J. Sci. & Devel., Vol. 11, No.

3: 364-372

Tp ch Khoa hc v Pht trin 2013, tp 11, s 3: 364-372 www.hua.edu.vn

HOT TNH CHNG OXI HA V C CH ENZYME POLYPHENOLOXIDASE CA MT S LOI THC VT N C VIT NAM
Nguyn Xun Duy1* v H B Vng2
2

Khoa Cng ngh Thc phm, Trng i hc Nha Trang Hc vin lp cao hc CNSTH2012, Trng i hc Nha Trang Email*: duy.ntu.edu@gmail.com

Ngy gi bi: 24.04.2013

TM TT

Ngy chp nhn: 12.06.2013

Su loi th c vt n c Vit Nam c la chn nghin c u hot tnh ch ng oxi ha v c ch enzyme polyphenoloxidase (PPO), gm: L tr xanh (TX), l tru khng (TK), l i (LO), l khoai lang (KL), l lt (LL) v l nhu (LN). Hot tnh chng oxi ha c nh gi da vo kh nng kh gc t do DPPH, nng l c kh v kh nng c ch s oxi ha cht bo trn m hnh d u-n c. Bn cnh , hm l ng polyphenol ca su loi th c vt cng c xc nh. K t qu nghin cu cho thy c su lo i thc vt u th hin kh nng chng oxi ha thng qua cc php th v kh nng ch ng oxi ha ph thuc theo loi. Hm lng polyphenol ca su lo i thc vt khc nhau theo loi v dao ng trong khong 11,73 n 188,19 mg GAE/g cht kh. Hot tnh c ch PPO ca su lo i th c vt ph thuc theo loi, cao nht thuc v LO (64,77%), theo sau bi TK (61,66%), TX (43,87%), LL (25,59%), LN (21,93%) v KL (21,42%). T kha: Cht chng oxi ha, hot tnh chng oxi ha, polyphenoloxidase, hot tnh c ch polyphenoloxidase, th c vt n c.

Antioxidant Activity and Polyphenoloxidase Inhibitory Activity of Edible Plants in Vietnam

ABSTRACT
Leaves of six edible plants in Vietnam, including Camellia sinensis, Piper betle, Psidium guajava, Ipomoea batatas, Piper lolot, and Morinda citrifolia L. were selected to investigate antioxidant and polyphenoloxidase inhibitory activity. Antioxidative activities were evaluated by DPPH free radical scavenging capacity, reducing power capacity and inhibitory of lipid oxidation in oil-in-water model. In addition, polyphenol content in leaves of six edible plants were also determined. Research results indicated that all of plants had antioxidative activity via antioxidant tests and the antioxidant ability depended on species. Polyphenol contents were different from species to species. Their values varied from 11.73 to 188.19 mg GAE/g db. Polyphenoloxidase (PPO) inhibitory activity differed with species. Psidium guajava leaves had the highest activity of PPO inhibitory (64.77%), followed by Piper betle (61.66%), Camellia sinensis (43.87%), Piper lolot (25.59%), Morinda citrifolia (21.93%), and Ipomoea batatas (21.42%). Keywords: Antioxidant, antioxidant activity, edible plant, polyphenoloxidase, polyphenoloxidase inhibitory.

1. T V N
Thc vt l mt ngun cha cc cht chng oxi ha tuyt vi (Huda-Faujan v cng s, 2009). Cc hp cht polyphenol l nhng cht chng oxi ha t nhin, c pht hin ph bin trong cc loi thc vt n c v khng n c. Chng c nhiu chc nng sinh hc bi v

chng c kh nng tr hon hiu qu qu trnh oxi ha v v vy gp phn ci thin cht lng v dinh dng ca thc phm (Marja v cng s, 1999). Nhiu nghin cu c thc hin cho thy trong cc phn ca thc vt cha nhiu cc cht chng oxi ha nh: Phenolics, flavonoids, tanins, vitamins, quinines, coumarins, lignans, ligin (Cai v cng s, 2004;

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Amarowicz v cng s, 2004). V vy, thc vt s l mt ngun nguyn liu tt thu nhn v ng dng cc cht c hot tnh chng oxi ha v hot tnh sinh hc. Vit Nam nm vng kh hu nhit i thuc khu vc ng Nam . H thc vt v cng phong ph v a dng vi xp x 2.500 loi thc vt c nhn din (Chi, 1997). Nhiu loi thc vt trng Vit Nam c s dng trong y hc, dc liu t lu i v nhng c tnh sinh hc a dng ca n (Doan v cng s, 1992; Nguyen v cng s, 2006; Nguyen v cng s, 2010; Hue v cng s, 2008). Thc vt dc liu trng Vit Nam cng nhn c s quan tm c bit ca cc nh nghin cu trong vi thp k qua (Yasuko v cng s, 2011). C th ni Vit Nam c ngun thc vt di do phc v tt cho lnh vc thc phm cng nh dc phm. Mc d vy, cho n nay nhng nghin cu v thc vt trng Vit Nam ch yu l khm ph cc c tnh sinh hc phc v cho mc ch dc liu. Nhng nghin cu ny ch gii hn trong mt s loi thc vt dc liu. Trong khi , nghin cu v hot tnh chng oxi ha v c ch enzyme nu ha ca thc vt n c trng Vit Nam vn cha c nghin cu mt cch y . Thng tin v hot tnh chng oxi ha v c ch enzyme polyphenoloxidase ca mt s loi thc vt n c trng Vit Nam vn cn rt hn ch. V vy, mc tiu ca nghin cu ny l xc nh hot tnh chng oxi ha v c ch enzyme polyphenoloxidase ca mt s loi thc vt n c trng Vit Nam.

Nha Trang trong thng 3/2013, trng thi ti. Ngay sau khi mua, nguyn liu c vn chuyn v phng th nghim khng qu mt gi tin hnh cc x l tip theo. 2.2. Ha cht 1,1-diphenyl-2-picrylhydrazyl (DPPH), axt Gallic, L-3,4-dihydroxyphenylalanine (LDOPA) mua t Sigma Aldrich (USA). K3(Fe[CN]6), AlCl3, axt trichloracetic (TCA), NaH2PO4, Na2HPO4, Na2CO3, thuc th FolinCiocalteu, Tween 40, Ethanol t hng phn tch ca Merck (c), enzyme Tyrosinase ca hng Worthington, Biochemical Corporation (NJ, USA) cha 836 U/mg DW tng ng vi 100.000 n v hot . 2.3. Chun b dch chit Mi i tng thc vt s dng trong nghin cu c khi lng khong 1,5-2kg ti (mu ln). T mu ln, ly ngu nhin ba mu nh cho mi loi nguyn liu, qu trnh chit c thc hin trn mi mu nh, dch chit t mi mu nh c phn tch lp li t nht hai ln (2-3 ln). Kt qu bo co cui cng l gi tr trung bnh t cc mu nh, dng nh gi kt qu cho mu ln. Nguyn liu ti c bm nh bng my ct (Super Blender, MX T2GN, Matsushita Electric Industrial Co., Ltd, Japan) trc khi tin hnh chit. Nc c s dng lm dung mi chit vi t l nguyn liu/dung mi l 1/15 (w/v), nhit v thi gian chit ln lt l 90oC v 30 pht (Dng Th Kim Nguyn v cng s, 2012). Qa trnh chit c thc hin trong b n nhit (Elma, S 300H, Elmasonic, Germany). Dch lc trong thu c sau qu trnh ly tm 4oC, tc 5.000 rpm trong 15 pht (Centrifuge, Labentech, Mega 17R, Germany), c s dng tin hnh cc phn tch tip theo. 2.4. Xc nh hm lng polyphenol Hm lng polyphenol c xc nh theo phng php ca Singleton v cng s (1999). Kt qu c bo co bi mg axt gallic tng ng (GAE)/g cht kh.

2. V T LI U V PHNG PHP
2.1. Vt liu Su loi thc vt c tuyn chn trong nghin cu da vo cc tiu ch: D kim, r, c trng ph bin Vit Nam v c th n c, bao gm: L tr xanh (Camelliasinensis), l lt (Piper lolot), l nhu (Morinda citrifolia L.), l i (Psidium guajava), l khoai lang (Ipomoea batatas) v l tru khng (Piper betle). Tt c cc nguyn liu c mua ti ch a phng TP.

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Hot tnh ch ng oxi ha v c ch enzyme polyphenoloxidase ca mt s loi thc vt n c Vit Nam

2.5. Xc nh hot tnh chng oxi ha 2.5.1. Kh nng kh gc t do DPPH Kh nng kh gc t do DPPH c xc nh theo phng php ca Fu v cng s (2002) vi mt vi hiu chnh nh. Khong 20l n 140l dch chit trn vi nc ct t th tch tng cng 3ml. Sau thm 1ml dung dch DPPH 0,2mM, lc u v yn trong bng ti 30 pht. hp thu quang hc c o bc sng 517nm (Spectrophotometer, Carry 50, Varian, Australia). Kh nng kh gc t do DPPH c xc nh theo cng thc sau: DPPH (%) = 100 (ACT - ASP)/ACT. Trong : ACT: hp thu quang hc ca mu trng khng cha dch chit; ASP: hp thu quang hc ca mu c cha dch chit. Kt qu bo co bi gi tr IC50 l nng ca dch chit kh c 50% gc t do DPPH iu kin xc nh. Gi tr IC 50 cng thp th hot tnh kh gc t do DPPH cng cao. 2.5.2. Nng lc kh Nng lc kh c xc nh theo phng php ca Oyaizu (1986) vi mt vi hiu chnh nh. Nhiu th tch khc nhau ca dch chit c trn vi m phosphate pH = 6,6 t th tch cui cng 1,5ml trc khi thm 0,5 ml K3(Fe[CN]6) 1%. Hn hp c 50oC trong 20 pht, sau thm 0,5ml TCA 10% v 2 ml nc ct, cui cng 0,4ml AlCl3 0,1% c thm vo. hp thu quang hc c xc nh ti bc sng 700nm. hp thu quang hc cng cao th nng lc kh cng mnh. Kt qu c tnh ton bi gi tr IC50, l lng mu lm tng hp thu quang hc ln 0,50. 2.6. Xc nh kh nng chng oxi ha trn m hnh du-nc H nh tng du-nc c chun b gm: 10% du Olive, 85% nc v 0,5% Tween 40. Hn hp c ng ha tc 10.000rpm trong 5 pht (IKA, T18B, Ultra - Turax, Germany). Chnh xc 2ml dch chit c trn u vi 10ml h nh tng du-nc cha trong ng nha 50ml c np y, t trong t n nhit 50oC, qu trnh oxi ha cht bo c quan st

hng ngy. Hm lng hydroperoxide c xc nh theo phng php ca Richards v Hultin (2002). Hm lng hydroperoxide c xc nh trn dch chit cht bo theo phng php ca Bligh and Dyer (1959). Kt qu tnh ton hm lng hydroperoxide t ng chun Cumene hydroperoxide (HPO) nng t 0-120 nmol/ml. 2.7. Xc nh hot tnh c ch enzyme polyphenoloxidase bi dch chit Hot tnh c ch enzyme polyphenoloxidase c thc hin theo phng php ca Fu v cng s (2005) vi mt vi hiu chnh nh. Nhiu th tch dch chit khc nhau c trn vi dung dch m phosphate pH = 6,6 t th tch cui cng 2,8ml. Sau , 0,05ml enzyme PPO (1 mg/ml) c thm vo, gi hn hp 2 pht nhit phng trc khi thm 0,2ml LDOPA (0,4 mg/ml). hp thu quang hc c xc nh sau mi 0,5 pht bc sng 475nm. 2.8. Phng php x l s liu Cc phn tch c tin hnh lp li m bo thc hin phn tch ANOVA. Hm lng polyphenol, gi tr IC50 ca kh nng kh gc t do DPPH, nng lc kh, mc oxi ha cht bo v kh nng c ch enzyme PPO c thc hin t nht hai ln lp li. S liu c phn tch trn phn mm Statistica 8.0 (Stasoft, Tulsa, Ok, USA). Kim nh Tukey c thc hin sau phn tch ANOVA nh gi s khc nhau ca cc gi tr vi mc ngha P < 0,05.

3. K T QU V TH O LU N
3.1. Hm lng polyphenol Hm lng polyphenol ca su loi thc vt c tuyn chn c trnh by trong Hnh 1. Kt qu cho thy hm lng polyphenol ph thuc theo loi (P < 0,05). Trong s su loi thc vt c nghin cu th l Tru khng (TK) c hm lng polyphenol cao nht (188,19mg GAE/g cht kh), theo sau bi l i (LO) v l tr xanh (TX) vi hm lng tng ng l 146,5 v 84,5mg GAE/g cht kh. L nhu (LN), l lt (LL) v l khoai lang (KL) c hm lng

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200
Polyphenol (mg GAE/g khi lng cht kh)

f e

180 160 140 120 100 80 60 40 20 0 TX TK LO KL LL LN d c b a

Loi thc vt Hnh 1. Hm lng polyphenol ca su loi thc vt c tuyn chn (Ch ci trn ct khc nhau ch ra s khc nhau c ngha P < 0,05) polyphenol thp nht, gi tr ca chng tng ng l 11,7; 39,3 v 60,7mg GAE/g cht kh. Truong v cng s (2007) cng b hm lng polyphenol ca l i, l tr xanh, l khoai lang v l lt ln lt l 122,8; 84,8; 68,4 v 19,6mg GAE/g cht kh. Kt qu nghin cu ca Marja v cng s (1999) khi nghin cu hm lng polyphenol ca 92 loi thc vt n c v khng n c bo co rng hm lng polyphenol ca chng cng ph thuc vo loi v gi tr dao ng kh rng trong khong t 0,2 n 155,3mg GAE/g cht kh. Theo nhm tc gi ny nhng loi thc vt c hm lng polyphenol ln hn 20mg GAE/g cht kh th c hot tnh chng oxi ha mnh. Kt qu nghin cu ca chng ti cho thy hu ht cc loi thc vt c tuyn chn trong nghin cu (ngoi tr LN) u c hm lng polyphenol cao hn mc khuyn co ca Marja v cng s (1999) t 1,97 n 9,41 ln. 3.2. Kh nng kh gc t do DPPH Kh nng kh gc t do DPPH l mt trong nhng php phn tch nh gi hot tnh chng oxi ha trong vitro thng s dng nht trong nghin cu, c n 90% cc nghin cu v cht chng oxi ha s dng php phn tch ny (Joon-Kwan v Takayuki, 2009). Kh nng kh gc t do DPPH ca dch chit t su loi thc vt tuyn chn c th hin trong hnh 2. Kt qu cho thy hot tnh chng oxi ha ca l tr xanh (TX), l i (LO) v l tru khng (TK) cao hn ng k so vi l khoai lang (KL), l lt (LL) v l nhu (LN), P < 0,05. Phn tch thng k cho thy hot tnh chng oxi ha ca TX, TK v LO khng khc bit ng k (P > 0,05). Gi tr IC50 ca chng ln lt l 0,49; 0,84 v 1,18g cht kh/ml. KL c hot tnh chng oxi ha cao hn LL v LN vi gi tr IC50 l 10,08 so vi LL v LN l 13,52 v 14,08g cht kh/ml. Mi lin quan gia hot tnh chng oxi ha th hin qua kh nng kh gc t do DPPH vi hm lng polyphenol c trnh by trong hnh 3. Phn tch tng quan hi qui cho thy h s R2 = 0,716 ch ra mi tng quan cht ch gia hm lng polyphenol vi kh nng kh gc t do DPPH. Kt qu ny cng ph hp vi kt qu cng b bi Truong v cng s (2007). Cng t kt qu ny cho thy polyphenol l thnh phn chnh gp phn to nn kh nng chng oxi ha ca cc thc vt c tuyn chn trong nghin cu.

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Hot tnh ch ng oxi ha v c ch enzyme polyphenoloxidase ca mt s loi thc vt n c Vit Nam

16 14 12 b

Gi tr IC50 (g)

10 8 6 4 2 0 TX TK LO KL LL LN c c c

Loi thc vt Hnh 2. Nng lc kh gc t do DPPH ca su loi thc vt c tuyn chn (Ch ci trn ct khc nhau ch ra s khc nhau c ngha P < 0,05)

Hm lng polyphenol (mg GAE/g cht kh)

200 180 160 140 120 100 80 60 40 20 0 0 5

Gi tr IC50 (g/ml)

10

15

Hnh 3. Mi lin quan gia kh nng thu gc t do DPPH vi hm lng polyphenol 3.3. Nng lc kh Nng lc kh cng l mt php phn tch nhm nh gi kh nng chng oxi ha trong vitro. Hnh 4 trnh by kt qu nh gi nng lc kh ca dch chit t su loi thc vt tuyn chn. Kt qu cho thy nng lc kh th hin qua gi tr IC50 ca l tr xanh (TX), l tru khng (TK) v l i (LO) thp hn ng k so vi ba loi l cn li (P < 0,05), iu cng c ngha l dch chit t ba loi l ny c hot tnh chng oxi ha cao hn ng k so vi ba loi l cn li. Gi tr IC50 ca chng ln lt l 1,94; 1,40 v 2,28g cht kh/ml. L lt (LL, IC 50 = 16,45g cht kh/ml) c hot tnh chng oxi ha cao hn l nhu (LN, IC50 = 43,84g cht kh/ml) v l khoai lang (KL, IC50 = 112,99g cht kh/ml) c nng lc kh thp nht.

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120 100

Gi tr IC50 (g)

80 60 b 40 20 0 d TX d TK d LO KL LL LN c

Loi thc vt Hnh 4. Nng lc kh ca su loi thc vt c tuyn chn (Ch ci trn ct khc nhau ch ra s khc nhau c ngha P < 0,05) 3.4. Kh nng hn ch s oxi ha cht bo trn m hnh du-nc Dch chit t l tru khng (TK) c ch ng k s hnh thnh hydroproxide trong m hnh du-nc (P<0,05) so vi dch chit t nm loi l cn li (Hnh 5). Hm lng hydroperoxides (HPO) ca mu TK sau 4 ngy bo qun l 7,86nmol HPO. Trong khi gi tr ny ca nm mu dch chit nm loi l cn li dao ng trong khong 16,18-17,36nmol HPO. Hm lng HPO ca mu i chng (CT) v mu BHA sau 4 ngy bo qun ln lt l 22,25 v 15,25nmol. Kt qu cng cho thy hiu qu c ch s oxi ha cht bo ca TK cao hn ng k (P<0,05) so vi BHA nng 100 g/ml. Hui-Yin v Gow-Chin (2007) bo co rng dch chit trong nc ca l i c th c ch 94,4-96,2% trong m hnh axt linoleic nng 100 g/ml. Lakshmi v cng s (2006) cng bo co rng dch chit ethanol ca l tru khng c kh nng hn ch s oxi ha cht bo tt hn BHA trong m hnh du da v du c. Kt qu nghin cu ca Nabasree v Bratati (2004) ch ra rng dch chit trong nc ca l tru khng c tim nng c ch s oxi ha cht bo tt hn l tr xanh. 3.5. Hot tnh polyphenoloxidase c ch enzyme

Enzyme polyphenoloxidase (PPO) l mt loi enzyme gy hin tng bin en cho mt s loi rau, qu v gip xc (tm, gh), gy nn nhng tn tht cht lng khng mong mun cho nguyn liu v sn phm thc phm. Chnh v vy, nghin cu c ch enzyme ny nhn c s quan tm c bit t cc nh nghin cu. Kt qu nghin cu hot tnh c ch PPO ca dch chit t su loi thc vt u th hin kh nng c ch PPO ng k (P<0,05) so vi mu i chng (CT). Dch chit t LO th hin kh nng c ch hot tnh PPO cao nht 64,77%, theo sau bi dch chit t TK (61,66%), TX (43,87%), LL (25,59%), LN (21,93%) v KL (21,42%) (Hnh 6). Kh nng c ch hot tnh PPO ca cc dch chit c th c l gii l do trong dch chit cha cc polyphenol, c bit l cc flavonoid, nhng cht ny c kh nng to phc hp vi ng trong trung tm hot ng ca PPO. V vy, chng c kh nng c ch PPO (Donghyun v cng s, 2006). Nhng pht hin ny v kh nng c ch enzyme PPO c th l nhng cng b u tin

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Hot tnh ch ng oxi ha v c ch enzyme polyphenoloxidase ca mt s loi thc vt n c Vit Nam

35 30
Hydroperoxide (nmol)

CT BHA TX TK LO KL LL

25 20 15 10 5 0 0 1 2 Thi gian (ngy) 3 4

LN

Hnh 5. S c ch hnh thnh hydroperoxide ca dch chit t su loi thc vt c tuyn chn trong m hnh du-nc 0.7 0.6 0.5
Abs 475 nm

0.4 0.3 0.2 0.1 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

LO TK TX KL LL LN CT

Thi gian (pht)

Hnh 6. Hot tnh c ch enzyme polyphenoloxidase ca su loi thc vt c tuyn chn v kh nng c ch hot tnh PPO t mt s loi thc vt n c Vit Nam. Kt qu ny cng m ra tim nng s dng dch chit t mt s loi thc vt trong vic hn ch s bin en ca mt s loi rau qu, tri cy v trong mt s loi gip xc.

4. K T LU N
Hm lng polyphenol, hot tnh chng oxi ha v hot tnh c ch enzyme polyphenoloxidase ca su loi thc vt n c trng Vit Nam ph thuc theo loi. Hm lng polyphenol dao ng t 11,73-188,19mg

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GAE/g cht kh. Hot tnh chng oxi ha da vo kh nng thu gc t do DPPH (IC50) thay i t 0,49-14,08g cht kh/ml, i vi nng lc kh l 1,94-112,99g cht kh/ml. Tt c su loi thc vt u th hin s c ch qu trnh hnh thnh hydroperoxide trn m hnh du-nc. Hot tnh c ch enzyme polyphenoloxidase xp theo th t gim dn nh sau: LO > TK > TX > LL > LN > KL. Nhng kt qu nghin cu ca chng ti cho thy dch chit t cc loi thc vt n c l ngun cht chng oxi ha v cht c ch polyphenoloxidase t nhin v c tim nng s dng trong lnh vc thc phm. Do , cc nghin cu tip theo nn tp trung vo vic ti u ha qu trnh chit v ng dng dch chit t cc loi thc vt n c.

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