Analytica Chimica Acta 445 (2001) 67–77

Recovery of phenolic compounds fromOlea europaea

Danielle Ryan, Helen Lawrence, Paul D. Prenzler,Michael Antolovich, Kevin Robards∗

Faculty of Science and Agriculture, School of Science and Technology, Charles Sturt University,Locked Bag 588, Wagga Wagga 2678, NSW, Australia

Received 28 March 2001; accepted 29 June 2001

Abstract

The recovery of biophenols from olive drupes by simple extraction (free phenolics) was compared to that from acid- andbase-treated extracts. Quantitative data are presented for levels of phenols obtained by the different procedures. The majorphenol in green olives, oleuropein, was significantly affected by both acid- and base-treatment, and resulted in the liberationof hydroxytyrosol glucoside. The level of hydroxytyrosol glucoside produced after 24 h of acid-treatment was approximately50 times that produced in base-treated extracts. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Olive; Phenolic compounds; Hydrolysis; Liquid chromatography

1. Introduction

The nutritional, functional and “organoleptic” qual-ity of olive products is closely related to the phenoliccontent of the fruit. Phenolic profiles have been de-termined on many varieties of olive (e.g. [1–3]) butquantitative data are less common [4,5]. Extraction ofphenolic compounds from natural matrices is compli-cated by their diversity and sensitivity to oxidationand hydrolysis. Extraction procedures must allow forthese intrinsic differences but are often neglected, withemphasis being placed upon the highly sophisticatedinstrumental end of the analysis.

Methods used for phenolic isolation vary greatly,and range from those that incorporate precautionarymeasures to protect the phenolic compounds [6], toquite simple solvent extractions [7]. The method used

∗ Corresponding author. Tel.:+61-2-6933-2539;fax: +61-2-6933-2737.E-mail address: krobards@csu.edu.au (K. Robards).

[8] is dependent upon the particular class of phenoliccompound and, to a lesser extent, on the nature of thematrix (fruit, seed, leaf). In olive, for example, totalphenolics were recovered [4,9,10] from olive powderusing aqueous ethanol in the presence of metabisul-fite (disulphite). The extract was concentrated undervacuum and washed with petroleum ether to removelipids and pigments, followed by ethyl acetate washesin the presence of ammonium sulfite, metaphosphoricacid and methanol. In contrast, Vlahov [11] isolatedflavonoids from olive fruit by extraction with aqueousmethanol and precipitation of interfering substancesby addition of acetic acid.

These extraction procedures provide information onthe level of free phenolic compounds in the fruit. How-ever, in many instances as, for example, in studies ofdietary intake, the total phenolic content is of poten-tially more interest. Hydrolysis has been used (e.g.[12]) to provide data on total phenolic contents in otherfruit, particularly citrus, but has received little atten-tion in olives. This is surprising given the importance

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0003-2670(01)01241-7

68 D. Ryan et al. / Analytica Chimica Acta 445 (2001) 67–77

of the olive in the Mediterranean diet and the physi-ological activity of phenols as the main driving forcedetermining the directions of phenolic research. Apartfrom its fundamental importance, the effect of base onphenolic compounds is also commercially importantbecause of the use of base-treatment in preparation oftable olives [3].

This paper reports the results of a systematic studyof the isolation of phenolic compounds from olive dru-pes. Phenolic profiles obtained by simple extraction(free phenolics) are compared with data obtained byacid and base hydrolysis and extraction. Quantitativedata are reported for the more significant phenols.

2. Experimental

2.1. Reagents

Reagents from the following sources were usedwithout further purification: methanol (EM Sci-ence), acetic acid (Ajax Chemicals), and hydrochloricacid (32%) (Ajax Chemicals). Phenolic standardswere obtained as follows: oleuropein, luteolin, api-genin, apigenin-7-glucoside, luteolin-7-O-glucosideand cyanidin chloride from Extrasynthese (Genay,France); caffeic, chlorogenic,o-coumaric,p-coumaric,ferulic, gallic, homovanillic, sinapic, syringic andvanillic acids from Sigma Chemical Co.; and tyrosolfrom Aldrich Chemical Co. All water used for re-verse phase liquid chromatography (RPLC) analyses,sample and standard preparation was purified usingreverse osmosis. Acetone supplied by Ajax Chemicalswas used for cleaning purposes.

2.2. Olive samples

Olive samples (cv Manzanillo) were obtained fromthe Wagga Wagga olive grove that was established in1898 as an experimental orchard. Samples were col-lected at green maturation for the acid/base hydrolysisand extraction study, or following green maturation(purple skin) (harvested 17 June 1999) for selectionof extracting solvent. Fruits (500 g) were selectedrandomly from around the tree and were then imme-diately freeze dried. After freeze drying, the pulp wasseparated from the olive stone and blended into anhomogenous powder that was stored in a freezer at–18◦C prior to analysis.

2.3. Choice of extraction solvent

Dried olive powder (0.25 g) was extracted twicewith either methanol (5 ml), water, various mixturesof methanol and water or aqueous carbonate solu-tion [13]. The effect of extraction time was examinedover the range 15–480 min. The extracts were com-bined and diluted with water (to produce a solutionwith a minimum 20% aqueous content) prior to wash-ing with hexane (5 ml). The extract was then filteredthrough 0.45�m filters and analysed by LC (gradient1). Alternatively, the inclusion of a solid phase extrac-tion (SPE) clean-up step following the hexane washwas also investigated. The C18 SPE cartridge (Wa-ters) was conditioned first with methanol (6 ml) thenwater (6 ml). The phenolics retained on the cartridgewere eluted in a stepwise gradient elution beginningwith water, through various mixtures of methanol andwater to pure methanol. Each eluate was filtered andanalysed as before.

2.4. Optimised extraction procedure — control

Dried olive (0.25 g) and methanol+ water (50+ 50v/v; 5 ml) were blended using an Ultra Turrax highspeed blender for 20 s. The solution was left to standfor 30 min and filtered using GF/F filter paper. Afterhexane washing (5 ml), 1/3 of the extract by mass wasmade up to a final mass of 3 g with methanol+ water(50+ 50 v/v), filtered through a 0.45�m filter, and aportion injected into the chromatography. The remain-ing extract was stirred for 2 h, and another third of theoriginal extract was made up to 3 g with methanol+water (50+ 50 v/v) and filtered through a 0.45�mfilter, for subsequent LC analysis. The final third ofthe original extract was stirred, and after a total of24 h, was also made up to 3 g with methanol+ water(50+ 50 v/v), filtered (0.45�m) and analysed by LC.

2.5. Acid and base hydrolysis

Dried olive (0.25 g) was blended with methanol+water (50+ 50 v/v; 5 ml) using an Ultra Turrax highspeed blender for 20 s. The solution was left to standfor 30 min and was filtered using GF/F filter paper.After hexane washing (5 ml), hydrochloric acid (1 M,2 ml) was added to the aqueous phase, and this solutionwas stirred for 2 h. Half of the acidified extract was

D. Ryan et al. / Analytica Chimica Acta 445 (2001) 67–77 69

neutralised (pH 7) with NaOH (5 M), and the remain-ing extract was neutralised after 24 h stirring. Both ex-tracts were made up to a final mass of 4.5 g and wereimmediately filtered (0.45�m) and analysed by LC.

The same procedure was used in the base hydrolysisexperiments, however NaOH (1 M, 2 ml) was addedto the extract after hexane washing, and HCl (5 M)was used to neutralise the extracts after 2 and 24 h,respectively.

The effect of nitrogen blanketing and air saturationof hydrolysed extracts was also investigated. Threeacidic, three basic and three control extracts wereset-up in scintillation vials (10 ml headspace) so thatone of each extract was exposed to nitrogen, com-pressed air, or atmospheric conditions, respectively, bybubbling for 24 h. Both the nitrogen and compressedair were saturated with methanol+water (25+75 v/v)prior to use.

2.6. Acid and base extraction

Dried olive (0.25 g) and methanol+ water (50+ 50v/v; 5 ml) were blended using an Ultra Turrax highspeed blender for 20 s. After standing for 30 min, hy-drochloric acid (1 M; 2 ml) was added, and the solutionwas stirred for 2 h. Half of the extract was filtered us-ing GF/F filter paper, and washed with hexane (5 ml).The aqueous extract was neutralised with NaOH (5 M),made up to a final mass of 4.5 g and filtered (0.45�m)prior to analysis by LC. The remaining half of the ex-tract was stirred for a total of 24 h and was subjected tothe same clean-up procedure as that of the 2 h extract.

The method above was also used for base hydrolysisexperiments, however NaOH (1 M, 2 ml) was addedafter standing the extract for 30 min, and HCl (5 M)was used to neutralise the extracts after 2 and 24 h,respectively.

2.7. LC analysis

LC analyses were performed using a Perkin-Elmerbinary LC pump 250 equipped with a 20�l loop in-jector. A Perkin-Elmer LC-235 Diode Array detector(280 and 240 nm) and a Perkin-Elmer LC-240 Fluores-cence detector (excitation 280 nm, emission 340 nm)connected in series served to monitor the column elu-ent. Separation was achieved on an Alltech AlltimaC18 column (5�m, 150 mm× 4.6 mm) with gradi-

ent elution. Various gradients were compared but thethree most suitable were based on those described byServili et al. [6] and Brenes et al. [2]. The LC systemwas interfaced to a DCM-1488E Lasernet ComputerSystems computer with a 200MB hard disk. Chro-matograms were generated using an LX-300 printer.The mobile phases were degassed under vacuum us-ing Alltech Nylon 66 membranes and continuouslysparged with high purity helium during analysis toprevent resaturation by air.

Gradient 1 [6] used solvent A (water+ acetic acid;100+ 1 v/v) and solvent B (methanol+ acetonitrile+acetic acid; 95+ 5 + 1 v/v/v) with a 2 min isocraticelution (5% solvent B) followed by a stepwise linearincrease to 100% solvent B at 45 min. Gradient 2 [2]used methanol as solvent A with water+ acetic acid(0.02%) as solvent B. Gradient 3 employed the samesolvents as for gradient 1 but with a much shallowergradient comprising a linear increase from 10% to30% solvent B at 10 min followed by a 5 min isocraticelution and then linear increases to 40% solvent B (at25 min), 50% at 40 min and 100% at 50 min with afinal 5 min isocratic run and return to initial conditionsover 25 min.

3. Results and discussion

The phenolic fraction of olive represents a com-plex mixture that is difficult to resolve without com-plex gradients and ternary mobile phases. All threegradients used in this study provided adequate separa-tions (Figs. 1–3) although resolution of some critical“pairs” required use of the shallower gradient (gra-dient 3) at the expense of analysis time. The latter,when used with an Alltech Alltima C18 column (5�m,150 mm× 4.6 mm), was more suited to resolution ofthe range of phenols encountered in drupes prior to,and post, green maturation, and has since been used forroutine phenolic profiling. A high speed C18 column(Alltech Alltima C18 Rocket 3�m, 50 mm× 7 mm)did not provide adequate separation relative to the con-ventional column. Chromatograms obtained with theRocket column exhibited significant baseline noise,and significantly fewer peaks (about 30) were resolvedcompared to chromatograms obtained with the con-ventional C18 column whereby some 60 peaks weredetected via UV monitoring at 280 nm. Retention data

70 D. Ryan et al. / Analytica Chimica Acta 445 (2001) 67–77

Fig. 1. Chromatograms comparing the effect of different mixtures of aqueous methanol on extraction selectivity from olive drupes (purplefruit). Conditions: gradient 1 with detection at 280 nm. Chromatograms are at a fixed attenuation. Extracting solvents were (a) methanol(100%) and (b) methanol+ water (50+ 50 v/v).

using gradient 3 are presented in Table 1 togetherwith an indication of the suitability of fluorescence asan alternative mode of detection. Detection at 240 nmprovided lower selectivity particularly as seen in thedetection of a split peak at 15.0 min (Fig. 3b and e).

This peak, which was not detected by either fluores-cence or absorption at 280 nm, was present in con-trol chromatograms but increased significantly in ex-tracts treated with acid or base, particularly after 2 h.The increase in this peak coincided with a decrease

D. Ryan et al. / Analytica Chimica Acta 445 (2001) 67–77 71

in oleuropein. The identity of this peak has not beenconfirmed but previous studies suggest that it is in-completely resolved elenolic acid and elenolic acidglucoside.

The carbonate extraction method previously re-ported [13] for the extraction of phenolic compoundsfrom olive drupes gave quantitative recovery of oleu-ropein, the major phenolic compound, from spiked

Fig. 2. Chromatograms illustrating the effect of acid and base hydrolysis on the phenolic profile of green olives. Conditions: gradient 3with detection at 280 nm. The different treatments were: (a) control; (b) acid hydrolysis for 2 h, and (c) base hydrolysis for 2 h.

samples. However, extraction from synthetic mixturesof phenols particularly at lower levels than thosepreviously encountered produced variable recoveries.There are wide variations in the levels of variousphenolics in olive which when combined with sea-sonal and varietal differences mandates a more robustextraction procedure. Methanol has been widely usedfor the extraction of phenolic compounds from a va-

72 D. Ryan et al. / Analytica Chimica Acta 445 (2001) 67–77

Fig. 2. (Continued).

Table 1Retention data for selected olive biophenols using gradient 3

Number Phenolic compound Retention time (min) Fluorescence at 340 nm (excitation 280 nm)

1 Gallic acid 5.4 Yes2 Hydroxytyrosol glucoside 8.2 Yes3 Hydroxytyrosol 8.7 Yes4 Tyrosol 11.8 Yes5 Chlorogenic acid 14.6 Yes6 Homovanillic acid 15.0 Yes7 Vanillic acid 15.4 No8 Caffeic acid 16.4 No9 Syringic acid 16.6 Yes

10 p-Coumaric acid 22.7 Weak11 Ferulic acid 24.4 No12 Sinapic acid 24.8 No13 o-Coumaric acid 29.7 No14 Cyanidin chloride 29.9 No15 Luteolin-7-O-glucoside 31.1 No16 Oleuropein 33.3 Yes17 Apigenin-7-glucoside 35.5 No18 Luteolin 47.0 No19 Apigenin 49.0 No

riety of matrices [14] and its use for olive fruit wasexamined in this study.

The effect on selectivity of incorporating water inthe extracting solvent was monitored by LC analysisusing gradient 1. Profiles were generally similar but

significant quantitative differences were observed asillustrated in Fig. 1. A solvent comprising methanol+ water (50+ 50 v/v) provided maximum relative re-covery of phenolics including two strong peaks elutingat 16.8 and 22.2 min (Fig. 1). Thus, methanol+ water

D. Ryan et al. / Analytica Chimica Acta 445 (2001) 67–77 73

Fig. 3. Chromatograms illustrating the effect of acid (a, b and c) and base extraction (d, e and f) on phenolic profile of green olives usingdifferent modes of detection as follows: (a) and (d) absorption at 280 nm; (b) and (e) absorption at 240 nm; and (c) and (f) fluorescenceat 340 nm. The same intensity scales are used in (a), (b), (d) and (e) and in (c) and (f) (time in min). In each case, the chromatogram inthe foreground is shown after 2 h treatment and that in the rear at 24 h.

74 D. Ryan et al. / Analytica Chimica Acta 445 (2001) 67–77

Fig. 3. (Continued).

D. Ryan et al. / Analytica Chimica Acta 445 (2001) 67–77 75

(50+ 50 v/v) was determined to be the best extractingsolvent with respect to selectivity and extraction ef-ficiency. Similarly, an extraction time of 30 min gaveoptimum recovery as assessed by comparison of chro-matographic peak heights. The inclusion of a clean-upstep involving SPE did not improve chromatographicperformance (peak shape, column lifetime) and, insome instances, led to selective losses of phenolics.On the other hand, a simple hexane wash was efficientin removing lipoidal materials without loss of phenolsand was more rapid than SPE.

Profiles of free phenolics recovered from drupes atgreen maturation by aqueous methanol extraction weredominated by the oleuropein peak (Fig. 2a: peak 16)which was present as a minor component in the sam-ples following green maturation (Fig. 1). In the lat-ter, the quantitatively most significant phenols elutedat 16.8 and 22.2 min. These peaks exhibited pseudo-molecular ions in positive and negative ion electro-spray mass spectrometry corresponding to molecularmasses of 624 and 540, respectively. A molecular massof 624 corresponds to that of verbascoside but themass spectra (particularly negative ion) showed sig-nificant differences from that of verbascoside [15] inthat the intensity of peaks atm/z 461 and 161 was verymuch weaker than seen for verbascoside and “acteo-side isomer” [15]. Both verbascoside and the “acteo-side isomer” eluted much later than the 16.8 min seenfor this compound which exhibited an absorption max-imum at 331 nm with a shoulder at 298 nm. The posi-tive and negative ion mass spectra of the later-elutingpeak (retention time 22.2 min) were very similar tothose of oleuropein [15] which eluted, however, at26.3 min under Gradient 1 conditions. Savournin et al.[16] have observed that oleuroside (which is isomericto oleuropein) eluted after oleuropein under reversedphase conditions, and hence the peak at 22.2 min doesnot correspond to oleuroside. The unidentified peakexhibited absorption maxima at 233 and 284 nm.

Oleuropein is generally considered the most signif-icant olive phenol [4] and hence drupes at green mat-uration were used in experiments on acid/base hydrol-ysis. Stability of the control extract at room temper-ature was examined over 24 h and there was no sig-nificant reduction in the oleuropein content over 2 hbut a 14.8% loss after 24 h storage (Table 2). On theother hand, the level of some of the minor componentsdecreased after 2 h storage and thus extracts should

generally be examined with minimal delay or storedat low temperature in the dark. This variation in sta-bility of the various phenols is not surprising in viewof their different antioxidant capacities. Extraction un-der a nitrogen atmosphere did not significantly al-ter the profiles relative to normal atmosphere. Indeed,bubbling air through the extracts showed no apparentenhancement in the rate of decomposition althoughsome early-eluting (before 15 min) peaks appeared tobe air-sensitive under these conditions. However, de-composition (oxidation, hydrolysis) of glycosides thatare generally later-eluting may enhance the content ofsimpler early-eluting phenols. This situation is furthercomplicated in UV detection by the great differencesin molar absorptivities of the various phenols.

4. Effects of acid- and base-treatment on oliveextracts

Acid- and base-treatment of the aqueous methanol-extractable fraction induced hydrolysis or breakdownof the more complex, later-eluting phenolic speciesand enhanced the number and size of the early-elutingpeaks (Figs. 2 and 3). The most notable effect seen inthe UV (240 and 280 nm absorption) and fluorescencechromatograms (280 nm excitation, 340 nm emission)(Fig. 2) was the reduction in the amount of oleuropeinin both acid- and base-treated extracts. Acid-treatment(extraction and hydrolysis) produced a 40 and 99%loss in oleuropein after 2 and 24 h, respectively. Thecorresponding values for base-treatment (extractionand hydrolysis) were 98 and 100%, respectively. Caf-feic acid is of particular interest as it is one of the ma-jor phenolic acids in plants. The level of caffeic acid incontrol samples decreased significantly after 2 h, con-sistent with the reactivity of caffeic acid inferred byits o-diphenolic grouping. However, the rise in levelof caffeic acid at 24 h can be attributed to degradationof caffeic containing species. In contrast, the additionof base to olive extracts resulted in an increase in caf-feic acid concentration after 2 h (compared to controlextracts at 2 h), followed by a complete disappearanceafter 24 h. The levels of caffeic acid in acid-treatedextracts were more variable (Table 2).

An other significant difference between acid- andbase-treatment of extracts was seen in the formationof an unknown compound B (retention time 22.9 min)

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in base-treated extracts (see Table 2, Fig. 2). Levelsof this compound were very similar in base-treatedextracts after 2 and 24 h. A further unknown com-pound A (Table 2, Fig. 2), retention time 9.6 min,was produced in both acid- and base-treated extracts.The amount of hydroxytyrosol produced was minimalalthough higher in base-treated than acid-treated ex-tracts. Tyrosol was detected in acid- and base-treatedextracts, but was higher in the latter. The origin of thisis unclear but it may arise from ligstroside.

The major degradation product from oleuropeinin both acid- and base-treated extracts was hydrox-ytyrosol glucoside. The amount of this compoundproduced after 2 h of treatment with base was ap-proximately 0.50 mg g−1 (as tyrosol) (Table 2). Nofurther hydroxytyrosol glucoside was produced after24 h consistent with its production from oleuropeinand the complete destruction of oleuropein after 2 htreatment with base. The amount of hydroxytyrosolglucoside produced by acid-treatment (extraction andhydrolysis) increased significantly between 2 and 24 hconsistent with the extracts containing residual oleu-ropein at 2 h (Table 2). The amount of hydroxytyrosolglucoside that was produced after 24 h acid-treatmentwas approximately 50 times that produced in basicextracts (Table 2).

It is particularly notable that the level of phenolswas generally the same for both extraction and hy-drolysis treatments. This is in sharp contrast to citrusfruits where base hydrolysis, particularly, liberates ad-ditional phenolic acids from the cell walls. This wasevidenced by Fallico et al. [17] and Peleg et al. [18] inthat the level of free acids as determined by direct ex-traction of orange juice was very low compared withthat of bound phenolic acids released by hydrolysis.This suggests that the phenolic components of cellmembranes in olive drupes are resistant to both acidand base.

Olive products are regarded as important com-ponents of a healthy diet because of their phenoliccontent. There are limited data on the absorption andmetabolism of plant phenols and it is unclear whetherfree or total phenols is important in dietary studies.These results suggest that the amount of total phenols

in olive products will not vary greatly from that offree phenols. However, further studies are clearly in-dicated.

Acknowledgements

The assistance of Dr. Daniel Jardine, Department ofChemistry, Macquarie University in providing LC–MSdata for compounds in Fig. 1 is gratefully acknowl-edged.

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