Food Chemistry. Lijun You a, Mouming Zhao a, *, Joe M. Regenstein b, Jiaoyan Ren a. abstract. Contents lists available at ScienceDirect

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1 Food Chemistry 124 (211) Contents lists ville t ScienceDirect Food Chemistry journl homepge: In vitro ntioxidnt ctivity nd in vivo nti-ftigue effect of loch (Misgurnus nguillicudtus) peptides prepred y ppin digestion Lijun You, Mouming Zho, *, Joe M. Regenstein, Jioyn Ren College of Light Industry nd Food Sciences, South Chin University of Technology, Gungzhou 5164, Chin Deprtment of Food Science, Cornell University, Ithc, NY , USA rticle info strct Article history: Received 5 Novemer 29 Received in revised form 19 April 21 Accepted 3 June 21 Keywords: Loch peptide Antioxidnt ctivity Anti-ftigue effect Free rdicls Blood glucose The in vitro ntioxidnt ctivity nd in vivo nti-ftigue ctivity of loch peptide (LP) were determined. Results showed tht LP contined the mino cids, which were expected to contriute to its ntioxidnt nd nti-ftigue ctivities. LP could scvenge 1,1-diphenyl-2-picrylhydrzyl (DPPH) (IC ±.54 mg/ ml) nd hydroxyl rdicls (IC ±.29 mg/ml). It could chelte cupric ion nd inhiit the lipid peroxidtion in linoleic cid emulsion system. It lso prolonged the swimming time to exhustion of mice y 2 28% compred to the control. It incresed the levels of lood glucose (28 42% increse) nd liver glycogen ( fold increse). It decresed the levels of lctic cid nd lood ure nitrogen y % nd %, respectively. It lso improved the endogenous cellulr ntioxidnt enzymes in mice y incresing the ctivities of superoxide dismutse (SOD), ctlse (CAT) nd glutthione peroxidse (GSH-Px). Therefore, LP cn increse n endurnce cpcity nd fcilitte recovery from ftigue. Ó 21 Elsevier Ltd. All rights reserved. 1. Introduction Ftigue is est defined s the difficulty in inititing or sustining voluntry ctivities, nd cn e clssified into mentl nd physicl ftigue. Physicl ftigue is thought to e ccompnied y deteriortion in performnce (Tnk et l., 28). There re severl theories out the mechnisms of exercise-induced ftigue: exhustion theory, clogging theory, rdicl theory, homeostsis disturnce theory, protective inhiition theory, nd muttion theory (Wng et l., 28). The exhustion theory suggests tht during exercise, mny energy sources, such s glucose (GLU) nd liver glycogen, will e exhusted, thus leding to physicl ftigue. Severl reports showed tht post-exercise nutrition through the dministrtion of proteins, peptides or mino cids cn fcilitte recovery from ftigue (Wng et l., 28). Compred with proteins, peptides, s nutrition supplements, not only re sored quickly nd esily without competition from mino cids, ut lso promote the use of mino cids, proteins nd glucose (vn Loon, Sris, Kruijshoop, & Wgenmkers, 2). Therefore, peptides my e useful in ssisting in countercting nd meliorting physicl ftigue. According to the clogging theory, the over ccumultion of serum lctic cid (LA) nd lood ure nitrogen (BUN) will lso result in metolic disorders leding to ftigue. * Corresponding uthor. Tel./fx: E-mil ddress: femmzho@scut.edu.cn (M. Zho). Among the ftigue mechnisms, the rdicl theory hs een ttrcting more interest. Free rdicls re intermedite metolites of mny vitl iochemicl events in the ody, nd they re in dynmic lnce etween their production nd clernce. Hrmn s clssicl rdicl theory suggests tht intense exercise cn produce n imlnce etween the ody s oxidtion system nd its nti-oxidtion system. The ccumultion of rective free rdicls will put the ody in stte of oxidtive stress nd ring injury to the ody y ttcking lrge molecules nd cell orgns (Wng et l., 28). Muscle cells contin complex endogenous cellulr defense mechnisms to eliminte rective oxygen species, such s superoxide dismutse (SOD), glutthione peroxidse (GSH-Px) nd ctlse (CAT), nd to protect mong other things ginst exercise-induced oxidtive injury. Moreover, some reports (Yu et l., 26) showed tht exogenous dietry ntioxidnts cn lso decrese the contriution of exercise-induced oxidtive stress nd improve the niml s physiologicl condition. The reson my e tht exogenous ntioxidnts cn promote or interct with endogenous ntioxidnts to form coopertive network of cellulr ntioxidnts (Mizuno et l., 28). However, the mechnisms hve not een fully elucidted. Nevertheless, consumers re seeking more nturl ntioxidnt components in their diet to reduce oxidtive dmge nd fight ginst ftigue, without the side effects of other nti-ftigue drugs. Such ntioxidnt components hve een found in plnts, such s red mold rice (Wng, Shieh, Kuo, Lee, & Pn, 26) nd tissue culture extrcts of Sussure (Ji & Wu, 28), nd in hers, such s Trichopus zeylnicus (Thrkn, Dhnsekrn, & Mnym, 25). However, there re few reports /$ - see front mtter Ó 21 Elsevier Ltd. All rights reserved. doi:1.116/j.foodchem

2 L. You et l. / Food Chemistry 124 (211) regrding ntioxidnt peptides from niml sources hving ntiftigue ctivity. Loch (Misgurnus nguillicudtus) is common freshwter fish in Est Asi. It hs een fmilir to the Chinese since ncient times, for its desirle tste nd flvour. It hs lso trditionlly een considered s folk remedy for physicl nd mentl ftigue in the southern prt of Chin. However, the mechnisms of its nti-ftigue ctivity hve not een explored. In this study, we investigted the in vitro ntioxidnt ctivities nd the in vivo nti-ftigue effect of loch peptide (LP) previously prepred y ppin digestion. The in vitro ntioxidnt ctivities were studied y determining the 1,1-diphenyl-2-picrylhydrzyl (DPPH) nd the hydroxyl rdicls scvenging ctivities, the chelting ility with Cu 2+ ions, nd the lipid peroxidtion inhiitory ctivity in linoleic cid emulsion system, using glutthione (GSH) s control. In the susequent in vivo study, mice were fed with high or low doses of LP for 4 weeks. Then, n exhustive swimming time ws recorded nd severl iochemicl prmeters relted to ftigue [GLU, liver glycogen, LA, BUN, lctte dehydrogense (LDH), cretine kinse (CK), SOD, CAT, GSH-Px] were lso determined to explore the nti-ftigue ctivity of loch peptides. 2. Mterils nd methods 2.1. Loch peptide preprtion Live loch (Misgurnus nguillicudtus, 8.4 ± 1.1 g ody weight nd 9.8 ± 3 cm ody length) were purchsed from locl mrket in Gungzhou, Chin, nd trnsported to the l within 1 min. After killing, the met (without hed, til, skin, one nd lood) ws collected nd ground twice through met grinder with plte with 4 mm holes (MM12, Shogun Food Mchine Co., Shogun, Chin). The ground met ws stored in polyethylene g t 18 C until use, for mximum of 4 months. Fifty grms of loch met were mixed with 1 ml of distilled wter nd homogenised t speed of 1, rpm for 1 min using sic homogeniser (T25, Ik, Stufen, Germny). The homogente ws hydrolyzed with ppin (6 1 5 U/g) (Bio Biochemistry Co., Jingmen, Chin) t 55 C for 4.5 h. The enzyme to sustrte rtio ws 3:1 (w/w). The hydrolysis ws conducted t ph 7. (SL1-PHS-3B ph-metre, Wuhn Midwest Instrument Co. Ltd., Wuhn, Chin) in wter th shker (New Brunswick Scientifics C24, Jintn, Chin). After hydrolysis, the enzyme ws inctivted y plcing the smples in oiling wter for 15 min. The hydrolystes were centrifuged in GL-21M refrigerted centrifuge (Xingyi Instrument Co. Ltd., Chngsh, Chin) t 5g for 2 min, nd the superntnts were frctionted through ultrfiltrtion memrnes using iorector system (Vivflow 2, Vivscience, Srtorius, Goettingen, Germny) with moleculr weight cutoff (MWCO) of 5 kd. The frction with moleculr weight less thn 5 kd ws lyophilised (R2L- 1KPS, Kyow Vcuum Engineering, Tokyo, Jpn) nd stored in desicctor for further use. The estimted protein content of the LP powder ws 85%, determined y the Kjeldhl method (AOAC, 1995) using conversion fctor of The frctions in the LP with moleculr weight <1 kd, 1 3 kd, 3 5 kd nd >5 kd were 32%, 48%, 18% nd 2%, respectively, determined y gel permetion chromtogrphy, s descried in our previous report (You, Zho, Cui, Zho, & Yng, 28) Animls All the in vivo tests were crried y the School of Phrmceuticl Sciences of Southern Medicl University (Gungzhou, Chin), which otined the permission for performing the reserch protocols nd ll niml experiments conducted during the present study from the ethics committee of Southern Medicl University. Fifty-four mle NIH mice (18 22 g, specific pthogen-free grde, SPF, Approvl No. 27A58) were purchsed from the Acdemy of Experimentl Animl Center of Southern Medicl University (Gungzhou, Chin). They were housed in SPF level lortory in the School of Phrmceuticl Sciences of Southern Medicl University. All experimentl procedures were conducted under the oversight nd pprovl of the Acdemy of Experimentl Animl Center of Southern Medicl University nd in strict ccordnce with the NIH Guide for the Cre nd Use of Lortory Animls (NIH, 22). Animls were llowed to dpt to their surroundings for 1 week efore strting the experiments. Mice were housed 5 6 per cge t room temperture (22 ± 2 C) nd moderte humidity (5 ± 1%), with 12/12-h light drk cycle; noise ws <6 db. They were fed lnced murine diet provided y the Acdemy of Experimentl Animl Center of Southern Medicl University, nd hd drinking wter ville d liitum. After dpttion, the 54 mice were rndomly divided into six groups: two control groups, two LP tretment groups t high dose (LP-H), nd two groups t low dose (LP-L), with 9 mice ech. One group from ech set ws used for the exhustive swimming test. The other group ws used for collecting the lood to determine iochemicl prmeters relted to ftigue, fter swimming for 3 min. For 4 week, the LP-H mice were given 5 mg/(g d) of LP, nd the LP-L mice were given 1 mg/(g d) y intrperitonel injection every dy t 1: 3: pm. The control groups received distilled wter. After ech tretment, ll groups of the mice were llowed to rest 3 min nd were forced to swim for 2 min to ecome ccustomed to swimming (see elow) Amino cid nlysis The mino cid profile of LP ws determined ccording to the method of You et l. (28). The mino cid composition ws determined y high-performnce liquid chromtogrphy (Wters, Milford, MA, USA) equipped with PICO.TAG column. The totl mino cids (except for tryptophn) were determined fter hydrolysis t 11 C for 24 h with 6 M HCl, prior to the derivtistion with phenyl isothiocynte. Alkline hydrolysis ws lso done for the determintion of tryptophn level. The mino cid stndrds (Sigm Aldrich, St. Louis, MO, USA) were nlysed using the sme conditions s for the smples Determintion of in vitro ntioxidnt ctivities of LP Hydroxyl rdicl scvenging ctivity ssy The hydroxyl rdicl scvenging ctivity ws ssyed ccording to the method of Li, Jing, Zhng, Mu, nd Liu (28), with some modifictions. A mix of 6 ll of 1,1-phennthroline (5. mm), 6 ll of FeSO 4 (5. mm) nd 6 ll of ethylenediminetetrcetic cid (EDTA) (15 mm) were mixed with 4 ll of sodium phosphte uffer (.2 M, ph 7.4). Then, 6 ll of LP ( 25 mg/ml) nd 8 ll of H 2 O 2 (.1%) were dded. The mixture ws incuted t 37 C for 6 min, nd the sornce ws mesured t 536 nm (UV754, Xinjin Scientific Instrument Co., Shnghi, Chin). GSH (Sigm Aldrich, St. Louis, MO, USA) t 1 mg/ml ws used s control. The following eqution ws used: Hydroxyl rdicl scvenging ctivity ð%þ ¼ðA s A Þ1=ðA c A Þ where A s is the sornce of the smple; A is the sornce of the lnk solution using distilled wter insted of smple; nd A c is the sornce of control solution in the sence of H 2 O 2. The plot of scvenging ctivity ginst the concentrtion of the hydroly-

3 19 L. You et l. / Food Chemistry 124 (211) ste ws prepred, nd the IC 5 (concentrtion of smples to decrese the scvenging ctivity y 5%) otined DPPH rdicl scvenging ctivity ssy The DPPH rdicl scvenging ctivity ws determined y the method of Wu, Chen, nd Shiu (23), with slight modifiction. Two millilitres of LP ( 5 mg/ml) were mixed with 2. ml of.15 mm DPPH tht ws dissolved in 95% ethnol. The mixture ws then shken vigorously using mixer (QT-1 Mixer, Tinchen Technologicl Co. Ltd., Shnghi, Chin), nd ws kept for 3 min in the drk. The sornce of the resulting solution ws recorded t 517 nm. GSH t 5 mg/ml ws used s control. The scvenging ctivity ws clculted using the following eqution: Scvenging ctivity ð%þ ¼ðA DPPH smple A smple control Þ 1=A DPPH lnk where A DPPH smple is the vlue for the 2 ml of smple solution mixed with the DPPH solution; A smple control is the vlue for the 2 ml of smple solution mixed with the 2 ml of 95% ethnol; nd A DPPH lnk is the vlue for the 2 ml of 95% ethnol mixed with the DPPH solution. The plot of the scvenging ctivity ginst the concentrtion of smple ws prepred, nd the IC 5 otined Cupric ion chelting ctivity The ility of LP to chelte pro-oxidtive Cu 2+ ws investigted ccording to Zhu, Chen, Tng, nd Xiong (28). In the cheltion test, 1 ml of 2 mm CuSO 4 ws mixed with 1 ml of pyridine (ph 7.) nd 2 ll of.1% pyroctechol violet. After the ddition of 1 ml of LP ( 5 mg/ml), the disppernce of the lue color, due to dissocition of Cu 2+, ws recorded y mesuring the sornce t 632 nm fter 5 min of rection. An equivlent volume of distilled wter insted of the smple ws used for the lnk. GSH t 5 mg/ml ws used s control. The Cu 2+ chelting ctivity of the GI digests were clculted s: Cu chelting ctivity ¼½ðA A s Þ=A Š1% where A s is the sornce of the smple nd A is the sornce of the lnk solution using distilled wter insted of smple. The plot of the scvenging ctivity ginst the concentrtion of smple ws prepred, nd the IC 5 otined Mesurement of the lipid peroxidtion inhiition ctivity in linoleic cid emulsion system The lipid peroxidtion inhiition ctivity of LP ws mesured in linoleic cid emulsion system ccording to the methods of Qin, Jung, Byun, nd Kim (28). Briefly, 2. ml of LP ( 2 mg/ml) ws mixed with 2 ml of 2.5% linoleic cid dissolved in 95% ethnol. Then, 4 ml of 5 mm sodium phosphte uffer (ph 7.) nd 2 ml of distilled wter were dded. The mixture ws incuted in 5 ml conicl flsk with screw cp t 4 ± 1 C in drk room, nd the degree of oxidtion ws evluted y mesuring the FeSCN vlues descried elow. The rection solution (1 ll), incuted in the linoleic cid model system, ws mixed t different intervls during the incution period with 9.7 ml of 75% ethnol,.1 ml of 3% NH 4 SCN, nd.1 ml of 2 mm FeCl 2 solution in 3.5% HCl. After 3 min, the SCN vlue ws mesured y reding the sornce t 5 nm. An equivlent volume of distilled wter ws used s lnk. GSH t 2 mg/ml ws used s control. The lipid peroxidtion inhiition ctivity ð%þ ¼½1 ða s;t¼144 h A s;t¼ h ÞŠ 1=ðA ;t¼144 h A ;t¼ h Þ where A s,t=144 h nd A s,t= h re the sornces for the smple t 144 h nd h, respectively & A,t=144 h nd A,t= h re the sornces for the lnk t 144 h nd h, respectively. The plot of the scvenging ctivity ginst the concentrtion of smple ws prepred, nd the IC 5 otined In vivo nti-ftigue effect of LP Exhustive swimming test After the finl tretment with LP or distilled wter, the mice were llowed to rest for 3 min. Then, they were plced in the swimming tnk (5 cm 5 cm 4 cm) with 3 cm deep wter t 25 ± 1 C. The current in the pool ws generted y circulting wter with pump, nd the strength of the current ws djusted to 8 l/min with wter flow metre (type F455, Blue White Co., Westminster, CA, USA). The wter ws gitted to mke the mice lims keep moving. The mice were determined to e exhusted when they filed to rise to the surfce to rethe fter 7 s (Jung, Hn, Kwon, Lee, & Kim, 27) Mesuring iochemicl prmeters relted to ftigue After the finl tretment with LP or distilled wter, the mice were llowed to rest for 3 min. Then they were plced in the swimming tnk (5 cm 5 cm 4 cm) with 3 cm deep of wter (temperture: 25 ± 1 C). After swimming for 3 min, they were tken out. Blood ws collected from the oritl sinus to determine GLU, LA nd BUN content nd CK, SOD, GSH-Px, LDH nd CAT ctivity (Jung et l., 27). The livers of the mice were lso tken to determine the content of liver glycogen (see elow). All of the iochemicl prmeters were determined y using n utomted Biochemistry Anlyzer (76-1, Hitchi, Jpn). The glucose, BUN, CK nd LDH ctivities were determined using commercil dignostic kits (Product Nos. 611, 28, 9 nd 131, respectively, Biosino Bio-technology nd Science Inc., Beijing, Chin). LA ws determined using commercil dignostic kit (Product No. LC6351, Beijing Ledmn Biochemistry Technology Co. Ltd., Beijing, Chin). The SOD ctivity ws determined using SOD Assy Kit A1 (Institute of Biologicl Engineering of Nnjing Jinchen, Nnjing, Chin). The GSH-Px ctivity ws determined with GSH-Px Assy Kit A5 (Institute of Biologicl Engineering of Nnjing Jinchen, Nnjing, Chin). The GSH-Px hs the ility to decompose hydrogen peroxide (H 2 O 2 ) nd other orgnic hydroperoxides (ROOH). The rection uses GSH to complete the rection using H 2 O 2, s the sustrte. The consumption of nicotinmide denine dinucleotide phosphte (NADPH) is used to determine the GSH-Px ctivity. The ctlse ctivity ws determined colorimetriclly with CAT Assy Kit A7 (Institute of Biologicl Engineering of Nnjing Jinchen, Nnjing, Chin). The test is sed on the decomposition of the H 2 O 2 opticl density t 415 nm y CAT. The livers of the mice were dissected immeditely fter tking them out, wshed with.9% sline, nd lotted dry with filter ppers. Liver smples (1 mg) were ccurtely weighed, nd homogenised in 8 ml of homogenistion uffer from Liver Glycogen Assy Kit A43 (Institute of Biologicl Engineering of Nnjing Jinchen, Nnjing, Chin). The liver glycogen ws determined ccording to the recommended procedures Sttisticl nlysis All the tests were conducted in triplicte. The experimentl dt were expressed s men ± stndrd error. The results were sujected to one-wy nlysis of vrince (ANOVA). LSD nd Dunnett s T3 tests were performed to determine the significnt difference etween smples within the 95% confidence intervl, using SPSS 13. softwre (SPSS Inc., Chicgo, IL, USA).

4 L. You et l. / Food Chemistry 124 (211) Results nd discussion 3.1. Amino cid composition of LP The mino cid compositions of the protein hydrolystes re directly relted to their ioctive ctivities. Histidine or histidinecontining peptides hve chelting nd lipid rdicl trpping ility due to the imidzole ring (Uchid & Kwkishi, 1992). Aromtic mino cids, such s tryptophn exhiit ntioxidnt ctivity since they cn donte protons esily to electron-deficient rdicls. Severl mino cids, such s tyrosine, methionine, histidine, lysine, nd tryptophn, hve generlly een considered s ntioxidnts (Chen, Murmoto, Ymuchi, & Nokihr, 1996). As shown in Tle 1, the loch peptide contins 24.8% of the ove five mino cids, suggesting tht it might hve potentil ntioxidnt ctivity. Amino cids lso ply n importnt role in the regultory metolism involved in musculr ctivity. Some mino cid, especilly the rnched chin mino cids, cn improve the exercise cpility nd mrkedly retrd the ctolism of protein in the muscle during exercise (Blomstrnd & Newsholme, 1992). Bzzrre, Murdoch, Wu, Herr, nd Snider (1992) reported tht the mount of mino cids, especilly lnine, glycine, vline, isoleucine, threonine, serine nd tyrosine in the plsm will decrese rpidly during n endurnce test. As shown in Tle 1, the loch peptide contins 32.2% of the ove mino cids, suggesting tht it might enhnce exercise cpility. Glutmic cid ws found to hve very positive effect on the nervous system nd would lso e helpful during exercise (Guezennec et l., 1998). Asprtic cid ws considered to e helpful in the oxidtive demintion nd could lower the lood mmoni concentrtion, therefore delying the occurrence of ftigue (Mrquezi, Roschel, Cost, Swd, & Lnch, 23). The loch peptide contins 16.7% glutmic cid nd 8.74% sprtic cid, suggesting tht it might hve potentil nti-ftigue effect In vitro ntioxidnt ctivities of LP Tle 1 The mino cid composition of LP. Amino cid Concentrtion (mg mino cid residues/g loch peptide powder) Asprtic cid Glutmic cid Serine Glycine Histidine Arginine Threonine Alnine Proline Tyrosine Vline Methionine Cystine.2.2 Isoleucine Leucine Tryptophn Phenyllnine Lysine Totl Composition (%) One grm of loch protein from to 5 kd frction contined 85 mg of totl estimted protein. Thus, the pproximte yield of the identified mino cid residues (mino cids minus H 2 O) ccounted for 798/85, or pproximtely 94% of the nominl protein. Normlised so tht the oserved mino cid residues dd up to 1% of the totl mino cid residues. As one of rective oxygen species generted in the humn ody, hydroxyl rdicls cn rect esily with iomolecules, such s mino cids, proteins nd DNA. This cn led to physiologicl disorders (Ccciuttolo, Trinh, Lumpkin, & Ro, 1993). DPPH hs een used extensively s the free rdicl to evlute reducing sustnces. As shown in Tle 2, LP hd hydroxyl IC 5 vlue of 17. ±.54 mg/ml nd DPPH IC 5 vlue of 2.64 ±.29 mg/ml, which ws 2.6-fold nd 2.4-fold higher thn tht of GSH, respectively. The IC 5 vlue for the cheltion ctivity of Cu 2+ ion ws 2.89 ±.33 mg/ml, which ws 1.8-fold higher thn tht of GSH. The lipid peroxidtion is thought to proceed vi rdicl-medited strction of hydrogen toms from methylene crons in polyunsturted ftty cids (Rjpkse, Mendis, Jung, Je, & Kim, 25). The lipid peroxidtion inhiitory ctivity of LP of the linoleic cid system gve n IC 5 vlue of 12.3 ±.98 mg/ml, which ws not sttisticl different from tht of GSH (P >.5). Nevertheless, the verge moleculr weight of LP ws four times more thn tht of GSH (dt not shown). Tht is to sy tht LP hd stronger ntioxidnt ctivities thn GSH t the sme moleculr concentrtion. Therefore, LP hs in vitro ntioxidnt ctivities, which indictes tht it my hve the potentil to reduce the oxidtive stress in vivo nd to fight ftigue LP prolonged the exhustive swimming time A direct mesure of n nti-ftigue effect is the increse in exercise tolernce. Swimming to exhustion is n experimentl exercise model to evlute nti-ftigue; it works well for evluting the endurnce cpcity of mice, nd gives high reproduciility (Zhng, Yo, Bo, & Zhng, 26). Reduced susceptiility to ftigue is correlted with longer swimming times. Both the high dose (5 mg/(g d)) nd low dose (1 mg/(g d)) of LP tretments prolonged the swimming time of the mice. As shown in Fig. 1, the medin exhustion time of the LP-H mice ws 241 min (28% greter thn tht of the wter-injected control group); the medin exhustion time of the LP-L mice ws 226 min (2% greter thn tht of the control) (P <.5), indicting tht LP possesses n nti-ftigue effect. To explore the mechnism(s), some iochemicl prmeters, such s GLU, liver glycogen, LA, BUN contents nd LDH, CK, SOD, CAT, GSH-Px ctivities were determined in the mice fter they swm for 3 min LP incresed lood GLU nd liver glycogen The homeostsis of lood glucose plys n importnt role in prolonging endurnce exercise (Wgenmkers et l., 1991). Hypoglycemi cn suppress the ctive functioning of the rin during exercise, nd this often leds to the inility to continue exercise (Wng et l., 26). Thus, the mount of lood glycogen cn illustrte the speed nd degree of ftigue development (Wng et l., 28). As shown in Tle 3, the lood GLU levels of LP-H nd LP- L mice were oth significntly higher thn tht of the control group (P <.5); 28% nd 42% higher, respectively. However, there is no ovious dose-dependence etween the high nd low doses groups Tle 2 In vitro ntioxidnt ctivity of LP. Smple Antioxidnt ctivity (IC 5 ) (mg/ml) Hydroxyl DPPH Cupric ion c Lipid peroxidtion inhiition d LP 17. ± ± ± ±.98 GSH 4.7 ± ± ± ± 1.47 c d The scvenging ctivity for the hydroxyl rdicl. The scvenging ctivity for the DPPH rdicl. The chelting ctivity of Cu 2+ ion. The lipid peroxidtion inhiition ctivity in linoleic cid emulsion system.

5 192 L. You et l. / Food Chemistry 124 (211) Swimming time (min) Growing evidence indictes tht rective oxygen species re responsile for exercise-induced protein oxidtion, nd contriute strongly to muscle ftigue (Powers, DeRuisseu, Quindry, & Hmilton, 24). Muscle cells contin two mjor clsses (non-enzymic nd enzymic) of endogenous cellulr defense mechnisms to elimcontrol LP-L LP-H Group (P >.5). This indicted tht the lood-glucose-regulting ility of the tretment groups ws higher thn tht of the control. Liver glycogen is nother index of ftigue. The role of heptic glycogen is to complement the consumption of lood glucose nd mintin the lood glucose in the physiologic rnge. Ftigue will hppen when the liver glycogen is mostly consumed (Ji nd Wu, 28). As shown in Tle 3, the liver glycogen levels of LP-H nd LP-L mice were oth significntly higher thn tht of the control group (P <.5), 2.3-fold nd 3.-fold higher, respectively. Moreover, there ws positive dose-dependent effect etween LP-H nd LP-L (P <.5). The results show tht the nti-ftigue ctivity of LP my e relted to the improvement in the metolic control of exercise nd the ctivtion of energy metolism (Wng et l., 26) LP decresed LA nd BUN in the lood Fig. 1. Effect of loch peptide on the swimming time in mice. The error rs represent one stndrd devition. Different letters indicte significnt differences etween groups (P <.5). Tle 3 The contents of glucose, liver glycogen, lctic cid nd lood ure nitrogen in the mice (mmol/l). Groups Control LP-L LP-H Glucose 5.6 ± 1.,* 6.47 ± ±.98 Liver glycogen 3.44 ±.28 c 11.4 ± ± 1.74 Lctic cid 11.4 ± ± ±.59 c Blood ure nitrogen 9.98 ± ± ±.36 c * Different letters indicte significnt differences etween groups for the sme. The response to exercise in mmmls egins with n increse in eroic musculr ctivity, which switches over to neroic metolism if the exercise is intense, which leds to the ccumultion of LA (Evns, Surmonim, Rjsekhrn, & Pushpngdn, 22). So, the ccumultion of lood serum LA is n importnt cuse of ftigue. With intense exercise, O 2 nd pyruvic cid re reduced y LDH to LA, which decreses the ph, ffecting oth the crdio-circulting system nd the skeletl muscle system function. The decrese in the contrctive strength of the muscle eventully induces ftigue (Ji nd Wu, 28). As shown in Tle 3, the LA levels of LP-H nd LP-L mice were oth significntly lower thn tht of the control group (P <.5), 1.9% nd 27.5% decreses, respectively. Moreover, there is dose-dependent effect etween LP-H nd LP-L (P <.5), which is nother confirmtion tht LP hs n nti-ftigue effect. Ure is formed in the liver s the end product of protein metolism. During digestion, protein is roken down into smll peptides nd mino cids. The mino cid nitrogen is removed s NH þ 4, while the rest of the molecule is used to produce energy or other sustnces needed y the cell (Koo, Lee, Hong, & Kim, 24). Serum ure nitrogen, which is product of energy metolism, is nother sensitive index of ftigue sttus. There ws positive correltion etween ure nitrogen in vivo nd exercise tolernce. The less n niml is dpted to exercise, the more the ure nitrogen level increses (Zhng et l., 26). As shown in Tle 3, the BUN levels of the mice were significntly lower y 17.5% in the LP-H group compred to the control (P <.5), while the decrese of 8.6% in the LP- L group ws not significntly different to the control (P >.5). The reduced protein metolism of the high dose of LP is indictive of enhnced endurnce (Ji nd Wu, 28; Wng et l., 28) LP incresed LDH ctivity ut decresed CK ctivity Serum CK nd LDH re known to e ccurte indictors of muscle dmge. The norml function of CK in cells is to dd phosphte group to cretine, turning it into the high-energy molecule phosphocretine, which is urned s quick source of energy y the cells (Coomes & McNughton, 2). However, the norml function of CK is not s relevnt here s wht hppens to CK when the muscle is dmged. During the process of muscle degenertion, the muscle cells lyse nd their contents find their wy into the loodstrem. Becuse most of the CK in the ody normlly exists in the muscle, n increse in CK in the lood indictes tht muscle dmge hs occurred or is occurring. LDH ctlyses the interconversion of pyruvte nd lctte (Koo et l., 24). As shown in Fig. 2, the LDH level of mice ws significntly incresed for LP-H mice (P <.5), while the serum CK level decresed (P <.5) compred to the control. There were no significnt chnges for the LDH nd CK levels of LP-L. However, the sttisticl differences were significnt etween the high nd low LP (P <.5) for these two iochemicl prmeters LP enhnced the ntioxidtive enzymes in mice LDH ctivity (U/L) LDH CK x control LP-L LP-H Group Fig. 2. Effect of loch peptide on LDH nd CK ctivities in mice. LDH refers to lctte dehydrogense nd CK refers to cretine kinse. The error rs represent one stndrd devition. Different letters indicte significnt differences etween groups within the sme procedure (P <.5). x y 5 CK ctivity (U/L)

6 L. You et l. / Food Chemistry 124 (211) Antioxidnt enzymes ctivity (U/L) inte rective oxygen species. The primry ntioxidnt enzymes include SOD, GSH-Px nd CAT (Thrkn et l., 25). SOD dismuttes superoxide rdicls to form H 2 O 2 nd O 2. GPH-Px is n enzyme responsile for reducing H 2 O 2 or orgnic hydroperoxides to wter nd lcohol, respectively. CAT ctlyses the rekdown of H 2 O 2 to form wter nd O 2. These ntioxidnt defense mechnisms ecome weker during chronic ftigue nd other disese conditions (Powers & Lennon, 1999). So, the improvement in the ctivities of these defense mechnisms cn help to fight ginst ftigue. As shown in Fig. 3, the CAT ctivities of the LP tretment groups (oth high nd low doses) were significntly higher thn tht of the control (49% nd 36% greter, respectively) (P <.5), while there ws no sttisticl difference etween LP-H nd LP-L. The GSH-Px nd SOD ctivities of LP-H incresed y 27% nd 8.5%, respectively, compred to the control (P <.5), while those for the low dose of LP tretment incresed y 13% nd 1.7%, respectively; however, oth were without sttisticl significnce (P >.5), compred to the control. The results show tht LP cn promote increses in the ctivities of these ntioxidnt enzymes, gin supporting tht LP hs n nti-ftigue effect. 4. Conclusions y # CAT GSH-Px SOD control LP-L LP-H Group Fig. 3. Effect of loch peptide on the ctivities of ntioxidnt enzymes in mice. SOD refers to superoxide dismutse; GSH-Px refers to glutthione peroxidse; nd CAT refers to ctlse. The error rs represent one stndrd devition. Different letters indicte significnt differences etween groups within the sme procedure (P <.5). The results show tht loch peptide prepred y ppin digestion hs not only in vitro ntioxidnt ctivities, ut lso n in vivo nti-ftigue effect in mice. It contined the mino cids, which were expected to contriute to its ntioxidnt nd nti-ftigue ctivities. It cted s the scvenger for DPPH nd hydroxyl rdicls. It hd the ility to chelte Cu 2+ ion nd to inhiit lipid peroxidtion in linoleic cid emulsion system. The in vivo study showed tht LP prolonged the exhustion swimming time of the mice. It improved the metolic control of exercise nd ctivted the energy metolism y incresing the levels of lood GLU nd liver glycogen. It helped to eliminte the ccumulted products of metolism y decresing the levels of LA nd BUN. It lso improved the endogenous cellulr ntioxidnt enzymes in mice y incresing the ctivities of SOD, CAT nd GSH-Px. Therefore, LP cn elevte the endurnce cpcity nd fcilitte recovery from ftigue. The results provide n importnt sis for developing the loch peptide s novel ntioxidnt nd nti-ftigue compound. 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