Figure 1
Chemical structures of the triterpenes tested for trypanocidal activity: (1) α/β-amyrin, (2) 3-O-fumaryl-α/β-amyrin, (3) 3-O-succinyl-α/β-amyrin, (4) 3-O-acetyl-α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin, (7) 3-O-succinyl-11-oxo-α/β-amyrin, (8) 3-O-phthaloyl-11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one, (10) lup-20(29)-ene-3b,6b,15b-triol, (11) betulinic acid and (12) lanosta-8,24-dien-3-one; a isomer: R3 = CH3, R4 = H; b isomer: R3 = H, R4 = CH3.
Figure 2
Semi-synthesis of triterpene derivatives, reagents and conditions: (a) maleic anhydride, DMAP, CH2Cl2, reflux, 24 h; (b) succinic anhydride, DMAP, CH2Cl2, reflux, 24 h; (c) acetic anhydride, DMAP, reflux, 30 min; (d) phthalic anhydride, DMAP, CH2Cl2, reflux, 24 h; and (e) acetic anhydride, acetic acid, tert-butyl chromate, reflux, 6 h.
Figure 3
Combinatorial effects between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin and benznidazole with (4) 3-O-acetyl-α/β-amyrin on amastigotes of T. cruzi and LLCMK2 cells. Isobolograms (a) and (b) show the combinatorial effects of (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin and (4) 3-O-acetyl-α/β-amyrin with benznidazole against amastigotes, respectively; (c) and (d) illustrate combination between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin and (4) 3-O-acetyl-α/β-amyrin with benznidazole on LLCMK2 cells, respectively. Points below the additivity effect line indicate a synergistic effect and above indicate an antagonistic effect (combination index (CI)).
Figure 4
Transmission electron images of amastigotes forms of T. cruzi treated with triterpenes for 24 h. (A) Control; (B)-(G) correspond to treated-amastigotes with IC50 of the compounds (1) α/β-amyrin (20.2 µM), (5) 3-O-phthaloyl-α/β-amyrin (6.1 µM), (6) 3-O-acetyl-11-oxo-α/β-amyrin (55.3 µM), (7) 3-O-succinyl-11-oxo-α/β-amyrin (9.7 µM), (9) lup-20(29)-en-3-one (21.8 µM), and drug combination between (1) α/β-amyrin (7.1 µM) with (6) 3-O-acetyl-11-oxo-α/β-amyrin (14.5 µM), respectively; n: nucleus, k, kinetoplast, m, mitochondrion, and f, flagellum. Bar = 1 µm.
Figure 5
Transmission electron images of amastigotes forms of T. cruzi treated with triterpenes for 24 h. (A)-(F) correspond to treated-amastigote with an IC90 of the compounds (1) α/β-amyrin (97.3 µM), (5) 3-O-phthaloyl-α/β-amyrin (86.3 µM), (6) 3-O-acetyl-11-oxo-α/β-amyrin (112.5 µM), (7) 3-O-succinyl-11-oxo-α/β-amyrin (90.6 µM), (9) lup-20(29)-en-3-one (95.9 µM), and drug combination between (1) α/β-amyrin (38.3 µM) with (6) 3-O-acetyl-11-oxo-α/β-amyrin (47.2 µM), respectively. Key: n, nucleus, k, kinetoplast, and m, mitochondrion. Bar = 1 µm.
Figure 6
Cell membrane integrity assay by flow cytometry in amastigote forms of T. cruzi treated with triterpenes for 24 h and stained with PI. (a) Untreated amastigotes; (b) positive control; histograms (c), (e), (g), (i) (k) and (m) correspond to treated-amastigotes with IC50, and (d), (f), (g), (h), (j) (l) and (n) treated-amastigotes with IC90 of the compounds (1) α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin, (7) 3-O-succinyl-11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one and drug combination between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin, respectively. Percentage of PI-stained positive cells is shown in the upper right and left quadrants. Typical histograms of at least three independent experiments are shown.
Figure 7
Mitochondrial membrane potential assay in amastigote forms of T. cruzi treated with IC50 and IC90 of the triterpenes for 24 h and stained with Rh123. (a)-(f) Amastigotes treated with triterpenoids (1) α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin, (7) 3-O-succinyl-11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one and drug combination between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin, respectively. Arrows correspond to the concentration tested and control group (untreated parasites) that are also shown. Typical histograms of at least three independent experiments are presented.
Figure 8
Cell volume determination of amastigotes treated with triterpenoids for 24 h and analyzed by flow cytometer. The histograms show the relationship between the number of cells (counts) and FSC considered as a function of cell size. Gray-filled areas represent untreated cells. In each plot, the black line with unfilled areas represents treated-parasites. Histograms (a), (c), (e) (g), (i) and (k) correspond to treated-amastigotes with IC50 and (b), (d), (f), (h), (j) and (l) treated-amastigotes with IC90 of the compounds (1) α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin, (7) 3-O-succinyl-11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one and drug combination between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin, respectively.
Table 1
Trypanocidal activity and cytotoxicity on mammalian cells of twelve triterpenoid compounds (with standard deviation): (1) α/β-amyrin, (2) 3-O-fumaryl-α/β-amyrin, (3) 3-O-succinyl-α/β-amyrin, (4) 3-O-acetyl-α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyL-11-oxo-α/β-amyrin, (7) 3-O-succinyL-11-oxo-α/β-amyrin, (8) 3-O-phthaloyL-11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one, (10) lup-20(29)-ene-3β,6β,15β-triol, (11) betulinic acid and (12) lanosta-8,24-dien-3-one
Table 2
Mitochondrial potential assay in parasitic forms of Trypanosoma cruzi treated with following triterpenoids for 24 h and stained with Rh123: (1) α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyL-11-oxo-α/β-amyrin, (7) 3-O-succinyL-11-oxo-α/β-amyrin, (9) lup 20(29)-en-3-one and drug combination between (1) α/β-amyrin with (6) 3-O-acetyL-11-oxo-α/β-amyrin
