Original Paper Hypoxia Tolerance and Fatigue Relief Produced By

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Original paper

Hypoxia Tolerance and Fatigue Relief Produced by Lepidium meyenii and its Water-soluble Polysaccharide in Mice

1 2 1 1 1 1 Xiao-Feng Chen , Yan-Yun Liu , Min-Jie Cao , Ling-Jing Zhang , Le-Chang Sun , Wen-Jin Su and 1* Guang-Ming Liu

1College of Food and Biological Engineering, Xiamen Key Laboratory of Marine Functional Food, Fujian Provincial, Engineering Technology Research Center of Marine Functional Food, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Jimei University, 43 Yindou Road, Xiamen, Fujian, 361021, China 2Xiamen Everbright Biotechnology Ltd, 68 Hubinbei Road, Xiamen, Fujian, 361001, China

Received November 14, 2015 ; Accepted January 24, 2016

The aim of the present study was to investigate the effects of Lepidium meyenii (maca) on hypoxia tolerance and fatigue relief, and to determine its active constituents. The results showed that, in the mouse model, maca powder could significantly prolong hypoxia time (HT) and forced swim time (FST) and optimize blood sugar ratio (BSR), liver glycogen (LG), muscle glycogen (MG), blood lactic acid, and lactic dehydrogenase. Based on these results, aqueous extracts and maca water-soluble polysaccharide (MWP) were isolated. The total sugar content of MWP is 90.41 ± 2.55%, and the main monosaccharide component is glucose (79.88%). In the mouse model, HT (40.76 ± 7.97 min), FST (48.32 ± 10.76 min), BSR (4.49 ± 4.04%), LG (11.27 ± 0.61 mg/g), and MG (1.45 ± 0.17 mg/g) were all significantly enhanced in groups given maca powder compared to the control group (p < 0.05). In summary, maca is effective in improving hypoxia tolerance and relieving fatigue, and MWP is the active substance.

Keywords: maca (Lepidium meyenii), maca aqueous extracts, maca water-soluble polysaccharide, relieve fatigue, hypoxia tolerance, BALB/c mice

Introduction Hyung, Rogers, Shao, Huang, Lu, Qien and Zheng, 2000). Maca (Lepidium meyenii) is a plant that belongs to the Brassica Maca has been utilized for almost 2000 years (Ganzera, Zhao family, Lepidium genus (Gustavo, 2012), which is native to areas and Khan), with people mainly consuming its tubers for extra in the Andes in South America with elevations over 4000 m. It is nutrition and treating diseases (Gonzales-Castañeda, Rivera, mainly cultivated in Peru (Gonzales, Gonzales. and Gonzales, Chirinos, Evelson and Gonzales, 2011). Modern technology and 2009). In dried form, maca has abundant carbohydrates (59.0%), methods have been employed to confirm or explore the different proteins (10.2%) and lipids (2.2%) (Dini, Migliuolo, Rastrelli, kinds of bioactive effects of maca. Rubio et al. established an Saturnino and Schettino, 1993), and contains amino acids, minerals animal model via ovariectomy, and found that black maca could (Gustavo, 2012), and secondary metabolites (Zheng, He, Kim, improve experimental memory impairment induced by ovariectomy

Abbreviations: BLA, blood lactic acid; BSR, blood sugar ratio; FST, forced swim time; HT, hypoxia time; LDH, lactic dehydrogenase; LG, liver glycogen: MAE, maca aqueous extracts; MG, muscle glycogen; MWP, maca water-soluble polysaccharide;

*To whom correspondence should be addressed. E-mail: [email protected] 612 X.-F. Chen et al.

(Rubio, Qiong, Liu, Jiang, Dang, Chen and Gonzales, 2011). Zheng a BALB/c mouse model in which hypoxia time could be tested to et al. found that lipid extracts of maca could enhance the sexual evaluate hypoxia tolerance induced by maca, and forced swim time function of mice and rats (Zheng, He, Kim, Hyung, Rogers, Shao, (FST) could be used as the exercise index with the biochemical Huang, Lu, Qien and Zheng, 2000). Ai et al. established a mouse parameters blood sugar ratio (BSR), LG, MG, LDH, BLA, and model of chronic unpredictable mild stress (CUMS), and BUN measured to investigate the fatigue-relieving activity of demonstrated the antidepressant-like effect of the petroleum ether maca. Furthermore, the active components could be isolated by extract of maca (Ai, Cheng, Yu, Yu and Jin, 2014). Furthermore, extraction and purification, to provide theoretical support for the anti-oxidant effects (Večeřa, Orolin, Škottová, Kazdová, Oliyarnik, use of maca as a functional food. Ulrichová and Simanek, 2007), and decreases in serum IL-6 levels (Gonzales, Gasco and Lozada, 2013) produced by maca have been Materials and Methods reported. Maca Maca powder used in this study was cultivated in Muscles experience a progressive decline in performance when Lijiang city, Yunnan province, China, and was provided by used intensively, but normally recover after a period of rest. This Xiamen Everbright Biotechnology Ltd, Fujian, China reversible phenomenon is denoted muscle fatigue (Westerblad, et Chemicals and reagents Ethyl alcohol, anthrone, sulfuric acid, al. 2003). Fatigue may decrease work efficiency and reduce quality soda lime, arabinose, galactose, xylose, and glucose were of life. Available therapies in modern medicine for relieving or purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, preventing fatigue are limited; hence, more and more people are China). NaOH was purchased from Guanghua Sci-tech Co., Ltd. turning to traditional medicine (Zhang, Ren, Huang, Ding, Zhou (Shantou, Guangdong, China). Vaseline was purchased from and Liu, 2010). Methods of evaluating fatigue relief are various. Shanghai Reagent Factory (Shanghai, China). Trifluoroacetic acid,

For example, the forced swim test is directly related to the capacity trichloroacetic acid, BaCl2, and K2SO4 were purchased from Xilong to relieve fatigue in mice (Wang, Yan and Cai, 2014). A previous Chemical Co., Ltd. (Shantou, Guangdong, China). Gelatin was study speculated that depletion of energy storage and accumulation purchased from Genebase Gene-tech Co., Ltd. (Shanghai, China); of metabolic products in the body may lead to fatigue (Zhang, Liu, nitrogen gas was purchased from Linder Gas (Xiamen, Fujian, Zhou, Wang and Sun, 2014). Blood sugar is the most preferred China). Cellulase was purchased from Novozymes (Tianjin, energy source that is mobilized during exercise. Extensive exercise China). often leads to a decrease in blood sugar level, and weakens exercise Animals Female BALB/c mice were purchased from Slaccas performance of the body (Kumar, Anand, Singsit, Khanum and Lab Animal Ltd. (Shanghai, China) at 6 _ 8 weeks old. Mice were Anilakumar, 2013). During extensive exercise, the body cannot get housed in a specific pathogen-free (SPF) barrier room (room enough energy via aerobic respiration only, so glycolysis becomes temperature 23 ± 2℃, relative humidity 50% _ 70%, 12/12 h light/ an important energy source. In the glycolysis pathway, lactate dark cycle), and food and water were available ad libitum. All dehydrogenase (LDH) is the key enzyme. An increase in LDH experiments were carried out following the institutional and would accelerate the glycolysis process, producing more energy as national guidelines, SCXK 2012-0005, and all animals were used well as blood lactic acid (BLA). Lactic acid may cause fatigue for academic research. according to Hill and Kupalov’s research (Hill, et al., 1929), so it Use of the BALB/c mouse model to determine hypoxia is also often detected as an indicator in anti-fatigue tests (Hao, Cao, tolerance and fatigue- relieving activities of maca powder Hao and Zhang, 2013; Xu, Lv, Lo, Cui, Hu and Fan, 2012). Mouse model design. After 1 week of adaption, 48 selected Glycogen, including liver glycogen (LG) and muscle glycogen mice were randomly divided into four groups (12 mice in each (MG), is an important energy source stored in the body. Glycogen group): a high-dose group, a medium-dose group, and a low-dose could be used to complement the consumption of blood sugar (Ren, group, which were treated with maca powder at doses of 1, 0.5, or Zhao, Wang, Cui and You, 2011; Shao, Wang and Zheng, 2013). 0.1 g/kg body weight, respectively; a control group, which was Because of its importance as an energy source, glycogen is often treated with distilled water. Maca samples were dispersed in selected as an index for evaluating relief of fatigue (Lin, Liu, Fang, distilled water and given by oral administration continuously for Yu, Xiong and Ke, 2014). Proteins and amino acids are other 30 days. Body weight was measured using an electronic balance sources of energy, and blood urea nitrogen (BUN) reflects the (Yingpai, Shanghai, China). production of proteins and amino acid metabolism (Wang, Shieh, Hypoxia tolerance test. Hypoxia tolerance was measured Kuo, Lee and Pan, 2006). It has been reported that BUN is 30 minutes after the latest treatment, following the method of Luo significantly positively correlated with exercise and endurance time (Luo, Cai and Hu, 2014), with modifications. In brief, three mice (You, Zhao, Regenstein and Ren, 2011). from each group were placed individually into a 250 mL sealable If the hypoxia tolerance and fatigue-relieving activities of maca glass bottle containing 15 g of soda lime, and vaseline was smeared could be established, then its active substances could be around the cap to enhance the seal. Each mouse remained in the determined. Therefore, the aim of the present study was to establish bottle until it succumbed to oxygen deficiency and died. The The Activities of Relieving Fatigue of Lepidium meyenii 613 survival time was recorded as the hypoxia time (HT). Because the Extraction and purification of maca water-soluble mice in HT test were expected to die after the experiment, polysaccharide A certain amount of MAE was dissolved with considering the welfare of mice, fewer mice were used in HT test distilled water, and ethyl alcohol was added to a final concentration than that in forced swim test. of 80%, with stirring for 6 h. The mixture was centrifuged Forced swim test. This was carried out following the method of (Beckman Coulter. 8000×g, 30 min), and the sediment was Bo (Bo, et al., 2014), with modifications. In brief, 30 minutes after collected and dissolved with distilled water. This alcohol the latest treatment, the blood glucose level was detected in the tail precipitation step was repeated five times. Finally, the sediment vein using a glucometer (Roche, Mannheim, Germany). After a was collected as maca water-soluble polysaccharide (MWP) and 5-minute rest, six mice from each group were dropped individually freeze-dried (Boyikang). into a water tank (water depth 40 cm, water temperature 30 ± 3℃) Analysis of the total sugar content and the monosaccharide with a load (4% of body weight) attached to the tail. Exhaustion composition The total sugar contents of MAE and MWP were was determined by observing when the mice sank below the determined by the anthrone-sulfuric acid method (Morris, 1948). surface of the water without being able to return to the surface MAE and MWP powders were dissolved with distilled water, within 10 s. The forced swim time (FST) was defined as the time ensuring the sample concentrations were 0.5 mg/mL. Anthrone span from when the mouse was dropped into water until 0.2 g was diluted with sulfuric acid to 100 mL, and 5 mL of exhaustion. anthrone-sulfuric acid were added to 20 mL of sample (maca Detection of plasma biochemical parameters. Mice were extract or glucose standard), and boiled for 10 min. After cooling, removed from the water tank and dried by filter paper. Blood was the absorbance was measured at 630 nm, using a UV-visible collected immediately from the eye socket of the mice, by spectrophotometer (PerkinElmer, Massachusetts, USA). Glucose removing the eyeball, and EDTA·K2 was used as a blood was used as the standard. anticoagulant. Blood glucose was detected again after the forced The monosaccharide composition of MWP was analyzed by swim test. BSR was calculated from the equation: ion chromatography (IC. Dionex, California, USA). In brief, 9 mg of sample were put into an Ann cutting bottle, then 3 mL of 4 mol/ BSR (%) = [(C1-C2)/(C1)] ×100% ······Eq. 1 L trifluoroacetic acid were added. After filling with nitrogen gas, In the equation, C1 is blood sugar content before the forced the Ann cutting bottle was put into a 100℃ water bath (Memmert) swim test and C2 is blood sugar content after the forced swim test. to digest the sample for 3 h. The sample was then dried by vacuum Plasma was separated by centrifugation (Eppendorf, Hamburg, evaporation (Thermo, Massachusetts, USA) and 300 mL of distilled Germany. 4℃, 3500×g, 7 min) to determine BLA, BUN, and LDH water were added. Arabinose, galactose, glucose, and xylose were by using an automatic biochemistry analyzer (Beckman Coulter, used as standards. IC parameters were as follows: Dionex California, USA). CarboPac PA-10 chromatographic column (4 × 250 mm); pulse Detection of tissue glycogen. Liver and gastrocnemius muscle ampere detector; gold electrode; mobile phase, 0.02 M NaOH; flow were taken from 3 executed mice from each group that had not rate, 0.6 mL/min; column temperature, 30℃; and injection volume, undergone the forced swim test, 30 min after the latest treatment. 25 μL. 2_ Liver glycogen (LG) and muscle glycogen (MG) were determined The SO4 content was analyzed following the method of following the methods of Jung (Jung, Han, Lee and Kim, 2007), Kawai (Kawai, Seno and Anno, 1969). with modifications. In brief, liver and gastrocnemius muscles were Hypoxia tolerance and fatigue-relieving activities of MAE and hydrolyzed with 2 M NaOH at 100℃ for 30 min. The hydrolysate MWP was centrifuged (Beckman Coulter, California, USA. 8000×g, Mouse model design. After one week of adaption, 84 selected 10 min) and extracted four times with anhydrous ethanol, and the mice were randomly divided into seven groups (12 mice each supernatants were discarded. The sediment was dissolved in group): MAE high-dose group, MAE medium-dose group, and distilled water and diluted to 1 mL, then 4 mL of 0.2% anthrone- MAE low-dose group, which were treated with MAE at doses of 1, sulfuric acid were added. Tubes were placed into a 100℃ water 0.5, and 0.1 g/kg body weight, respectively; MWP high-dose bath for 20 min. Absorbance was measured at 630 nm using a UV- group, MWP medium-dose group, and MWP low-dose group, visible spectrophotometer. which were treated with MWP at doses of 1, 0.5, and 0.1 g/kg body Preparation of maca aqueous extracts Maca powder was weight, respectively; control group, which was treated with immersed in ten-fold distilled water, and cellulase was added to distilled water. disrupt the cell membranes at 40℃ for 1 h, then extracted with Dose administration, hypoxia tolerance test, forced swim test, 80℃ water bath (Memmert, Frankfurt, Germany) for another 1 h. and the detection of plasma biochemical parameters and tissue The mixture was centrifuged (Beckman Coulter) 8000×g, 30 min, glycogen were the same as described in the Section “Use of the and the supernatant was collected as maca aqueous extracts (MAE) BALB/c mouse model to determine hypoxia tolerance and fatigue- and freeze-dried (Boyikang, Beijing, China). relieving activities of maca powder”. 614 X.-F. Chen et al.

Fig. 1. HT and FST analysis of maca powder groups Values are expressed as mean±S.D.. A HT of maca powder groups (n=3). B FST of maca powder groups (n=6). Data are expressed as mean ± SD. ** denotes p < 0.01 compared with the control group.

Statistical analysis Data were expressed as means ± standard medium-dose groups were very significantly longer than that of the deviation (SD), and were analyzed using SPSS version 17.0 (IBM control group. This indicates that maca significantly prolongs the Corp., New York, USA). The differences between groups were hypoxia time of mice. Furthermore, dose dependence of the assessed by analysis of variance (ANOVA); p < 0.05 was hypoxia tolerance was observed, as the ANOVA p value of the _ considered statistically significant, and p < 0.01 was considered high-dose group compared to the low-dose group was 4.6 × 10 4 statistically very significant. (<0.01). Effect of maca powder in relieving fatigue Results Forced swim test. FST results for mice administered maca Effect of maca powder on hypoxia tolerance Results for HTs powder are shown in Figure 1-B. FSTs of the three treated groups of mice in the present study are shown in Figure 1-A. HT of the were all longer than that of the control group. Furthermore, FST of high- and medium-dose groups were 47.19 ± 2.03 and 44.65 ± 1.63 the high-dose group (32.84 ± 6.73 min) was very significantly min, respectively. When compared with the control group longer than that of the control group (14.06 ± 3.10 min), as the _ (22.41 ± 2.96 min), the ANOVA p values were 2.4 × 10 4 and ANOVA p value was 0.002 (<0.01) and the increased proportion _ 3.6 × 10 4, respectively, indicating that HT of the high- and was 110.24%. This indicates that maca can significantly enhance The Activities of Relieving Fatigue of Lepidium meyenii 615

the exercise tolerance of mice. Furthermore, significant differences in FSTs between the high-dose group and the medium- and low- dose groups were observed, as the ANOVA p values were 0.04 and 0.002, respectively, meaning that the effect of maca in prolonging FST was dose-dependent. Biochemical parameters. Results for BSR, LDH, BLA, BUN, group Control Control 9.8 ± 0.56 445 ± 63 4.84 ± 0.07 8.17 ± 0.55 1.08 ± 0.03 LG, and MG of groups administered maca powder are shown in 34.18 ± 10.73 Table 1. BSR values of the treated groups were all lower than that of the control group, and the p value comparing the high-dose and control groups was 0.024 (<0.05), indicating that the difference group 9.2 ± 1.65 Low-dose 554 ± 43 4.49 ± 4.04**

1.23 ± 0.16 between them was significant. Maca powder significantly stabilized 11.09 ± 1.11** 11.09 10.48 ± 1.62 the blood sugar level of mice during the forced swim test. LDH values of the three treated groups were all higher than that of the control group. Furthermore, the difference between the MWP group high-dose group and the control group was very significant, as the 10.5 ± 2.76 9.56 ± 1.05 1.25 ± 0.02 1156 ± 1091 1156 Medium-dose 11.32 ± 3.14* 11.32

11.27 ± 0.61** 11.27 _ p value between them was 1.75 × 10 5 (<0.01). BLA values of the three treated groups were all lower than that of the control group as well. And the ANOVA results revealed that group High-dose 845 ± 681 1.45 ± 0.17* 9.55 ± 1.36** the p values comparing the medium-, low-dose, and control groups 20.22 ± 12.33 11.67 ± 1.79 11.67 10.98 ± 1.81 were 0.024 and 0.001 (<0.05), indicating that the differences between them were all significant and maca powder could dramatically inhibit the production of BLA. group Low-dose 652 ± 106 6.29 ± 4.13* 8.90 ± 3.20 5.99 ± 0.36** BUN of maca powder treated mice were all lower than that of 1.26 ± 0.01** 12.06 ± 0.01** the control group. And the their p values of three treated groups _ _ compare to the control group were 3.14 × 10 4, 2.12 × 10 5, _ 2.60 × 10 4 respectively, all lower than 0.01, mean that after the MAE group 454 ± 46

1.43 ± 0.15* consuming of maca, the body gain the ability to fulfill the 7.61 ± 0.56 9.40 ± 0.89** Medium-dose 15.00 ± 4.29 12.70 ± 1.55 requirement of energy in vigorous exercise so that there is no need to use energy sources such as protein and aminol acid. These results indicate that maca powder can very significantly group

High-dose promote the accumulation of LG (the p values between the high-, 1.18 ± 0.13 6.12 ± 0.09* 9.02 ± 1.23** 1008 ± 802 18.86 ± 11.09 15.27 ± 0.58 _ medium-, low-dose, and control groups were 5.27 × 10 10, _ _ 2.02 × 10 10, and 5.91 × 10 10 (<0.01) and MG (the p values between the high-, medium-, low-dose, and control groups were group _8 _9 _9 Low-dose 650 ± .12 4.64 × 10 , 1.72×10 , and 4.35 × 10 (<0.01). 5.60 ± 0.25** 1.67 ± 0.05** 5.42 ± 0.23** 8.04 ± 0.24** Effects of maca powder, MAE and MWP on biochemical parameters MAE and MWP of maca powder, 1. Effects Table 21.97 ± 6.21 Total sugar content and monosaccharide composition of MAE and MWP MAE and MWP in this study were extracted by boiling-water bath, indicating that they are heat resistant. The total group 7.5 ± 1.27**

714 ± 870 sugar contents of MAE and MWP are shown in Figure 2-A. The 1.74 ± 0.01** 4.95 ± 0.03** Medium-dose 16.10 ± 16.04 11.39 ± 0.31** 11.39 Maca Powder total sugar content of MAE was 69.32 ± 0.23%. After several purifications by alcohol precipitation, the total sugar contents were very significantly increased to 76.19 ± 0.12% (after the first group _8

High-dose precipitation, ANOVA p with MAE was 1.03 × 10 ), 8.71 ± 4.89* 8.20 ± 1.27 5.77 ± 0.63** 1.52 ± 0.01** 1388 ± 149** 10.64 ± 0.12** 83.31 ± 0.37% (after the third, ANOVA p compared to the first was _ 2.41 × 10 8), and 90.41 ± 2.6% (after the fifth, ANOVA p compared to the third was 0.01). The monosaccharide composition of MWP is shown by Values are expressed as mean±S.D. n=6 in each group. BSR=blood sugar ratios, BLA=blood lactic acid, LDH=lactic dehydrogenase, BUN= are expressed as mean±S.D. n=6 in each group. BSR=blood sugar ratios, BLA=blood lactic blood urea nitrogen, LG= liver glycogen, Values MG= muscle glycogen. * denotes p < 0.05 compared with the control group; ** denotes p < 0.01 compared with the control group. BSR (%) LDH (IU/L) BLA (mmol/L) BLA BUN (mmol/L) MG (mg/g) LG (mg/g) comparing Figures 2-B and 2-C. Figure 2-B is the chromatogram of four standard monosaccharides, in which peak 1 is arabinose, peak 2 is galactose, peak 3 is glucose, and peak 4 is xylose. Figure 2-C is the chromatogram of MWP, in which only one peak near 17 616 X.-F. Chen et al.

Fig. 2. Analysis of MAE and MWP A Total sugar content of MAE and MWP, where 0 is MAE, 1 is MWP after the first alcohol precipitation, 3 is MWP after the third alcohol precipitation, and 5 is MWP after the fifth alcohol precipitation. B IC chromatogram of four standard monosaccharides, in which peak 1 is arabinose, peak 2 is galactose, peak 3 is glucose, and peak 4 is xylose. C Chromatogram of MWP, in which peak 3 is glucose. Data are expressed as mean ± SD. ** denotes p < 0.01 compared to the control group. min was observed, which is close to the glucose peak in Figure compared to the control group were 0.026 and 0.006, respectively, 2-B, indicating that the main monosaccharide composition of meaning that HT values of the high- and medium-dose groups were 2_ MWP is glucose, and the content is 79.88%. SO4 content in MAE significantly longer than that of the control group. This indicates was found to be 14.36 ± 3.82%, but null in MWP. that MAE can significantly prolong the HT of mice. Effect of MAE on hypoxia tolerance HT results for mice in the Effect of MAE on relief of fatigue Forced swim test. FST MAE groups are shown in Figure 3-A. HT of the high-, medium-, results for mice administered MAE are shown in Figure 3-B, in and low-dose groups and the control group were 51.91 ± 2.11, which values for the high-, medium-, and low-dose mice were 68.58 ± 16.33, 44.30 ± 3.97, and 14.06 ± 3.10 min, respectively. 47.50 ± 30.04, 35.25 ± 14.06, and 30.78 ± 4.64 min, respectively, The ANOVA p values of the high- and medium-dose groups all longer than that of the control group (14.06 ± 3.10 min). The The Activities of Relieving Fatigue of Lepidium meyenii 617

Fig. 3. HT and FST analysis of MAE groups Values are expressed as mean±S.D.. A HT of MAE groups (n=3). B FST of MAE groups (n=6). Data are expressed as mean ± SD. * denotes p < 0.05 compared with the control group; ** denotes p < 0.01 compared with the control group.

difference between the medium-dose group and the control group groups were 0.004, 0.003, and 0.001 [<0.01]), and MG (the p was significant, as the ANOVA p value was 0.025 (<0.05). That values comparing the medium-, low-dose, and control groups were indicated MAE could significantly enhance the exercise tolerance 0.004 and 0.003 [<0.01]). of mice. But as the p value for the high-dose group was high, the Effect of MWP on hypoxia tolerance Figure 4-A shows the result for this group was considered to be not significant, although HT results for the groups administered MWP. HT values of the its mean value was the highest among the three groups. high-, medium-, and low-dose groups and the control group were Biochemical parameters. For mice administered MAE, the 40.76 ± 7.97, 36.92 ± 2.26, 46.87 ± 15.13, and 14.06 ± 3.10 min, results for biochemical parameters are shown in Table 1. According respectively. And the ANOVA p values of the high-, medium-, and to the ANOVA analysis, significantly better results were observed low-dose groups compared to the control group were 0.103, 0.171, for BSR (the p value comparing the low-dose and control groups and 0.048, respectively, which means that HT of the low-dose was 0.023 [<0.05]), BUN (the p values comparing the high-, low- group was significantly longer than that of the control group. This dose, and control groups were 0.012 and 0.008 [<0.05]), LG (the p indicates that MWP can prolong the HT of mice. values comparing the high-, medium-, low-dose, and control Effect of MWP on relief of fatigue 618 X.-F. Chen et al.

Fig. 4. HT and FST analysis of MWP groups Values are expressed as mean±S.D.. A HT of MWP groups (n=3). B FST of MWP groups (n=6). Data are expressed as mean ± SD. * denotes p < 0.05 compared with the control group; **

Forced swim test. Figure 4-B shows the results of FSTs for the and control groups were 0.005, 0.002, and 0.002 [<0.01]) and MG groups administered MWP, in which the values for high-, medium-, ( p value comparing the high-dose and control groups was 0.031 and low-dose groups were 40.76 ± 7.97, 36.92 ± 2.26, and [<0.05]). And other three parameters: LDH, BLA and BUN were 46.87 ± 15.13 min, respectively, all longer than that of the control not optimized significantly. group (14.06 ± 3.10 min). Furthermore, the ANOVA p value of the low-dose group compared to the control group was 0.048 [<0.05]. Discussion This indicates that MWP can significantly enhance the exercise In the pilot study, the HT and fatigue-relieving activities of ability of mice. maca powder, MAE, maca alcohol extracts, and maca chloroform Biochemical parameters. Results for biochemical parameters extracts were investigated, and the results showed that only maca of the groups administered MWP are shown in Table 1. Three of powder and MAE could significantly prolong HT and enhance the the six biochemical parameters were detected to be significantly athletic ability of mice. Therefore, maca powder and MAE were better than that of the control group: BSR ( p values comparing the selected for further research. Furthermore, previous studies found medium-, low-dose, and control groups were 0.011 and 0.002 that the carbohydrate content of maca is approximately 59.0% [<0.05]), LG ( p values comparing the high-, medium-, low-dose, (Dini, Migliuolo, Rastrelli, Saturnino and Schettino, 1993), and the The Activities of Relieving Fatigue of Lepidium meyenii 619 total sugar content of MAE in this study was found to be (Wang, Li, Fan, Chen, Liu, Cheng, Gao and Zhou, 2010), 69.32 ± 0.23%. Various studies have shown that polysaccharides Hippophae rhamnoides L., Lycium barbarum L., Lycium demonstrate biological activities (Jin, Huang, Ke and Peng, 2013; ruthenicum Murr, and Nitraria tangutorum Bobr (Ni, Gao, Wang, You, Yin, Zhang and Jiang, 2014), which makes polysaccharides Du, Li, Li, Wei and Bi, 2013). increasingly attractive as therapeutic agents. Hence we focused on Furthermore, of the six measured biochemical parameters the water-soluble polysaccharide components in tracking down the (BSR, LDH, BLA, BUN, LG, and MG) mice in the maca powder active substances in maca, which is effective in improving HT and group tested positive, which may indicate the mechanism by which relieving fatigue in mice. maca powder relieves fatigue is to enhance energy storage within In order to investigate its biological activities, highly purified the body, including LG and MG, and then to provide a long-term, MWP was essential. However, the existing methods for extracting continuous energy supply. Meanwhile, the increase in LDH may and purifying polysaccharides are complicated, and generally accelerate the glycolysis process to produce more ATP, and to microwave, ultrasonic, and chromatographic methods are needed. digest BLA more quickly after exercise, allowing the body to Hence we developed a simple method to extract and purify MWP, recover normal conditions and prepare for the next exercise. At the which only requires five steps of alcohol precipitation, and the final meantime, maca has the potential to inhibit the production of lactic purity of MWP was up to 90.41%. This simple method equates to a acid and protect cells from low pH conditions. And could relatively low cost process that is appropriate for industrial significantly reduce the consumption of protein and amino acid, applications. thus less BUN were produced and no harm would be introduced by With respect to hypoxia tolerance, previous studies showed BUN. that a lack of oxygen would lead to low oxygen pressure in MWP may be the active substance in maca because of its mitochondria, which may cause a shortage of energy that then ability to significantly optimize plasma biochemical parameters and affects the activity of cellular ion pumps, disturbs the gradients that enhance energy storage. In brief, MAE could stabilize BSR, control homeostasis, and kills the cell (Zhang et al., 2011). And increase LDH, reduce BUN, and increase LG and MG, but it did studies have demonstrated that polysaccharides extracted from not inhibit the production of BLA. And BSR, LG, and MG values Sipunculus nudus L (Zhang et al., 2011), Brassica rapa L (Yue, of the MWP group were positive. Jiang, Su, Pi, Chao and Ping, 2010), and Schisandra chinensis As described above, besides its high purity and simple (Zhang, et al., 2011) could significantly prolong hypoxia time. In extraction method, another characteristic of MWP is that it is heat- the present study, each mouse was put into a sealed bottle, which resistant, as it still has biological activity after 1 h in an 80℃ water contained a certain volume of air that was not replenished, to bath during the extraction process. More research could be carried simulate a low-oxygen environment to investigate the hypoxia out on MWP in the future, such as determining the structure- tolerance of mice. The longest HT values of the maca powder, function relationship between MWP and fatigue-relieving activity. MAE, and MWP groups were 47.19 ± 2.03, 68.58 ± 16.33, and Based on the results above, two kinds of new relevant products 46.87 ± 15.13 min, respectively. MAE performed better than maca were developed and patented. One was named “the essence of powder and MWP, the performances of maca powder and MWP maca and seaweed” (patent number CN104397707A), in which were about the same, and they all performed significantly better maca powder and MWP were the main constituents, and kelp, than the control group. Considering the progressive relationship collagen peptide, and Long-Yan were also added. The other was between the sources of these three samples, we may speculate that named “yogurt with maca” (patent number CN104304454A), in maca improves hypoxia tolerance, and MWP is one of the active which MWP was added to fresh milk that then underwent a substances, but not the only one. Some other active constituents fermentation process to produce a delicious and nutritious yogurt. such as benzylglucosinolate (Ikeuchi, Koyama, Takei, Kino and These two products were investigated and found to be effective in Yazawa, 2009) may be eliminated during the alcohol precipitation relieving fatigue. steps, as they may be soluble in 80% alcohol solution. In conclusion, we can confirm that maca is effective in The forced swim test was carried out as an exercise model to enhancing hypoxia tolerance and relieving fatigue and may evaluate fatigue-relieving activity. FST values of the maca powder, speculate that MWP is at least one of the active substances. MAE, and MWP groups were 32.84 ± 6.73, 35.25 ± 14.06, and Furthermore, the role of MWP is to maintain the energy supply of 48.32 ± 10.77 min, respectively, so that a trend of increasingly the body, including blood sugar and glycogen (LG and MG). good performance was observed with successive extraction steps. However, it is probably not the only active substance. Some other Hence it seems that MWP is at least one of the active substances substances that give maca powder the ability to enhance LDH and that gives maca powder and MAE the ability to improve FST inhibit the production of BLA may be insoluble in water. results of mice. Previous studies have demonstrated that polysaccharides extracted from other species are effective in Ackenowledgrments This work was supported by the Grant from enhancing FST results of mice, such as Panax ginseng C. A. Meyer the National Natural Scientific Foundation of China (31171660, 620 X.-F. Chen et al.

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