Antiamoebic Activity of Adenophyllum aurantium (L.) Strother and Its Effect on the Actin Cytoskeleton of Entamoeba histolytica

Abstract

In Mexico, the Adenophyllum aurantium (L.) Strother plant is consumed as an infusion to treat intestinal diseases such as amoebiasis, which is an endemic health problem in Mexico and other countries. However, the effect of A. aurantium on Entamoeba histolytica, the causative agent of amoebiasis, is unknown. An aerial part methanolic extract (AaMeA), a root methanolic extract (AaMeR) and a root ethyl acetate extract (AaEaR) were tested on E. histolytica trophozoites. AaMeA and AaMeR did not show antiproliferative activity; however, AaEaR exhibited an in vitro GI50 of 230 μg/ml, and it was able to inhibit the differentiation of Entamoeba invadens trophozoites into cysts. The intraperitoneal administration of AaEaR (2.5 or 5 mg) to hamsters that were infected with E. histolytica inhibited the development of amoebic liver abscesses in 48.5 or 89.0% of the animals, respectively. Adhesion to fibronectin and erythrophagocytosis were 28.7 and 37.5% inhibited by AaEaR, respectively. An ultrastructure analysis of AaEaR-treated trophozoites shows a decrease in the number of vacuoles but no apparent cell damage. Moreover, this extract affected the actin cytoskeleton structuration, and it prevented the formation of contractile rings by mechanism(s) that were independent of reactive oxygen species and RhoA activation pathways. (13)C NMR data showed that the major compounds in the AaEaR extract are thiophenes. Our results suggest that AaEaR may be effective in treatments against amoebiasis, nevertheless, detailed toxicity studies on thiophenes, contained in AaEaR, are required to avoid misuse of this vegetal species.

Keywords: Adenophyllum aurantium; Entamoeba histolytica; actin; amoebiasis; cytoskeleton; encystation.

FIGURE 1

In vitro activity of AaEaR against Entamoeba histolytica trophozoites. The trophozoites (1 × 10⁴) were treated (200 μg/ml) with aerial part methanolic extract (AaMeA), root methanolic extract (AaMeR), root ethyl acetate extract (AaEaR) and MTZ (0.25 μg/ml) for 48 h to obtain the % of growth inhibition by using the formula (1-[Extract/Control]) × 100. The figure shows the graphical and corresponding images of the different treatments (A). The trophozoites (1 × 10⁴) were treated with AaEaR (B) or MTZ (C) at different concentrations. The data are representative of three independent experiments that were performed in duplicate, error bars represent SD. ANOVA analysis was used to compare among treatments (B,C). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 2

AaEaR prevents the differentiation of Entamoeba invadens. E. invadens trophozoites were induced to encyst in LG medium diluted at 47% with 5% ABS, in the presence of AaEaR (230 μg/ml) for 72 h. The trophozoites, precysts and cysts were counted, and the cell percentage was obtained using 100% as the total cell number under the control condition without treatment. Representative images of cells that were stained with Calcofluor White m2r are shown below the graph, and the cysts are more fluorescent than the trophozoites. Two independent experiments were performed in duplicate (n = 4), error bars represent SD. ^∗∗∗p < 0.001.

FIGURE 3

AaEaR inhibits the development of amebic liver abscesses. Trophozoites (1.5 × 10⁶) were inoculated in hamsters, and 4 days post-infection, animals were treated over the 4 days with vehicle, MTZ (0.1 mg) or AaEaR (2.5 and 5 mg) by intraperitoneal route (A). Representative images and graphics are shown (B). Two independent experiments were performed using four animals per experiment, error bars represent SD. ANOVA analysis was used to compare among treatments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (n = 8).

FIGURE 4

AaEaR affects adhesion and erythrophagocytosis in E. histolytica. The trophozoites (2 × 10⁵) were treated with AaEaR (230 μg/ml) for 48 h. The treated cells were incubated on fibronectin-coated coverslips (100 μg/ml) for 15 min and the adhered amoebas were counted (A). The amoebas were incubated with erythrocytes (1:50 ratio) for 15 min, and after washing off the non-ingested erythrocytes, the total hemoglobin content was measured (B). The data are representative of three independent experiments that were performed in duplicate, error bars represent SD. ^∗∗∗p < 0.001.

FIGURE 5

AaEaR does not induce cell death characteristics. The trophozoites (1 × 10⁴) were treated with AaEaR (230 μg/ml) and MTZ (0.25 μg/ml) for 48 h. An ultrastructure analysis by transmission electron microscopy (A) and the number of vacuoles per cell are shown (B). The data were collected from five images per condition. Amoebas (1 × 10⁴) were treated for 48 h with AaEaR (230 μg/ml) or MTZ (0.25 μg/ml), washed and reincubated in fresh TYI-S-33 medium without treatment, and their proliferation was determined at different times (C). The data are representative of three independent experiments that were performed in duplicate, error bars represent SD. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 6

AaEaR altered the formation of contractile rings in E. histolytica through reactive oxygen species and RhoA GTPase activity independent pathways. The trophozoites (2 × 10⁵) were treated with AaEaR (230 μg/ml) for 1 h, and filamentous actin was stained with rhodamine phalloidin (red); and the AaEaR extract exhibits auto-fluorescence (yellow; A). The contractile ring number was determined in 20 amoebas from three different optical fields with the ImageJ program (B). The amoebas (2 × 10⁵) were incubated with AaEaR (230 μg/ml) for 72 h and the cell number was determined at different times (C). The ROS production was evaluated by DCF-DA test, and the percentage was obtained by comparing them with untreated amoebas (D). The active RhoA was measured by G-LISA cytoskeleton kit (E). The data are representative of two independent experiments that were performed in triplicate or duplicate, error bars represent SD. ^∗p < 0.05, ^∗∗∗p < 0.001.

FIGURE 7

¹³C NMR spectra of Adenophyllum aurantium extracts. NMR measurements were performed in a Bruker ASCENDTM 400 (400 MHz proton frequency) spectrometer at 298 K. Chemical shifts (δ) are reported in ppm from tetramethylsilane (TMS). Solvents used were CD₃OD (A,B) and DMSOd6 (C).

FIGURE 8

α-Terthienyl and 5-(4′′-Hydroxy-1′′-butynyl)-2-2′- bithiophene were isolated from AaEaR. Ethyl acetate extract (14.36 g) was subjected to column chromatography (SiO₂) different mixing ratios of hexane-ethyl acetate. Eluted fractions between 94:6 and 92:8 yielded the following brown solids: (1) α-Terthienyl, and (2) 5-(4′′-Hydroxy-1′′-butynyl)- 2-2′-bithiophene.