Key words
Entamoeba histolytica
-
Tabernaemontana arborea
- Apocynaceae - ibogaine - voacangine - alkaloids - anti-amoebic - amoebic liver abscess
Introduction
Amoebiasis caused by the enteric protozoan parasite Entamoeba histolytica is a cosmopolitan disease that infects millions of people, making it a leading cause
of diarrhoea, mainly in
developing countries, and estimated to cause the death of more than 55 000 people
each year [1 ], [2 ], [3 ].
In a global burden of diseases study from 1990 to 2010, worldwide deaths due to
amoebiasis were reported to be around 1 per 100 000 people, causing a loss of disability
life-years (DALY) of 32
years on average [4 ]. Amoebic trophozoites invade the intestinal mucosa, producing dysentery, and sporadically
migrate to the liver resulting in abscesses, which is
the main cause of death by this parasite [3 ]. Nitroimidazole derivatives such as metronidazole (MTZ) are the drugs of choice
for treatment. However, MTZ is
inefficient against the transmission stage of cysts, and it has the disadvantage
of inducing many side effects that may result in the abandonment of therapy before
eradication of the infection
[5 ]. Moreover, genotoxicity in human cells, mutagenicity in bacteria, and carcinogenicity
in rodents have been reported for MTZ [6 ], [7 ]. In addition, in vitro resistance to MTZ by E. histolytica trophozoites has been observed when parasite cultures are exposed to
increasing concentrations of the drug [8 ], [9 ]. These results increased awareness about the development of MTZ resistance in field
conditions when this drug is indiscriminately used [3 ], [10 ].
Therefore, it is imperative to identify or develop novel anti-amoebic compounds with
low toxicity in humans and at low cost, since producing a new drug can be prohibitive,
exceeding one
billion dollars [11 ]. In this sense, plant extracts are considered a natural and affordable source of
new compounds with powerful anti-parasitic activity and safe
use in humans [12 ].
Tabernaemontana (Apocynaceae) genus includes nearly 100 species with pantropical distribution, and
are found in Asia, Africa, Australia, North and South America, as well as in a
variety of oceanic islands [13 ], [14 ]. The plants are evergreen shrubs and small trees that grow between 1 and 15 m tall.
The leaves
are opposite, 3 to 25 cm long, with milky sap, commonly called “milk wood”. The
flowers are fragrant, white and 1 to 5 cm in diameter [13 ]. Plants within the
Tabernaemontana genus are characterized by a high alkaloid content, usually displaying several commonly
known pharmacological activities including antimicrobial, antioxidant,
anti-inflammatory, anticancer, antidiabetic, antihypertensive, antineurodegenerative,
antivenom, wound healing, analgesic, and others [13 ], [14 ], [15 ].
México presents a particularly high number (18) of Tabernaemontana species, such as T. arborea Rose ex J. D.Sm. [14 ]. To the best of our knowledge,
T. arborea has not been recorded to have any ethnomedical application in México, but other species
of this genus are used in Latin America and other countries in traditional medicine,
and active extracts and compounds have been reported. Therefore, we followed a
chemotaxonomic approach. For instance, T. alba Mill., a species found from the south of México to Panama,
is used in traditional medicine to treat skin conditions due to their anti-inflammatory,
analgesic, and anti-microbial properties [15 ]. In addition to the
well-known antibacterial effect of extracts and alkaloid fractions of several
Tabernaemontana species plants [16 ], [17 ], [18 ], [19 ], [20 ], [21 ], anti-parasitic effects have also been reported,
including extracts and alkaloids from T. citrifolia on the gastrointestinal nematode Haemonchus contortus
[22 ], T. elegans, T. pachysiphon, T. peduncularis and T. macrocarpa on the malarial protozoan Plasmodium falciparum
[23 ], [24 ], [25 ], [26 ], T. pandacaqui on the protozoan Trypanosoma
cruzi , causal agent of Chagas disease [27 ] and T. longipes on Trypanosoma brucei , the causal agent of African sleeping sickness [28 ]. Previous phytochemical research has shown T. arborea root bark to be particularly rich in monoterpenoid indole alkaloids (MIAs), including
ibogaine,
voacangine, and vobasine, as well as the bis-indole type alkaloid voacamine [21 ], [29 ].
Due to the clear need for new drugs to treat amoebiasis, this study was aimed at determining
the effect of the alkaloid fraction of T. arborea root bark, and its ibogaine and
voacangine MIAs ([Fig. 1 ]), on the viability of E. histolytica trophozoite cultures and their effect on the development of amoebic liver abscesses
in
hamsters.
Fig. 1 Alkaloids of T. arborea from the alkaloid fraction of the root bark methanolic extract.
Results
Details on the identification and isolation of ibogan-type alkaloid from root bark
of T. arborea methanol extract was previously reported by our group [21 ]. This alkaloid fraction contains three MIAS – ibogaine, voacangine and vobasine
– identified by GC-MS with 33.74, 53.58, and 62.67 mg/g, respectively. It also includes
the
bis-indole type alkaloid voacamine, detected only by HPLC-UV probably due to its
high molecular weight, and it was not quantified ([Fig. 1 ]).
The T. arborea alkaloid fraction and pure alkaloids ibogaine and voacangine were tested for their
amoebicidal effect in cultures of 24, 48 and 72 h of exposure. Results showed that
the
alkaloid fraction significantly decreased the viability of E. histolytica trophozoites after 24 h of exposure at all concentrations tested in a dose-dependent
manner ([Fig. 2 a ]; p < 0.05). When amoebae were exposed for 48 and 72 h to the alkaloid fraction,
only the higher concentrations tested, of 1 and 10 µg/mL, significantly
affected the viability, killing almost all parasites at 10 µg/mL ([Fig. 2 a ]; p < 0.01). Like the alkaloid fraction, ibogaine and voacangine significantly
decreased the amoebic viability in all cultures, but only at the highest concentration
of 10 µg/mL ([Fig. 2 b ] and [c ]; P < 0.01).
No clear differences in the anti-amoebic activity between the two alkaloids were
observed. In agreement, the half-maximal inhibitory concentration (IC50 ) values for the alkaloid
fraction and both alkaloids were quite similar over cultures treated for 24 h,
ranging between 1.4 µg/mL (4.5 µM) for ibogaine and 3.0 µg/mL (8.1 µM) for voacangine
([Table 1 ]). IC50 median values obtained were very close to the metronidazole IC50 of 1.17 µg/mL (6.8 µM) previously reported by us [30 ]. Considering the CC50 values on VERO cells previously reported by our group [21 ], the SI obtained for the alkaloid fraction of T.
arborea root bark and the isolated alkaloids ibogaine and voacangine were of 10.14, 252.8
and 13.6, respectively ([Table 1 ]).
Fig. 2 Effect of T. arborea alkaloids on the amoebae viability. E. histolytica trophozoites (105 /mL) were cultured in the presence of 0.01 to 10 µg/mL of
alkaloid fraction of the root bark methanolic extract (a ) and isolated ibogaine (b ) and voacangine (c ) alkaloids, for 24, 48, and 72 h at 37 °C. Viability was
determined by MTT measuring the absorbance at 595 nm. Vehicle treatment controls
(DMSO 0.2%) that do not affect viability are also included. Values are presented as
means ± SD of three
independent experiments. The asterisks mark the conditions where a significant
difference was found (* p < 0.01).
Table 1 IC50 values of T. arborea alkaloid fraction and alkaloids ibogaine and voacangine on the viability of E. histolytica
cultures.
Alkaloid
Molar mass (g/mol)
IC50 (µg/mL)
IC50 (µM)
CC50 * (µg/mL)
SI
* Cytotoxic Concentration 50 previously reported in [21 ], except for metronidazole, which was previously reported by us in [30 ]; SI: Selectivity Index (CC50 /IC50 ); ND: Not determine
Alkaloid fraction
ND
3.5
ND
35.5
10.14
Ibogaine
310.44
1.4
4.5
354
252.8
Voacangine
368.48
3.0
8.1
40.8
13.6
Metronidazole
171.16
1.17
6.8
> 1000
> 854.7
To determine the changes on the amoebic morphology and the type of death induced by
the treatments, we analyzed amoebic trophozoites exposed for 24 h to the IC50 . [Fig. 3 ] shows that trophozoites treated with the T. arborea alkaloid fraction or the pure alkaloids exhibited a slight increase in granularity
when compared to
the DMSO-treated control (from 13% to 16 – 20%; [Fig. 3 e ] vs. [f–h ]). This increase was small when compared to the increase in
granularity induced by the heat and metronidazole treatments (76 and 31.8%, respectively;
[Fig. 3 c ] and [d ]). On the other hand,
increase in cellular size was only observed in heat-treated trophozoites ([Fig. 3 c ]). No differences in size and granularity were observed between non-treated
amoebae (stained with Annexin or propidium iodide; [Fig. 3 a ] and [b ], respectively) and the vehicle control (DMSO; [Fig. 3 e ]). [Fig. 4 ] shows that trophozoites treated with the alkaloid fraction exhibit a significant
percentage of cells dying by
apoptonecrosis (late apoptosis) and necrosis (11.5 and 7.35%, respectively; [Fig. 4 f ]) when compared to the DMSO treated control (0.84 and 1.10%; [Fig. 4 e ]), and even when compared with the heat and metronidazole treated amoebae ([Fig. 4 c ] and [d ]). In
contrast, the treatment of trophozoites with the pure alkaloids did not induce
a clear pattern of cell death ([Fig. 4 g ] and [h ]).
However, ibogaine induced the same level of apoptonecrosis that metronidazole
(around 1.8%), suggesting a similar mechanism of action ([Fig. 4 g ] vs. [d ]).
Fig. 3 Changes on amoebic trophozoites morphology induced by T. arborea alkaloid fraction and isolated ibogaine and voacangine evaluated at 24 h post-exposure.
Trophozoites
(105 /mL) were cultured in the presence of previously determined IC50 values for 24 h at 37 °C and then analyzed by flow cytometry in a FACSCalibur. a and
b untreated amoebae stained with Annexin or propidium iodide, respectively. c and d amoebae exposed to heat or metronidazole, respectively. e amoebae exposed
to DMSO as vehicle control. f – h amoebae treated with T. arborea alkaloid fraction or the pure alkaloids ibogaine and voacangine, respectively. The
results shown are
representative of three independent experiments.
Fig. 4 Evaluation of apoptosis/necrosis death induced by T. arborea alkaloid fraction and isolated ibogaine and voacangine evaluated at 24 h post-exposure.
Type of cell
death was determined using the FITC Annexin V-Sytox green assay. Trophozoites
(105 /mL) were cultured in the presence of previously determined IC50 values for 24 h at
37 °C and then analyzed by flow cytometry in a FACSCalibur. a and b untreated amoebae stained with Annexin or Sytox-green for basal autofluorescence
and necrosis,
respectively. c and d amoebae exposed to heat or metronidazole, respectively. e amoebae exposed to DMSO as vehicle control. f – h , amoebae treated with
T. arborea alkaloid fraction or the pure alkaloids ibogaine and voacangine, respectively. The
results shown are representative of three independent experiments.
Considering that the alkaloid fraction from T. arborea and ibogaine and voacangine showed a potent in vitro anti-amoebic activity comparable to that of metronidazole, we decided
to evaluate its effect in vivo on the development of amoebic liver abscesses in hamsters, an animal model widely
used for this purpose [31 ]. Intraperitoneal
administration to hamsters of each treatment one day previous, and two days after
the intraportal challenge did not inhibit the development of liver abscesses in any
animal ([Fig. 5 ]). In detail, the 15 treated hamsters (5 per group) developed numerous large abscesses
throughout the four liver lobules, indistinguishable from those of
non-treated, but infected, animals. No differences were also scored by weighing
the complete livers nor evaluating the abscesses dissected from them (data not shown).
However,
histopathological analysis on tissue sections from treated hamsters revealed lower
number of trophozoites in the alkaloid fraction-treated and alkaloids-treated animals
compared to the control
DMSO-treated animals (Compare [Fig. 5 a ] vs. [b–d ]). Despite this, tissue sections of all treated hamsters showed large necrotic zones
surrounded by a ring of intense inflammatory infiltrate mainly composed of neutrophils,
lymphocytes and few macrophages, a pattern like that observed in the tissue sections
of untreated, but
infected, animals ([Fig. 5 ]).
Fig. 5 Effect of T. arborea alkaloids on the infectivity of amoebae. Golden hamsters were intraperitoneally treated
with T. arborea alkaloid fraction (20 mg/Kg) or
isolated alkaloids ibogaine and voacangine (10 mg/Kg) every other day, for 3
days, starting 1 day prior to infection. Animals were infected by intraporal route
with E. histolytica
trophozoites (106 parasites) and the development of amoebic liver abscesses evaluated 7 days later.
Tissue sections from livers were obtained and stained with
haematoxylin-eosin. Representative images of livers from DMSO (a ), T. arborea alkaloid fraction (b ), ibogaine (c ), and voacangine (d ) treated hamsters
are shown. Arrows show amoebic trophozoites; N: necrotic areas; I: inflammatory
infiltrates. All images were taken at 10 × magnification. Bars: 100 µm.
Discussion
Amoebiasis continues to be one of the most frequent parasitic diseases in the world
and a public health problem in developing countries. Although treatment with metronidazole
is usually
highly efficient, it has the disadvantage of causing many secondary effects that
promote its abandonment [5 ]. Furthermore, the appearance of drug-resistant strains
of amoebae is a latent reality supported by the ease with which resistant cultures
are obtained by continuous exposure to increasing concentrations of the drug [9 ].
As such, amoebiasis requires more efficient control mechanisms, mainly based on
the development of new drugs of natural origin.
In this paper, we show that both the alkaloid fraction and its alkaloids ibogaine
and voacangine, from the plant T. arborea, exhibit a potent in vitro anti-amoebic activity,
almost comparable with metronidazole, the drug of choice for treatment of intestinal
amoebiasis. Anti-parasitic properties of plant extracts of several species of the
genus
Tabernaemontana have previously been reported as mentioned in the introduction. However, few studies
on the anti-amoebic activity of Tabernaemontana extracts are available. In
a survey of herbalists in a central Kenyan town, it was reported that based on
their empirical knowledge, extracts of Tabernaemontana pachysiphon are effective against amoebiasis [32 ]. Studies in India [33 ] demonstrated that Tabernaemontana spp. roots have also been reported to be anti-amoebic, although no
information was provided on effective concentrations for the extracts. In a unique
experimental antique report on the effect of ethanolic extracts from 15 Tabernaemontana species from
Africa, Asia, and Guyana, on E. histolytica DU strain, it was found that extracts from leaves and root and stem barks of all
species showed significant activity killing most amoebae at
4.5 mg/mL, whereas extracts from only three species, stem barks of T. contorta , stem barks of T. penduliflora, and leaves of T. psorocarpa , showed some activity at
0.45 mg/mL [34 ]. They showed that the active substance from leaves, which does not contain alkaloids,
was the seco-iridoid sweroside. In contrast, the compounds
responsible for pharmacological activity in the extracts of stem barks were not
identified; however, they suggested these could be the alkaloids [34 ].
To our knowledge, our work is the first report regarding the anti-parasitic activity
of T. arborea . Here we show that the alkaloid fraction and two MIAS from the root bark have potent
anti-amoebic activity, inhibiting the growth of cultures after 24 h post-exposure
with a striking IC50 lower than 4 µg/mL (4.5 and 8.1 µM for ibogaine and voacangine, respectively).
In addition, the alkaloid ibogaine showed a selectivity index (SI) of 252.8, which
was very high in comparison to the SI of the alkaloid fraction and voacangine, suggesting
that ibogaine is of
low toxicity to mammalian cells. Although our study is not completely comparable
with those mentioned above, since different strains of amoebae and extracts of different
plant species were
used, alkaloids of T. arborea seem to be much more active in killing amoebae at concentrations that were 2 to 3
orders of magnitude lower. It is worth emphasizing that both the alkaloid
fraction and the isolated MIAs ibogaine and voacangine exerted anti-amoebic effects
almost comparable to that of metronidazole (IC50 6.8 µM), the drug of choice for the treatment of
amoebiasis. However, the SI of metronidazole was much higher than that of the
compounds tested, suggesting that it is even less toxic to VERO cells. There are few
cases in the literature of
compounds with amoebicidal activity equal to or close to metronidazole, with auranofin
being perhaps the most recently known compound [35 ], which has even passed
phase I clinical trial [36 ].
In a previous report, our group identified MIAs ibogaine and voacangine in T. arborea root bark methanol extract [21 ]. In contrast to the moderate
antimycobacterial activity shown by these alkaloids (IC50 between 16 and 40 µg/mL), both showed potent amoebicidal activity, causing the death
of all parasites in 24 h at a
concentration of 10 µg/mL and yielding an IC50 between 1.4 and 3.5 µg/mL, as mentioned above. It is worth mentioning that the anti-amoebic
effect was lost between 48 and 72 h
post-exposure at concentrations below 10 µg/mL, suggesting their processing to
inactive metabolites. Anti-bacterial and anti-fungal activity of ibogaine and voacangine
has been widely reported
[15 ]. In contrast, anti-parasite activity has been scarcely studied. As with the amoebae,
voacangine and ibogaine have been shown to be highly effective against
microfilariae and male adults of Onchocerca ochengi
[37 ]. Moreover, it has been shown that a Tabernaemontana catharinensis extract, rich in voacangine (53%) exhibited potent antileishmanial activity against
L. amazonensis
[38 ]. Other alkaloids of natural origin, such as those related to emetine, have been
shown to have a powerful anti-amoebic effect, but most of their studies have
focused only on their in vitro effects [39 ]. Of them, for instance, 3-isocorynantheol has been recorded in PubChem as anti-amoebic
with the AID 1 097 036
(https://pubchem.ncbi.nlm.nih.gov/bioassay/1097036 ).
Our results also showed that ibogaine and voacangine from T. arborea can cause the death of E. histolytica trophozoites by rapid or late necrosis (apoptonecrosis), suggesting
that they could cause damage to the cell membrane. However, the cell targets through
which they exert their anti-amoebic effect are unknown and further studies are needed
to identify the
possible receptor(s) for the alkaloids, as well as the intracellular changes they
induce. Similar alkaloids, like cinchona, has been reported to possess anti-malarial
activity acting by a
mechanism alike to chloroquine, inhibiting the crystallization of the heme group
in the digestive vacuole of the parasite [40 ]. Taken together, these findings
point to the possibility of using alkaloids as anti-parasitic agents, particularly
as anti-amoebics.
Since the effect of T. arborea alkaloid fraction, ibogaine, and voacangine was comparable to that of metronidazole,
we decided to evaluate the ability of these compounds to inhibit the
development of amoebic liver abscesses in hamsters. The results showed that although
ibogaine and voacangine appear to reduce the number of parasites seen in histological
sections of livers,
abscess formation from the infection was not affected. This suggests that the
remaining amoebic trophozoites were enough to promote abscesses by triggering and
inflammatory responses that
usually contribute to the pathology, as previously reported [41 ]. The reason for the lack of protection is unknown but could be related to the speed
at which the
alkaloids are catabolized in the blood of animals before they can reach the liver
from the peritoneal cavity, where they were inoculated. In this regard, it has been
reported that the
half-life of ibogaine in rat plasma is only 2 h, rapidly o-demethylated to form
the metabolite 12-hydroxyibogamine (noribogaine) [42 ]. Conversely, a low
bioavailability of voacangine on rats has also been reported [43 ]. With this possibility in mind, we conducted trials administering the alkaloid fraction
of T.
arborea root bark, ibogaine, and voacangine via the vena cava using an indwelling catheter.
However, the results were like those obtained when the compounds were administered
by
intraperitoneal route (data not shown). Since ischemia is characteristic of amoebic
liver abscesses [44 ], studies on the bioavailability of these compounds to the
liver and the possible processing of them by amoebae are warranted to understand
the cause of their lack of protective effect. Furthermore, trials on the effect of
these compounds on
intestinal amoebiasis and in vivo cytotoxicity studies on human cells are also encouraging, to determine the potential
of T. arborea MIAs ibogaine and voacangine as a new
anti-amoebic drug.
Material and Methods
Plant material and preparation of alkaloid fraction
Tabernaemontana arborea Mill. was collected from the roads leading to the “Estación de Biología Tropical
Los Tuxtlas” in the state of Veracruz, México. A voucher specimen was
deposited at the herbarium of the Facultad de Ciencias (FCME), UNAM (voucher
number 133 359) [29 ].
Crude extracts from the root bark were obtained by extraction with methanol, from
which the alkaloid fraction was obtained by acid-base extraction and analyzed by GC-MS,
and HPLC as
described in [21 ], and where the chromatograms can be consulted. Pure ibogaine and voacangine kindly
donated by Phytostan Enterprises, Inc. were used as external
standards in these analyses.
Stock solutions
The stock solutions used in this work were prepared by solubilizing 10 mg of alkaloid
fraction, ibogaine or voacangine in 1 mL of DMSO. All subsequent dilutions were also
made in DMSO,
which never exceeded 0.2% in the tests, a concentration previously shown that
does not affect the growth of E. histolytica trophozoites (data not shown).
Cultures of Entamoeba histolytica trophozoites
Axenic E. histolytica trophozoites of HM1:IMSS strain (originally isolated from a patient in México) were
maintained in TYI-S-33 medium supplemented with 15% adult bovine serum
(Microlab) and 3% Diamond vitamins (SAFC, Biosciences) at 37 °C in anaerobic
conditions. Trophozoites grown for 72 h (log-phase) were harvested by ice-chilling
for 5 min and centrifugation
at 150 × g for 5 min at 4 °C. Trophozoiteʼ virulence was maintained through successive
passages in golden hamster livers and then recovering parasites from the induced amoebic
abscesses
[31 ].
Evaluation of activity on trophozoite cultures
Viability of E. histolytica trophozoites was performed, as previously described [30 ]. In brief, trophozoites were seeded in 96-well culture plates at
104 cells/100 µL of fresh TYI-S-33 medium. Tabernaemontana arborea alkaloid fraction, ibogaine, or voacangine were added at concentrations between 0.1 – 10 µg/mL.
Once
added, the plates were incubated for 72 h at 37 °C and the viability determined
each 24 h by taken an aliquot and performing an MTT assay. In brief, 80 µL of MTT/well
(1 mg/mL) was added and
the plates were incubated for 1 h at 37 °C in darkness. Thereafter, the plates
were centrifuged at 400 × g for 5 min and the supernatant removed to preserve a volume
of 100 µL per well. Each
well was added with 100 µL of 60 °C heated sodium dodecyl sulphate : hydrochloric
acid (15% SDS, 0.01 N HCl) and then homogenized to dissolve formazan salt. Finally,
the absorbance was
measured within 15 min at 595 nm using a Synergy HTX (BioTek) plate reader. The
IC50 was obtained using the web IC50 calculator AAT Bioquest, at https://www.aatbio.com/tools/ic50-calculator (version 2021). The SI was obtained by dividing the CC50 of the alkaloid
fraction of T. arborea root bark and the isolated alkaloids ibogaine and voacangine on VERO cells at 24 h
over the respective IC50 .
Apoptosis/necrosis analysis by flow cytometry
Flow cytometry studies were performed to determine whether T. arborea alkaloid fraction, ibogaine, and voacangine alkaloids lead to amoebic death by apoptosis
or necrosis. In brief,
E. histolytica trophozoites were cultured at 104 cells per 100 µL of fresh TYI-S-33 medium in 96-well culture plates. Amoebae were
treated with the IC50 for
the alkaloid fraction, ibogaine, or voacangine determined in the viability assays
and cultured during 24 h at 37 °C. Trophozoites cultured with metronidazole (10 µg/mL)
for 24 h at 37 °C or
incubated for 30 min at 56 °C were used as positive controls of death. Treated
amoebae were harvested by chilling on ice for 5 min and centrifugation (150 × g for
5 min) followed by two
washes with PBS. The parasites were resuspended in 50 µL of 1 × Annexin V binding
buffer (BD Pharmingen), and 5 µL of rh Annexin V-FITC (Enzo) and 500 nM Sytox Green
(Thermo Fisher) were
added. After 20 min at room temperature in darkness, 450 µL of 1 × Annexin V
binding buffer was added and the samples analyzed using a FACSCalibur (Beckton Dickinson)
flow cytometer. At
least 104 gated events of each sample were considered. Data were analyzed using software package
FlowJo v10 (BD Biosciences) to determine changes in size, granularity, and
fluorescence intensity.
Evaluation of activity on amoebic liver abscesses
Male hamsters (Mesocricetus aureus ) of 6 weeks old and weighing about 100 g were divided into four groups of five animals
each (provided by the animal facility of the Faculty of
Medicine, UNAM). One group was intraperitoneally injected with 100 µL T. arborea alkaloid fraction (20 mg/Kg) and the other two groups with 100 µL ibogaine (10 mg/Kg)
or 100 µL
voacangine (10 mg/Kg), every other day, for 3 days, starting 1 day prior to infection
(− 1, 1 and 3 day). The vehicle in which the samples were prepared (DMSO) was administered
to the fourth
group as control. Hamsters were then infected with E. histolytica trophozoites as described below. In brief, animals were anesthetized (Anestesal,
60 mg/kg) and the peritoneal cavity
opened by surgical laparotomy. The portal vein was exposed and 106 axenic trophozoites resuspended in 100 µL PBS were injected into the portal vein
bloodstream. The inoculation
site was immediately occluded with sterile gel foam, the intestines returned
to the peritoneum and the abdominal walls sutured (Vicryl, 4 – 0). Hamsters were sacrificed
seven days
post-infection using excess of anaesthesia, and the livers weighed. Fragments
of the liver containing abscesses were fixed in 4% paraformaldehyde in PBS and stored
in 30% sucrose for
histology. Tissue sections of 5 µm were obtained in a microtome and haematoxylin-eosin
stained for microscope analysis.
All animals were maintained in a common room under controlled temperature and a 14 h
dark/10 h light cycle, and were inspected by the Internal Committee for the Care and
Use of Laboratory
Animals (CICUAL), Biomédicas, UNAM, ID 273 (date of approval May 2th, 2017).
and Governmental agencies to ensure compliance with institutional and federal regulations
and international
guidelines.
Statistical analysis
Differences between the controls and experimental groups were determined using the
software PAST 3.05 (http://www.toyen.uio.no/~ohammer/ )
with Tukeyʼs and Dunettʼs tests, whereas the differences between the tested substances
and the reference drug were examined by Tukeyʼs test. The IC50 and confidence intervals were
calculated using the software GraphPad Prism 8.
Contributorsʼ Statement
Data collection: J. C. Carrero, V. Curay-Herrera, L. Chacón-Niño, F. Krengel, S. L.
Guzmán-Gutiérrez, M. Silva-Miranda; design of the study: J. C. Carrero, C. Espitia,
R. Reyes-Chilpa, J. P.
Laclette; statistical analysis: L. C. González-Ramírez, R. Bobes-Ruíz; analysis
and interpretation of the data: J. C. Carrero, V. Curay-Herrera, R. Reyes-Chilpa;
drafting the manuscript: J. C.
Carrero, C. Espitia, R. Reyes-Chilpa; critical revision of the manuscript: J. C.
Carrero, R. Reyes-Chilpa, J. P. Laclette.
