Doseresponse effect of black maca (Lepidium meyenii) in mice with memory impairment induced by ethanol

Abstract

Previous studies have shown that black variety of maca has beneficial effects on learning and memory in experimental animal models. The present study aimed to determine whether the hydroalcoholic extract of black maca (BM) showed a dose-response effect in mice treated with ethanol 20% (EtOH) as a model of memory impairment. Mice were divided in the following groups: control, EtOH, ascorbic acid (AA) and 0.125, 0.25, 0.50 and 1.00 g/kg of BM plus EtOH. All treatments were orally administered for 28 days. Open field test was performed to determine locomotor activity and water Morris maze was done to determine spatial memory. Also, total polyphenol content in the hydroalcoholic extract of BM was determined (0.65 g pyrogallol/100 g). Mice treated with EtOH took more time to find the hidden platform than control during escape acquisition trials; meanwhile, AA and BM reversed the effect of EtOH. In addition, AA and BM ameliorated the deleterious effect of EtOH during the probe trial. Correlation analyses showed that the effect of BM a dose-dependent behavior. Finally, BM improved experimental memory impairment induced by ethanol in a dose-response manner due, in part, to its content of polyphenolic compounds.

Full text

1
Introduction
Maca (Lepidium meyenii Walp.) grows over 4000 m alti-
tude in the Central Peruvian Andes, particularly in Junin
plateau. According to the color of its hypocotyls, ~13 vari-
eties of maca have been described ranging from white to
black (Tello, 1992). Also, dierent biological properties
have been observed among varieties of maca (Gonzales
et al., 2005; Gonzales et al., 2006; Rubio et al., 2006). From
these, black maca (BM) presented the greatest eect on
cognitive function (Rubio et al., 2006) in dierent animal
models of memory impairment (Rubio et al., 2007, 2008).
In fact, it have been demonstrated by others that maca
shows neuroprotective activity (Pino-Figueroa et al.,
2010).
One related explanation to the benecial eect of maca
was related to its antioxidant properties in vitro and in
vivo (Sandoval et al., 2002; Lee et al., 2005). For instance,
BM was able to reduced malonaldehyde brain levels, a
marker related to lipid peroxidation, in ovariectomized
mice (Rubio et al., 2008) supporting the fact that maca
show the capacity to reduce oxidative stress as sug-
gested previously (Rubio et al., 2008; Pino-Figueroa et al.,
2010).
Ethanol administration has been used previously as a
model to induce cognitive impairment in experimental
animal models in order to describe dierent mecha-
nisms related to memory processes (de Oliveira and
Nakamura-Palacios, 2003; Miller and Mooney, 2004;
Izumi et al., 2005; Mameli et al., 2005; Self et al., 2005)
and to evaluate the potential benecial eects of dier-
ent compounds and drugs (Singh et al., 2003; Bao et al.,
2005; Baydas et al., 2005; Khalil et al., 2005; Pinto et al.,
2006). In fact, ethanol is a drug that is rapidly absorbed
and produces eects only in specic brain regions,
including the hippocampus (Matthwes and Silvers,
2004). e neurotoxic eects of ethanol administration
are mainly related to its capacity to induce oxidative
stress (Pinto et al., 2006).
RESEARCH ARTICLE
Dose–response eect of black maca (Lepidium meyenii) in
mice with memory impairment induced by ethanol
Julio Rubio, Sandra Yucra, Manuel Gasco, and Gustavo F. Gonzales
Department of Biological and Physiological Sciences, Faculty of Sciences and Philosophy and Instituto de Investigaciones
de la Altura, Universidad Peruana Cayetano Heredia, Lima, Peru
Abstract
Previous studies have shown that black variety of maca has benecial eects on learning and memory in experimental
animal models. The present study aimed to determine whether the hydroalcoholic extract of black maca (BM) showed
a dose–response eect in mice treated with ethanol 20% (EtOH) as a model of memory impairment. Mice were
divided in the following groups: control, EtOH, ascorbic acid (AA) and 0.125, 0.25, 0.50 and 1.00 g/kg of BM plus EtOH.
All treatments were orally administered for 28 days. Open eld test was performed to determine locomotor activity
and water Morris maze was done to determine spatial memory. Also, total polyphenol content in the hydroalcoholic
extract of BM was determined (0.65 g pyrogallol/100 g). Mice treated with EtOH took more time to nd the hidden
platform than control during escape acquisition trials; meanwhile, AA and BM reversed the eect of EtOH. In addition,
AA and BM ameliorated the deleterious eect of EtOH during the probe trial. Correlation analyses showed that the
eect of BM a dose-dependent behavior. Finally, BM improved experimental memory impairment induced by ethanol
in a dose–response manner due, in part, to its content of polyphenolic compounds.
Keywords: Black maca, ethanol, memory and learning, total polyphenols
Address for Correspondence: Julio Rubio, Department of Biological and Physiological Sciences, Faculty of Sciences and Philosophy and
Instituto de Investigaciones de la Altura, Universidad Peruana Cayetano Heredia, 430 Honorio Delgado Ave. Lima 31, Peru. Tel: +511 319-
0000, extension: 2515. E-mail: julio.rubio.m@upch.pe
(Received 17 January 2011; revised 18 April 2011; accepted 18 April 2011)
Toxicology Mechanisms and Methods, 2011, 1–7, Early Online
© 2011 Informa Healthcare USA, Inc.
ISSN 1537-6516 print/ISSN 1537-6524 online
DOI: 10.3109/15376516.2011.583294
Toxicology Mechanisms and Methods
2011
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17 January 2011
18 April 2011
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© 2011 Informa Healthcare USA, Inc.
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2 J. Rubio
UTXM 583294 Toxicology Mechanisms and Methods
To our knowledge, there is no scientic study that
evaluates the eect of BM on memory in a dose–re-
sponse manner. For this reason, the present study aims
to determine if the hydroalcoholic extract of BM has a
dose–response eect in mice with memory impairment
induced by ethanol administration, as a model of mem-
ory impairment related to oxidative stress. Ascorbic
acid (AA) was used as a positive control (Kumar et al.,
2009) to compare the eect of BM. In addition, the
total polyphenolic content in BM hypocotyls will be
assessed.
Materials and methods
Animals
ree-month-old male mice from the Swiss strain
obtained from the animal house of the Universidad
Peruana Cayetano Heredia were used for the study.
Mice were housed ve per cage and maintained at
room temperature (22°C) with a 12:12 h light/dark
cycle in the animal house at the Universidad Peruana
Cayetano Heredia. Mice were fed Purina laboratory
chow (Agribrands Purina Peru S.A., Lima, Peru) and tap
water ad libitum. Purina is a standard laboratory food
containing protein 18%, carbohydrates 50%, fat 3.5%,
bre 6%, calcium 0.8%, phosphorus 0.8%, vitamins (A,
D, B12, K, E, riboavine, niacin, panthotenic acid, cho-
line chloride, piridoxine, thiamine, biotin, folic acid)
and minerals (copper, Manganese, zinc, iodine and
selenium).
Experimental design
For the present study, 66 male mice (initial body weight:
31.88 ± 0.57 g) were randomized divided in seven groups
(10 or 9 per group) according to treatment: 1) mice
treated with vehicle (Control group); 2) mice treated with
vehicle and a 20% ethanol solution (EtOH); and mice that
received a 20% ethanol solution plus 3) AA (250 mg/kg)
(Kumar et al., 2009); 4) 0.125 g/kg (EtOH + BM 0.125);
5) 0.25 g/kg (EtOH + BM 0.25); 6) 0.50 g/kg (EtOH + BM
0.50), and 7) 1.00 g/kg (EtOH + BM 1.0) of a hydroalco-
holic extract of BM.
Vehicle (distilled water), EtOH, AA and BM were orally
administered for 28 days using an intubation needle No
18 (Fisher Scientic, Pittsburgh, Pennsylvania). EtOH
concentration and administration pathway used in this
study were chosen from a preliminary dose–response
study performed previously by the researchers where
three dierent concentrations (10, 15 and 20% of etha-
nol solution dissolved in distilled water) of ethanol were
studied (data not shown). Also, previous studies demon-
strated that a 20% ethanol solution produced negative
eects on memory and learning tasks (Lukoyanov et al.,
2003; Assunção et al., 2007). Ethanol was administered to
rats, from groups 2 to 7, 1 h before the open eld (day 22)
test and water Morris maze (from day 23 to 28).
All animal procedures were conducted in compliance
with “Guide of the care and use of laboratory animals”
(National Research Council, 1996). e Institutional
Review Board of the Scientic Research Oce from the
Universidad Peruana Cayetano Heredia approved the
study (SIDISI-UPCH: 52763, 2007).
Plant material
e dried hypocotyls of BM were obtained in 2007 from
Carhuamayo, Junin at 4000 m altitude in the Central
Peruvian Andes where it is traditionally cultivated (Valerio
and Gonzales, 2005). Irma Fernandez, a Botanist of the
Department of Pharmaceutical Sciences, Universidad
Peruana Cayetano Heredia, authenticated the identity of
the plant. e voucher (IFV 1885) was deposited at the
Department.
Preparation of hydroalcoholic extract of BM
Hydroalcoholic extract of BM was prepared with aqueous
ethanol (60%, v/v) by percolation at room temperature for
24 h and concentrate at low pressure to constant weight.
e extract was prepared by Eng. Alfonso Higa from
Agroindustrial Chanchamayo (Lima, Peru). One gram of
dried BM hypocotyls produced 0.22 g of hydroalcoholic
of BM. is extract was further diluted in distilled water
to obtain dierent concentrations in 1 ml. Solutions were
placed in vials and kept in a refrigerator at 4°C until use.
Determination of polyphenol content
Total polyphenol content was assessed according to
Folin-Ciocalteu described previously by Kähkönen et al.
(1999). e analysis was made by triplicate. Briey, 1.5 ml
of Folin-Ciocalteu reagent (1:10 v/v) and 1.2 ml of 7.5%
sodium carbonate solution were mixed with 300 µl of the
extract and kept in a dark room for 30 min at room tem-
perature. Pyrogallol (50 µg/ml) was used as a standard.
e absorbance of the sample was measured at 760 nm.
e results are expressed as g pyrigallol/100 g of hydroal-
coholic extract.
Open field test
e open eld test was performed to evaluate the inu-
ence of treatments on locomotor activity. e apparatus
consisted in a square with 90 × 90 cm white oor, which
was divided into 81 equal squares (10 × 10 cm) by black
lines, and surrounded by white walls, 10 cm high. On day
22, mice were placed in the middle of the arena and the
number of crossings with four paws (from one square to
another) was recorded for 5 minutes as a measure for
locomotor activity.
Water Morris maze
is task was adapted for mice from the paradigm orig-
inally described by Morris (1984). e water maze was
a circular pool (65 cm in diameter, 25 cm high), lled
with water (26 ± 1°C) and made opaque with black ink,
to the depth of 20 cm. e pool was divided into four
quadrants. An escape platform (6 cm in diameter, 19 cm
high) was placed in the middle of one quadrant, 1.0 cm
below the water surface, equidistant from the sidewall
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and middle of the pool. e platform providing the
only escape from the water was located in the same
quadrant on every trial. ree dierent starting points
for mice were placed around the perimeter of the pool.
On each of the four training days, all three start points
were used once each in a pseudo-random sequence
so the starting point was dierent every session. e
water maze was always located in a large room with
a number of extra-maze visual cues including (lights,
desks, personal computer and video equipment, etc.).
e experimenter was always sat at the same position.
All experiments were carried out between 10:00 h and
16:00 h.
Escape acquisition
A trial began by placing the animal in the water fac-
ing the wall of the pool at one of the starting points.
If the animal failed to escape on the platform within
120 s it was gently placed there by the experimenter
and allowed to stay for 15 s. e inter-trial interval was
5–10 min. ree escape trails were given to all mice per
day for ve consecutive days (days 23–27 of each treat-
ment). e escape latency was recorded during these
trials.
Spatial memory test
Twenty-four hours after the last training trial (day 28) in
the escape acquisition test, mice were submitted to the
probe trial in which the platform was removed. In the
60-s probe trial, the time in the target quadrant (s) was
obtained as a measure for spatial memory.
Escape latency is dened as the time (s) that mice
required to reach the hidden platform during the escape
acquisition sessions; meanwhile, the target quadrant
is referred to the quadrant in which the platform was
located during the escape acquisition sessions.
Statistical analyses
Data were analyzed using the statistical package
STATA (version 8.0) for personal computers (Stata
Corporation, 702 University Drive East, College Station,
TX). Homogeneity of variances was assessed using a
Bartlett test. If variances were homogeneous, dier-
ences between groups and treatment were assessed by
one-way or two-way analysis of variance (ANOVA). If
the p value, in the ANOVA test, was signicant, the dif-
ferences between pair of means were assessed by the
Schee’s test. Body weight and the time in the target
quadrant were analyzed using one-way ANOVA; mean-
while, escape latency during the water Morris maze
was analyzed using two-way ANOVA. Data are pre-
sented as mean ± standard error of the mean (SEM).
When variances were not homogeneous, dierences
between groups were assessed using Mann–Whitney U
nonparametric test. Data are presented as Median and
interquartile range. Data from the open eld test was
analyzed using this nonparametric test. Data are pre-
sented as Median and interquartile range. In general,
a value of p < 0.05 was considered to be statistically
signicant.
Results
Total polyphenols content in the hydroalcoholic
extract of BM
e total polyphenols content found in the hydroal-
coholic extract of BM was 0.65 g pyrogallol/100 g.
us, the doses of polyphenols were 3.13, 6.25, 12.5
and 25 mg pyrogallol/kg body weight for 0.125, 0.25,
0.50 and 1.00 g hydroalcoholic extract of BM/kg body
weight, respectively.
Body weight
At the beginning, no dierences between groups were
observed regarding to body weight (Control: 31.90 ± 1.64 g;
EtOH: 31.50 ± 1.99 g; AA: 31.56 ± 0.85 g; EtOH + BM 0.125:
31.11 ± 0.87 g; EtOH + BM 0.25: 32.78 ± 1.82 g; EtOH +
BM 0.50: 30.78 ± 0.81 g; EtOH + BM 1.00: 33.10 ± 0.89 g;
p > 0.05). Also, there were no statistical dierences
at end of the study between control (33.90 ± 1.377 g),
EtOH (30.30 ± 1.21 g), AA (32.33 ± 0.88 g) and BM-treated
mice plus EtOH (0.125 g/kg: 32.33 ± 0.94 g; 0.25 g/
kg: 33.78 ± 1.53; 0.50 g/kg: 31.44 ± 0.84; and, 1.00 g/kg:
34.10 ± 0.89; p > 0.05).
Effect of hydroalcoholic extract of BM on locomotor
activity during the open field test
No statistical dierences between control and EtOH
groups were found (194.5 [160.3–235.5] vs. 235.5 [202.5–
270.0]; p = 0.615) regarding to the number of crossings.
In addition, there were no dierences between con-
trol, EtOH, AA (201.0 [181.0–239.0]; p > 0.05) and 0.125
(253.0 [188.5–273.0]; p > 0.05), 0.25 (210.0 [191.0–253.0];
p > 0.05), 0.50 (227.0 [211.0–266.5]; p > 0.05) and 1.00
(215.5 [164.8–271.3]; p > 0.05) g/kg of BM. No dierences
between BM-treated groups were observed.
Effect of hydroalcoholic extract of BM on escape
acquisition and spatial memory
Figure 1 shows the escape latency of mice with memory
impairment induced by ethanol and treated with 0.125,
0.25, 0.50 and 1.00 g/kg of hydroalcoholic extract of
BM. Two-way ANOVA analyses revealed an eect of the
number of days (F4,295 = 193.29, p < 0.001) and groups
(F6,295 = 33.23, p < 0.001). In addition, an eect of the
interaction days x groups was observed (F24,295 = 2.17,
p < 0.01). No dierences between groups were observed
in the rst day of the escape acquisition trial. From day
2 to day 5, control group showed a better performance
than ethanol-treated mice (p < 0.01). In addition, mice
treated with 0.125, 0.25, 0.50 and 1.00 g/kg of BM reach
shorter escape latencies than ethanol group in days 2
(p < 0.05), 3 (p < 0.001), 4 (p < 0.001) and 5 (p < 0.001).
ere were no dierences in mice treated with any dose
of BM (p > 0.05) or control group (p > 0.05) regarding to
escape latency.
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4 J. Rubio
UTXM 583294 Toxicology Mechanisms and Methods
Figure 2 shows the time in the target quadrant during
the probe trial of the water Morris maze. Ethanol resulted
in a reduction in the time spent by the mice in the target
quadrant when compared to control group (p < 0.001).
Mice treated with ethanol also spent a signicantly
shorter time than those mice treated with AA (p < 0.001)
and BM at 0.125 (p < 0.05), 0.25 (p < 0.01), 0.50 (p < 0.001)
and 1.00 g/kg (p < 0.001). In addition, mice treated with
0.125 g/kg showed lower values than controls and those
treated with AA (p < 0.01) and 1.00 g/kg of BM (p < 0.01).
No dierences between control and with 0.25, 0.50 and
1.00 g/kg BM groups were observed (p > 0.05).
No correlation between the number of crossings in the
open eld test and time in the target quadrant (r = 0.29,
Figure 1. Eect of black maca on escape latency (s) in mice with ethanol-induced memory impairment in the water Morris maze. Male
mice received vehicle (Control), ethanol (EtOH), ascorbic acid (AA), and 0.125, 0.25, 0.50 and 1.00 g/kg of BM plus EtOH (BM 0.125, BM
0.25, BM 0.50, BM 1.00, respectively). All treatments were orally administered for 28 days. Escape acquisition trials were performed from
days 23 to 27. Mice were submitted to three trials for 5 consecutive days. Ethanol was administered 1 h before each acquisition session.
Data are mean values ± SEM.
Figure 2. Eect of black maca on the time in the target quadrant during the probe trial in the water Morris maze. Groups: vehicle (Control),
ethanol (EtOH), ascorbic acid (AA) and 0.125, 0.25, 0.50 and 1.00 g/kg of BM plus EtOH (BM 0.125, BM 0.25, BM 0.50, BM 1.00, respectively).
All treatments were orally administered for 28 days. Probe trials were performed on day 28. Ethanol was administered 1 h before the probe
trial. Data are mean values ± SEM. *p < 005 vs. Control group, ap < 005 vs. EtOH group, bp < 005 vs. AA group and cp < 005 vs. BM 0.125
group.
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Dose–response effect of black maca on memory 5
© 2011 Informa Healthcare USA, Inc. UTXM 583294
p = 0.137) and escape latency during the last day of the
escape acquisition session (r = –0.04, p = 0.854) were
found. Moreover, there was a positive correlation between
treatment and the time spent by the mice in the target
quadrant during the probe trial (r = 0.42, p < 0.01) and
negative correlation between treatment and the escape
latency at the 5th day of the escape acquisition session
(r = –0.43, p < 0.01) suggesting a dose–response eect of
BM on spatial memory.
Discussion
It is known that ethanol is able to alter cognitive and
behavioral performance in both humans and labora-
tory animals (Gönenç et al., 2005). In fact, one of the
principal cognitive eects of ethanol is disruption of
learning and memory (Gönenç et al., 2005) by induc-
ing oxidative stress in brain (Pinto et al., 2006) due
its capacity to cross cell membranes, including the
blood-brain barrier (Mansouri et al., 2001). Ethanol
preferentially impairs hippocampal-dependent learn-
ing and memory tasks (Acheson et al., 2001). In fact,
both ethanol and hippocampal lesions impair water
maze performance on spatial learning and memory
tasks (Matthews et al., 1999). Furthermore, ethanol
administration produces lipid peroxidation, which is
an indicator of oxidative stress, in the brain (Mansouri
et al., 2001; Assunção et al., 2007). e outcomes of this
study support the fact that ethanol consumption can
cause memory impairment during the water Morris
maze by increasing escape latency and reducing the
time in the target quadrant during the acquisition trials
and probe trial, respectively. e results observed dur-
ing the water maze in ethanol-treated mice may be due
to its oxidative capacity in brain as mentioned above.
All doses of hydroalcoholic extract of BM showed
inhibitory eects against ethanol-induced memory
impairment during the escape acquisition trials; that
is, mice treated with BM showed shorter escape latency
than mice treated with ethanol. In addition, BM-treated
mice showed a similar behavior than those treated with
AA in the escape acquisition sessions. During the probe
trial, AA- and BM-treated mice showed an increase in
the time spent in the target quadrant in a dose–re-
sponse manner. It is important to notice that the water
Morris maze investigated spatial learning and memory
(D’Hooge and De Deyn, 2001) and it is especially sen-
sitive to impaired hippocampal function (Gage and
Bjorklund, 1986). e latter suggests that BM improved
spatial learning and memory in male mice treated with
ethanol. Also, this is the rst study that demonstrated
that BM eect on spatial learning and memory follows
a dose–response behavior.
Outcomes from the present study showed that poly-
phenol content in BM was 0.65 g pyrogallol/100 g of
hydroalcoholic extract. Polyphenolic constituents in
plants enhance the cognitive performance of rats dur-
ing memory tasks, especially those related to spatial
learning and memory including water Morris maze (Kim
et al., 2004; Barros et al., 2006). e neuroprotective
eects of plants containing polyphenolic compounds
such as quercetin (Naidu et al., 2004; Andres-Lacueva
et al., 2005) and anthocyanins (Ramirez et al., 2005;
Barros et al., 2006; Shin et al., 2006) have been previ-
ously reported. Moreover, the antioxidant eects on
quercetin (Naidu et al., 2004; Farombi and Onyema,
2006) and anthocyanins (Cho et al., 2003; Shih et al.,
2010) on brain have been previously demonstrated.
Previous studies demonstrated that maca hypocotyls
contain quercetin (Lee et al., 2004) and anthocyanins
(Valerio and Gonzales, 2005). For these reasons, the
eect of BM on memory and learning may be due to its
content of polyphenolic compounds and their antioxi-
dant activity as suggested previously (Rubio et al., 2006,
2007, 2008; Pino-Figueroa et al., 2010). In fact, black
was able to reduce brain lipid peroxidation in ovariec-
tomized mice, a model related to estrogen deciency.
Another possibility related to the eect of maca may be
related to the antiapoptotic capacity of its polyphenolic
compounds (i.e., quercetin and anthocyanins). In fact,
other authors found that quercetin could blockade the
activation of caspase cascade in neurons exposed to
amyloid beta toxicity in vivo and in vitro (Wang et al.,
2001).
Also, epidemiological studies found a signicant
correlation between dietary intake of vegetables and
improvement in cognitive function in elderly people (Lee
et al., 2001). In fact, aging women consuming cruciferous
vegetables (e.g., broccoli and cauliower) showed less
cognitive decline than those not consuming them (Kang
et al., 2005). Members of the genus Lepidium, including
maca, belong to the cruciferous (Brassicaceae) family
and it is possible that this plant may have eects on cog-
nitive functions.
In addition, some novel compounds have been
recently identied, as two new imidazole alka-
loids (lepidine A and B) (Cui et al., 2003). Also, a
benzylated product, named Macaridine, deriva-
tive of 1,2-dihydro-N-hydroxypyridine, together
with the benzylated alkamides (Macamides),
N-benzyl-5-oxo-6E,8Eoctadecadienamide and
N-benzylhexadecanamide, as well as the acyclic keto
acid, 5-oxo-6E,8E-octadecadienoic acid have been
described (Muhammad et al., 2002). However, the
eect of these compounds has not been assessed for
any of the functions described for maca including
learning and memory. For these reasons, future studies
are necessary to elucidate the chemical composition of
BM and the components related to its neuroprotective
eect.
Correlation analyses showed that the eect of BM
on escape latency and time in the target quadrant is no
related to locomotor activity. ese outcomes demon-
strated that the eect of BM on memory and learning
is not related to locomotor activity as showed by others
(Rubio et al., 2006, 2007, 2008).
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6 J. Rubio
UTXM 583294 Toxicology Mechanisms and Methods
Conclusion
Finally, the hydroalcoholic extract of BM inhibits the eth-
anol-induced memory impairment in a dose–response
manner in male rats. Also, the total content of polyphe-
nols such as quercetin and anthocyanins may be related
to the eect of BM on cognitive function.
Acknowledgment
Sandra Barrueta and Sonia Davila are acknowledged for
their help in this study.
Declaration of interest
e present study was supported by a grant obtained from
Peruvian National Council of Sciences, Technology and
Innovation (CONCYTEC) through the grant PROCOM
2008. All authors participated in the experimental design
and development of the study. Also, the authors report
no conicts of interest. e study was performed in
2008. Since 2009, Dr Gustavo F. Gonzales is in charged of
CAYENATUR EIRL an enterprise that develops products
based on Maca.
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