Fig 1.
Flow diagram of the chemical and biological steps to identify active compounds in Connarus suberosus.
(A) 9 different extracts from Brazilian Cerrado Biome Plant Extract Bank. (B) statistical grouping of extracts using chemical and biological data, with the RBH (root bark hexane) extract containing ion m/z 391.2284, important for differentiation within the active group. (C) Classical isolation in L. amazonensis promastigotes. (D) NMR (nuclear magnetic resonance), HRESIMS (high resolution electrospray ionization mass spectrometry), IR (infrared), and VCD (vibrational circular dichroism). (E) 3 isolated compounds. (F) Extract and compound activity. (G) Compound 2 and 3 studies: ROS (reactive oxygen species) production, ΔΨm (mitochondrial membrane potential) and neutral lipid body accumulation. (H) TEM (transmission electron microscopy).
Fig 2.
Statistical analysis produced with metabolomics data.
(A) PCA (principal component analysis) does not consider differences between active and inactive samples to group them, (Fig B) PLS-DA (partial least squares discriminant analysis) considers the difference and maximizes it, (C) PCA–loading, and (D) VIP (Variable importance projection) scores show the different important ions presented in the chemical profile of 9 different C. suberosus extracts used to create active and inactive groups, both pinpointing ion m/z 391.2284 as important for differentiation, and associated L. amazonensis promastigote mortality.
Fig 3.
Isolated compounds from C. suberosus.
C. suberosus compounds: hemileiocarpin (1) and leiocarpin (2), isolated due to the simple fraction chromatographic profiles; connarin (3), the ion pinpointed by metabolomics as important for activity, not found in the SciFinder database; and the quinones: rapanone (4), embelin (5) and suberonone (6) previously isolated by our group [12].
Table 1.
Activity of SPE-Diol fractions from C. suberosus Root Bark Hexane (RBH) extract against L. amazonensis promastigotes, after 24 h.
Table 2.
1H (600 MHz) and 13C (150 MHz) NMR data assignments for compound 3– chemical shifts (ppm) and coupling constants.
Table 3.
In vitro antileishmanial activity, cytotoxicity in murine peritoneal macrophages, and selectivity index of a C. suberosus Root Bark Hexane (RBH) extract and compounds 1–6.
Fig 4.
Mechanism of action of compounds 2 and 3 against L. amazonensis promastigotes after 24 h exposure.
(A) Evaluation of mitochondrial membrane potential; positive control: FCCP. (B) Reactive oxygen species (ROS) detection; positive control: miltefosine. Fluorescence intensity expressed in arbitrary units (A.U.). (C) Plasma membrane integrity evaluation; positive control: parasites heated at 65°C. (D) Neutral lipid quantification; negative control: untreated parasites. P< 0.001 (***) and 0.01 (**), significant difference compared with the control (Dunnett’s test).
Fig 5.
Micrographs of Leishmania amazonensis promastigotes.
(A) Control parasites incubated with Warren’s medium; (B) Control parasites incubated with 0.01% DMSO; (C-D) L. amazonensis treated with connarin (3) showing (L) lipid bodies. (F) Flagellum; (Gc) Golgi complex; (K) Kinetoplast; (M) Mitochondria; (N) Nucleus; (Nu) Nucleolus; (V) Vacuole.