Abstract of Master’s Dissertation
No.1
CourseHealth Innovation (MSc)NameQuankai Mu

ThesisInsights into Anti-Trypanosomal Mechanisms of Coptisine derived from Kampo

Title

Medicine

Abstract of Master’s Dissertation
Background: Chagas disease, also called American trypanosomiasis, is caused by theprotozoan parasite Trypanosoma cruzi. About 6–7 million people worldwide, mainly in Latin
America, are estimated to be infected with the parasite. There are two phases of infection, acuteand chronic. After the invasion of infection from the parasite, intracellular propagation of theparasite occurred locally. The acute phase persists for one to two weeks and is usuallytransected to the chronic phase characterized by asymptomatic and systematic persistentinfection. Currently, two widely used anti-T. cruzi drugs, nifurtimox and benznidazole, areeffective in the acute phase but not the chronic phase. When used for chronic, significant side
effects and variable efficacy are observed. Therefore, there is an urgent need to produce new,effective drugs to treat Chagas disease. One of the efforts to find a seed medicine is to use aresource of traditional herbal medicine, which we call Kampo in Japan. In previous studies,
coptisine, one of the main protoberberine-type alkaloids of Coptidis Rhizoma, has been shownanti protozoan parasites activity, including anti-malaria and anti-T. cruzi potential.
Objectives: In this study, we investigated the possible mechanisms of coptisine on theanti-trypanosomal effect in vitro
Methods: The possible molecular targets of coptisine and the drug-responsible metabolites

profile are assessed with the Agilent Seahorse Extracellular Flux Technology and the capillaryelectrophoresis-mass spectrometry analysis. The Agilent Seahorse Extracellular Flux technology
allows examining real-time changes in oxygen consumption rate due to ATP turnover, proton
leak, and electron transfer capacity to assess mitochondrial respiratory function in living cell. Dueto the addition of a proton (H+) detection probe, independently senses the pH in each well of theplate to measure extracellular acidification rate in test T. cruzi cells, making it possible toinvestigate changes in glycolytic as well as oxidative metabolic flux. At the same time, the fourports in the assay cartridge above each well can inject different drugs to detect quantitativechanges in oxygen consumption rate and extracellular acidification rate. The capillaryelectrophoresis–mass spectrometry, the combination of the liquid separation process of capillaryelectrophoresis with mass spectrometry, is an analytical chemistry technique for the efficientprofiling of polar and ionizable compounds in the given biological samples, notably for＊ The abstract, containing background, objectives, methods, results and conclusion should notexceed 300-500words and printed double sided on A4 paper)

Abstract of Master’s Dissertation
No.2
CourseHealth Innovation (MSc)NameQuankai Mu

compound classes such as nucleotides, sugar phosphates, organic acids, nucleotides, andamino acids. This approach has been successfully applied for the comprehensive profile ofintracellular metabolites under various conditions, including drug treatment and parasite
infection.
Results : In the extracellular flux assay, using oligomycin A, a classical bc1 complex (orcomplex Ⅲ) inhibitor,

as a control, coptisine and benznidazole (reference compound)showed any effect on mitochondrial respiration, as indicated by unaffected oxygen
consumption rate, whilst oligomycin A completely inhibited parasite respiration. Interestingly,
coptisine presented an inhibitory effect on the extracellular acidification rate which was notobserved in benznidazole. While using glucose, pyruvate and glutamine as carbon sources toprobe possible target metabolic pathways, it could still stably and similarly reduce
extracellular acidification rate in multiple experiments without causing changes in oxygen
consumption rate. This result was consistent with the intracellular metabolomics profiles,
which showed no significant difference in the production of various metabolites of
mitochondrial respiration and glycolysis between treated and untreated groups with coptisine.
Intotal,379

intracellular

metabolites

weresuccessfully

detectedbycapillaryelectrophoresis–mass spectrometry which may relate to 11 metabolic pathways including
carbohydrate metabolism, glucose metabolism/gluconeogenesis, energy storage/conversion by
TCA, glutamate metabolism, choline metabolism, nucleic acid metabolism, aromatic aminoacid metabolisms (tryptophan metabolism, phenylalanine metabolism, tyrosine metabolism).
Among these, 7.1% of metabolites (27/379) altered significantly in response to the coptisine
treatment. Interestingly, the glycerophospholipid synthesis pathway, including choline,
phosphorylcholine, ethanolamine phosphate, and glycerophosphocholine, is profoundly
affected by coptisine treatment, implying a unique metabolic feature in the anti-trypanosomal
effect of this compound.
Conclusion : In this report, we investigated the inhibition efficiency and the possible

mechanism of action of coptisine against T. cruzi using extracellular flux technology andmetabolomic analysis. We found that coptisine significantly inhibits the T. cruzi growth invitro. A significant effect on the extracellular acidification rate and the glycerophospholipid
synthesis of the parasites indicated the possible drug targets on the energy and lipid
metabolism of cells. Our works provide new insights into the development ofanti-trypanosomal drugs and the research of therapeutic targets.