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Mortality and morphological changes in Giardia duodenalis induced by exposure

to ethanolic extracts of Justicia spicigera

Article in Proceedings of the Western Pharmacology Society · February 2001

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Proc. West. Pharmacol. Soc. 44: 151-152 (2001)
Mortality and Morphological Changes in Giardia duodenalis Induced by Exposure to
Ethanolic Extracts of Justicia spicigera
MARTHA PONCE-MACOTELA1*, YADIRA RUFINO-GONZÁLEZ1, JOSÉ I. DE LA MORA-DE LA MORA1,
ANGÉLICA GONZÁLEZ-MACIEL2, RAFAEL REYNOSO-ROBLES2 & MARIO N. MARTÍNEZ-GORDILLO1
1
Parasitología experimental del Instituto Nacional de Pediatría, Insurgentes Sur No. 3700-C, CP 04530 México, D.F., México;
2
Microscopia electrónica del Instituto Nacional de Pediatría, Mexico
Giardia duodenalis is one of the most commonly iso-
lated intestinal parasites associated with diarrhea. It has a
word-wide distribution and is especially prevalent in chil-
dren in developing countries [1]. G. duodenalis may pro-
duce acute or chronic diarrhea, malabsorption and failure
to thrive [2]. Treatment used against Giardia including
drugs related to 5-nitroimidazole, (such as metronidazole
and tinidazole) benzimidazole (albendazole and mebenda-
zole), nitrofurans (furazolidone) and quinacrine [3]. How-
ever, some of these antiparasitic drugs have undesirable
side effects [4]. There can be treatment failures, selection
of resistant strains [5,6] and potential genetic injury [7].
Alternative therapies are desirable. In a previous study,
we found activity against G. duodenalis in a crude extract
from Justicia spicigera [8]. This plant has been used in
traditional medicine for diarrhea treatment. The aim of
this paper is report the J. spicigera ethanol extracts con-
centration at which G. duodenalis was unable to grow in
fresh TYI-S-33 medium, and to show ultra-structural
changes in trophozoites exposed to these plant extracts.
MATERIALS AND METHODS:
Biologic material. G. duodenalis isolates INP231087MM from a
symptomatic pediatric patient and INP020300B2 from a ram were
cultured in TYI-S-33 medium supplemented with bile and 10% of a
mix of human sera [9]. After 72 h, the cells were harvested by chilling
culture tubes in an ice bath for 15 min, and inverting gently to detach
the monolayer. Parasites were concentrated by centrifugation at 800 g
for 5 min, pellets were washed two times with cold PBS and the popu-
lation size was counted on a haematocytometer slide.
Ethanol extract. Justicia spicigera leaves were collected in the
winter of 1999, dried and identified to species level. Dried leaves were
frozen in liquid nitrogen and macerated with a mortar. Broken leaves
were placed in ethanol, in a concentration of 120 mg/ml, for 48 h at
4 °C. The ethanol solution was decanted and centrifuged at 2000 g to
remove leaf debris. Solution was sterilized by filtration across a 0.22
µm membrane, under sterile environment in a laminar flow cabinet.
The alcohol extracts samples were lyophilized, weighted and stored at
4°C in amber bottles.
Assays against Giardia. Trophozoites were exposed to different
concentrations (41 µg - 250 µg ) of J. spicigera ethanol extracts for 2 h
at 37°C. After being washed twice with PBS, pelleted cells were placed
in fresh medium TYI-S-33 and incubated at 37°C. Culture tubes were
examined every 24 h for a week, growing trophozoites were centri-
fuged and the size of the populations counted in an haematocytometer.
MTT-tetrazolium salts reduction to MTT-formazan. One mil-
lion and half trophozoites were placed in 1.5 ml tubes in PBS pH 7.2
151
and exposed to J. spicigera, from 41.7 µg/ml to 417 µg/ml, for 2 h at
37 °C. In all experiments the solvent concentration (ethanol) did not
exceed 0.5%. Cell viability was tested by MTT-tetrazolium salt (3[4,5
dimethilthiazol-2-yl]-5,5 diphenyl tetrazolium bromide) reduction to
MTT-formazan, which is catalyzed by (PMS) phenazine methosulfate
[10]. Briefly, after parasites were exposed to ethanol extracts, tropho-
zoites were washed with PBS, and incubated for 30 min at 37 °C in a
solution that contained 40 µL of MTT-tetrazolium (5 mg/mL) plus 20
µL of PMS (2.5 mg/mL). Formazan synthesized, a purple dye soluble
in isopropanol/hydrochloric acid, was extracted from cells. Concentra-
tion was measured in a spectrophotometer at 570 nm. Lethal dose at
100% was used for ultra-structural experiments. Cells exposed to etha-
nol extracts and control samples were fixed in 2.5% glutaraldehyde in
phosphate buffered saline 0.1 M, pH 7.2, postfixed in osmium tetroxide
1%, dehydrated in ethanol and embedded in LR white resin. Thin sec-
tions (60-90 nm) were mounted on formvar-coated cooper grids and
stained with led citrate-uranyl acetate. Thin sections were observed,
structural changes were seen and recorded in a Carl Zeiss EM-109
transmission electronic microscopy.
Controls were: (i) live parasites grown in TYI-S-33; (ii) tropho-
zoites exposed to tinidazole, a common antigiardia drug; and (iii) cells
killed by three cycles of freezing and thawed.
All experiments were carried out in a blind design. Data were ana-
lyzed by Student t test.
RESULTS: From reculture experiments we found that
the concentration at which trophozoites failed to grow in
fresh TYI-S-33 were for B2 isolate 125 ± 20 µg/mL and
84 ± 20 µg/mL for MM isolate. However, in the viability
assays performed with the reduction of MTT-tetrazolium
salts, it was necessary to increase the extract concentra-
tion to 417 µg/mL to kill 97 ± 2% of the population.
Untreated parasites from isolates B2 and MM showed
classic structures and shape. For G. duodenalis tropho-
zoites exposed to ethanol extracts, 98% of cells lose their
size and shape. Cells appear swollen, nuclear and cellular
membranes were damaged, structures like holes were seen
on nucleus and cytoplasm, ribosomes were displaced and
in some places they gave rise to ribosome cumulus
(Fig. 1).
DISCUSSION: In re-culture experiments we found irre-
versible damage in trophozoites at concentration ranging
between 125 ± 20 µg/mL for B2 isolate obtained from
ram and 84 ± 20 µg/mL for MM isolate from human. In
these concentrations ranges G. duodenalis trophozoites
were unable to grow and repopulate the culture tubes after
72 h at 37°C in fresh TYI-S-33. Some experiments were
maintained for a week, and no trophozoites were seen
growing. On the other hand, there was a differential sus-
ceptibility to plant extract, ram isolate needing a higher
concentration than human isolate. We found a similar
phenomenon of differential susceptibility between G. duo-
denalis isolates from dog, cat and humans [11]. One
explanation of this behavior perhaps is related to the ali-
mentary characteristics of the host. Rams eat grasses, and
it could be that some compounds from grasses have a
similar chemical structure to J. spicigera ethanol extract-
able substances.
Figure 1. Transmission electron micrographs of G. duodenalis tropho-
zoites. Panel A-B control trophozoites, showed normal structure: ri-
bosomes are homogeneous in all cytoplasm, adhesive disk (d),
axonemes (a), ventral lateral flange (f) and nucleus (n). Panel C-D,
trophozoites isolates B2 and MM incubated with J spicigera appeared
swollen with holes in cytoplasm, ribosomes formed cumulus (small
arrows), the nucleus membrane was broken (arrows) and lacunas were
seen. Cal: 1 µm.
The experimental findings are important as they sug-
gest that extractable products from leaves caused an irre-
versible damage in a short time. Ultra-structural data
showed a strong interaction between compounds from the
ethanolic extract and the membranes system. These con-
152
tacts gave rise to changes that finished in nuclear spilling,
due to lost integrity of the nuclear membrane.
In some areas there were ribosome cumulus. These
may be due to either some substance from plant extract
stopping ribosome synthesis or to induction of polyri-
bosome complexes by the plant extract, to synthesis en-
zymes, proteins or other substances to prevent or to com-
pensate for metabolic injury.
The injury mechanisms are quite different from those
observed with derivatives of benzimidazole, which clearly
prevent microtubules polymerization [12]. Nitroimida-
zoles and nitrofurans need to be reduced to give rise to
short-lived cytotoxic intermediates that interact with DNA
and produce loss of helix, strand breakage, and impair-
ment of its template function [3,13]. The mechanism of
action proposed for quinacrine is binding to DNA [14].
With the ethanol extracts from J. spicigera there were
many changes at different cellular levels. First, the glyco-
calyx was destroyed. In some places dorsal membrane
was dissolved and extra-cellular liquid entered, leading to
cell swelling. The nuclear membrane was also broken,
allowing contact between plant extract and cell cytoplasm
and nucleoplasm.
Other species of the genus Justicia have been exam-
ined, and some substances have been isolated and charac-
terized. Some of these compounds have anti-viral activity
[15], or inhibit platelet aggregation [16].
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