1.1.

8 Cc phng php xc nh hot tnh khng oxy ha ca dch chit t thc vt:
Phng php TEAC (Trolox equivalent antioxidant capacity): Xc nh hot tnh chng oxi ha so vi kh nng chng oxi ha ca Trolox. Phng php DPPH (Scavenging ability towards DPPH radicals): Kh nng kh gc t do DPPH. Phng php ORAC (oxygen radical absorbance capacity): xc nh kh nng hp th gc t do cha oxy hot ng. Phng php TRAP (total radical-trapping antioxidant potential): Kh nng chng oxi ha bng cch by cc gc t do. Phng php FRAP (ferric reducing-antioxidant power): lc chng oxi ha bng phng php kh st. Phng php reducing power: lc chng oxy ha bng phng php kh Fe Phng php FTC: (ferric thiocyanat ): Phng php Conjugated Diene: kho st ni i lin hp. Phng php TEAC: Cation ABTS+[2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate)(ABTS)] l mt gc t do bn. y l mt cht pht quang mu xanh, c c trng hp thu 734 nm. Khi cho cht chng oxi ha vo dung dch cha ABTS+, cc cht chng oxi ha s kh ion ny thnh ABTS. o gim hp thu ca dung dch bc sng 734nm xc nh hot tnh ca cht chng oxi ha trong s so snh vi cht chun Trolox[6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid]. Trong mi trng kali persulfate, gc ABTS+ c th bn 2 ngy nhit phng trong ti. Phng php DPPH: 1,1-Diphenyl-2-picrylhydrazyl (DPPH) l mt gc t do bn, c mu ta v c hp thu cc i bc sng 517 nm. Khi c mt cht chng oxi ha, n s b kh thnh 2,2Diphenyl-1-picrylhydrazine(DPPH-H), c mu vng. o gim hp thu bc sng 517nm xc nh kh nng kh gc DPPH ca cht chng oxi ha.

Hnh 7: Phn ng gia DPPH v mt cht chng oxi ha

Hot tnh sng lc gc t do c tnh theo phng trnh sau: Hot tnh kh gc t do DPPH(%) = Asample ( 30 pht ) Ablank ( 30 pht ) Asample ( 30 pht ) Ablank ( 30 pht ) Acontrol ( 0 pht ) x100 1 x100 Acontrol (30 pht ) Acontrol (30 pht ) Acontrol(0 pht) = hp thu ca mu i chng ngay sau khi pha ch. Acontrol(30 pht) = hp thu ca mu i chng sau 30 pht . Asample(30 pht) = hp thu ca mu th nghim sau 30 pht . Ablank(30 pht) = hp thu ca mu trng sau 30 pht . Lng mu cn thit phn ng vi mt na lng DPPH (hay hp thu 50%) c gi l lng tng i Trolox phn ng. Khi , hot tnh chng oxi ha ca mu c th din t bng thut ng l s micromole bng Trolox/100g mu hay n v Trolox/100g hay TE/100g. (TE: trolox equivalent) hay IC50. Phng php ORAC: Phng php ny o mc phn hy do b oxi ha ca fluorescein khi c s hin din ca gc peroxy. Phn ng trong iu kin ny c so snh vi phn ng trong s hin din ca cht chun Trolox (hay vitamin E) v trong hin din ca mu cha cht chng oxi ha cn xc nh hot tnh. Khi fluorescein b oxi ha, cng pht hunh quang s gim i. Tin hnh o gim cng pht quang ny lin tc trong 35 pht sau khi thm cht oxi ha vo. Khi c mt cht chng oxi ha, s phn r fluorescein s chm hn. Xy dng ng cong biu din s ph thuc gim pht hunh quang theo thi gian v vng di ng cong dng tnh ton. Kt qu tnh ton l mmol Trolox/g mu. u im ca phng php ORAC l xc nh c c hoc khng c s tr pha trong mu cha cc cht chng oxi ha. y l mt iu rt thun li khi o cc mu thc phm cha c nhng hp cht chng oxi ha c tc phn ng khc nhau nhiu.

Hnh 8 : th m t gim pht hunh quang theo thi gian Phng php TRAP:

Phng php TRAP s dng gc peroxyl c to thnh t 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH). Khi cho AAPH vo mi trng plasma, cc cht kh s b oxi ha. Qu trnh oxi ha ny c o t thng qua hm lng oxi tiu th bng mt in cc. Khi c mt cht chng oxi ha trong mi trng plasma, qu trnh oxi ha s xy ra chm hn. Gi tr TRAP ca mu th nghim c tnh ton da vo di pha lag ca mu so vi di pha lag ca mu trng v di pha lag ca cht chun l dung dch Trolox. Kt qu tnh ton l mmol Trolox/kg mu rn hoc mmol Trolox/L mu lng. Phng php (FRAP): Nguyn tc xc nh hot tnh chng oxi ha ca phng php ny l da trn kh nng ca cc cht chng oxi ho trong vic kh phc Fe 3+-TPTZ [2,4,6-tripyridyl-s-triazine (TPTZ)] (mu ta) thnh phc Fe2+-TPTZ (mu xanh) pH thp. Khi , tng cng mu xanh t l vi hm lng cht chng oxi ha c trong nguyn liu. Mc tng cng mu ny c o bc sng 593nm trong s so snh vi cht chun l dung dch FeSO4 hay BHT (Butylated Hydroxy Toluene). Khi cho phc Fe3+-TPTZ vo mi trng cha cht chng oxi ha, cc cht chng oxi ha s nhng in t cho phc ny v sinh ra Fe 2+-TPTZ. Kt qu tnh ton l mmol Fe2+/ g cht kh. Do , khi kt qu tnh ton ra ln th chng ta c th suy on rng trong mi trng phn ng , s lng cc phn t c th nhng in t l cao. Tuy nhin, iu ny khng hon ton ng v mt phn t cht chng oxi ha c th kh nhiu phc Fe 3+-TPTZ cng lc. y l mt hn ch ca phng php FRAP. Lipid assay: Phng php ny nh gi s khc bit trong tc oxi ha acid linoleic bi gc ABAP trong mi quan h vi cht chun l tocopherol. Trc v trong sut qu trnh xy ra phn ng, nhit ca hn hp{[70 L of linoleic acid (2,3 mmol/L)]; 100 mL m phosphate 0,05M (Natri phosphate ha tan trong nc; 2.88 g SDS; pH 7.4)} c iu chnh v duy tr 40oC. Ly 2l ca dung dch ny cho vo 0,01 ml ABAP(0,04M). Sau 2-5 pht phn ng, ly 0,02 ml dch trch ly cho vo hn hp ny. o hp thu ca hn hp 236nm. Xy dng ng chun l tocopherol. Phng php FTC: Mc ch ca phng php ny l kho st kh nng lm gim s peroxide ha lipid ca cht khng oxy ha. Thng thng, cc gc oxi t do s peroxide ha lipid, to thnh peroxide. Peroxide s oxi ha Fe2+ thnh Fe3+, phc ca Fe3+ vi SCN- c gi tr hp thu ti a ti 500nm. Gi tr hp thu cao ng ngha vi s peroxide ha lipid cao trong sut qu trnh dung dch. Mu dung dch c cha cht khng oxy ha s cho gi tr hp thu thp hn mu trng khng c cht khng oxy ha. Reducing power: Nguyn tc xc nh hot tnh chng oxi ha ca phng php ny l da trn kh nng ca cc cht chng oxi ho trong vic kh phc K3Fe(CN)6 thnh phc K4Fe(CN)6, phc ny tc dng vi FeCl3 thnh KFe[Fe(CN)6] (xanh Pruss). Khi , tng cng mu xanh t

l vi hm lng cht chng oxi ha c trong nguyn liu. Mc tng cng mu ny c o bc sng 690nm trong s so snh vi cht chun l dung dch acid ascorbic. Conjugated Diene: Mc ch ca phng php ny l kho st kh nng lm gim s peroxide ha lipid ca cht khng oxy ha. Bng 2: tm tt cc c im cc phng php o hot tnh khng oxy ha STT 1 2 3 4 5 6 7 Phng php c dim phng php ORAC TRAP FRAP TEAC DPPH FTC Reducing power ++ --+++ + + +++ +++ Trang thit b + -+++ + + +++ +++ + Tnh kh thi trong cc h thng sinh hc +++ +++ -

+, + +, + + + mc n gin tng dn -, - -, - - mc phc tp tng dn