http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(5): 551–559 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.850518
ORIGINAL ARTICLE
Effect of hydroalcoholic extract of Aegle marmelos fruit on radical scavenging activity and exercise-endurance capacity in mice Division of Biochemistry and Nanosciences Discipline, Defence Food Research Laboratory (DFRL), Defence Research & Development Organization, Siddharthanagar, Mysore, India Abstract
Keywords
Context: Aegle marmelos L. Corr (Rutaceae) is an important Indian Ayurvedic medicinal plant used for the treatment of various ailments. However, little information is available on the antifatigue properties of its fruit. Objective: Evaluation of the physical endurance and exercise-induced oxidative stress modulating properties of A. marmelos fruit in mice. Material and methods: Radical scavenging activity of the fruit hydroalcoholic extract was evaluated using in vitro systems. The extract was further evaluated for its endurance-enhancing properties at three oral doses (100, 200 and 400 mg/kg b.wt) in BALB/c mice for 21 d using a swimming test. Results and discussion: The extract exhibited significant scavenging activity against DPPH (IC50, 351 37 mg/ml) and ABTS radicals (IC50, 228 25 mg/ml), respectively, with the polyphenol content of 95 mg/mg extract. It also inhibited AAPH radical-induced oxidation of biomolecules such as BSA protein (63%), plasmid DNA (81%) and lipids (80.5%). Administration of extract resulted in an increase in the duration of swimming time to exhaustion by 23.4 and 47.5% for medium and higher doses, respectively. The extract significantly normalized the fatigue-related biochemical parameters and also down-regulated the swim stress-induced over-expression of heat shock protein-70 and up-regulated the skeletal muscle metabolic regulators (GLUT-4 and AMPK1-a) by 2- and 3-fold, respectively, at the higher dose in muscle tissues. Conclusion: Our study demonstrates the anti-fatigue properties of A. marmelos fruit, most probably manifested by delaying the accumulation of serum lactic acid, increasing the fat utilization and up-regulating the skeletal muscle metabolic regulators.
Anti-fatigue, forced swimming test, heat shock protein-70, lactate, polyphenols
Introduction Physical fatigue is a sensation most often experienced by every one of us during strenuous exercise and is accompanied by a feeling of physical tiredness. During exercise, there is an increase in the utilization of carbohydrates and lipids in the skeletal muscles. Initially, supply of energy to the muscle is provided from immediate energy sources such as ATP and phosphocreatine, and later from the anaerobic glycolysis and other metabolic pathways. Rapid depletion of energy reserves of muscle and liver tissues results in accumulation of lactate in the blood before exhaustion, which in turn affects the homeostasis of the internal environment of the cells (Gobatto et al., 2001). Moreover, fatigue also results in exerciseinduced oxidative stress due to excessive generation of reactive oxygen species (ROS) that causes tissue damage (Armstrong, 1990).
Correspondence: Ilaiyaraja Nallamuthu, Division of Biochemistry and Nutrition, Defence Food Research Laboratory (DFRL), Siddharthanagar, Mysore 570011, India. Tel: +91-821-2579486. Fax: +91-821-2473468. E-mail: [email protected]
History Received 8 March 2013 Revised 3 September 2013 Accepted 26 September 2013 Published online 5 February 2014
A large number of pharmacological studies have revealed that the extracts or bioactive compounds of plants such as Allium sativum L. (Amaryllidaceae) (Morihara et al., 2006), Bacopa monniera L. (Scrophulariaceae) (Anand et al., 2012a), Panax ginseng C.A. Meyer (Araliaceae) (Tang et al., 2008), Rubus coreanus (Rosaceae) (Jung et al., 2007), Pseudosasa japonica, (Poaceae) (You et al., 2006), Ocimum sanctum L. (Lamiaceae) (Prasad & Khanum, 2012) and mycelia of Cordyceps sinensis Berk (Ophiocordycipitaceae) (Koh et al., 2003), have increased endurance capacity as well as ameliorated exercise-induced oxidative stress in animal models after a Forced Swimming Test (Huanga et al., 2011). However, the available supplements for increasing endurance capacity from natural sources are very limited and therefore there has been a significant effort in search of novel anti-fatigue agents as an alternative to their synthetic counterparts. Aegle marmelos L. Corr (Rutaceae), commonly known as Bael, is an Indian Ayurvedic medicinal plant (Chanda et al., 2008). It is a mid-sized, slender, aromatic, armed, gumbearing tree growing up to 18 m tall and has a leaf with three leaflets. It is also known as Bengal quince, golden apple,
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Ilaiyaraja Nallamuthu, Anand Tamatam, and Farhath Khanum
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stone apple and bili. Traditionally, various parts of this plant, such as fruit, leaf and seeds, have been used to cure a variety of diseases. Literature available over the past few decades demonstrates the diverse pharmacological properties of the fruit, in particular, for its antioxidant, anti-inflammatory (Baliga et al., 2011), anti-ulcerative (Dhuley, 2007), antidiabetic (Kamalakkannan & Prince, 2003), anxiolytic, antidepressant (Kothari et al., 2010), immunomodulatory activity (Patel & Asdaq, 2010), etc. It contains a myriad of phytochemicals including polyphenols, tannins, alkaloids, marmelosin, aurapten, psoralen, etc. (Rajan et al., 2011). Although many medicinal attributes have been explored, scanty information is available on the role of A. marmelos fruit in improving physical performance. The present study evaluates the antioxidant as well as endurance-enhancing properties of the fruit.
Material and methods Plant material Aegle marmelos fruit (100–120 g) was purchased from the local market in Mysore. The identification and authentication of the plant was done by Prof. N. Yasodamma, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. A herbarium of the plant has been submitted with the Voucher Specimen No. AM-25. Fruit pulp was removed and dried in a hot air oven at 40 C. The powdered material was extracted with 70% alcohol. The flash evaporated and lyophilized sample (AME) yielded 14.5% extract on a dry weight basis, and used for the in vitro and in vivo studies. In vitro radical scavenging activity The concentration of total polyphenol was estimated by the Folin–Ciocalteu method using gallic acid as standard (Slinkard & Singleton, 1977). DPPH and ABTS radical scavenging activity was determined as per the method described (Braca et al., 2001; Re et al., 1996). Superoxide radical scavenging activity was measured according to Liu et al. (1997). The inhibitory effect of the extract on protein oxidation was carried out as described by Kwon et al. (2000). Briefly, 20 mM of AAPH was used to induce oxidative damage in 5 mg of BSA. After 2 h of incubation at 37 C, the protein was assayed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). For the DNA damage assay, 10 mM AAPH was used to induce strand breakage in the pBR332 plasmid DNA (Lee et al., 2002). After a 30-min incubation period, the sample was electrophoresed. The band intensity of the gel was quantified by Syngene Genetools software (Syngene, Cambridge, UK). Inhibition of lipid peroxidation in rat liver homogenate was evaluated by measuring the thiobarbituric acid reactive substances (TBARS) at 532 nm (Ardestani & Yazdanparast, 2007). High-performance liquid chromatography determination of polyphenols Polyphenols in the AME were extracted using a solid-phase extraction cartridge (Briviba et al., 2002). Briefly, the cartridge (Sep-Pak C18, 0.5 g, Waters, Milford, MA) was washed with
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methanol and equilibrated with 2 ml of water. The AME was then applied to the cartridge. After removing the water-soluble compounds, polyphenols were eluted in methanol. Compounds were analyzed by high-performance liquid chromatography (HPLC) system using photodiode array detector (JASCO, Tokyo, Japan). The sample (20 ml) was injected into the RP-C18 column (JASCO, Tokyo, Japan). The mobile phase consisted of 0.1% formic acid and methanol, and was run in a gradient cycle for 57 min at the flow rate of 0.8 ml/min. Anti-fatigue experiment Laboratory bred BALB/c male mice, weighing 22–25 g, were housed in acrylic cages under standard conditions of temperature (22 0.5 C) and humidity (50%) and a 12/12 h light/dark cycle. These animals had free access to stock diet and tap water and were maintained in accordance with the guidelines of National Institute of Nutrition, India and approved by Institutional Animal Ethics Committee (No. 28 CPCSEA/IAEC). As the physical activity of the mice is affected by circadian variation, the experiments were carried out from 1100 to 1700 h, a period in which minimal variation of swimming capacity has been reported. The Forced Swimming Test was conducted as per the standard protocols reported earlier using a water pool with slight modifications (Matsumoto et al., 1996). In an acrylic plastic tank (90 45 45 cm), water was filled to a depth of 37 cm. Initially, all the mice were allowed to swim for the period of 15 min to accustomize the mice to the swimming condition. Thirty mice with almost equal mean swimming time were selected and randomized into five groups with the same mean swimming capacity as: sedentary control, exercise control (water), three AME treated groups (100, 200, 400 mg/kg b.wt). The extract was orally administered for the consecutive period of 21 d. Exercise control and AME groups were subjected to the swimming test 1 h after administration on the 7th, 14th and 21st day (once in a week). During the test, mice were placed separately in the swimming tank against a running water current at the flow rate of 15 l/min and a temperature of 34 C. The total duration of swimming period untill exhaustion was measured. Mice were assumed to be fatigued when they failed to rise to the surface of the water to breathe within 7 s as the index of swimming capacity. Biochemical analyses On the 22nd day the mice were forced to swim for 10 min and, after 60 min of recess, all the mice were sacrificed by cervical dislocation and whole-blood samples were collected in tubes by heart puncture. The liver and gastrocnemius muscles were dissected out quickly from the mice, and washed with ice-cold physiological saline. Serum was prepared by centrifugation at 3000 rpm for 10 min at 4 C. Serum L-lactic acid was determined spectrophotometrically using a Randox Kit. Serum free fatty acid was determined as per the method described by Hron and Menahan (1981). Glycogen in muscle and liver were estimated using the anthrone method of Nicholas et al. (1955). BUN (blood urea nitrogen) was determined using Commercial Diagnostic Kits from Agappe Diagnostics, India.
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Assay of antioxidant enzymes Liver tissue (1 g) was homogenized in 0.2 M phosphate buffer, pH 7.4, and then centrifuged at 12 000g for 30 min. The supernatant was used for the measurement of antioxidant enzyme activities. Catalase activity was assayed using the method describe by Cohen et al. (1970). The activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) was determined using a commercially available kit (Randox, Antrim, UK). Total protein was estimated using total protein kit (Agappe Diagnostics, Kerala, India).
Figure 1. Inhibitory effect of AME extract on AAPH (20 mM) induced pBR322 DNA damage. Lane 1, plasmid only (negative control); Lane 2, plasmid þ AAPH (positive control); Lane 3–5, plasmid DNA þ AAPH þ extract (25, 50 and 100 mg); Lane 6, chlorogenic acid (5 mg).
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Western blot analysis The expression levels of heat shock protein (HSP) (Hsp-70) in liver tissues and GLUT-4 and AMPK-1a in muscle tissues were analyzed by SDS–PAGE electrophoresis followed by western blot (Jayaraj et al., 2006). Tissues were homogenized in 10 volumes of lysis buffer containing 10 mM HEPES pH 7.4, 42 mM KCl, 50 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 5 mm DTT, 2 mM PMSF and 1 complete protease inhibitor cocktail. Homogenate was centrifuged at 10 000g for 20 min and the supernatant was used for electrophoresis. Proteins (50 mg) were electrophoresed and transferred on to a nitrocellulose membrane. The membrane was probed with primary antibodies (1:1000 dilution, H-5147, clone BRM, Sigma-Aldrich, St. Louis, MO) followed by secondary antibody (1:150 000 dilution, rabbit anti-mouse). The detection was done using chemiluminescent peroxidase substrate and visualized on X-ray films. Statistical analysis All the data were expressed as mean standard deviation (SD). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s honestly significant difference (HSD). A level of p50.05 was used as the criterion for statistical significance.
Results In vitro antioxidant activity The IC50 value for the radical scavenging activities against DPPH and ABTS were 351 37 and 228 25 mg/ml, respectively, for AME extract, and were 11.8 0.5 and 25.26 0.6 mg/ml, respectively, for the vitamin C (standard). Further, it showed a marked superoxide radical scavenging activity with IC50 values of 375 21 and 23 2 mg/ml, respectively, for the extract and standard. The observed antioxidant activity can be correlated with the polyphenol content of 95 mg/mg extract. AME was further evaluated for its inhibitory effect on the oxidation of biomolecules such as DNA, protein and lipid. Figure 1 shows that the presence of AAPH radical resulted in the introduction of strand breakage in pBR322 plasmid DNA that led to the conversion of supercoiled form (SC) into open circular form (OC) in positive control (Lane 2). This strand breakage was dosedependently inhibited by radical scavenging activity of the extract at 65.5, 75.4 and 81%, respectively, for 25, 50 and 100 mg/ml concentration (Lane 3–5). Chlorogenic acid was used as a positive control (Lane 6). Figure 2 shows that a similar pattern was also obtained for the AAPH-induced
Figure 2. Inhibitory effect of AME extract on AAPH (20 mM) induced oxidative fragmentation of BSA. Lane 1, BSA only; Lane 2, BSA þ AAPH (positive control); Lane 3–7, BSA þ AAPH þ extract (100, 80, 60, 40 and 20 mg); Lane 8, chlorogenic acid (5 mg).
oxidative damage of BSA protein. In the presence of increasing concentration of AME extract (20, 40, 60, 80 and 100 mg/ml) the degradation of BSA was inhibited in a dosedependent manner (Lane 3–7) and maximum protection (63.5%) was observed at the concentration of 100 mg/ml (Lane 3). In the lipid oxidation assay, the addition of AAPH to the mouse liver homogenate significantly extended TBARS formation. AME extract (0.1–0.6 mg/ml) inhibited this lipid peroxidation in a concentration-dependant manner up to 80.5% as shown in Figure 3. HPLC analysis was carried out to determine the bioactive compounds responsible for the observed antioxidant activity of the extract. Peaks for the compounds such as chlorogenic acid, tannic acid, ferulic acid, gallic acid and quercetin were identified as a major polyphenols in the extract (Figure 4). Exercise-endurance test in mice Effect of AME on bodyweight change The weights of the mice were measured after they were treated with three different doses of the AME extract for 21 d. The increased weights of the animals in the treated group did not show any significant difference when compared to control groups throughout the experimental period which means that AME had no effect on body weight (Figure 5). Effect of AME on swimming capacity The maximum swimming times of the mice were recorded to determine the effect of AME on anti-fatigue activity. Figure 6 shows that the mouse group treated with AME extract remarkably prolonged the swimming time to exhaustion compared with the exercise control group. The results indicate that swimming time of the mice increased significantly (p50.05) after 2 weeks only for the higher dose whereas after 3 weeks of treatment, both the medium (200 mg/kg b.wt) and higher dose (400 mg/kg b.wt) group increased by 23.4% (p50.05) and 47.5% (p50.01) respectively. The effect was insignificant at the lower dose of 100 mg/kg b.wt.
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Figure 3. Inhibitory effect of AME extract on AAPH (20 mM) induced lipid peroxidation.
Figure 4. HPLC chromatogram of AME extract. Retention time in min (RT): gallic acid 9.0, chlorogenic acid 20.4, tannic acid 25, ferulic acid 30.6 and quercetin 44.7.
Effect of AME on serum biochemical parameters As shown in Table 1, the swimming exercise led to an increase in the levels of blood lactic acid and BUN compared to the control group (p50.01). Lactic acid was significantly reduced by the AME extract at both the medium and higher doses (p50.01) whereas BUN was reduced only at the higher dose of 400 mg/kg b.wt (p50.05). Moreover, the extract at the higher dose also resulted in increased availability of serum free fatty acid (1.7 0.1 mmol/l) compared to the exercise control group (1.33 0.1 mmol/l). Effect of AME on glycogen content The content of glycogen in muscle and liver tissues was decreased by swimming exercise to 0.14 0.04 and 1.23 0.12 mg/g, respectively (p50.01), compared to sedentary control of 0.17 0.09 and 2.7 0.17 mg/g, respectively.
Figure 7 shows that treatment with low and medium doses tended to increase these levels, but not significantly. However, these levels were significantly elevated at the higher dose than the exercise control. Effect of AME on lipid peroxidation Figure 8 shows that swimming exercise results in an increased level of malondialdehyde, an indirect indicator of lipid peroxidation, in liver and muscle tissues than the sedentary group (p50.01). At higher dose of AME, the MDA concentration was significantly lowered to 0.7 0.06 and 0.4 0.07 nmol/mg proteins respectively for liver and muscle tissues than in the exercise control group (0.92 0.07 and 0.53 0.09 nmol/mg proteins). Thus, it is evident that AME reduced the generation of lipid peroxide through an antioxidant mechanism.
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Figure 5. Effect of AME on body weight of mice. 1: sedentary control, 2: exercise control, 3:100 mg AME/kg b.wt, 4: 200 mg AME/kg b.wt and 5: 400 mg AME/kg b.wt AME.
Figure 6. Effect of AME on swimming duration to exhaustion of mice. As compared with exercise control: ap50.05, b p50.01.
Table 1. Effect of AME on the blood parameters. L-Lactic
Groups Group Group Group Group Group
I II III IV V
acid (mg/dl)
11.67 2.8 67.50 5.9a 70.68 6.3 55.17 6.2c 50.98 7.0c
Free fatty acid (mmol/l)
BUN (U/dl)
0.87 0.08 1.33 0.10a 1.30 0.14 1.40 0.14 1.70 0.10c
11.22 1.8 15.53 1.4a 14.52 1.6 15.15 1.8 12.02 1.1b
Values are expressed as mean SD (n ¼ 6). Group I (sedentary control), Group II (exercise control), Group III (100 mg AME), Group IV (200 mg AME), Group V (400 mg AME). As compared with sedentary control: ap50.01. As compared with exercise control: bp50.05, c p50.01.
Effect of AME on hepatic antioxidant enzymes It was observed that the activities of SOD and catalase enzymes were lesser (p50.01) in the swimming exercise group than the sedentary group (Table 2). At the higher dose, AME significantly (p50.05) increased the enzyme activities relative to the control group, whereas the medium dose could
increase the enzyme activities only slightly. The activity of GPx was insignificant in all the groups. Effect of AME on HSP-70 and skeletal muscle metabolic regulators Swimming stress caused an over-expression of Hsp-70 protein in muscle tissues of mice whereas AME treatment significantly (p50.01) down-regulated to almost normal level at the higher dose of 400 mg/kg b.wt compared to exercise control (Figure 9). Exercise stress also significantly increased the expression of GLUT-4 and AMPK-1a in muscle tissues compared to sedentary control. Supplementation of extract further increased their levels by 2- and 3-fold, respectively, compared to exercise control indicating that the extract, at the higher dose, up-regulated these metabolic regulators in skeletal muscle.
Discussion The present study demonstrates the antioxidant as well as anti-fatigue properties of A. marmelos fruit. Three doses of
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Figure 7. Effect of AME on glycogen content. 1: sedentary control, 2: exercise control, 3: 100 mg AME/kg b.wt, 4: 200 mg AME/ kg b.wt and 5: 400 mg AME/kg b.wt AME. As compared with sedentary control: a p50.01. As compared with exercise control: b p50.05, cp50.01.
Figure 8. Effect of AME hepatic lipid peroxidation. 1: sedentary control, 2: exercise control, 3: 100 mg AME/kg b.wt, 4: 200 mg AME/kg b.wt and 5: 400 mg AME/ kg b.wt. As compared with sedentary control: a p50.01. As compared with exercise control: b p50.05, cp50.01.
Table 2. Effect of AME on the hepatic antioxidant enzymes.
Groups Group Group Group Group Group
I II III IV V
GPx Catalse (nmol H2O2/min/mg (nmol ADPH/min/mg SOD protein) protein) (U/mg protein) 14.90 2.1 9.53 1.3a 9.13 1.4 12.08 1.1b 12.23 1.3b
56.70 4.5 46.22 3.3a 42.18 5.2 45.38 5.3 53.90 3.6b
5.3 0.74 6.4 0.75 6.1 0.84 5.2 0.50 5.9 0.80
Values are expressed as mean SD (n ¼ 6). Group I (sedentary control), Group II (exercise control), Group III (100 mg AME), Group IV (200 mg AME), Group V (400 mg AME). As compared with sedentary control: ap50.01. As compared with exercise control: bp50.05.
Figure 9. Western blots analysis of Hsp-70 and skeletal muscle metabolic regulators in muscle tissues of mice. 1: exercise control, 2: sedentary control, 3: 100 mg AME/kg b.wt, 4: 200 mg AME/kg b.wt and 5: 400 mg AME/kg b.wt AME.
AME were evaluated in mice by Forced Swimming Test, which is a valid animal model for screening anti-fatigue potency of various bioactive compounds (Choi et al., 2012). As shown in Figure 5, AME extract prolonged the swimming time of treated mice group to exhaustion and thereby indicating that AME possesses an anti-fatigue effect (Figure 5). The swimming exercise is known to induce biochemical changes in the blood and therefore we have further measured the effect of AME on FST-induced fatigue-relevant parameters. It has been reported that strenuous exercise alters the levels of blood parameters particularly causing an increased accumulation of lactate, a biomarker for fatigue, in the muscle and plasma compared to resting conditions (Ren et al., 2011;
Tan et al., 2012). Lactate is a catabolic product of glycolysis and therefore can be used as an index of anaerobic metabolism. The accumulation of lactate eventually leads to the occurrence of fatigue and exhaustion in the exercising subject (Jung et al., 2004). Anti-fatigue agents have been shown to effectively work by delaying lactate accumulation either by reducing the glycolytic process or by removing the blood lactate (Evans et al., 2002; Li et al., 2008). The results of present study also showed that FST increased serum lactate levels significantly in the exercise control group compared with the sedentary control and this effect was significantly attenuated by AME administration. The glycogen content of liver and muscle tissue were determined after the swimming exercise. Glycogen is the
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DOI: 10.3109/13880209.2013.850518
main source of energy for intensive exercise in a short time and it is catabolized under anaerobic condition to produce lactates through the glycolytic pathway. In the exercise control group, liver and muscle glycogen were decreased by swimming exercise. As shown in Figure 4, AME supplementation significantly elevated these carbohydrates relative to the control group. These results suggest that AME has the capacity to decrease or slow the utilization of carbohydrate sources, otherwise physical fatigue occurs by depletion of glycogen. The increased level of muscular and hepatic glycogen in treated groups in this study indicate the glycogen sparing effect of AME extract and is well concordant with the earlier published reports (Anand et al., 2012a; Tan et al., 2012). BUN is a sensitive biomarker to determine the bearing capacity of a person subjected to physical load. It has been reported that there is a direct correlation between exercise tolerance and lower level of BUN. Protein, an energy source, upon digestion produces the amino acid monomers which degraded to ammonia and the rest of the carbon skeleton is used as an energy source. Urea is generated as the end product of protein metabolism in the body. The results of the present study also indicated that AME extract possessed the ability to lower the generation of BUN during exercise. Free fatty acids are a major source of energy in skeletal muscle that generates a larger quantity of ATP via the tricarboxylic acid (TCA) cycle of aerobic respiration. Fushiki et al. (1995) have shown that chronic consumption of medium-chain fatty acid increases the swimming endurance capacity of mice. It suggests that there is a close association between endurance capacity and fatty acid oxidation. Results of our study also showed the high level of free fatty acid availability in serum of AME-treated group at the higher dose which might have increased the mobility of fatty acid to the site of utilization. This could be a possible explanation for the prolonged swimming duration observed following AME administration in mice as reported for green tea polyphenols by Murase et al. (2005). Intense physical exercise also causes oxidative stress in the body due to excessive generation of radicals. During exercise, a large amount of oxygen is consumed and 4–5% of the total oxygen consumed during respiration is incompletely reduced to water and therefore results in the acceleration of radical generation. These radicals, in turn, oxidatively degrade biomolecules such as lipids, proteins and nucleic acids and therefore affect the homeostatic environment of cells. For this reason, in the present study, the protective effect of AME on oxidative damage of biomolecules was determined using in vitro systems. The results exhibited the scavenging activities of the extract against various radicals and inhibited the radical-induced oxidative damage on DNA, protein and lipids and thereby demonstrate its antioxidant activity. Earlier studies have shown that lipid peroxidation in liver and muscle tissues increases during intense physical exercise (Wang et al., 2006). Peroxiation is as an important indicator of oxidative stress that results from degradation of cell membrane by radicals. MDA is one of the end products in this process that is measured as TBARS. Results of the present study also indicated that the extract significantly reduces peroxidation.
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We examined further the anti-oxidative effect of AME extract in liver and muscle tissues during swimming exercise. Antioxidant enzymes such as SOD, catalase and GPx are regarded as the first line of the antioxidant defense system against radicals. SOD protects cells by catalyzing the conversion of superoxide radicals to O2 and H2O2. This toxic H2O2 is further decomposed into O2 and H2O by catalase. GPx catalyzes the reduction of hydroperoxides by glutathione. The enhanced production of superoxide and peroxide radicals causes a decrease in the activity of these enzymes in liver. Our findings showed that activities of SOD and catalase in liver tissues of AME treated groups were significantly higher than in the exercise control. Thus, it is evident that AME extracts possess the ability to suppress tissue damage in response to exercise by reducing the lipid peroxidation and up-regulating the antioxidant enzymes. These modulatory effects are in consistent with the earlier report of Wang et al. (2010), which demonstrated the antifatigue activity of P. ginseng. Studies have revealed that ROS induces HSPs synthesis in cells (Gorman et al., 1999). It is a family of highly conservative stress proteins regarded as molecular chaperons. Particularly, the 70 kDa Hsp-70 contributes to stress tolerance by increasing its chaperone activity. The role of this protein has also been implicated in physical stress like swimming (Anand et al., 2012b), and therefore we have measured its expression in liver tissues. Our results indicated that AME extract down-regulates HSP expression that was over-expressed by swimming stress through various physiological responses. Moreover, the supplementation of the extract also up-regulated endurance responsive skeletal muscle regulators such as GLUT-4 and AMPK in muscle tissues compared to exercise control and thereby favored better glucose uptake. However, the exact mechanism is unclear and requires further studies. Polyphenols have been proved to prevent various oxidative stress-related diseases by their antioxidant mechanism. Their dietary supplementation also plays a vital role in reducing exercise-induced oxidative stress (Clarkson & Thompson, 2000; Morillas-Ruiz et al., 2006; Williams, 2004). Recently, the anti-fatigue role of ferulic acid, epigallocatechin gallate and other flavonoids and polyphenolics has been proven experimentally (Dhankhar et al., 2011; Kang et al., 2012; Tanaka et al., 2008; You et al., 2009). HPLC analysis of the AME extract in the present study showed that the extract contains bioactive compounds such as polyphenols such as chlorogenic acid, tannic acid, ferulic acid, gallic acid and quercetin, and therefore the stimulatory effect of AME on endurance capacity could be attributed to the comprehensive effect of these polyphenolic compounds (Yadav et al., 2011). The molecular mechanism of the anti-fatigue effect of the bioactive compounds/extracts has not been well established so far. Emerging evidences have shown for few agents that endurance exercise up-regulate the skeletal muscle metabolic regulators (AMPK, GLUT-4, PGC-1a and PPAR-d), antioxidant, lipogenic genes, and also oxidative stress responsive transcription factor NRF-2 in rats and mice (Kumar et al., 2011; Suwa et al., 2008). The mechanisms for other endurance-enhancing compounds at
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the cellular level are very complex and have yet to be confirmed and remain unclear.
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Conclusion Taking all these results together, our study shows that A. marmalus fruit does possess an anti-fatigue effect by extending the swimming time for the mice. The possible underlying mechanisms for anti-fatigue activity of AME could be mediated by delaying the accumulation of serum lactic acid, increasing the fat utilization and up-regulating skeletal muscle metabolic regulators. In addition, the results suggested that supplementation of A. marmalus alleviates exercise-induced oxidative damage through antioxidant potential. However, further investigations are necessary to elucidate the molecular mechanisms at the cellular level including the effect of bioactive constituents on the expression of fatigue responsive metabolic regulators and genes.
Declaration of interest The authors report no conflicts of interest.
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ORIGINAL ARTICLE
Effect of hydroalcoholic extract of Aegle marmelos fruit on radical scavenging activity and exercise-endurance capacity in mice Division of Biochemistry and Nanosciences Discipline, Defence Food Research Laboratory (DFRL), Defence Research & Development Organization, Siddharthanagar, Mysore, India Abstract
Keywords
Context: Aegle marmelos L. Corr (Rutaceae) is an important Indian Ayurvedic medicinal plant used for the treatment of various ailments. However, little information is available on the antifatigue properties of its fruit. Objective: Evaluation of the physical endurance and exercise-induced oxidative stress modulating properties of A. marmelos fruit in mice. Material and methods: Radical scavenging activity of the fruit hydroalcoholic extract was evaluated using in vitro systems. The extract was further evaluated for its endurance-enhancing properties at three oral doses (100, 200 and 400 mg/kg b.wt) in BALB/c mice for 21 d using a swimming test. Results and discussion: The extract exhibited significant scavenging activity against DPPH (IC50, 351 37 mg/ml) and ABTS radicals (IC50, 228 25 mg/ml), respectively, with the polyphenol content of 95 mg/mg extract. It also inhibited AAPH radical-induced oxidation of biomolecules such as BSA protein (63%), plasmid DNA (81%) and lipids (80.5%). Administration of extract resulted in an increase in the duration of swimming time to exhaustion by 23.4 and 47.5% for medium and higher doses, respectively. The extract significantly normalized the fatigue-related biochemical parameters and also down-regulated the swim stress-induced over-expression of heat shock protein-70 and up-regulated the skeletal muscle metabolic regulators (GLUT-4 and AMPK1-a) by 2- and 3-fold, respectively, at the higher dose in muscle tissues. Conclusion: Our study demonstrates the anti-fatigue properties of A. marmelos fruit, most probably manifested by delaying the accumulation of serum lactic acid, increasing the fat utilization and up-regulating the skeletal muscle metabolic regulators.
Anti-fatigue, forced swimming test, heat shock protein-70, lactate, polyphenols
Introduction Physical fatigue is a sensation most often experienced by every one of us during strenuous exercise and is accompanied by a feeling of physical tiredness. During exercise, there is an increase in the utilization of carbohydrates and lipids in the skeletal muscles. Initially, supply of energy to the muscle is provided from immediate energy sources such as ATP and phosphocreatine, and later from the anaerobic glycolysis and other metabolic pathways. Rapid depletion of energy reserves of muscle and liver tissues results in accumulation of lactate in the blood before exhaustion, which in turn affects the homeostasis of the internal environment of the cells (Gobatto et al., 2001). Moreover, fatigue also results in exerciseinduced oxidative stress due to excessive generation of reactive oxygen species (ROS) that causes tissue damage (Armstrong, 1990).
Correspondence: Ilaiyaraja Nallamuthu, Division of Biochemistry and Nutrition, Defence Food Research Laboratory (DFRL), Siddharthanagar, Mysore 570011, India. Tel: +91-821-2579486. Fax: +91-821-2473468. E-mail: [email protected]
History Received 8 March 2013 Revised 3 September 2013 Accepted 26 September 2013 Published online 5 February 2014
A large number of pharmacological studies have revealed that the extracts or bioactive compounds of plants such as Allium sativum L. (Amaryllidaceae) (Morihara et al., 2006), Bacopa monniera L. (Scrophulariaceae) (Anand et al., 2012a), Panax ginseng C.A. Meyer (Araliaceae) (Tang et al., 2008), Rubus coreanus (Rosaceae) (Jung et al., 2007), Pseudosasa japonica, (Poaceae) (You et al., 2006), Ocimum sanctum L. (Lamiaceae) (Prasad & Khanum, 2012) and mycelia of Cordyceps sinensis Berk (Ophiocordycipitaceae) (Koh et al., 2003), have increased endurance capacity as well as ameliorated exercise-induced oxidative stress in animal models after a Forced Swimming Test (Huanga et al., 2011). However, the available supplements for increasing endurance capacity from natural sources are very limited and therefore there has been a significant effort in search of novel anti-fatigue agents as an alternative to their synthetic counterparts. Aegle marmelos L. Corr (Rutaceae), commonly known as Bael, is an Indian Ayurvedic medicinal plant (Chanda et al., 2008). It is a mid-sized, slender, aromatic, armed, gumbearing tree growing up to 18 m tall and has a leaf with three leaflets. It is also known as Bengal quince, golden apple,
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Ilaiyaraja Nallamuthu, Anand Tamatam, and Farhath Khanum
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stone apple and bili. Traditionally, various parts of this plant, such as fruit, leaf and seeds, have been used to cure a variety of diseases. Literature available over the past few decades demonstrates the diverse pharmacological properties of the fruit, in particular, for its antioxidant, anti-inflammatory (Baliga et al., 2011), anti-ulcerative (Dhuley, 2007), antidiabetic (Kamalakkannan & Prince, 2003), anxiolytic, antidepressant (Kothari et al., 2010), immunomodulatory activity (Patel & Asdaq, 2010), etc. It contains a myriad of phytochemicals including polyphenols, tannins, alkaloids, marmelosin, aurapten, psoralen, etc. (Rajan et al., 2011). Although many medicinal attributes have been explored, scanty information is available on the role of A. marmelos fruit in improving physical performance. The present study evaluates the antioxidant as well as endurance-enhancing properties of the fruit.
Material and methods Plant material Aegle marmelos fruit (100–120 g) was purchased from the local market in Mysore. The identification and authentication of the plant was done by Prof. N. Yasodamma, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. A herbarium of the plant has been submitted with the Voucher Specimen No. AM-25. Fruit pulp was removed and dried in a hot air oven at 40 C. The powdered material was extracted with 70% alcohol. The flash evaporated and lyophilized sample (AME) yielded 14.5% extract on a dry weight basis, and used for the in vitro and in vivo studies. In vitro radical scavenging activity The concentration of total polyphenol was estimated by the Folin–Ciocalteu method using gallic acid as standard (Slinkard & Singleton, 1977). DPPH and ABTS radical scavenging activity was determined as per the method described (Braca et al., 2001; Re et al., 1996). Superoxide radical scavenging activity was measured according to Liu et al. (1997). The inhibitory effect of the extract on protein oxidation was carried out as described by Kwon et al. (2000). Briefly, 20 mM of AAPH was used to induce oxidative damage in 5 mg of BSA. After 2 h of incubation at 37 C, the protein was assayed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). For the DNA damage assay, 10 mM AAPH was used to induce strand breakage in the pBR332 plasmid DNA (Lee et al., 2002). After a 30-min incubation period, the sample was electrophoresed. The band intensity of the gel was quantified by Syngene Genetools software (Syngene, Cambridge, UK). Inhibition of lipid peroxidation in rat liver homogenate was evaluated by measuring the thiobarbituric acid reactive substances (TBARS) at 532 nm (Ardestani & Yazdanparast, 2007). High-performance liquid chromatography determination of polyphenols Polyphenols in the AME were extracted using a solid-phase extraction cartridge (Briviba et al., 2002). Briefly, the cartridge (Sep-Pak C18, 0.5 g, Waters, Milford, MA) was washed with
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methanol and equilibrated with 2 ml of water. The AME was then applied to the cartridge. After removing the water-soluble compounds, polyphenols were eluted in methanol. Compounds were analyzed by high-performance liquid chromatography (HPLC) system using photodiode array detector (JASCO, Tokyo, Japan). The sample (20 ml) was injected into the RP-C18 column (JASCO, Tokyo, Japan). The mobile phase consisted of 0.1% formic acid and methanol, and was run in a gradient cycle for 57 min at the flow rate of 0.8 ml/min. Anti-fatigue experiment Laboratory bred BALB/c male mice, weighing 22–25 g, were housed in acrylic cages under standard conditions of temperature (22 0.5 C) and humidity (50%) and a 12/12 h light/dark cycle. These animals had free access to stock diet and tap water and were maintained in accordance with the guidelines of National Institute of Nutrition, India and approved by Institutional Animal Ethics Committee (No. 28 CPCSEA/IAEC). As the physical activity of the mice is affected by circadian variation, the experiments were carried out from 1100 to 1700 h, a period in which minimal variation of swimming capacity has been reported. The Forced Swimming Test was conducted as per the standard protocols reported earlier using a water pool with slight modifications (Matsumoto et al., 1996). In an acrylic plastic tank (90 45 45 cm), water was filled to a depth of 37 cm. Initially, all the mice were allowed to swim for the period of 15 min to accustomize the mice to the swimming condition. Thirty mice with almost equal mean swimming time were selected and randomized into five groups with the same mean swimming capacity as: sedentary control, exercise control (water), three AME treated groups (100, 200, 400 mg/kg b.wt). The extract was orally administered for the consecutive period of 21 d. Exercise control and AME groups were subjected to the swimming test 1 h after administration on the 7th, 14th and 21st day (once in a week). During the test, mice were placed separately in the swimming tank against a running water current at the flow rate of 15 l/min and a temperature of 34 C. The total duration of swimming period untill exhaustion was measured. Mice were assumed to be fatigued when they failed to rise to the surface of the water to breathe within 7 s as the index of swimming capacity. Biochemical analyses On the 22nd day the mice were forced to swim for 10 min and, after 60 min of recess, all the mice were sacrificed by cervical dislocation and whole-blood samples were collected in tubes by heart puncture. The liver and gastrocnemius muscles were dissected out quickly from the mice, and washed with ice-cold physiological saline. Serum was prepared by centrifugation at 3000 rpm for 10 min at 4 C. Serum L-lactic acid was determined spectrophotometrically using a Randox Kit. Serum free fatty acid was determined as per the method described by Hron and Menahan (1981). Glycogen in muscle and liver were estimated using the anthrone method of Nicholas et al. (1955). BUN (blood urea nitrogen) was determined using Commercial Diagnostic Kits from Agappe Diagnostics, India.
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Assay of antioxidant enzymes Liver tissue (1 g) was homogenized in 0.2 M phosphate buffer, pH 7.4, and then centrifuged at 12 000g for 30 min. The supernatant was used for the measurement of antioxidant enzyme activities. Catalase activity was assayed using the method describe by Cohen et al. (1970). The activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) was determined using a commercially available kit (Randox, Antrim, UK). Total protein was estimated using total protein kit (Agappe Diagnostics, Kerala, India).
Figure 1. Inhibitory effect of AME extract on AAPH (20 mM) induced pBR322 DNA damage. Lane 1, plasmid only (negative control); Lane 2, plasmid þ AAPH (positive control); Lane 3–5, plasmid DNA þ AAPH þ extract (25, 50 and 100 mg); Lane 6, chlorogenic acid (5 mg).
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Western blot analysis The expression levels of heat shock protein (HSP) (Hsp-70) in liver tissues and GLUT-4 and AMPK-1a in muscle tissues were analyzed by SDS–PAGE electrophoresis followed by western blot (Jayaraj et al., 2006). Tissues were homogenized in 10 volumes of lysis buffer containing 10 mM HEPES pH 7.4, 42 mM KCl, 50 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 5 mm DTT, 2 mM PMSF and 1 complete protease inhibitor cocktail. Homogenate was centrifuged at 10 000g for 20 min and the supernatant was used for electrophoresis. Proteins (50 mg) were electrophoresed and transferred on to a nitrocellulose membrane. The membrane was probed with primary antibodies (1:1000 dilution, H-5147, clone BRM, Sigma-Aldrich, St. Louis, MO) followed by secondary antibody (1:150 000 dilution, rabbit anti-mouse). The detection was done using chemiluminescent peroxidase substrate and visualized on X-ray films. Statistical analysis All the data were expressed as mean standard deviation (SD). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s honestly significant difference (HSD). A level of p50.05 was used as the criterion for statistical significance.
Results In vitro antioxidant activity The IC50 value for the radical scavenging activities against DPPH and ABTS were 351 37 and 228 25 mg/ml, respectively, for AME extract, and were 11.8 0.5 and 25.26 0.6 mg/ml, respectively, for the vitamin C (standard). Further, it showed a marked superoxide radical scavenging activity with IC50 values of 375 21 and 23 2 mg/ml, respectively, for the extract and standard. The observed antioxidant activity can be correlated with the polyphenol content of 95 mg/mg extract. AME was further evaluated for its inhibitory effect on the oxidation of biomolecules such as DNA, protein and lipid. Figure 1 shows that the presence of AAPH radical resulted in the introduction of strand breakage in pBR322 plasmid DNA that led to the conversion of supercoiled form (SC) into open circular form (OC) in positive control (Lane 2). This strand breakage was dosedependently inhibited by radical scavenging activity of the extract at 65.5, 75.4 and 81%, respectively, for 25, 50 and 100 mg/ml concentration (Lane 3–5). Chlorogenic acid was used as a positive control (Lane 6). Figure 2 shows that a similar pattern was also obtained for the AAPH-induced
Figure 2. Inhibitory effect of AME extract on AAPH (20 mM) induced oxidative fragmentation of BSA. Lane 1, BSA only; Lane 2, BSA þ AAPH (positive control); Lane 3–7, BSA þ AAPH þ extract (100, 80, 60, 40 and 20 mg); Lane 8, chlorogenic acid (5 mg).
oxidative damage of BSA protein. In the presence of increasing concentration of AME extract (20, 40, 60, 80 and 100 mg/ml) the degradation of BSA was inhibited in a dosedependent manner (Lane 3–7) and maximum protection (63.5%) was observed at the concentration of 100 mg/ml (Lane 3). In the lipid oxidation assay, the addition of AAPH to the mouse liver homogenate significantly extended TBARS formation. AME extract (0.1–0.6 mg/ml) inhibited this lipid peroxidation in a concentration-dependant manner up to 80.5% as shown in Figure 3. HPLC analysis was carried out to determine the bioactive compounds responsible for the observed antioxidant activity of the extract. Peaks for the compounds such as chlorogenic acid, tannic acid, ferulic acid, gallic acid and quercetin were identified as a major polyphenols in the extract (Figure 4). Exercise-endurance test in mice Effect of AME on bodyweight change The weights of the mice were measured after they were treated with three different doses of the AME extract for 21 d. The increased weights of the animals in the treated group did not show any significant difference when compared to control groups throughout the experimental period which means that AME had no effect on body weight (Figure 5). Effect of AME on swimming capacity The maximum swimming times of the mice were recorded to determine the effect of AME on anti-fatigue activity. Figure 6 shows that the mouse group treated with AME extract remarkably prolonged the swimming time to exhaustion compared with the exercise control group. The results indicate that swimming time of the mice increased significantly (p50.05) after 2 weeks only for the higher dose whereas after 3 weeks of treatment, both the medium (200 mg/kg b.wt) and higher dose (400 mg/kg b.wt) group increased by 23.4% (p50.05) and 47.5% (p50.01) respectively. The effect was insignificant at the lower dose of 100 mg/kg b.wt.
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Figure 3. Inhibitory effect of AME extract on AAPH (20 mM) induced lipid peroxidation.
Figure 4. HPLC chromatogram of AME extract. Retention time in min (RT): gallic acid 9.0, chlorogenic acid 20.4, tannic acid 25, ferulic acid 30.6 and quercetin 44.7.
Effect of AME on serum biochemical parameters As shown in Table 1, the swimming exercise led to an increase in the levels of blood lactic acid and BUN compared to the control group (p50.01). Lactic acid was significantly reduced by the AME extract at both the medium and higher doses (p50.01) whereas BUN was reduced only at the higher dose of 400 mg/kg b.wt (p50.05). Moreover, the extract at the higher dose also resulted in increased availability of serum free fatty acid (1.7 0.1 mmol/l) compared to the exercise control group (1.33 0.1 mmol/l). Effect of AME on glycogen content The content of glycogen in muscle and liver tissues was decreased by swimming exercise to 0.14 0.04 and 1.23 0.12 mg/g, respectively (p50.01), compared to sedentary control of 0.17 0.09 and 2.7 0.17 mg/g, respectively.
Figure 7 shows that treatment with low and medium doses tended to increase these levels, but not significantly. However, these levels were significantly elevated at the higher dose than the exercise control. Effect of AME on lipid peroxidation Figure 8 shows that swimming exercise results in an increased level of malondialdehyde, an indirect indicator of lipid peroxidation, in liver and muscle tissues than the sedentary group (p50.01). At higher dose of AME, the MDA concentration was significantly lowered to 0.7 0.06 and 0.4 0.07 nmol/mg proteins respectively for liver and muscle tissues than in the exercise control group (0.92 0.07 and 0.53 0.09 nmol/mg proteins). Thus, it is evident that AME reduced the generation of lipid peroxide through an antioxidant mechanism.
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Figure 5. Effect of AME on body weight of mice. 1: sedentary control, 2: exercise control, 3:100 mg AME/kg b.wt, 4: 200 mg AME/kg b.wt and 5: 400 mg AME/kg b.wt AME.
Figure 6. Effect of AME on swimming duration to exhaustion of mice. As compared with exercise control: ap50.05, b p50.01.
Table 1. Effect of AME on the blood parameters. L-Lactic
Groups Group Group Group Group Group
I II III IV V
acid (mg/dl)
11.67 2.8 67.50 5.9a 70.68 6.3 55.17 6.2c 50.98 7.0c
Free fatty acid (mmol/l)
BUN (U/dl)
0.87 0.08 1.33 0.10a 1.30 0.14 1.40 0.14 1.70 0.10c
11.22 1.8 15.53 1.4a 14.52 1.6 15.15 1.8 12.02 1.1b
Values are expressed as mean SD (n ¼ 6). Group I (sedentary control), Group II (exercise control), Group III (100 mg AME), Group IV (200 mg AME), Group V (400 mg AME). As compared with sedentary control: ap50.01. As compared with exercise control: bp50.05, c p50.01.
Effect of AME on hepatic antioxidant enzymes It was observed that the activities of SOD and catalase enzymes were lesser (p50.01) in the swimming exercise group than the sedentary group (Table 2). At the higher dose, AME significantly (p50.05) increased the enzyme activities relative to the control group, whereas the medium dose could
increase the enzyme activities only slightly. The activity of GPx was insignificant in all the groups. Effect of AME on HSP-70 and skeletal muscle metabolic regulators Swimming stress caused an over-expression of Hsp-70 protein in muscle tissues of mice whereas AME treatment significantly (p50.01) down-regulated to almost normal level at the higher dose of 400 mg/kg b.wt compared to exercise control (Figure 9). Exercise stress also significantly increased the expression of GLUT-4 and AMPK-1a in muscle tissues compared to sedentary control. Supplementation of extract further increased their levels by 2- and 3-fold, respectively, compared to exercise control indicating that the extract, at the higher dose, up-regulated these metabolic regulators in skeletal muscle.
Discussion The present study demonstrates the antioxidant as well as anti-fatigue properties of A. marmelos fruit. Three doses of
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Figure 7. Effect of AME on glycogen content. 1: sedentary control, 2: exercise control, 3: 100 mg AME/kg b.wt, 4: 200 mg AME/ kg b.wt and 5: 400 mg AME/kg b.wt AME. As compared with sedentary control: a p50.01. As compared with exercise control: b p50.05, cp50.01.
Figure 8. Effect of AME hepatic lipid peroxidation. 1: sedentary control, 2: exercise control, 3: 100 mg AME/kg b.wt, 4: 200 mg AME/kg b.wt and 5: 400 mg AME/ kg b.wt. As compared with sedentary control: a p50.01. As compared with exercise control: b p50.05, cp50.01.
Table 2. Effect of AME on the hepatic antioxidant enzymes.
Groups Group Group Group Group Group
I II III IV V
GPx Catalse (nmol H2O2/min/mg (nmol ADPH/min/mg SOD protein) protein) (U/mg protein) 14.90 2.1 9.53 1.3a 9.13 1.4 12.08 1.1b 12.23 1.3b
56.70 4.5 46.22 3.3a 42.18 5.2 45.38 5.3 53.90 3.6b
5.3 0.74 6.4 0.75 6.1 0.84 5.2 0.50 5.9 0.80
Values are expressed as mean SD (n ¼ 6). Group I (sedentary control), Group II (exercise control), Group III (100 mg AME), Group IV (200 mg AME), Group V (400 mg AME). As compared with sedentary control: ap50.01. As compared with exercise control: bp50.05.
Figure 9. Western blots analysis of Hsp-70 and skeletal muscle metabolic regulators in muscle tissues of mice. 1: exercise control, 2: sedentary control, 3: 100 mg AME/kg b.wt, 4: 200 mg AME/kg b.wt and 5: 400 mg AME/kg b.wt AME.
AME were evaluated in mice by Forced Swimming Test, which is a valid animal model for screening anti-fatigue potency of various bioactive compounds (Choi et al., 2012). As shown in Figure 5, AME extract prolonged the swimming time of treated mice group to exhaustion and thereby indicating that AME possesses an anti-fatigue effect (Figure 5). The swimming exercise is known to induce biochemical changes in the blood and therefore we have further measured the effect of AME on FST-induced fatigue-relevant parameters. It has been reported that strenuous exercise alters the levels of blood parameters particularly causing an increased accumulation of lactate, a biomarker for fatigue, in the muscle and plasma compared to resting conditions (Ren et al., 2011;
Tan et al., 2012). Lactate is a catabolic product of glycolysis and therefore can be used as an index of anaerobic metabolism. The accumulation of lactate eventually leads to the occurrence of fatigue and exhaustion in the exercising subject (Jung et al., 2004). Anti-fatigue agents have been shown to effectively work by delaying lactate accumulation either by reducing the glycolytic process or by removing the blood lactate (Evans et al., 2002; Li et al., 2008). The results of present study also showed that FST increased serum lactate levels significantly in the exercise control group compared with the sedentary control and this effect was significantly attenuated by AME administration. The glycogen content of liver and muscle tissue were determined after the swimming exercise. Glycogen is the
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DOI: 10.3109/13880209.2013.850518
main source of energy for intensive exercise in a short time and it is catabolized under anaerobic condition to produce lactates through the glycolytic pathway. In the exercise control group, liver and muscle glycogen were decreased by swimming exercise. As shown in Figure 4, AME supplementation significantly elevated these carbohydrates relative to the control group. These results suggest that AME has the capacity to decrease or slow the utilization of carbohydrate sources, otherwise physical fatigue occurs by depletion of glycogen. The increased level of muscular and hepatic glycogen in treated groups in this study indicate the glycogen sparing effect of AME extract and is well concordant with the earlier published reports (Anand et al., 2012a; Tan et al., 2012). BUN is a sensitive biomarker to determine the bearing capacity of a person subjected to physical load. It has been reported that there is a direct correlation between exercise tolerance and lower level of BUN. Protein, an energy source, upon digestion produces the amino acid monomers which degraded to ammonia and the rest of the carbon skeleton is used as an energy source. Urea is generated as the end product of protein metabolism in the body. The results of the present study also indicated that AME extract possessed the ability to lower the generation of BUN during exercise. Free fatty acids are a major source of energy in skeletal muscle that generates a larger quantity of ATP via the tricarboxylic acid (TCA) cycle of aerobic respiration. Fushiki et al. (1995) have shown that chronic consumption of medium-chain fatty acid increases the swimming endurance capacity of mice. It suggests that there is a close association between endurance capacity and fatty acid oxidation. Results of our study also showed the high level of free fatty acid availability in serum of AME-treated group at the higher dose which might have increased the mobility of fatty acid to the site of utilization. This could be a possible explanation for the prolonged swimming duration observed following AME administration in mice as reported for green tea polyphenols by Murase et al. (2005). Intense physical exercise also causes oxidative stress in the body due to excessive generation of radicals. During exercise, a large amount of oxygen is consumed and 4–5% of the total oxygen consumed during respiration is incompletely reduced to water and therefore results in the acceleration of radical generation. These radicals, in turn, oxidatively degrade biomolecules such as lipids, proteins and nucleic acids and therefore affect the homeostatic environment of cells. For this reason, in the present study, the protective effect of AME on oxidative damage of biomolecules was determined using in vitro systems. The results exhibited the scavenging activities of the extract against various radicals and inhibited the radical-induced oxidative damage on DNA, protein and lipids and thereby demonstrate its antioxidant activity. Earlier studies have shown that lipid peroxidation in liver and muscle tissues increases during intense physical exercise (Wang et al., 2006). Peroxiation is as an important indicator of oxidative stress that results from degradation of cell membrane by radicals. MDA is one of the end products in this process that is measured as TBARS. Results of the present study also indicated that the extract significantly reduces peroxidation.
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We examined further the anti-oxidative effect of AME extract in liver and muscle tissues during swimming exercise. Antioxidant enzymes such as SOD, catalase and GPx are regarded as the first line of the antioxidant defense system against radicals. SOD protects cells by catalyzing the conversion of superoxide radicals to O2 and H2O2. This toxic H2O2 is further decomposed into O2 and H2O by catalase. GPx catalyzes the reduction of hydroperoxides by glutathione. The enhanced production of superoxide and peroxide radicals causes a decrease in the activity of these enzymes in liver. Our findings showed that activities of SOD and catalase in liver tissues of AME treated groups were significantly higher than in the exercise control. Thus, it is evident that AME extracts possess the ability to suppress tissue damage in response to exercise by reducing the lipid peroxidation and up-regulating the antioxidant enzymes. These modulatory effects are in consistent with the earlier report of Wang et al. (2010), which demonstrated the antifatigue activity of P. ginseng. Studies have revealed that ROS induces HSPs synthesis in cells (Gorman et al., 1999). It is a family of highly conservative stress proteins regarded as molecular chaperons. Particularly, the 70 kDa Hsp-70 contributes to stress tolerance by increasing its chaperone activity. The role of this protein has also been implicated in physical stress like swimming (Anand et al., 2012b), and therefore we have measured its expression in liver tissues. Our results indicated that AME extract down-regulates HSP expression that was over-expressed by swimming stress through various physiological responses. Moreover, the supplementation of the extract also up-regulated endurance responsive skeletal muscle regulators such as GLUT-4 and AMPK in muscle tissues compared to exercise control and thereby favored better glucose uptake. However, the exact mechanism is unclear and requires further studies. Polyphenols have been proved to prevent various oxidative stress-related diseases by their antioxidant mechanism. Their dietary supplementation also plays a vital role in reducing exercise-induced oxidative stress (Clarkson & Thompson, 2000; Morillas-Ruiz et al., 2006; Williams, 2004). Recently, the anti-fatigue role of ferulic acid, epigallocatechin gallate and other flavonoids and polyphenolics has been proven experimentally (Dhankhar et al., 2011; Kang et al., 2012; Tanaka et al., 2008; You et al., 2009). HPLC analysis of the AME extract in the present study showed that the extract contains bioactive compounds such as polyphenols such as chlorogenic acid, tannic acid, ferulic acid, gallic acid and quercetin, and therefore the stimulatory effect of AME on endurance capacity could be attributed to the comprehensive effect of these polyphenolic compounds (Yadav et al., 2011). The molecular mechanism of the anti-fatigue effect of the bioactive compounds/extracts has not been well established so far. Emerging evidences have shown for few agents that endurance exercise up-regulate the skeletal muscle metabolic regulators (AMPK, GLUT-4, PGC-1a and PPAR-d), antioxidant, lipogenic genes, and also oxidative stress responsive transcription factor NRF-2 in rats and mice (Kumar et al., 2011; Suwa et al., 2008). The mechanisms for other endurance-enhancing compounds at
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the cellular level are very complex and have yet to be confirmed and remain unclear.
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Conclusion Taking all these results together, our study shows that A. marmalus fruit does possess an anti-fatigue effect by extending the swimming time for the mice. The possible underlying mechanisms for anti-fatigue activity of AME could be mediated by delaying the accumulation of serum lactic acid, increasing the fat utilization and up-regulating skeletal muscle metabolic regulators. In addition, the results suggested that supplementation of A. marmalus alleviates exercise-induced oxidative damage through antioxidant potential. However, further investigations are necessary to elucidate the molecular mechanisms at the cellular level including the effect of bioactive constituents on the expression of fatigue responsive metabolic regulators and genes.
Declaration of interest The authors report no conflicts of interest.
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