CONICET - Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias, Hospital de Clínicas José de San Martín, Buenos Aires, Argentina.
Universidad de Buenos Aires, Cátedra de Farmacognosia, Facultad de Farmacia y Bioquímica, Junín 956 2 P (1113). Buenos Aires, Argentina.
Phytomedicine. 2019 Mar 15;56:27-34. doi: 10.1016/j.phymed.2018.10.015. Epub 2018 Oct 11.
Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model.
Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets.
The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine.
Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy.
Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.
去氧米坎酮是从微花耳草（Mikania micrantha）和微花耳草（M. variifolia）中分离出的一种倍半萜内酯，先前已证明其对克氏锥虫具有体外活性，对感染的小鼠模型具有体内活性。
基于这些有希望的发现，本研究旨在研究该化合物对不同寄生虫靶标的作用机制。
在还原和非还原条件下，通过测量血红素的 Soret 吸收带的变化来检查去氧米坎酮与血红素的相互作用；通过测定 5,5'-二硫代双-2-硝基苯甲酸与谷胱甘肽存在时的巯基相互作用来确定；通过测量超氧化物歧化酶和三肽还原酶的活性以及通过流式细胞术评估寄生虫抗氧化系统的活性，同时评估细胞内氧化状态。超氧化物歧化酶和三肽还原酶的活性通过分光光度法进行测试。通过碘化丙啶、膜联蛋白-V 和罗丹明 123 染色分别评估细胞活力、磷脂酰丝氨酸暴露和线粒体膜电位；通过 TLC 定性和定量测试甾醇；通过透射电子显微镜分析超微结构变化。通过用单丹磺酰尸胺染色检测自噬细胞。
去氧米坎酮降低了寄生虫中还原巯基的数量，从而使它们随后容易受到氧化应激的影响。用该化合物处理寄生虫会导致线粒体膜去极化，尽管质膜通透性不受影响。去氧米坎酮不影响细胞内氧化还原状态，因此，不能将该化合物产生的线粒体功能障碍归因于 ROS 的产生。抗氧化防御系统在处理 24 小时后受到去氧米坎酮的影响，观察到氧化应激增加和超氧化物歧化酶和三肽还原酶活性降低（分别为 40%和 60%）。氧化应激和线粒体功能障碍通过凋亡和自噬诱导寄生虫死亡。
基于我们的结果，去氧米坎酮将通过作为一种强大的巯基阻断剂和产生线粒体功能障碍来发挥其抗 T 克氏锥虫活性。
