Autores
Marinho Bouty, L.F. (UECE) ; Carneiro de Freitas, J.C. (FATENE) ; Pinto Vieira, I.G. (PADETEC) ; Teixeira, M.J. (UFC) ; Moreira Pinto, C.E. (UECE) ; Farias, D.F. (UFPB) ; Amaral de Abreu, C.R. (VERTRAUM) ; Maia de Morais, S. (UECE) ; Fontenele Urano Carvalho, A. (UFC)
Resumo
The search for new antileishmanial compounds is constant, since the drugs used to
the treatment are limited and toxic. Plants constitutes are important
alternatives. To evaluate the toxicity in vivo and leishmanicidal activity in
vitro of an herbal mixture of A. muricata and D. gardneriana extracts.
METHODS: A. muricata extract was mixed with the D. gardneriana extract to prepare
the Murigard®. To evaluate the anti-leishmanial activity, L. amazonensis
promastigotes were incubated with the Murigard®. For the acute toxicity test, a
single oral dose, in increasing concentrations, was performed. For hematology and
biochemistry, blood samples were collected, on the 14th day. The animals were
euthanized and dissected to observe the anatomical-morphological condition.
Palavras chaves
Anona muricata; Dimorphandra gardneriana ; Leishmanicidal activity
Introdução
Leishmaniosis is a complex of chronic infectious zoonoses caused by protozoa of
the genus Leishmania(1). They can present from mild and asymptomatic forms to
severe and symptomatic forms(2). The clinical signs onset is mainly due to
changes in the immune system,(3,4) by the increase of the free radical’s
production, such as nitric oxide.(5,6) Leishmaniosis are among the most
important diseases in Brazil, due to its wide distribution throughout the
country, the occurrence, the clinical severe forms and difficulties related to
both the diagnosis and the treatment.(7)
The search for new antileishmanial agents is constant, since the arsenal of
drugs is limited and presents undesirable side effects.(8) Several compounds
isolated from plants, such as terpenes, sterols, flavonoids, alkaloids,
naphthoquinones and phenolics compounds and have been studied to assess their
effects on Leishmania spp.(9,10)
Phenolic compounds, such as flavonoids, stand out for their good inhibition of
free radicals.(11) Rutin and quercetin isolated from Dimorphandra gardneriana
are known antioxidants and also presented antileishmanial action against both
forms of L. infantum.(12)
Annona muricata acetogenins were also tested against the promastigotes of L.
donovani, L. major and L. mexicana, showing significant activity.(13) Hexane,
ethyl acetate and methanol extracts of A muricata pericarp were tested in vitro
against L. braziliensis and L. panamensis promastigotes, being the ethyl acetate
extract more effective than the reference drug.(14)
Thus, the objective of this work was to evaluate the toxicity in vivo and
leishmanicidal activity in vitro of the Annona muricata and Dimorphandra
gardneriana herbal mixture.
Material e métodos
Plant Material - A. muricata leaves were collected from the Ceará State
University campus in Fortaleza, and the beans of D. gardneriana were collected
in the Crato city, Ceará, Brazil. The several parts of the plants were deposited
in the Prisco Bezerra Herbarium under reference numbers 43951 and 32339,
respectively.
Preparation of the herbal mixture: Preparation of the Annona muricata
hydroalcoholic extract - A. muricata leaves were dried at room temperature and
triturated in knife mills to obtain a powder. The extract of the leaf powder
(1000g) was prepared by maceration using as solvent ethyl alcohol (70%) for a
period of 24 hours. This process was repeated 15 times to obtain the maximum
possible of a crude hydro-alcoholic extract, then the solvent was subjected to
evaporation using roto-evaporator, recovering a crude extract of A. muricata,
approximately 100g (10%) which was used in the preparation of the herbal
complex. Preparation of the herbal mixture: Characterization of acetogenins from
A. muricata by spectroscopic analysis - The plant materials (2kg) were powdered
and immersed in a methanol-H2O solution (80:20, 3.0 L).Preparation of the herbal
mixture: Preparation and analysis of the Dimorphandra gardneriana flavonoid rich
extract - Methanol (400 mL) was added to D. gardneriana seed kernel (50 g) and
submitted to boiling then filtration (while still hot), the extraction is
repeated 6 times. Activity of the herbal mixture on L. amazonensis promastigotes
- To evaluate the anti-leishmanial activity of the herbal mixture, L.
amazonensis promastigotes in early stationary phase were incubated with the
extracts at concentrations of 50, 25, 12.5, and 6.125 g/mL, in a final volume of
500 µL per well. The promastigote cultures containing 5x105 parasites/mL were
incubated during 48 hours at 24 °C and then the parasites were counted.
Laboratory animals and accommodations - Twenty-five female conventional mice
(Swiss), three weeks old, were obtained from the Central Animal Laboratory of
the Universidade Federal do Ceará (UFC-Biocen). The animals were housed at the
Regional Resource Bioprospecting Experimental Animal Laboratory (Bioprospec),
Department of Biology of the UFC, with temperature conditions (23.0 ± 2.0 °C),
photoperiod (12 h light/12 h dark) and humidity (45-55%) controlled. Acute
toxicity test - For the acute toxicity test, a single oral dose of the herbal
complex was performed according to protocol nº 420 of the Organization for
Economic Cooperation and Development.(16) Female mice (n = 5 per group) weighing
between 20 and 24g were used. The sample was resuspended in distilled water and
administered orally at doses of 5, 50, 300 and 2000 mg per kg of body weight,
thus forming 4 groups. Determination of hematological parameters - For
evaluation of hematological parameters, blood samples were collected, on the
14th day of the experiment, via retro-orbital plexus with heparinized capillary
tube aid and
Resultado e discussão
D gardneriana flavonoid rich extract was obtained with 16.0% yield. The HPLC
fingerprint of D gardneriana extract revealed the presence of rutin in higher
yield (78.92%), followed by isoquercetrin (19.75%) and kaempferol 3-rutinoside
(1.33%).
The leaf hydroethanolic extract of A. muricata, after column chromatography, led
to the isolation of a compound which spectroscopic data was compared and show
similar data of Annonacin, main compound previously found in A. muricata leaves.
(15) The toxicology of the plant which has been shown to be due to the presence
of neurotoxic annaceous acetogenins and benzyltetrahydro-isoquinoline alkaloids
has also been updated to provide recent information on its safety aspects.(17)
Based on these data, an herbal mixture with an ethanolic extract from A.
muricata leaves, which contains Annonacin and extract rich in flavonoids from D.
gardneriana seeds was developed. This herbal mixture was named Murigard®.
Murigard® was tested against L. amazonensis promastigote and presented relevant
leishmanicidal activity, with IC50 value of 27.15 µg/mL.
In hematological analysis, there were discrete changes in white blood cells, red
blood cells and platelets counts. The white blood cells count was increased in
50 mg/kg, 300 mg/kg and 2000 mg/kg groups, when compared to control and 5 mg/kg
groups. The red blood cells count was reduced in the group treated with 300
mg/kg, when compared to other groups. Control, 5 mg/kg, 50 mg/kg and 2000 mg/kg
groups did not show significant differences between them. The platelet counts of
the animals that received the 50 mg/kg dose of Murigard® was significantly
higher than control, 300 mg/kg and 2000 mg/kg groups. Control, 300 mg/kg and
2000 mg/kg groups did not show significant differences between them.
The other hematological parameters (hemoglobin, hematocrit, mean corpuscular
volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration,
red blood cells distribution width) did not show significant differences between
the groups tested.
Regarding the biochemical parameters evaluated in this study, differences were
observed, in the serum albumin dosage, in the groups treated with the 50 mg/kg
and 300 mg/kg concentrations of the Murigard®, demonstrating a significant
reduction, when compared to the other groups, including the control group. In
the same way, it happened in the alkaline phosphatase serum dosage, in the group
that received the 50 mg/kg concentration of the Murigard®.
The serum urea and creatinine dosage, in the groups that received the 300 mg/kg
and 2000 mg/kg concentrations of the Murigard®, respectively, showed a
significant increase, when compared to the other groups. However, in the serum
creatinine dosage, the group that received the 300 mg/kg concentration of the
Murigard® showed a significant reduction, when compared to the other groups,
including the control group.
Regarding the organs relative weight evaluation, the thymus showed a significant
reduction in its relative weight in the group that received a 5 mg/kg
concentration of Murigard®, when compared to the other groups.
Similarly, the spleen showed a significant decrease of relative weight, in the
same group, and an increase in its relative weight in the group receiving 2000
mg/kg of Murigard®, when compared to the other groups.
The pancreas, on the other hand, presented an increase in relative weight, in
all groups that received the herbal mixture, when compared to the control group.
Moreover, it was observed that the relative weight did not differ significantly
between the tested groups.
In the duodenum relative weight evaluation, only the group treated with 2000
mg/kg Murigard® was significantly higher than other groups, including the
control. There were no significant differences of jejunum relative weight, but
in relation to the ileum relative weight, 5 mg/kg and 50 mg/kg doses groups,
were significantly lower, and 2.000mg/kg dose group was significant higher, when
compared to other groups.
The bladder and ovaries relative weights, in all treated groups, were
significantly higher than the control group, but uniform among them. Since the
uterine tube + uterus relative weights were significantly lower in the groups
that received 50 mg/kg and 2000 mg/kg Murigard® doses, when compared to control
group and groups that received 5 mg/kg, 300 mg/kg Murigard® doses.
The other evaluated organs (brain, heart, lungs, liver, kidneys, stomach and
cecum) showed no significant differences in mean relative weight among the
groups.
In the histopathological evaluation, architectural alterations were not evident
in cardiovascular structures. Atria, ventricles, valves and major vessels showed
no microscopic changes, as there was no evidence of cytotoxicity in muscle
fibers.
With regard to the digestive tract organs, in relation to the stomach, gastro-
esophageal junction, gastric cardia, body/antrum and pylorus did not have
microscopic changes. There was no evidence of epithelial cytotoxicity, including
the small and large intestines. There were no other significant changes in any
of the doses administered. In the spleen histopathological analysis, a discrete
extramedullary hematopoiesis was observed in all groups.
In the lung, there was no morphological changes in most structures in all groups
analyzed. Bronchial, bronchiolar, bronchioloalveolar transition branches and
alveolar sacs showed no microscopic alterations. However, it was found
atelectasis in all animals, at all doses tested, including the control group. In
the lungs of one animal, treated with 2000 mg/kg of the Murigard®, was found a
calcified thrombus in blood vessel. There were no other significant changes.
In the liver of tested animals, hepatic acini, structures found in the hepatic
portal space and in the central-lobular veins, were structurally unchanged.
There was cloudy swelling (hydropic degeneration) in all animals, including the
control group. Despite being considered a cytotoxic degeneration, it appeared
lightly, not changing the liver markers serum concentrations and it
had no association with the herbal mixture administration/dose. There were still
a small lymphoplasmacytic inflammatory foci, irregularly distributed by the
hepatic lobules.
Kidneys and pancreas were free of morphological changes. Glomerular and tubular
structures, pancreatic acini and Langerhans islets were structurally preserved.
There were no significant microscopic changes.Leishmanicidal activity of the
flavonoids rutin and quercetin, obtained from D gardneriana, was prviously
evaluated. These phenolic compounds showed similar results as the standard drug
Pentamidine, against L. infantum promastigotes forms, and comparable results as
the standard drug Amphotericin B, against amastigote form.(12) Acetogenins of A
muricata leaves were effective against the promastigotes of Leishmania sp.(13)
The ethyl acetate extract of pericarp also showed satisfactory leishmanicidal
activity against Leishmania sp.(14)
Annona glauca (Annonaceae) seeds dichloromethane extract was active against
three Leishmania species (L. donovani, L. braziliensis and L. amazonensis)
strains. Nine know acetogenins were isolated and identified and then evaluated
in vitro against Leishmania species and the bloodstream forms of Trypanosoma
cruzi. Annonacin A and goniothalamicin showed activity against Leishmania.(18)
Corossolone and anonacinone, extracted from A. muricata seeds, showed IC50
values with 25.9 and 37.6 µg/mL against promastigote forms and 28.7 and 13.5
µg/mL against amastigote forms of L. infantum.(19)
The host response to the Leishmania spp presence triggers an increase in
oxidative stress, which can cause serious damage to the organism.
Conclusões
Based on what has been exposed and due to the high concentrations of acetogenins,
in A muricata leaves, and flavonoids, in D gardneriana seeds, the Murigard® has
shown low toxicity and promising action for the leishmaniasis treatment. However,
future studies, with in vivo tests performed on naturally infected dogs by
Leishmania spp, are necessary to prove the efficacy of leishmanicidal activity,
presenting itself as an alternative in the control and treatment.
Agradecimentos
Preparation of herbal mixture – LFMB, IGPV, DFF, CEMP, AFUC, SMM; Activity of
herbal mixture on Leishmania amazonensis promastigotes – LFMB, AASS, MJT, FSB,
AFUC, SMM; Acute toxicity test – LFMB, DAV, AASS, MJT, FSB, CEMP, DFF,
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