Phylobioactive hotspots in plant resources used to treat Chagas disease

Abstract

Globally, more than six million people are infected with Trypanosoma cruzi, the causative protozoan parasite of the vector-borne Chagas disease (CD). We conducted a cross-sectional ethnopharmacological field study in Bolivia among different ethnic groups where CD is hyperendemic. A total of 775 extracts of botanical drugs used in Bolivia in the context of CD and botanical drugs from unrelated indications from the Mediterranean De Materia Medica compiled by Dioscorides two thousand years ago were profiled in a multidimensional assay uncovering different antichagasic natural product classes. Intriguingly, the phylobioactive anthraquinone hotspot matched the antichagasic activity of Senna chloroclada, the taxon with the strongest ethnomedical consensus for treating CD among the Izoceño-Guaraní. Testing common 9,10-anthracenedione derivatives in T. cruzi cellular infection assays demarcates hydroxyanthraquinone as a potential antichagasic lead scaffold. Our study systematically uncovers in vitro antichagasic phylogenetic hotspots in the plant kingdom as a potential resource for drug discovery based on ethnopharmacological hypotheses.

Keywords: Bioactive Plant Product; Biological Sciences; Ethnobotany; Ethnopharmacology; Natural Product Chemistry; Plant Biology; Plants.

Graphical abstract

Figure 1

Ethnopharmacological survey in Bolivia (A) The three different municipalities (orange), surveyed communities (red dots), and cities (stars) where the field study was conducted are shown. (B) Graphical summary of the ethnomedical data. The number of informants from the four ethnic groups (Ayoreo, Chiquitano, Guaraní, and Quechua) reporting knowledge of medicinal plants/agents for CD treatment (CD medplant knowledge) and the reported occurrence of CD in the family (CD in family) are shown. See also Figure S1.

Figure 2

Biological profiling of the CD botanical drug library collected in Bolivia Few EtOAc extracts showed selective toxicity toward T. cruzi epimastigotes (Y strain) with IC₅₀ values below 20 μg/mL (arrows). A high number of extracts were toxic for procyclic T. brucei, and many showed antiproliferative effects in HeLa and Raw 264.7 cells. The Leguminosae was the only subfamily cluster (top) showing no activities up to 25 μg/mL. In the sesquiterpene lactone-rich family Asteraceae, only Acanthostyles buniifolius showed selective antitrypanosomal effects. Data represent profiling values (based on IC₅₀ values) from at least two independent screening assays, each performed in triplicates. See also Table S1 and Figure S2.

Figure 3

FACS parasite release assay in 0.5% FBS and serum-free host cell conditions (A) Representative FACS histograms of the FL-1 channel (488/530 nm) showing parasites released into the medium from host cells in no FBS and 0.5% hiFBS cells infected with wild-type trypomastigotes. The vehicle control, benznidazole (BZN) treatment at 20 μM, and no infection control are shown. All samples were stained with the SYTO9 dye. Data are representative of at least six independent experiments. (B) Measurement of reactive oxygen species in CHO-K1 cells cultured in the different FBS conditions. ROS levels were measured using the indicator DCFDA by FACS after 24 hr of exposure to different medium conditions. Control cells were cultured in complete medium. Bar graphs represent the mean fold change in the geometric mean of fluorescence intensity ±SD and were analyzed using FlowJo from three independent experiments performed in triplicate. Statistical significance was calculated with t test. ∗∗∗∗P < 0.0001.

Figure 4

Comparative profiling of the DMM library from the Mediterranean Phylobioactivity tree displaying phylogenetic relationships associated with bioactivities of EtOAc extracts. The outer ring shows growth inhibition on T. cruzi epimastigotes, and the inner ring shows growth inhibition of HeLa cells (both at 25 μg/mL; only most active plant part shown). A hypothetical coumarin cluster (^aRuta chalepensis root, Levisticum officinale seeds, and Seseli tortuosum root) and the anthraquinone cluster (Rumex crispus and Rheum rhaponticum rhizoma) are visible. Laurus nobilis root and fruits and Sium sisarum root (microfractionated) are indicated in the phylogenic tree. Detailed data on plant species and activities are shown in Table S2.

Figure 5

Microfractionation of selected extracts and identification of antitrypanosomal natural products Bioactivity-guided microfractionation is exemplified with R. crispus using liquid chromatography and photodiode array (PDA) and evaporative light scattering detectors (ELSD). Isolation of antichagasic metabolites was based on epimastigote proliferation inhibition. False negatives are a limitation of this qualitative approach (shown here with chrysophanol) due to low concentrations. Nepodin and emodin/torachrysone were identified and isolated from fully active fractions (0% cell viability). The moderately active chrysophanol was identified in a negative fraction. Controls: BZN, benznidazole; Nifx, nifurtimox.

Figure 6

FACS parasite release assay using the GFP-expressing T. cruzi strain and reduced potency of benznidazole in serum-free culture conditions (A) Representative FACS histograms of the FL1 channel (488/530 nm) showing parasites released into the medium from host cells in 0.5% hiFBS cells infected with GFP-expressing trypomastigotes. The samples were prepared by fixing the released trypomastigotes in 4% paraformaldehyde as described in transparent methods. The vehicle control, benznidazole (BZN) treatment at 20 μM, and no infection control are shown. Data show mean values ± SD of at least 6 independent experiments. (B) Dose-dependent inhibition of parasite release by BZN under low and high ROS conditions. Data show mean values ±SD of at least three independent experiments performed in triplicate. See also Figure S3.

Figure 7

Emodin potency is dependent on serum and host cell ROS concentration (A) Dose-dependent inhibition of parasite release by emodin under serum and no serum (low and high ROS) conditions. (B) Dose-dependent inhibition of parasite release by hydroxyanthraquinones which inhibited more than 25% release at 5 μM using 0.5% hiFBS (low ROS): purpurin, aloe-emodin, quinizarin, and disperse Red11. Data represent the average ± SD of three independent experiments each performed in triplicate.