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Bioavailability of the phenolic compounds of the fruits (drupes) of Olea europaea (olives): impact on plasma antioxidant status in humans.

Abstract

To examine the bioavailability of olive polyphenols and to correlate it with their antioxidant efficacy, plasma and urine from healthy volunteers who had consumed 20 olives were subjected to (a) GC-MS analysis for individual phenolics, (b) estimation of plasma total polyphenol content and (c) estimation of plasma total antioxidant potential. Olive polyphenols were absorbed and metabolized within the body, occurring in plasma mainly in the conjugated form with glucuronic acid and reaching [C.sub.max] in 1-2 h. Excretion rates were maximum at 0-4 h. Tyrosol and hydroxytyrosol increased in plasma after intervention. Total antioxidant potential increased (p < 0.05). The results indicate that olive polyphenols possess good bioavailability, which is in accordance with their antioxidant efficacy.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Olea europaea (olives); Polyphenols; Bioavailability; Antioxidant status in humans; Hydroxytyrosol; Tyrosol; Metabolites

Introduction

Potential systemic effects of food components are dependent on their behavior in the digestive tract (Bravo, 1998). By definition, food components must be bioavailable in some form to exert their biological effects (Williamson and Manach, 2005). Bioavailability and metabolic kinetics in vivo studies are critical in order to (a) clarify the mode of action of a compound, (b) determine which components are better absorbed, leading to the formation of active metabolites, and (c) demonstrate a direct link among food components, metabolites and biological effects.

Because numerous studies have clearly demonstrated that polyphenolic compounds (PP) exhibit beneficial health effects due to their antioxidant (Andrikopoulos et al., 2002), anti-inflammatory (Visioli et al., 1998a, b) and cancer-preventive (Fabiani et al., 2006) activities, measurement of plasma concentrations and urinary excretion among subjects provided with pure PP, plant extracts or whole food/beverages rich in PP is increasingly a component of studies (Manach et al., 2005).

The olive is the fruit of an evergreen olive tree (Olea europaea, Oleaceae) growing in the temperate climate of the Mediterranean basin (Gruenwald, 1998). Table olives, as well as olive oil and olive products are an important part of the Mediterranean diet, the greatest value of which is due to olive PP. Olive oil stability and modulation of the oxidative balance (Visioli et al., 1998a, b) are attributed to these antioxidant components.

The main complex phenolic compounds are oleuropein, verbascoside and ligstroside, the contents of which decrease drastically during maturation of the fruit and give rise to hydroxytyrosol (HT) and other simple phenolics (Bianci, 2003). Simple phenolics such as HT, tyrosol (T), homovanillic alcohol (HVAlc), 3,4-di-hydroxyphenylacetic acid (DHPAC), caffeic acid, p-coumaric acid, phloretic acid and vanillic acid are also present in the mature fruit (Boskou et al., 2006). To date, studies on the absorption and metabolism of these phenolic compounds have been conducted after oral or intravenous administration of individual phenols (Tuck et al., 2001) or after olive oil administration (Visioli et al., 2003). The present study is the first in which concentrations of individual PP, either in their initial form or as active metabolites, as well as total polyphenol content in plasma and plasma total antioxidant potential (TAP), have been measured after oral administration of table olives of Greek origin.

Materials and methods

Materials

Commercially available Greek Kalamon olives packed in olive oil and vinegar were purchased at a local market in Athens.

All solvents, salts and acids were purchased from Merck (Darmstadt, Germany). T, DHPAC, caffeic acid and protocatechuic acid were obtained from Fluka (Buchs, Germany). HVAlc, oleanolic acid and 3-(4-hydroxyphenyl)-l-propanol (as internal standard (IS)) were from Aldrich (Steinheim, Germany). HT was purchased from Extrasynthese. All other phenolic standards, as well as [beta]-glucuronidase, were obtained from Sigma (Steinheim, Germany). Solid phase extraction (SPE) columns, Isolute C8 (EC) (500 mg, 3 ml), were purchased from International Solbent Technologies (Mid Glamorgan, UK). The purity of the compounds was over 90%. Bis-(trimethylsilyl)-trifluoro-acetamide (BSTFA), 99% with 1% trimethylchlorosilane was obtained from Aldrich (Steinheim, Germany).

Extraction and pretreatment of olive fruit phenols

Phenolic compounds were extracted and quantified in olive fruits as described previously (Boskou et al., 2006). Briefly, approximately 0.5 g of dry sample was extracted five times with 5 ml methanol, and then the total of all extracts was evaporated under a stream of nitrogen. The residue was dissolved in 5 ml methanol and stored at -40[degrees]C. Total phenolic content was estimated photometrically (Uvikon 931, Contron, Milan, Italy) using the Folin-Ciocaltaue reagent (Gutfinger, 1980). The results were expressed as T equivalents (mg T/100 g olives). For gas chromatography/mass spectrometry (GC/MS) analysis, 0.5 ml of methanol extract was evaporated till dryness in a speed vacuum evaporator. Derivatization prior to GC/MS analysis was performed with 250 [micro]l of BSTFA, vigorous vortexing and incubation at 70 [degrees]C for 20 min.

Study design and sample collection

The Bioethics Committee of Harokopio University approved the protocol. Eight male healthy subjects aged between 30 and 40 years were recruited for the study. As assessed by a medical history questionnaire, physical examination and biochemical and hematological indices, all subjects included in the study were healthy. Exclusion criteria were a BMI value greater than 29, alcohol or drug abuse, any medication or antioxidant vitamin/mineral supplementation, or alternative diet (vegetarian, macrobiotic, etc.) prior to the study. An additional exclusion criterion was preexistence or clinical evidence of any gastrointestinal disease, such as inflammatory bowel disease, gastric ulcer, and stomach or intestinal cancer. The nutritional habits of the subjects were assessed using a Food Frequency Questionnaire and a 3-day food record, with emphasis on vegetable, fruit and olive, and olive oil consumption. All subjects who participated in the study had similar eating habits. After a full explanation of the tasks, all volunteers provided written consent.

Blood and urine samples were collected after overnight fasting (control, C) prior to the study. Next, the volunteers were subjected to a 3-day wash-out (WO) diet, meaning they were instructed to abstain from PP-rich foods, listed by an experienced nutritionist. To evaluate compliance to the protocol, volunteers were subjected to 24 h recall interviews every morning during the WO period. One of the eight participants was excluded due to lack of compliance. On day 4, following overnight fasting, blood and urine samples were obtained from volunteers (WO) prior to olive administration. Next, each volunteer consumed a meal of 20 olives (approximately 100g) alone. Blood samples were collected in EDTA tubes 1, 2, 3 and 4h after meal consumption. Accordingly, urine samples were collected during the 24 h following meal consumption, in four individual tubes containing 0-4 h urines, 4-8 h urines, 8-12 h and 12-24 h urines. Plasma was isolated and aliquots (1ml) were acidified immediately using 36 [micro]l HC1 (4mol/l) at pH 3.0. All samples, acidified plasma and urine, were stored at -80 [degrees]C until analysis. During the experiment, subjects were not allowed to eat or drink anything except water. After the last blood collection and until the end of urine collection, they were instructed to avoid olive oil and olives, as well as other foods rich in PP. Compliance was controlled by a self-estimated 24 h record. A graphical representation of the study is shown in Fig. 1.

[FIGURE 1 OMITTED]

Plasma total polyphenol assay

Total phenolic compounds in plasma were measured using the Folin-Ciocaltaue reagent (Arendt et al., 2005). For determination of total PP in plasma, T was employed as a calibration standard and results were expressed as T equivalents ([micro]g T/ml of plasma). All photometric data were acquired using an Uvikon 931 (Contron, Milan, Italy) UV-vis spectrophotometer. Experiments were performed in duplicate.

Plasma total antioxidant potential assay

TAP in plasma was assessed using a colorimetric, quantitative assay for TAP in aqueous samples (Oxis-Research Portland, USA) according to the manufacturer's instructions. The results of the assay were expressed as "mM uric acid equivalents". Sensitivity of the assay was 30 [micro]mol/l uric acid equivalents.

Calibration curves and control samples

For GC, stock solutions of 15 individual PP were prepared at 2mg/ml each in methanol and stored at 2-4 [degrees]C in the dark. Additionally, a working methanolic solution was prepared, adding 0.2 ml of individual PP stock solution to a final volume of 50 ml.

Plasma and urine calibration curves were prepared by adding a suitable volume of the working solution to 1 ml of pooled blank plasma (WO) or urine (WO), free of tested compounds. Working ranges for calibration curves were 30-2000 ng/ml for plasma and 160-2000 ng/ml for urine. Calibration standards were then treated the same as biological samples and were analyzed by GC/MS.

Preanalytical treatment of biological samples

Individual aliquots of plasma or urine were thawed and treated according to one of the procedures listed below:

Non-enzymatic treatment

Plasma (1 ml) was deproteinized by the addition of 3 volumes of ethanol. After vortexing, the sample was centrifuged for 5min (17,500g, 4[degrees]C). The supernatant was collected and the protein pellet was resuspended in ethanol, vortexed and centrifuged for another 5min (17,500g, 4[degrees]C). The two supernatants were pooled and dried in a speed vacuum evaporator. The dried residue obtained was dissolved in 1 ml of distilled water and vortexed (Nardini et al., 2002). After adjusting pH to 3.0, the samples were injected into a preconditioned SPE column for the exclusive retention of PP.

As in the case of urines, prior to SPE, 0.25 ml of 1 mol/1 sodium acetate buffer (pH 5.0) was added to 1 ml of urine (Watson and Oliveira, 1999).

Enzymatic treatment

Plasma was deproteinized with ethanol as above. The dried residue was dissolved in 1 ml of 0.1 mol/l sodium acetate buffer (pH 5.0) and vortexed. Next, [beta]-glucuronidase (4000 units) was added and incubated for 2 h (37 [degrees]C). At the end of incubation, the pH was adjusted to 3.0. Finally, PP were extracted using an SPE column as above.

As regards urines, 100 units of [beta]-glucuronidase in 0.25 ml of potassium phosphate buffer (1 mol/1, pH 6.8) was added in individual urine samples (1ml). The samples were incubated for 1 h (37 [degrees]C). Finally, prior to SPE extraction the urine samples were acidified with 1 ml HC1 (1 mol/l, pH 1-2).

SPE extraction and derivatization of PP from the biological samples

SPE extraction of PP was done according to Soleas et al. (1997) with modifications. C8 cartridges were preconditioned with 3 ml ethyl acetate, followed by 3 ml methanol, and 5 ml of deionized water. The samples were then injected onto the preconditioned Sep Pack and allowed to dry in vacuo for approximately 30min. An SPE vacuum device, which allowed handling 20 samples simultaneously, was used.

Phenolic compounds were extracted by eluting the dry cartridges with 3 ml methanol. To ensure complete removal of water, approximately 5mg of anhydrate sodium sulfate was added to the methanolic fraction and the sample was filtered. Then, 60 [micro]l (480 ng) of IS in methanol was added and the samples were evaporated till dryness in a speed vacuum evaporator. Samples were further dried (70 [degrees]C, 15 min) in order to exclude any remaining moisture and derivatized with 250 [micro]l of BSTFA, vigorous vortexing and incubation at 70 [degrees]C for 20 min.

GC-MS analyses of phenolic compounds in olives and biological samples

An Agilent (Wallborn, Germany) series GC 6890 N coupled with an HP 5973 Mass Spectrometer detector (EI, 70eV), split-splitess injector and an HP 7683 autosampler were used for analysis. An aliquot (1 [micro]l) from 250 [micro]l PP derivatized sample (olives, plasma or urine) was injected into the GC at a split ratio of 1:20. GC separation was achieved using an HP-5 MS capillary column (5% phenyl -95% methyl siloxane, 30 mm x 0.25 mm x 250 [micro]m). Helium was used as a carrier gas at a flow rate of 0.6 ml/min. The injector and transfer line temperatures were set at 280 and 300 [degrees]C, respectively. The oven temperature program was initial temperature 70 [degrees]C for 5 min, 70-130 [degrees]C at 15 [degrees]C/min, 130-160 [degrees]C at 4 [degrees]C/min, held for 15 min, 160-300 [degrees]C at 10 [degrees]C/min, and finally held at 300 [degrees]C for 15 min. A selective ion monitoring GC-MS method was applied for the detection of 15 target PP compounds. Detection of PP was based on the [+ or -]0.05 RT presence of target and qualifier ions of the standard PP at the predetermined ratios. Target and qualifier ions (T, Q1 and Q2) for the 15 PP compounds were set as follows: vanillin: 194 and 209; T: 179, 267, 282; p-hydroxy-benzoic acid: 267, 223, 193; p-hydroxy-phenylacetic acid: 252, 296, 281; phloretic acid: 192 and 310; vannilic acid: 297, 267, and 312; protocatechuic acid: 193, 355, and 370; DHPAC: 384, 267, and 179; ferulic acid: 338, 323, and 308; syringic acid: 327, 342, and 312; caffeic acid: 396, 219, and 381; HT: 267 and 370; HVA: 326, 267, and 311; HVAlc: 209 and 179; quercetin: 647, 559, and 575; and terpenoid oleanolic acid: 203, 320, and 482 (Kaliora et al., 2004).

Identification of chromatographic peaks was accomplished by comparing the retention times and abundance ratios of three fragment ions of each PP compound with those of reference compounds, while quantification was carried out using 3-(4-hydroxyphenyl)-l-propanol as IS at target ion m/z 206 and qualifiers 191 and 179. Quantification of PP was performed using an 8-point linear calibration curves constructed in matrix for each sample treatment as mentioned above, while in olive extracts quantification of PP was performed by IS technique using linear reference compounds calibration curves. Linearity was obtained for all compounds with correlation coefficients varying from 0.992 to 0.999.

Statistical analysis

Statistical analysis was performed using the statistical package SPSS 12.0 for Windows. The effects of olive consumption on the investigated parameters were evaluated by comparing the values obtained at different time intervals with WO using the Wilcoxon signed rank test. Correlation was assessed using the Pearson correlation test. A p value <0.05 indicated a statistical significance difference or correlation.

Results

Olive fruit PP

Table 1 shows the GC-MS analysis of olive phenolic compounds. Among the phenolics detected, HT and T were present at the highest concentrations (76.73 and 19.48 mg/100 g olives, respectively). Other phenolics such as phloretic acid vanillic acid and quercetin were also present in considerable amounts.

Plasma TAP and total PP

As shown in Table 2, following a PP-free diet, a statistically significant decrease (p = 0.028) on total PP and a marginal decrease on TAP (p = 0.063) was managed. The increase in TAP was statistically significant 2 h after olive administration (p = 0.043), while the increase in total PP 3 h was significant after olive administration (p = 0.028). Alterations in TAP were in the same order of magnitude with alterations in total PP. Significant correlation was observed between total PP and TAP at 4h (p = 0.04).

Quantification of plasma individual phenols

Figs. 2 and 3 present the concentrations of T, pHPAC, pHBEN, HT and of the respective metabolites (HVAlc, HVA and DHPAC) in plasma ([micro]g/ml) after enzymatic hydrolysis with [beta]-glucuronidase. All PP reached maximum concentrations ([C.sub.max]) 1 h after olive consumption, with the exception of pHBEN, which reached [C.sub.max] 2 h after the consumption. After 1 h, plasma concentration of T, pHBEN and pHPAC was 0.169 ([+ or -]0.027), 1.324 ([+ or -]0.207) and 8.828 ([+ or -]0.926), respectively, while for HT, HVAlc, HVA and DHPAC it was 3.145 ([+ or -]0.341), 0.122 ([+ or -]0.013), 2.263 ([+ or -]0.170) and 14.742 ([+ or -]0.941) [micro]g/ml plasma, respectively. Statistically significant increases were observed in HT between WO and 1 h (p = 0.028) and 2h (p = 0.063) after administration, as well as in DHPAC between WO and 1 (p = 0.028) 2h (p = 0.043), 3h (p = 0.028) and 4h (p = 0.028) after administration. GC-MS analysis of plasma samples without enzymatic hydrolysis showed that approximately 15% of T and 16% of HT was in free form; approximately 5% of p-HBEN, p-HPAC and DHPAC, 17% of HVAlc and 18% of HVA were also in free form. Protocatechuic acid, HVAlc, syringic acid and quercetin were detected only in the conjugated form. A statistically significant correlation was found among T and p-HBEN (r > 0, p = 0.017) and p-HPAC (r > 0, p = 0.008), as well as among HT and HVA (r > 0, p = 0.025) and DHPAC (r > 0, p = 0.005) on [beta]-glucuronidase-treated samples (Figs. 2 and 3). No significant results were observed when checking the correlation of (a) individual polyphenol species and TAP, (b) the sum of T, pHBEN and pHPAC and TAP, and (c) HT metabolites and TAP, checked by the Pearson correlation test.

[FIGURE 2 OMITTED]

Excretion rate of PP

Figs. 4 and 5 present the excretion rate of PP after enzymatic treatment. Excretion rates for T, pHBEN and pHPAC were maximum at 0-4 h, being 0.008 ([+ or -]0.004), 0.611 ([+ or -]0.200) and 5.778 ([+ or -]2.062) mg/h, respectively, while for HT, HVA, HVAlc and DHPAC the maximum excretion rates at 0-4h were 4.762 ([+ or -]1.296), 9.116 ([+ or -]3.596), 0.025 ([+ or -]0.004) and 17.866 ([+ or -]11.275) mg/h, respectively.

Discussion

We investigated the bioavailability of olive PP in healthy volunteers. As shown by TAP values and total plasma PP prior to WO, volunteers had similar antioxidant/PP intake, while decrease after WO implies that all volunteers complied well with the protocol (Table 2).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

An increase in plasma PP content was observed following olive administration, indicating that olive PP are bioavailable. We focused on T and HT, the main olive phenolics, on the minor p-HBEN, p-HPAC, and the HT metabolites HVA, HVAlc and DHPAC (D' Angelo et al., 2001), which showed significant increases after olive consumption (Figs. 2 and 3). Other phenolics detected in olives, including vanillin, phloretic acid, vanillic acid, protocatechuic acid, syringic acid, ferulic acid, quercetin and caffeic acid, were also determined in plasma, as well as in urine, especially in their conjugated form as glucuronides. However, their concentrations were constant after olive consumption (data not shown). Our results are in agreement with those of Corona et al. (2006), who studied olive oil PP metabolism in vitro.

HVA does not exist in olive drupes. However, after HT administration, HVA occurred in plasma and in urines, ensuring the catechol-O-methyl transferase action on HT in the liver and kidneys. A significant linear regression of HT administered and HVA detected in urine has also been reported (Caruso et al., 2001). In the present study, a significant correlation between HT ingested from olives (Table 1) and HVA detected in plasma (Fig. 3) and in urines at 0-4 h (Fig. 5) (p = 0.004) was detected. Oleuropein, assessed by HPLC, was under quantification limits in olives (data not shown). Usually, the content of oleuropein in olives is minimal, while the content of HT is very high, showing a possible breakdown of oleuropein to HT under hydrolytic conditions during olive fermentation (Armandodoriano and Uccella, 2000). Corona et al. (2006) found that oleuropein was also not transferred across small intestine segments. In the same study, they reported that after perfusion with T a "new substance" appeared in the gut serosal fluid, showing spectral properties similar to T. This, in comparison with the significant correlation observed in the present study between T, pHBEN and pHPAC in plasma as well as in urine, indicates that either pHBEN or pHPAC is the so-called "new substance". Whether the ingested olive PP that remain undetected in plasma and urine are accumulated in tissues, metabolized or degraded to other PP or compounds from the colonic microflora, or are excreted in a different way, requires further investigation. D'Angelo et al. (2001) studied the pharmacokinetics and metabolism of labeled HT in rats, and detected metabolites in plasma and urine, as well as in the brain, heart, kidney, liver and lung.

[FIGURE 5 OMITTED]

Although dietary control and fasting resulted in HT decrease, elimination could not be attained, in accordance with other studies (Caruso et al., 2001). Since HT and the respective metabolites have been identified as catecholamine metabolites in dopaminergic cell lines, it is unlikely that they ever eliminate into the body. HT, as well as HVA, DHPAC and HVAlc are products of a dopamine metabolite 3,4-dihydroxyphenylacetaldehyde reduction by the human liver alcohol dehydrogenase isoenzyme (Eisenhofer et al., 2004).

PP absorption was rather rapid. Increases of 15% for T, pHBEN, pHPAC, HVA and HVAlc and 50% for HT and DHPAC were observed in plasma 1 h after intervention (Figs. 2 and 3). In all plasma samples, we found 17% HT as itself and the remainder as HT metabolites, namely, 15% HVA, 67% DHPAC and 1% HVAlc (Fig. 6A).

Significant correlation was observed between total PP and TAP at 4 h (p = 0.04), meaning a possible contribution of olive PP in enhancement of antioxidant status. No significant correlation was found between subtotal or individual PP and TAP. However, TAP values increased after olive ingestion (WO vs. 2h, p = 0.043) (Table 2). TAP increase is therefore in agreement with that reported by Serafini et al. (1998), who demonstrated a statistically significant increase in antioxidant capacity after alcohol-free red wine ingestion. Because TAP measurement is an assay that is not selective for dietary antioxidants, but for total plasma antioxidants, it is likely that TAP increase is due to induction of the synthesis of endogenous antioxidants in the presence of olive PP. Generally, PP have been shown to induce the synthesis of endogenous intracellular antioxidants in cell culture studies (Jiao et al., 2003). Studies conducted on the antioxidant activity of olive oil PP have not all shown an effect. In two studies reviewed by Vissers et al. (2004), when olive oil was administered to healthy subjects a protective effect against oxidation was observed. Weinbrenner et al. (2004) showed that the phenolic content of the olive oils administered may account for protection of the endogenous antioxidant defenses in the postprandial state after ingestion of moderate and high phenolic content olive oils, but not those of low phenolic content. Generally, among the PP detected in olive oil, HT has been shown to possess strong antioxidant properties, attenuating oxidative stress via the decrease of reactive oxygen species and the increase of glutathione (Goya et al., 2007).

Regarding PP kinetics in plasma, intraindividual variability was observed, which has also been reported previously (Cerda et al., 2005). Five out of seven volunteers showed a peak at 1 h, while the rest were at 2 h (data not shown). This individual variability was observed in [beta]-glucuronidase-treated samples, while the untreated samples were more homogeneous, all of them appearing in a peak at 1 h (data not shown). In the study of Miro Casas (2003) on olive oil bioavailability, a peak at 30min after oil ingestion was observed. The different peak time could be ascribed to the different food matrix. In the present study, a rise of PP 4 h next to administration (Fig. 2) indicates a possible enteropatic recycling; PP are reabsorbed and occur in the body longer (Manach et al., 2004).

The conjugation levels of PP with glucuronic acid were in agreement with those of Miro Casas et al. (2003). Nevertheless, in our study, the percentage of conjugation was different among subjects. T in the free form was found around 8% in three volunteers and 18% in four. It is possible that intraindividual variability is due to different

conjugation enzyme expressions. Corona et al. (2006) noted that olive oil PP conjugation, such as HT O-methylation, HT and T glucuronidation, and HT-glutathionyl conjugation, occurs mainly in the small intestine. Genetic polymorphism of the enzymes involved in the PP conjugation might be responsible for this intraindividual variability (Manach et al., 2004).

In urine, 14.8% was HT itself and the rest were HT metabolites, namely 48.3% HVA, 36.5% DHPAC and 0.3% HVAlc (Fig. 6B). The highest percentage of HVA in urine as compared with plasma (Fig. 6A) indicates a possible high catechol-O-methyl transferase activity in the kidneys. The increased activity of catechol-O-methyl transferase in kidney and liver has been reported (Manach et al., 2004). With the exception of HVAlc, the percentages of HT and HVA, mainly in the 0-4 h urine samples (Fig. 6B), in our study were similar to those reported by Caruso et al. (2001) for olive oil. The above findings indicate that HT is excreted mainly in the form of HVA, followed by DHPAC and HVAlc.

Conclusions

We have shown that olive administration leads to PP increase in plasma and in TAP. PP are extensively metabolized in the human body and appear in biological fluids, mainly as glucuronides. Among 15 phenolic compounds detected in olives, seven (T, HT and their possible metabolites) were increased in biological fluids after administration.

Acknowledgments

We thank the volunteers for their participation. AMK was funded by the Ministry of National Education and Religious Affairs, Program "Herakleitos 2002-2006".

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A.M. Kountouri, A. Mylona, A.C. Kaliora, N.K. Andrikopoulos*

Laboratory of Chemistry-Biochemistry-Physical Chemistry of Foods, Department of Science of Dietetics-Nutrition, Harokopio University, 70 El. Venizelou Ave., 176 71 Kallithea, Athens, Greece

Received 9 February 2007; accepted 5 June 2007

*Corresponding author. Tel.: + 302109549303; fax: + 302109577050.

E-mail address: nandrikop@hua.gr (N.K. Andrikopoulos).
Table 1. Polyphenol content of Kalamon olives by GC/MS analysis

No. Phenolics Abbreviation mg/100 g olives

 1 Tyrosol T 19.48
 2 p-Hydroxy-benzoic acid p-HBEN 0.44
 3 p-Hydroxy-phenylacetic acid p-HPAC 0.30
 4 Homovanillic alcohol HVAlc 0.55
 5 Homovanillic acid HVA nd
 6 3,4-Di-hydroxy-phenylacetic acid DHPAC 0.14
 7 Hydroxytyrosol HT 76.73
 8 Vanillin - 0.95
 9 Phloretic acid - 6.37
10 Vanillic acid - 1.32
11 Protocatechuic acid - 0.49
12 Ferulic acid - 0.06
13 Syringic acid - 0.18
14 Caffeic acid - 0.48
15 Quercetin - 3.12

Table 2. Total polyphenols expressed as tyrosol (T) equivalents
([micro]g/ml plasma) and total antioxidant potential (TAP) in plasma
expressed as "mmol/l uric acid" equivalents

 Total plasma PP
Collection time ([micro]g T/ml plasma) TAP (mmol/l uric acid)

Control 24.76 [+ or -] 2.36 0.221 [+ or -] 0.17
Wash out (0 h) 20.74 [+ or -] 0.99 (a) 0.071 [+ or -] 0.05
1h 22.14 [+ or -] 2.50 0.108 [+ or -] 0.04
2h 23.74 [+ or -] 2.22 0.109 [+ or -] 0.07 (a)
3h 22.46 [+ or -] 1.45 0.103 [+ or -] 0.07
4h 22.84 [+ or -] 1.80 0.108 [+ or -] 0.07

Values are means [+ or -] SD of the total plasma PP and TAP of the seven
volunteers.
(a) Statistically significant differences (p < 0.05).

A

HT 17%
HVA 15%
HV Alc 1%
DHPAC 67%

B

HT 14.81%
HVA 48.31%
HV Alc 0.37%
DHPAC 36.51%

Fig. 6. Percentages of HT and respective metabolites (HV Alc, HVA and
DHPAC) in (A) plasma and (B) urine samples. Percentages were extracted
by the mean polyphenol concentrations on plasma samples of all seven
individuals.

Note: Table made from pie chart.
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Author:Kountouri, A.M.; Mylona, A.; Kaliora, A.C.; Andrikopoulos, N.K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Geographic Code:4EUGR
Date:Oct 1, 2007
Words:5454
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