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Journal of Health Science, 55(2) 178–182 (2009)

Effects of Benzylglucosinolate on Endurance Capacity
in Mice
Mayumi Ikeuchi, a Tomoyuki Koyama, a Shin Takei, b Takao Kino, band Kazunaga Yazawa∗, aaLaboratory of Nutraceuticals and Functional Foods Science, Graduate School of Marine Science and Technology, Tokyo Universityof Marine Science and Technology, 4–5–7 Konan, Minato-ku, Tokyo 108–8477, Japan and b KINOS INC., Times Bldg. 5F, 1–16–9,Shintomi, Chuo-ku, Tokyo 104–0041, Japan(Received May 16, 2008; Accepted December 22, 2008; Published online January 28, 2009)

The present study was designed to determine the effects of benzylglucosinolate on endurance capacity in mice.
Mice were orally administered either vehicle or benzylglucosinolate (0.015 or 0.03 mg/kg) via stomach intubation
for a 6-week period. Benzylglucosinolate-treated mice showed a significantly increased endurance exercise capacity. Benzylglucosinolate significantly decreased blood lactate concentrations and significantly elevated plasma
non-esterified fatty acid (NEFA) during exercise. Lastly, benzylglucosinolate treatment significantly decreased fat
accumulation. These data suggest that benzylglucosinolate enhanced swimming endurance due to increased fatty
acid utilization as an energy source.
Key words —— benzylglucosinolate, endurance capacity, Lepidium meyenii (Maca)

INTRODUCTION
Exercise-induced fatigue has been attributed toseveral factors. First, cells and tissues are damaged by exercise resulting in leakage of myoglobin
and metabolic coenzymes into the blood, as well as
the destruction of red blood cells. Second, exercise requires consumption of energy sources suchas glycogen, and maximally induces metabolism
of internal energy stores thereby depleting thisenergy source. Third, through these processes,
exercise causes production and accumulation ofmetabolism-related substances such as lactic acidin the body.1–3) Therefore recovery from exerciseinduced fatigue requires repair of damage that hasoccurred in the body. Specifically, re-synthesis of
the consumed energy sources and leaked cell andtissue components is needed, as are degradation
and removal of internal reduction substances. Thus
early-stage recovery from exercise-induced damage
and minimizing damage can be considered antifatigue mechanisms; it can be said that anti-fatigue∗To whom correspondence should be addressed: Laboratory of
Nutraceuticals and Functional Foods Science, Graduate Schoolof Marine Science and Technology, Tokyo University of Marine Science and Technology, 4–5–7 Konan, Minato-ku, Tokyo
108–8477, Japan. Tel.: +81-3-5463-0621; Fax: +81-3-54630417; E-mail: yazawa@kaiyodai.ac.jp

functionality is the promotion of recovery from fatigue or minimizing fatigue development. We consider increased resistance to the development of fatigue to be synonymous with increased endurance
capacity.
Lepidium meyenii (Maca) has become increasingly popular as a nutritional supplement, with its
use spreading to medical practice.4) Maca has traditionally been used by Peruvians living at high altitudes as a nutrient, energizer, aphrodisiac, and/orfertility enhancer and physical endurance enhancer,
as well as for male impotence and female hormonal imbalances.5–8) Maca is capable of attenuating stress responses by decreasing changes of corticosterone and glucose levels, as well as reducing
or abolishing stress-induced ulcers and increases ofadrenal gland weight.7)Benzylglucosinolate is one of the constituentsof Maca and is thought to be its active ingredient. However, few studies have examined benzylglucosinolate, and its effects on endurance capacity
have not been previously quantified. The aim of thisstudy was to determine the effects of oral administration of benzylglucosinolate on endurance capacity in male mice.

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Benzylglucosinolate —— Benzylglucosinolate waspurchased from C2.Bioengineering Glucosinolates.com, Copenhagen, Denmark (Purity, 98.9%).
All chemicals used were of the highest analyticalgrade.
Animals —— Four-week-old male ddY mice (SLC,Shizuoka, Japan) were housed in standard cages(21.5 × 32 × 14 cm, 5 mice/cage) under controlled
conditions:temperature (24 ± 1◦ C), humidity(50 ± 2%), and lighting (lights on from 08:00 to20:00). The mice were provided a normal diet (MRstock, NIHON NOUSAN, Kanagawa, Japan) andwater ad libitum.
The animal studies were undertaken according
to the regulations of our laboratory and were in linewith the 1980 guideline titled Notification No. 6 of
the Prime Minister’s Office of Japan.
Swimming Exercise Test Protocol —— The mice
were habituated to laboratory housing for 1 weekprior to the start of the experiments. Thirty mice
were divided into three groups (n = 10 each) thatwere given either vehicle (distilled water) or 0.015
or 0.03 mg/kg benzylglucosinolate via stomach intubations. Intubations were performed at 17:00,
5 days/week for 6 weeks. Swimming exercise
was conduced with mice supporting constant loads(lead fish sinkers, attached to the tail) corresponding to 10% of their body weight. The animals
were considered fatigued when they were unable torise to the surface for 5 seconds.9) The mice performed swimming exercise tests once weekly for
4 weeks. Swimming exercise was carried out in atank (28 × 46 × 29 cm), filled with water to a depthof 26 cm and maintained at 30 ± 1◦ C. To avoid theinfluence of circadian variations in physical activity,10) swimming exercise was done from 11:00 to17:00, a period in which minimal variation of endurance capacity has been confirmed in rats.
After a period of 5 weeks, the mice were madeto swim for 15 min supporting loads corresponding
to 5% of their body weight. Blood samples for lactate, glucose, and non-esterified fatty acid (NEFA)
determinations were collected from the tail vein atseven-time points: prior to exercise, during exercise(at 5, 10, and 15 min), and at 10, 30, and 60 min
after exercise. Lactic acid concentration was determined by Kyowa Medex Kit (Determiner LA,Tokyo, Japan). NEFA was measured by acyl-CoAsynthetase and acyl-CoA-oxidase enzyme method(NEFA C-test Wako, Wako Pure Chemical Indus-tries, Osaka, Japan). Glucose was assayed by acombination of mutase and glucose oxidase (Glucose CII test Wako, Wako Pure Chemical Industries,Osaka, Japan).
One week later, the groups were further subdivided into no-exercise and exercise groups. Exercise groups were made to swim for 15 min with aload of 5% body weight and immediately thereafter
were killed by cervical dislocation. Liver samples
were removed and stored at −20◦ C. Liver glycogen content was subsequently determined using themethod of Lo et al.11)
Statistical Analysis —— Data are expressed asmean ± S.E. Comparisons of swimming capacitybetween control and treated groups and data on
metabolic parameters and tissue weight were assessed by one-way analysis of variance (ANOVA)
and Tukey’s multiple comparison test. Data on liver
glycogen concentration were assessed by two-way
ANOVA. A p-value < 0.05 was used as criterion for
statistical significance.

RESULTS
Effect of Benzylglucosinolate on Exercise Capacity
Mice receiving benzylglucosinolate (0.03 mg/kg)
showed a significant increase of exercise capacityfrom the first week onward (Fig. 1). However,0.015 mg/kg benzylglucosinolate administrationdid not significantly increase exercise capacity.

Fig. 1. Effect of Benzylglucosinolate on Swimming Capacity
in Mice
Swimming capacity was determined in mice administered vehicle ( , control) or 0.015 () or 0.03 mg/kg benzylglucosinolate ().
Swimming was performed with a 10% body weight load attached tothe tails of the mice. Each value represents mean ± S.E. (n = 10).
∗ Significantly different from control group (p < 0.05).
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Effect of Benzylglucosinolate on Glycometabolism during ExerciseIn control animals, 15 min of exercise decreased plasma glucose levels with plasma glucose levels recovering after the end of the exercise period (Fig. 2A). However, in mice administered 0.03 mg/kg benzylglucosinolate, plasma glucose levels were maintained throughout the exercise period (Fig. 2A). In all groups, exercise
increased blood lactate concentrations.
However, blood lactate concentrations were significantly
lower in mice administered benzylglucosinolate(Fig. 2B). Although exercise significantly decreased
liver glycogen content in all treatment groups, liver
glycogen content following exercise was significantly higher in mice administered benzylglucosinolate (Fig. 3).
Effect of Benzylglucosinolate on Lipid
Metabolism during Exercise
In the control mice, plasma NEFA concentrations were unaffected by 15 min of exercise. However, in mice administered 0.03 mg/kg benzylglucosinolate, exercise caused a significant increase ofplasma NEFA levels (Fig. 2C).
No significant differences in body weight
between the experimental groups were observed
during the course of the experiment (control,42.1 ± 1.0 g; benzylglucosinolate 0.015 mg/kg, 42.9 ±1.1 g; benzylglucosinolate 0.03 mg/kg, 42.3 ± 1.2 g).
However, in mice administered 0.03 mg/kg benzylglucosinolate, epididymal adipose tissue weight
was significantly (p < 0.05) decreased (Fig. 4).

DISCUSSION

Fig. 2. Effect of Benzylglucosinolate on Plasma Glucose (A),Blood Lactate (B), and Non-esterified Fatty Acids (C)during Exercise and at Rest
Mice were administered vehicle ( , control) or 0.015 () or
0.03 mg/kg benzylglucosinolate (). Swimming (15 min) was performed with a 5% body weight load attached to the tails of the mice.
Each value represents mean ± S.E. (n = 10); ∗ p < 0.05; ∗∗ p < 0.01 vs.
control group.
In the present study, the effect of benzylglucosinolate supplementation on endurance capacityand fatigue was evaluated in male mice subjectedto swimming exercise. Here, we report that administering benzylglucosinolate significantly prolonged
swimming time. Additionally, we further sought toclarify the mechanism of this effect.
Exercise decreased plasma glucose in control
animals, whereas in mice receiving benzylglucosinolate, plasma glucose levels were maintained. No
difference of amount of glycogen was noted; no exercise groups. Hence benzylglucosinolate is considered not to participate in synthetic promotion of
the glycogen in liver. Whereas, mice receiving benzylglucosinolate exhibited significantly higher liver

glycogen content compared with control animals
following swimming. These results indicate thatbenzylglucosinolate supplementation can moderateNo. 2Fig. 3. Effect of Benzylglucosinolate on Liver Glycogen after
15 min of Exercise
Mice were administered vehicle (control) or 0.015 or 0.03 mg/kg
benzylglucosinolate. Half the animals in each group were subjected
to 15-min exercise (swimming with a 5% body weight load attached totheir tails) and liver glycogen was subsequently determined. : no exercise; : exercise. Each value represents mean ± S.E. (n = 5). ∗ p < 0.05;∗∗ p < 0.01 vs. post-exercise control group. # p < 0.05, ## p < 0.01 vs.
no-exercise group.

Fig. 4. Effect of Benzylglucosinolate on Epididymal Adipose
Tissue Weight

Each value represents mean ± S.E. (n = 10). ∗ p < 0.05 vs. control.

the supply of glucose and/or decrease glucose utilization as an energy source during exercise. In
addition, benzylglucosinolate supplementation significantly decreased exercise-induced blood lactate
concentrations. Since lactic acid is produced as aresult of carbohydrate metabolism, these results areconsistent with the hypothesis that benzylglucosinolate decreases glucose utilization as an energy
source during exercise.
Increasing fatty acid utilization during exercise reduces the glycogen depletion rate and improves endurance exercise performance.12) Therefore increased fatty acid utilization is thought important for endurance performance. Caffeine is ameaningful example of this because caffeine in-181

creases plasma free fatty acid (FFA) concentrations
and reduces glycogen depletion.13) Oral administration of capsaicin successfully improves endurance
capacity during prolonged exercise.14, 15) This increase was associated with enhanced lipolysis andsparing of stored glycogen, resulting in delayed
glycogen depletion by increasing circulating catecholamines. The enhanced availability of NEFA
is thought to cause greater fat metabolism in active muscles, which in turn decreases carbohydrate
utilization and leads to increased exercise capacity.16) In the control animals, exercise decreased
plasma NEFA concentrations, whereas in mice receiving benzylglucosinolate, exercise significantly
increased plasma NEFA levels. These results indicate that it is likely that benzylglucosinolate favors
lipid utilization more than glucose utilization as anenergy source during exercise. While benzylglucosinolate supplementation had no significant effecton body weight, mice receiving 0.03 mg/kg benzylglucosinolate had significantly decreased adipose
tissue weights. Benzylglucosinolate is thought toincrease hormone-sensitive lipase activity and enhance fat mobilization from adipose tissues, resulting in increased plasma NEFA levels. We intend to investigate this mechanism in detail. The
metabolic effects of benzylglucosinolate on increasing endurance performance are consistent with thehypothesis that increasing fatty acid utilization as
an energy source leads to glycogen sparing. Thus,
preserved glycogen would be available as an energy
source during prolonged exercise, delaying the onset of fatigue.
The experimental dose used in this study waspreviously determined (during the swimming endurance test) as the effective quantity (Ikeuchi,et al., unpublished results). Generally, in commonlyused dietary supplement (extract of Maca), about
2 mg of benzylglcosinolate is contained. This quantity can sufficiently be taken in by human. We thinkthat the same effect as observed in our animal experiment may be transferable to human.
In conclusion, our study suggests that benzylglucosinolate may have beneficial effects on endurance exercise capacity. Administration of benzylglucosinolate causes increases of fatty acid utilization as an energy source, leading to sparing ofliver glycogen. However, comprehensive chemicaland pharmacological research is required to determine the precise mechanism of benzylglucosinolate
in enhancing endurance exercise capacity.

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