Lepidium meyenii (Maca) Varieties Did Not Alter Female Reproductive Parameters in Adult Intact Rats

Abstract

The main goal of this study was to evaluate the effect of three different varieties of Lepidium meyenii (Black, Yellow and Red Maca) during seven estrous cycles in rats. Adult female rats (n=8 per group) were treated with freeze-dried aqueous extract of each maca variety (1 g/kg) during 28 days. The duration of each phase of the estrous cycle and body weight was determined. Animals were sacrificed in first estrous phase after seven estrous cycles and the number of ova from oviduct, wet uterine and body weight and estradiol levels were recorded. Total polyphenols content found in black, yellow and red maca freeze-dried was 0.56, 0.57 and 0.58 g/100g of lyophilized respectively. The maca varieties did not affect any phase of estrous cycle, number of ova recovered within oviduct, serum estradiol levels, wet uterine and body weights as compared with control group (vehicle). Three varieties of maca did not present effects on the regulation of the number of ova released at ovulation in adult intact female rats.

Full text

Volume 5, Issue 1 2008 Article 15
Journal of Complementary and
Integrative Medicine
Lepidium meyenii (Maca) Varieties Did Not
Alter Female Reproductive Parameters in
Adult Intact Rats
Manuel Gasco, Universidad Peruana Cayetano Heredia
Sandra Yucra, Universidad Peruana Cayetano Heredia
Julio Rubio, Universidad Peruana Cayetano Heredia
Gustavo F Gonzales, Universidad Peruana Cayetano
Heredia
Recommended Citation:
Gasco, Manuel; Yucra, Sandra; Rubio, Julio; and Gonzales, Gustavo F (2008) "Lepidium
meyenii (Maca) Varieties Did Not Alter Female Reproductive Parameters in Adult Intact Rats,"
Journal of Complementary and Integrative Medicine: Vol. 5: Iss. 1, Article 15.
DOI: 10.2202/1553-3840.1121
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Lepidium meyenii (Maca) Varieties Did Not
Alter Female Reproductive Parameters in
Adult Intact Rats
Manuel Gasco, Sandra Yucra, Julio Rubio, and Gustavo F Gonzales
Abstract
The main goal of this study was to evaluate the effect of three different varieties of Lepidium
meyenii (Black, Yellow and Red Maca) during seven estrous cycles in rats. Adult female rats (n=8
per group) were treated with freeze-dried aqueous extract of each maca variety (1 g/kg) during 28
days. The duration of each phase of the estrous cycle and body weight was determined. Animals
were sacrificed in first estrous phase after seven estrous cycles and the number of ova from
oviduct, wet uterine and body weight and estradiol levels were recorded. Total polyphenols
content found in black, yellow and red maca freeze-dried was 0.56, 0.57 and 0.58 g/100g of
lyophilized respectively. The maca varieties did not affect any phase of estrous cycle, number of
ova recovered within oviduct, serum estradiol levels, wet uterine and body weights as compared
with control group (vehicle). Three varieties of maca did not present effects on the regulation of
the number of ova released at ovulation in adult intact female rats.
KEYWORDS: Lepidium meyenii, estrous cycle, oocytes, uterine wet weight, body weight, serum
estradiol levels, estrous phase, female rats
Author Notes: This study was financially supported by a grant from International Foundation for
Science (F/3895-1).
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Introduction
Lepidium meyenii (Maca) grows exclusively in the Peruvian Andes over 4000
m altitude. It has been used traditionally as a nutrient and to increase fertility.
Although yellow maca is most popular and preferable for natives, the plant is
observed in several varieties characterized by different colours of the
hypocotyls (Valerio and Gonzales, 2005). Previous scientific studies have
demonstrated that maca improves spermatogenesis in rats (Gonzales et al,
2001, 2003, 2004, 2006; Chung, 2005; Gonzales et al, 2006), mice (Bustos-
Obregon, 2005) and humans (Gonzales et al 2001).
Studies related to the effect of maca in female reproduction are scarce
(Chacon, 1961; Oshima, et al, 2003; Ruiz-Luna et al, 2005). Chacon (Chacon,
1961) described that treatment with maca in female rats resulted in a high
number of oocytes released to oviduct. However the study design has been
criticized due to the small number of animals. Up to date there is not any
scientific study designed to demonstrate the effect of maca on reproductive
parameters in non pregnant adult female rats.
The present study aims to investigate the effects of three different varieties
of maca (red, yellow and black maca) on estrous cycle, number of oocytes
recovered from the oviduct, and wet uterine and body weights. In addition,
serum estradiol level was measured at the end of each treatment (7 estrous
cycles).
Material and Methods
Animals
Three-month-old Holtzman female rats were used. Rats were housed 4 per
cage and maintained at 22°C with a 12:12 light/dark cycle. Rats w ere provi ded
with Purina laboratory chow and tap water ad libitum. The animals were
treated according to the standards of the National Institute of Health for the
care and use of laboratory animals (National Research Council, 1996). The
I n s t i t u t i o n a l R e v i e w B o a r d o f t h e S c i e n t i f i c R e s e a r c h O f f i c e f r o m t h e
Universidad Peruana Cayetano Heredia approved the study (SIDISI-UPCH:
50682).
Experimental prot ocol
Rats received freeze-dried extracts of Black maca (BM), Yellow maca (YM)
and Red maca (RM), in a single daily dose of 1 g of dried hypocot yls/Kg body
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weight suspended in 2 ml of water. An additional group received 2 ml of
vehicle (distilled water). Each group of treatment included 8 animals. Black
maca, YM, RM or vehicle were administered orally from day 1 to day 28 (7
estrous cycles ) usin g an intubation needle No 18 (Fisher Scientific, Pittsburgh,
Pennsylvania). Rats were treated with maca or vehicle for 28 days because
previous studies demonstrated that after 28 days of maca treatment increased
litter size in pregnant female mice (Ruiz-Luna et al., 2005). The phases of the
estrous cycle were determined by daily vaginal smears,
and all females were
used after they had shown at least two
regular 4-day cycles. The estrus
was
designed as D1, metaestrus and diestrus designed as D2 and proestrus phase as
D3.
At the end of each treatment (at D1 of the next estrous cycle), blood
was collected by cardiac puncture. Rats were sacrificed by excess of ether
inhalation. Uterus was carefully dissected out, cleaned of the adhering
connective tissues and accurately weighed. Also, body weight was recorded at
the end of each treatment.
Preparation of aqu eous extract of the varieti es of Maca
Hypocotyls of Lepidium meyenii, which is cultivated by farmers, were
obtained from Carhuamayo at 4000 m altitude in Junin (Central Andes of
Peru). The plant was authenticated by Irma Fernandez, Botanist from the
Department of Pharmaceutical Sciences, Universidad Peruana Cayetano
Heredia. The hypocotyls correspond to the BM, YM, RM varieties. The
vouchers for black maca (IFV 2374), yellow maca (IFV 1885) and red maca
(IFV 2378) were deposited at the Department.
An aqueous extract of the hypocotyls was prepared for each variety. In
brief, 100 g of the dried pulverized hypocotyls were placed in a container with
600 ml of water, and boiled for 60 minutes. Each preparation was left standing
to cool, filtered, and freeze-dried. One gram of dried hypocotyls of BM, YM
and RM produced, 0.56, 0.36 and 0.40 grams of freeze-dried aqueous extract,
respectively. The freeze-dried maca extracts were further diluted to obtain a
dose equivalent to 1g raw material/Kg BW in 2ml of vehicle. These solutions
were placed in small vials and kept in 4° C refrigerator until use.
Assessment of the number of oocytes recovered from the oviduct
Animals were sacrificed at D1 of estrous cycle, post treatment with each of the
three varieties of maca or vehicle. The genital tract was removed and oviducts
were flushed separately with a needle (26G x ½ inch) containing saline (1 ml).
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DOI: 10.2202/1553-3840.1121
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The flushings were examined under low-power magnification. The number of
oocytes in the oviduct was recorded.
Ho rmone assays
After blood collection, serum was obtained by centrifugation at 3000rpm for
10 minutes. Serum estradiol concentrations of rats treated for 28 days with
vehicle, or yellow maca were measured by radioimmunoassay using
commercial kits (Diagnostic Products Co, Los Angeles, USA). The hormone
labeled with iodine-125 was used as radioactive marker. Samples were run in
the same assay to avoid inter-assay variation. The intra-assay variation was
6.42% for estradiol. The minimum estradiol detection level was 0.9pg/ml.
Total Polyphenol Assay
Sample: 50 mg of dry extract was suspended in 30 ml of water, heated on a
water-bath for 30 minutes at 50o C, cooled under running water and transferred
quantitatively to a 100 ml volumetric flask and diluted to 50 ml with water.
After solid material had settled out of the solution the liquid was filtered
through an Ashless filter paper (ALBET ®, DP150 125, Lot: 2164/54/73). The
first 10 ml of the filtrate was discarded. This is a procedure routinely
performed to allow a more homogeneous and clean sample.
The next 5 ml of the filtrate was diluted to 25 ml with water (sample).
Then, 1 ml of this solution was mixed with 0.5 ml of Folin Ciocalteu phenol
reagent and shook for 5 min, after which 4 ml of a 7.5% sodium carbonate
solution was added. Following mixing, the solution was incubated in the dark
for 5 min at 50 0C. After incubation the absorbance was measured at 760 nm
using a spectrophotometer.
Standard: 50 mg of pyrogallol was dissolved in water and diluted to
100 ml with the same solvent. 5 ml of this solution was diluted to 100 ml with
water (standard solution).
One ml of the sample or standard solution was mixed with 0.5 ml of
Folin Ci ocalt eu rea gent an d 4 ml 7.5% w/ v of so di um car b onate. T hen, it was
incubated in the dark for 5 min at 50 0C. Absorbance was read, at 760 nm (A1
or
A3), using water as a blank. The equation was used to calculate the
percentage content of polyphenols expressed as pyrogallol:
12.5 ( A1) m2
A3 x m
1
m 1=Mass of the sample that was analyzed, in grams
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m 2= Mass of pyrogallol, in grams
A1 = Total polyphenols absorbance
A3 = Standard absorbance
Statistical analysis
Data were analyzed using the statistical package STATA (version 8.0) for
personal computer (Stata Corporation, 702 University Drive East, College Station,
TX, USA). Data are presented as mean ± standard error of the mean (SEM).
Homogeneity of variances was assessed by the Bartlett test. If variances were
homogeneous, differences between groups were assessed by two-way analysis of
v a r i a n ce ( A N O V A ) t e s t . D i f fe r e n c e s b e t w e e n p a i r o f m e a n s w e r e a s s e s s e d b y t h e
Scheffé test. When variance was not homogeneous a non-parametric analysis was
performed. A value of P< 0 . 0 5 w a s c o n s i d e r e d t o b e s t a t i s t i c a l l y s i g n i f i c a n t .
Results
Estrous Cycle
The phases of estrous cycle were similar between groups; treatment with three
different varieties (BM, YM or RM) did no alter D1, D2 and D3 during the
twenty-eight days of treatment with maca (Table 1).
Table 1. Effect of three different varieties of Lepidium meyenii on the duration of
the phases of the estrous cycle in intact Holtzman female rats.
Treatment Dose
(g/kg)
No. of da ys in estrus
(D1)
No. of da ys in
diestrus (D2)
No. of da ys in
proestrus (D3)
Control - 7.43±0.28 15.81±0.34 6.57±0.33
Black Maca 1 6.89±0.20 15.89±0.51 5.78±0.43
Yellow Maca 1 6.20±0.73 17.40±0.45 6.00±0.56
Red Maca 1 8.08±0.47 15.92±0.53 6.00±0.39
Values are mean±SEM. n=8 in each group.
P>0.05 respect to control.
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Body Weight
According to records all animals showed increase in body weight after 28 days
of treatment (7 estrous cycles) with vehicle (control) or each variety of maca.
Mean weight gain was 19.95 g in all groups and there were no differences
between groups. Final body weights were not different in rats treated with
maca (RM, YM or BM) or vehicle (Table 2).
Table 2. Effect of three different varieties of Lepidium meyenii on body
weightg in intact Holtzman female rats
Treatment Dose
(g/kg)
I n i t i a l w e i g h t
(IW)
Final w eight
(FW)
I n c r e a s e i n b o d y
weight
Control - 254±4 277±5 24±3
Bla ck M ac a 1 251±6 270±9 19±4
Yellow Maca 1 250±4 267±5 19±2
Red Maca 1 248±3 266±3 18±2
Values are mean±SEM. n=8 in each group.
P>0.05 respect to control.
Number of oocytes
Regarding to the mean number of oocytes recovered from the oviducts in D1
of estrous cycle, no differences between RM, YM, BM and control group were
observed(P>0.05) (Figure 1).
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F i g u r e 1 . Effect of three varieties of maca (Black Maca, Yellow Maca and
Red Maca) on number of oocytes recovered from rat oviduct on Day 1 (D1) of
estrous cycle. The number of animals was 8 per group. Data are Mean ± SEM.
There were no significant differences between groups (P>0.05)
0
2
4
6
8
10
12
Co n t r ol Black Maca Yellow Maca R e d M a c a
N u m b e r o f o v a p e r g r o u p
Ut erine wet weight
After 28 days of treatment, all variety of Lepidium meyenii (BM, YM or RM)
did not modify uterine wet weight as compared with control group (P>0.05)
(Figure 2).
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F i g u r e 2 . Effect of three varieties of maca on uterine wet weight recovered at
Day 1 (D1) of estrous cycle. Control (vehicle) or Black Maca, Yellow Maca
and Red Maca (1 g/kg). The number of animals was 8 per group. Data are
Mean ± SEM. P>0.05 between different groups of treatment and controls.
20 0
21 5
23 0
24 5
26 0
27 5
29 0
C o n t r o l Black Maca Yellow Maca R e d M a c a
u t e r i n e w e t w e i g h t ( m g )
Serum estradiol levels
The rats were sacrificed at Day 1 (D1) of estrous cycle post treatment with
maca (yellow maca) or vehicle (control group). Treatment with yellow maca
did not affect estradiol levels when compared with control group (P>0.05)
(Figure 3).
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F i g u r e 3 . Effect of Lepidium meyenii (maca) on serum estradiol levels in
intact Holtzman female rats.
0.0
10.0
20.0
30.0
40.0
50.0
60.0
Control M a c a
p g / m l
Total Polyphenol Assay
Total polyphenols content found in black, yellow and red maca freeze-dried
extracts was 0.56, 0.57 and 0.58 g of pyro gallol/100g, respectivel y.
Di scussion
Lepidium meyenii (maca) is a plant in which hypocotyls have been
traditionally employed for its supposed fertility-improving properties since it
was described for the first time in the Century XVII by Bernabe Cobo, a
Chronicler of the Conquest of Peru (Valerio and Gonzales, 2005). Chacon in
her bachelor thesis in 1961 claimed that alkaloids from maca increased the
number of ovarian follicles, affected pituitary function and increased estrogen
levels (Chacon, 1961). The design of the study was based on observation of
histological pictures without any quantitative measurements. Also, in this
study animals were not controlled for day of the estrous cycle. Moreover,
neither pituitary function nor estrogen levels were assessed. The final
conclusion was that number of offsprings increased when maca was
administered. Others authors have demonstrated that after 28 da ys of treatment
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with maca increased litter size in pregnant female mice (Ruiz-Luna et al.,
2005).
The present study demonstrated that maca did not affect the number of
oocytes recovered from rat oviduct after 7 estrous cycles rejecting the
hypothesis from Chacon (1990) who suggested that maca increased the
number of follicles (Chacon, 1990). The data collected under the experiment
conditions (dose, duration of treatment, age and strain of animals, etc.) of the
present study also eliminated the possibility that alkaloids were active
principles responsible for increasing ovarian function. Moreover, our findings
demonstrated that serum estradiol levels were not affected by administration
of maca. Different factors may alter the estrous cycle like stress, light,
hormones and temperature (Consoli, 2005; Vinogradova, 2006). Maca
varieties did not modify any phase of estrous cycle during treatment; therefore,
we can suggest that maca did not exert stressor and hormonal effects in intact
female rats.
I n m a l e s , i t h a s b e e n o b s e r v e d t h a t b l a c k m a c a a n d y e l l o w m a c a
increase spermatid count in testis and epididymis sperm count (Gonzales,
2006). However, in the present study maca did not affect ovarian function in
female rats. Instead maca affected on the survival of embryos as demonstrated
previously by Ruiz-Luna et al (2005).
The roles of gonadotrophins on follicular growth and oocyte
maturation (Kurowicka et al, 2006; Mihn et al, 2006; Hillier, 2004) and
acceleration of oviductal transport of oocytes induced by estradiol in cycling
and mating rats (Shoham, 2002; Orihuela et al, 1999, 2001; Rios et al 1997)
have been widely studied. Data from the present study suggest that estrogen-
like activity is not present in the extracts of different varieties of maca. This is
confirmed by the failure to increase uterine weight after treatment with maca.
In conclusion, our results demonstrated that black maca, yellow maca
and red maca administer ed during seven estrous cycles (28 da ys) did not affect
the phases of estrous cycle, number oocytes recovered from the of oviduct,
uterine wet and body weight.
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