Antioxidant and Immunostimulating Activities of Yeast (Saccharomyces cerevisiae) Autolysates

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1 Worl Appli Sins Journl 15 (8): , 2011 ISSN IDOSI Pulitions, 2011 Antioxint n Immunostimulting Ativitis of Yst (Shromys rvisi) Autolysts Hzm M.M. Hssn Dprtmnt of Biohmistry, Fulty of Agriultur, Ciro Univrsity, Giz, Egypt Astrt: Th ojtiv of th prsnt stuy ws to invstigt th ntioxint n immunostimulting tivitis of two yst utolysts (kr's yst n tiv ry yst) using vrious mthos. In this stuy, totl glutthion ontnt ws trmin in oth yst utolysts. Antioxint tivity of oth yst utolysts ws vlut y vrious ssys, inluing totl ntioxint pity, ruing powr, DPPH ril svnging, nitri oxi svnging, hyroxyl ril svnging n mtl ion hlting tivitis.th immunostimulting tivity of oth yst utolysts ws vlut using rt splnoyt prolifrtion y th olorimtri MTT ssy. Th otin rsults show tht tiv ry yst utolyst (ADYA) ontins highr onntrtion of glutthion ompr with kr's yst utolyst (BYA). Th ADYA possss ntioxitiv tivity highr thn BYA. Both yst utolysts h stimulting tivity of splnoyt prolifrtion. Thrfor, th yst utolysts hv th potntil to xplor s novl nturl ntioxints n immunostimulting gnts for using in funtionl foos or miin. Ky wors: Antioxint Immunostimulting tivity Bkr's yst Ativ ry yst Yst utolyst INTRODUCTION Yst-sonry n yst-riv prouts r vlop y-y-y n ysts r now stnr Mny isss r us y oxitiv strss. mol orgnisms for th isstion of mny iologil Alrt ll oxition ontriuts to riovsulr mhnisms, s wll s othr importnt funtions in our iss, tumor growth, wrinkl skin, nr, Alzhimr's lif. Yst utolysts r onntrts of th solul iss n vn lin in nrgy n nurn [1-3]. omponnts of yst lls n r gnrlly prou y Antioxints r sustns tht ly or prvnt th utolysis. In othr wors, n utolyst is th totl oxition of llulr oxiizl sustrts. Thy xrt thir ontnt of th yst following utolysis, whih is fft y svnging rtiv oxygn spis, tivting ssntilly grtiv pross rri out y tivting uttry of toxifying protins or prvnting th th yst s own grtion nzyms to soluiliz th ll gnrtion of rtiv oxygn spis [4, 5]. In rnt omponnt foun in th ll. Yst utolysts r lso yrs, thr hs n n inrsing intrst in fining known unr th nm of yst xtrts. Thy r minly nturl ntioxints, whih n prott th humn oy us in th frmnttion inustry s sustrts n in th from fr rils n rtr th progrss of mny hroni foo inustry s flvor improvrs (flvoring gnts). isss [6, 7]. Th immun systm is vulnrl to Simply, utolysis of yst is fin s slf igstion of oxitiv strss. During rtin iss stts, s wll s yst lls. Tht is to sy, tht ll hyrolysis is uring ging, thr is n to oost th ntioxint prform without ition of othr nzyms. Autolysis ilitis, thry potntiting th immun mhnism [8]. iffrs from hyrolysis, in tht th rkown of th ll Th ntioxints prsrv n qut funtion of onstitunts suh s protins n nuli is is immun lls ginst homostti isturns [9, 10]. hiv y th tion of nzyms prou y th During th lst fiv s, spil intrst yst ll itslf [11, 12]. Yst xtrt (YE) ompriss tths to ysts, us yst onstitunts r th wtr-solul omponnts of th yst ll, th onsir s ompouns of nutritionl vlu to omposition of whih is primrily mino is, pptis, humn n highr nimls. Yst lls ontin lot of rohyrts n slts. Nitrogn omponnts n protin, rohyrts, lipi, vitmins n minrls. vitmins r th vlu of yst xtrt us of thir Corrsponing Author: Hzm M.M. Hssn, Dprtmnt of Biohmistry, Fulty of Agriultur, Ciro Univrsity, Giz, Egypt. 1110

2 Worl Appl. Si. J., 15 (8): , 2011 nutritionl hrtristis. Hn, YE hs n minly Th im of this stuy ws to invstigt th us in th foo inustry, s flvoring gnt in soup, ntioxint n immunostimulting tivitis of sus, grvis, stws, snk foo n nn foo, s utolysts otin from kr's yst n tiv ry wll s in pt foos n osmti mtrils n s plnt yst using vrious mthos. nutrint. Othr pplitions inlu vitmin n protin supplmnts in hlth foos n s sour of nutrints MATERIALS AND METHODS in miroiologil mi [13-17]. Mny workrs hv shown th growth promoting proprtis of yst xtrts Mtrils (YE) on tril growth [18-21]. Yst smpls: In this invstigtion, two yst Rgring th yst utolysts (Shromys (Shromys rvisi) smpls wr us. rvisi), th litrtur ws poor onrning in th t Frsh kr s yst n tiv ry yst wr otin of ntioxint n immunostimulting tivitis of ths from lol mrkt t Giz, Egypt. yst utolysts. Th ntioxitiv proprtis of yst xtrts om from thir ontnts of glutthion, Animls: Ml Wistr whit rt, wighing out 80 g ws Millr rtion prouts n sulfur-ontining mino purhs from Rsrh Institut of Ophthlmology, is [12, 22]. Wng t l. [23] trmin th possil in Giz, Egypt. situ ntioxint tivity of two ommril yst xtrts in ook f pttis. Thy osrv tht th yst Chmils: Asori i, 1, 1-iphnyl-2-pirylhyrzyl xtrts supprss thiorituri i-rtiv (DPPH), sulfnilmi, N-(1-nphthyl) thylnimin sustns (TBARS) formtion in ook f pttis ihyrohlori, 2-oxyrios, frrous sulpht, uring storg. Jung t l. [24] vlut th ril RPMI-1640 mium, 3-(4,5-imthylthizol-2-yl)-2,5- svng n gluos tolrn of yst hyrolyst with iphnylttrzolium romi (MTT) n high ontnt of Cylo-His-Pro (CHP). lipopolyshri (LPS) wr purhs from Th rsults show tht yst hyrolyst intns Sigm Chmil Co., USA. Glutthion ru svnging ilitis of oth 1, 1-iphnyl-2-pirylhyrzyl (GSH) kit ws otin from Bio-Dignosti Co., Giz, (DPPH) n 2, 2'-zino-is (3-thylnzthizolin-6- Egypt. All othr hmils wr of nlytil sulphoni i) (ABTS) rils. In vitro n in vivo rgnt gr. stuis in nimls n humns show tht -gluns riv from fungi n ysts hv immunomoulting Mthos proprtis. Most frquntly vlut r thir ffts Prprtion of Yst Autolysts: Yst utolyst on lukoyt tivity. Immun rspons is influn solutions of frsh kr's yst n tiv ry y oth prntrl n ntrl ministrtion of -glun yst wr prpr oring to th mtho [25-27]. Jng t l. [28] vlut th omprtiv sri y Sommr [12] s follows: On prt of immunomoultory ffts of -gluns isolt from yst ws mix wll with two prt of istill wtr mushroom fungi (Coriolus vrsiol), yst (DW). Th suspnsion ws thn inut t room (Shromys rvisi) n tri (Agrotrium) tmprtur (25 C) for 72 h. Aftr inution, th on th mjor funtions of mrophgs. Th rsults solution ws ntrifug t 4000 rpm for 10 min. init tht ths -gluns, isolt from thr Th rsius wr r-xtrt twi with istill iffrnt sours, hv iffrnt ffts on mrophg wtr thn ntrifug t th sm rpm for 10 min. funtion n thrfor, my hv iffrnt linil uss in Th otin yst utolyst solutions wr iffrnt for vrious typs of isss. Ylin t l. [29] omin in volumtri flsk (500 ml) n omplt to trmin th ffts of itry yst utolyst on th mrk with istill wtr. Th rsulting utolyst prformn, gg trits, gg holstrol ontnt, gg yolk solution ws us s kr's yst utolyst (BYA) ftty i omposition, lipi oxition of gg yolk, som or tiv ry yst utolyst (ADYA). Th totl solis loo prmtrs n humorl immun rspons of lying (%) in oth utolysts wr trmin y th ry hns uring 16 wk prio. Th rsults show tht wights following rying t 100 C until onstnt mss itry yst utolyst t lvls of 2, 3 n 4 g/kg h ws hiv. In ll ssys, onntrtions of yst nfiil ffts on prformn, gg holstrol ontnt utolysts (BYA or ADYA) wr lult s n humorl immun rspons. yst quivlnt. 1111

3 Worl Appl. Si. J., 15 (8): , 2011 Dtrmintion of Totl Glutthion: Th ru Assy of DPPH Ril Svnging Ativity: glutthion (GSH) ontnt ws trmin in yst Th ntioxint tivity of yst utolysts, s on utolysts using th prour sri y th svnging tivity of th stl DPPH fr Butlr t l. [30]. Th sulfhyril groups prsnt in ril, ws trmin y th mtho sri y th glutthion forms olor omplx with DTNB L t l. [33] s follows: Known volums ( µl) of [5, 5 ithiois (2-nitronzoi i)], whih ws yst utolyst wr iniviully to tst tus msur olorimtrilly t 405 nm. thn omplt to known volum (1.0 ml) y DW. 1.0 ml of DPPH solution (0.2 mm in thnol) ws to h Dtrmintion of Totl Antioxint Cpity: Totl tu thn mix wll n inut t room tmprtur ntioxint pity of yst utolysts ws ssy y for 30 min. Control ws prpr y th sm prour th phosphomolynum mtho s sri y without yst utolyst. Asori i solution (0.03%, Kumrn n Krunkrn [31] s follows: Known w/v) ws us s positiv ontrol. Th sorn (A) volums ( ml) of h yst utolyst wr of th solution ws msur t 517 nm using Jnwy to tst tu thn omplt to onstnt volum (0.3 ml) 6300 sptrophotomtr. Inhiition of DPPH fr ril with DW. 3.0 ml of rgnt solution (0.6 M sulfuri i, in prnt (I%) ws lult from th following 28.0 mm soium phospht n 4.0 mm mmonium qution: molyt) wr to h tu n mix wll thn inut t 95 C for 90 min. Blnk ws prpr y th I% = [(A ontrol A smpl)/a ontrol] x100 sm prour without yst utolyst. Asori i solution (0.03%, w/v) ws us s positiv ontrol. Assy of Nitri Oxi Svnging Ativity: Aftr ooling to room, th sorn of th solution ws Th svnging tivity of nitri oxi y yst msur t 695 nm using Jnwy 6300 utolysts ws trmin y th mtho sri y sptrophotomtr ginst lnk. Inrs sorn Kumrn n krunkrn [31] s follows: In ln tst of th rtion mixtur init inrs totl tus, 0.5 ml of soium nitroprussi solution (10 mm in ntioxint pity. 0.1 M phospht uffr slin, ph 7.4) ws mix with iffrnt volums of yst utolyst ( ml) thn DW Dtrmintion of Ruing Powr: Th ruing powr ws to h tu to omplt th solution to of yst utolysts ws trmin y th mtho of known volum (1.0 ml). Th tst tus wr inut t Mthw n Arhm [32] s follows: In ln tst tus, room tmprtur for 150 min thn 0.5 ml of Griss rgnt sril of known volums ( ml) of h yst (1% sulfnilmi, 2% H3PO 4 n 0.1% N-(1-nphthyl) utolyst wr. Th solutions wr omplt thylnimin ihyrohlori) ws to h tu to 1.0 ml with DW. 2.5 ml of phospht uffr solution n mix wll. Th sorn (A) ws msur t 546 (0.2 M, ph 6.6) n 2.5 ml of potssium frriyni nm using Jnwy 6300 sptrophotomtr. Control ws solution (1%, w/v) wr to h tu thn mix prpr y th sm prour without yst utolyst. wll. Th mixturs wr inut t 50 C for 20 min. Asori i solution (0.03%, w/v) ws us s positiv Aftr inution, 2.5 ml of trihloroti i ontrol. Svnging tivity of nitri oxi ws lult solution (10%, w/v) wr to h mixtur thn from th following qution: ntrifug t 5,000 rpm for 10 min. A known volum (2.5 ml) of h lr solution otin ftr ntrifugtion Svnging tivity (%) = [(A ontrol A smpl)/a ontrol] x 100 (suprntnt) ws tkn in nothr ln tst tu thn 2.5 ml of DW n 0.5 ml of frri hlori solution (0.1%, Assy of Hyroxyl Ril Svnging Ativity: w/v) wr n mix wll. Th sorn ws Th svnging tivity of hyroxyl ril y yst msur t 700 nm using Jnwy 6300 utolysts ws ssy y oxyrios mtho s sptrophotomtr. Blnk ws prpr y th sm sri y Ngi t l. [34] s follows: In ln tst prour without yst utolyst. Asori i tus, 0.45 ml of soium phospht uffr solution solution (0.03%, w/v) ws us s positiv ontrol. (0.2 M, ph 7.0), 0.15 ml of 2-oxyrios solution (10 mm), Inrs sorn of th rtion mixtur init 0.15 ml of FSO4-EDTA solution (10 mm FSO 4, 10 mm inrs ruing powr. EDTA), 0.15 ml of H O solution (10 mm) n yst

4 Worl Appl. Si. J., 15 (8): , 2011 utolyst ( µl) wr. Th solutions wr t 3,000 rpm for 10 min thn wsh twi with RPMI-1640 omplt to finl volum (1.5 ml) with DW thn mium. Th viility of splnoyts ws trmin y inution t 37 C for 4 h. Aftr inution, th rtion stining th lls with trypn-lu y xlusion. ws stopp y ing 0.75 ml of trihloroti i Ltr, ths splnoyts wr suspn in omplt solution (2.8%, w/v) n 0.75 ml of thiorituri i RPMI-1640 mium (Th RPMI-1640 mium solution (1% in 50 mm NOH solution) thn th supplmnt with ftl lf srum (10%), 100 units of solutions wr oil for 10 min n ool in wtr. pniillin n 100 µg of strptomyin) n ll Th sorn (A) of th solutions ws msur t onntrtion ws just to 1 10 lls/ml. nm using Jnwy 6300 sptrophotomtr. Control ws prpr y th sm prour without yst utolyst. Prour: Known volums (8, 16 n 20 µl) of h Asori i solution (0.03%, w/v) ws us s positiv yst utolyst wr iniviully to mixtur ontrol. Inhiition of oxyrios grtion in prnt ompos of 20 µl of th splnoyt suspnsion (I%) ws lult using th following qution: 6 (1x10 lls/ml) n 40 µl of RPMI-1640 mium in 96-wll plt. Phospht uffr slin solution (PBS; 0.1 I% = [(A ontrol A smpl)/a ontrol] x100 M phospht uffr, ph 7.4 n 0.9% NCl, w/v) ws in orr to omplt th mixtur of h wll to Assy of F Chlting Ativity: Th ility of yst known volum (80 µl). PBS n Lipopolyshri (LPS) utolyst to hlt frrous (F ) ion ws trmin solution (5 µg/ml) wr us s ngtiv n positiv using moifi mtho of Minotti n Aust [35] s ontrol, rsptivly. Aftr inution t 37 C in sri y Ooh t l. [36]. In ln tst tu, 150 µl humiifi 5% CO 2 tmosphr for 72 h, 20 µl of MTT of frshly prpr frrous sulpht solution (500 µm) solution (5 mg/ml in PBS) n 40 µl of RPMI-1640 mium wr to rtion mixtur onsist of 168 µl of wr. Th ll suspnsions wr ultur t 37 C Tris HCl uffr solution (0.1 M, ph 7.4) n yst for 4 h. 100 µl of HCl in isopropnol solution (0.04 M) wr utolyst (10-25 µl). Th solution ws omplt y to ll wlls n mix thoroughly thn 100 µl of slin solution (0.9% NCl, w/v) to known volum DW wr to ilut th solution. Th optil nsity (561 µl). Th rtion mixtur ws inut for 5 min (OD) ws msur t 570 nm using Jnwy 6300 t room tmprtur for th ition 13 µl of 1, sptrophotomtr. Th prntg of prolifrtion ws 10-phnnthrolin solution (0.25%, w/v). Th sorn lult y th following qution: (A) ws msur t 510 nm using Jnwy 6300 sptrophotomtr. Control ws prpr y th sm ODsmpl - ODngtiv ontrol Prolifrtion (%)= 100 prour without yst utolyst. Asori i ODngtiv ontrol solution (0.03%, w/v) ws us s positiv ontrol. Th F hlting tivity (%) ws lult from th Sttistil Anlysis: Th rsults wr nlys y n following qution: nlysis of vrin (P<0.05) n th mns sprt y Dunn s multipl rng tst. Th rsults wr pross F hlting tivity (%) = [(A ontrol A smpl)/aontrol] y CoStt omputr progrm. x100 RESULTS AND DISSCUSION Assy of Mitogn-Inu Splnoyt Prolifrtion: Th lymphoyt prolifrtion ssy ws rri out Antioxint Ativity of Yst Autolysts: Rtiv oring to th MTT mtho s sri y Mnosroi oxygn spis n othr hrmful ompouns r t l. [37] s follows: prou uring th norml growth of roi lls n ths my inhiit ll growth. Dfn systms suh s Prprtion of Rt Splnoyts: Rt ws kill y rvil ntioxint n rox nzyms r rquir for th norml islotion. Th spln ws sptilly rmov n growth of th lls. GSH, known s mjor ntioxint, is pl in 5.0 ml of stril RPMI-1640 mium. Th singl prsnt in high onntrtions (up to 10 mm in th livr) ll suspnsion ws prpr y isrupting spln into in most living lls, from miroorgnisms to humns n smll pis. Th r loo lls wr lys using 3.0 ml is known to involv in llulr rsponss to vrious of lysis uffr solution (155 mm NH4Cl, 0.1 mm EDTA strsss [38, 39]. So tht totl glutthion ontnt ws n 10 mm KHCO 3, ph 7.4) for 5 min. Aftr r loo lls hmilly trmin in oth yst utolysts. lyss, th splnoyts wr sprt y ntrifugtion Th ntioxint tivity of yst utolysts ws 1113

5 Worl Appl. Si. J., 15 (8): , 2011 vlut using vrious ntioxint ssys, inluing totl ntioxint pity, ruing powr, DPPH ril svnging, nitri oxi svnging, hyroxyl ril svnging n mtl ion hlting tivitis. Th vrious ntioxint tivitis of yst utolysts wr ompr to stnr ntioxint (sori i). Glutthion Contnt: Glutthion is th trippti of glutmt, ystin n glyin. GSH plys n importnt rol in mintining th intrllulr thiol rox stt n protting lls ginst oxitiv mg, xnoioti orgni hmils n hvy mtls [40, 41]. Glutthion ontnt ws olorimtrilly trmin in oth yst utolysts. Th otin rsults in Tl 1 show tht tiv ry yst utolyst ontins highr onntrtion of glutthion (17.26 mg/100 ml yst utolyst) ompr with kr's yst utolyst (1.77 mg/100 ml yst utolyst). Totl solis of h utolyst wr ADYA 48% n BYA 15.60%. Th rsults r support y Sommr [12] who foun tht frsh krs yst ontins pproximtly 0.65% (ry sis) glutthion. By frmnttions of slt yst strins glutthion ontnts up to 5% n hiv. This is th sis for high glutthion ysts xtrts (with up to 15% glutthion). Sllh t l. [42] trmin th fft of oprting prmtrs suh s th yst onntrtion, prossing tmprtur n isoltion tim on th isoltion of GSH using utolysis mtho. Th rsults otin show tht th mximum vlu of GSH onntrtion (1.38 µmol/ml) ws isolt from th utolysis pross with th following oprting prmtrs; yst onntrtion of 45 wt%, prossing tmprtur of 28 C n th isoltion tim of 3 h. Totl Antioxint Ativity: Th ntioxint pity of yst utolysts otin from kr's yst n tiv ry yst ws msur sptrophotomtrilly through phosphomolynum mtho, whih ws s on th rution of Mo (VI) to Mo (V) y th smpl nlyt n th susqunt formtion of grn phospht/mo (V) ompouns with mximum sorption t 695 nm. Dt in Tl (2) show th totl ntioxint pity of oth yst utolysts ompr to sori i. Th otin t rvl tht th ntioxint tivity of th oth yst utolysts is in th inrsing trn with th inrsing onntrtion of th yst utolyst. Th tiv ry yst utolyst h highr pity thn kr's yst utolyst. Th ntioxint tivitis of oth yst utolysts n stnr wr in th following orr: sori i > ADYA (75 mg/ml) > ADYA (50 mg/ml) > ADYA (25 mg/ml) > BYA (75 mg/ml) > BYA (50 mg/ml) > BYA (25 mg/ml). Tl 1: Totl solis n glutthion ontnt of yst utolysts Glutthion Autolyst Totl solis (%) (mg/100 ml yst utolysts) Bkr's yst ± ±0.13 Ativ ry yst 48 ± ±0.87 L.S.D Vlus r mns of thr rplits ± SE. Numrs in th sm olumn follow y th sm lttr r not signifintly iffrnt t P<0.05. Tl 2: Totl ntioxint pity of kr's yst utolyst (BYA), tiv ry yst utolyst (ADYA) n sori i Trtmnt Con. (mg/ml) Totl ntioxint pity (O.D 695 nm) BYA 25 f ± ± ±0.005 ADYA ± ± ±0.006 Asori i ±0.016 L.S.D Vlus r mns of thr rplits ± SE. Numrs in th sm olumn follow y th sm lttr r not signifintly iffrnt t P<0.05. Tl 3: Totl rution pility of kr's yst utolyst (BYA), tiv ry yst utolyst (ADYA) n sori i Trtmnts Con. (mg/ml) Totl rution pility (O.D 700 nm) BYA 50 g ± f ± ±0.002 ADYA ± ± ±0.006 Asori i ±0.001 L.S.D Vlus r mns of thr rplits ± SE. Numrs in th sm olumn follow y th sm lttr r not signifintly iffrnt t P<0.05. Ruing Powr: Th rutiv pility of oth yst utolysts n sori i is givn in Tl 3. In this ssy, th olor of th tst solution hngs to vrious shs of grn n lu, pning on th ruing powr of h ompoun. Th prsn of rurs 3+ uss th rution of th F /frriyni omplx to th frrous form. Thrfor, y msuring th formtion of Prl's Prussin lu t 700 nm, w n monitor th F onntrtion. Th ruing proprtis r gnrlly ssoit with th prsn of rutons [43], whih hv n shown to xrt ntioxint tion y rking th fr ril hin y onting hyrogn tom [44]. It ws foun tht th ruing powr of yst utolyst ws onntrtion pnnt. Thr ws positivly linr rltionship twn th ruing powr n th onntrtion of yst utolyst. Th otin rsults rvl tht th ruing powr of ADYA ws foun to signifintly mor pronoun thn tht BYA t onntrtion rngs of mg/ml. Th sorn t 700 nm ws foun to for BYA ut up to for ADYA t onntrtion lvl of 250 mg/ml. 1114

6 Worl Appl. Si. J., 15 (8): , 2011 Tl 4: Svnging tivity of kr's yst utolyst (BYA), tiv ry yst utolyst (ADYA) n sori i ginst DPPH ril Trtmnt Con. (mg/ml) Svnging tivity (%) BYA ± ±1.22 ADYA ± ±0.68 Asori i ±1.43 L.S.D Vlus r mns of thr rplits ± SE. Numrs in th sm olumn follow y th sm lttr r not signifintly iffrnt t P<0.05. Tl 5: Nitri oxi svnging tivity of kr's yst utolyst (BYA), tiv ry yst utolyst (ADYA) n sori i Trtmnt Con. (mg/ml) Svnging tivity (%) BYA ± ± ±1.78 ADYA ± ± ±0.15 Asori i ±3.59 L.S.D Vlus r mns of thr rplits ± SE. Numrs in th sm olumn follow y th sm lttr r not signifintly iffrnt t P<0.05. Tl 6: Hyroxyl ril svnging tivity of kr's yst utolyst (BYA), tiv ry yst utolyst (ADYA) n sori i Trtmnt Con. (mg/ml) Svnging tivity (%) BYA ± ± ±2.48 ADYA ± ± ±2.48 Asori i ±2.04 L.S.D Vlus r mns of thr rplits ± SE. Numrs in th sm olumn follow y th sm lttr r not signifintly iffrnt t P<0.05. DPPH Ril Svnging Ativity: Th DPPH ril svnging tivity of th yst utolysts ws vlut using n thnoli solution of th stl fr ril, DPPH. A frshly prpr DPPH solution xhiit p purpl olor with mximum sorption t 517 nm. This purpl olor ispprs whn n ntioxint is prsnt in th mium. Thus, ntioxints moluls n qunh DPPH fr rils n onvrt thm to olorlss prout, rsulting in rs in sorn t 517 nm. Th DPPH ril svnging tivity vlus of ADYA, BYA n sori i r prsnt in Tl 4. In gnrl, th svnging ility of th DPPH fr-ril inrs with th inrs in onntrtion of yst utolyst. Th svnging tivity of h yst utolyst t highr onntrtion (25 mg/ml) n sori i stnr solution with th DPPH ril ws in th following orr: ADYA (98.19%) > BYA (69.06%) > sori i (49.69%). It hs n foun tht ystin, glutthion, sori i, toophrol, flvonois, tnnins n romti mins (p-phnyln imin, p-minophnol, t.), ru n olouris DPPH y thir hyrogn onting ility [31]. Cystin n glutthion ompouns of th yst utolysts r proly involv in thir ntiril tivity. Nitri Oxi Svnging Ativity: Th utolysts of kr's yst n tiv ry yst show mort nitri oxi-svnging tivity twn 25 n 125 mg/ml in os pnnt mnnr (Tl 5). In ition to rtiv oxygn spis, nitri oxi is lso implit in inflmmtion, nr n othr pthologil onitions [45]. Both yst utolysts my hv th proprty to ountrt th fft of NO formtion n in turn my of onsirl intrst in prvnting th ill ffts of xssiv NO gnrtion in th humn oy. Furthr, th svnging tivity my lso hlp to rrst th hin of rtions initit y xss gnrtion of NO tht r trimntl to humn hlth. Dt s shown in Tl 5 rvl tht th ility of ADYA on nitri oxi svnging tivity ws highr thn tht of BYA. Th svnging tivity (%) ws inrs with inrsing onntrtion of th yst utolyst. As shown in Tl (5), lowr onntrtion of ADYA (25 mg/ml) lmost posssss nitri oxi svnging tivity qul out to highr onntrtion of BYA (125 mg/ml). Hyroxyl Ril Svnging Ativity: Using th Fnton rtion systm, th hyroxyl ril svnging tivity of oth yst utolysts ws msur n rsults r init s th svnging tivity (Tl 6). Eh utolyst shows hyroxyl ril svnging tivity t onntrtions twn 12.5 n 25 mg/ml. Th otin rsults rvl tht th ADYA ws mor fftiv thn BYA in hyroxyl ril svnging tivity. Th svnging tivity ws inrs with inrsing th onntrtion of yst utolyst. From th otin rsults, it oul rrng ths trtmnts oring to thir hyroxyl ril svnging tivitis in th following rsing orr: 25 mg/ml of ADYA (91.07%) > 25 mg/ml of BYA (76.52%) > mg/ml of ADYA (74.17%) > of BYA (68.07%) > 12.5 mg/ml of ADYA (59.15%) > 12.5 mg/ml of BYA (50.23%) 1115

7 Worl Appl. Si. J., 15 (8): , 2011 > 0.03 mg/ml of sori i (23.47%). Gnrlly, oth yst utolysts monstrt th ntioxint ffts ginst proxition of iomoluls to svng th hyroxyl rils n suproxi nions t th stg of initition n trmintion of proxyl rils. Mtl Chlting Ativity: Th hlting of frrous ions y oth yst utolysts ws stimt y th mtho of Minotti n Aust [35]. 1,10-phnnthrolin n quntittivly form omplxs with F. In th prsn of othr hlting gnts, th omplx formtion is isrupt with th rsult tht th r olour of th omplxs rss. Msurmnt of th rt of olour rution thrfor llows stimtion of th hlting tivity of th oxisting hltor [46]. In this ssy, oth yst utolysts, intrfr with th formtion of frrous n 1,10-phnnthrolin omplx, suggsting tht it hs hlting tivity n pturs frrous ion for 1,10-phnnthrolin (Tl 7). Th sorn of F 1,10-phnnthrolin omplx is linrly rs with th os tkn os (from 2.5 to 6.25 mg/ml). Mtl hlting pity ws signifint sin th yst utolyst ru th onntrtion of th tlyzing trnsition mtl in lipi proxition [47]. It ws rport tht hlting gnts, who form ó-ons with mtl, r fftiv s sonry ntioxints us thy ru th rox potntil thry stilising th oxiis form of th mtl ion [44]. Th t otin from Tl 7, rvl tht oth yst utolysts otin from kr's yst n tiv ry yst hv n fftiv pity for iron ining, suggsting tht its tion s n ntioxint my rlt to its iron ining pity. In gnrl, th utolysts otin from kr's yst n tiv ry yst show strong ntioxint tivity, ruing powr, DPPH ril svnging, nitri oxi svnging, hyroxyl ril svnging n mtl hlting tivitis whn ompr to stnrs suh sori i. Th ntioxitiv fft of oth yst utolysts is my u to thir ontnts of ntioxint gnts, inluing glutthion, Millr rtion prouts, sulfur-ontining mino is n polyshris [12, 48]. Th highr ntioxitiv tivity of tiv ry yst utolyst in omprison with kr's yst utolyst ws ssoit with its ontnt of totl glutthion (Tl 1). Glutthion hs n rport to hv multipl iologil tivitis, inluing ntioxint tivity. Th ntioxint tivity of glutthion is minly u to thir rox proprtis, whih n ply n importnt rol in th ntioxition of rtiv oxygn spis n fr rils. By ting s n ntioxint or y ining Tl 7: Mtl hlting tivity of kr's yst utolyst (BYA), tiv ry yst utolyst (ADYA) n sori i Trtmnt Con. (mg/ml) % F Chltion BYA ± ± ±3.48 ADYA ± ± ±1.68 Asori i ±2.19 L.S.D Vlus r mns of thr rplits ± SE. Numrs in th sm olumn follow y th sm lttr r not signifintly iffrnt t P<0.05. with llulr mutgns, glutthion n srv s n ntirinogn [12]. Wng t l. [23] osrv tht th yst xtrts supprss thiorituri i-rtiv sustns (TBARS) formtion in ook f pttis uring storg. Thy init tht th ntioxint tivity prsnt in oth yst xtrts hv potntil to improv th oxitiv stility of foos, whih is n nfit to thir min rol s flvor nhnrs. Jung t l. [24] show tht yst hyrolyst intns svnging ilitis of oth 1,1-iphnyl-2-pirylhyrzyl (DPPH) n 2,2'-zino-is(3-thylnzthizolin-6-sulphoni i) (ABTS) rils. Th IC (50) vlus of yst hyrolyst on DPPH n ABTS rils wr 1.9 n 0.9 mg/ml, rsptivly. Immunostimulting Ativity of Yst Autolysts: Th utolysts of kr's yst n tiv ry yst wr xmin for thir immunostimulting tivitis y in vitro mtho. Stimultion of Splnoyt Prolifrtion: In this stuy, lipopolyshri (LPS), mitogn for T-ll inpnnt B-ll prolifrtion, ws us s rfrn for stimultion of splnoyt prolifrtion (in vitro). LPS (0.001 mg/ml) n oth yst utolysts t iffrnt onntrtions (10, 20 n 25 mg/ml) nhn th prolifrtion of splnoyts ompr to th ontrol (Tl 8). Dt rvl tht ADYA n BYA t iffrnt onntrtions h stimulting tivity mor thn tht of rfrn mitogn-inu splnoyt prolifrtion (i.. LPS). Th prolifrtion (%) of kr's yst utolyst n tiv ry yst utolyst t highr onntrtion (25 mg/ml) n LPS s mitogn ws 21.04, n 8.82%, rsptivly. A os rspons rltionship of oth yst utolysts on splnoyt prolifrtion ws osrv. In othr wors, thr is positiv orrltion twn prolifrtion (%) n onntrtion of oth yst utolysts (BYA n ADYA). 1116

8 Worl Appl. Si. J., 15 (8): , 2011 Tl 8: Efft of lipopolyshri (LPS), kr's yst utolyst (BYA), tiv ry yst utolyst (ADYA) on splnoyt prolifrtion Trtmnt Con. (mg/ml) Prolifrtion (%) f LPS ±0.29 BYA ± ± ±0.51 ADYA ± ± ±0.91 L.S.D Vlus r mns of thr rplits ± SE. Numrs in th sm olumn follow y th sm lttr r not signifintly iffrnt t P<0.05. Du to th similr fft on splnoyt prolifrtion stimultion of th mitogn LPS, th oth utolysts might ontin tiv omponnts tht involv qully oth to T- n B-ll prolifrtion stimultion. This xplntion is support y th suggstion of Mnosroi t l. [37]. In gnrl, it ppr tht th utolysts otin from kr's yst n tiv ry yst ontin tiv prinipls, my glutthion n/or polyshri(s), with immunonhning ffts on oth phgoyti n lymphoyti systms [12, 25, 28, 49-51]. In vitro stuis hv suggst tht lrg molulr wight or prtiulr yst -gluns (suh s zymosn) n irtly tivt lukoyts, stimulting thir phgoyti, ytotoxi n ntimiroil tivitis [52, 53]. Howvr th mhnism on how -glun promots lymphoyt prolifrtion is not lr yt. Snul t l. [54] isolt prtiult -D-glun from kr's yst using utolysis n lipiiztion of th lls, follow y lklin n i trtmnt. Thy invstigt th immunomoultory tivity of ths prprtions in mitogni n o-mitogni tsts on rt thymoyts. Thy show highr stimultion inis ompr with th known immunomoultor zymosn. Ylçin t l. [29] show tht itry yst utolyst t lvls of 2, 3 n 4 g/kg h nfiil ffts on humorl immun rspons. CONCLUSION Finlly, it oul onlu tht th utolysts of kr's yst n tiv ry yst hv th potntil to xplor s novl nturl ntioxints n immunostimulting gnts for using in funtionl foos or miin. REFERENCES 1. Finkl, T. n N.J. Holrook, Oxints, oxitiv strss n th iology of ging. Ntur, 408: Risky, D.C., D.D. Lvy, L.E. Littlpg, E.L. Gon, D.G. Alrtson, M.A. Nito, Z. Wr n M.J. Bissl, R1 n rtiv oxygn spis mit MMP-3-inu EMT n gnomi instility. Ntur, 436(7047): Frsqut, F., F. Pourgu, V. Llis, R.P. Brns, J.P. Svinu, R. Mrthn n B. Mullr, Rol of rtiv oxygn spis n gp91phox in nothlil ysfuntion of pulmonry rtris inu y hroni hypoxi. British Journl of Phrmology, 148: Hlliwll, B., Antioxint in humn hlth n iss. Annul Rviw of Nutrition, 16: Hlliwll, B. n J.M.C. Guttrig, Fr r Rils in Biology n Miin. 3 E. Oxfor Univrsity Prss, Oxfor, Unit Kingom. 6. Kur, C. n H.C. Kpoor, Antioxints in fruits n vgtls th millnnium s hlth. Intrntionl J. Foo Sin n Thnol., 36(7): Mili, S. n M. Dmk, In vitro ntioxint tivitis of lo vr lf skin xtrts. J. l Soiété Chimiqu Tunisi, 10: Dvsgym, T.P.A. n K.B. Sinis, Immun systm n ntioxints, spilly thos riv from Inin miinl plnts. Inin J. Exprimntl Biol., 40: D l Funt, M. n M. Vitor, Antioxints s moultors of immun funtion. Immunology n Cll Biol., 78: Dsgupt, N. n B. D, Antioxint tivity of som lfy vgtls of Ini: A omprtiv stuy. Foo Chmistry, 102(2): Brry, D.R., Ysts in Inustry. In Biology of Yst. E. Ewr, A., Lonon, pp: Sommr, R., Yst utolyst. Th 9 th Intrntionl Symposium on Ysts, Syny. 13. Ppplr, H.J., Yst Extrts. In Frmnt Foos. E. Ros, A.H., Ami Prss, Lonon, pp: Slmr-Olsn, E. n T. Sorhug, Comprtiv stuis of th growth of Ltoillus plntrum in why supplmnt with utolyst from rwry yst iomss or ommril yst xtrt. Milhwissnshft, 53(7):

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