Natural terpenoids from Ambrosia species are active in vitro and in vivo against human pathogenic trypanosomatids

Abstract

Among the natural compounds, terpenoids play an important role in the drug discovery process for tropical diseases. The aim of the present work was to isolate antiprotozoal compounds from Ambrosia elatior and A. scabra. The sesquiterpene lactone (STL) cumanin was isolated from A. elatior whereas two other STLs, psilostachyin and cordilin, and one sterol glycoside, daucosterol, were isolated from A. scabra. Cumanin and cordilin were active against Trypanosoma cruzi epimastigotes showing 50% inhibition concentrations (IC50) values of 12 µM and 26 µM, respectively. Moreover, these compounds are active against bloodstream trypomastigotes, regardless of the T. cruzi strain tested. Psilostachyin and cumanin were also active against amastigote forms with IC50 values of 21 µM and 8 µM, respectively. By contrast, daucosterol showed moderate activity on epimastigotes and trypomastigotes and was inactive against amastigote forms. We also found that cumanin and psilostachyin exhibited an additive effect in their trypanocidal activity when these two drugs were tested together. Cumanin has leishmanicidal activity with growth inhibition values greater than 80% at a concentration of 5 µg/ml (19 µM), against both L. braziliensis and L. amazonensis promastigotes. In an in vivo model of T. cruzi infection, cumanin was more active than benznidazole, producing an 8-fold reduction in parasitemia levels during the acute phase of the infection compared with the control group, and more importantly, a reduction in mortality with 66% of the animals surviving, in comparison with 100% mortality in the control group. Cumanin also showed nontoxic effects at the doses assayed in vivo, as determined using markers of hepatic damage.

Figure 1. Chemical structures of the sesquiterpene lactones cumanin, psilostachyin and cordilin and the sterol glycoside daucosterol.

Figure 2. Inhibition of T. cruzi epimastigotes by cumanin, cordilin and daucosterol.

Growth inhibition of parasites was evaluated by a [3H] thymidine uptake assay. Epimastigotes of (A) RA and (B) K98 strain, were adjust at 1.5×10⁶/ml and cultured for 72 h at 28°C in the presence of the compounds at a final concentration ranging from 100 - 1 µg/ml. The percentage of inhibition was calculated as 100−[(cpm of treated parasites)/(cpm of untreated parasites)]×100. Values represent mean ± SEM from three independent experiments carried out in triplicate.

Figure 3. Isobologram describing the effect of the combination of (A) cumanin and psilostachyin and, (B) cumanine and benznidazole, on epimastigotes of T. cruzi.

Growth inhibition of epimastigotes was evaluated by a [3H] thymidine uptake assay. RA epimastigotes were cultured for 72 h in the presence of different combinations of both compounds ranging from 0–5 µg/ml. The fractional inhibitory concentrations of drugs (FIC) were calculated for each point, and an isobologram was plotted. The fractional inhibitory concentration index (FICI) was interpreted as follows: FICI≤0.5 synergy, FICI>4.0 antagonism, FICI = 0.5–4 addition.

Figure 4. Effect of cumanin, cordilin and daucosterol against T. cruzi trypomastigotes.

Bloodstream trypomastigotes diluted at 1.5×10⁶/ml in complete liver infusion tryptose medium, were seeded by duplicate in the presence of different concentration of each compound (0–376 µM, 0–375 µM and 0–174 µM, for cumanin, cordilin and daucosterol, respectively) and incubated at 4°C for 24 h. The remaining live parasites of (A) RA strain, and (B) K98 strain, in each sample was determined in 5 µl of cell suspension diluted 1/5 in lysis buffer (0.75% NH4Cl, 0.2% Tris, pH 7.2) and counted in a Neubauer chamber. Values represent mean ± SEM from three independent experiments carried out in duplicate.

Figure 5. Inhibition of T. cruzi amastigotes by cumanin, psilostachyin, cordilin and daucosterol.

Vero cells (5×10³ p/well) were seeded in a 96 well plates and infected 24 h later with transfected trypomastigotes expressing beta-galactosidase at a parasite cell ratio 10∶1. After 24 h of coculture, plates were washed and drug compounds were added at 0–94 µM, 0–96 µM, 0–96 µM and 0–43 µM, for cumanin, psilostachyin, cordilin, and daucosterol, respectively, in 150 µl of RPMI medium without phenol red. On day 6 p.i., the assays were developed by addition of CPRG (100 mM) and Nonidet P-40 (1%). Plates were incubated for 4–6 h at 37°C and quantitated at 570 nm. Controls included infected untreated cells (100% infection control). The percentage of inhibition was calculated as 100−{[(Absorbance of treated infected cells)/(Absorbance of untreated infected cells)]×100}.

Figure 6. Inhibition of Leishmania promastigotes grown by cordilin, psilostachyin, cumanin and daucosterol.

Promastigotes of (A) L. amazonensis and (B) L. braziliensis, were adjust at 1.5×10⁶/ml and cultured for 72 h at 26°C in the presence of 1–100 µg/ml of the compounds. [3H] thymidine was added for the last 16 h of culture. The percentage of inhibition was calculated as 100−[(cpm of treated parasites)/(cpm of untreated parasites)]×100. Result is representative of two independent experiments carried out in triplicate.

Figure 7. In vivo trypanocidal activity of cumanin: parasitemia levels (A) and survival curve (B) during the acute infection period.

C3H/HeN female mice infected with 500 trypomastigotes were treated daily, by intraperitoneal route, with PBS, cumanin or benznidazole (1 mg/kg of body weight/day), starting on day 5 postinfection. Levels of parasitemia were monitored every 2 days in 5 µl of fresh blood from the tail vein, by counting parasites in a Neubauer chamber. The number of dead mice was recorded daily. Results are representative of three independent experiments.