Kaempferol-3-O-α-(3,4-di-E-p-coumaroyl)-rhamnopyranoside from Nectandra oppositifolia releases Ca2+ from intracellular pools of Trypanosoma cruzi affecting the bioenergetics system.

Phytochemical analysis of EtOH extract from leaves of Nectandra oppositifolia afforded three flavonoids: kaempferol (1), kaempferol-3-O-α-rhamnopyranoside (2) and kaempferol-3-O-α-(3,4-di-E-p-coumaroyl)-rhamnopyranoside (3), which were characterized by NMR and ESI-HRMS. When tested against the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, flavonoids 1 and 3 were effective to kill the trypomastigotes with IC50 values of 32.0 and 6.7 µM, respectively, while flavonoid 2 was inactive. Isolated flavonoids 1-3 were also tested in mammalian fibroblasts and showed CC50 values of 24.8, 48.7 and 153.1 µM, respectively. Chemically, these results suggested that the free aglycone plays an important role in the bioactivity while the presence of p-coumaroyl unities linked in the rhamnoside unity is important to enhance the antitrypanosomal activity and reduce the mammalian cytotoxicity. The mechanism of cellular death was investigated for the most potent flavonoid 3 in the trypomastigotes using fluorescent and luminescent-based assays. It indicated that this compound induced neither permeabilization of the plasma membrane nor depolarization of the membrane electric potential. However, early time incubation (20 min) with flavonoid 3 resulted in a constant elevation of the Ca2+ levels inside the parasite. This effect was followed by a mitochondrial imbalance, leading to a hyperpolarization and depolarization of the mitochondrial membrane potential, with reduction of the ATP levels. During this time, the levels of reactive oxygen species levels (ROS) were unaltered. The leakage of Ca2+ from the intracellular pools can affect the bioenergetics system of T. cruzi, leading to the parasite death. Therefore, flavonoid 3 can be a useful tool for future studies against T. cruzi parasites.

Metabolomics reveals biomarkers in human urine and plasma to predict cytochrome P450 2D6 (CYP2D6) activity.

BACKGROUND AND PURPOSE: Individualized assessment of cytochrome P450 2D6 (CYP2D6) activity is usually performed through phenotyping following administration of a probe drug to measure the enzyme's activity. To avoid any iatrogenic harm (allergic drug reaction, dosing error) related to the probe drug, the development of non-burdensome tools for real-time phenotyping of CYP2D6 could significantly contribute to precision medicine. This study focuses on the identification of markers of the CYP2D6 enzyme in human biofluids using an LC-high-resolution mass spectrometry-based metabolomic approach. EXPERIMENTAL APPROACH: Plasma and urine samples from healthy volunteers were analysed before and after intake of a daily dose of paroxetine 20 mg over 7 days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants. KEY RESULTS: We report four metabolites of solanidine and two unknown compounds as possible novel CYP2D6 markers. Mean relative intensities of these features were significantly reduced during the inhibition session compared with the control session (n = 37). Semi-quantitative analysis showed that the largest decrease (-85%) was observed for the ion m/z 432.3108 normalized to solanidine (m/z 398.3417). Mean relative intensities of these ions were significantly higher in the CYP2D6 normal-ultrarapid metabolizer group (n = 37) compared with the poor metabolizer group (n = 6). Solanidine intensity was more than 15 times higher in CYP2D6-deficient individuals compared with other volunteers. CONCLUSION AND IMPLICATIONS: The applied untargeted metabolomic strategy identified potential novel markers capable of semi-quantitatively predicting CYP2D6 activity, a promising discovery for personalized medicine.

Metabolite profile of Nectandra oppositifolia Nees & Mart. and assessment of antitrypanosomal activity of bioactive compounds through efficiency analyses.

EtOH extracts from the leaves and twigs of Nectandra oppositifolia Nees & Mart. shown activity against amastigote forms of Trypanosoma cruzi. These extracts were subjected to successive liquid-liquid partitioning to afford bioactive CH2Cl2 fractions. UHPLC-TOF-HRMS/MS and molecular networking were used to obtain an overview of the phytochemical composition of these active fractions. Aiming to isolate the active compounds, both CH2Cl2 fractions were subjected to fractionation using medium pressure chromatography combined with semi-preparative HPLC-UV. Using this approach, twelve compounds (1-12) were isolated and identified by NMR and HRMS analysis. Several isolated compounds displayed activity against the amastigote forms of T. cruzi, especially ethyl protocatechuate (7) with EC50 value of 18.1 µM, similar to positive control benznidazole (18.7 µM). Considering the potential of compound 7, protocatechuic acid and its respective methyl (7a), n-propyl (7b), n-butyl (7c), n-pentyl (7d), and n-hexyl (7e) esters were tested. Regarding antitrypanosomal activity, protocatechuic acid and compound 7a were inactive, while 7b-7e exhibited EC50 values from 20.4 to 11.7 µM, without cytotoxicity to mammalian cells. These results suggest that lipophilicity and molecular complexity play an important role in the activity while efficiency analysis indicates that the natural compound 7 is a promising prototype for further modifications to obtain compounds effective against the intracellular forms of T. cruzi.