tumor part of Aralia elata leaves and 6 kinds of triterpene saponins can reduce the P450 enzyme activity,wherein the least value of IC50 is 0.981?g/mL.However,the anti-tumor part of Aralia elata leaves had comparatively smaller inhibitory effect---only a little inhibitory effect on CYP2A6 and CYP2B6,and the value of IC50 for them are 159.7?g/mL and 69.36?g/mL.This experiment reminds us of the need to completely assess the risk of drug combination when we are doing the clinical research of the anti-tumor part of Aralia elata leaves.4.The study of antitumor mechanisms of the anti-tumor part of Aralia elata leaves to three tumor cells based on cell metabonomicsOn the platform of LC-MS/MS technology,the thesis used multivariate statistical analysis to explore the anti-tumor part of Aralia elata leaves working on three tumor cells and later changes in intracellular metabolites.Meanwhile,substances with significant differences was rapidly assessed and identified by means of Progenesis QI 2.0 software.Here's the result,the methionine,8,11,14 eicosenoic acid,9(10)-Epoxy octadecadienoic acid and hexadecenoic acid etc.altogether 20 kinds of differential metabolism had been identified in HepG-2 cells;2(R)-hydroxy docosanoic acid,9-hydroxyl docosanoic acid and 7,10,13,16,docosatetraenoic acid-altogether 9 kinds of differential metabolism had been identified in MCF-7 cells;15(s)-hydroxy eicosatrienoic acid,docosatrienoic acid and 3(R)-hydroxy myristic acid-altogether 9 kinds of differential metabolism had been identified in BGC-823 cells.According to the biological analysis of differential metabolism,it can be predicted that the anti-tumor part of A ralia elata leaves might inhibit the tumor's growth and reproduction by inhibit their energy metabolism.5.Anti-tumor Effect of the anti-tumor part of Aralia elata leaves on the Energy Metabolism of 3 kinds of Cancer CellsThis paper uses bio-energy analyzer to detect the level of oxygen consumption rate of the blank group and drug group of 3 kinds of cancer cells.The result shows that compared with the blank group,energy produced by mitochondrial oxidation phosphate and non-mitochondrial of the cancer cells in the drug group show significant declination(P<0.01),as the drug dose increases,this trend becomes more obvious,in the meanwhile,the anti-tumor effective fractions of Aralia elata leaves can effectively reduce the amount of ATP and the mitochondrial reserves energy in BGC-823 cells and MCF-7 cells(P<0.01),but have no obvious effect on the amount of ATP and the mitochondrial reserves energy of HepG-2 cellsThis paper also respectively studies the effect of the anti-tumor effective fractions of Aralia elata leaves on the ATP level,ROS level,activity of mitochondrial breathing chain enzyme composite ?,?,? and ? and the mitochondrial membrane potential level of 3 kinds of cancer cells,the results show that the anti-tumor effective parts of Aralia elata leaves can significantly reduce the ATP level of 3 species of cancer cells,and with more drug dose the declination is more obvious;other group can significantly improve ROS level except low dose group,which has no statistics meaning on HepG-2 cell ROS;Compared with the blank control group,the respiratory chain enzyme complex activity of each group was decreased,and the differences were significant,among which the declination amplitude of composite ?,? and ? is more obvious.Though each drug group can reduce the activity of composite ?,but compared with aforesaid 3 kinds of complexes,its declination trend is not as obvious as first three kinds;the declination trend of the mitochondrial membrane potential of each drug group is obvious,and has significant difference with blank control groupThe results of above experiment show that the influence that the anti-tumor effective fraction of Aralia elata leaves exerts on energy metabolism might have something to do with accumulation of ROS.In conclusion,this paper established the determination method of Aralia elata leaves and their anti-tumor fractions.Besides,this paper also conduct systematic research over pharmacokinetics of their anti-tumor effective fractions,thus,the pharmacokinetics characteristics of the anti-tumor effective fractions inside the body of rats and their influence over CYP450 enzyme activity are clarified preliminarily,which provide the pharmacokinetics basis for the clinical safety and effectiveness of anti-tumor effective fractions.Through the research over cell metabolomics,the anti-tumor mechanism of Aralia elata leaves is found,which is verified preliminarily with such ways as Seahorse XF Extracellular Flux Analyzer.Therefore,this paper lays the foundation for the deeper research to the anti-tumor function of Aralia elata leaves. |
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