Effects of Ganoderma lucidum and Essence of Chicken on Physical Fatigue Recovery and Exercise Performance Improvement

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1 Chinese Journl of Physiology 61(6): , 218 DOI: 1.477/CJP.218.BAH646 Effects of Gnoderm lucidum nd Essence of Chicken on Physicl Ftigue Recovery nd Exercise Performnce Improvement HuShui Li 1, Ying-Ju Chen 2, Yi-Ju Hsu 1, Ming-Fng Wu 1, 3, Chien-Cho Chiu 1, Yu-Tng Tung 5, Wei-Jern Tsi 6, Wen-Ching Hung 4, nd Chi-Chng Hung 1, 5 1 Grdute Institute of Sports Science, Ntionl Tiwn Sport University, Toyun 3331, 2 Deprtment of Food nd Nutrition, Providence University, Tichung 4331, 3 Z-Plus Biotechnology Co., Ltd., Hmi St., Dtong Dist., Tipei City 1367, 4 Deprtment of Exercise nd Helth Science, Ntionl Tipei University of Nursing nd Helth Sciences, Tipei 11219, 5 Grdute Institute of Metolism nd Oesity Sciences, Tipei Medicl University, Tipei 1131, nd 6 Ntionl Reserch Institute of Chinese Medicine, Ministry of Helth nd Welfre, Tipei 11221, Tiwn, Repulic of Chin Astrct A fst-pced lifestyle, pressure from the environment nd hevy work lod often cuse extreme tiredness in modern life. Different kinds of nutritionl supplements in the form of functionl foods or trditionl Chinese medicine, such s essence of chicken nd Gnoderm lucidum hve een climed to enefit physicl performnce nd promote helth. Previous studies hve reveled tht essence of chicken or G. lucidum hve wide spectrum of iologicl ctivities. In this study, we comined these two ingredients together (designted s CEG) to evlute their synergistic effects on physiologicl dption on exercise ftigue nd physicl ctivities. The ICR strin mice were llocted s, 833, 1666, nd 4165 mg/kg dose groups nd dministrted y orl gvge consecutively for 4 weeks. Physicl ctivities including grip strength nd eroic endurnce were evluted. Vrious ftigue-ssocited iochemicl vriles such s lctte, BUN or CK were lso evluted. The levels of liver nd muscle glycogen were mesured s n indictor of energy storge t the end of the experiment. Sfety ssessments for supplementtion were lso evluted. CEG supplementtion significntly incresed the endurnce nd grip strength nd demonstrted eneficil effects on lctte production nd clernce rte fter n cute exercise chllenge. The CEG supplementtion significntly mitigted the BUN nd CK indexes fter extended exercise nd elevted the glycogen content in the liver nd muscle tissues. According to ody composition, iochemicl nd histopthologicl dt, dily dministrtion of CEG for over 28 dys (sucute toxicity) lso demonstrted resonle sfety results for supplementtion. Comined G. lucidum nd essence of chicken cn significntly increse the exercise performnce nd improve ftigue recovery. It my lso provide vile lterntive nutritionl supplement for helth promotion. Key Words: clernce rte, essence of chicken, exercise ftigue, physicl ctivities, sfety Corresponding Authors: : [1] Dr. Wen-Ching Hung, Deprtment of Exercise nd Helth Science, Ntionl Tipei University of Nursing nd Helth Sciences, Tipei 11219, Tiwn, ROC, E-mil: wenching@ntunhs.edu.tw, Tel: ext. 7721; [2] Dr. Chi-Chng Hung, Grdute Institute of Sports Science, Ntionl Tiwn Sport University, Toyun 3331, Tiwn, ROC, E-mil: john5523@ntsu. edu.tw, Tel: ext Received: Octoer 24, 218; Revised: Decemer 9, 218; Accepted: Decemer 18, y The Chinese Physiologicl Society. ISSN :

2 The Chicken Essence nd G. lucidum on Anti-ftigue Activities 373 Introduction Physiologicl ftigue relevnt to exercise physiology occurs through two different mechnisms, centrl mechnism involving proximl motor neurons (minly in the rin) nd peripherl mechnism within the motor units (i.e., motor neurons, peripherl nerves, motor endpltes, nd muscle fiers) (1). The centrl ftigue hypothesis suggests tht n incresed rtio of centrl serotonin to dopmine is ssocited with feelings of tiredness nd lethrgy, ccelerting the onset of ftigue (25). In contrst to the centrl ftigue, the processes in the motor neurons, neuromusculr junctions, srcolemml memrne, excittion-contrction coupling, ccumultion of metolites, or depletion of fuel re involved in peripherl ftigue (2). Peripherl nd centrl ftigue my hppen seprtely or together, depending on the specific sitution or condition. Ftigue cn e lso further ctegorized in other wys, such s secondry, nd chronic ftigue. Secondry ftigue is cused y n underlying medicl condition nd my lst one month or longer, ut less thn six months. If the ftigue syndrome lsts longer thn 6 months, it cn e defined s chronic ftigue (5). Gnoderm lucidum, clled Lingzhi in Mndrin, is well-known trditionl medicinl fungus tht hs een shown to hve mny physiologicl effects. For hundreds of yers, this mushroom hs een used s folk medicine nd pplied for the prevention nd tretment of vrious humn diseses, such s heptitis, hypertension, chronic ronchitis, ronchil sthm, nd cncer (3). In evidence-sed reports, G. lucidum demonstrted heptoprotective effects on CCL4-induced liver injury model resulting in significnt decrese in the heptic indexes (13). In ddition, exhustive exercise-induced musculr oxidtive stress could e ttenuted y supplementtion with G. lucidum polyscchrides (47). The immune-suppression cused y long-term nd high intensity exercise could e mitigted y G. lucidum supplementtion vi nonspecific nd specific immune response improvement (31) nd it could lso significntly improve eroic endurnce, lower ody flexiility, nd velocity (1). Furthermore, G. lucidum could improve chemotherpy-induced ftigue vi regultion of inflmmtory responses, oxidtive stress nd reduction of nephrotoxicity (28) nd lso showed eneficil effects on qulity of life nd immune mrkers pertining to cncer-relted ftigue (46). Essence of chicken is liquid nutritionl supplement mde y cooking whole chickens y high-temperture extrction, centrifugtion, vcuum concentrtion, nd steriliztion. It contins some specil nutrients such s imidzole dipeptide (crnosine nd nserine), turine, polypeptides, minerls, trce elements, nd multiple mino cids (23). Mentl ftigue often occurs in popultions who sit in meetings, red, or tke exms for long periods nd dily intke of essence of chicken my e effective for recovery (43). The imidzole dipeptide, re nturl ntioxidnts tht hve een reported to inhiit tissue dmge nd suppress the reduction in performnce level induced y mentl ftigue (4). In ddition, essence of chicken hs een proved to reduce hyperglycemi nd the L-Crnosine component helps to regulte lood sugr levels (36, 44). Chicken essence (CE) supplementtion lso hs een found to hve other eneficil effects on mood (45), cognition (22), circdin clock resetting process (41), hypertension (32) nd exercise dption (18). The food nd nutrition industry hs grown rpidly in recent yers nd mny different functionl or medicl food comintions re eing tried to otin etter helth enefits. In the current study, we investigted the effects of G. lucidum nd CE comintion (CEG) for on exercise physiologicl dption using slightly modified niml pltform. We lso evluted the sfety of this supplementtion using sucute toxicity test to understnd the potentil risk with consecutive nd dose-effect supplementtion. Mterils Mterils nd Methods The supplementtion contined CE nd extrcts of G. lucidum fruiting ody. The CE nd extrcts of G. lucidum, designted s CEG, were provided y Tin Yun Xing Co. Limited (Tinn City, Tiwn). The dried fruiting odies of G. lucidum were extrcted y sequentil procedures, first with lcohol t room temperture (12 h) nd then wter, nd comined these extrctions for lyophiliztion. The CEG included the 9% CE nd 1% G. lucidum extrcts s proportion for current study. The CEG ws freeze dried for comprle nd comptile dosge conversion for niml experimentl designs nd the verged lyophilized powder weight ws out 4.1 grms for humn recommended CEG dily serving (6 ml). The clorie count ws out 15.6 Kcl/serving s nlyzed y SGS Tiwn, Ltd. Animls nd Experimentl Designs Forty mle ICR mice (6 weeks old) from AAALAC certified y BioLASCO Tiwn (Yi-Ln, Tiwn) were used in this study. Mice were cclimtized nd llowed food d liitum for 2 weeks

3 374 Li, Chen, Hsu, Wu, Chiu, Tung, Tsi, Hung nd Hung Grip Strength (Dy 28) Endurnce Test (Dy 29) Lctte/CK Test (Dy 31) Glycogen Assy (Dy 36) BUN Test (Dy 33) Weight (n = 1) Vehicle (n = 4) (n = 1) (n = 1) CEG-1X CEG-2X (n = 1) CEG-5X -14 Adption Supplementtion Test (Dy) Fig. 1. Experimentl designs for the effects of CEG on exercise dption. The nimls were rndomly ssigned to the indicted 4 groups (Vehicle, CEG-1X, CEG-2X, nd CEG-5X) nd consecutively supplemented the CEG until the end of the experiments. The physicl cpcities nd relted iochemistries were ssessed within test durtion. prior to experiments. All nimls were given stndrd lortory diet # 51 (PMI Nutrition Interntionl, MO, USA) nd distilled wter d liitum, nd mintined in constnt photoperiod, temperture, nd humidity (12-h light/12-h drk cycle, 24 ± 2 C, nd 55% 65%, respectively). Routine clening ws conducted twice per week. The Institutionl Animl Cre nd Use Committee (IACUC) of Ntionl Tiwn Sport University pproved ll niml experiments in this study, nd the study conformed to the guidelines of protocol IACUC-1513 pproved y the IACUC ethics committee. The recommended CEG dosge ws 4.1 g per dy (lyophilized powder), which is equivlent to CEG t 6 ml/serving/dy (18). The mouse CE dose (845 mg/kg) we used ws converted from humn equivlent dose (HED) sed on ody surfce re s CEG-1X y the formul from the US Food nd Drug Administrtion. The detiled formul ws s descried in previous study (18) nd we designed the different dependence-dosges (vehicle, CEG-1X, CEG-2X, nd CEG-5X groups). The detiled experimentl procedure is illustrted in Fig. 1. All nimls were given n cclimtion period of two weeks to dpt to the environment nd diet. The weight, dietry, nd socil ehviors were monitored during the supplementtion period nd the dily supplementtion egn t the regulr time with fresh CEG smple preprtions. Tretment dosges of, 833, 1666, nd 4165 mg/kg/dy were designted s vehicle, CEG-1X, CEG-2X, nd CEG-5X nd were dministrted y orl gvge with volume 1 ml/kg BW. Physicl fitness ws evluted y grip strength nd eroic endurnce cpcities nd the exercise-relted iochemistry ws immeditely ssessed fter fixed exercise time/intensity. Exercise Endurnce Performnce Test Exercise performnce ws sed on the survivl motives to ssess the eroic endurnce cpcities. The nimls were loded with weight equivlent to 5% individul ody weight nd forced to swim in tnk until exhustion. The persistent time from eginning to exhustion ws recorded s endurnce index. The detiled procedures nd protocol were descried in our previous rticle (35). Forelim Grip Strength A low-force testing system (Model-RX-5, Ai-

4 The Chicken Essence nd G. lucidum on Anti-ftigue Activities 375 koh Engineering, Ngoy, Jpn) for forelim grip strength ws used to mesure the grip strength. The detils hve een descried previously (8). Determintion of Ftigue-Associted Biochemicl Vriles Bsed on our previous reports (15), the effect of CEG supplementtion on ftigue-ssocited iochemicl indexes ws slightly modified to ccurtely demonstrte the physiologicl sttus. The lood ws smpled y sumndiulr collection for further iochemicl nlysis. For the lctte metolites profile, the lood smpling time points were pre-exercise, immeditely fter 1 min cute exercise, nd then fter 2 min rest. The other indexes, such s lood urine nitrogen (BUN) nd cretine kinse (CK), were immeditely ssessed t the 6 min rest time point fter 9 min extended exercise. The lood smples were centrifuged t 1 g nd 4 C for 15 min fter complete clotting for serum seprtion nd determined y n utonlyzer (Hitchi 76, Hitchi, Tokyo, Jpn). Clinicl Biochemicl Profiles CEG supplementtion ws dministrted continuously until niml scrifice. All mice were euthntized y 95% CO 2 sphyxition one hour fter the lst tretment nd immeditely smpled lood y crdic puncture. Serum ws seprted y centrifugtion nd clinicl iochemicl vriles, including sprtte minotrnsferse (AST), lnine trnsminse (ALT), mmoni (NH 3 ), CK, glucose (GLU), BUN, cretinine (CREA), uric cid (UA), totl cholesterol (TC), triglycerides (TG), lumin (ALB), nd totl protein (TP) were mesured y use of n utonlyzer (Hitchi 76). Body Composition nd Glycogen Content Anlysis After scrifice, the importnt viscerl orgns, including liver, kidney, hert, lung, muscle, epididyml ft pd (EFP), nd rown dipose tissue (BAT), were ccurtely excised nd weighed. Then, the orgns were sved into 1% formlin for further histopthology. Prt of muscle ws kept in liquid nitrogen for glycogen content nlysis s descried previously (9). Histopthology The viscerl orgns preserved in 1% formlin were trimmed nd emedded in prffin for tissue sections with 4 μm thickness slices. Tissue sections were further stined with hemtoxylin nd Time to Exhustion (min) eosin (H&E) stining nd exmined under light microscope equipped with CCD cmer (BX-51, Olympus, Tokyo, Jpn) y veterinry pthologist. Sttisticl Anlysis Trend Anlysis (P <.1) 2.32 The dt were represented s men ± stndrd devition (SD). The sttisticl difference mong groups in physicl ctivities, iochemistry, ody composition, dietry nd glycogen content were nlyzed y one-wy nlysis of vrince (ANOVA) nd the Cochrn-Armitge test for dose-effect trend nlysis with use of SAS v. 9. (SAS Inst., Cry, NC, USA). A mixed design two-wy ANOVA (supplementtion time) ws lso pplied to the supplementtion effects on lctte metolite profiles nd growth curve within repeted time points. Dt were considered sttisticlly significnt when the proility of type I error ws less thn.5. Results Fig. 2. Effect of 4-week CEG supplementtion on exhustive swimming time. Dt re men ± SD for n = 1 mice per group. Columns with different superscript letters (, ) re significntly different t P <.5. Numers ove the columns re the fold increse from vehicle. Effects of CEG Supplementtion on Endurnce Cpcity The endurnce cpcity ws mesured y exhustive swimming test nd it showed significnt difference mong groups (F(3,36) = 11.19, P <.1). The CEG supplementtion groups (CEG-1X, CEG- 2X, nd CEG-5X) were significntly higher thn the vehicle group showing 2.32-, 2.8-, nd fold increse nd demonstrted the significnt dosedependent effects (P <.1) in trend nlysis (Fig. 2).

5 376 Li, Chen, Hsu, Wu, Chiu, Tung, Tsi, Hung nd Hung Tle 1. The effects of CEG on lctte metolite profiles during cute exercise chllenge Time point Lctte (mmol/l) Before swimming (A) 2.6 ± ± ± ±.3 After swimming (B) 9. ± ± ± ±.6 After 2-min rest (C) 8.5 ± 1.3 c 6.1 ± ± ±.8 Rte of lctte production nd clernce Production rte = B/A 3.57 ± ± ± ±.43 Clernce rte = (B-C)/B.6 ±.3.15 ±.2.19 ±.6 c.21 ±.8 c The lctte metolites were ssessed for the 4 groups llocted s vehicle, CEG-1X, CEG-2X, nd CEG-5X t three time points. Lctte production rte ws clculted s, fter exercise divided y efore exercise (B/A) nd the lctte difference etween fter exercise nd fter rest divided y fter rest ws defined s the clernce rte. Vlues in the sme row with different superscript letters (,, c) differ significntly, P <.5. A Trend Anlysis (P <.1) B Trend Anlysis (P <.1) Grip Strength (g) c 1.31 Reltive Grip Strength (%) c 1.29 Fig. 3. Effect of 4-week CEG supplementtion on solute forelim grip strength (A) nd forelim grip strength (%) reltive to ody weight (B). Dt re men ± SD for n = 1 mice per group. Columns with different superscript letters (,, c) re significntly different t P <.5. Numers ove the columns re the fold increse from vehicle. The Effects of CEG Supplementtion on Grip Strength The grip strength showed significnt differences mong groups in solute strength (F(3,36) = 2.87, P <.1) nd reltive strength djusted y individul weight (F(3,36) = 43.11, P <.1). The CEG supplementtion groups (CEG-1X, CEG- 2X, nd CEG-5X) were significntly higher thn vehicle group (P <.1) with regrd to solute strength (1.22-, 1.22-, nd 1.31-fold, respectively) nd reltive strength (1.2-, 1.22-, nd 1.28-fold, respectively). Both grip strength mesures lso showed significnt CEG dose-dependent effects (P <.1) in trend nlysis (Fig. 3). The Effects of CEG Supplementtion on Exercise-relted Biochemicl Indexes fter Exercise Chllenge The lctte metolite ws ssessed preexercise, immedite post- exercise, nd fter rest (3 time points) with the different CEG tretments (Tle 1). It showed significnce difference in supplementtion min effect (F(3,36) = 23.75, P <.1) nd time min effect (F(2,72) = 966.9, P <.1). The interction effects ws lso significnt (F(6,72) = 21.82, P <.1). Further nlysis of the 3 time points showed significnt difference in the post-exercise (F(3,36) = 19.74, P <.1) nd fter rest (F(3,36) = 3.84, P <.1) time points. The vehicle group ws significntly higher thn other CEG tretment groups t time points of postexercise nd fter rest y one-wy ANOVA. The self-comprison indexes, such s lctte production rte nd clernce rte, lso demonstrted significnt eneficil effects with CEG supplementtion (P <.5). The other metolic indictor, BUN, demonstrted significnce fter extended exercise (F(3,36)

6 The Chicken Essence nd G. lucidum on Anti-ftigue Activities 377 A Trend Anlysis (P <.214) B Trend Anlysis (P <.1) 5 4 1% 82.7% 82.19% 8.41% % BUN (mg/dl) CK (U/L) % 4.15% 34.75% Fig. 4. Effect of 4-week CEG supplementtion on the serum BUN (A) nd CK (B) levels fter extended exercise chllenge. The indicted 4 groups underwent 9 min swimming exercise nd lood ws smpled fter 6 min rest. Dt re men ± SD for n = 1 mice per group nd the columns with different superscript letters (, ) re significntly different t P <.5. Numers ove the columns re the fold-decrese from vehicle. A Glycogen (mg/g liver) Trend Anlysis (P <.1) c 1.86 B Glycogen (mg/g muscle) Trend Anlysis (P <.1) c 2.54 Fig. 5. Effect of 4-week CEG supplementtion on heptic (A) nd muscle (B) glycogen level. Dt re men ± SD for n = 1 mice per group. Brs with different superscript letters (,, c) re significntly different t P <.5. = 4.62, P =.8) (Fig. 4A). CEG supplementtion significntly decresed the exercise-induced BUN elevtion y 18-2% (P <.5) showing dosedependent trend (P =.2). The other importnt injury index, CK, showed significnt difference mong groups (F(3,36) = 25.74, P <.1) (Fig. 4B). The CEG supplementtion could mitigte the CK increse y 49.3, 39.2, nd 65%, respectively, nd showed dose-dependent trend (P <.1). The Effects of CEG Supplementtion on Glycogen Content Glycogen is minly stored in the liver nd muscle tissue for the purpose of energy homeostsis. CEG supplementtion ws shown to ffect the glycogen content in the liver (Fig. 5A; F(3,36) = 9.51, P <.1). Heptic glycogen significntly incresed 1.44-, 1.5-, nd 1.86-fold, respectively, s compred to the vehicle group. Musculr glycogen lso showed significnt difference mong the groups ((F(3,36) = 22.75, P <.1). The musculr glycogen significntly incresed 1.85-, 1.96-, nd 2.54-fold, respectively, s compred to the vehicle group (Fig. 5B). Both the heptic nd musculr glycogen showed significnt dose-dependent trend (P <.1). Sucute Orl Toxicity Evlution of CEG Supplementtion Sucute toxicity evlution following OECD Guideline 47 ws performed not only for physiologicl exercise dption ut lso to ssess the

7 378 Li, Chen, Hsu, Wu, Chiu, Tung, Tsi, Hung nd Hung Tle 2. Growth curve nd dietry profiles during experiments Time point Initil BW (g) 35.1 ± ± ± ±.7 1st wk BW (g) 35.9 ± ± ± ± 1.1 2nd wk BW (g) 36.6 ± ± ± ± 1. 3rd wk BW (g) 37.5 ± ± ± ± 1. 4th wk BW (g) 38. ± ± ± ± 1.1 Finl BW (g) 39.3 ± ± ± ±.9 Wter intke (ml/mouse/dy) 8.8 ± ± ± ± 2.4 Chow 51 (g/mouse/dy) 6.9 ± ± ± ±.8 *CEG supplement Diet (g/mouse/dy) The weight nd diet were mesured regulrly for the 4 groups llocted s vehicle, CEG-1X, CEG-2X, nd CEG-5X. All the dt re represented s men ± SD nd nlyzed y one-wy ANOVA. Vlues in the sme row with different superscript letters (, ) differ significntly, P <.5. (*) CEG supplement ws clculted s niml verged weight s 4 grms. BW, ody weight. Tle 3. Effects of CEG on ody compositions Chrcteristic Liver (g) 2.38 ± ± ± ±.23 Muscle (g).36 ±.2.37 ±.3.39 ±.2.39 ±.2 Kidney (g).62 ±.3.62 ±.4.65 ±.4,.66 ±.3 Hert (g).24 ±.3.28 ±.2.28 ±.3.29 ±.3 Lung (g).24 ±.1.25 ±.1,.26 ±.4,.27 ±.4 EFP (g).38 ±.8.33 ±.1,.31 ±.5,.3 ±.9 BAT (g).12 ±.2.15 ±.1.15 ±.2.16 ±.2 Reltive liver weight (%) 6.5 ± ± ± ±.46 Reltive muscle weight (%).9 ±.4.92 ±.4.99 ±.2.99 ±.2 Reltive kidney weight (%) 1.58 ± ± ± ±.6 Reltive hert weight (%).61 ±.6.7 ±.4.69 ±.5.72 ±.6 Reltive lung weight (%).6 ±.2.62 ±.2,.64 ±.8,.68 ±.9 Reltive EFP weight (%).96 ± ±.22,.77 ±.9.75 ±.22 Reltive BAT weight (%) 2.1 ± ± ± ±.21 Dt re men ± SD for n = 1 mice in ech group. Dt in the sme row with different letters (, ) differ significntly t P <.5 y one-wy ANOVA; EFP: epididyml ft pd; BAT: rown dipose tissue. sfety of the supplementtion. Severl indexes including ehvior, diet, growth curve, orgn weight, iochemicl ssessments nd histopthology were evluted for the sucute toxic effects of CEG supplementtion. Behvior ws monitored with dily CEG dministrtion nd no norml ehvior ws seen mong the groups. As shown in Tle 2, the tretment min effect did not show n significnt difference (F(3,36) =.7, P =.976) ut the time min effect showed significnt difference (F(3.33,199.9) = 725.5, P <.1) which indicted time-dependent growth. However, the interction effect (supplement time) ws not significntly different (F(1,199.9) = 1.26, P =.261) nd further nlysis t different time points y one wy ANOVA showed no significnt difference mong groups. Diet nd wter intke lso did not significntly differ mong groups with dose-dependent CEG supplementtion (F(3,12) =.5, P =.999; F(3,12) =.26, P =.994). Body composition my lso reflect the effect of supplementtion on different orgns. As shown in Tle 3, the muscle weight ws significntly different mong groups (F(3,36)=6.96, P =.1) nd the CEG supplementtion groups (CEG-2X nd CEG-5X) were significntly higher thn ve-

8 The Chicken Essence nd G. lucidum on Anti-ftigue Activities 379 Tle 4. The effects of CEG on clinicl iochemicl nlysis t the end of the experiment Prmeter AST (U/L) 89 ± 15 8 ± 12, 78 ± 14, 77 ± 13 ALT (U/L) 68 ± ± 1 55 ± ± 1 NH 3 (µmol/l) 199 ± ± 9, c 159 ± 4, c 147 ± 27 CK (U/L) 319 ± ± ± ± 93 GLU (mg/dl) 148 ± 8 c 137 ± 11, 143 ± 1, c 13 ± 8 CREA (mg/dl).21 ±.4.23 ±.4.34 ±.9.38 ±.7 BUN (mg/dl) 22.2 ± ± ± ± 1.8 UA (mg/dl) 1.6 ± ±.2 1. ±.2 1. ±.2 TC (mg/dl) 163 ± 9 c 142 ± ± ± 15 TG (mg/dl) 182 ± ± ± ± 18 ALB (g/dl) 2.6 ± ± ±.1 3. ±.2 TP (g/dl) 4.9 ± ±.1, 5.1 ± ±.2 Dt re men ± SD for n = 1 mice in ech group. Vlues in the sme row with different superscript letters (,, c) differ significntly, P <.5, y one-wy ANOVA; AST, sprtte minotrnsferse; ALT, lnine trnsminse; NH 3, Ammoni; CK, Cretine kinse; GLU, glucose; CREA, Cretinine; BUN, lood ure nitrogen; UA, uric cid; TC, totl cholesterol; TG, tricylglycerol; ALB, lumin; TP, totl protein. hicle group (P <.5). The EFP nd BAT weight were significntly different mong groups (F(3,36) = 1.77, P =.45 nd F(3,36) = 5.42, P =.3, respectively). The EFP of the CEG-5X group ws significntly lower thn vehicle (P <.5) nd the BAT ws significntly higher in CEG groups thn vehicle group (P <.5). The reltive orgn weight djusted y individul weight showed similr sttisticl results which lso demonstrted tht the CEG supplementtion could increse the len mss nd BAT nd decrese the EFP weight in the current study. Clinicl iochemistry ws lso used to evlute the effects of supplementtion on physiologicl sttus. As shown in Tle 4, the indexes were relevnt to the heptic function, lood lipid, renl function, injury nd metolites index pplied to the current iochemicl ssessment. The indices of heptic function, AST, nd ALT, were significntly different mong the groups (F(3,36) = 1.93, P =.42 nd F(3,36) = 5.88, P =.2, respectively) nd the higher CEG doses (CEG-5X) could significntly decrese the AST nd ALT levels s compred to the vehicle group (P <.5). The lood lipid indexes, TC nd TG, were significntly different (F(3,36) = 14.2, P <.1 nd F(3,36) = 4.4, P =.1, respectively) nd the CEG supplementtion could significntly decrese those indexes s compred to vehicle (P <.5). For the renl function index, the CREA showed significnt difference (F(3,36) = 16.41, P <.1) nd there ws not significnt difference in the BUN index (F(3,36) =.132, P =.94). The CEG higher dose (CEG-2X nd CEG- 5X) dministrtion could significntly increse the CREA levels s compred to vehicle group (P <.5). As shown in Fig. 6, the pthologicl results did not show normlities ttriuted to CEG supplementtion s oserved y clinicl veterinrin. Discussion In the current study, we used comined G. lucidum nd essence of chicken (CEG) supplementtion to investigte whether it could improve the exercise ftigue recovery nd performnce. The physicl ctivities, endurnce cpcities nd grip strength significntly incresed with CEG supplementtion without trining intervention. The iochemicl vriles including the lctte, BUN, nd CK indexes showed eneficil effects with CEG supplementtion fter intervention of exercise chllenges. Furthermore, we found tht CEG supplementtion could not only increse the physiologicl dption ut lso improve the exercise performnce possily vi glycogen content increse in the liver nd muscle. The sucute orl toxicity of CEG ws lso evluted for sfety nd the results demonstrted eneficil effects on the ody composition nd iochemistry in resonle rnge. The tredmill nd swimming methods hve een widely pplied to ssess exercise endurnce cpcity (7, 17). However, the tredmill is generlly equipped with n electric shock stimultor to enforce continued exercise. The shock pprtus cn

9 38 Li, Chen, Hsu, Wu, Chiu, Tung, Tsi, Hung nd Hung A B C D E Fig. 6. Effect of CEG supplementtion on histomorphologic fetures of the liver (A), hert (B), soleus muscle (C), lung (D) nd dipocyte tissue (E) in mice. Specimens were photogrphed under light microscope. (H&E stin, mgnifiction: 2 ; r, 4 or 8 μm). interfere with quntittion of running endurnce, s well s confound mesurements of post-exercise serum hormone nd cytokine levels (11). The swimming test (21) could ensure the higher differentil cpcity with stronger survivl instinct s compred to the tredmill methods (16). Therefore, in the current study we chose to ssess endurnce with CEG supplementtion using the swimming method. CEG supplementtion in the current study showed incresed endurnce cpcity s compred to previous CE tretment study (18). Grip strength cn potentite initil motor ctivity nd e relted to neuromusculr dmittnce (26). In previous report, grip strength ws found to e strongly ssocited with gender nd weight in young popultion (29). CEG my hve positive effects on motor control for etter strength performnce ccording to the current results (Fig. 3). With regrd to exercise-relted iochemistry, lctte, ws positively correlted with exercise durtion nd intensity, resulting in the relese of hydrogen ions. Incresed hydrogen ions cuse cidity, which ffect physiologicl metolism nd clcium relese to musculr contrctions (6), nd eventully contriute to the senstion of ftigue. Lctte cn lso e metolized s energy nd it cn e ccumulted when the production rte exceeds the clernce rte. In the current study, CEG could decrese the production rte nd increse the clernce rte significntly (Tle 1). Sufficient energy is criticl to physiologicl mintennce nd the energy molecule (ATP, ADP, nd AMP) is under homeostsis under norml conditions. ATP is consumed y exercise demnd nd ADP is converted to AMP for

10 The Chicken Essence nd G. lucidum on Anti-ftigue Activities 381 ATP replenishment. AMP cn e further metolized to IMP nd mmoni y deminse nd the mmoni could e metolized s BUN vi the ure cycle. Therefore, BUN cn e considered to e iomrker for ATP metolism (14). In previous study, CE supplementtion improved the lctte nd mmoni levels in the recovery phse fter exhustive exercise (24). CEG could keep the efficient energy supplement so the BUN index could e meliorted with dose-dependence in current study (Fig. 4A). Oxidtive stress, such s rective oxygen species nd free rdicls, induced y prolonged/ exhustive exercise cn injure the cell memrne permeility nd lctic dehydrogense (LDH) nd CK cn lek out into the lood nd ct s dmge mrkers (19, 33). The nti-oxidtion cpcity of CE ctive peptides nd the totl triterpene frction isolted from G. lucidum hve een reported (34, 4). In our study, CEG could significntly decrese the CK levels cused y exercise-induced oxidtion (Fig. 4B). Glycogen is n importnt fuel during exercise from the point view of exercise physiology (39) nd is considered s crohydrte energy store for ATP production. Intermyofirillr, intrmyofirillr, nd susrcolemml glycogen, out 75% of totl ody glycogen, is stored in musculr cells (27). The positive correltion etween exercise endurnce nd glycogen content ws reveled in the 196s (2). In our previous study on CE supplementtion, glycogen ws significntly incresed out 7% nd 3% in liver nd muscle, respectively (18). The musculr glycogen content with CEG dose-dependent supplementtion ws lso significntly elevted more thn 8%, which ws more thn with CE tretment only. G. lucidum hs een reported to up-regulte glycogen synthesisrelted genes (GS2 nd GYG1) (38) nd cted synergisticlly with CE to contriute to the incresed glycogen content. The efficient glycogen elevtion with CEG tretment my explin the higher endurnce cpcities thn those found previously with CE. G. lucidum hs een reported to hve potentil toxicity nd side effects in previous reports, including dizziness, dry nose nd throt, hedche, nd skin irrittion (12). Therefore, the sucute orl toxicity of CEG supplementtion ws ssessed. CEG supplementtion ws not oserved to cuse normlity in socil ehvior, ppernce, or diet. With respect to ody composition, our previous report showed tht CE tretment did not result in significnt difference, ut in the current study CEG supplementtion cused significnt difference in muscle, ft, nd BAT tissues. CE comined with green te showed positive clinicl effect y reducing ody ft (37) nd G. lucidum hs een reported to up-regulte lipid metolism-relted genes (38). Ft composition nd lipid iochemistry (TG nd TC) ws significntly improved possily vi the comintion of the effects of CE nd G. lucidum. Interestingly, rown dipocyte tissue (BAT) showed significnt increse with CEG supplementtion which ws different s compred to our previous CE tretment only study (18). Exercise might ctivte nd recruit BAT through the ctivtion of the sympthetic nervous system, hert nd skeletl muscle (3). The possile effects of CEG on BAT ctivtion relevnt to exercise physiology will e worth further investigtion. The effect of CE tretment on iochemistry ws reported in our previous study (21). However, we found the effect of CEG on severl indexes were different thn in the previous CE study. The liver indexes, AST nd ALT, showed eneficil effects fter supplementtion G. lucidum (42). It is rther remrkle tht consecutive CEG supplementtion resulted in significntly higher CREA index with dose-dependence trend, which is consistent with previous study. The CEG supplementtion my therefore pose potentil risk for specific popultions of ptients, such s those with nephropthy. Conclusions CE is widely cceptle s nutritionl supplement. From the perspective of dietry therpy nd nourishment, comintions of different functionl or medicl food re eing investigted nd developed for etter efficcy. However, evidencesed study is still needed to investigte the efficcy nd possile toxicity. In this study, we demonstrted tht G. lucidum comined with CE could synergisticlly improve the exercise ftigue recovery nd exercise performnce within helthy popultion of mice. Acknowledgments This study ws sed on the University-Industry Coopertion Fund #1515 (Ntionl Tiwn Sport University, Toyun, Tiwn) nd finiclly supported y the Ministry of Science nd Technology of Tiwn (No. MOST H-227-7). The uthors re grteful to the grdute students t the Sports Nutrition Lortory, Ntionl Tiwn Sport University, for their technicl ssistnce in niml experiments. Competing Interests The uthors declre no conflict of interest.

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