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Anti-fatigue activity of a novel polysaccharide conjugates from Ziyang green tea
Accepted Manuscript Title: Anti-fatigue activity of a novel polysaccharide conjugates from Ziyang green tea Author: Aiping Chi Hong Li Chenzhe Kang Huanhuan Guo Yimin Wang Fei Guo Liang Tang PII: DOI: Reference:
S0141-8130(15)00458-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.06.055 BIOMAC 5201
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
27-2-2015 12-6-2015 29-6-2015
Please cite this article as: A. Chi, H. Li, C. Kang, H. Guo, Y. Wang, F. Guo, L. Tang, Anti-fatigue activity of a novel polysaccharide conjugates from Ziyang green tea, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.06.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Anti-fatigue activity of a novel polysaccharide conjugates from
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Ziyang green tea
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Aiping Chi , Hong Li, Chenzhe Kang, Huanhuan Guo, Yimin Wang, Fei
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Guo, Liang Tang
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Laboratory of Nutrition and Hygiene, Shaanxi Normal University, Xi’an
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710119, China.
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* Corresponding author.
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Phone: +86-29-13008408400
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Fax: +86-02985310156
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E-mail: [email protected]
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Abstract The aim of this study was to investigate the anti-fatigue activity of
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polysaccharides from Ziyang green tea. Polysaccharides were isolated
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from Ziyang green tea and its physicochemical properties were analyzed.
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Meanwhile, a 4-week weight-loaded swimming test of mice was
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established and polysaccharides were orally administrated during exercise.
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The biochemical parameters related to fatigue were determined, such as
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exhaustive time, blood urea nitrogen (BUN), blood lactate acid (Bla)
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levels and lactic dehydrogenase (LDH) activity in serum, Superoxide
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dismutase
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Malondialdehyde (MDA) and glycogen levels in skeletal muscle. The
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results demonstrated that polysaccharide from Ziyang green tea was a
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selenium–polysaccharide–protein
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administration significantly prolonged exhaustive time and increased
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glycogen level and GSH-Px activity in muscle, in addition, markedly
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decreased BUN, Bla levels and LDH activity in serum and MDA level in
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muscle. In conclusion, Se-TP treatment can significantly improve
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exercise-induced fatigue and decrease the oxidative stress induced by the
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exhaustive exercise.
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Keywords: Selenium; Polysaccharide; Exercise-induced fatigue
Glutathione
peroxidase
(GSH-Px)
activities,
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(SOD),
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conjugate
(Se-TP),
and
Se-TP
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1. Introduction Fatigue, including mental and physical fatigue, was not only associated
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with the elevated stress level caused by a modern life style, but also
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thought to be accompanied by deterioration in exercise performance [1].
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There were some reasons to induce exercise-induced fatigue, such as the
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consumption and depletion of energy sources [2], the production and
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accumulation of metabolic products [3], the dysfunction of immune
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system [4], and excessive generation of Reactive Oxygen Species (ROS),
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which are highly reactive molecules that can attack and damage cellular
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structure [5]. Therefore, recovery from exercise-induced fatigue requires
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repairing the damage that has occurred in the body and/or prompting
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elimination of the metabolic products accumulated during exercise. The
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positive effects of nutrient supplementation on exercise capacity are well
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known. Many researchers have attempted to seek natural anti-fatigue
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compounds without adverse effect to improve athletic ability, postpone
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fatigue and accelerate the elimination of fatigue in human beings [6].
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However, it is important to develop a safe and effective anti-fatigue food
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and people tend to take dietary supplements or “tonics” as an alternative.
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The function of polysaccharides has been well documented, such as
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lowering blood cholesterol, protecting against infections, modulating
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immune and improving antioxidant capacity [7-9]. Especially, some
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polysaccharides were considered to be a new sort of natural anti-fatigue 3
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substances [10,11]. In addition, Selenium (Se) is not only an essential
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element for human body but also an active center of Glutathione
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peroxidase [12,13]. Wang et al. reported that Se-polysaccharide
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conjugates possess more or stronger biological activities in comparison
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with polysaccharide [14].
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Ziyang green tea is widely distributed in Ziyang County, which is the
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famous selenium-enriched region in China. Previous researches have
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reported that the polysaccharides from Ziyang green tea have many kinds
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of activities, such as antioxidant, anti-radiation, immunomodulation and
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anti-cancer [15]. Nevertheless, there is no available information regarding
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its anti-fatigue activity and physicochemical properties. In present study,
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we investigated physicochemical properties of polysaccharides from
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Ziyang green tea and the effects of polysaccharides supplementation on
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exercise-induced fatigue, and we try to elucidate the relationship between
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its structure and anti-fatigue activity.
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2. Materials and methods
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2.1. Extraction and purification of Se-TP
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Ziyang green tea was purchased in local tea market in Ziyang county,
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Shaanxi province, China, and was identified by botanist Prof. X. Yang
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(College of Food Engineering and Nutritional Science, Shaanxi Normal
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University). The sample (1000 g) was air- and oven-dried at 45 ℃ until a
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constant weight (755 g) and then mashed into powder, and soaked in 95% 4
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alcohol (1: 5, w/v) at room temperature for 2 h. After the mixture was
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filtered, the residues were dried by airing and then extracted in hot water
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(1:10, w/v) at 80 ℃ three times, 1 h each time. The extracted solution was
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concentrated to 40% of the original volume in a rotary evaporator under
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reduced pressure, and the free proteins were deproteinized 7 times using
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the Savage method [16], and then polysaccharides were precipitated with
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4-fold volume of ethanol at 4℃ for 24 h, then dialysis against running
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water for 24 h using the membranes with molecular weight cut-off of 10
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kDa. The retentate was collected and lyophilized to afford crude
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polysaccharide. The latter was dissolved in distilled water (1:10, w/v) and
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then injected into a column (2.0 × 80 cm) of DEAE-52 cellulose, and
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eluted with distilled water at rate of 0.8 ml/min and collected with
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automatic fraction collector (5.0 ml per tube). Polysaccharides were
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monitored using the phenol-sulfuric acid method while protein was
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detected using an ultraviolet detector at 280 nm. The elution peaks were
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collected and the main fraction was further purified using Sephadex
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G-150 column (2.0 cm × 40 cm), and eluted with 0.1 mol/l NaCl solution.
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The purified ingredient was collected and named Se-TP.
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2.2. General analysis of Se-TP
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Total carbohydrate content was determined according to the
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phenol-sulfuric acid method using glucose as a standard and the
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carbohydrate recovery assay was carried out with adding a certain amount
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of glucose into sample before hydrolysis [17]. Selenium content analysis
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was performed by a graphite furnace atomic absorption spectroscopy
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(ZA3000, Hitachi, Japan) as described by Deaker and Maher [18]. Se-TP
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was ground with KBr powder and then pressed into pellets for Infrared
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(IR) measurement using a Tensor 27 Bruker instrument in the frequency
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range of 400-4000 cm-1. The ultraviolet spectrogram of Se-TP was
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measured from 200 to 800 nm of wavelength with an ultraviolet and
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visible spectrophotometer (U-3900, Hitachi, Japan). Moisture of Se-TP
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was determined by drying at 110
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temperature drying oven (202-2A, Tianjin Taisite Instrument Co., LTD,
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China) and calculating the amount of evaporated water [19]. The ash
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amount was measured by incinerating sample overnight in a muffle
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furnace (SX-12-10, Shanghai Hongji Instrument Co., LTD, China) at 550
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and weighing the residue [20]. The content of protein was quantified
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for 2 h using an electric constant
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according to the Bradford method with BSA as a standard [21].
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2.3. Determination of the molecular
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Molecular mass of Se-TP was determined as our previous study [22].
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Se-TP was diluted to 10 mg/mL with 0.1 mol/L NaNO3 solution. The
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mixture was subjected to centrifuge (1,000rpm) for 10 min to give the
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supernatant. Measurements were performed on a HPGPC with an
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Ultrahydrogel linear column. Preliminary calibration of the column was 6
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conducted using T-series dextrans of different molecular weight. NaNO3
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(0.1 mol/L) was used the mobile phase at a flow rate of 0.9 mL/min. The
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molecular mass of Se-TP was calculated using the retention time based
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on the standard curve.
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2.4. Monosaccharide compositions analysis
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The monosaccharide compositions were analyzed according to the
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previous method [23]. Briefly, Se-TP was hydrolyzed into 2 ml of 3 M
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TFA at 100 ℃ for 6 h to release monosaccharides, which were
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derivatized with 40 L of 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP)
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and 40 L of 0.3 M NaOH at 70 ℃ for 2 h. The analysis of the PMP
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derivatives was performed by an HPLC (LC-2010A, Shimadzu, Japan)
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system and a RP-C18 column (4.6 mm × 250 mm, 5 m, Venusil, USA)
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was used for the separation of monosaccharide. The HPLC conditions
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were as following: the column temperature was 25 ℃, the UV absorbance
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was measured at 250 nm, and the flow phase was 0.02 M sodium acetate,
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the flow rate was 1 ml/min.
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2.5. Amino acid analysis of Se-TP
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20 mg of Se-TP was hydrolyzed under vacuum at 110℃ in 6 M
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hydrochloric acid for 24 h. The hydrolysis products were detected with an
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amino acid analysis analyzer (Hitachi L-8900, Japan) with a group of
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standard amino acids as the markers. The content of each kind of amino 7
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acids was calculated using the standard curves.
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2.6. Exercise model of mice 100 male Kunming mice (20-23g), 60-day-old, were purchased from
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Experimental Animal Centre of Xi'an Jiaotong University (Xi'an, China)
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and maintained in a temperature-controlled environment (23 ± 2 ℃) with
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humidity 50% and 12 h light-dark cycle with free access to water and
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standard rodent chow. Animals were randomly divided into 5 groups with
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20 mice each as follows: Exercise control group (EC), Fatigue control
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group (FC), three Se-TP groups with three different dose (100, 200 and
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400 mg/kg/day).
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After 1 week acclimation, exercise was carried out as described in our
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previous study [24] and made slight adjustment. Briefly, from Monday to
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Saturday in each week (total 4 weeks), all of mice were swum for 90 min
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in a pool (length 65 cm, width 50 cm, depth 50 cm, water depth 30 cm
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and temperature 30℃). Mice in FC and Se-TP groups were tied a wire of
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5% body weight on their tails whereas those in EC were only swum
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without loads. Mice in Se-TP groups were administrated with
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corresponding dose of Se-TP as well as those in EC and FC were given
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vehicle alone by gavage every morning.
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2.7. Assay of the biochemical parameters related fatigue
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On the last day of the 4th week, all of mice were performed exhausting
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exercise and time to exhaustion was recorded by a trained experimenter,
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blind to the experimental conditions. Exhaustion criterion: mice ceased
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struggling and went under water for 5 s. After exhaustion test
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(post-exhaustion), half of mice in each group were immediately
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anesthetized for the collection of blood to prepare serum, and then killed
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by decapitation for the preparation of liver and skeletal muscle
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homogenates to measure the glycogen content. The remainder mice were
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allowed to recover for 24 h and given the same treatment as above.
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Levels of blood urea nitrogen (BUN), blood lactate acid (Bla) and
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lactic dehydrogenase (LDH) in serum were determined using an
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autoanalyzer (Hitachi 7020, Hitachi, Japan). The levels of superoxide
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dismutase (SOD), glutathione peroxidase (GSH-Px), malonaldehyde
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(MDA) and glycogen in skeletal muscle, and liver glycogen were
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determined using commercially available kits from the Nanjing Jiancheng
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Biocompany (Nanjing, China).
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2.8. Statistical analysis
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Data are presented as the mean ± standard deviation (SD). Statistical
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differences between groups were evaluated using a t-test. P values＜0.05
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were considered significant.
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3. Results
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3.1. Fractionation and purification of Se-TP
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The crude polysaccharides were isolated from the dried sample using a 9
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multistep purification procedure including hot-water extraction and
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repeated ethanol precipitation with a yield of 3.46% (w/w). And then the
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crude polysaccharides were fractionated on DEAE-52 cellulose column.
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Three peaks (peak 1, 2 and 3) were obtained (shown in Fig. 1 A) with
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yields of 12.34%, 53.69% and 16.51%, respectively. Peak 2 was the main
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part of the crude polysaccharides and purified further using Sephadex
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G-150 column. The purified component was collected and named Se-TP,
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and total 12.27 g Se-TP was obtained using this method. Fig. 1 B showed
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that Se-TP was a symmetrical peak, and the curves of polysaccharide and
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protein were superposed.
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3.2. Composition of Se-TP
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The results showed that the contents of total carbohydrate, protein,
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selenium, ash and moisture in Se-TP were 83.51%, 4.76%, 2.06%, 2.15%
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and 6.33%, respectively. The result of the carbohydrate recovery assay
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showed that the average recovery was 98.92%.The results also showed
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that Se-TP was light gray powder and not soluble in organic solvents such
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as ethanol, ether, acetone, and chloroform but easily soluble in water.
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3.3. UV and IR spectra of Se-TP
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The absorption peak at 285.5 nm in the ultraviolet scan spectrogram
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(shown in Fig. 2) further demonstrated that Se-TP contained protein
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portions.
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IR spectrum of Se-TP exhibited some characteristic absorption peaks of 10
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polysaccharide (shown in Fig. 3). The peak at 3438.22 cm−1 was
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attributed to the stretching vibration of O–H and/or N–H. The peak at
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2926.21 cm−1 was attributed to the stretching vibration of C–H. The band
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at 1639.09 cm−1 would be due to the stretching vibration of C=O and/or
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the variable-angle vibration of N–H. The band at 1449.49 cm−1～1393.81
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cm−1, 1111.16 cm−1 would be due to the stretching vibration of C–O, and
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the band at 1336.28 cm−1～1233.30 cm−1 would be due to the stretching
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vibration of O–H (Chi, et al. 2015; He, et al., 2013; Wang, et al., 2013).
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3.4. Molecular weight determination of Se-TP
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As shown in Fig. 4, Se-TP was eluted as a single symmetrical peak,
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corresponding to a molecular weight of 150 kDa, which indicated that the
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polysaccharide was homogeneous.
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3.5. Monosaccharide composition of Se-TP
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Monosaccharide composition of Se-TP was determined by HPLC. The
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result from Fig. 5 suggests that Se-TP was composed of Man, Rha, Glu,
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Gal, Ara and Galacturonic acid in the molar percentages of 3.57%, 1.69%,
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32.35%, 25.81%, 30.64% and 2.26%, respectively.
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3.6. Amino acid analysis of Se-TP
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As shown in Fig. 6, amino acid analysis indicated that the protein of
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Se-TP consisted of 11 amino acids (μg/mg): Asp 4.75, Ser 4.14, Glu 5.37,
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Ala 1.69, Cys 2.83, Val 3.49, Ile 5.15, Leu 1.43, Lys 4.76, His 3.87, Arg
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8.35, respectively. Total protein content of Se-TP was determined as
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4.58% by above analysis of the amino acids.
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3.7. Effect of Se-TP on the biochemical parameters related fatigue As shown in Table 1 and 2, mice in FC, Se-TP groups, which tied
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additional load, were carried out intense exercise than those in EC. For
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this reason, the exhausting time of FC mice was significantly shortened in
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comparison with those of EC (P < 0.01). Moreover, the extent of fatigue
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was also confirmed by the increase in BUN, Bla, LDH and the decrease
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in muscle glycogen level (P < 0.05 or P < 0.01) at the time periods of
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Post-exhaustion or Recover. However, administration of Se-TP, to a
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certain extent, not only significantly prolonged the exhausting time but
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also decreased the levels of BUN and Bla as well as LDH activity in
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comparison with FC.
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In addition, as shown in Table 2, the content of muscle glycogen was
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decreased whereas MDA level was increased in skeletal muscle in FC
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mice in comparison with EC group. However, treatments of Se-TP (200
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and 400 mg/kg) significantly recovered muscle glycogen level, Se-TP
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(400 mg/kg) markedly increased the activity of GSH-Px, and Se-TP
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significantly decreased the level of MDA in skeletal when compared with
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FC group (P < 0.05 or P < 0.01).
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4. Discussion
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A water-soluble polysaccharide coded Se-TP was isolated from Ziyang 12
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green tea. It exhibited a single and symmetrical peak in the HPGPC
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analysis, which indicated its homogeneity based on the distribution of
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molecular weight. Meanwhile, the result showed that Se-TP was a novel
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conjugate with portions of polysaccharide and protein. The contents of
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total carbohydrate in Se-TP were determined using phenol-sulfuric
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method which has higher recovery rate and less loss of monosaccharide
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during hydrolysis. The protein content of Se-TP was determined using
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Bradford method and the total amino acid analysis method. The result
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showed that the protein content of Se-TP in former was bigger than the
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latter. The reason was related the loss of part amino acid during protein
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hydrolysis.
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Swimming with weight loading of mice is an ideal experimental model
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to evaluate the capacity of anti-fatigue [11]. In the present study, a
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four-week swimming model of mice was carried out. The results
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demonstrated that the exhausting time of FC (loaded 5% weight of body)
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mice was significantly shortened in comparison with those of EC
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(without load). Whereas, Se-TP treatment prolonged the exhausting time
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of mice, indicating that Se-TP possess an anti-fatigue effect.
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BUN, a metabolic product of protein and amino acid, is one of blood
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biochemical parameters related to fatigue. The less an animal is adapted
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to exercise, the more the BUN level increases [6]. The result showed
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Se-TP treatment reduced serum BUN levels and indicated its positive 13
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effect on enhancing endurance. As an important parameter of fatigue, Bla
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is produced by anaerobic glycolysis, which can be further degraded via
277
the tricarboxylic acid cycle for the production of ATP or removed to other
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tissue for oxidization or gluconeogenesis [25]. So there are two dynamic
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balances between aerobic oxidation of glycogen and anaerobic glycolysis
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and between the accumulation and elimination of Bla. Whereas, it was
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unbalanced in a high-intensity exercise [26]. The increase of Bla
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concentration and consequent lactic acidosis observed in skeletal muscles
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during exercise were major cause of muscle fatigue. The result showed
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Se-TP treatment reduced Bla levels and indicated its can improve
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glycogen metabolism.
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In exercise-fatigue conditions, the activities of SOD and GSH-Px are
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generally considered indicators of the capacity of the antioxidant
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defensive system [27]. Malondialdehyde is an oxidative degradation
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product of cell-membrane lipids, and its level is an indicator of lipid
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peroxidation [28]. These conditions are also marked by the release of
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LDH into the serum, serving as an indirect index of the damage of
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membrane structure [29]. Energy use leads to reduction of glycogen in
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muscle and the level of muscle glycogen is one of key factors of
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evaluating exercise time to exhaustion [24]. As indicators for oxidative
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stress, the present study showed that the muscle could have been
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assaulted by the ROS for higher activity of LDH in mice serum after
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exhaustive exercise. It also provided some evidence such as lower activity
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of SOD and GSH-Px and higher level of malondialdehyde in muscle. The
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results suggested that there was a closed relationship between free-radical
300
attack and exhaustive exercise. In the current study, we found that all effects were blocked just after
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Se-TP administration. These results include the lower activity of LDH in
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serum, a higher activity GSH-Px and glycogen levels, and the decrease of
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malondialdehyde content in muscle. This is similar to results of other
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works [26] and suggested that the anti-fatigue effect of Se-TP probably
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occurred through protection of corpuscular membrane by preventing lipid
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oxidation via modifying GSH-Px activity. Considerable evidence has
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indicated that the role of polysaccharide in scavenging ROS is likely
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associated with hydrogen and hydroxyl of the polysaccharide chain [24],
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and
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polysaccharide also significantly affect the role of polysaccharide in
312
scavenging ROS [30]. Taken together, our results showed that Se-TP
313
supplementation is effective in improving exercise fatigue. However,
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further research on structure of Se-TP is needed in order to expose more
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details on its anti fatigue mechanism.
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their
monosaccharide
composition
and
characterization
in
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Acknowledgements This work was supported by the Fundamental Research Funds for the
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Central Universities (GK201402046), Young Teacher Research Funds for
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School of Sports, Shaanxi Normal University (No. 20151601) and Shanxi
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Province Sports Bureau Project (No.14046).
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References
322
[1] M. Tanaka, Y. Baba, Y. Kataoka, N. Kinbara, Y.M. Sagesaka, T.
326 327 328
Molecules, 16 (2010) 28–37.
ip t
325
[2] X.L. Zhang, F. Ren, W. Huang, R.T. Ding, Q.S. Zhou, X.W. Liu,
[3] T.H. Pedersen, O.B. Nielsen, G.D. Lamb, D.G. Stephenson, Sci. 305
cr
324
Kakuda, Nutr. 24 (2008) 599–603.
(2004) 1144–1147.
us
323
[4] A.D.H. Smith, S.L. Coakley, M.D. Ward, A.W. Macfarlane, P.S. Friedmann, N.P. Walsh. Brain Behav. Immun. 25 (2011) 1136–1142.
330
[5] K. Azizbeigi, S.R. Stannard, S. Atashak, M.M. Haghighi, J. Exerc. Sci.
333
M
[6] C. Xu, J. Lv, Y.M. Lo, S.W. Cui, X. Hu, M. Fan, Carbohydr. Polym. 92 (2013) 1159–1165.
d
332
Fit. 12(2014) 1–6
Ac ce pt e
331
an
329
334
[7] X.Q. Han, B.C.L. Chen, C.X. Dong, Y.H. Yang, C.H. Ko, G.G.L. Yue,
335
C. Chen, C.K. Wong, C.B.S. Lau, P.F. Tu, P.C. Shaw, K.P. Fung, W.L.
336 337 338 339 340 341 342
Hsiao, Q.B. Han, J. Agric. Food Chem. 60 (2012) 4276–4281.
[8] W. Nan, Y. Jingyue, L. Jianguo, Q. Qing, W. Tao, D. Xilin, B. Guoqiang, H. Xianli, Carbohydr. Polym. 111 (2014) 47–55.
[9] A.P. Chi, J.P. Chen, Z.Z. Wang, Z.Y. Xiong, Q.X. Li, Carbohydr. Polym. 74 (2008) 868–874. [10] J. Wang, S. Li, Y. Fan, Y. Chen, D. Liu, H. Cheng, X. Gao, Y. Zhou, J. Ethnopharmacol.130 (2010) 421–423. 17
Page 17 of 29
348 349 350 351 352
[13] C. Sanmartín, D. Plano, A.K. Sharma, J.A. Palop, Int. J. Mole. Sc. 13
ip t
347
81–110.
(2012) 9649–9672.
cr
346
[12] A.F. Martinez, L. Charlet, Rev. Environ. Sci. and Biotechnol. 8 (2009)
[14] Y. Wang, J. Chen, D. Zhang, Y. Zhang, Y. Wen, L. Li, L. Zheng, Carbohydr. Polym. 98 (2013) 1186–1190.
us
345
Biol. Macromol. 50 (2012) 59– 62.
[15] J. Wang, B. Zhao, X. Wang, J. Yao, J. Zhang, Int. J. Biol. Macromol.
an
344
[11] W. Tan, K. Yu, Y. Liu, M Ouyang, M. Yan, R. Luo, X. Zhao, Int. J.
51 (2012) 987–991.
M
343
[16] A.M. Staub, Methods in Carbohydrate. Chem.5 (1965) 5–6.
354
[17] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal.
356 357 358 359 360 361
Chem. 28 (1956) 350–356.
Ac ce pt e
355
d
353
[18] M. Deaker, W. Maher. Analytica. Chimica. Acta. 350 (1997) 287–294.
[19] X. Yang, Y. Zhao, Y. Yang, Y. Ruan, J. Agric. Food Chem. 56 (2008) 6905–6909.
[20] X. Yang, Y. Lv, L. Tian, Y. Zhao, J. Agric. Food Chem. 58 (2010) 6075–6080.
362
[21] M.M. Bradford,. Anal. Biochem. 72 (1976) 248–254.
363
[22] A.P. Chi, J.P. Chen, Z.Z. Wang, Z.Y. Xiong, Q.X. Li, Carbohydr.
364
Polym. 74(2008) 868–874. 18
Page 18 of 29
365 366 367 368
[23] A.P. Chi, C. Kang, Y. Zhang, L. Tang, H. Guo, H. Li, K. Zhang, Carbohydr. Polym. 122 (2015) 189–196. [24] A.P. Chi, L. Tang, J. Zhang, K. Zhang, Int. J. Sport Nutr. Exerc. Metab. 22 (2012) 479–485. [25] J. M. Jia, C. F. Wu, Pharm. Biol. 46 (2008) 433–436.
370
[26] A. Niu, J. Wu, D. Yu, R. Wang, Int. J. Biol. Macromol. 42 (2008)
cr
447–449.
us
371
ip t
369
[27] G. Lenaz, Biochim. Biophys. Acta. 1366 (1998) 53–67.
373
[28] H.M. Alessio, A.H. Goldfarb, J. Appl. Physiol. 64, (1988)
376 377
M
[29] S. Passarella, L. de Bari, D. Valenti, R. Pizzuto, G. Paventi, A. Atlante, FEBS Lett. 582 (2008) 3569–3576.
d
375
1333–1336.
[30] A. Kardosova, E. Machova, Fitoterapia, 77 (2006) 367–373.
Ac ce pt e
374
an
372
19
Page 19 of 29
cr us
Dose
Exhausting Time
(n=20)
(mg/kg)
(min)
EC
-
153.04±16.58
FC
-
129.76±11.19**
Se-TP
100
138.52±12.14#
Se-TP
200
143.64±14.05#
Se-TP
400
142.64±11.34#
BUN (mmol/L)
M
Group
an
Table 1 Comparison of biochemical markers related fatigue in serum
Post-exhaustion
Recover
Bla (mmol/L) Post-exhaustion
LDH(U/L)
Recover
Post-exhaustion
△△
353.74±22.19
224.85±27.73
14.08±2.25*
13.42±1.78**
368.85±26.97
316.73±29.64*
12.72±2.28
10.90±0.46#
△
334.38±26.91
279.63±48.37
11.47±1.85#
10.34±0.52#
△
299.25±31.67#
232.42±64.13#
12.03±2.76
10.54±2.16#
△
303.85±27.12#
253.09±25.56#
pt
ed
11.34±2.18
ce
378
9.17±1.05
Vs. EC, *: p < 0.05, **: p < 0.01.
380
Vs. FC, #: p < 0.05, ##: p < 0.01.
381
Vs. Post-exhaustion, △: p < 0.05, △△: p < 0.01.
806.19±17.36
792.43±15.58
1102.47±43.26**
903.52±27.82**
964.68±29.77
824.69±25.78#
△△
△
930.57±24.86#
846.25±19.78#
△△
△
929.71±28.54#
829.76±22.11#
△
△
Ac
379
△
Recover
382
20
Page 20 of 29
△
△△
cr us
Group
Dose
(n=20)
(mg/kg)
EC
-
10.19±2.23
FC
-
9.94±2.17
Se-TP
100
9.92±1.73
Se-TP
200
10.35±2.26
Se-TP
400
an
Table 2 Comparison of biochemical markers related fatigue in muscle
Post-exhaustion
Recover
14.27±2.81
Post-exhaustion
△
Recover
GSH-Px (U/mg protein) Post-exhaustion
MDA(nmol/mg protein)
Recover
Post-exhaustion △
4.71±0.86
△△
9.33±1.87*
5.06±0.32
△
△
7.62±1.37#
4.57±0.26#
△
△
7.58±2.31#
4.39±0.14#
△
6.94±1.15#
3.88±1.02##
58.64±7.47
47.95±5.78
98.62±11.85*
106.43±17.89*
52.17±5.26*
46.51±6.02*
104.35±17.64
122.73±13.41
59.54±10.67
46.76±5.15
14.82±2.13#
118.71±14.98
129.84±18.27
61.50±4.44
50.49±6.33
14.64±1.47#
112.43±13.19
125.37±16.79
69.86±8.75#
57.34±4.66#
ed
133.14±11.53
10.62±3.24*
13.51±3.17
Vs. EC, *: p < 0.05, **: p < 0.01.
384
Vs. FC, #: p < 0.05, ##: p < 0.01.
385
Vs. Post-exhaustion, △: p < 0.05, △△: p < 0.01.
△
△
Ac
383
△
21
Page 21 of 29
Recover
8.37±1.45
115.47±10.38
pt
10.54±1.61
SOD (U/mg protein)
M
Glycogen (mg/g protein)
ce
382
△
△
386
Figure captions
387 388
Figure 1. Fractionation and purification profiles of Se-TP A: Fractionation of the crude polysaccharides in DEAE-52 cellulose
390
column; B: Purification of Se-TP in Sephadex G-150 column
ip t
389
Figure 2. UV spectrum of Se-TP
us
392
cr
391
393
Figure 3. IR spectrum of Se-TP
an
394
396
M
395
Figure 4. HPGPC profile of Se-TP
Figure 5. The HPLC chromatograms of SE-TP
Ac ce pt e
398
d
397
399
a: Standard (1 Man; 2 Rib; 3 Rha; 4 GlucA; 5 GalcA; 6 Glu; 7 Xyl; 8
400
Gal; 9 Ara; 10 FUC). b: Monosaccharide of SE-TP
401 402 403 404
Figure 6. Amino acid composition of SE-TP
Highlight:
405 406 407
1. Polysaccharide from Ziyang green tea was a selenium–enriched polysaccharide conjugate (Se-TP).
22
Page 22 of 29
409 410 411 412
2. Se-TP administrations significantly prolonged exhaustive time and increased glycogen level and GSH-Px activity in muscle. 3. Se-TP administrations markedly decreased BUN, Bla levels and LDH activity in serum and MDA level in muscle. 4. Se-TP treatments can significantly improve exercise-induced fatigue.
ip t
408
cr
413
Ac ce pt e
d
M
an
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414
23
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pt
ce
Ac M
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i
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pt
ce
Ac M
an
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i
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ed
pt
ce
Ac M
an
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i
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ed
pt
ce
Ac M
an
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S0141-8130(15)00458-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.06.055 BIOMAC 5201
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
27-2-2015 12-6-2015 29-6-2015
Please cite this article as: A. Chi, H. Li, C. Kang, H. Guo, Y. Wang, F. Guo, L. Tang, Anti-fatigue activity of a novel polysaccharide conjugates from Ziyang green tea, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.06.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Anti-fatigue activity of a novel polysaccharide conjugates from
2
Ziyang green tea
3
Aiping Chi , Hong Li, Chenzhe Kang, Huanhuan Guo, Yimin Wang, Fei
5
Guo, Liang Tang
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4
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6
Laboratory of Nutrition and Hygiene, Shaanxi Normal University, Xi’an
8
710119, China.
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9
M
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12
d
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* Corresponding author.
14
Phone: +86-29-13008408400
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Fax: +86-02985310156
16
E-mail: [email protected]
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17
Abstract The aim of this study was to investigate the anti-fatigue activity of
19
polysaccharides from Ziyang green tea. Polysaccharides were isolated
20
from Ziyang green tea and its physicochemical properties were analyzed.
21
Meanwhile, a 4-week weight-loaded swimming test of mice was
22
established and polysaccharides were orally administrated during exercise.
23
The biochemical parameters related to fatigue were determined, such as
24
exhaustive time, blood urea nitrogen (BUN), blood lactate acid (Bla)
25
levels and lactic dehydrogenase (LDH) activity in serum, Superoxide
26
dismutase
27
Malondialdehyde (MDA) and glycogen levels in skeletal muscle. The
28
results demonstrated that polysaccharide from Ziyang green tea was a
29
selenium–polysaccharide–protein
30
administration significantly prolonged exhaustive time and increased
31
glycogen level and GSH-Px activity in muscle, in addition, markedly
32
decreased BUN, Bla levels and LDH activity in serum and MDA level in
33
muscle. In conclusion, Se-TP treatment can significantly improve
34
exercise-induced fatigue and decrease the oxidative stress induced by the
35
exhaustive exercise.
36
Keywords: Selenium; Polysaccharide; Exercise-induced fatigue
Glutathione
peroxidase
(GSH-Px)
activities,
Ac ce pt e
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M
(SOD),
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18
conjugate
(Se-TP),
and
Se-TP
2
Page 2 of 29
37
1. Introduction Fatigue, including mental and physical fatigue, was not only associated
39
with the elevated stress level caused by a modern life style, but also
40
thought to be accompanied by deterioration in exercise performance [1].
41
There were some reasons to induce exercise-induced fatigue, such as the
42
consumption and depletion of energy sources [2], the production and
43
accumulation of metabolic products [3], the dysfunction of immune
44
system [4], and excessive generation of Reactive Oxygen Species (ROS),
45
which are highly reactive molecules that can attack and damage cellular
46
structure [5]. Therefore, recovery from exercise-induced fatigue requires
47
repairing the damage that has occurred in the body and/or prompting
48
elimination of the metabolic products accumulated during exercise. The
49
positive effects of nutrient supplementation on exercise capacity are well
50
known. Many researchers have attempted to seek natural anti-fatigue
51
compounds without adverse effect to improve athletic ability, postpone
52
fatigue and accelerate the elimination of fatigue in human beings [6].
53
However, it is important to develop a safe and effective anti-fatigue food
54
and people tend to take dietary supplements or “tonics” as an alternative.
Ac ce pt e
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38
55
The function of polysaccharides has been well documented, such as
56
lowering blood cholesterol, protecting against infections, modulating
57
immune and improving antioxidant capacity [7-9]. Especially, some
58
polysaccharides were considered to be a new sort of natural anti-fatigue 3
Page 3 of 29
substances [10,11]. In addition, Selenium (Se) is not only an essential
60
element for human body but also an active center of Glutathione
61
peroxidase [12,13]. Wang et al. reported that Se-polysaccharide
62
conjugates possess more or stronger biological activities in comparison
63
with polysaccharide [14].
ip t
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Ziyang green tea is widely distributed in Ziyang County, which is the
65
famous selenium-enriched region in China. Previous researches have
66
reported that the polysaccharides from Ziyang green tea have many kinds
67
of activities, such as antioxidant, anti-radiation, immunomodulation and
68
anti-cancer [15]. Nevertheless, there is no available information regarding
69
its anti-fatigue activity and physicochemical properties. In present study,
70
we investigated physicochemical properties of polysaccharides from
71
Ziyang green tea and the effects of polysaccharides supplementation on
72
exercise-induced fatigue, and we try to elucidate the relationship between
73
its structure and anti-fatigue activity.
74
2. Materials and methods
75
2.1. Extraction and purification of Se-TP
Ac ce pt e
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76
Ziyang green tea was purchased in local tea market in Ziyang county,
77
Shaanxi province, China, and was identified by botanist Prof. X. Yang
78
(College of Food Engineering and Nutritional Science, Shaanxi Normal
79
University). The sample (1000 g) was air- and oven-dried at 45 ℃ until a
80
constant weight (755 g) and then mashed into powder, and soaked in 95% 4
Page 4 of 29
alcohol (1: 5, w/v) at room temperature for 2 h. After the mixture was
82
filtered, the residues were dried by airing and then extracted in hot water
83
(1:10, w/v) at 80 ℃ three times, 1 h each time. The extracted solution was
84
concentrated to 40% of the original volume in a rotary evaporator under
85
reduced pressure, and the free proteins were deproteinized 7 times using
86
the Savage method [16], and then polysaccharides were precipitated with
87
4-fold volume of ethanol at 4℃ for 24 h, then dialysis against running
88
water for 24 h using the membranes with molecular weight cut-off of 10
89
kDa. The retentate was collected and lyophilized to afford crude
90
polysaccharide. The latter was dissolved in distilled water (1:10, w/v) and
91
then injected into a column (2.0 × 80 cm) of DEAE-52 cellulose, and
92
eluted with distilled water at rate of 0.8 ml/min and collected with
93
automatic fraction collector (5.0 ml per tube). Polysaccharides were
94
monitored using the phenol-sulfuric acid method while protein was
95
detected using an ultraviolet detector at 280 nm. The elution peaks were
96
collected and the main fraction was further purified using Sephadex
97
G-150 column (2.0 cm × 40 cm), and eluted with 0.1 mol/l NaCl solution.
98
The purified ingredient was collected and named Se-TP.
99
2.2. General analysis of Se-TP
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100
Total carbohydrate content was determined according to the
101
phenol-sulfuric acid method using glucose as a standard and the
5
Page 5 of 29
carbohydrate recovery assay was carried out with adding a certain amount
103
of glucose into sample before hydrolysis [17]. Selenium content analysis
104
was performed by a graphite furnace atomic absorption spectroscopy
105
(ZA3000, Hitachi, Japan) as described by Deaker and Maher [18]. Se-TP
106
was ground with KBr powder and then pressed into pellets for Infrared
107
(IR) measurement using a Tensor 27 Bruker instrument in the frequency
108
range of 400-4000 cm-1. The ultraviolet spectrogram of Se-TP was
109
measured from 200 to 800 nm of wavelength with an ultraviolet and
110
visible spectrophotometer (U-3900, Hitachi, Japan). Moisture of Se-TP
111
was determined by drying at 110
112
temperature drying oven (202-2A, Tianjin Taisite Instrument Co., LTD,
113
China) and calculating the amount of evaporated water [19]. The ash
114
amount was measured by incinerating sample overnight in a muffle
115
furnace (SX-12-10, Shanghai Hongji Instrument Co., LTD, China) at 550
116
and weighing the residue [20]. The content of protein was quantified
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102
Ac ce pt e
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M
for 2 h using an electric constant
117
according to the Bradford method with BSA as a standard [21].
118
2.3. Determination of the molecular
119
Molecular mass of Se-TP was determined as our previous study [22].
120
Se-TP was diluted to 10 mg/mL with 0.1 mol/L NaNO3 solution. The
121
mixture was subjected to centrifuge (1,000rpm) for 10 min to give the
122
supernatant. Measurements were performed on a HPGPC with an
123
Ultrahydrogel linear column. Preliminary calibration of the column was 6
Page 6 of 29
conducted using T-series dextrans of different molecular weight. NaNO3
125
(0.1 mol/L) was used the mobile phase at a flow rate of 0.9 mL/min. The
126
molecular mass of Se-TP was calculated using the retention time based
127
on the standard curve.
128
2.4. Monosaccharide compositions analysis
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The monosaccharide compositions were analyzed according to the
130
previous method [23]. Briefly, Se-TP was hydrolyzed into 2 ml of 3 M
131
TFA at 100 ℃ for 6 h to release monosaccharides, which were
132
derivatized with 40 L of 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP)
133
and 40 L of 0.3 M NaOH at 70 ℃ for 2 h. The analysis of the PMP
134
derivatives was performed by an HPLC (LC-2010A, Shimadzu, Japan)
135
system and a RP-C18 column (4.6 mm × 250 mm, 5 m, Venusil, USA)
136
was used for the separation of monosaccharide. The HPLC conditions
137
were as following: the column temperature was 25 ℃, the UV absorbance
138
was measured at 250 nm, and the flow phase was 0.02 M sodium acetate,
139
the flow rate was 1 ml/min.
140
2.5. Amino acid analysis of Se-TP
Ac ce pt e
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141
20 mg of Se-TP was hydrolyzed under vacuum at 110℃ in 6 M
142
hydrochloric acid for 24 h. The hydrolysis products were detected with an
143
amino acid analysis analyzer (Hitachi L-8900, Japan) with a group of
144
standard amino acids as the markers. The content of each kind of amino 7
Page 7 of 29
145
acids was calculated using the standard curves.
146
2.6. Exercise model of mice 100 male Kunming mice (20-23g), 60-day-old, were purchased from
148
Experimental Animal Centre of Xi'an Jiaotong University (Xi'an, China)
149
and maintained in a temperature-controlled environment (23 ± 2 ℃) with
150
humidity 50% and 12 h light-dark cycle with free access to water and
151
standard rodent chow. Animals were randomly divided into 5 groups with
152
20 mice each as follows: Exercise control group (EC), Fatigue control
153
group (FC), three Se-TP groups with three different dose (100, 200 and
154
400 mg/kg/day).
M
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147
After 1 week acclimation, exercise was carried out as described in our
156
previous study [24] and made slight adjustment. Briefly, from Monday to
157
Saturday in each week (total 4 weeks), all of mice were swum for 90 min
158
in a pool (length 65 cm, width 50 cm, depth 50 cm, water depth 30 cm
159
and temperature 30℃). Mice in FC and Se-TP groups were tied a wire of
160
5% body weight on their tails whereas those in EC were only swum
161
without loads. Mice in Se-TP groups were administrated with
162
corresponding dose of Se-TP as well as those in EC and FC were given
163
vehicle alone by gavage every morning.
164
2.7. Assay of the biochemical parameters related fatigue
165
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On the last day of the 4th week, all of mice were performed exhausting
8
Page 8 of 29
exercise and time to exhaustion was recorded by a trained experimenter,
167
blind to the experimental conditions. Exhaustion criterion: mice ceased
168
struggling and went under water for 5 s. After exhaustion test
169
(post-exhaustion), half of mice in each group were immediately
170
anesthetized for the collection of blood to prepare serum, and then killed
171
by decapitation for the preparation of liver and skeletal muscle
172
homogenates to measure the glycogen content. The remainder mice were
173
allowed to recover for 24 h and given the same treatment as above.
us
cr
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166
Levels of blood urea nitrogen (BUN), blood lactate acid (Bla) and
175
lactic dehydrogenase (LDH) in serum were determined using an
176
autoanalyzer (Hitachi 7020, Hitachi, Japan). The levels of superoxide
177
dismutase (SOD), glutathione peroxidase (GSH-Px), malonaldehyde
178
(MDA) and glycogen in skeletal muscle, and liver glycogen were
179
determined using commercially available kits from the Nanjing Jiancheng
180
Biocompany (Nanjing, China).
181
2.8. Statistical analysis
Ac ce pt e
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182
Data are presented as the mean ± standard deviation (SD). Statistical
183
differences between groups were evaluated using a t-test. P values＜0.05
184
were considered significant.
185
3. Results
186
3.1. Fractionation and purification of Se-TP
187
The crude polysaccharides were isolated from the dried sample using a 9
Page 9 of 29
multistep purification procedure including hot-water extraction and
189
repeated ethanol precipitation with a yield of 3.46% (w/w). And then the
190
crude polysaccharides were fractionated on DEAE-52 cellulose column.
191
Three peaks (peak 1, 2 and 3) were obtained (shown in Fig. 1 A) with
192
yields of 12.34%, 53.69% and 16.51%, respectively. Peak 2 was the main
193
part of the crude polysaccharides and purified further using Sephadex
194
G-150 column. The purified component was collected and named Se-TP,
195
and total 12.27 g Se-TP was obtained using this method. Fig. 1 B showed
196
that Se-TP was a symmetrical peak, and the curves of polysaccharide and
197
protein were superposed.
198
3.2. Composition of Se-TP
M
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188
The results showed that the contents of total carbohydrate, protein,
200
selenium, ash and moisture in Se-TP were 83.51%, 4.76%, 2.06%, 2.15%
201
and 6.33%, respectively. The result of the carbohydrate recovery assay
202
showed that the average recovery was 98.92%.The results also showed
203
that Se-TP was light gray powder and not soluble in organic solvents such
204
as ethanol, ether, acetone, and chloroform but easily soluble in water.
205
3.3. UV and IR spectra of Se-TP
Ac ce pt e
d
199
206
The absorption peak at 285.5 nm in the ultraviolet scan spectrogram
207
(shown in Fig. 2) further demonstrated that Se-TP contained protein
208
portions.
209
IR spectrum of Se-TP exhibited some characteristic absorption peaks of 10
Page 10 of 29
polysaccharide (shown in Fig. 3). The peak at 3438.22 cm−1 was
211
attributed to the stretching vibration of O–H and/or N–H. The peak at
212
2926.21 cm−1 was attributed to the stretching vibration of C–H. The band
213
at 1639.09 cm−1 would be due to the stretching vibration of C=O and/or
214
the variable-angle vibration of N–H. The band at 1449.49 cm−1～1393.81
215
cm−1, 1111.16 cm−1 would be due to the stretching vibration of C–O, and
216
the band at 1336.28 cm−1～1233.30 cm−1 would be due to the stretching
217
vibration of O–H (Chi, et al. 2015; He, et al., 2013; Wang, et al., 2013).
218
3.4. Molecular weight determination of Se-TP
an
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210
As shown in Fig. 4, Se-TP was eluted as a single symmetrical peak,
220
corresponding to a molecular weight of 150 kDa, which indicated that the
221
polysaccharide was homogeneous.
222
3.5. Monosaccharide composition of Se-TP
Ac ce pt e
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219
223
Monosaccharide composition of Se-TP was determined by HPLC. The
224
result from Fig. 5 suggests that Se-TP was composed of Man, Rha, Glu,
225
Gal, Ara and Galacturonic acid in the molar percentages of 3.57%, 1.69%,
226
32.35%, 25.81%, 30.64% and 2.26%, respectively.
227
3.6. Amino acid analysis of Se-TP
228
As shown in Fig. 6, amino acid analysis indicated that the protein of
229
Se-TP consisted of 11 amino acids (μg/mg): Asp 4.75, Ser 4.14, Glu 5.37,
230
Ala 1.69, Cys 2.83, Val 3.49, Ile 5.15, Leu 1.43, Lys 4.76, His 3.87, Arg
11
Page 11 of 29
231
8.35, respectively. Total protein content of Se-TP was determined as
232
4.58% by above analysis of the amino acids.
233
3.7. Effect of Se-TP on the biochemical parameters related fatigue As shown in Table 1 and 2, mice in FC, Se-TP groups, which tied
235
additional load, were carried out intense exercise than those in EC. For
236
this reason, the exhausting time of FC mice was significantly shortened in
237
comparison with those of EC (P < 0.01). Moreover, the extent of fatigue
238
was also confirmed by the increase in BUN, Bla, LDH and the decrease
239
in muscle glycogen level (P < 0.05 or P < 0.01) at the time periods of
240
Post-exhaustion or Recover. However, administration of Se-TP, to a
241
certain extent, not only significantly prolonged the exhausting time but
242
also decreased the levels of BUN and Bla as well as LDH activity in
243
comparison with FC.
Ac ce pt e
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234
244
In addition, as shown in Table 2, the content of muscle glycogen was
245
decreased whereas MDA level was increased in skeletal muscle in FC
246
mice in comparison with EC group. However, treatments of Se-TP (200
247
and 400 mg/kg) significantly recovered muscle glycogen level, Se-TP
248
(400 mg/kg) markedly increased the activity of GSH-Px, and Se-TP
249
significantly decreased the level of MDA in skeletal when compared with
250
FC group (P < 0.05 or P < 0.01).
251
4. Discussion
252
A water-soluble polysaccharide coded Se-TP was isolated from Ziyang 12
Page 12 of 29
green tea. It exhibited a single and symmetrical peak in the HPGPC
254
analysis, which indicated its homogeneity based on the distribution of
255
molecular weight. Meanwhile, the result showed that Se-TP was a novel
256
conjugate with portions of polysaccharide and protein. The contents of
257
total carbohydrate in Se-TP were determined using phenol-sulfuric
258
method which has higher recovery rate and less loss of monosaccharide
259
during hydrolysis. The protein content of Se-TP was determined using
260
Bradford method and the total amino acid analysis method. The result
261
showed that the protein content of Se-TP in former was bigger than the
262
latter. The reason was related the loss of part amino acid during protein
263
hydrolysis.
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Swimming with weight loading of mice is an ideal experimental model
265
to evaluate the capacity of anti-fatigue [11]. In the present study, a
266
four-week swimming model of mice was carried out. The results
267
demonstrated that the exhausting time of FC (loaded 5% weight of body)
268
mice was significantly shortened in comparison with those of EC
269
(without load). Whereas, Se-TP treatment prolonged the exhausting time
270
of mice, indicating that Se-TP possess an anti-fatigue effect.
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BUN, a metabolic product of protein and amino acid, is one of blood
272
biochemical parameters related to fatigue. The less an animal is adapted
273
to exercise, the more the BUN level increases [6]. The result showed
274
Se-TP treatment reduced serum BUN levels and indicated its positive 13
Page 13 of 29
effect on enhancing endurance. As an important parameter of fatigue, Bla
276
is produced by anaerobic glycolysis, which can be further degraded via
277
the tricarboxylic acid cycle for the production of ATP or removed to other
278
tissue for oxidization or gluconeogenesis [25]. So there are two dynamic
279
balances between aerobic oxidation of glycogen and anaerobic glycolysis
280
and between the accumulation and elimination of Bla. Whereas, it was
281
unbalanced in a high-intensity exercise [26]. The increase of Bla
282
concentration and consequent lactic acidosis observed in skeletal muscles
283
during exercise were major cause of muscle fatigue. The result showed
284
Se-TP treatment reduced Bla levels and indicated its can improve
285
glycogen metabolism.
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In exercise-fatigue conditions, the activities of SOD and GSH-Px are
287
generally considered indicators of the capacity of the antioxidant
288
defensive system [27]. Malondialdehyde is an oxidative degradation
289
product of cell-membrane lipids, and its level is an indicator of lipid
290
peroxidation [28]. These conditions are also marked by the release of
291
LDH into the serum, serving as an indirect index of the damage of
292
membrane structure [29]. Energy use leads to reduction of glycogen in
293
muscle and the level of muscle glycogen is one of key factors of
294
evaluating exercise time to exhaustion [24]. As indicators for oxidative
295
stress, the present study showed that the muscle could have been
296
assaulted by the ROS for higher activity of LDH in mice serum after
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297
exhaustive exercise. It also provided some evidence such as lower activity
298
of SOD and GSH-Px and higher level of malondialdehyde in muscle. The
299
results suggested that there was a closed relationship between free-radical
300
attack and exhaustive exercise. In the current study, we found that all effects were blocked just after
302
Se-TP administration. These results include the lower activity of LDH in
303
serum, a higher activity GSH-Px and glycogen levels, and the decrease of
304
malondialdehyde content in muscle. This is similar to results of other
305
works [26] and suggested that the anti-fatigue effect of Se-TP probably
306
occurred through protection of corpuscular membrane by preventing lipid
307
oxidation via modifying GSH-Px activity. Considerable evidence has
308
indicated that the role of polysaccharide in scavenging ROS is likely
309
associated with hydrogen and hydroxyl of the polysaccharide chain [24],
310
and
311
polysaccharide also significantly affect the role of polysaccharide in
312
scavenging ROS [30]. Taken together, our results showed that Se-TP
313
supplementation is effective in improving exercise fatigue. However,
314
further research on structure of Se-TP is needed in order to expose more
315
details on its anti fatigue mechanism.
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their
monosaccharide
composition
and
characterization
in
316
15
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316
Acknowledgements This work was supported by the Fundamental Research Funds for the
318
Central Universities (GK201402046), Young Teacher Research Funds for
319
School of Sports, Shaanxi Normal University (No. 20151601) and Shanxi
320
Province Sports Bureau Project (No.14046).
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321
References
322
[1] M. Tanaka, Y. Baba, Y. Kataoka, N. Kinbara, Y.M. Sagesaka, T.
326 327 328
Molecules, 16 (2010) 28–37.
ip t
325
[2] X.L. Zhang, F. Ren, W. Huang, R.T. Ding, Q.S. Zhou, X.W. Liu,
[3] T.H. Pedersen, O.B. Nielsen, G.D. Lamb, D.G. Stephenson, Sci. 305
cr
324
Kakuda, Nutr. 24 (2008) 599–603.
(2004) 1144–1147.
us
323
[4] A.D.H. Smith, S.L. Coakley, M.D. Ward, A.W. Macfarlane, P.S. Friedmann, N.P. Walsh. Brain Behav. Immun. 25 (2011) 1136–1142.
330
[5] K. Azizbeigi, S.R. Stannard, S. Atashak, M.M. Haghighi, J. Exerc. Sci.
333
M
[6] C. Xu, J. Lv, Y.M. Lo, S.W. Cui, X. Hu, M. Fan, Carbohydr. Polym. 92 (2013) 1159–1165.
d
332
Fit. 12(2014) 1–6
Ac ce pt e
331
an
329
334
[7] X.Q. Han, B.C.L. Chen, C.X. Dong, Y.H. Yang, C.H. Ko, G.G.L. Yue,
335
C. Chen, C.K. Wong, C.B.S. Lau, P.F. Tu, P.C. Shaw, K.P. Fung, W.L.
336 337 338 339 340 341 342
Hsiao, Q.B. Han, J. Agric. Food Chem. 60 (2012) 4276–4281.
[8] W. Nan, Y. Jingyue, L. Jianguo, Q. Qing, W. Tao, D. Xilin, B. Guoqiang, H. Xianli, Carbohydr. Polym. 111 (2014) 47–55.
[9] A.P. Chi, J.P. Chen, Z.Z. Wang, Z.Y. Xiong, Q.X. Li, Carbohydr. Polym. 74 (2008) 868–874. [10] J. Wang, S. Li, Y. Fan, Y. Chen, D. Liu, H. Cheng, X. Gao, Y. Zhou, J. Ethnopharmacol.130 (2010) 421–423. 17
Page 17 of 29
348 349 350 351 352
[13] C. Sanmartín, D. Plano, A.K. Sharma, J.A. Palop, Int. J. Mole. Sc. 13
ip t
347
81–110.
(2012) 9649–9672.
cr
346
[12] A.F. Martinez, L. Charlet, Rev. Environ. Sci. and Biotechnol. 8 (2009)
[14] Y. Wang, J. Chen, D. Zhang, Y. Zhang, Y. Wen, L. Li, L. Zheng, Carbohydr. Polym. 98 (2013) 1186–1190.
us
345
Biol. Macromol. 50 (2012) 59– 62.
[15] J. Wang, B. Zhao, X. Wang, J. Yao, J. Zhang, Int. J. Biol. Macromol.
an
344
[11] W. Tan, K. Yu, Y. Liu, M Ouyang, M. Yan, R. Luo, X. Zhao, Int. J.
51 (2012) 987–991.
M
343
[16] A.M. Staub, Methods in Carbohydrate. Chem.5 (1965) 5–6.
354
[17] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal.
356 357 358 359 360 361
Chem. 28 (1956) 350–356.
Ac ce pt e
355
d
353
[18] M. Deaker, W. Maher. Analytica. Chimica. Acta. 350 (1997) 287–294.
[19] X. Yang, Y. Zhao, Y. Yang, Y. Ruan, J. Agric. Food Chem. 56 (2008) 6905–6909.
[20] X. Yang, Y. Lv, L. Tian, Y. Zhao, J. Agric. Food Chem. 58 (2010) 6075–6080.
362
[21] M.M. Bradford,. Anal. Biochem. 72 (1976) 248–254.
363
[22] A.P. Chi, J.P. Chen, Z.Z. Wang, Z.Y. Xiong, Q.X. Li, Carbohydr.
364
Polym. 74(2008) 868–874. 18
Page 18 of 29
365 366 367 368
[23] A.P. Chi, C. Kang, Y. Zhang, L. Tang, H. Guo, H. Li, K. Zhang, Carbohydr. Polym. 122 (2015) 189–196. [24] A.P. Chi, L. Tang, J. Zhang, K. Zhang, Int. J. Sport Nutr. Exerc. Metab. 22 (2012) 479–485. [25] J. M. Jia, C. F. Wu, Pharm. Biol. 46 (2008) 433–436.
370
[26] A. Niu, J. Wu, D. Yu, R. Wang, Int. J. Biol. Macromol. 42 (2008)
cr
447–449.
us
371
ip t
369
[27] G. Lenaz, Biochim. Biophys. Acta. 1366 (1998) 53–67.
373
[28] H.M. Alessio, A.H. Goldfarb, J. Appl. Physiol. 64, (1988)
376 377
M
[29] S. Passarella, L. de Bari, D. Valenti, R. Pizzuto, G. Paventi, A. Atlante, FEBS Lett. 582 (2008) 3569–3576.
d
375
1333–1336.
[30] A. Kardosova, E. Machova, Fitoterapia, 77 (2006) 367–373.
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cr us
Dose
Exhausting Time
(n=20)
(mg/kg)
(min)
EC
-
153.04±16.58
FC
-
129.76±11.19**
Se-TP
100
138.52±12.14#
Se-TP
200
143.64±14.05#
Se-TP
400
142.64±11.34#
BUN (mmol/L)
M
Group
an
Table 1 Comparison of biochemical markers related fatigue in serum
Post-exhaustion
Recover
Bla (mmol/L) Post-exhaustion
LDH(U/L)
Recover
Post-exhaustion
△△
353.74±22.19
224.85±27.73
14.08±2.25*
13.42±1.78**
368.85±26.97
316.73±29.64*
12.72±2.28
10.90±0.46#
△
334.38±26.91
279.63±48.37
11.47±1.85#
10.34±0.52#
△
299.25±31.67#
232.42±64.13#
12.03±2.76
10.54±2.16#
△
303.85±27.12#
253.09±25.56#
pt
ed
11.34±2.18
ce
378
9.17±1.05
Vs. EC, *: p < 0.05, **: p < 0.01.
380
Vs. FC, #: p < 0.05, ##: p < 0.01.
381
Vs. Post-exhaustion, △: p < 0.05, △△: p < 0.01.
806.19±17.36
792.43±15.58
1102.47±43.26**
903.52±27.82**
964.68±29.77
824.69±25.78#
△△
△
930.57±24.86#
846.25±19.78#
△△
△
929.71±28.54#
829.76±22.11#
△
△
Ac
379
△
Recover
382
20
Page 20 of 29
△
△△
cr us
Group
Dose
(n=20)
(mg/kg)
EC
-
10.19±2.23
FC
-
9.94±2.17
Se-TP
100
9.92±1.73
Se-TP
200
10.35±2.26
Se-TP
400
an
Table 2 Comparison of biochemical markers related fatigue in muscle
Post-exhaustion
Recover
14.27±2.81
Post-exhaustion
△
Recover
GSH-Px (U/mg protein) Post-exhaustion
MDA(nmol/mg protein)
Recover
Post-exhaustion △
4.71±0.86
△△
9.33±1.87*
5.06±0.32
△
△
7.62±1.37#
4.57±0.26#
△
△
7.58±2.31#
4.39±0.14#
△
6.94±1.15#
3.88±1.02##
58.64±7.47
47.95±5.78
98.62±11.85*
106.43±17.89*
52.17±5.26*
46.51±6.02*
104.35±17.64
122.73±13.41
59.54±10.67
46.76±5.15
14.82±2.13#
118.71±14.98
129.84±18.27
61.50±4.44
50.49±6.33
14.64±1.47#
112.43±13.19
125.37±16.79
69.86±8.75#
57.34±4.66#
ed
133.14±11.53
10.62±3.24*
13.51±3.17
Vs. EC, *: p < 0.05, **: p < 0.01.
384
Vs. FC, #: p < 0.05, ##: p < 0.01.
385
Vs. Post-exhaustion, △: p < 0.05, △△: p < 0.01.
△
△
Ac
383
△
21
Page 21 of 29
Recover
8.37±1.45
115.47±10.38
pt
10.54±1.61
SOD (U/mg protein)
M
Glycogen (mg/g protein)
ce
382
△
△
386
Figure captions
387 388
Figure 1. Fractionation and purification profiles of Se-TP A: Fractionation of the crude polysaccharides in DEAE-52 cellulose
390
column; B: Purification of Se-TP in Sephadex G-150 column
ip t
389
Figure 2. UV spectrum of Se-TP
us
392
cr
391
393
Figure 3. IR spectrum of Se-TP
an
394
396
M
395
Figure 4. HPGPC profile of Se-TP
Figure 5. The HPLC chromatograms of SE-TP
Ac ce pt e
398
d
397
399
a: Standard (1 Man; 2 Rib; 3 Rha; 4 GlucA; 5 GalcA; 6 Glu; 7 Xyl; 8
400
Gal; 9 Ara; 10 FUC). b: Monosaccharide of SE-TP
401 402 403 404
Figure 6. Amino acid composition of SE-TP
Highlight:
405 406 407
1. Polysaccharide from Ziyang green tea was a selenium–enriched polysaccharide conjugate (Se-TP).
22
Page 22 of 29
409 410 411 412
2. Se-TP administrations significantly prolonged exhaustive time and increased glycogen level and GSH-Px activity in muscle. 3. Se-TP administrations markedly decreased BUN, Bla levels and LDH activity in serum and MDA level in muscle. 4. Se-TP treatments can significantly improve exercise-induced fatigue.
ip t
408
cr
413
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414
23
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pt
ce
Ac M
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i
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pt
ce
Ac M
an
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i
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pt
ce
Ac M
an
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i
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pt
ce
Ac M
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pt
ce
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pt
ce
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