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Analysisi of Catechin in Grape Seed Extract

Analysisi of Catechin in Grape Seed Extract

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Analysis of Catechins and Proanthocyanidins in Grape Seeds byHPLC with Photodiode Array Detection
E. Revilla 1. / M. Bourzeix 2 / E. Alonso 11Universidad Autdnoma de Madrid, Departamento de Qu/mica Agr/cola, Geologfa y Geoqufmica, 28049 Madrid, Spain2Institut National de la Recherche Agronomique, Station d'Oenologie et de Technologic des Produits V6g6taux, 11100Narbonne, France
Key Words
Column liquid chromatographyCatechinsGrape seedsProanthocyanidinsUV-visible spectroscopy/diode array detection
Summary
Reversed-phase, high-performance liquid chromatogra-phy coupled to photodiode array detection has been used toanalyse catechins and proanthocyanidins extracted fromgrape seeds. Results show that the ethyl acetate fractionobtained by passing extracts of samples adjusted to pH 7.0through preconditioned C~8 SEP-PAK cartridges containsseveral catechins and proanthocyanidins. Seven peaks havebeen assigned to standard catechins and proanthocyanidinson the basis of their retention times and UV spectra. Otherpeaks which appear in the chromatogram show spectralbehaviour similar to that of standard catechins and proan-thocyanidins.
Introduction
Grapes are the edible fruits which probably contain thelargest amounts of catechins (flavan-3-ols) and their oli-gomers, known as proanthocyanidins. The study of thesecompounds in grapes and wines has become a topic ofinterest as a consequence of their positive role in humannutrition as captors of free radicals [1-2] and in relation tocertain vascular diseases [3]. Catechins and proanthocya-nidins are more abundant in lignified tissues than in otherplant tissues; thus, grape seeds and cluster stems are richerin them than grape skins and pulps [4-5]. The amount ofcatechins and proanthocyanidins in the different parts ofthe grape cluster greatly affects the content of those sub-stances in wines, which is in turn dramatically influenced bywinemakingprocedures [4]. Nevertheless, a variable amountof catechins and proanthocyanidins remains in grape seedsafter winemaking, depending on the technological processesused; hence, this winemaking by-product may be used as asource of catechins and proanthocyanidins for pharmaco-logical and cosmetic purposes.Traditionally, grape and wine phenolics have been separat-ed by conventional techniques, such as paper, thin-layerand column chromatography, which are time-consumingand do to permit quantitative analysis. In recent years,reversed-phase, high-performance liquid chromatographyhas proved to be a useful technique for the analysis ofphenolic compounds, and several methods have been de-veloped to analyse catechins and proanthocyanidins ingrapes, in wines, and in other beverages [5-9]. In order toavoid interference by other phenolic compounds in HPLCand also to reduce the time of analysis, several attemptshave been made to perform a simple and rapid fractiona-tion of grape and wine phenolics before HPLC analysis [10-12].The UV-visible spectra of catechins and proanthocyanidinsgive an absorption maximum between 270 and 280 nm,which is remarkably different to those of most plant phe-nolics which present two or more absorption maxima be-tween 250 and 400 nm [13-14]. Some phenols and phenolicacids with very simple substitution patterns are the only on eto present a unique absorption maximum between 270 and280 nm [15]. Thus, if these substances are removed from thesample before HPLC analysis, the UV-visible spectra ofpeaks in the chromatogram may be used for their identifi-cation. For these reasons, photodiode array detectioncoupled with HPLC may permit rapid and simultaneousseparation, identification and quantitation of catechins andproanthocyanidins after the fractionation of grape andseed phenolics.In this paper, a liquid chromatograph equipped with aphotodiode array detector was used to analyse catechinsand proanthocyanidins extracted from grape seeds afterpurification of the extracts by reversed-phase silica mini-columns, and to achieve the spectral purity of differentpeaks in the chromatogram.Chromalographia Vol. 31, No. 9110, May 1991 Originals4650009-5893/91/5 0465-04 $ 03.00/0 9 1991 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
 
ExperimentalPreparation of Samples
Chardonnay grapes were collected at Pech Rouge Experi-mental Vineyard (INRA, France) during the 1989 harvest.Once in the laboratory, grape seeds were separated frompulp, and dried under CO 2 to elimin ate surface water. 10.00 gseeds were placed in a flask and 40 mL methanol wasadded. Then, samples were pulped under CO 2 with an U1-tra-Turrax T25 mixer, and flasks kept at - 24 ~ for onenight. Afterwards, the samples were centrifuged at 3000 gfor 10 rain in a Jouan GR 2000S centrifuge and the super-natant liquids were stored. The residues were then, extract-ed sequentially with 80 % methanol at room temperaturefor 4 hours, with 50 % methanol at room temperature for 4hours, with distilled water at - 24 ~ for 15 hours and with75 % acetone at room temperature for 1 hour. Afterwards,all the extracts were mixed and the volume raised to 250mL. Lastly, the methanolwas removed from 25 mL aliquotsby vacuum at 30 ~ and their volume restored to 25 mLwith distilled water.
Fractionation of Phenolic Compounds
The fractionation of phenolic compounds was carried outon two Waters C18 SEP-PAK cartridges connected by rub-ber tube (preconditioned by sequentially passing 10 mLmethanol and 2.5 mL distilled water adjusted to pH 7.0 withNaOH dropwise), by injecting 0.5 mL of the alcohol-freesample of the grape seed extract adjusted to pH 7.0 toabsorb neutral phenolics. Then, 10 mL distilled water ad-justed to pH 7.0 was passed through the cartridges to eluteacidic phenolic compounds. The cartridges were driedunder N2, and 10 mL ethyl acetate passed through them toelute catechins and proanthocyanidins. Afterwards, thecartridges were washed by sequentially passing 2.5 mLdistilled water adjusted to pH 2.0, 2.5 mL 16 % acetonitrileadjusted to pH 2.0, and 10 mL methanol through them toelute other phenolics, mainly anthocyanins and red andbrown polymers. The ethyl acetate fraction was dried undervacuum at 30 ~ and the residue dissolved in 0.5 mL 50 %methanol and stored 0 ~
HPLC Analysis
A Hewlett-Packard liquid chromatograph equipped withan HI) 1050 series quaternary pump, a Rheodyne 7125injection valve furnished with a 20 gL sample loop, an HP1040A diode array detector and an HP 9000 series 300computer was used. The separation of catechins and proan-thocyanidins was carried out with an Altech column(100 x 2.1 ram) packed with 30-38 gm CO:PELL ODS
Table
I Solvent gradient used in HPLC analysis.Time (minutes) % A % B0 10 9047 82 1855 100 065 100 0(Whatman Ltd., England) and a pre-packed analyticalcolumn (250 x 4.6 mm) of Nucleosil Ct8, 5 #m (S.EC.C.,Neuilly, France), using a linear gradient of 10 % acetic acid(solvent A) in water (solvent B), as shown in Table I, at aflow rate of 0.8 mL rain -1. Columns were placed in a waterbath at 32 ~
TLC Analysis
In dubious cases, the effluents corresponding to severalpeaks in the chromatograms were collected, to follow theirbehaviour in two-dimensional cellulose MN-300 TLC, us-ing 20 x 20 cm plates, which were developed with tert-butanol/acetic acid/water (3:1:1 v/v) and water/acetic (85:15v/v). The components were detected by spraying the plateswith 1% p-vanillin in concentrated hydrochloric acid, giv-ing pink-coloured spots.
Standards
(+)-catechin and (-)-epicatechin were from Fluka AS(Switzerland) and Aldrich Chemical Co. (USA), respec-tively. Procyanidins B 1, B2, B3, B4 and C1 were supplied byDr. M. Mountounet, Institut de Produits de la Vigne,Montpellier, France.
Results and Discussion
The chromatographic analysis of solutions containing (+)-catechin, (-)-epicatechin, and procyanidins B1, B2, B3, B4
LC 280 nm or CHFIP,,DONNFIY SEEDS. 25.00-37.O0 m~n.16
1412109
64
a~ " .--,; " " 3~ " ~5 " 3;, " ~;
Tlrr, m (m~rl, '~k~,._LC 21~O turn o4' CHARDONNFIY SEEDS, 37.00-B3.O8 rnln.16
1412"I0"64'1510
~ l~12~j~j 16
20
2 17 |8
19
413 45 5{3 55 60Ttma /mira. )
Figure 1
Chromatogram of Chardonnay grape seeds extracts recorded at 280
rim.
466
Chromatographia Vol. 31, No. 9/10, May 1991 Originals
 
and C1 allows a convenient separation of all these substances,whose retention times are listed in Table II. All the peaks inthe chromatograms show similar UV spectra at the upslope,at the apex and at the downslope of the peak in the range250 to 400 nm. Absorption maxima were close to 277 nm ineach case, and spectral peak purity was greater than 95 %.To standardise this methodology for quantitative estimationof catechins and proanthocyanidins in plant extracts, foods,and beverages; solutions of the above-mentioned com-pounds at increasing concentrations were injected into thechromatograph. The calibration curves, giving the peakareas against the concentration of the solutions injected,are straight lines for all compounds.Chromatograms of the extracts of Chardonnay grape seedsshow the presence in that material of a number of com-pounds whose chromatographic and spectral characteristicsare close to those of standard catechins and proanthocya-nidins. Figure 3 shows the chromatogram [at 280 nm] be-tween 25 and 37 min and between 37 and 63 rain and at least22 different peaks can be seen. The four major peaks (8, 9,15 and 21) were assigned to procyanidin B1, (+)-catechin,procyanidin B2 and (-)-epicatechin, respectively, on thebasis of their retention times and UV-visible spectra. TheUV-visible spectra between 250 and 400 nm of (+)-catechin,procyanidin B2, peak 9 and peak 15 are in Figure 4 and, asseen, their spectra are close to those of standard materials.Retention times and spectra of another three peaks (4, 10and 22) are close to those of procyanidin B3, procyanidinB4 and procanidin C1, and may be assigned to these sub-stances. The spectral peak purity of these seven peaks wasgreater than 95 %. Further analysis involving TLC co-chromatography of the effluents containing these peakswith standard proantocyanidins shows their identity withthem.Some other peaks show spectral characteristics close tothose of catechins and proantocyanidins: their UV Xmax isclose to 280 nm, as shown in Figure 5 for peaks 2 and 3, theirabsorption spectra do not present any other )~rnax at higherwavelengths and the spectral peak purity was greater than90 %. TLC analysis carried out with the HPLC effluent ofpeak 1 demonstrates that it contains a compound whosebehaviour in the TLC system used is similar to that of thecatechins and the proanthocyanidins available in our lab-oratories, giving a pink-coloured spot when the plate wassprayed with 1% p-vanillin in c. HCI. Thus, the majorsubstance contained in peak 1 probably belongs to this
Table !1 Retention times of some catechins and proantho-cyanidins.
Substance Relative retention times(minutes)procyanidin B3 31.6procyanidin B1 35.0(+)-catechin 38.2procyanidin B4 38.3procyanidin B2 44.6(-)-epicatechin 56.2procyanidin C1 60.9
1801
/%
(+)-CRTECHIN
8@
388
350
408
Ldavelength (nm)
's~ PRoc, , io, o..
9,30 3-=;0
488
1.4a v e. 1 e. n _q t.h ( ,Trn )ZICE"l /'\
Pe a
9
t
2,elot \ ......3oL3 35~
1,4ave 1 ei'tgt-t7 ( r, rt',)
, , '7'" , I
480
4811 Pe~k 15
31~O 35~
40~
Na,,el ei-,gt.l-, ~
r,m)
Figure 2
UV-visible spectra of (+)-catechin, procyanidin B2, peak 9 and peak 15.
Peak
2,
t 0 .f--..
300 358 400
61a,~ I ength (nm)
Pe ak 3
15-1
0 .... , .... ,
300
14ave Iength (rim)
Figure 3
UV-visible spectra of peaks 2 and 3.
 , , , , -,--i
~50
400
Chromatographia Vol. 31, No. 9/10, May 1991 Originals
467

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