Computational protocol: In vivo validation of anti-malarial activity of crude extracts of Terminalia macroptera, a Malian medicinal plant

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Protocol publication

[…] Metabolite profiles of TMR ethanol extract (1 mg/mL) were acquired using a UHPLC-DAD-CAD-LTQ Orbitrap XL instrument (Thermo Fisher Scientific, UK) equipped with an electrospray ionization source (ESI). The UHPLC system consisted of an Ultimate 3000 UHPLC (Thermo Fisher Scientific, UK) equipped with a Acquity BEH C18 column (100 × 2.1 mm i.d., 1.7 µm, Waters, USA). The mobile phase was composed of solvent A (0.1% formic acid–water) and solvent B (0.1% formic acid-acetonitrile) with a gradient elution (0–0.5 min, 95% A; 0.5–12 min, 95–5% A; 12–15 min, 5% A; 15–15.5 min, 5–95% A; 15.5–19 min, 95% A). The flow rate of the mobile phase was 0.45 mL/min. The injection volume was 4 µL and the column temperature was maintained at 40 °C. Electrospray ionization was applied in negative ion (NI) and positive ion (PI) mode under the following conditions: capillary voltage at 3.0 and 4.2 kV for NI and PI respectively, and capillary temperature at 300 °C. The UV detection was performed by a diode array detector (DAD) from 210 to 400 nm. Full mass spectra were recorded between 100 and 1500 Da. Collision Induced Dissociation mass spectra were obtained using the following parameters: 35% normalized collision energy, isolation width 2 Da, activation Q 0.250. External mass calibration was accomplished before starting the experiment. Hyphenation with Charged Aerosol Detector (CAD), Thermo Fisher Scientific, UK) was performed after DAD using a split 1:1 between CAD and Orbitrap.Data processing and statistical analysis were performed as previously described []. Briefly, the UHPLC-HRMS raw data were processed with MS-Dial v.2.56 for mass signal extraction and peaks alignment []. Molecular formula prediction and compound annotation of significant features (m/z, RT pairs) were calculated with MS-FINDER 2.10 []. Only natural product databases focused on plants were selected [i.e. Universal Natural Products Database (UNPD), KNApSAc, PlantCyc, Dictionary of Natural Products (DNP, CRC press, v26:1) and CheBI]. Database interrogation was performed following a three-step process. Already known compounds belonging to the plant species were first analysed, then those belonging to the botanical family, and finally, molecules present in all plant databases were interrogated. Results were presented as a list of compounds sorted according to a score value for each match. This value encompassed uncertainty on accurate mass, the isotopic pattern score and the experimental MS/MS fragmentation mirrored to in silico fragmentation of the candidate structure. […]

Pipeline specifications

Software tools MS-DIAL, MS-FINDER
Databases ChEBI PMN DNP UNPD
Application MS-based untargeted metabolomics
Organisms Plasmodium falciparum, Homo sapiens, Mus musculus, Plasmodium chabaudi chabaudi, Plasmodium berghei ANKA
Diseases Infection, Malaria, Malaria, Cerebral, Drug-Related Side Effects and Adverse Reactions
Chemicals Chloroquine