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Anti-fatigue effect of Amarkand on endurance exercise capacity in rats
© Narkhede et al. 2016
Received: 17 August 2015
Accepted: 12 January 2016
Published: 22 January 2016
Amarkand tubers are routinely used by many Indian tribes as a specialized food for health and longevity but so far there is no scientific evidence for their activities. Taxonomically, Amarkand belong to genera Eulophia and Dioscorea.
In this communication, comparative antifatigue potential of Amarkand was analyzed using forced swimming model in rats and evaluated using biomarkers of physical fatigue.
Methanol extracts of tubers of D. bulbifera, E. ochreata, E. leghapanensis and bulbils of D. bulbifera exhibited rich polyphenolic content. D. bulbifera bulbils and E. ochreata significantly prolonged the swimming endurance time. Creatine kinase and urea nitrogen were significantly reduced by treatment of D. bulbifera bulbils and E. ochreata as compared to negative control. D. bulbifera bulbils effectively increased creatine (p < 0.001), lactate dehydrogenase (p < 0.01) and hemoglobin (p < 0.001) compared to negative control. D. bulbifera bulbils and E. ochreata treatments significantly increased glycogen (p < 0.05, p < 0.01) and lowered malondialdehyde levels (p < 0.001) in muscles and in liver tissue compared to negative control.
These results indicate that a treatment with D. bulbifera bulbils and tubers of E. ochreata facilitates aerobic glucose metabolism and endurance by improving various impairments associated with fatigue.
Fatigue is a complex metabolic phenomenon, which involves listless conditions. During fatigue, body faces difficulty in initiating or sustaining voluntary activities. It induces alteration in performance leading to decrease in muscular power and endurance, decreased motor skill performance and diminished physical as well as mental functions . Accumulation of metabolic by-products like magnesium, inorganic phosphates, lactate, reactive oxygen species etc. can potentially contribute to fatigue during exercise and limit the performance. Physiological fatigue may also result due to metabolic disorders like diabetes, hyperthyroidism, anemia, high Body Mass Index, liver diseases etc. . Regular exercise helps the body to function better, but strenuous or high intensity exercise may cause reactive oxygen species (ROS) production and exhaustion leading to oxidative stress, which often overwhelms body’s capacity to detoxify them. Fatigue is worthy of attention since it may cause various disorders related to bio-regulatory and immune system. Also, these disorders cause reduction in exercise intensity or even interruption of activity .
Tribal populations are rich in traditional knowledge about the plants available in their surrounding and their medicinal as well as food values . There are certain groups of plants locally identified by a particular vernacular name but botanically they belong to different genera and family. Amarkand is one of such groups. Tubers of Amarkand are extensively utilized by many Indian tribes as a source of food and especially as medicine . Ethanobotanical survey in Maharashtra conducted during 2004 to 2009 by Medicinal Plants Conservation Centre (MPCC) revealed that, there are multiple species recognized as Amarkand especially from genus Eulophia (Family Orchidaceae) and one from genus Dioscorea (Family Dioscoreaceae). However, with respect to taxonomical knowledge, there is still confusion in identification of species of Eulophia under the name Amarkand, which has rich food and medicinal value .
About 32 species of Eulophia, spread all over India, along with Dioscorea bulbifera are recognized as Amarkand by tribes, of which 21 species are medicinally important. Eulophia species are used as medicine fairly throughout the greater part of India; most of them are diverse but mainly tropical found up to 2200 m above sea level . According to ayurvedic perspectives, these species have wide spectrum of uses like expectorant, anabolic, tonic, diuretic, astringent, digestive and soft purgative. It promotes strength and alleviates all the three ‘doshas’. It has also been used in ear discharge, blood clotting, joint edema and debility also . According to ethno-botanical reports, tubers of these plants are described as appetizer, anthelmintic, aphrodisiac, stomachic, alterative and commonly given to stimulate appetite and to purify blood in heart troubles [9–11]. Pharmacologically, E. ochreata and D. bulbifera are reported for their antioxidant , anti-inflammatory , nutritional content , antihyperglycemic, antidyslipidemic , analgesic  activity.
In our earlier study, out of 21 species, 11 species of Amarkand were prioritized and studied for their free radical quenching potential, of which four species namely, bulbils of D. bulbifera and tubers of E. ochreata, E. leghapanensis, D. bulbifera have been prioritized and selected in this study . Polyphenolic contents from plants are known to be helpful for extensive exercise , thus initially polyphenolic content of these selected species of Amarkand were analyzed. These three plants are still used by different tribes as they believe that these bulbils and tubers would maintain as well as increase their endurance for hard work and thereby extend their longevity [4, 17].
These species are used in routine diet and as a medicine by tribal people, but still it is less explored in urban lifestyle. Though the literature reveals, its high potential as a medicine as well as food, no study evidence has been reported on these species individually or comparatively concerning their anti-fatigue activity. Thus, in view to search complementary and/or alternative therapy from medicinal plants, it is obligatory to resolve the abstraction for Amarkand plants with reference to endurance or postpone the appearance of fatigue for its societal significance. In the present study, methanol extracts of Amarkand species were investigated for their anti-fatigue activity which may further provide scientific evidence for development of Amarkand species as a potential natural anti-fatigue agent. Considering the limitations of available therapies for fatigue in modern medicine , potential alternatives from traditional medicine and their mechanisms of action are worth investigating.
Plant material and extraction
Fresh tubers of Eulophia ochreata Lindl. (EO), Eulophia leghapanensis (Noven) (EL), Dioscorea bulbifera L. (DBT) and bulbils of Dioscorea bulbifera L. (DBB) were identified and collected by Dr. Suresh Jagtap, Botanist, from Leghapani (Nandurbar) and Bhimashankar (Pune) of Maharashtra, India. Plants were authenticated at Medicinal Plants Conservation Centre (MPCC), Pune with voucher specimen numbers, MPCC3125 for EO, MPCC3468 for EL and MPCC906 for D. bulbifera. The tubers were cut into small pieces and shade dried. The dried material was then pulverized using an electric blender and stored in an air tight polythene bags for further use. Methanol extract was prepared from these samples using hot extraction method followed by concentrating to dryness under reduced pressure using rotary evaporator.
Determination of total phenolic, flavonoids and proanthocyanidin contents
Total phenol contents of all Amarkand tuber extracts were quantified using folin-ciocalteu reagent method mentioned in Singleton and Rossi, . All four samples were analyzed at a final concentration of 50 μg/ml. Results are expressed as mg/g of gallic acid equivalent. Total flavonoid content was assessed according to . Evaluation was carried out at the concentration of 1 mg/ml for all the samples. The flavonoid content is expressed as mg of quercetin equivalents/g of dry matter. Total proanthocynidin content was determined using procedure followed by . All the extracts were evaluated at 1 mg/ml concentration and results are expressed as catechin equivalent (mg/g).
Approval for the experimental protocol from Institutional animal ethical committee was obtained before initiation of the study (CPCSEA/7/2679/2012-2013) from Bharati Vidyapeeth Deemed University, Medical College, Pune. Wistar rats (Female) weighing approximately 100–120 g were used for the study. They were housed for 2 weeks in solid bottomed polypropylene cages for acclimatization before use, maintained under standard conditions and fed standard rat chow with water ad libitum. Guidelines laid down by the CPCSEA were observed throughout the study for animal handling and experimentation. Animals were randomly divided into 14 groups having six rats per group. Low-100 mg/kg bw (L), Medium 300 mg/kg bw (M) and High 500 mg/kg bw (H) dose of plant extracts including sedentary and control group. Sedentary and control group were fed with distilled water by gavage and other 12 treated groups were force fed with different doses of EO, EL, DBT and DBB (100, 300 and 500 mg/kg bw) by gavage for 2 weeks.
Weight loaded forced swimming test
Weight loaded forced swimming test (FST) was performed according to Venuprasad et al.,  with some modification. Amarkand treated and control rats were subjected to FST every alternate day supporting constant load with a lead block weighing approximately 10 % of their body weight attached to their tails. The attached weight forced these animals to maintain rapid continuous leg movements . Exhaustion was determined by observing their loss of coordinated movements and failure to swim. Endurance time was recorded until the rat was completely exhausted and failed to return to surface to breathe within 10s . FST was performed in small tank of 30 cm deep, containing water at temperature 25 ± 2 °C.
After a period of 4 weeks, animals were sacrificed. Blood samples were collected from heart immediately after last exercise. It was collected by heparinized syringe into vacutainers. Whole blood was used to analyze hemoglobin, which was expressed in gm% and serum was separated by centrifugation at 3000 rpm at 4 °C for 10 min. Serum was used to analyze Serum Urea Nitrogen (SUN), Lactate Dehydrogenase (LDH), Creatine Kinase (CK) and Creatine (Cr). All these parameters were assessed according to the procedure provided by commercially available kits manufactured by Span diagnostic Ltd., India. SUN and Cr were expressed in mg/dl while LDH and CK were expressed in lu/L. Liver and muscle tissue from hind limb were collected and frozen immediately at −80 °C for estimation of MDA and glycogen content. MDA content was performed according to the method described by Vinodini et al.,  and it is expressed in nm/g of tissue, whereas glycogen level in muscle/liver were analyzed using a kit (Cayman chemical company, USA) and it indicated as mg/g of tissue.
The values are presented as mean ± SD. One way ANOVA was used to determine significant differences in groups and negative control group. All analyses were performed using Graph pad Prism 5 software and p values <0.05 were accepted as statistically significant.
Result and discussion
Polyphenolic content of methanolic extract of selected Amarkand plants
Polyphenolic content assays
mg/g dry mass
1.29 ± 0.03
1.43 ± 0.06
3.53 ± 0.15
1.45 ± 0.23
0.39 ± 0.06
3.80 ± 0.10
1.72 ± 0.03
0.82 ± 0.09
9.24 ± 0.50
0.61 ± 0.07
0.18 ± 0.02
0.23 ± 0.03
Effect of Amarkand plants on Creatine content, Hemoglobin concentration and Serum urea nitrogen in rats
Serum Urea Nitrogen (SUN)
1.01 ± 0.122
13.16 ± 0.917
23.58 ± 2.916
0.95 ± 0.052
12.28 ± 0.966
26.43 ± 4.928
1.05 ± 0.074
13.62 ± 1.137
21.20 ± 1.513*
1.21 ± 0.045***
14.13 ± 0.771**
19.46 ± 0.884***
0.81 ± 0.085
13.72 ± 0.608*
20.66 ± 1.676**
1.06 ± 0.080
13.28 ± 0.722
27.53 ± 3.794
1.09 ± 0.111
13.10 ± 1.203
21.20 ± 1.513*
1.14 ± 0.083*
13.37 ± 0.504
30.80 ± 2.343
1.04 ± 0.175
13.75 ± 0.187*
26.20 ± 1.600
1.27 ± 0.088***
13.35 ± 0.736
22.40 ± 4.498
1.22 ± 0.172***
13.30 ± 0.328
19.71 ± 2.761**
1.01 ± 0.080
13.18 ± 0.865
25.83 ± 2.163
0.96 ± 0.075
13.32 ± 0.708
23.13 ± 1.420
1.14 ± 0.027**
13.27 ± 0.671
28.98 ± 3.950
Creatine in the form of phosphocreatine provides a quick energy source for the cells during exercise. Creatine is often taken as an energy-enhancing, fatigue-reducing supplement . In the Table 2, DBB and EO at 300 mg/kg treated groups have shown significant increase in creatine content as compared to negative control group (p < 0.001), which indicate that EO and DBB treatments may boost energy level and increase resistance to muscular fatigue. Similarly, hemoglobin concentration below normal levels results in impaired oxygen delivery to the tissues and negatively affects endurance exercise capacity . EO and DBB treated groups have shown significant increase in hemoglobin concentration, which is 14.13 ± 0.771 gm% (p < 0.01), 13.75 ± 0.187 gm% (p < 0.05), respectively as compared to negative control group, improving their efficacy to recover from fatigue.
On the contrary, exercise bouts leads to increase serum CK levels which gradually go back to basal level. Serum CK level is a marker of the functional status of muscle tissue, which also indicate cellular necrosis and acute or chronic muscular injuries. Significant decrease (p < 0.001) in CK level was also observed in EL and DBB treated groups in comparison with negative control group (Fig. 2). Venuprasad et al.  indicated lowering of CK levels in O. sanctum extract treated rats. This was attributed with the antifatigue activity of these extracts. These results indicate the beneficial effect of EL in fatigue by accelerated clearing lactate from cells as well as efficient energy utilization with potential scavenging of free radicals.
In summary, our data suggested that methanol extracts of DBB, EO and EL has highest polyphenolic content. Presence of polyphenols may suggest their involvement in the anti-fatigue effects in rats. Further, DBB, EL and EO treatment extend the swimming time to exhaustion by activating the energy metabolism by increasing or maintaining the content of glycogen in liver and muscle. They help eliminate the accumulated products like SUN, lactic acid and decrease the CK activity as well as inhibit lipid peroxidation in liver and muscle tissues. They also maintain the Cr and Hb content, which may facilitate recovery from fatigue.
Our data validated unexplored Amarkand group of plants, which are rich in components having various health benefits. Among four selected Amarkand species, DBB and EO supplementation may decrease the contribution of exercise-induced oxidative stress, and effectiveness to combat fatigue. Further studies are needed at the cellular and molecular levels to know their mechanism by means of understanding their role in regulation of glucose transport genes or through inflammatory pathways against fatigue and exercise endurance. Such type of study is required for societal benefit and sustaining the knowledge of tribal communities.
Authors are thankful to Department of Science and Technology (DST), India for the research grant. We are also thankful to Prof. Sharma G. D., Principal Rajiv Gandhi Institute of Biotechnology and Director, IRSHA for their support and encouragement.
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