Food Chemistry 212 (2016) 443–452

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Morinda citrifolia leaf enhanced performance by improving angiogenesis,

mitochondrial biogenesis, antioxidant, anti-inflammatory & stress
responses
Nor Aijratul Asikin Mohamad Shalan a, Noordin M. Mustapha b, Suhaila Mohamed a,⇑
a
UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
b
Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Morinda citrifolia fruit, (noni), enhanced performances in athletes and post-menopausal women in clinical
Received 23 September 2015 studies. This report shows the edible noni leaves water extract enhances performance in a weight-loaded
Received in revised form 23 May 2016 swimming animal model better than the fruit or standardized green tea extract. The 4 weeks study
Accepted 30 May 2016
showed the extract (containing scopoletin and epicatechin) progressively prolonged the time to exhaus-
Available online 02 June 2016
tion by threefold longer than the control, fruit or tea extract. The extract improved (i) the mammalian
antioxidant responses (MDA, GSH and SOD2 levels), (ii) tissue nutrient (glucose) and metabolite (lactate)
Keywords:
management, (iii) stress hormone (cortisol) regulation; (iv) neurotransmitter (dopamine, noradrenaline,
Morinda citrifolia leaves
Exercise performance
serotonin) expressions, transporter or receptor levels, (v) anti-inflammatory (IL4 & IL10) responses; (v)
Fatigue skeletal muscle angiogenesis (VEGFA) and (v) energy and mitochondrial biogenesis (via PGC, UCP3,
Epicatechin NRF2, AMPK, MAPK1, and CAMK4). The ergogenic extract helped delay fatigue by enhancing energy pro-
Scopoletin duction, regulation and efficiency, which suggests benefits for physical activities and disease recovery.
Mitochondrial biogenesis Ó 2016 Elsevier Ltd. All rights reserved.
Angiogenesis

1. Introduction
Morinda citrifolia fruit, (called noni in USA, Mengkudu in Malay-
sia) has been traditionally consumed by Polynesians to maintain
health and vigor besides combating fatigue or diseases (Thaman,
1990). Two clinical studies on athletes and post-menopausal
women demonstrated the noni juice effect on improving endur-
ance (Langford, Doughty, Wang, Clayton, & Babich, 2004; Palu,
Seifulla, & West, 2008). Another in vivo study on aged mice, given
Abbreviations: AMPK, AMP-activated protein kinase; CAMK4, calcium/
calmodulin-dependant protein kinase IV; DRD2, dopamine receptor D2; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathione; H6PD, hexose-6-
phosphate dehydrogenase; HPA, hypothalamic–pituitary–adrenal axis; HSP90AB1,
heat shock protein 90 kDa alpha (cytosolic) class B member 1; IL10, interleukin-10;
IL4, interleukin-4; MAPK1, mitogen-activated protein kinase 1; MDA, malondialde-
hyde; NADPH, nicotinamide adenine dinucleotide phosphate-oxidase. NR3C1;
NRF2/NFE2L2, nuclear factor erythroid derived 2 like 2; PGC, peroxisome prolifer-
ative activated receptor gamma; PRKAA1, protein kinase AMP-activated alpha 1
catalytic subunit; ROS, reactive oxygen species; SLC6A2, solute carrier family 6
(neutransmitter transporter noradrenaline) member 2; SLC6A4, solute carrier
family 6 (neutransmitter transporter serotonin) member 4; SOD2, superoxide
dismutase 2; UCP3, uncoupling protein 3; VEGFA, vascular endothelial growth
factor A.
⇑ Corresponding author.
E-mail address: mohamed.suhaila@gmail.com (S. Mohamed).
increasing doses of Tahitian Noni Juice orally (10, 20 and 40 mL/kg
body weight) showed significantly longer average time in both the
swim test and the rotarod test when compared with young and
aged control (Ma et al., 2007). However, the fruit have been asso-
ciated with liver toxicity (Millonig, Stadlmann, & Vogel, 2005),
while the leaves are consumed as vegetables after blanching since
ancient times. M. citrifolia leaves reportedly have antioxidant, liver-
protective and wound healing properties without any acute, sub-
acute and sub-chronic oral toxicity (West, Tani, Palu, Tolson, &
Jensen, 2007). The M. citrifolia leaves extract oral no observed-
adverse-effect level (NOAEL) is 1000 mg/kg (Lagarto, Bueno, &
Merino, 2013).
Ergogenic functional foods help improve physical performance
or suppress fatigue, by enhancing energy production, regulation
or efficiency. It is not only useful in sports but also for recovery
from illnesses. Compounds with ergogenic potential include vita-
mins, protein, amino acid, sodium bicarbonate (Shelton & Kumar,
2010), caffeine, creatine monohydrate and herbs (Chen,
Muhamad, & Ooi, 2012). Caffeine for example has anti-fatigue
and alertness effects and the ergogenic anabolic steroids such as
amphetamines have been abused and banned for athletes.
Fatigue may be influenced or caused by (i) excessive levels of
reactive oxygen species (ROS) accumulating in the contracting
muscles, that inhibit force production (Reid, 2001), (ii) muscle

http://dx.doi.org/10.1016/j.foodchem.2016.05.179
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
444 N.A.A. Mohamad Shalan et al. / Food Chemistry 212 (2016) 443–452
contraction-associated pro-inflammatory cytokines increase
(Radak, Naito, Taylor, & Goto, 2012), (iii) energy source depletion
and excess metabolite accumulation (You, Zhao, Liu, &
Regenstein, 2011), or (iv) de-regulation of neuro-immune-
endocrine dysfunction and (v) alteration of Hypothalamic–pitui
tary–adrenal (HPA) axis activity (Gupta, Aslakson, Gurbaxani, &
Vernon, 2007; Rajeevan et al., 2007; Watanabe, Evengård,
Natelson, Jason, & Kuratsune, 2008). This research studies the ergo-
genic and anti-fatigue properties of M. citrifolia leaves water
extract and the molecular mechanisms of actions involved,
through weight-loaded mice swimming test.
2. Material and methodology
2.1. Plant material, aqueous extraction and High-pressure liquid
chromatography (HPLC) analysis
Morinda citrifolia leaves (MCL) were obtained from the Institute
of Bioscience, Universiti Putra Malaysia. A voucher specimen
SK2322/14 was deposited at the Laboratory of Natural Products,
Institute of Bioscience, Universiti Putra Malaysia. Leaves of M. citri-
folia were oven-dried at 60 °C for 24 h, ground and extracted by
boiling in distilled water (1:10, w/v) for 3 h. The resulting suspen-
sions were filtered and evaporated to dryness at 60 °C. Standard-
ized green tea water extract containing 95% polyphenol, 5%
caffeine and 40% epigallocatechingallate, (Seamax Resources Sdn
Bhd, Kajang, Malaysia) were used as the positive control for
comparison.
The extracts were analyzed by HPLC (Waters 2996, Milford,
MA). Epicatechin, scopoletin, HPLC grade methanol (MeOH), ace-
tonitrile (MeCN) and analytical grade trifluoroacetic acid (TFA)
were obtained from Merck (Darmstadt, Germany). Chromato-
graphic separation was equipped with an Atlantis C18 column
(4.6 mm  250 mm; 5 lm, Waters Corporation, Milford, MA,
USA). The pump was connected to a mobile phase system com-
posed of three solvents: A; MeCN, B; MeOH, and C; 0.1 TFA% in
H2O (v/v). The mobile phase was programmed consecutively in lin-
ear gradients as follows: 0 min, 10% A, 10% B, and 80% C; 15 min,
20% A, 20% B, and 60% C; 26 min, 40% A, 40% B, and 20% C; 28–
39 min, 50% A, 50% B, and 0% C; and 40–45 min, 10% A, 10% B,
and 80% C. The elution was run at a flow rate of 1.0 mL/min. The
UV spectra were monitored in the range of 210 and 450 nm. The
injection volume was 50 lL for each of the sample solutions. The
marker compounds scopoletin and epicatechin were present at
6 mg and 9 mg respectively per g of the leaf extract.
2.2. Animals and treatments
Female ICR (Imprinting Control Region) mice, aged 6–7 weeks,
were provided by the Faculty of Veterinary Medicine, Universiti
Putra Malaysia, and acclimatized for a week to the laboratory con-
ditions before the experiment. Animal were housed in an environ-
mentally controlled animal laboratory and maintained on a 12-h
light/dark cycle at 25 ± 2 °C. They were given standard pellet food
(Gold Coins from A Sapphire Enterprise, Malaysia) and water ad li-
bitum. The experimental protocol was approved by the Institu-
tional Animal Care and Use Committee, Universiti Putra Malaysia
(Ref: UPM/IACUC/AUP-R022/2013).
Their baseline initial swimming time endurance capacities were
measured after acclimatization. The mice were divided into 5
groups (n = 10, 5 mice/cage): (i) MCL200 group received 200 mg
M. citrifolia leaves extract/kg bw, (ii) MCL400 group received
400 mg extract/kg bw, (iii) GT positive control group received
200 mg green tea extract/kg bw, (iv) negative control with exercise
(CE) group received the vehicle only and (v) negative control (C)
without exercise group also received the vehicle only. The treat-
ments were given by oral gavage daily for 28 days.
2.3. Weight-loaded swimming test
The swimming endurance capacity was assessed weekly for
4 weeks as the swimming time (s) from start to fatigue. A 5%
weight was tied at each mice tail, to force it to keep swimming
or submerge at fatigue point (and immediately rescued). The
swimming pool water was maintained at 25 ± 2 °C. In the final
week, the mice were sacrificed under ether anesthesia immedi-
ately after exercise. Blood samples were taken by cardiac puncture,
then centrifuged at 2000g for 15 min to separate the serum and
stored at 80 °C until further analysis. The skeletal muscle and
liver tissues were isolated and stored at 80 °C for mRNA gene
expression and histopathological observations.
2.4. Sample analysis
Blood glucose and blood lactate was measured using an autoan-
alyzer (Brand, country). Blood plasma, skeletal muscle and liver
glutathione (GSH), blood cortisol and serum malondialdehyde
(MDA) levels were analyzed using commercial ELISA kits (Cayman
Chemical Company), according to the manufacturer’s instructions.
2.5. mRNA gene expression
Quantitative RT-PCR analysis was performed using the compar-
ative threshold cycle method to calculate fold change in gene
expression of anti-inflammatory cytokines [IL-4 (Interleukin-4)
and IL-10 (Interleukin-10)], angiogenesis marker [VEGFA (vascular
endothelial growth factor A)], antioxidant enzyme [SOD2 (Super-
oxide dismutase 2)], mitochondrial biogenesis or fatty acid meta-
bolism [UCP3 (Superoxide dismutase 2), PGC (Peroxisome
proliferative activated receptor, gamma, coactivator 1 alpha),
NFE2L2 (Nuclear factor, erythroid derived 2, like 2)], anabolic
kinase signalling [MAPK1 (Mitogen-activated protein kinase 1),
PRKAA1 (Protein kinase, AMP-activated, alpha 1 catalytic subunit)],
Ca2+ anabolic signalling [CAMK4 (Calcium/calmodulin-dependent
protein kinase IV)], hypothalamic–pituitary–adrenal (HPA) axis
function [NR3C1 (Nuclear receptor subfamily 3, group C, member
1), H6PD (Hexose-6-phosphate dehydrogenase)], and neurotrans-
mitter [DRD2 (Dopamine receptor D2), SLC6A4 (serotonin trans-
porter: Solute carrier family 6, member 4), SLC6A2(noradrenalin
transporter: Solute carrier family, member 2)], normalized to
beta-actin, GAPDH and HSP90AB1 (Heat shock protein 90 kDa
Gene ID: 3326) as the reference housekeeping gene.
RNA was isolated using Trizol (Invitrogen, Carlsbad, CA). Cus-
tom RT2 Profiler PCR Array (CAPM11988), RT2 SYBR Green
qPCRMastermix, RT2 First Strand Kit and RNase-Free DNase Set
were from SuperArray Bioscience Corporation (Frederick, MD).
Quantitative RT-PCR array for differentially expressed genes was
performed utilizing RT2 Profiler PCR Array Data Analysis version
3.5 (SABiosciences; Fredrick, MD, USA), which normalized to
beta-actin (NM_009793), HSP90AB1 (NM_008302) and GAPDH
(NM_008084) as housekeeping genes. RT-PCR data is represented
as the average relative mRNA gene expression of each experimen-
tal group (n = 3). The p values only for RT-PCR are calculated based
on a t-test of the replicate 2^( DCt) values for each gene in the
control group and treatment groups.
2.6. Statistical analysis
All data are expressed as mean ± S.D. The significance of differ-
ences between groups was determined using one or 2-way analysis
of variance (ANOVA). Differences between groups were further
 N.A.A. Mohamad Shalan et al. / Food Chemistry 212 (2016) 443–452 445

evaluated by Duncan’s post hoc test and considered significant at week 4. The lower dose of 200 mg/kg produced better effects than
p < 0.05. All statistical analysis was performed using SPSS 21.0 the higher 400 mg/kg dose indicating that the optimum dose is clo-
(SPSS Inc., Chicago, IL, USA). ser to 200 mg/kg and the 400 mg/kg may be closer to an overdose.
The results indicate that M. citrifolia leaves water extract signif-
3. Results icantly and progressively extended the duration time to exhaustion
three times longer than the control mice in weight-loaded swim-
3.1. Effects of M. citrifolia leaves extract on time to exhaustion in a ming test, which indicated that MCL have ergogenic and anti-
weight-loaded swimming test fatigue properties. It was much more effective than the green tea
extract which contained 5% caffeine, 95% polyphenol and 45% Epi-
Fig. 1 shows there was no significant difference in the mean gallocatechingallate or the fruit extract.
baseline initial swimming time (D0) between the groups, but sig-
nificant ergogenic effects were already seen by the first week of 3.2. Effect of M. citrifolia leaves extract on the blood glucose, blood
treatment. Treatment with 200 and 400 mg extract/kg BW (Fig 1B) lactate and plasma cortisol levels
significantly increased the exhaustive swimming time by 40% and
28% in the first week, 20% and 14% in the second week, and 50% and The treatments produced no significant effects on blood glucose
41% in the 4th week, respectively, as compared to control CE group. and lactate levels in the exercise groups even though the MCL mice
MCL group given 200 and 400 mg extract/kg also showed signifi- swam three times longer than the other mice groups (Fig. 2A and
cantly longer swimming time as compared to GT group, by 23% B). Blood cortisol levels after exhaustive swimming were signifi-
and 10% in week 1, 20% and 17% in week 2, and 43% and 33% in cantly lower in both the 200 mg extract/kg (2.3 ± 0.4 ng/ml) and

A 3000
d
2500
c
CONTROL
Swimming me (s)

2000 cd
ab GT (100mg/kg)
b
cd b
1500 ab b COFFEE (100mg/kg)
bc ab
bc MCF (100mg/kg)
a a a a
1000 ab a MCF (200mg/kg)
ab a
MCL (100mg/kg)
500 a a a a a a a a
MCL (200mg/kg)

0
W0 W1 W2 W4
Week of treatment

B 4000
c
3500

3000 b
Swimming me (s)

2500
c b C
2000 b
GT (200mg/kg)
b
a a
1500 b a a MCL(200mg/kg)
a MCL(400mg/kg)
1000
a a a a
500

0
W0 W1 W2 W4
Week of treatment

Values are expressed as mean ± SD (n=10). Means with different superscript letters within the
same graph are significantly different (p<0.05).
Fig. 1. Effect of M. citrifolia leaves (MCL) water extract and green tea (GT; positive control) (A) compared to coffee, M. citrifolia fruit extract (preliminary trial); (B) repeated
trials with higher green tea extract dose, on weight-loaded swimming duration to exhaustion.
446 N.A.A. Mohamad Shalan et al. / Food Chemistry 212 (2016) 443–452

Values are expressed as mean ± SD (n=3). Means with different superscript letters within the
same graph are significantly different (p<0.05).
Fig. 2. Effects of MCL supplementation on (A) blood glucose, (B) blood lactate levels and (C) plasma cortisol levels after swimming exercise to exhaustion.

400 mg extract/kg (0.3 ± 0.1 ng/ml) than in the GT (2.9 ± 0.9 ng/
ml), the CE (3.6 ± 0.5 ng/ml), and were nearer to values of the con-
trol without exercise (0.4 ± 0.1 ng/ml) mice (Fig. 2C). Excessive cor-
tisol levels ultimately cause a variety of physiologic problems,
including fatigue.
3.3. Effects of M. citrifolia leaves extract on oxidative status
Fig. 3A, shows supplementation with both 200 and 400 mg/kg
MCL significantly reduced the plasma MDA concentrations by
48% and 59%, respectively as compared to control with exercise
group. Fig. 3B shows the blood plasma GSH concentration in the
200 mg MCL/kg group (8.9 ± 0.9 lM) were twice as high as the C
control without exercise group (4.4 ± 0.2 lM), CE control with
exercise (3.9 ± 0.3 lM), GT (4.4 ± 0.2 lM) and 400mg MCL/kg
(3.9 ± 0.3 lM). However in the skeletal muscle and liver tissue,
the GSH concentrations between the treatments were insignifi-
cantly different in spite of having different swimming time dura-
tions (Fig. 3C and D). GSH is the major non-enzymatic
intracellular redox homeostasis regulator, ubiquitously present in
all cell types. The presence of GSH is essential to prevent cytotox-
icity due to ROS (reactive oxygen species).
 N.A.A. Mohamad Shalan et al. / Food Chemistry 212 (2016) 443–452 447

Values are expressed as mean ± SD (n=3). Means with different superscript letters within the
same graph are significantly different (p<0.05).
Fig. 3. Effects of MCL on (A) MDA level, GSH level in (B) blood plasma, (C) skeletal muscle, and (D) liver tissue after swimming exercise.

3.4. Effects of M. citrifolia leaves extract on anti-inflammatory, (UCP3, PGC, NFE2L2 upregulated about 2-fold in the muscles and
angiogenesis, antioxidant enzyme, and mitochondrial biogenesis up to 7.6-fold in the liver).
markers/fatty acid metabolism mRNA expressions in muscle and liver
tissues
3.5. Effects of M. citrifolia leaves extract on anabolic kinase signalling,
stress hormones, and Neurotransmitter receptor or Neurotransmitter
Fig. 4 shows that the 200 mg/kg MCL up-regulated the mRNA
transporter mRNA expressions in muscle and liver tissues
expression of the anti-inflammatory cytokines (IL-4 up-regulated
2-fold in the muscle and 8-fold in the liver; and IL-10 up-
Fig. 5 shows that the anabolic kinase signalling markers
regulated 3-fold in the muscles and 7-fold in the liver); the vascu-
(MAPK1, PRKAA1), Ca2+ signalling (CAMK4) were upregulated in
lar endothelial growth factor A (VEGFA upregulated 3-fold in the
the muscles and liver (except MAPK1 in the liver). The stress corti-
muscles and 2-fold in the liver); the endogenous antioxidant
sol (H6PD) expression of the hypothalamic–pituitary–adrenal
response elements (SOD2 upregulated 2-fold in the muscles);
(HPA) axis function was downregulated for both the muscle and
and the fatty acid metabolism/mitochondrial biogenesis markers
liver, in agreement with the blood cortisol levels. Additionally
448 N.A.A. Mohamad Shalan et al. / Food Chemistry 212 (2016) 443–452

3.5
3 GT
‘ * * * * * * * * * * * * * * *
2.5 MCL200
2 MCL400
1.5
1
0.5
0
-0.5 IL4 IL10 VEGFA SOD2 UCP3 PGC NFE2L2
-1 Anti-inflammatory
Angiogenesis
Antioxidant
Mitochondrial biogenesis/fatty acid met
-1.5 MUSCLE
-2

10
*
* GT
8 *
*
MCL200
*
6 MCL400
*
* *
4 * * * *
* * * *

0
IL4 IL10 VEGFA SOD2 UCP3 PGC NFE2L2
-2
Anti-inflammatory
Angiogenesis
Antioxidant
Mitochondrial biogenesis/fatty acid me
LIVER

Values represent fold change between control and treatment group, and differed significantly
at p<0.05, represented by ‘*’.
Fig. 4. Effects of M. citrifolia leaves extract on inflammatory, angiogenesis, antioxidant, mitochondrial biogenesis and fatty acid metabolism genes expressions in the muscles
and liver.

the stress hormone nuclear receptor mRNA (NR3C1) were also
downregulated in the liver. The dopamine and noradrenalin neuro-
transmitters (DRD2, SLC6A2), were downregulated in the muscles.
In the liver the dopamine receptor and serotonin transporter
mRNA (SLC6A4) were upregulated.
4. Discussion
Fatigue is influenced by the central nervous system (CNS) such
as alterations in hypothalamic–pituitary–adrenal (HPA) axis, levels
of CNS neurotransmitters (such as serotonin, dopamine and nora-
drenalin) in various brain regions, along with the neuromodulators
cytokines secreted by immune cells. Peripheral factors including
depleted energy and oxygen source for the active muscle fibers,
as well as increases in blood and muscle lactate (metabolic by-
product) levels.
M. citrifolia leaves water extract may alleviate fatigue through
both central and peripheral mechanisms. Glucose as the main
energy source, and lactate as the major metabolite that accumu-
lates during exercise, were measured after the exhaustive exercise.
Although the MCL mice swam three times longer than the control
or green tea mice, their final blood glucose and lactate accumula-
tion level were similar to the control exercise mice. The result
showed that MCL may help improve the blood glucose or nutrient
and lactate levels management to enable them to swim three times
longer than the other mice, before reaching the exhaustive levels.
The improvement is progressive which indicate some anabolic
development including cellular mitochondrial biogenesis or angio-
genesis (new blood vessel formation).
The oxygen free radicals and lipid peroxidases production
increases with the rise in oxygen consumptions during exercise.
Lipid peroxidation which is initiated by the free radicals attacking
 N.A.A. Mohamad Shalan et al. / Food Chemistry 212 (2016) 443–452 449

10

8
GT
6
MCL200
4 * * * * * * * * * * * * * * * * *
MCL400
2

0
MAPK1 PRKAA1 CAMK4 NR3C1 H6PD DRD2 SLC6A4 SLC6A2
-2 Anabolic kinase HPA-axis function Neurotransmitter receptor/transporter
MUSCLE
-4

-6

12

10

8 GT
6 MCL200
4 MCL400
* * * * * * * * * * * * * * * * * * * *
2

0
MAPK1 PRKAA1 CAMK4 NR3C1 H6PD DRD2 SLC6A4 SLC6A2
-2
Anabolic kinase HPA-axis funcon Neurotransmier
-4 receptor/transporter
-6 LIVER

Values represent fold change between control and treatment group, and differed significantly
at p<0.05, represented by ‘*’.
Fig. 5. Effects of M. citrifolia leaves extract on anabolic kinase signalling (cellular energy metabolic pathway), stress hormones, Neurotransmitter receptor & Neurotransmitter
transporter genes expressions in the muscles and liver.

polyunsaturated fatty acids in membranes as a chain reaction,
causes oxidative damage, which affects membrane stability
(Ayala, Muñoz, & Argüelles, 2014). The lipid peroxidation marker
MDA (malondialdehyde), increased after exhaustive exercise due
to the high ROS production (Tsai, Kan, Liu, & Jeng, 2004). The
MDA was significantly reduced by both doses of MCL compared
to the control exercise group, suggesting its anti-oxidative proper-
ties. MCL at 200 mg/kg dose also increased blood GSH (reduced
glutathione) levels which is the endogenous antioxidant defense
response molecule. GSH help scavenge free radicals and other reac-
tive oxygen species directly, and indirectly through enzymatic
reactions (Fang, Yang, & Wu, 2002). Additionally, the MCL caused
up-regulation of the SOD2 in the skeletal muscles. The SOD (super-
oxide dismutase) is a major endogenous antioxidant enzyme that
helps remove ROS. Among the isoforms of SOD, manganese super-
oxide dismutase (SOD2) is the primary mitochondria antioxidant
enzyme (Mlakar, Osredkar, Prezelj, & Marc, 2012). The above
results showed that one of the anti-fatigue mechanisms of MCL
is by increasing the endogenous and exogenous antioxidant
responses as evidenced by the reduced plasma MDA level,
increased blood GSH, and up-regulated SOD2 mRNA expression.
The fatigue associated with central proximal regulation is
mainly influenced by neurotransmitters and hypothalamic–pitui
tary–adrenal (HPA) axis activities. The activation of HPA axis under
stressful events, such as strenuous exercises, causes increased
stress hormone production such as cortisol (Coiro et al., 2011).
Here, both doses of MCL supplementation reduced plasma cortisol.
The reduction of stress hormone, help delay fatigue by improving
motivation, effort and mood (Davis & Brown, 2001).
In support of this data, the MCL treatment caused down-
regulation of the H6PD expressions in both the skeletal muscle
and liver tissues. Increased H6PD expression is associate with
increased NADPH levels and NADPH is utilized to convert inactive
cortisone to active cortisol (White, Rogoff, McMillan, & Lavery,
450 N.A.A. Mohamad Shalan et al. / Food Chemistry 212 (2016) 443–452
2007). Furthermore, MCL (400 mg/kg) significantly down regulated
the glucocorticoid receptor NR3C1 expression in the liver. NR3C1 is
believed to contribute to the pathophysiology of patients subgroup
with chronic fatigue (Smith, White, Aslakson, Vollmer-Conna, &
Rajeevan, 2006). The results show that another anti-fatigue
mechanism of MCL may be through the suppression of stress
responses.
In this study, both doses of MCL significantly up-regulated the
expressions of anti-inflammatory cytokines; IL-4 and IL-10 in the
skeletal muscle and liver tissues. Strenuous exercise elevates
proinflammatory cytokines levels and induces immune changes.
Immune dysregulation with high levels of proinflammatory cytoki-
nes is associated with fatigue induction. Strenuous exercise has
been associated with decreased resistance to upper respiratory
tract infections such as the common cold in the days after exercise,
whereas moderate exercise seems to offer some protection against
infections (Nieman, 2000).
Capillaries growth (angiogenesis) in skeletal muscles is an
adaptation to tissue hypoxia during exercise training, to sustain
oxygen and nutrients supply, and to remove metabolic by-
products that can contribute to fatigue. VEGF is the main angio-
genic factor in skeletal muscle capillary growth, and is stimulated
by mechanical forces during muscle activity (shear stress and
stretch) (Hoier & Hellsten, 2014). The skeletal muscle fibers con-
tain large stores of VEGF within vesicles, and during exercise, these
vesicles are secreted into the extracellular fluid (Hoier & Hellsten,
2014). Enhanced VEGF mRNA expression after exercise may be a
mechanism for replenishing the depleted VEGF stores (Hoier &
Hellsten, 2014). Initial VEGF concentrations is positively correlated
to antioxidant protection and trained athletes have higher VEGF
levels than untrained healthy individuals (Vinnichuck & Gunina,
2015). Micronutrients may affect VEGF expression and the
400 mg MCL/kg significantly up-regulated the VEGFA (pro-
angiogenic) to above the normal adaptive exercise responses in
the muscle, but suppressed VEGFA expression (anti-angiogenic)
in the liver (Fig. 4). Many anti-angiogenic reports on Morinda citri-
folia were based on the fruit and none were done on the leaf under
exercise training conditions (Beh et al., 2012; Jang, 2012). The Mor-
inda leaf was anti-angiogenic under lung cancer metastasis condi-
tions (Lim, Mustapha, Goh, Bakar, & Mohamed, 2016).
In the skeletal muscle and liver tissue, MCL apparently up-
regulated the gene expressions for mitochondrial biogenesis and
cellular energy metabolic pathway namely PGC, UCP3, NFE2L2
(synonym to NRF2: nuclear respiratory factor 2), PRKAA1 (syn-
onym to AMPK: adenosine monophosphate-activated protein
kinase), MAPK1, and CAMK4. The PGC-1 coactivators serve as an
inducible nuclear receptor boosters to equip the organism to meet
the energy demands of diverse physiologic and dietary conditions
(Finck & Kelly, 2006). CAMK (Schaeffer et al., 2004), p38 MAPK
(Akimoto et al., 2005) and AMPK (Jäger, Handschin, St-Pierre, &
Spiegelman, 2007) signalling pathways influence PGC-1 activity
directly or via PGC-1 coactivation. The PGC-1 coactivates NRF2,
the protein that will bind to and interact with a number of mito-
chondrial genes in the cell nucleus, resulting in increased mito-
chondrial biogenesis (Lin, Handschin, & Spiegelman, 2005). PGC-
1a also co-activate UCP3, a protein that mediate the up-
regulation of energy expenditure through increased fatty acid oxi-
dation and thermogenic uncoupling (Cha, Rodgers, Puigserver,
Chohnan, & Lane, 2006).
The expression of DRD2 (dopamine receptor) and SLC6A2 (nora-
drenaline transporter) were upregulated in the liver but down-
regulated by MCL in the skeletal muscles. Dopamine and nora-
drenaline are catechol-aminergic neurotransmitters that have
been implicated in prolonging the duration to exhaustion.
Serotonin affects emotional, motor, cognitive and autonomic
behaviors specifically or generally via coordinating the nervous
system activities proportional to the degree of stimulation. Sero-
tonin is believed to help integrate behavioral output functions by
suppressing other sensory activities not relevant to the ongoing
performance (Frazer & Hensler, 1999). Activation of serotonergic
transmission impedes information processing in afferent systems
(increases focus). MCL supplementation, caused upregulation of
the serotonin transporter mRNA in the liver tissues. The results
here indicate that another anti-fatigue mechanism of MCL is via
modulating the neurotransmitter (stress) responses that may help
the mammal to focus the energy for the activity.
Fig. 6 summarizes the proposed pathways by which the Mor-
inda leaf extract improved performance. Skeletal muscle contrac-
tile activity is activated by elevated intracellular Ca2+ and Ca2+
sensitive signalling molecules such as calcium/calmodulin depen-
dant protein kinase (CaMK). ATP utilization which causes the rise
in AMP will trigger AMP-activated protein kinase (AMPK). Exercise
enhances ROS production, which initiates the redox-sensitive tran-
scription factors, such as mitogen activated protein kinase (MAPK)
and AMPK. These signalling kinases translocate to the nucleus and
affects gene transcription through the interactions with peroxi-
some transcriptional proliferator-activated receptor gamma, coac-
tivator 1 alpha (PGC1-a). The PGC1-a auto-regulates its
expression, and the expression of nuclear respiratory factor-2
(NRF-2), peroxisome proliferator-activated receptor alpha (PPARa)
and estrogen-related receptor alpha (ERRa). NRF-2 and NRF-1 acti-
vates nuclear genes that encode mitochondrial proteins. It includes
the transcription, translation, and repair of mitochondrial tran-
scription factor A (TFAM), TFB1M, and TFB2M, which control mito-
chondrial DNA (mtDNA). Reactive oxygen species (ROS) and ROS
by-products can be reduced by the uncoupling protein 3 (UCP3)
by inducing UCP3 uncoupling (Schrauwen & Hesselink, 2004).
Additionally UCP3 export unoxidized fatty acids (FA) from the
mitochondrial matrix, to regulate fatty acid metabolism via activa-
tion of PPARa (Hood, Irrcher, Ljubicic, & Joseph, 2006). PGC1-a
stimulates the secretion of angiogenic factors, vascular endothelial
growth factor (VEGFA) through co-activation of ERRa (Arany et al.,
2008; Eivers et al., 2012).
Immune cells are mobilized and activated by exercise in
response to muscle stress or damage and through the release of
stress hormones (catecholamines, growth hormone, cortisol) in
response to growing metabolic demands and body temperature.
The immune cells interacts with stress hormones to alter cytokines
production (Peake, Suzuki, & Coombes, 2007). MCL supplementa-
tion increased the anti-inflammatory cytokines (IL4 and IL10)
expression, and upregulated CAMKIV, AMPK, MAPK, PGC, NRF2,
UCP3, SOD2, while downregulating the stress hormone production
and related genes expression.
The choice of a suitable positive control was quite difficult
because the anabolic steroids such as amphetamines are banned
for ergogenic uses and have different mechanisms of action. The
branched amino acids do not produce consistent results, while caf-
feine which is often used as a positive control, is naturally present
in the standardized green tea extract, which is used here for com-
parison. Morinda citrifolia leaf reportedly also contained 63.5 mg/g
(+) catechin and 6 mg/g rutin that inhibited lipoprotein lipase
in vitro, better than the fruit or green tea.
In conclusion, the present study demonstrates the performance
enhancing and anti-fatigue effects of MCL, and the supportive evi-
dences from mRNA gene expressions to indicate the possible
mechanisms involved. The suggested mechanisms include (i)
enhancing the mammalian antioxidant responses, (ii) improving
tissue nutrient and metabolite management (probably related to
angiogenesis) (iii) suppressing stress hormone and regulating neu-
rotransmitter expression levels (iv) increasing mitochondrial bio-
genesis (v) augmenting skeletal muscle angiogenesis and (v)
boosting anti-inflammatory responses.
 N.A.A. Mohamad Shalan et al. / Food Chemistry 212 (2016) 443–452
Fig. 6. A summary of the suggested pathways by which the Morinda leaf extract may have improved performance (based on mRNA expression results).
Acknowledgment
This study is supported by the Herbal Development Office, Min-
istry of Agriculture (Grant: 6372200).
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