HQI NGHI KHOA HQC CONU NtiMi; ^mr\ n y i ; T ( - j y - « U ' . I V * ; . : k «
HOAT TiNH C H 6 N G OXY HOA C O A HOP C H A T POLYPHENOL TRONG QUA TRiNH NAY M A M C O A M Q T S O LOAI HAT OAU
D& Tin Khang, Ph?m Duy KhSnh
van Nghiin cOu vi Phat triin Cdng nghe sinh hgc. Tnrdng d?i hgc Cin Tha
T 6 M T A T
Cic cay hp d|u tu xua da dugc bilt nhu ngu&i dinh duang quj gia cho sire khoe. Ngiy nay, cac nha kboa h^c da chung minh dugc trong dSa co chiia cic hgp chat c6 the ng3n ngia cac bfnh nguy hilm 6 con ngudi, tiSu^biSu nhSt Ui polyphenol PolyphenoUa hgp ch^ c6 nguon gdc tv nhien, h i ^ di§n 6 rit nhieu loa thuc v^ nhirng chiing co nhifiu nhat d cac cSy h* d|u. Dva tr6n phuong pfa^
xAc dinh nong do polyjAeno cua 6 lo^d d§u trong 5 ngay nay mlini bing duong chuan acid gallic, xac dinh dupc 6 lop d$u dugc khio sit d» him hi9ng polyjAenol cao nhit vio ngiy tiiu 5, din ph$ng Ii loai dJu co h ^ lupng polyphaiol^cao nhat Thyc hien MC fti n g h i ^ khio sit ho^t tinh ch6ng oxy hoa 6 loju dau d^a tren phan fog kh* ion Fe vi phan fog cat goc t? do cua DPPH, kit qui xic djnh dugc diu phgng la lo?i d|u c6 hoat tinh chfing oxy h6a cao nhat trong 6 lo^ d|u dirge khao s i t Thyc hi^n phin tich tiiinh phan polyphenol trong cac loai d?u bing [Auong phip s3c ky 16ng hi$u nimg cao (HPLC) vdi hai chat chuan li acid gallic va add fciTulic, k6t qui dio thiy d|u ph^ng c6 chiia nhiHi lo^i polyphenol nhit, hai peak xuat hi§n 6 tiidi diem phut tiiii 1, 5 vi 2,4 d^i di(n cho hai thanh phin chmb dia polyphenol trong 6 lo^ dfiu.
Tir khoa: DPPH, d|u, HPLC, hop tinh chfing oxy h6a, polyphenol
Mir Dku
Hgp chSt polyphenc^ cd kha nSng chdng sg oxy h6a vi bao v# giup ng^n ngfra nguy ca mic cac b^nh v^ thodi h6a.
Djnh nghTa t ^ g qudt vk hgp chit polyphenol la bit cii hg'p chit nao trong c i u t^o co chiJa m$t vong benzene duvc gin vdi mOt ho$c nhifiu nhdm hydroxyl nhuphenoiic acid (hydroxybenzoic acid, hydroxydnnamic acid), flavonoid (flawnd, flavone, isoflavone. flavanone, anthocyanin. v^ flavanoi), tannin co d$c, coumarin, va alkyl-feson:inol. NhOng thge phim o5 ngu&n g6c tir thgc v$t deu c6 chira phenol, chit nay Snh hu-dng d4n di$n m^o bgn ngodi, mfii ^^, vi sif dn djnh khi H oxy h6a (Naczk vi Shahkli, 2004). Trong cdc h^t ngu c6c, nhirng hgp chat ndy hi$n di$n chu yeu d phan vo h^t. Chiing cd thi dirge thu h^ tif vd hgt bong qua b-inh san xuat dm, ti> d6 cd t h i thSm chiing vio nhO'ng thi,rc phim chOng ta si>
dgng hdng ngdy nhir bdnh ml, tidnh ngpt, tidnh bip... Diiu ddcd thi Idm tdng lu-gng chitxfftiSu hda vanhOngdlctft' hi b$nh cua thgc fAi^tn. Da s i cdc tdi li§u vilt v i polyphenol d thgc vgt diu t§p trung tren trdi cay, lau cdi, nrqnj vari vd hd. Tuy nhidn, hiu hit cdc h ^ chit polyphenol c6 trong trdi cdy vd rau cdi diu du^c ttm thiy d cdc lo^i d§u. Oili mOt vdi polyphenol cd d duy nhit mOt lodi, vi dy nhu- avenanthramide trong yin mach (Katarina, 2003).
Nhdn thiy hgp chit pdyptieno! cd nhieu Igi fch cho con ngirdi vcrimgc dfch nghiin ciru ro hern v i hgp chit polyphend trong cdc lo^i hgt dgu , chung tdi Bin hdnh khdo sat hogt tlnh ching oxy hda cua hgp chit polyphenol h> dd dua n:
duvc nhOng s i ti$u quan bgng v i hdm iirgng polyphenol vd hogt Hnh ching oxy hda cua cdc logi hgt ddu hong qudi blnh ndy mam.
VAT u^u vk PHirarNG PHAP v|it ii$u
Cdc logi ddu: ddu xanh {V7gna radiata), ddu b^ng {Vigna unguiculata), dgu den (Wgna cylindrica), ddu dd {VigJ angularis). ddu ndnh {Glydne max), ddu ph^ng {Arachls hypogaea) dirge mua tir chg Xuan Khdnh, Thdnh p h i Cin Tto.
Chuin bj hgt ndy mim
Ngdm diu vdl- nir*c d 35°C trong 5 gidr. sau dd nit hit nuwc. Rdi deu ddu vdo hOp nh^ra cd Idt s i n vai mim. Bpc h^pM bdng giiy nhom d i dam bdo miu du'gc giO trong toi vd ddt vdo mdy ij d 35°C cho ddu nay mim.
Cdc nghi§m thiJc dugc bo bf nglu nhidn. theo thirl gian nay mim cua hat ddu: 0 , 1 , 2, 3,4, 5 ngdy.
&.U khi ddu ^ "^j;^-^^^^°fl^l^ ^. hdt vd mim ddu. chf chgn nhi>ng hgt nay mim. logi bd cac hgt hdng hojc khdng^nay mim. Bdo qudn ddu d -20=C trong 24 gia rii siy ddng kho. Sau khi siy xong, nghiin mlu thdnh b^t mjn vi
Tdch chiit hg^ chat polyphenol (Lin vd Lai, 2006) ' DCing 5 g bjt da nghiin mjn hda v<« 100 mL dung djch methanol 80%. Sau dd dem di u vd lie d eO^C trong 2 gi* Ssi
khi u xong, hin hgp dirge lgc qua giiy lgc Whatman d l loai bd phin can, thu lly phin djcrtrfch vd bdo qudn d .20°C !
Ham Iirgng polyphenol tong (Taga vi ddng tic gia, 1984) '
Ddnh gid kha ndng ching oxy hda
KhS nang ch6ng oxy hte cOa miu au.or<: ainh g,a «,6ng qua thf nghiim . 4 kha nang khu.. v4 khi nang c4t cSc g & |
3 a
1" - ' O C CONG NGHe SINH HQC TOAN QUOC 2013
do, trong dd cd sir dgng 2,2-diphenyl-1-picrylhydrazyl (DPPH) Ddnh gia khd nang khCr (Lin vd Lai, 2006)
1 mL miu dugc hda tan vdi 2,5 mL phosphate buffer (pH 6,6) vd 2,5 mL K3[Fe(CN)6]1%. H5 hgp phdn irng u d STfC trang 20 phiit Thim vao hfin hgp 2,5 mL TCA 10% vd ly tam 3000 vdng/phOt trong lOphuL Djch nii (2,5 mL) du'gc hoa v6i 2,5 mL nirerc c i t va 0,5 mL dung djch FeCIs 1%. Do d5 h i p thg buifc sdng 700 nm.
Cdng thCrc tfnh: [(ODai^^g -ODmiu)/ODa4,chOn9l x 100%
Miu doi chirng Id mlu thay djch chiit ddu bdng nu'dc cat vd Bin hdnh M nghigm nhu- tren.
Sdnh gid khd ndng cat cdc goc t v do (Siddiqua vd ding tdc gid, 2010)
Hiit vdo mil ing nghidm 3 mL methanol 100% vd 150 IJL thuic thir DPPH 0,1%, d l yen 30 phiit d nhidt dO phong. Sau dd thdm 1 mL djch d i i l t ddu vd lie deu, d l ydn trong 30 phCit cho hin hgp phan irng. Dg hip thg cOa tiSn hgp dirge do vdi bird^ sdng 519 nm. Kha ndng khir gie tg' do eua m l u dirge tfnh dga trdn cdng thirc:
%DPPH biw& = [(ODdichftns- ODmSu)/ODaiichi,nQl X 100%
Phdn tich thanh p h i n polyphenol
Polyphenol todn phan dugc Igc bang mdng Igc n^on (0,45 nm) tnidc khi phdn tfch bing h$ thong sac k;? ldng hidu nang cao Hitachi HPLC apparatus (Hitachi, Tokyo, Japan), hg thing bao gim bam Model L-6200, bO phdn tiem mau Rheodyne Model 7125, bO phdn dgc tfn hi§u Model L-4000 UV-Vis {birdc sdng 270 nm), b5 phdn trfeh xuit dO' li$u Model D-2500 Chromaha-integrator. COt RP C18 Luna (150 mm x 4,6 nm ID x 5 pm dugc sir dgng cho phdn tdch. Qud hinh rira gidi bit d i u vdi methanol ning dd tdng din tip 0-30% trang 50 phiit d i u tiin vdi tic dO ddng 0,7 mL/phiit, sau dd giii' 6n djnh trong 15 phut tiip theo. TO- phiit 65 din phut 75, tdng din ning dO methanol tip 30% len 100%. Hal chit chuin duge sir dyng la acid gallic va aeid fenoilie vdi cdc nong dg 10,50 vd 100 pg/mL.
X i > i $ s i i i # u
Cdc s6 li$u du-gc bieu diinblng s i lidu bung blnh ± dd l#eh chuin, xt> ly thing Kd bang phin mim Excel vd Statgraphics XV, Diing phdp thir LSD d i xdc djnh khdc bigt cd y nghTa eho cde s i lidu (p<0,05).
K ^ QUA VA T H A O LUAN Hdm lipgng polyphenol tong
Phirong phdp Folin-Ciocalteu Id mdt trortg nhu'ng phuvng phdp dirge sir dgng d i ^nh lu'gng polyphenols tdng so.
Polyphenol phdn Ceng vdi thuie ttiu Folin-Ciocalteu (dung djch mau vdng cua phosphotungstenate va molybdate) trong mii tru'd'ng kilm y i u tgo thdnh mdu xanh duong. hap thu d- budc sdng 750 nm. Hdm lugng polyphenoltfing dugc tinh dga vdo dud'ng chuin acid gallic. K i t qua ^nh lirgng eua polyphenol ting eOa edc logi ddu qua cdc ngdy ndy mim dugc trlnh bdy trong Bdng 1.
Bing 1. Him lirgng polyphenol ting (pg/mL) die cdc lo;il d$u sau 5 ng^y nay mim
0 1 2 3 4 5 Trunn blnh
E>3uxant)
3.03 5,79 6,11 6.19 6.2B 7,50 6 8 2 *
Q ^ d e n
5 93 6,62 6,84 7 06 7,07 8 2 3 6 , 9 6 ' '
D ^ p T i O n g
9.07 13J>S 14.1B 14,51 14,97 18 5 0 1 4 . 0 9 ^
O ^ u d o
6.11 6.85 7.80 79A 8,28 8,47 7.S7«
£ > | u n i n n
7.01 10,65 10 73 1 1 8 5 1 2 2 5 13 97 1 1 , 0 f l ' '
Diu Uang
4.00 5 72 J 7,05 7 3 7 8.14 9.68 8,99'^
Tning blnh
5 , 8 6 ' 8 . 1 6 * 8 . 7 9 ' ' 9,1S«
9,S0'*
1 1 , 0 6 ^
Ghi chu- Gld trj trung binh theo sau bd cic chd cdi khSc nhau Sii khdc bi$t c6 y nghTa th6ng ki & mdc 5%.
Xdt v i thdi gian ndy mim, hdm lugng polyphenol trcing cdc logi ddu diu tang len theo thdi gian ndy mim. Cg t h i , ham lugng polyphenol tdng din tir ngdy 0 (5,86 pg/mL ) din ngdy 5, ngdy 5 Id ngdy cd hdm lugng polyphenol eao nhlt trang cde ngdy khdo sdt (11,06 pg/mL). TCr ngdy 0 din ngdy thir 4, hdm lugng polyphenol faxxig hgt tdng din nhung khong diu. D i n ngdy thir 5, hdm lugng polyphenol cd su tdng mgnh tir 9,50 len 11,06 pg/mL (tuo'ng duo'ng aeid gallic), khac bi§t cd ^ nghTa thing kd d mil-c 5%. Diiu ndy c6 t h i do sg tdng cudng h-ao doi chit mgnh dgt ngOt trong hat din din hdm lugng polyphenol tdng nhiiu.
Khi so sdnh hdm lugng polyphenol glira ede logi dgu, so ligu eho thiy ddu phdng cd chifa ham lugng polyphenol cao nhlt trang 6 logi ddu dirge khdo sdt (14,09 pg/mL), ke din Id dau ndnh, ddu do, ddu trang, ddu den vd ddu xanh, hlu hit edc trung blnh diu khdc bl§t cd y nghTa thing ke muc y nghTa 5%, frir ddu den vd ddu trang. Cac nghien ciru hirdc ghi nhdn, trong qud trinh nay mim ciia dgu ph$ng, resveratral dugc tdng hgp vd'i hdm lugng Idn (Win vd ding tdc gid, 2011). Resveratrol chinh la m^t loai polyphenol hign didn rat nhieu trong ddu ph$ng, nhlt la khi dgu phOng ndy mim (Win vd ding tdc gld, 2011). Bin cgnh do, flavonoid vd epicatechln cung Id hai logi polyphenol cd rit nhieu trang dgu phdng (Francisco vd Resurreccion. 2009). Cdn d dgu ndnh, polyphenol chO yiu chlnh Id flavonoid trong khi d ddu xanh, ddu dd, ddu den vd dgu trang, thdnh phdn chu y i u gim phenolic acid (Sosulski vd dong tdc gia, 1982). Mgt s i nghidn
«.i •,4„ i,«.nn oohmhenol diKic Iv mdi phu thupc vSo dung m6i lHf(rc sCr dgng va thanh phjn polvphenol~^
'=*' ' = ^ " ; S „ ^ J J ^ » ^ ? S ^ d u ^ I i methanol phu hqrp d * ly trich cic miu cb chfta resveratrol va slilhe»
S f ' i ^ ^ i r ^ S * . S T l S S i U t dalhim i w i g cao. dung m61 thirftng dirgrc sir dgng \i ethanol (SosuteW. 1982^
S f ' n i S ^ ^ E j S ^ S ^ l r , S , thu a Z o ham liw-g polyphenoltoSn phSn c i a dau ph«ng cao hem cac lo*
ddu khdc Id mdt kit qud phu hgp.
Khd ndng ching oxy h6a
Bing 2. Khd nteg ching oxy hoa cua cac lo?i d^u Loai dau
eiu«
e^uden E ) $ u t r ^ D^u ndnh 0$uph$ng
KhS nana khu he*-!'*) 64,92 ± 0,57^
61,53*0,68'^
26.45 ± 0.95*
42.51 ± 1.24®
75,08*1.18'' 84.48 ±1,24^
nPPHbikhi>(%l 20.80 ± 0,39*^
17,46 ± 0,60^
7,44 ± 0,39*
11,17±0,63^
26,94 ±0,71'' 32,51 ± 0,54^
Ghi did: Giitii tmng blnh theo sau bd cd: chir cai khic nhau trong ciing md c& thi khic d& c6 j? nghia thing ke d mCec 5%.
Kit qud bdng 2 cho thiy ddu ph^ng cd khd ndng khu ion Fe^* cao nhlt, lin din 84,5%. Tiip theo Id ddu ndnh vdi 75,1%, vd cuii eiing Id ddu den (26.4%), khde bi#t cd y nghTa thing k i vdi mi>c •j nghia 5%.
Polyphenol cua ddu phOng cd hogt tfnh ching oxy hda cao nhlt frong cde logi d|u dugc khdo sdt (32,5% gic ^r do bi ell), dimg thir hai Id ddu ndnh vdi 26,9% gic hr do bi cat. khdc biit c6 •} nghTa thing k i vcri mirc J nghTa 5%. Ddu den ed hogt tfnh ching oxy hda thip nhat (7,4% gic oxy hda bj cit), Dilu ndy hodn todn phi^ hgp vdi kit qua ire khd ndng khu ton Fe**. Khd ndng ching oxy hda c6 lidn quan chgt che din hdm lugng polyirfienol ting (Lee va dong tic gii.
2004). Nhu kit qud thu dugc * thi nghigm xac dinh ning d i polyphenols, ddu (rtiOng Id ddu chira hdm lugng polyphenols cao nhlt nfin trong phdn i>ng ddnh gid kha ndng diing oxy hda, ddu phdng cd hogt tinh cao nhlt Id dilu phll hgp vdi kit qud ttif nghiim ddnh gid khd ndng khCr Fe3+ vd thi nghiim d t gic tv do DPPH. Ngodi ra, ttieo Corral- Aguayo vd ding tdc gid (2008), niu so sdnh v i khd nang ching oxy hda, resveratrol ed hogt tlnh manh hom so vdi flavonoid, vd hogt tfnh thip nhit Id phenolic acid. Ben canh dd, thdnh phin polyphenol chii ylu trong dgu ndnh Id flavonoid cdn d cic logi ddu cdn lgi (ddu xanh, den, do. hang) thdnh phin chu y i u Id phenolic acid (Sosulski va dong tic gii, 1982). Diiu ndy cd thi gidi thfch dugc vi sao dgu nanh cd hogt tlnh ching oxy hda mgnh han 4 logi ddu cdn Igi.
Thdnh phin polyphenol
Sau khi phdn tfch thdnh phin polyphenol trong cdc logi ddu bang phuong phdp HPLC, nhin chung dgu phgng xuit hiOn nhiiu dinh nhit (Bdng 3), Diiu dd chirng td rang hong ddu phdng chi>a nhilu logi polyphenol han ede logi ddu khdc, vd k l cd mdt s i thdnh phin polyphenol khdng <^ d 5 logi ddu eon lai Cg thi. cdc dinh 3, 4, 6, 7 vd 8 cd xuit hign d sac k^
d i ciia ddu phdng nhirng Igi khdng xuit hi^n d cdc logi dgu cdn lgi Mgt khdc, dinh 1 vd dinh 2 xuit hidn d t i t ca cac sic ky d i ciia 6 logi ddu (Hlnh 1). Didn tfc*i hai iSnh ndy eung Idn hen han diin tich cdc peak cdn Igi. Do dd cd the ddy Id hai logi polyphenol ca bin. Id thdnh phin chCi yiu cua polyphenol trong 6 logi ddu ndy. Bin cgnh hai dinh ndy, edn cd hai dinh xudt hidn trong hlu hit cde sic ky d i Id dinh 5 vd dtnh 9 (xuit hidn d 4/6 logi ddu).
» w T 1 A 1
]
,
\ , I
, . .--.-=
Hlnh 1 . S i c k;; d i phSn tich HPLC d$u ph$ng (A) va d^u xanh (B) B i n g 3. B i n g so s i n h t h i n h p h i n polyphenol trong cac loai dau
Dlnh1 Dinh 2 Ellnh3 Olnh 4 Olnh 5 Olnh 6 Dfnh 7 CHnhS Mnh a
Diu xanh Diu Hen oaufld Oau prifinq Dju nanh D i u tring
Ghi chii; + • Ohh xuit hiin trin sic ky d6. -: Dinh khdng xuit hi$n trdn sic ky d6
K ^ L U A N
C d e loai d d u d u g c khdo sdt d i u ed h d m l u g n g polyphenol t a n g d d n theo t h g i gian n d y m i m , d a t c a o n h l t vdo n g a y thi>
5 trong k h o a n g t h a i g i a n khdo sdt. Logi d d u c d c h u a h d m l u g n g polyphenol cao n h l t b'ong c d c logi d d u d i r g e khao sdt Id d d u p h $ n g d g t 14,1 p g / m L . Polyphenol trong d d u p h g n g c u n g Id polyphenol e d hogt tfnh c h i n g oxy hda cao n h l t T i l p d i n Id d g u n d n h , d d u h a n g , ddu d d , d g u d e n , d g u x a n h . D d u phgng c 6 chi>a nhieu t h d n h p h i n polyphenol n h l t trong 6 logi d d u , x u i t h i i n 7 d i n h h o n g t i n g s i 9 dinh da d u g c khdo sdt.
Chung lot xm chdn thdnh cdm an Chuong trinh hoc bong Australia cho su phdi triin tai VietNam da ho trv todn bp lanh phi da da an, trdn trpng cdm an Vi4n nghien cm vd phdt trien Cong nghe sink hpc dd giup do ve ddet bj, cam tm PGS. IS. Todor Vasiljevic da dng hp vi0c thuc kien nghien
TAI LIEU THAM KHAO
Conal-Aguayo RD, Yahia AM, Can-illo-Lopez A, Gonzalez-Aguilar G (2008) Correlation between some nuhiUonal components and the total antioxidant capacity measured witii six different assays in eight horticultural crops. J Agn f^ood Chem 56.10498-10504.
Katarina B, Sunnerheim K, Bryngelsson S, Fagertund A, Engman L, Andeisson RE, D'imberg LH (2003) Avenanthramides in oats (Avena sativa L,) and structure-antioxidant activity relationships. J Agric Food Chem. 51 (3), 594-600,
Lln PY, Lai HM (2006) Bioaclive compounds in legumes and their germinated products JAgri Food Chem. 24.3807-3814 Naczk M, Shahldi F (2004) Extraction and analysis of phenolics in food. J Chromatogr, A1054-95.
Sosulski F, Krygier K, Hogge L (1982) Free, esteiified, and insoluble txHind phenolic acids. Composition of phenolic acids in cereal and potato flours. J Agric Food Chem 30:337-340.
Taga MS. Miller EE, Pratt DE (1984) Chia seeds as a source of natural antioxidants. J / ( m Oil Chem Soc 61:928-931,
Win MM, Midul-Hamid A, Bahann BS, Anwar F, Sabu MC, Pak-Dek MS (2011) Phenolic compounds and antioxidant activity of peanut's skin, hull, raw kemel and roasted kemel flour. Pak J Bd. 43(3): 1635-1642.
STUDYING THE ANTIOXIDANT CAPACITY OF POLYPHENOLS DURING GERMINATION OF SOME LEGUME GRAINS
Do Tan Khang, Pham Duy Khanh
Biotechnology research and development institute. Can Tho university
SUMMARY
Legumes have traditionally been known as a source of valuable nutrients for health. Today, scientists have proven that legumes contam many compounds that can help prevent serious diseases in human, the most typical compound is polyphenols. Polyphenols are compounds of natural origin, present in many plant species but diey are most abundant m legumes. Based on the method of determining the concentration of polyphenols of 6 types of bean in 5 day-germinating by gallic acid calibration curve, the results showed that 6 legume species contained the highest concentration of polyphenols in the 5"* day. The peanut was the species containing highest concentration of polyphenols among 6 species. Doing experiments to survey the antioxidant ct^acity of six species of legumes based on Fe^ reduction reaction and the DPPH radical scavenguig reaction, the results showed diat peanut had the strongest antioxidant capacity among 6 species. There were totally nine peaks after HPLC analysis. Peanut contained the most components of polyphenols compared to other samples. The two peaks presented in all sanples.
Keywords: antioxidative activity, bean, DPPH, HPLC, polyphenol
• AuthOT for corresspondence: Tel: +84-7103.730271 ; Email- [email protected] vn
