Morinda citrifolia leaf enhanced performance by improving angiogenesis, mitochondrial biogenesis, antioxidant, anti-inflammatory & stress responses

Food Chemistry 212 (2016) 443–452

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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 b

UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

a r t i c l e

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Article history: Received 23 September 2015 Received in revised form 23 May 2016 Accepted 30 May 2016 Available online 02 June 2016 Keywords: Morinda citrifolia leaves Exercise performance Fatigue Epicatechin Scopoletin Mitochondrial biogenesis Angiogenesis

a b s t r a c t Morinda citrifolia fruit, (noni), enhanced performances in athletes and post-menopausal women in clinical studies. This report shows the edible noni leaves water extract enhances performance in a weight-loaded swimming animal model better than the fruit or standardized green tea extract. The 4 weeks study showed the extract (containing scopoletin and epicatechin) progressively prolonged the time to exhaustion 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) management, (iii) stress hormone (cortisol) regulation; (iv) neurotransmitter (dopamine, noradrenaline, serotonin) expressions, transporter or receptor levels, (v) anti-inflammatory (IL4 & IL10) responses; (v) skeletal muscle angiogenesis (VEGFA) and (v) energy and mitochondrial biogenesis (via PGC, UCP3, NRF2, AMPK, MAPK1, and CAMK4). The ergogenic extract helped delay fatigue by enhancing energy production, regulation and efficiency, which suggests benefits for physical activities and disease recovery. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Morinda citrifolia fruit, (called noni in USA, Mengkudu in Malaysia) 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 endurance (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-6phosphate 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, malondialdehyde; NADPH, nicotinamide adenine dinucleotide phosphate-oxidase. NR3C1; NRF2/NFE2L2, nuclear factor erythroid derived 2 like 2; PGC, peroxisome proliferative 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: [email protected] (S. Mohamed). http://dx.doi.org/10.1016/j.foodchem.2016.05.179 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

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 associated 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, liverprotective and wound healing properties without any acute, subacute and sub-chronic oral toxicity (West, Tani, Palu, Tolson, & Jensen, 2007). The M. citrifolia leaves extract oral no observedadverse-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 vitamins, 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

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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-immuneendocrine 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 ergogenic 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. citrifolia 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 suspensions were filtered and evaporated to dryness at 60 °C. Standardized 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), acetonitrile (MeCN) and analytical grade trifluoroacetic acid (TFA) were obtained from Merck (Darmstadt, Germany). Chromatographic 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 composed of three solvents: A; MeCN, B; MeOH, and C; 0.1 TFA% in H2O (v/v). The mobile phase was programmed consecutively in linear 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 conditions before the experiment. Animal were housed in an environmentally 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 libitum. The experimental protocol was approved by the Institutional 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 treatments 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 immediately 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 autoanalyzer (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 comparative 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 (Superoxide dismutase 2)], mitochondrial biogenesis or fatty acid metabolism [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 neurotransmitter [DRD2 (Dopamine receptor D2), SLC6A4 (serotonin transporter: 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). Custom 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 experimental 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 differences between groups was determined using one or 2-way analysis of variance (ANOVA). Differences between groups were further

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week 4. The lower dose of 200 mg/kg produced better effects than the higher 400 mg/kg dose indicating that the optimum dose is closer to 200 mg/kg and the 400 mg/kg may be closer to an overdose. The results indicate that M. citrifolia leaves water extract significantly and progressively extended the duration time to exhaustion three times longer than the control mice in weight-loaded swimming test, which indicated that MCL have ergogenic and antifatigue properties. It was much more effective than the green tea extract which contained 5% caffeine, 95% polyphenol and 45% Epigallocatechingallate or the fruit extract.

evaluated by Duncan’s post hoc test and considered significant at p < 0.05. All statistical analysis was performed using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Effects of M. citrifolia leaves extract on time to exhaustion in a weight-loaded swimming test Fig. 1 shows there was no significant difference in the mean baseline initial swimming time (D0) between the groups, but significant ergogenic effects were already seen by the first week of treatment. Treatment with 200 and 400 mg extract/kg BW (Fig 1B) significantly increased the exhaustive swimming time by 40% and 28% in the first week, 20% and 14% in the second week, and 50% and 41% in the 4th week, respectively, as compared to control CE group. MCL group given 200 and 400 mg extract/kg also showed significantly longer swimming time as compared to GT group, by 23% and 10% in week 1, 20% and 17% in week 2, and 43% and 33% in

A

3.2. Effect of M. citrifolia leaves extract on the blood glucose, blood lactate and plasma cortisol levels The treatments produced no significant effects on blood glucose and lactate levels in the exercise groups even though the MCL mice swam three times longer than the other mice groups (Fig. 2A and B). Blood cortisol levels after exhaustive swimming were significantly lower in both the 200 mg extract/kg (2.3 ± 0.4 ng/ml) and

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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.

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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 control without exercise (0.4 ± 0.1 ng/ml) mice (Fig. 2C). Excessive cortisol 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 insignificantly different in spite of having different swimming time durations (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 cytotoxicity due to ROS (reactive oxygen species).

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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, angiogenesis, antioxidant enzyme, and mitochondrial biogenesis markers/fatty acid metabolism mRNA expressions in muscle and liver tissues Fig. 4 shows that the 200 mg/kg MCL up-regulated the mRNA expression of the anti-inflammatory cytokines (IL-4 up-regulated 2-fold in the muscle and 8-fold in the liver; and IL-10 upregulated 3-fold in the muscles and 7-fold in the liver); the vascular endothelial growth factor A (VEGFA upregulated 3-fold in the muscles and 2-fold in the liver); the endogenous antioxidant response elements (SOD2 upregulated 2-fold in the muscles); and the fatty acid metabolism/mitochondrial biogenesis markers

(UCP3, PGC, NFE2L2 upregulated about 2-fold in the muscles and up to 7.6-fold in the liver). 3.5. Effects of M. citrifolia leaves extract on anabolic kinase signalling, stress hormones, and Neurotransmitter receptor or Neurotransmitter transporter mRNA expressions in muscle and liver tissues Fig. 5 shows that the anabolic kinase signalling markers (MAPK1, PRKAA1), Ca2+ signalling (CAMK4) were upregulated in the muscles and liver (except MAPK1 in the liver). The stress cortisol (H6PD) expression of the hypothalamic–pituitary–adrenal (HPA) axis function was downregulated for both the muscle and liver, in agreement with the blood cortisol levels. Additionally

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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 neurotransmitters (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 noradrenalin) 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 byproduct) 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 accumulates 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 accumulation 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 angiogenesis (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

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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 properties. 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 reactive 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 (superoxide dismutase) is a major endogenous antioxidant enzyme that helps remove ROS. Among the isoforms of SOD, manganese superoxide 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 downregulation 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,

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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 cytokines 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 byproducts that can contribute to fatigue. VEGF is the main angiogenic 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 contain 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 (proangiogenic) 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 citrifolia were based on the fruit and none were done on the leaf under exercise training conditions (Beh et al., 2012; Jang, 2012). The Morinda leaf was anti-angiogenic under lung cancer metastasis conditions (Lim, Mustapha, Goh, Bakar, & Mohamed, 2016). In the skeletal muscle and liver tissue, MCL apparently upregulated the gene expressions for mitochondrial biogenesis and cellular energy metabolic pathway namely PGC, UCP3, NFE2L2 (synonym to NRF2: nuclear respiratory factor 2), PRKAA1 (synonym 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 mitochondrial genes in the cell nucleus, resulting in increased mitochondrial biogenesis (Lin, Handschin, & Spiegelman, 2005). PGC1a also co-activate UCP3, a protein that mediate the upregulation of energy expenditure through increased fatty acid oxidation and thermogenic uncoupling (Cha, Rodgers, Puigserver, Chohnan, & Lane, 2006). The expression of DRD2 (dopamine receptor) and SLC6A2 (noradrenaline transporter) were upregulated in the liver but downregulated by MCL in the skeletal muscles. Dopamine and noradrenaline 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. Serotonin 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 Morinda leaf extract improved performance. Skeletal muscle contractile activity is activated by elevated intracellular Ca2+ and Ca2+ sensitive signalling molecules such as calcium/calmodulin dependant 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 transcription 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 peroxisome transcriptional proliferator-activated receptor gamma, coactivator 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 activates nuclear genes that encode mitochondrial proteins. It includes the transcription, translation, and repair of mitochondrial transcription factor A (TFAM), TFB1M, and TFB2M, which control mitochondrial 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 activation 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 supplementation 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 caffeine which is often used as a positive control, is naturally present in the standardized green tea extract, which is used here for comparison. 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 evidences 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 neurotransmitter expression levels (iv) increasing mitochondrial biogenesis (v) augmenting skeletal muscle angiogenesis and (v) boosting anti-inflammatory responses.

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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, Ministry of Agriculture (Grant: 6372200).

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