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e Scientic World Journal
Volume , Article ID , pages
http://dx.doi.org/.//
Research Article
Acute Toxicity and Genotoxic Activity of Avocado Seed Extract
(Persea americana Mill., c.v. Hass)
Eduardo Padilla-Camberos, Moisés Martínez-Velázquez,
José Miguel Flores-Fernández, and Socorro Villanueva-Rodríguez
Centro de Investigaci´
on y Asistencia en Tecnolog´
ıa y Dise˜
no del Estado de Jalisco, A.C., Avenida Normalistas 800,
Colonia Colinas de la Normal, Guadalajara 44270, JAL, Mexico
Correspondence should be addressed to Eduardo Padilla-Camberos; epadilla@ciatej.net.mx
Received August ; Accepted September
AcademicEditors:D.MatthopoulosandI.V.Sharakhov
Copyright © Eduardo Padilla-Camberos et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
e use of vegetal extracts requires toxicological and genotoxic evaluations to establish and verify safety before being added to
human cosmetic, pharmaceutical medicine, or alimentary products. Persea americana seeds have been used in traditional medicine
as treatment for several diseases. In this work, the ethanolic seed extract of Persea americana was evaluated with respect to its
genotoxic potential through micronucleus assay in rodents. e frequency of micronuclei in groups of animals treated with avocado
seed extract showed no dierences compared to the negative control (vehicle); therefore, it is considered that the avocado seed
extract showed no genotoxic activity in the micronucleus test.
1. Introduction
e fruit of Persea americana, commonly known as avocado,
is an edible fruit from Central America which is easily
adaptable in tropical regions []. e avocado has an olive-
green peel and thick pale yellow pulp that is rich in fatty
acids such as linoleic, oleic, palmitic, stearic, linolenic, capric,
and myristic acids. is fruit is normally used for human
consumption, but it also has been used as a medicinal plant
in Mexico and elsewhere in the world [].
e avocado seed represents –% of the fruit, and it
is a byproduct generally not utilized. Normally, the seed is
discarded during the processing of the pulp. e seed waste
may represent a severe ecological problem []. However,
atthesametime,itmaybeofinteresttoindustryasa
source of bioactive compounds. Its chemical composition is
comprised of phytosterols, triterpenes, fatty acids, and two
new glucosides of abscisic acid [].
Several biological activities of the avocado seed have
been reported such as antioxidant, antihypertensive, larvi-
cidal, fungicidal, hypolipidemic, and recently amoebicidal
and giardicidal activities [–]. Additionally, several studies
havefocusedontheevaluationofacutetoxicityofthefruit
and leaves []. Avocado leaves showed cardiotoxic eects in
mammals and birds [–]. Similarly, the mutagenicity of
fruit and leaves extracts in human lymphocytes has been
assessed []. However, no study has been done to examine
the possible genotoxic activity of avocado seed extract. In
this study, we evaluate the genotoxic eect of a P. americana
seed extract in vivo, by induction of micronuclei in blood
polychromatic erythrocytes of BALB/c mice.
2. Materials and Methods
2.1. Plant Material. Avocados were purchased from Micho-
ac´
an, Mexico. e seeds were dried at ∘Candvacuum
packaged until use.
2.2. Avocado Seed Extract. e P. americana seed extract is an
ethanolic extract obtained through soxhlet reux equipment
and evaporated on a rotary evaporator as mentioned by
Ramos et al. in []. Dried avocado seeds ( g) were
ground to powder in a laboratory mill, defatted with petrol
ether (–∘C),andmaceratedwithfreshlydistilledethanol
e Scientic World Journal
until exhaustion. Aer ltration, extracts were concentrated
under vacuum at ∘C.
2.3. Animals. Forty-ve, eight-week-old male BALB/c mice
( ± g) were purchased from the Zooterio of the University
of Guadalajara. e mice were fed Standard Diet
Tekland and water. ey were kept at room temperature
under a hours of light and hours of dark cycle at ∘C.
irtymicewereemployedforacutetoxicitytestandeen
mice for genotoxicity test. Animals were handled following
the animal care guidelines in accordance with regulations
enacted by the Federal Government of Mexico (NOM--
ZOO- and NOM--ZOO-).
2.4. Acute Toxicity Test. To determine the median lethal dose
(LD50)oftheP. americana seed extract, six groups of mice
each were administered one by one by oral gavage at dierent
dosesconsistingof,,,,andmg/kg
using an orogastric tube (Popper). Mortality was recorded
hours aer the administration of the extract. Animals were
observed during one week to detect signs of delayed toxicity.
2.5. Genotoxicity Test. e genotoxicological study of the P.
americana seed extract was carried out using an identi-
cation and quantication on the erythrocyte micronucleus
formation test. According to the acute toxicity test results,
the most appropriate extract dose for genotoxic study was
selected. ree groups of mice each were employed in
the experiment. e rst group was given P. americana seed
extract at a dose of mg/kg, dissolved in : alcohol-water
solution; the second group was given colchicine dissolved in
physiological saline at a dose of mg/kg and was designated
as the positive control, while the third group was assigned
as the negative control and received the vehicle ( : alcohol-
water solution), all solutions in an amount of mL/kg. At
hours aer administration of treatments, peripheral blood
samples were collected by perforating the caudal vein, and
drops were placed at the prestained slides with acridine
orange as described by Hayashi and Sofuni, [].
e micronucleated cells were scored under a uorescence
microscope. peripheral reticulocytes per mouse were
analyzed, and the frequencies of micronucleated peripheral
reticulocytes were scored in three slides per animal [].
2.6. Statistical Analyses. LD50 value was determined through
Probit analysis []. One-way ANOVA multicomparisons
tests were used to identify any signicant dierence among
genotoxicity tests between animal groups, and Fisher’s least
signicant dierence (LSD) was used to compare signicant
dierences between groups. A 𝑃value <. was considered
statistically signicant. All data was analyzed using the
soware Statgraphics Version XVI.I.
3. Results
3.1. Acute Toxicity. e P. americana seed extract admin-
istrated at doses of , , and mg/Kg showed a
mortalityof,,and%,respectively.eanimalgroups
0
0 125 250 500 1000 2000
10
20
30
40
50
60
70
80
90
100
Mortality (%)
Dose (mg/Kg)
F : Percentage of mortality for determining the acute toxicity
of the P. americana seed extract.
treatedwithandmg/Kg,aswellasthecontrolgroup,
showed no mortality (Figure ). e LD50 calculated for the
avocado seed extract was . mg/kg. According to these
results, a concentration of mg/Kg was determined for
genotoxicity testing.
3.2. Genotoxicity Test. e animal groups administered with
mg/Kg of P. americana seed extract and the negative
control group showed a low amount of micronucleated
cells, while the positive control administered with colchicine
showed clear evidence of harm. ere is no statistically
signicant dierence between the group administered with
the extract of P. americana seed and control group; however,
there is a signicant dierence between both these groups in
regard to the positive control (Table ).
4. Discussion
e avocado (P. americana)isconsumedbyhumansbecause
of its organoleptic characteristics; furthermore, the pulp con-
tains up to % oil, rich in monounsaturated fatty acids [].
e avocado seed is discarded in the majority of countries,
although in some countries such as Niger, it is consumed
[,]. is waste may represent an ecological or human
contaminant.
In a study previously reported on acute and subacute
toxicity of a P. americana aqueous extract, it was not possible
to estimate the LD50 value with the doses tested (up to
g/kg). Also, in repeated doses, toxicity tests during
days showed no aectations in hematological and biochem-
ical parameters. erefore, the authors concluded that the
aqueous extract appears safe at least on an acute and sub-
acute basis []. We showed that the ethanolic extract of P.
americana seed presents acute toxicity with a LD50 value
e Scientic World Journal
T : Micronucleated peripheral reticulocytes (MNRET) formed by P. americana seed extract.
Samples Dose (mg/kg) MNRET/ RET
Number %
Alcohol-water solution 10.6 ± 1.1 1.06 ± 0.11
P. americana seed extract 16.4 ± 4.5 1.64 ± 0.45
Colchicine 189.2 ± 19.3 18.92 ± 1.93∗
e data are presented as mean standard deviation of three repetitions. Asterisks denote statistical dierences compared with control (∗𝑃 < 0.05).
of . mg/kg. e acute toxicity dierences found in
theaqueousandethanolicextractsmaybeduetochemical
components obtained by dierent extraction methods used.
Micronucleus is an excellent genotoxic biomarker [,];
therefore, the staining technique with acridine orange helps
dierentiate micronucleated cells. e evaluation of micronu-
cleus frequencies in vivo is one of the primary genotoxicity
tests recommended internationally by regulatory agencies for
product safety assessment [].Basedontheinternational
working group that evaluates the micronucleus test, this
should be used when no signs of toxicity at maximal possible
concentration are seen [,]. Accordingly, from the acute
toxicity test, the dose of mg/kg was selected for the
genotoxicity test [].
isistherststudyonthegenotoxicityoftheethanolic
avocado seed extract. e micronucleus induction with the
P. americana seed extract showed no statistical dierence
with regard to the negative control, but with regard to the
positive control it did. erefore, it is considered that the P.
americana seed extract showed no genotoxic activity with
the micronucleus test. ere is a study that demonstrates the
genotoxicity of the avocado fruit and leaf extracts in human
peripheral lymphocytes []; however, this study was carried
out in vitro. ere are dierent toxic eects in vitro and in
vivo, especially when it is administered orally.
Reportsofgenotoxicityhaverevealedthatmanyplants
used as food or in traditional medicine have cytotoxic,
mutagenic, and genotoxic properties []. is indicates the
need to understand the toxicological prole of substances that
are in direct or indirect contact with humans.
To complement the toxicological prole of the avocado
seed extract, it is also necessary to test for other areas of
potential damage, such as those related to the immune system
and those that alter endocrine function.
5. Conclusion
e ethanolic extract of the P. americana seed showed an
acute toxic eect at concentration starting at mg/kg. In
vivo mutagenicity on peripheral blood cells of the seed extract
was not observed. However, this study needs to be supported
with experimental toxicity studies using isolated compounds.
e lack of in vivo genotoxic activity of the extract allows us
to hope that the P. americana seedextractcouldbeusedasa
possible food, cosmetic, or pharmaceutical material.
Conflict of Interests
e authors declare that they have no nancial and commer-
cial interests. No conict of interests has been declared.
Acknowledgment
e authors would like to thank Tina Coop for helping in the
translation of this paper into English.
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