J Sci Food Agric 1998, 77, 368È372

Isolation, Characterisation and Determination ofBiological Activity of Coþee ProanthocyanidinsNe� lida Gonza� lez de Colmenares,1 Jose� R Ram•� rez-Mart•�nez,2 Jose� O Aldana,2Mar•�a E Ramos-Nin8 o,2 Michael N Cliþord,3* Sara Pe� kerar4 and Bernardo Me� ndez5

1 Centro de Qu•�mica de los Productos Naturales, Facultad de Humanidades y Educacio� n, Universidadadel Zulia, Venezuela2 Nu� cleo de Fitoqu•�mica, Departamento de Bioqu•�mica, Universidad Nacional Experimental del Ta� chira(UNET), Apartado Postal 436, San Cristo� bal, Venezuela3 Food Safety Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, GU2 5XH,UK4 Centro de Qu•�mica, Instituto Venezolano de Investigaciones Cient•� Ðcas (IVIC), Caracas, Venezuela5 Escuela de Qu•�mica, Universidad Central de Venezuela (UCV), Caracas, Venezuela

(Received 7 April 1997 ; revised version received 11 November 1997 ; accepted 19 November 1997)

Abstract : Two proanthocyanidin-containing preparations were isolated from thepulp of ripe Co†ea canephora co†ee cherries. The preparations were partiallypuriÐed by Sephadex LH-20 column chromatography and characterised bymeans of infrared and 13C NMR spectroscopy, and reactions with several func-tional group-speciÐc reagents. These proanthocyanidin-rich extracts from co†eepulp, obtained also from co†ee leaves, inhibit the germination in vitro of Hemi-leia vastatrix race 2 uredospores. Greater proanthocyanidin content was associ-ated with greater potency, and the leaves of Co†ea arabica, a co†ee speciessusceptible to rust, contained less proanthocyanidins than the leaves of C canep-hora, a rust-resistant co†ee. 1998 SCI.(

J Sci Food Agric 77, 368È372 (1998)

Key words : co†ee pulp ; proanthocyanidins ; condensed tannins ; antifungal activ-ity ; co†ee rust ; Hemileia vastatrix

INTRODUCTION

Phenolic compounds are some of the most widespreadmolecules in nature. They are especially associated withplants where they are found as simple phenols, phenolicacids, hydroxycinnamic acids derivatives, Ñavonoidsand tannins (Ho 1992). Tannins have relatively largemolecular mass and complex strongly with carbo-hydrates and proteins (Porter 1989). Several tannin sub-groups are known: one of these, condensed tannins or

* To whom correspondence should be addressed.Contract/grant sponsor : EC.Contract/grant number : C11*-CT92-0018.Contract/grant sponsor : Decanato de Investigacion UNET.Contract/grant number : 04-005-93.

proanthocyanidins, is polyÑavonoid in nature, consist-ing of chains of Ñavan-3-ol units with C-4 to C-8 or C-4to C-6 linkages (Czochanska et al 1980).

Plants have developed a range of strategies fordefence against their predators, one of which is com-pounds with antimicrobial activity. Numerous studieshave described the inhibitory e†ect of plant extracts onmicrobial growth (Payne et al 1989 ; Evert-Ting andDeibel 1992 ; Hefnawy et al 1993 ; Pandit and Shelef1994). The proanthocyanidins are one class of second-ary metabolite to which antimicrobial activity has beenassigned (Scalbert 1991). It has been reported that theyform complexes with enzymes produced by pathogenicfungi to digest the cell wall of plants (Laks et al 1988 ;Porter and Hemingway 1989).

3681998 SCI. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain(

Biological activity of co†ee proanthocyanidins 369

Proanthocyanidins are found in relatively high quan-tities in co†ee pulp. Some reports on their quantiÐca-tion have been published previously (Zuluaga-Vascoand Tabacchi 1980 ; Cli†ord and Ram•� rez-Mart•� nez1991 ; Cli†ord et al 1991 ; Gonza� lez de Colmenares et al1994), but there have been no reports on their isolationand characterisation, or of their occurrence in co†eeleaves. In this study two proanthocyanidin-rich frac-tions have been isolated from co†ee pulp, and partiallycharacterised by spectroscopic and chemical methods.These, and similar material isolated from co†ee leaves,have been tested for their ability to inhibit the germi-nation of co†ee rust uredospores in vitro.

MATERIALS AND METHODS

Materials

Isolation and characterisationFreshly harvested ripe Co†ea canephora co†ee cherrieswere supplied by the Estacio� n Experimental Agr•� cola deBramo� n, Venezuela. Cyanidin-3-glucoside was obtainedfrom Apin Chemicals (Abingdon, Oxon, UK). All otherreagents were standard analytical grade items fromreputable commercial sources.

Biological activityRipe co†ee cherries of Co†ea canephora ; leaves of Ccanephora and C arabica var caturra ; and uredosporesof the co†ee rust fungus (Hemileia vastatrix, race 2) werecollected from the Estacio� n Experimental Agr•� cola deBramo� n, Venezuela. Peptone (Merck) and Tween-80(Farmacia Ta� chira) were used in the biological testing.

Methods

Isolation of co†ee pulp proanthocyanidinFresh pulp from ripe C canephora co†ee cherries (100 g)was mashed and extracted (5] 100 ml) with aqueousacetone (700 ml litre~1). The pooled extracts were con-centrated by low pressure evaporation at \30¡C toeliminate acetone. The concentrated aqueous solutionwas extracted again with petroleum ether (5] 100 ml)to remove lipids and fat-soluble pigments, with ethylacetate (11] 100 ml) to solubilise simple phenols, Ñavo-noids, proanthocyanidin dimers and oligomers andÐnally, with dichloromethane (5] 100 ml) to eliminateca†eine. One-third of this crude aqueous extract wasmixed with an equal amount of ethanol and added tothe top of an open Sephadex-LH 20 column(40 ] 2 cm) activated previously with aqueous ethanol(500 ml litre~1). The crude extract was drawn 6 cm intothe packing by allowing the column to drain. Thecolumn was washed with 500 ml of distilled water fol-lowed by equal volumes of aqueous ethanol (100, 200,

300, 400, 600 and 800 ml litre~1) and Ðnally ethanolalone. The column was eluted with 175 ml aqueousacetone (800 ml litre~1) to remove the proanthocyani-dins (PA) and seven 25-ml fractions were collected.These seven fractions are referred to subsequently asPA1, PA2, . . . , PA7. The ethanol washings and theaqueous acetone fractions were subjected to TLC, RP-HPLC, and autooxidation reactions with n-BuOHÈHClÈFe(III) in order to locate proanthocyanidin-richfractions and assess their homogeneity.

Crude fractions were similarly isolated from theleaves of Co†ea arabica (susceptible to co†ee rust) andC canephora (resistant to co†ee rust).

Characterisation of proanthocyanidinsThe aqueous acetone fractions PA2 and PA3 elutedfrom the Sephadex LH-20 column were lyophilised andsubjected to PorterÏs reaction (Porter et al 1986), andthe visible absorbance spectrum (400È600 nm) of thered pigment produced was determined in a Perkin-Elmer spectrometer model Lambda 2. The red pigmentsobtained by the autooxidation of the proanthocyanidinsand hydrolysis of cyanidin-3-glucoside were also sub-jected to paper chromatography on 3 MM Ðlter paperusing tolueneÈacetoneÈformic acid (6 : 6 : 1, v/v/v) aseluent (Hussein et al 1990) and RP-HPLC. TheRP-HPLC was carried out in Waters equipment con-sisting of a sample injector (model U6K), two pumps(model 510) and a photodiode array detector (model996). A Hichrom (Theale, Berkshire, UK) Spherisorb S5ODS2 column (250 ] 4É6 mm) was used with a Ñowrate of 0É5 ml min~1 and a linear gradient from solventA (aqueous phosphoric acid 5 ml litre~1) to solvent B(methanol) over 40 min, followed by isocratic elutionfor 40 min, and reset. Infrared (IR) spectra were deter-mined using KBr discs (1È2 mg of sample in a 13 mmKBr disc) (Foo 1981) in a Perkin-Elmer FTIR spectro-meter (model 1600). 13C NMR spectra were determinedin a 67 MHz JOEL spectrometer by dissolving thesample in (1 : 1) (Sun et al 1988 ; Porteracetone-d6/D2Oet al 1991 ; Eberhardt and Young 1994).

Functional group analysis included nitrogen by semi-micro analysis (Meites 1963), protein nitrogen by theninhydrin reaction (Jayaraman 1981), carbonyl groupdetection through osazone formation (Shriner et al1966), detection of intact Ñavanol A-ring using 4-dimethyl-amino-cinnamaldehyde (McMurrough andMcDowell 1978) and the detection of 1,2-dihydroxyphenyl groups with sodium molybdate (Cli†ord andStaniforth 1977).

Determination of biological activityLyophilised preparations of the crude proanthocyan-idin-rich extract, fraction PA2, and crude proantho-cyanidin-rich extracts from leaves of co†ee rust-resistant(C canephora) and co†ee rust-susceptible (C arabica varcaturra) co†ee trees were tested for their ability to

370 N Gonza� lez de Colmenares et al

inhibit germination of co†ee rust uredospores in vitro.The uredospores of the co†ee rust fungus (Hemileia vas-tatrix, race 2) were collected from naturally infectedtrees by gently scraping using the edge of an Eppendorftube. The spores were stored at 4¡C and 50% relativehumidity as recommended by Stahmann et al (1976)and used within 2 weeks of harvest. Solutions(4 mg ml~1) of the lyophilised fractions were preparedin peptone water (1 g litre~1) containing Tween-80 (5 glitre~1). Three serial dilutions were then made with thesame media.

Using a spatula, a small quantity of uredospores(about 5 mg) was diluted in 10 ml of peptone water toobtain a concentration of 75 000 uredospores per mland 100 ll introduced to the cavity of a dry concaveslide within a Petri dish above 2É5 ml of water, in quad-ruplicate. Then 100 ll of each serial dilution of extractsand a control containing only peptone water andTween-80 was carefully added to each slide using amicropipette. Counts of the number of spores with germtubes were made after 6 h of incubation in the dark at23¡C (Stahmann et al 1976). The experiment was repli-cated three times. About 400È500 spores were countedon each slide and the inhibitory e†ect of the extractswith respect to the control was reported as :

% Inhibition \

(Control count [ Treatment count)(Control count)

] 100

RESULTS AND DISCUSSION

Isolation and characterisation of co†ee pulpproanthocyanidins

The paper chromatograms of the fractions eluted fromthe Sephadex LH-20 column with aqueous ethanol (upto and including 400 ml litre~1) showed elongatedbands, whereas those obtained with aqueous ethanol atconcentrations of 500 ml litre~1 or more showed slight-ly elongated spots at the origin. The HPLC chromato-grams of the fractions eluted with relatively diluteethanol presented multiple peaks superimposed on anapproximately Gaussian hump, whereas those elutedwith more concentrated ethanol tended to progressivelysharper peaks with several small subsidiary spikes onthe ascending and descending slopes (cf Bailey andNursten 1994).

Out of the seven 25-ml fractions eluted with aqueousacetone from the Sephadex LH-20 column, only twofractions (PA2 and PA3) were homogeneous by paperchromatography and HPLC criteria. After lyophi-lisation and treatment with PorterÏs reagents PA2 andPA3 gave RP-HPLC chromatograms with abundantpeaks at 280 nm (29 for PA2 and 37 for PA3), few ofwhich absorbed in the visible region. This multiplicity

of products indicates the structural complexity and/orheterogeneity of PA2 and PA3. The UV-Vis spectrumof the pigmented PorterÏs transformation products hadseveral including one at 547 nm which does notjmaxcoincide with the spectra of the common condensedtannin-derived anthocyanidins (pelargonidin, cyanidinand delphinidin). These observations suggest that eitherthe anthocyanidins once released are atypical, or theyare labile, being transformed during treatment withPorterÏs reagents or during chromatography.

However, if the crude products obtained by treatingPA2 and PA3 with PorterÏs reagents were diluted withn-BuOHÈHCl then only one absorbance maximum wasobserved in the vicinity of 550 nm which could beattributed to cyanidin and this is consistent with pre-vious reports (Cli†ord and Ramirez-Martinez 1991).

The most striking di†erence between the IR spectraof these two fractions is that PA2 shows a signal at1706 cm~1 (carbonyl) and signals at 1534, 1520 and732 cm~1 which suggest the presence of prodelphinidin(PD), whereas PA3 presents bands at 1524 and782 cm~1 which correspond to procyanidin (PC)according to Foo (1981). The presence of prodelphini-dins has previously been suggested in some (Cli†ordand Ram•� rez-Mart•� nez 1991), but not all, studies(Gonzalez de Colmenares et al 1994).

PA2 contains non-protein nitrogen (positive to Kjel-dahl and negative ninhydrin reaction). Both PA2 andPA3 contain a structure equivalent to a Ñavanol A-ring,as shown by their positive reaction to dimethyl-aminocinnamaldehyde, and a 1,2-dihydroxyphenylstructure equivalent to a Ñavanol B-ring as shown bytheir response to sodium molybdate. Both responses areconsistent with the presence of procyanidins and/orprodelphinidins. PA2 gave positive reaction with 2,4-dinitro-phenylhydrazine consistent with the IR spec-trum carbonyl signal. Such carbonyls are not normallya feature of proanthocyanidins.

The 13C NMR spectra for PA2 and PA3 are similarto the proÐles shown by complex proanthocyanidins(Porter 1989). In the spectrum for PA2, condensedtannin and carbohydrate portions can be inferred fromthe resonances (ppm) at : 27É54 (C-4 of terminal unit) ;36É60, 38É43 (C-4 of extending PC and PD units) ; 60É88(C-6 of b-glucose) ; 71É95, 76É21, 78É87, 82É36(heterocyclic ring carbons C-2 and C-3) ; 95É48(unsubstituted C-6 and C-8) ; 103É79 (C-1 of b-glucose) ;106É42 (C-4a of 2,3-cis PC and PD units) ; B-ring signalsat 114É81 (C-2@ and C-6@ of PD units) ; 118É37, 119É99 (C-2@, C-5@ and C-6@ of PC units) ; 129É38, 129É93, 131É03 (C-1@ of PC and PD units and C-4@ of PD units) ; 144É58 (C-3@ and C-4@ of PC and PD units) ; 155É67 (C-5, C-7 andC-8a of all units) ; 176É71 (carbonyl group). Alkalinehydrolysis under nitrogen followed by HPLC demon-strated a peak indistinguishable from glucose standard.

Similarly, from the spectrum for PA3, portions corre-sponding to proanthocyanidin and a methoxylated

Biological activity of co†ee proanthocyanidins 371

TABLE 1Percentage inhibition of co†ee rust uredospore germination

by proanthocyanidin-containing extracts of co†ee pulp

Concentration Percentage inhibition (mean^SD)(g litre~1)

C canephora C canephora(crude extract) (PA2)

2É00 97a ^ 2 100a ^ 01É00 66a ^ 10 99a ^ 20É50 2a ^ 5 99a ^ 70É25 1a ^ 1 49a ^ 12

a Short germination tubes.

compound(s) can be deduced from the following reso-nances : (ppm) : 16É91 36É06, 36É48 (C-4 of PC(ÈCH3) ;and PD units) ; 54É85, 57É41 69É86 (C-3 ter-(ÈOCH3) ;minal unit) ; 71É63 (C-3 of PC and PD units ; 81É93 (C-2of 2,3-trans PC and PD units) ; 95É09 (unsubstituted C-6and C-8 of all units) ; 105É27 (C-4a of 2,3-cis PC and PDunits) ; 115É49, 115É63 (C-2@ and C-6@ of PD units) ;117É95, 118É89, 119É69 (C-2@, C-5@ and C-6@ of PC units) ;

TABLE 2Percentage inhibition of co†ee rust uredospore germinationby proanthocyanidin-containing extracts of leaves from resist-

ant and susceptible species of co†ee

Concentration Percentage inhibition (mean^SD)(g litre~1)

C canephora C arabica(leaf extract) var caturra

(leaf extract)

2É00 100a ^ 0 100a ^ 01É00 99a ^ 10 98a ^ 20É50 76a ^ 7 9a ^ 100É25 0a ^ 0 0a ^ 0

a Short germination tubes.

TABLE 3Proanthocyanidin content of lyophilised proanthocyanidin-

rich extracts from co†ee leaves and pulp

Source Procyanidinequivalents (%)a

Mean^ SD, N P 4

C canephora (leaves : 15É27 ^ 0É12crude extract)

C arabica var caturra (leaves : 12É00 ^ 0É08crude extract)

C canephora (pulp : PA2) 56É16 ^ 0É72

a The equivalents were obtained using for oligo-E5501† \ 470mers (Porter et al 1986 ; Porter 1989 ; Cli†ord and Ram•� rez-Mart•� nez 1991 ; Gonza� lez de Colmenares et al 1994).

130É55, 131É21, 131É82 (C-1@ of PC and PD units andC-4@ of PD units) ; 144É27, 144É37 (C-3@ and C-4@ of PDunits) ; 154É26, 154É62, 155É35 (C-5, C-7 and C-8a of allunits).

It is apparent that these two preparations (PA2 andPA3) isolated from the pulp of ripe red C canephoracherries are complex mixtures. PA2 contains condensedtannins, carbohydrate and some nitrogenous substit-uent other than protein or an a-amino acid, whereasPA3 is composed of condensed tannins and meth-oxylated Ñavonoids, methyl ethers and/or lignans. It isnecessary to improve chromatographic systems to beable to resolve these complex proanthocyanidins beforeelucidating their structures.

Biological activityTables 1 and 2 report the inhibitory e†ect of theextracts from co†ee pulp and leaves respectively on thegermination of uredospores of co†ee rust. The proanth-ocyanidins content of the fractions tested are reportedin Table 3.

All proanthocyanidin-rich preparations tested weree†ective inhibitors of uredospore germination at2 mg ml~1. Those preparations shown to have a greaterproanthocyanidins content were signiÐcantly morepotent, with PA2 causing B50% inhibition even at0É25 mg ml~1. It is interesting to note that the crudeproanthocyanidin preparation from the leaves of rust-resistant C canephora was more potent than that fromthe rust-sensitive C arabica. This di†erence in proantho-cyanidin content/potency might be one factorresponsible for the di†ering susceptibility of these twospecies to rust.

ACKNOWLEDGEMENTS

This project was supported by EC Contract C11*-CT92-0018 and by the Decanato de Investigacion,UNET, project 04-005-03.

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