Use of Near-Infrared Spectroscopy for Screening Total and Individual Glucosinolates in Cabbage Leaves.

Rafael Font1*, Mercedes del Ro-Celestino1, Eduardo Rosa2 and Antonio de Haro-Bailn1 1 Depart ent of A!rono " and #lant Breedin!$ %nstitute of &ustaina'le A!riculture (C&%C)$ Ala eda del *'ispo s+n$ 1,-.- Crdo'a$ &pain$ 2 /ni0ersidade de 1ras-os-Montes e Alto Douro, 2--1-3-3 4ila Real$ #ortu!al$ *e- ail5 font6cica$es 1l$5 78, 329 ,33211: fa;5 78, 329 ,33222 Key words Ca''a!e leaf, Cancer c<e o-pre0ention, =lucosinolates, Hi!< perfor ance li>uid c<ro ato!rap<", Hu an nutrition, ?ear-infrared spectroscop" (?%R&) bstract 1<e potential of near infrared spectroscop" (?%R&) for screenin! t<e total !lucosinolates (t-=&@), !luconapin (=?A), !luconasturtiin (=?A&1) and neo!luco'rassicin (?=B&) contents of ca''a!e (Brassica oleracea @$) leaf, Aas assessed$ @ea0es of t<is species Aere anal"sed '" ?%R&, and t<eir reference 0alues of !lucosinolates o'tained '" <i!< perfor ance li>uid c<ro ato!rap<", Aere re!ressed a!ainst different spectral transfor ations '" odified partial least s>uares (M#@&) re!ression$ 1<e coefficients of deter ination in t<e cross-0alidation (r2) for t-=&@, =?A, =?A&1 and ?=B& Aere, respecti0el", -$.8, -$9-, -$B2 and -$B-$ 1<e standard de0iation to standard error of cross-0alidation (&EC4) ratios Aere, for t<ese constituents, as folloA5 t-=&@5 -$,2: =?A5 -$2,: =?A&15 -$B1: ?=B&5 -$B8$ An e;a ination of t<e M#@& loadin!s used in odelin! t<e t-=&@ e>uation, su!!ests t<at *-H !roups, C-H co 'inations of t<e et<"lene olecule and also, ?-H !roups of a ides, Aere t<ose olecular associations ore used in de0elopin! t<e first t<ree ter s of t<is e>uation$ 1<e results reported in t<is AorC <a0e de onstrated t<e successful application of t<e ?%R& tec<ni>ue to t<e screenin! of !lucosinolates in plant leaf, s<oAin! t<e loAer cost and considera'le reduction of t<e ti e re>uired, in contrast to t<ose of t<e standard tec<ni>ues of anal"sis$ Introduction *0er t<e past t<ree decades, Brassica production <as increased to 'eco e one of t<e ost i portant sources of oil and protein of plant ori!in for <u an and ani al nutrition, respecti0el"$ %n addition, so e species of t<e !enus are <i!<l" consu ed as !reen leaf" 0e!eta'les all o0er t<e Aorld$ %n t<e %'erian #eninsula, t<e <i!< consu ption of Brassica crops is reflected '" a lar!e use of floAer 'uds and lea0es of se0eral of t<ese species, includin! ca''a!e (Brassica oleracea @$)$ HoAe0er, t<e infor ation a0aila'le on t<e !lucosinolate (D-t<io!lucoside-N-<"dro;"sulp<ates) (Fi!ure 1) co position of t<is species is scarce (Rosa et al$, 1339)$ !igure ". Basic structure of !lucosinolates

=lucosinolate-containin! plants in t<e Brassicaceae fa il" represent a potential source of control for different soil-'orne pests (Fa<e" et al$, 2--1), and as cancer-c<e opre0ention a!ents (&<apiro et al$, 2--1)$ HoAe0er, t<e to;ic and anti-nutriti0e effects of !lucosinolates <a0e li ited t<e use of Brassica species for <u an and ani al feed (&orensen, 133-)$ 1<e deter ination of t<e !lucosinolate content '" <i!< perfor ance li>uid c<ro ato!rap<" (H#@C) is e;pensi0e and ti e-consu in!, and specialised personal is 1

needed$ %n contrast, t<e use of fast anal"tical tec<ni>ues suc< as near-infrared spectroscop" (?%R&) results in an" ad0anta!es, since anal"sis can 'e carried out Ait< a considera'le sa0in! of ti e, at a loA cost and Ait<out usin! c<e icals$ 1<e purpose of t<is AorC Aas to test t<e potential of ?%R& for screenin! t<e total !lucosinolate content (t-=&@), as Aell as so e aEor !lucosinolates e;<i'itin! different olecular for s of t<ose reported in ca''a!e leaf, i$e$, !luconapin (=?A, alip<atic), !luconasturtiin (=?A&1, aro atic) and neo!luco'rassicin (?=B&, indole)$ #aterials and $et%ods Plant material and HPLC analysis 1<e plant aterial used in t<is AorC is Aidespread in #ortu!al and ?ort< of &pain$ Fres<l" <ar0ested lea0es of ca''a!e Aere collected at different aturit" sta!es to increase c<e ical and p<"sical 0aria'ilit" in t<e sa ples$ 1<en sa ples Aere taCen to t<e la'orator" for i ediate anal"sis$ Desulp<o!lucosinolates Aere deter ined '" H#@C at t<e Field Crops Depart ent at t<e /ni0ersidade de 1rFs-os-Montes e Alto Douro, 4ila Real, #ortu!al, folloAin! t<e et<od proposed '" &pinCs et al$ (13.,)$ NIRS procedure: recording of spectra and processing of data FreeGe-dried and !round sa ples of lea0es of ca''a!e Aere anal"sed in an ?%R spectrop<oto eter (?%R&"ste s od$ B2--, Foss-?%R&"ste s, %nc$, &il0er &prin!, MD, /&A) in t<e reflectance ode$ &a ple spectra Aere re!istered as an indi0idual file, in t<e ran!e fro ,-- to 22-- n , at 2 n inter0als$ %n t<e second step, t<e indi0idual and total !lucosinolate contents, as t<e" Aere deter ined '" H#@C, Aere added to t<e file of spectra, t<us eac< spectru <a0in! an associated 0alue for eac< !lucosinolate$ Developing calibration e uations /sin! t<e application =@*BA@ 0$ 1$2- (H%?%&% %%, %nfrasoft %nternational, @@C, #ort Matilda, #A, /&A), different cali'ration e>uations for t-=&@, =?A, =?A&1, and ?=B& Aere de0eloped on t<e A<ole set$ Cali'ration e>uations Aere co puted usin! t<e raA optical data (lo! 1+R, A<ere R is reflectance), or first or second deri0ati0es of t<e lo! 1+R data$ 1o correlate t<e spectral infor ation and t<e content of t<e different !lucosinolates, odified partial least s>uares (M#@&) Aas used as a re!ression et<od$ %n addition, standard nor al 0ariate plus de-trendin! transfor ations (&?4-D1) (Barnes et al$, 13.3) Aere used to correct 'aseline offset$ !alidation of t"e e uations 1<e perfor ances of t<e different cali'ration e>uations o'tained Aere deter ined fro cross-0alidation$ 1<e et<od is carried out '" splittin! t<e cali'ration set into # se! ents and t<en cali'ratin! # ti es, eac< ti e testin! a'out a (1+#) part of t<e cali'ration set (Martens I ?aes, 13.3)$ The prediction ability of the equations obtained was determined on the basis of their coefficient of determination in the cross-validation (r 2) (Shenk and Westerhaus, 1996) and standard deviation (sd) to standard error of cross-validation (SECV) ratio (Williams & Sobering, 1996). &esults and discussion $lucosinolates in t"e samples =lucosinolate co position of t<e sa ples used in t<is AorC are su arised in 1a'le 1$ 1<e t-=&@ and indi0idual !lucosinolate contents found in t<e sa ples used in t<is AorC co0ered a Aide ran!e of 0alues, si ilar to t<ose contents c<aracteristic of t<is species, as it <as 'een pre0iousl" reported (Rosa, 1333)$

% uation performances 1<e 0alidit" of cross-0alidation to e0aluate t<e perfor ance of an ?%R e>uation <as 'een supported '" different researc<ers (&<enC I Hester<aus, 133B: Hillia s I &o'erin!, 133B), <a0in! 'een applied successfull" '" t<e aut<ors of t<is AorC to t<e anal"sis of !lucosinolates in a pre0ious report (Font et al$, 2--,)$ %n t<e present AorC on t<e 'asis t<e r 2 0alues, t<e four e>uations for !lucosinolate co position s<oAed fro !ood >uantitati0e infor ation (t-=&@ and =?A) to correct separation of t<e sa ples into loA, ediu and <i!< !roups (=?A&1 and ?=B&) (1a'le 1) (&<enC I Hester<auss, 133B)$ Table ". Cali'ration and cross-0alidation statistics for t<e different e>uations de0eloped (nJ 1--) (K ol 1-- !-1 dA)$ calibration cross-validation glucosinolate range $ean sd S'C &( sd S'C)-" r( nt t-=&@(2) ,.$9.-8,32$B2 9,1$1B B21$33 13B$28 -$32$8. -$.8 . =?A(1) --1821$B9 2-2$.8 2B.$-2 12,$23 -$9. 1$.2 -$9- 3 (1) =?A&1 --11.$2,9$33 8-$81,$21 -$99 1$B8 -$B2 . ?=B&(1) --1BB$12 ,1$.8,$91 18$3. -$.1$2. -$B- 3 (1) First deri0ati0e transfor ation (1,,,,,1: &?47D1) of t<e ori!inal spectral data: (2) &econd deri0ati0e transfor ation (2,2,2,2: &?47D1) of t<e ori!inal spectral data: sd5 standard de0iation of t<e reference c<e istr" data as t<e" Aere o'tained '" H#@C, &EC5 standard error of cali'ration: R25 coefficient of deter ination of t<e cali'ration: sd &EC4 -15 ratio of t<e standard de0iation of t<e reference c<e istr" data to t<e standard error of cross-0alidation: r 25 coefficient of deter ination of t<e cross-0alidation: nt5 nu 'er of ter s of t<e e>uation$
4000 3000 1600 1400 1200

GSL (predicted)

GNA (predicted)
0 1000 2000 3000 4000

1000 800 600 400 200 0 -200 -200 0 200 400 600 800 1000120014001600

2000 1000 0

GSL (laboratory)
140 120 200 175 150 125 100 75 50 25 0 0 20 40 60 80 100 120 140 0 25

GNA (laboratory)

GNAST (predicted)

80 60 40 20 0 -20 -20

NG S (predicted)

100

50

75 100 125 150 175 200

GNAST (laboratory)

NG S (laboratory)

!igure (. Cross-0alidation scatter plots for total !lucosinolates, !luconapin, !luconasturtiin and neo!luco'rassicin$ %n spite of t<e loA concentrations in A<ic< !lucosinolates are found in t<e lea0es, in co parison to t<ose contents of t<e seed of Brassica, t<e different e>uations s<oAed r2 and sd &EC4-1 0alues t<at Aere si ilar or sli!<tl" loAer t<an t<ose o'tained pre0iousl" '" us (Font et al$, 2--,) o0er t<e intact seed of %ndian ustard$ *n t<e ot<er <and, t<e decrease of t<e correlation e;istin! 'etAeen t<e spectral and c<e istr" data t<at is o'ser0ed in 1a'le 1, fro

t<e <i!<est of t-=&@ to t<e loAest e;<i'ited '" ?=B&, is e;plained <ere on t<e 'asis of t<e ean concentrations s<oAn '" t<ese olecular for s in t<e leaf sa ples$ 1<e results reported in t<is AorC s<oA t<at ?%R& is a'le to predict t<e t-=&@ and =?A in t<e lea0es of ca''a!e Ait< !ood precision$ %n addition, t<e =?A&1 and ?=B& e>uations could 'e used to identif" sa ples Ait< loA, ediu and <i!< contents of t<ese !lucosinolates$ 1<en, a proper selection of t<e indi0iduals of interest can 'e ade for ore accurate c<e istr" 0alues$ 1<e use of t<is et<odolo!" supposes and i portant reduction of ti e and cost of anal"sis in routine AorC$ Eac< sa ple of leaf t<at Ae anal"Ged '" usin! t<e ?%R& et<od, tooC us appro;i atel" 1$2 in, and prediction results for t<e indi0idual and total !lucosinolate content Aere onitored si ultaneousl"$ 1<e de0elop ent of t<ese cali'rations Aill alloA researc<ers to identif" >uicCl" t<ose indi0iduals of interest in t<e lea0es of ca''a!e, Ait<out t<e need of doin! H#@C anal"sis$ &eferences Barnes, R$ L$, M$ &$ D<anoa I &$ L$ @ister, 13.3$ &tandard nor al 0ariate transfor ation and de-trendin! of near-infrared diffuse reflectance spectra$ Appl$ &pectrosc$ ,85 992-999$ Fa<e", L$ H$, A$ 1$ Malc ann I #$ 1alala", 2--1$ 1<e c<e ical di0ersit" and distri'ution of !lucosinolates and isot<ioc"anates a on! plants$ #<"toc<e istr" 2B5 2-21$ Font, R$, M$ Del Ro, L$ Do n!ueG, L$ M$ FernFndeG-MartneG I A$ De Haro, 2--,$ /se of near-infrared spectroscop" for screenin! t<e indi0idual and total !lucosinolate content in %ndian ustard seed (Brassica &uncea @$ CGern$ I Coss$)$ L$ A!ri$ Food C<e $ 225 82B882B3$ Martens, H$ I 1$ ?aes, 13.3$ Multi0ariate cali'ration$ Lo<n Hile" I &ons, ?eA NorC$ Rosa, E$ A$ &$, R$ O$ Heanc", =$ R$ FenAicC I C$ A$ M$ #ortas, 1339$ =lucosinolates in crop plants$ Hort$ Re0$ 135 33-212$ Rosa, E$ A$ &$, 1333$ C<e ical Co position$ %n5 C$ = eG Ca po (Ed$), Biolo!" of Brassica Coenospecies, pp$ 812-829 $ Else0ier &cience B$ 4$, A nsterda $ &<apiro, 1$ A$, L$ H$ Fa<e", O$ @$ Hade, O$ O$ &tep<enson I #$ 1alala", 2--1$ C<e oprotecti0e !lucosinolates and %sot<ioc"anates of Broccoli sprouts5 eta'olis and e;cretion in <u ans$ Cancer Epide iolo!", Bio arCers I #re0ention 1-5 2-1-2-.$ &<enC, L$ &$ I M$ *$ Hester<aus, 133B$ Cali'ration t<e %&% Aa"$ %n5 A$ M$ C$ Da0ies I #$ C$ Hillia s (Eds$), ?ear %nfrared &pectroscop"5 1<e Future Ha0es, pp$ 13.-2-2$ ?ir #u'lications, C<ic<ester$ &orensen, H$, 133-$ =lucosinolates5 structure, properties, function$ %n5 F$ &<a<idi (Ed$), Canola and Rapeseed$ #roduction, c<e istr", nutrition and processin! tec<nolo!", pp$ 1,3192$ 4an ?ostrand Rein<old, ?eA NorC$ &pinCs, E$ A$, O$ &ones I =$ R$ FenAicC, 13.,$ 1<e >uantitati0e anal"sis of !lucosinolates in cruciferous 0e!eta'les, oilseeds and fora!es usin! <i!< perfor ance li>uid c<ro ato!rap<"$ Fette &eifen Anstric< ittel .B5 22.-281$ Hillia s, #$ C$ I D$ C$ &o'erin!, 133B$ HoA do Ae do it5 a 'rief su ar" of t<e et<ods Ae use in de0elopin! near infrared cali'rations$ %n5 A$ M$ C$ Da0ies I #$ C$ Hillia s (Eds$), ?ear %nfrared &pectroscop"5 1<e Future Ha0es$ ?ir #u'lications, pp$ 1.2-1..$ C<ic<ester$ HorC an, Lr$ L$ L$, 1332$ ?ir spectroscop" cali'ration 'asics$ %n5 D$ A$ Burns I E$ H$ CiurcGaC (Eds$), Hand'ooC of ?ear-%nfrared Anal"sis, pp$ 2,9-2.-$ DeCCer %nc$, ?eA NorC$