food &function human absorption and...

Mol. Nutr. Food Res. 2013, 57, 2079–2085 2079DOI 10.1002/mnfr.201200795

FOOD & FUNCTION

Human absorption and metabolism of oleuropein

and hydroxytyrosol ingested as olive (Olea europaea L.)

leaf extract

Martin de Bock1, Eric B. Thorstensen1, Jose G. B. Derraik1, Harold V. Henderson2,Paul L. Hofman1,3 and Wayne S. Cutfield1,3

1 Liggins Institute, University of Auckland, Auckland, New Zealand2 AgResearch, Ruakura Research Centre, Hamilton, New Zealand3 Gravida: National Centre for Growth and Development, Auckland, New Zealand

Phenolic compounds derived from the olive plant (Olea europaea L.), particularly hydroxyty-rosol and oleuropein, have many beneficial effects in vitro. Olive leaves are the richest sourceof olive phenolic compounds, and olive leaf extract (OLE) is now a popular nutraceutical takeneither as liquid or capsules. To quantify the bioavailability and metabolism of oleuropein andhydroxytyrosol when taken as OLE, nine volunteers (five males) aged 42.8 ± 7.4 years wererandomized to receive either capsulated or liquid OLE as a single lower (51.1 mg oleuropein,9.7 mg hydroxytyrosol) or higher (76.6 mg oleuropein, 14.5 mg hydroxytyrosol) dose, andthen the opposite strength (but same formulation) a week later. Plasma and urine sampleswere collected at fixed intervals for 24 h post-ingestion. Phenolic content was analyzed byLC-ESI-MS/MS. Conjugated metabolites of hydroxytyrosol were the primary metabolites re-covered in plasma and urine after OLE ingestion. Peak oleuropein concentrations in plasmawere greater following ingestion of liquid than capsule preparations (0.47 versus 2.74 ng/mL;p = 0.004), but no such effect was observed for peak concentrations of conjugated (sulfated andglucuronidated) hydroxytyrosol (p = 0.94). However, the latter peak was reached earlier withliquid preparation (93 versus 64 min; p = 0.031). There was a gender effect on the bioavailabilityof phenolic compounds, with males displaying greater plasma area under the curve for con-jugated hydroxytyrosol (11 600 versus 2550 ng/mL; p = 0.048). All conjugated hydroxytyrosolmetabolites were recovered in the urine within 8 h. There was wide inter-individual variation.OLE effectively delivers oleuropein and hydroxytrosol metabolites to plasma in humans.

Keywords:

Bioavailability / Hydroxytyrosol / Oleuropein / Olive leaf extract / Phenols

Received: December 2, 2012Revised: March 24, 2013Accepted: April 5, 2013

� Additional supporting information may be found in the online version of this article atthe publisher’s web-site

The health benefits associated with the Mediterranean dietare well established, including protection from cardiovascu-lar disease, age-related cognitive decline, and cancer [1]. Suchbenefits were thought to be associated with the consump-tion of MUFA, but have more recently been attributed tothe high intake of phenolic compounds that are abundant

Correspondence: Dr. Wayne S. Cutfield, Liggins Institute, Univer-sity of Auckland, Private Bag 92019, Auckland, New ZealandE-mail: [email protected]: +64-9-373-8763

Abbreviations: AUC, area under the curve; OLE, olive leaf extract

in olive oil [2]. Phenolic compounds derived from the oliveplant (Olea europaea L.) have a range of beneficial health ef-fects, with antioxidant, anti-inflammatory, antiatherogenic,and anticarcinogenic properties [3].

Previous literature on olive phenols has focused onolive oil consumption, as this is the most common di-etary source. However, phenolic compounds in the oliveplant are mostly concentrated in the leaves. These werepreviously discarded as byproducts of fruit harvesting, buthave recently emerged as commercially valuable nutraceuti-cals. Despite the growing market of olive leaf extract (OLE),there is scarce literature examining the consequent absorp-tion and metabolism of its principal phenols (oleuropein

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com

2080 M. de Bock et al. Mol. Nutr. Food Res. 2013, 57, 2079–2085

and hydroxytyrosol). In contrast, the literature on phenolicabsorption and metabolism after olive oil ingestion isextensive [4, 5].

The unique olive plant polyphenol is oleuropein, which ismost abundant in the leaves (up to 264 mg/g of dry leaf, whenexpressed as tyrosol equivalents) [6]. Oleuropein concentra-tions are comparatively lower in extra virgin olive oil [7], andespecially so in refined oil due to its hydrolysis into tyrosoland hydroxytyrosol during processing [4]. It is important toinvestigate the fate of oleuropein and hydroxytyrosol ingestedvia OLE rather than olive oil, due to different phenolic con-centrations, chemical forms, and modes of delivery (e.g. cap-sules versus liquid). All of these factors are likely to affectmetabolism and should be explored before any bioactivitycan be attributed to OLE consumption (as a nutraceutical).

Thus, we aimed to quantitatively determine the absorptionand metabolism of oleuropein and hydroxytyrosol in humans,following acute ingestion of different doses and formulationsof OLE. We used a previously validated technique [8], specifi-cally looking for oleuropein, hydroxytyrosol, and homovanillicacid. Further, we also adopted �-glucuronidase hydrolysis todetermine conjugated (sulfated and glucuronidated) metabo-lites of hydroxytyrosol.

Ethics approval for this study was provided by the North-ern Y Regional Ethics Committee, Ministry of Health, NewZealand (NTY/11/02/015). Written informed consent wasobtained from participants. Five male and five female sub-jects were recruited within the University of Auckland viaemail advertising. This sample size is comparable to previ-ous studies in this field [5, 9–13]. Subjects were aged 42.8 ±7.4 years (range 31.7–54.0) and with BMI 26.9 ± 1.9 kg/m2

(range 24.6–29.8). All subjects were healthy, free of chronicillnesses, and nonsmokers. Women were excluded if men-struating during the study. Note that to minimize the ef-fects of dietary intake, assessments were carried out after anovernight fast (≥8 h), and participants were also instructed toavoid all olive products and alcohol for at least 24 h prior toassessment (olive metabolites are eliminated well within thistime [14]).

Participants were randomized (Supporting Information)to receive OLE in either capsulated or liquid form (Comvita,Auckland, New Zealand) to assess effects of delivery method.A possible dose affect was also investigated. There is no es-tablished standard dose of oleuropein, and commercial OLEproducts suggest 20–100 mg/day. Each subject received twosingle doses of OLE within this range: either a lower (with51.1 mg oleuropein) or higher (76.6 mg) dose. Participantsreceived the opposite strength (but same formulation) a weeklater. The oleuropein dose was matched between the twodifferent preparations, but hydroxytyrosol concentration washigher in capsules than liquid (9.7 versus 5.4 mg for lowerdose, and 14.5 versus 8.1 mg for higher dose). Thus, oleu-ropein accounted for 82.6% of phenols in both preparations,but hydroxytyrosol accounted for 15.5 and 8.5% of phenols incapsules and liquid preparations, respectively. Phenolic con-tent in capsules and liquid preparations was independentlyverified (Conmac Laboratory Services, Queensland, Australia;Supporting Information Table 1). Minor changes in otherphenolic compounds present at much lower concentrationswere not examined.

Concentrations of oleuropein and its metabolites inplasma and urine were quantified by LC-ESI-MS/MS [8](Supporting Information). Data were analyzed using linearmixed models with repeated measures (SAS Institute, Cary,NC, USA). Models accounted for preparation (capsule versusliquid), dose (higher versus lower), sex (male versus female),and between-subject differences. Data are expressed as meansand 95% confidence intervals, adjusted for confounding fac-tors from multivariate models.

Nine patients completed both doses (one woman takingliquid formulation did not complete). No adverse effects werenoted, and liver function (aspartate aminotransferase, ala-nine aminotransferase, alkaline phosphatase, gamma glu-tamyl transferase, and international normalized ratio) wasunaltered. Conjugated metabolites of hydroxytyrosol (glu-curonidated and sulfated) made up 96–99% of OLE phenolicmetabolites detected in plasma, with much less oleuropeinrecorded. Hydroxytyrosol acetate sulfate (recently identified

Table 1. Bioavailability parameters of oleuropein and conjugated metabolites of hydroxytyrosol in plasma after OLE ingestion, accordingto preparation and dose

Biochemical Capsules Liquid preparation

Lower dose Higher dose Lower dose Higher dose

n 5 5 4 4Sex ratio (males/females) 3/2 2/3 1/3 3/1Oleuropein

Peak (ng/mL) 0.52 ± 0.24 0.60 ± 0.37 2.55 ± 2.39 3.55 ± 2.27AUC (ng/mL) 96 ± 44 101 ± 104 197 ± 160 214 ± 136Time to peak (min) 40 ± 27 38 ± 22 20 ± 12 20 ± 12

Conjugated metabolites of hydroxytyrosolPeak (ng/mL) 61 ± 69 67 ± 50 148 ± 154 149 ± 99AUC (ng/mL) 5565 ± 4864 6517 ± 5093 13 356 ± 11 678 13 112 ± 8784Time to peak (min) 90 ± 0 96 ± 13 53 ± 29 75 ± 17

Data are means and SDs.


Mol. Nutr. Food Res. 2013, 57, 2079–2085 2081

in plasma [15]) was not detected, but homovanillic acid was de-tected in trace amounts in urine and plasma. LC-ESI-MS/MSdata on recorded metabolites are provided (Supporting Infor-mation Fig. S1).

Table 1 provides descriptive data on observed plasma phe-nolic metabolites. Table 2 shows the effects of preparation,dose, and gender on phenolic bioavailability. Liquid prepa-ration led to peak plasma oleuropein concentrations sixfoldhigher than capsules (p = 0.004), and area under the curve(AUC) fourfold greater (p = 0.040; Table 2). No furtheroleuropein was recovered following �-glucuronidase hydrol-ysis. OLE dose did not significantly affect plasma parametersfor oleuropein or conjugated metabolites of hydroxytyrosol(Table 2), possibly due to sample size and/or marked in-terindividual variation.

Oleuropein and hydroxytyrosol metabolites were rapidlydetected in plasma after ingestion (23–93 min), witholeuropein concentrations peaking earlier than conjugates(Table 2; Fig. 1). Peak concentrations were reached earlierwith liquid than capsule preparations (Table 2; Fig. 1).

Peak plasma metabolite concentrations varied consider-ably among participants (coefficients of variation (SD/mean):46–122%), depending also on formulation and dose (datanot shown). Further, despite small sample size, there wasa marked gender effect. Males tended to have greater peakconjugated hydroxytyrosol concentrations than females (p =0.076; Table 2). Males also had plasma-conjugated hydroxy-tyrosol AUC 4.5× higher (p = 0.048), but plasma oleu-ropein AUC that tended to be threefold lower (p = 0.085;Table 2).

Conjugated metabolites of hydroxytyrosol were the pri-mary metabolites detected in urine, and concentrations wereunaffected by preparation (p = 0.33), dose (p = 0.70), or gen-der (p = 0.12). Most recovery occurred with in 8 h of ingestion(Supporting Information Fig. S2).

This is the first study to assess absorption and metabolismof oleuropein and hydroxytyrosol in human plasma followingOLE ingestion (and establish its time course). Predominantolive phenolic metabolites in plasma and urine were conju-gated (sulfated and glucuronidated) hydroxytyrosol metabo-lites. Table 3 compares our data to previous studies.

Our findings contrast to the only study that previouslyexamined oleuropein metabolism taken as OLE. After chronicingestion of OLE, phase II metabolites of oleuropein werealmost exclusively found in urine (glucuronidated at differentpositions), with no conjugated metabolites of hydroxytyrosol[9]. Phase II metabolites of oleuropein were detected here, butthese could not be quantified due to a lack of a standard. Ourcontrasting findings may result from subtle differences inphenolic composition of consumed extracts, which are alteredby agronomic and technological factors [16], methodologicalissues, and between-subject differences.

Main metabolites recorded here were sulfated and glu-curonidated conjugates of hydroxytyrosol, with only traceamounts of homovanillic acid and no hydroxytyrosol ac-etate sulfate observed. However, sulfated and glucuronidated T

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Figure 1. Plasma conjugated (glu-curonidated and sulfated) hydroxyty-rosol and oleuropein concentrationsover 8 h following OLE ingestion in cap-sule and liquid forms, at lower (64 mgtotal olive phenols, with 51.1 mg oleu-ropein) and higher (96 mg total, 76.6mg oleuropein) doses.

conjugates could not be individually quantified due to un-availability of specific standards. Our findings differ slightlyto previous studies (Table 3), two of which on rats [17, 18],and two human studies using olive cake to enrich theolive oil (altering the ratio of oleuropein derivatives in theconsumed sample) [5, 10]. Different forms of oleuropeinand derivatives consumed may influence the pathway ofconjugation [15].

In our study, mean time to peak for conjugated hydrox-ytyrosol metabolites following ingestion of liquid OLE was64 min. Peak plasma concentrations following olive oil in-gestion seem to occur much earlier (c.f. 30 min [10] and

32 min [13]). We observed large interindividual variation inabsorption and metabolism of phenolic compounds, possi-bly resulting from differences in human enzymatic activ-ity [19]. Importantly, despite our small sample size (thathinders statistical power), there were marked gender differ-ences, which is a novel finding. Males may be more effi-cient at conjugating oleuropein, which would explain theirlower AUC for oleuropein but higher AUC for hydroxyty-rosol metabolites. Although these observations need to beconfirmed by larger studies, pharmacokinetic and pharmaco-dynamic gender differences are well recognized [20]. Our ob-servations are important, suggesting effects of olive phenolic


Mol. Nutr. Food Res. 2013, 57, 2079–2085 2083Ta

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compounds may vary somewhat between males and females.Nonetheless, there could also be considerable daily varia-tion within individuals, which may have important clinicalimplications.

Compared to capsules, OLE in a liquid formulation ledto greater oleuropein peak levels and AUC in plasma. Therewas also an earlier metabolite peak associated with liquid for-mulation, also seen in other compounds primarily absorbedin the small intestine (e.g. paracetamol [21]). However, thesixfold higher peak oleuropein concentrations following in-gestion of OLE in liquid form were notable, even if they wereconsiderably lower than those of conjugated hydroxytyrosolmetabolites. Many factors may account for the observed dif-ferences, such as time taken for capsules to dissolve, thesuspension (glycerine versus safflower oil), or rapid deliv-ery of liquid formulations allowing escape from hydrolysis.Nonetheless, our results indicate that the OLE formulationis likely to be an important issue for nutraceutical compa-nies, and possibly influence choice among better informedconsumers.

Previous literature on the bioactivity of olive phenolshas focused on hydroxytyrosol and oleuropein. However,we showed that conjugated metabolites of hydroxytyrosolare the main olive phenolic metabolites found in biologi-cal fluids in vivo, corroborating previous findings (Table 3).There is emerging literature on bioactivity of these metabo-lites. For example, glucuronidated hydroxytyrosol is a free-radical scavenger five times more potent than the parent hy-droxytyrosol [18]. Although glucuronidated hydroxytyrosol isinactive against LDL-cholesterol oxidation [11], it protectedrenal tubular cells against oxidative damage in vitro [22].However, the long-held dogma that reactive oxygen speciesare associated with the progressive development of systemicdisease has been questioned, as superoxides are essential fornormal metabolome and physiological function [23]. Further,in vitro and animal experiments typically use phenolic dosesseveral fold higher or compounds that are not in their orig-inal form, which may lead to harmful effects that should beconsidered in future human studies [24]. Nonetheless, werecently showed improved insulin sensitivity in humans fol-lowing chronic ingestion of OLE (same lower dose liquidpreparation used here), without any harmful effects [25].

Alternate explanation for olive phenol bioactivity hasbeen proposed, with downregulation of proatherogenic genes(such as IFN-�, ARHGAP15, and IL7R) observed after olive oilconsumption [26]. There is also in vitro evidence of nutrige-nomic interaction on several cell lines covering cancer [27]and insulin sensitivity [28] genes.

In summary, conjugated (sulfated and glucuronidated)metabolites of hydroxytyrosol were the primary oleuropeinmetabolites recovered in plasma and urine following OLEconsumption. Absorption and metabolism of olive phe-nolic compounds to plasma is relatively rapid, as is renalclearance. Bioavailability and metabolism of oleuropeinare heterogeneous and highly dependent on a number offactors, including preparation (capsule/liquid) and gender,

which require verification in larger studies. The form ofoleuropein and the delivery method (OLE versus oil) alsoaffect absorption and metabolism. The bioactivity of the con-jugated metabolites needs to be further defined, and possibletoxicity levels explored. Chronic ingestion may influenceenzymatic activity and hence absorption and metabolism.Future research should also examine tissue distribution oflabeled olive phenolic compounds and identify individualswith greater absorption.

This study was supported by a TECHNZ grant (University ofAuckland–UniS 30475.001) through the New Zealand Ministryof Science and Innovation (MSI). TECHNZ grants are funded50% by the MSI and 50% by a commercial partner followingan extensive independent science review process. In this project,the commercial partner was the olive leaf extract manufacturer(Comvita). The first author (M. d. B.) was funded by the JoanMary Reynolds Trust.

Potential conflict of interest statement: All authors are com-pletely independent from the funders (Comvita and MSI), sothat neither the MSI nor the capsules supplier (Comvita) hadany role in study design, data collection and analysis, deci-sion to publish, or preparation of this manuscript. All au-thors have completed the Unified Competing Interest form atwww.icmje.org/coi_disclosure.pdf (available on request from thecorresponding author) and declared that (i) all authors have hadno relationships with Comvita that might have an interest in thesubmitted work; (ii) the authors’ spouses, partners, or childrenalso have no financial relationships that may be relevant to thesubmitted work; and (iii) none of the authors have any nonfinan-cial interests that may be relevant to the submitted work.

References

[1] Lopez-Miranda, J., Perez-Jimenez, F., Ros, E., De Caterina,R. et al., Olive oil and health: summary of the II interna-tional conference on olive oil and health consensus report,Jaen and Cordoba (Spain) 2008. Nutr. Metab. Cardiovasc.Dis. 2010, 20, 284–294.

[2] Covas, M. I., Nyyssonen, K., Poulsen, H. E., Kaikkonen, J.et al., The effect of polyphenols in olive oil on heart diseaserisk factors. Ann. Intern. Med. 2006, 145, 394–395.

[3] Cicerale, S., Lucas, L., Keast, R., Biological activities of phe-nolic compounds present in virgin olive oil. Int. J. Mol. Sci.2010, 11, 458–479.

[4] Vissers, M. N., Zock, P. L., Roodenburg, A. J. C., Leenen, R.et al., Olive oil phenols are absorbed in humans. J. Nutr.2002, 132, 409–417.

[5] Rubio, L., Valls, R. M., Macia, A., Pedret, A. et al., Impact ofolive oil phenolic concentration on human plasmatic pheno-lic metabolites. Food Chem. 2012, 135, 2922–2929.

[6] Preedy, V. R., Watson, R. R., Olives and Olive Oil in Health andDisease Prevention, Academic Press, San Diego, CA 2010.

[7] Montedoro, G., Servili, M., Baldioli, M., Miniati, E., Simpleand hydrolyzable phenolic compounds in virgin olive oil. 1.


Mol. Nutr. Food Res. 2013, 57, 2079–2085 2085

Their extraction, separation, and quantitative and semiquan-titative evaluation by HPLC. J. Agric. Food Chem. 1992, 40,1571–1576.

[8] Bazoti, F. N., Gikas, E., Tsarbopoulos, A., Simultaneous quan-tification of oleuropein and its metabolites in rat plasma byliquid chromatography electrospray ionization tandem massspectrometry. Biomed. Chromatogr. 2010, 24, 506–515.

[9] Kendall, M., Batterham, M., Callahan, D. L., Jardine, D. et al.,Randomized controlled study of the urinary excretion of bio-phenols following acute and chronic intake of olive leaf sup-plements. Food Chem. 2012, 130, 651–659.

[10] Suarez, M., Valls, R. M., Romero, M. P., Macia, A. et al.,Bioavailability of phenols from a phenol-enriched olive oil.Br. J. Nutr. 2011, 106, 1691–1701.

[11] Khymenets, O., Fito, M., Tourino, S., Munoz-Aguayo, D. et al.,Antioxidant activities of hydroxytyrosol main metabolites donot contribute to beneficial health effects after olive oil in-gestion. Drug Metab. Dispos. 2010, 38, 1417–1421.

[12] Garcıa-Villalba, R., Carrasco-Pancorbo, A., Nevedomskaya,E., Mayboroda, O. A. et al., Exploratory analysis of humanurine by LC–ESI-TOF MS after high intake of olive oil: un-derstanding the metabolism of polyphenols. Anal. Bioanal.Chem. 2010, 398, 463–475.

[13] Miro-Casas, E., Covas, M. I., Farre, M., Fito, M. et al., Hy-droxytyrosol disposition in humans. Clin. Chem. 2003, 49,945–952.

[14] Gonzalez-Santiago, M., Fonolla, J., Lopez-Huertas, E., Hu-man absorption of a supplement containing purified hydrox-ytyrosol, a natural antioxidant from olive oil, and evidencefor its transient association with low-density lipoproteins.Pharmacol. Res. 2010, 61, 364–370.

[15] Rubio, L., Macia, A., Valls, R. M., Pedret, A. et al., Anew hydroxytyrosol metabolite identified in human plasma:hydroxytyrosol acetate sulphate. Food Chem. 2012, 134,1132–1136.

[16] Montedoro, G., Servili, M., Olive oil quality parameters inrelationship to agronomic and technological aspects. Riv.Ital. Sostanze Gr. 1992, 69, 563–573.

[17] D’Angelo, S., Manna, C., Migliardi, V., Mazzoni, O. et al., Phar-macokinetics and metabolism of hydroxytyrosol, a naturalantioxidant from olive oil. Drug Metab. Dispos. 2001, 29,1492–1498.

[18] Tuck, K. L., Hayball, P. J., Stupans, I., Structural characteriza-tion of the metabolites of hydroxytyrosol, the principal phe-

nolic component in olive oil, in rats. J. Agric. Food Chem.2002, 50, 2404–2409.

[19] de Bock, M., Derraik, J. G. B., Cutfield, W., Polyphenols andglucose homeostasis in humans. J. Acad. Nutr. Diet 2012,112, 808–815.

[20] Morris, M. E., Lee, H. J., Predko, L. M., Gender differencesin the membrane transport of endogenous and exogenouscompounds. Pharmacol. Rev. 2003, 55, 229–240.

[21] Kelly, K., O’Mahony, B., Lindsay, B., Jones, T. et al., Compari-son of the rates of disintegration, gastric emptying, and drugabsorption following administration of a new and a conven-tional paracetamol formulation, using � scintigraphy. Pharm.Res. 2003, 20, 1668–1673.

[22] Deiana, M., Incani, A., Rosa, A., Atzeri, A. et al., Hydrox-ytyrosol glucuronides protect renal tubular epithelial cellsagainst H2O2 induced oxidative damage. Chem. Biol. Inter-act. 2011, 193, 232–239.

[23] Linnane, A. W., Kios, M., Vitetta, L., Healthy aging: regula-tion of the metabolome by cellular redox modulation andprooxidant signaling systems: the essential roles of super-oxide anion and hydrogen peroxide. Biogerontology 2007,8, 445–467.

[24] Acin, S., Navarro, M. A., Arbones-Mainar, J. M., Guillen, N.et al., Hydroxytyrosol administration enhances atheroscle-rotic lesion development in apo E deficient mice. J. Biochem.2006, 140, 383–391.

[25] de Bock, M., Derraik, J. G. B., Brennan, C. M., Biggs, J. B.et al., Olive (Olea europaea L.) leaf polyphenols improve in-sulin sensitivity in middle-aged overweight men: a random-ized, placebo-controlled, crossover trial. PLoS One 2013, 8,e57622.

[26] Konstantinidou, V., Covas, M. I., Munoz-Aguayo, D.,Khymenets, O. et al., In vivo nutrigenomic effects of vir-gin olive oil polyphenols within the frame of the Mediter-ranean diet: a randomized controlled trial. FASEB J. 2010, 24,2546–2557.

[27] Khymenets, O., Fito, M., Covas, M. I., Farre, M. et al.,Mononuclear cell transcriptome response after sustained vir-gin olive oil consumption in humans: an exploratory nutrige-nomics study. OMICS 2009, 13, 7–19.

[28] Konstantinidou, V., Khymenets, O., Covas, M. I., de la Torre,R. et al., Time course of changes in the expression of insulinsensitivity-related genes after an acute load of virgin oliveoil. OMICS 2009, 13, 431–438.


1

Online Supporting Information

Human absorption and metabolism of oleuropein and

hydroxytyrosol ingested as olive (Olea europaea L.)

leaf extract

Martin de Bock1, Eric B. Thorstensen

1, José G B Derraik

1, Harold V Henderson

2, Paul L

Hofman1,3

, Wayne S Cutfield1,3*

1 Liggins Institute, University of Auckland, Auckland, New Zealand

2 AgResearch, Ruakura Research Centre, Hamilton, New Zealand

3 Gravida – National Centre for Growth and Development, Auckland, New Zealand

*Author for correspondence: Liggins Institute, University of Auckland, Private Bag 92019,

Auckland, New Zealand; Ph: +64.9.923.5118; Fax: +64.9.373.8763; Email:

[email protected]

2

MATERIALS AND METHODS

Ethics approval

Ethics approval for this study was provided by the Northern Y Regional Ethics Committee

(Ministry of Health, New Zealand), approval number NTY/11/02/015. Written informed

consent was obtained from participants.

Subjects

Male and female subjects were recruited in equal numbers within the University of Auckland

via email advertising. Inclusion criteria required that all subjects were healthy, free of chronic

illnesses, and not regular drug users (including tobacco). Women were excluded if

menstruation occurred during the study. A total of five men and five women entered the study,

and were aged 42.8 ± 7.4 years (range 31.7–54.0 years) and with BMI 26.9 ± 1.9 kg/m2 (range

24.6–29.8 kg/m2).

Study design

The study was designed to evaluate the concentrations of olive phenolic metabolites in both

plasma and urine, following acute ingestion of OLE according to dose (lower vs higher) and

preparation (capsule vs liquid).

Using a random number generator, participants were randomly allocated at a 1:1 ratio to

receive either capsules or the liquid preparation throughout the study. Subsequently, each

participant was similarly assigned at random to receive doses in one of two sequences: either

lower-higher or higher-lower.

3

All clinical assessments were carried out at the Maurice & Agnes Paykel Clinical Research

Unit (Liggins Institute, University of Auckland). On arrival, participants were weighed and

measured, and asked to void urine. An intra-venous cannula was then placed, and the OLE

preparation was ingested.

Blood samples were drawn at 0, 10, 30, 60, 90, 120, 150, 180, 240, 360, 480 minutes, and 24

hours post-ingestion. Samples were collected into heparin tubes on ice, centrifuged, and plasma

was stored at -20°C until analysis. Urine samples were collected for 24 hours after OLE

ingestion. Once participants ingested the OLE preparation, they remained in the Maurice &

Agnes Paykel Clinical Research Unit fasted for the initial 8 hours, during which only water

was to be consumed. Subsequently, over the next 16 hours, participants could eat and drink ad

libitum, but were instructed to avoid alcohol and olive-containing products, and were instructed

to collect all urine.

Olive leaf extract

The olive phenolic content of the OLE (Comvita, Auckland, New Zealand) used in capsules

and liquid preparations were independently verified (Conmac Laboratory Services,

Queensland, Australia) (Table S1). Each OLE capsule contained 400 mg of the extract, as well

as 672.5 mg safflower oil, 150 mg lecithin, and 27.5 mg silica-colloidal anhydrous. The liquid

OLE preparation was suspended in glycerol. Participants taking capsules received either four

(51.1 mg oleuropein, 9.7 mg hydroxytyrosol) or six (76.6 mg oleuropein, 14.5 mg

hydroxytyrosol) capsules, taken a week apart. Those randomized to receive the liquid

preparation were matched to receive similar phenolic doses (Table S1).

4

Outcome measures

Parameters of interest were peak oleuropein, oleuropein area under the curve (AUC), peak

hydroxytyrosol, and conjugated metabolites of hydroxytyrosol AUC. For safety assessment,

plasma was collected for liver function tests at each assessment, with measurements of

aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase

(ALP), and gamma glutamyl transferase (GGT), and international normalised ratio (INR) for

liver synthetic function.

Chemical Analysis

Plasma and urine quantification of oleuropein and its metabolites were determined by LC-ESI-

MS/MS, as described previously [1]. Methodological exceptions were:

a) ß-glucuronidase hydrolysis: 0.05M acetate buffer used.

b) Solid phase extraction: Phenomenex (Auckland, New Zealand) Strata C18-E 100mg/3mL

cartridges were used, and supernatant transferred to tubes containing 25 ng/ml of internal

standard (2-Hydroxyphenylethanol (2-HPE), Sigma-Aldrich, St Louis, MO, USA); analytes

were eluted with 2.5 ml of ethyl acetate and evaporated to dryness under vacuum for several

minutes; residue was reconstituted with 9:1 H20 (pH 3.7):acetonitrile (Merck, KGaA,

Germany)

c) Instrumentation: all LC-HESI MS/MS analysis was performed on a Finnigan TSQ Quantum

Ultra Accurate Mass Triple-quadrupole system (Thermo Fisher Scientific, San Jose, CA,

USA); chromatographic separations were carried out using a Accela autosampler and 1250

pump (Thermo Fisher Scientific).

The separation was performed at 30oC on a Luna 2.5µm C18(2) – HST 100x3mm column

(Phenomenex) preceded by a KrudKatcher (Phenomenex) in-line filter. A gradient was used to

5

separate the analytes, solvent A was water (adjusted to pH 3.7) and solvent B was acetonitrile

(Merck, Darmstadt, Germany) (Table S2). A flow rate of 250 µl/min was maintained

throughout the separation.

Mass spectral analyses were carried out in negative ion mode for all analytes using the

following tune parameters: spray voltage 3500V, vaporiser temperature 320oC, and capillary

temperature 250oC. Nitrogen gas was used as sheath, ion sweep and auxiliary gas at 50, 1, and

2 psi, respectively, with argon as the collision gas. Single reaction monitoring (SRM)

parameters and the retention time were established for each compound, and their collision

pressures and energies are listed in Table S3. Quantification of the analytes was established by

assessing the peak area ratios of each analyte to that of the internal standard and comparing

them with the corresponding ratios of spiked control plasma (range 0.25–50 ng/ml), the intra

and inter-assay %CV’s for HT were 9 and 12%, respectively. Pre-filtered baseline and 8-hour

urine samples were injected unextracted into the mass spectrometer to identify the presence of

oleuropein and hydroxytyrosol metabolites following OLE consumption using single ion

monitoring (SIM).

AST, ALT, ALP, and GGT concentrations were measured on a Hitachi 902 autoanalyser

(Hitachi High Technologies Corporation, Tokyo, Japan) by enzymatic colorimetric assay

(Roche, Mannheim, Germany), with inter-assay CV’s of less than 2.5%. INR was assessed

using a point of care Coagucheck XS system (Roche Diagnostics, New Zealand).

Statistical analysis

Data were analysed in SAS v.9.2 (SAS Institute, Cary, NC, USA) using a linear mixed model

design with repeated measures. Models accounted for preparation (capsule vs liquid), dose

(higher vs lower), sex (male vs female), and between-subject differences. Models were also run

6

accounting for a possible interaction between preparation and dose. If necessary, data were log-

transformed to approximate normality. Data are expressed as the means and 95% confidence

intervals adjusted for confounding factors in the multivariate models (back-transformed where

appropriate).

Supplementary results

SIM results revealed the presence of hydroxytyrosol sulphate, hydroxytyrosol glucuronide, and

two open-ring oleuropein phase II metabolites (at m/z 553.15 and 555.17, respectively) (Figure

S1), which have been previously described [2]. SRM analysis further confirmed the identity of

the two hydroxytyrosol derivatives with parent – daughter ion transitions of 233-153 m/z and

329-153 m/z, respectively (Figure S3). It was not possible to determine the absolute amounts

of any of these individual metabolites, because of the lack of commercially available standards.

Note that there was a 6.9% difference in hydroxytrosol concentration between capsule and liquid

preparations. Thus, to ensure that this difference (although very small) would not affect the results, the

analyses were run with the liquid data adjusted (multiplied by approximately 1.08) to account for this

difference. However, as the results were mostly unchanged, these results are not reported.

REFERENCES

[1] Bazoti, F. N., Gikas, E., Tsarbopoulos, A., Simultaneous quantification of oleuropein and its

metabolites in rat plasma by liquid chromatography electrospray ionization tandem mass

spectrometry. Biomed Chromatography. 2010, 24, 506-515.

[2] Kendall, M., Batterham, M., Callahan, D. L., Jardine, D. et al., Randomized controlled study of the

urinary excretion of biophenols following acute and chronic intake of olive leaf supplements. Food

Chem. 2011.

7

Table S1. Phenolic content in lower and higher doses of capsules and liquid OLE preparations.

Lower Dose

Higher Dose

Polyphenol content (mg) 4 capsules 11.1 ml liquid

6 capsules 16.7 ml liquid

Oleuropein 51.124 51.030

76.600 76.600

Hydroxytyrosol 9.666 5.410

14.499 8.144

Rutin 0.150 0.418

0.225 0.627

Quercetin 0.038 0.517

0.057 0.776

Kaempferol 0.021 0.100

0.0315 0.15

Apigenenin 0.046 0.416

0.069 0.624

Flavonoid 0.028 0.254

0.042 0.381

Verbascoside 0.344 0.595

0.516 0.893

Phenolic acids1 0.233 0.075

0.350 0.113

Oleic acid 0.013 0.010

0.020 0.015

Luteolin 0.249 2.830

0.374 4.245

1 Phenolic acids are calculated as caffeic acid.

8

Table S2. Solvent gradient for the separation of oleuropein and hydroxytyrosol.

Time (min) % Solvent A % Solvent B

0.0 85 15

3.0 85 15

10.0 25 75

11.0 85 15

15.0 85 15

9

Table S3. Liquid Chromatography and mass spectrometer parameters

Oleuropein Hydroxytyrosol 2-hydroxyphenylethanol

Molecular weight (g/mol) 540.51 154.16 138.16

Single reaction monitoring (m/z) 539.11 ± 275.08 153.04 ± 123.10 137.06 ± 107.10

Retention time (min) 8.5 3.4 6.7

Collision E (V) 23 18 14

Collision P (mTorr) 1.2 1.2 1.2

Supporting Information Figure S1. Sample single ion monitoring (SIM) output showing urine

metabolites: A, sulphated hydroxytyrosol; B, glucuronidated hydroxytyrosol; C and D, open-ring

glucuronidated oleuropein metabolites.

A

B

C

D

Supporting Information Figure S2. Urine recovery of conjugated (glucuronidated and sulphated) hydroxytyrosol metabolites over 24 hours following olive leaf extract (OLE) ingestion in capsule and liquid forms, at lower (gray bars) and higher (black bars) doses.

Supporting Information Figure S3. Sample single reaction monitoring (SRM) from urine analyses confirming presence of sulphated (A) and glucuronidated (B) hydroxytyrosol metabolites.

A

B

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