A biochemical perspective on the fate of virgin olive oil phenolic compounds in vivo

Abstract The chemistry of the phenolic compounds found in virgin olive oil (VOO) is very complex due, not only to the different classes of polyphenols that can be found in it, but, above all, due to the existence of a very specific phenol class found only in oleaceae plants: the secoiridoids. Searching in the Scopus data base the keywords flavonoid, phenolic acid, lignin and secoiridoid, we can find a number of 148174, 79435, 11326 and 1392 research articles respectively, showing how little is devote to the latter class of compounds. Moreover, in contrast with other classes, that include only phenolic compounds, secoiridoids may include phenolic and non-phenolic compounds, being the articles concerning phenolic secoiridoids much less than the half of the abovementioned articles. Therefore, it is important to clarify the structures of these compounds and their chemistry, as this knowledge will help understand their bioactivity and metabolism studies, usually performed by researchers with a more health science’s related background. In this review, all the structures found in many research articles concerning VOO phenolic compounds chemistry and metabolism was gathered, with a special attention devoted to the secoiridoids, the main phenolic compound class found in olives, VOO and olive leaf.


Introduction
The regular consumption of virgin olive oil (VOO) has been associated with a lower incidence of diseases related to oxidative damage, such as coronary heart disease (Covas, de la Torre, et al. 2006;Covas, de la Torre, and Fitó 2015;Gaforio et al. 2019;López-Miranda et al. 2010;Parkinson and Cicerale 2016;Romani et al. 2019;Tresserra-Rimbau et al. 2014;Widmer et al. 2015) some types of cancer (Casaburi et al. 2013;Emma et al. 2021;Fabiani 2016;Psaltopoulou et al. 2011), and age-related cognitive decline (De La Cruz et al. 2015;Mete et al. 2018;Parkinson and Cicerale 2016;Solfrizzi, Panza, and Capurso 2003).The high content in oleic acid by itself, brings benefits to consumer health but several studies have shown that the consumption of olive oil rich in polyphenols produce higher antioxidant and anti-inflammatory effects than the consumption of low-polyphenol olive oils (Covas, de la Torre, et al. 2006;López-Miranda et al. 2010;Parkinson and Cicerale 2016;Tresserra-Rimbau et al. 2014;Weinbrenner et al. 2004).Therefore, it is well established that beneficial health effects of VOO consumption are partly due to the high content of polyphenols in this oil, which are known for their remarkable antioxidant activity (Gordon, Paiva-Martins, andAlmeida 2001, Paiva-Martins andGordon 2002).
Over the past years, there has been increasing interest in exploring nutrients that could be responsible for beneficial effects on the incidence of brain diseases in which oxidative stress plays an important role.The brain has a low level of endogenous antioxidants and is, therefore, particularly susceptible to oxidative damage, and oxidative stress is thought to have an important role in the development several neurological diseases such as Alzheimer's disease.For this reason, there is considerable interest in the possible role of VOO antioxidants as a potential therapeutic approach as some of the most important polyphenols in VOO, such as oleocanthal have demonstrated protection against the brain's inflammatory response to trauma (Mete et al. 2018), improved β-amyloid clearance in Alzheimer's disease (Qosa et al. 2015;Shinde et al. 2015) and neuroprotective effect as antioxidant agents (Bu et al. 2007;De La Cruz et al. 2015;Giusti et al. 2018;Sarbishegi, Mehraein, and Soleimani 2014;Sun et al. 2017).
Despite bioavailability controversies and their complex mechanism of action, VOO phenolic antioxidant intake exhibits promising results as an alternative to drug interventions for preventing many chronic diseases, such as CVD and NDD, in risk populations.Nevertheless, further studies are still needed for the complete understanding of the bioavailability and bioactivity of phenolic antioxidants in vivo and to relate this bioactivity with their metabolites and with the phenolic composition of VOO polyphenol extracts.
The bioactivity of polyphenols in vivo is dependent on their gastrointestinal stability, and on the extent of their absorption and metabolism.Before becoming bioavailable, VOO phenolic compounds also need to be released from food matrix and suffer several modifications in the gastrointestinal (GI) lumen.Therefore, any potential health effect given by the consumption of VOO phenols is dependent on the degree of absorption and on the extent of the first-pass metabolism before the substance reaches systemic circulation (Covas, de la Torre, et al. 2006;De La Cruz et al. 2015;Fabiani 2016;Rubió, Macià, et al. 2014).Several factors can influence the bioavailability of the ingested compounds, such as the phenolic compound concentration in the oil, each individual's characteristics, such as their enzymatic activity, habitual diet (Suárez et al. 2011), genomic profile and colonic microflora (Lozano-Castellón et al. 2020).On the other hand, the fraction of a compound released from the food matrix in the gut and the ability to pass through the intestinal barrier defines the bioaccessibility of a dietary compound (Rubió, Macià, et al. 2014).Moreover, the rate and extent of intestinal absorption and the nature of the metabolites circulating in the plasma are determined by the chemical structure of the polyphenols (Scalbert and Williamson 2000).
It is well known that VOO phenols suffer an extensive metabolism in the body, and their bioavailability seems to be poor with respect to their metabolites (Serreli and Deiana 2018).Therefore, the major limitation of in vitro studies on VOO phenols is to consider the bioactivity of parental compounds instead of their metabolites, which are often more concentrated in plasma and in specific tissues then the parental compounds.Nevertheless, little is known about the interaction of parental phenols with plasma and membrane proteins or their stability during analytical procedures, which may underestimate their concentration in the body.Moreover, recently, phase II metabolites have been regarded as possible temporary deposits of parental molecules (Bartholomé et al. 2010;Menendez et al. 2011;Patel et al. 2013;Sharan et al. 2012;Shimoi et al. 2001).These phase II metabolites can be excreted in the urine but they also transport more hydrophobic phenolic compounds trough the blood to several tissues, where, under oxidative stress, they can be hydrolyzed at the target site by the action of glucuronidases (Bartholomé et al. 2010;Menendez et al. 2011;Shimoi et al. 2001).Recent reports suggest that, during inflammation in the cardiovascular and central nervous systems, stimulated neutrophils and other injured cells can release glucuronidases, releasing the aglycone in situ (Bartholomé et al. 2010;Shimoi et al. 2001).Therefore, very little is still known about the underling mechanisms of the in vivo VOO polyphenol bioactivities and which compounds have actually a major contribution for the protective effect given by virgin olive oil consumption.
Another constraint in understanding VOO metabolism and activity in vivo is their particular chemistry.In fact, the main phenolic compounds found in VOO extracts belong to a particular class of polyphenols, only found in Olearaceae plants, the secoiridoids.As we will see further in this review, this class of polyphenols have a unique reactivity and only recently some light on their structures have been revealed.Therefore, the present review will focus on what is currently known about the major VOO polyphenols biochemical transformations that occurs in vivo as it is fundamental in understanding and evaluating the health benefits associated with VOO consumption.

Virgin olive oil phenols
Virgin olive oils are amongst the very few oils consumed without refining and, consequently, contain phenolic compounds together with tocopherols.In fact, its phenolic composition is different from that of any other virgin vegetable oil (Brenes et al. 2001;Costa and Paiva-Martins 2022;Del Monaco et al. 2015;García et al. 2001;Garcia et al. 2012;Ghanbari et al. 2012;Gómez-Rico, Fregapane, and Salvador 2008;Krichene et al. 2007;Servili et al. 1999;Kiritsakis, Kiritsakis, andTsitsipas 2020, Kiritsakis, Turkan, andKiritsakis 2020).The concentration in polyphenolic compounds can range between 50 and 1000 ppm depending on the cultivars used, age, maturity index, pedoclimatic and extraction conditions but usual values in commercial VOO range between 100 and 600 ppm.Nevertheless, in VOO obtained using an Abencor laboratory oil mill, up to 2150 ppm has already been reported in several studies (Brenes et al. 2001;Costa and Paiva-Martins 2022;Del Monaco et al. 2015;García et al. 2001;Garcia et al. 2012;Ghanbari et al. 2012;Gómez-Rico, Fregapane, and Salvador 2008;Krichene et al. 2007;Servili et al. 1999).Recently, a large-scale screening and statistical evaluation of 5764 VOO samples from Greece, coming from more than 30 varieties for an eleven-year period, showed that the mean concentration of total phenolic content was 483 mg/ kg, although a large variation among the different cultivars was observed, with some virgin olive oils reaching 4003 mg/kg (Diamantakos et al. 2021).
The presence of carbonyl groups in the secoiridoid structure confer to these compounds high reactivity against some solvents.For example, the reaction of oleacein and oleuropeidine with methanol (compounds 9, 10 and 33-36, Figure 2), which was found more recently to occur also with ethanol (Angelis et al. 2021), leading to the formation of hemiacetals has been well known for decades, first described by Montedoro et al in 1993 and confirmed more recently by other authors (Karkoula et al. 2012;Paiva-Martins et al. 2009).Usually, proper solvent evaporation reverses the reaction with recovery of the starting compound (Paiva-Martins and Gordon 2001).In addition, in the presence of water, oleacein easily forms hydrates reaching a quick equilibrium with a oleacein hydrate/ oleacein ratio of 2.2 at room temperature in a few minutes (Figure 1S and S2, Table S1, Supporting information) (Karkoula et al. 2012;Silva 2020).Although both oleacein carbonyl compounds could suffer this reaction, NMR studies confirmed that only the non-conjugated carbonyl is hydrated (Figure S1 and S2, Table S1, Supporting information) (Karkoula et al. 2012;Montedoro et al. 1993;Sánchez de Medina et al. 2017;Silva 2020).Moreover, in these NMR studies, possible pyrane ring formation via cyclization that could occur after addition of water or methanol has not been detected (Karkoula et al. 2012;Montedoro et al. 1993;Sánchez de Medina et al. 2017;Silva 2020).The addition of a water molecule to these compounds is of foremost importance in bioavailability studies as oleacein, oleocanthal, oleuropeidine and listrosidine in vivo are dissolved in an aqueous matrix and are probably naturally found mainly as hydrates in plasma, urine and other biological fluids.Therefore, these reactions need to be taken into consideration during the identification of metabolites by MS, both in olive extracts and in vivo, as some of the compounds detected may arise from reactions with solution and extraction solvents.
The second most important family of phenols in VOO are the phenylethanoids, mostly free HT and T (Costa and Paiva-Martins 2022).Their concentration is usually low in fresh VOO but increases during oil storage due to the hydrolysis of their acetates and glycosides, and secoiridoids (Brenes et al. 2001;Costa and Paiva-Martins 2022;Del Monaco et al. 2015;García et al. 2001;Garcia et al. 2012;Ghanbari et al. 2012;Gómez-Rico, Fregapane, and Salvador 2008;Krichene et al. 2007;Servili et al. 1999).
The lignans are the third family of compounds that are represented in important amounts in the oil.They are products of the dimerization of two phenylpropene or phenylpropene precursors by condensation of aromatic aldehydes.The furofuran L10 lignan type (+)-1-Acetoxypinoresinol and (+)-1-pinoresinol are the most concentrated lignans found in EVOO (Amiot, Fleuriet, and Macheix 1989;Del Monaco et al. 2015;Ghanbari et al. 2012;Gómez-Rico, Fregapane, and Salvador 2008;Vougogiannopoulou et al. 2014) and they seem to be among the most stable phenolic compounds during VOO storage and process (Silva, Garcia, andPaiva-Martins 2010a, Silva et al. 2010b).
Other families of phenolic compounds such the phenolic acids, including both hydroxybenzoic and hydroxycinnamic acid derivatives (protocatechuic acid, vanillic acid, gallic acid, syringic acid, p-hydroxybenzoic acid, caffeic acid, p-and o-coumaric acid, ferulic acid and cinnamic acid), and flavonoids (luteolin and apigenin) are also found in VOO but usually in a very low concentration when compared with the above-mentioned phenols (Costa and Paiva-Martins 2022).
The main phenols present in VOO, and their ranges of concentrations, can be found in Table 1.

Absorption and metabolism of olive oil phenols
The diary intake of VOO in Mediterranen countries has been estimated to be 25-50 mL, corresponding to around 9 mg of phenols, (de la Torre 2008; Williamson, Kay, and Crozier 2018).During the digestive process, important losses and transformations of these compounds with the rise in compounds with different structures and chemical properties may occur leading to differences in the amount and forms available in the intestinal tract for potential uptake.Moreover, several metabolic pathways will change the parental phenolic compounds into a further number of metabolites.Therefore, VOO phenolic compounds and their metabolites are apparently present in plasma and tissues at concentration more than 50-fold lower than the pool of endogenous antioxidants and their contribution for the overall body oxidative status is likely to be quite low when compared to other endogenous oxidizable substrates, including vitamin E, vitamin C, urate, tiois, bilirubin, proteins, enzymatic systems, and unsaturated fatty acids (Williamson, Kay, and Crozier 2018).Moreover, phenol conjugated metabolites have shown frequently a much lower radical scavenging capacity than the parental compounds and, unless some intracellular deconjugation happens, they are quite inactive as antioxidants (Fernandes et al. 2020;Khymenets et al. 2010;Paiva-Martins et al. 2013).However, and considering that VOO polyhenols are consumed throughout life, even a low radical scavenging capacity, may contribute for an overall antioxidant protective effect.In fact, clinical trials have shown that even short-term consumption of VOO (50 mL/day) could change several oxidative stress markers (Covas, de la Torre, et al. 2006;Weinbrenner, Fitó, et al. 2004), although the concentration of its phenols were lower than those required to show  1999).
in vitro bioactivity.The possibility of accumulation in a particular tissue (López-Yerena, Pérez, et al. 2021;López-Yerena, Vallverdú-Queralt, Lamuela-Raventós, et al. 2021;López de las Hazas et al. 2016, 2018) and their link to cell proteins and lipoproteins (de la Torre-Carbot et al., 2006;González-Santiago, Fonollá, and Lopez-Huertas 2010;Paiva-Martins et al. 2009) is not well known for most VOO phenolic antioxidants and are probably underestimated during in vivo studies.In fact, food and its components are ingested throughout a lifetime, during which even modest antioxidant effects may become noteworthy.Moreover, due to the strong possibility of additive or synergetic effects between phenols, it is difficult to predict the real contribution of VOO phenols to the human health.
In order to understand the health benefits associated with VOO consumption, evaluation of the bioavailability (absorption, metabolization, distribution and elimination) of VOO phenolic compounds is fundamental.After ingestion, VOO phenols must pass from the gut lumen into the circulatory system and the extent of this passage and the rate of absorption in the gut is mainly influenced by their chemical structure.The more hydrophilic glycosides such as oleuropein or ligtroside, seem to have difficulty in crossing through membranes and need to be hydrolyzed before absorption (Tomas-Barberan, González-Sarrías, and García-Villalba 2020).
The free hydroxyl groups in VOO phenolic compounds may easily undergo conjugation reactions with endogenous compounds to yield more polar and water soluble compounds, which can be transported from cells by active transport and excreted in the urine (Tomas-Barberan, González-Sarrías, and García-Villalba 2020).The principal expected conjugation reaction is the formation of glucuronides and the conjugation with the sulfonic acid moiety.Less polar conjugates are also expected to be formed by methylation catalyzed by the cathechol-1-O-methyl transferase (COMT) (Tomas-Barberan, González-Sarrías, and García-Villalba 2020).However, many unexpected metabolites may appear during in vivo studies and the lack of knowledge of their existence may lead to an underestimation of the absorption of the various phenols, on the one hand, and to a lesser understanding of the health effects of the metabolites produced by the body, on the other hand.

Biochemical transformations of phenolic compounds in the gastrointestinal tract
The digestive process is initiated by mastication in the mouth to release polyphenols from the food matrix and it continues in the stomach (Rubió, Macià, et al. 2014).Although the hydrolytic activity of saliva in the oral cavity can hydrolyze part of the glycosides, most pass through the stomach and reach the small intestine and colon to be hydrolyzed by intestinal enzymes (Vissers, Zock, and Katan 2004;Vissers et al. 2002) or metabolized by the colonic microbiota before absorption (Corona et al. 2006;López de las Hazas et al. 2016).Due to the special acid environment of the stomach, the more liposoluble phenols may cross the gastric mucosa, mainly by passive diffusion (Kay 2006;Kay et al. 2017), but due to relatively low time of residence, the absorption in the stomach is considered relatively low and untimely, their absorption will happen in a higher extent in the small intestine.Moreover, glucosylated secoiridoids, such as oleuropein and ligstroside, appear not to be well hydrolyzed in gastric conditions (Vissers et al. 2002) and enter the small intestine unmodified (Corona, Spencer, and Dessì 2009;Vissers et al. 2002, Vissers, Zock, andKatan 2004).Therefore, these more hydrophilic glycosides have difficulty in crossing through membranes and need to be hydrolyzed before absorption (Vissers et al. 2002).This hydrolysis may take place at the brush border of the enterocyte and is usually catalyzed by lactase phlorizin hydrolase (LPH), with the releasing of the much more lipophilic aglycones (Day et al. 2000).The aglycones are now able to enter the enterocyte by passive diffusion.Alternatively, VOO phenolic glycosides may be be transported into the enterocyte by active transport through a sodium-dependent glucose transporter (SGLT1) and then hydrolyzed inside the cell by cytosolic β-glucosidases, as observed for other polyphenol glycosides (Gee et al. 2000).
Besides the high stability shown during digestion in the mouth, Quintero-Flórez et al. (2018) reported important losses of secoiridoid aglycones (up to 84%) by a time-dependent hydrolysis in in vitro gastric conditions, with a significant increase in the amount of free HT and T. This decomposition of secoiridoid aglycones with the production of HT and T with increased gastric residency, was also reported by other authors (Corona et al. 2006;Pinto et al. 2011;Soler et al. 2010) although in a much less extent, with important fractions of the conjugated forms remaining unhydrolyzed.
Duodenal digestion greatly affects the recovery of phenolic compounds.Only HT and T showed higher recovery due to the hydrolysis of secoiridoids, with values of up to 440%.Among the secoiridoids, oleuropein derivatives seem to be less stable during the gastrointestinal digestive process than those of ligstroside (Quintero-Flórez et al. 2018), with important losses after the gastric and duodenal steps of between 66 and 93%.In these studies, oleuropein derivatives (oleacein and oleuropeidine) have shown a recovery index of only 1-6% in contrast with the 37-90% for ligstroside derivatives (oleocantal and ligstrosidine).This lower stability after the intestinal phase of oleuropein derivatives could be related with the lower stability of the catechol moiety to alkaline conditions (Pinto et al. 2011).However, in contrast with secoiridoids, hydroxytyrosol acetate (HTAc), also a catechol, has shown a remarkable stability under the same conditions (Chen et al. 2014), with recoveries of over 60%.Nevertheless, Pereira-Caro et al reported that only approximately one third of the HTAc remains unaltered (Pereira-Caro et al. 2012) in similar in vitro conditions.Some of the contradictory reports about the stability and bioaccessibility of phenolic compounds in the gastrointestinal media maybe related to the different phenolic composition and food matrix (Saura-Calixto, Serrano, and Goñi 2007).The lipids can improve the digestibility of phenolic compounds due to the better solubilization of compounds from the food matrix but they can also give a higher protection during the digestion process through their incorporation into mixed micelles (Ortega et al. 2009).In contrast with what happens to phenolic alcohols, after mouth digestion, the more hydrophobic phenolic compounds may precipitate, with consequent reductions in their recovery (Quintero-Flórez et al. 2018).Moreover, in most in vitro studies, the interactions of phenolic compounds with enzymes and proteins (González-Santiago, Fonollá, and Lopez-Huertas 2010; Paiva-Martins et al. 2009) from the digestive fluids used in the studies has not been taken into consideration as the pallet obtained after digestion has been discarded and only the aqueous suspension and oil phase of the digested fluids have been analyzed (Quintero-Flórez et al. 2018).
Another issue not well understood or studied, is the formation and fate of oxidized phenols, quinones, during digestion.Ortho-quinones are produced in vivo through the oxidation of catecholic substrates by enzymes such as tyrosinase and also by transition metal ions, usually present in foods, incubation media, and digestive juices.These ortho-quinones initially formed undergo extensive degradation with half-lives of up to 30 min at pH 6.8, and >100 min at pH 5.3 (Ito et al. 2016).However, the concentration on free metal ions in a food matrix is very difficult to predict as metal ions can either belong to the natural matrix.They can also be acquired during food processing and may vary widely even in simpler foods such as edible oils or drinking water (Silva, Garcia, andPaiva-Martins 2010a, Silva et al. 2010b).Moreover, even very low contamination by metallic ions (<0.02 ppm of iron for example) will have a major effect on the stability of phenolic compounds when tested in vitro as the oxidized metals are continually regenerated by the reaction of reduced metals with air oxygen (Paiva-Martins et al. 2007;Paiva-Martins and Gordon 2005).
The culture medium pH is also an important issue in this evaluation.Tyrosol, hydroxytyrosol, hydroxytyrosol acetate and oleuropein showed an important degree of degradation when they were incubated in culture medium (pH 7.4) (Paiva-Martins and Gordon 2005).Therefore, degradation of catecholics may be influenced by the composition not only of the food matrix but also of the media and digestive juices used in their evaluation (Markopoulos et al. 2009;Mosele et al. 2014).

Biochemical transformations performed by the microbiota
It is estimated that only 10-15% of polyphenols are absorbed in the small intestine (Williamson, Kay, and Crozier 2018).Therefore, most phenols reach the large intestine and, consequently, will suffer high exposure to colonic metabolism (Corona, Spencer, andDessì 2009, Corona et al. 2006), producing a wide number of metabolite compounds.Therefore, most of the observed bioactivity of phenols may result from the gut microbiota metabolism rather than the original dietary phenols.While polyphenols present in VOO have been associated with the promotion of intestinal health by the stimulation of a higher biodiversity of beneficial gut bacteria, enhancing their balance, very little is known about the chemical phenol transformations that may occur under the action of microbiota (Deiana, Serra, and Corona 2018;Gavahian et al. 2019;Marcelino et al. 2019;Markopoulos et al. 2009;Mitsou et al. 2017;Mosele et al. 2014;Rocchetti et al. 2022).
A number of microbiota enzymes are able to hydrolyze glycosides, to catalyze ring fissions leading to the production of smaller phenolic compounds, and to oxidase or reduce the non-phenolic moieties of phenolic compounds.These more lipophilic metabolites can now be absorbed and subjected to phase II metabolism, being responsible in large extent for the bioactivity of VOO phenolic compounds (Markopoulos et al. 2009).
It has been reported that secoiridoids that reach the large intestine are subjected to rapid degradation by the colonic microflora.Three major degradation products were produced from oleuropein, being one of them HT that can be now absorbed into the colon, increasing the overall HT bioavailability (Markopoulos et al. 2009;Vissers, Zock, and Katan 2004).In the presence of heat-inactivated microbiota, oleuropein was not degraded suggesting that the fecal matrix can protect VOO phenols from hydrolysis but, in the presence of colon microbiota, oleuropein was rapidly hydrolyzed to its aglycone.This compound was then further hydrolyzed to elenolic acid and HT, probably by microbial esterases (Markopoulos et al. 2009) (Figure 3).
The proposed metabolic pathways for the colonic metabolism of HT (40) and HTAc (41) can be observed in Figure 3. HTAc is likely to suffer the action of microbial esterases that cleave the ester bond from the molecule (Figure 3).Subsequently, the first catabolic reaction that HT and T suffer (Figures 3 and 4) is probably the oxidation of the alkyl hydroxyl group, originating hydroxylated phenylacetic acids (43, 51).There are two microbial enzymes involved in the oxidation of hydroxyl groups in the colon, namely alcohol dehydrogenase that oxidizes the hydroxyl group to an aldehyde group, and aldehyde dehydrogenase that transforms the aldehyde group into a carboxylic one, leading to the formation of hydroxyphenylacetaldehydes and hydroxyphenylacetic acids, respectively (Mosele et al. 2014;Rocchetti et al. 2022).As a consequence of colon bacteria dehydroxylations (Thomas et al. 2001), p-hydroxylphenylacetic acid (51) and phenylacetic acid (55) are probably formed from 3,4-dihidroxyphenyl acid (DOPAC, 43) and p-hydroxybenzoic acid (52), respectively.3-(4-Hydroxyphenyl)propionic acid (48) has also been detected in a HTAc fermentation medium, probably due to the action of non-substract-specific demethylases (Clavel, Borrmann, et al. 2006) that may demethylate hydroxytyrosol acetate to 3-(3,4-dihydroxyphenyl)propionic acid (47) as an intermediate product (Figure 3).However, in an human intervention study (Mosele et al. 2014), the analysis of the phenolic metabolites in fecal samples from ten volunteers, after 21 days of a 25 mL/day of a phenol-rich VOO intake, showed only a non-significant increase in the concentration of phenylacetic acid (55), p-hydroxyphenylacetic acid (51), and 3-(4′-hydroxyphenyl)-propionic acid (48) and also of 2-(2′-hydroxyphenyl)acetic acid (50), in contrast of what happen in in vitro digestion, where this metabolite was almost not detected.Although, so far, it has not been reported in VOO phenolic bioavailability studies, it has been found in studies concerning the bioavailability of other classes of phenols that p-hydroxyphenylacetic acid (51) can be oxidized into p-hydroxybenzoic acids (52) and, after absorption, be further converted into hippuric acids (53, 54) through glycination (Carregosa et al. 2020).The concentration of free hydroxytyrosol, although small, was found to be statistically higher than the control (Mosele et al. 2014).
After the sustained intake of a phenol-enriched olive oil by rats, the presence of oleuropein together with HT, elenolic acid and homovanillyl alcohol (HVA) (Lin et al. 2013) was detected in the rat feces.Nevertheless, the same was not observed in human fecal samples, and neither oleuropein nor HVA were present, probably due to differences in the gut metabolic pathways between rats and humans (Mosele et al. 2014) Lactobacillus plantarum, which is usually found in the human gastrointestinal tract, is the most effective bacteria converting OL into HT due to its β-glucosidase and esterase activity (Marsilio and Lanza 1998).This bacterium is also able to metabolize phenolic acids such as protocatechuic, ferulic, gallic and coumaric acids through inducible decarboxylase and reductase enzymes (Landete et al. 2008;Rodríguez et al. 2009;Tafesh et al. 2011).
The importance of microbiota toward the bioactivity of antioxidants has gained a huge interest in the last decade and it has shown to be one of the most important causes of inter-individual variations observed in the antioxidant bioavailability.Substantial inter-individual variation has been observed in the excretion recovery of VOO secoiridoids, ranging from 2 to almost 60% of the ingested phenolic compounds (Mosele et al. 2014).In fact, in many studies gender, body mass index, or even age and drug intake were Not only phenolic alcohols and secoiridoids are metabolized by gut microflora.Lignans, such as acetoxypinoresinol (60) and pinoresinol (61), are also metabolized in the proximal colon producing, through several metabolic pathways, enterodiol (67) and enterolactone (69), compounds known as "mammalian lignans" (Kezimana et al. 2018;Stevens and Maier 2016;Yoder et al. 2015) (Figure 5).Only small amounts of pinoresinol have been found in human urine after VOO consumption (Nurmi et al. 2003;Raffaelli et al. 2002).Therefore, serum enterolactone levels and urinary enterolactone excretion can be used as biomarkers for plant lignan intakes.However, differences in the enterolactone concentration may arise due to differences in: lignan composition of the oil; gut bacteria; individual intestinal transit time; redox level in the colon, and due to the use of antibiotics (Clavel, Borrmann et al. 2006, Clavel, Doré, andBlaut 2006;Kilkkinen et al. 2001).Enterolignans formed during the metabolic pathway such as secoisolariciresinol (63), enterodiol (67) and enterolactone (69), are now able to enter the enterohepatic circulation (Jansen et al. 2005).They are likely to undergo extensive first pass metabolism by phase II enzymes, resulting in glucuronides and/or sulfates, either in the gastric mucosa or in the liver prior to their appearance in the systemic circulation or simply be eliminated in the bile or urine (Adlercreutz et al. 1995 Rowland et al. 2003).Both enterodiol and enterolactone have shown to exert estrogenic effects in vivo and reduce breast and prostate cancer cell growth via both estrogen dependent (Carreau et al. 2008) and independent mechanisms (Ilbeigi et al. 2017).
Radiolabeled HT and T excretions were (D' Angelo et al. 2001;Tuck and Hayball 2002) investigated in vivo in male Sprague Dawley rats for 5 h following a single intravenous (i.v.) injection (in saline solution) or oral administration by gavage (in olive oil or aqueous solution).It was shown that most of total radioactivity was associated with sulfate conjugated forms of HT, confirming previous results, followed by 3′,4′-dihydroxyphenylacetaldehyde (41, DOPAL), HVA, DOPAC, HT and HVA, representing 28.8%, 25.6%, 23.6%, 12.7%, 6.2%, and 0.3% of total radioactivity in urine.The total radioactivity half-life in blood was estimated to be within minutes and the amount of radioactivity was highest after 5 min of administration followed by a gradual decrease.The radioactivity in the kidneys was about 10 times higher than in other organs.About 90% of the injected dose was excreted in urine collected up to 5 h after administration, indicating that renal excretion of hydroxytyrosol and its metabolites is the major elimination route.In these studies, the estimated bioavailability of hydroxytyrosol when given orally in oil and water was 99% and 75%, respectively.
Most studies agree in the identification of the several HT and T metabolites but the contribution of each compound to the total pool of metabolites in vivo is sometimes contradictory (González-Santiago, Fonollá, and Lopez-Huertas 2010;Tuck and Hayball 2002).Some of these contradictory reports maybe caused by the different dosage, different composition in phenolic compounds, different routes of administration or different food matrices, which can bypass or trigger activation of some metabolic pathways in some organs and tissues leading to sulfate or glucuronidation conjugation (Kotronoulas et al. 2013).In fact, it was reported by Kotronoulas et al. that the administration of low doses (1 mg/kg) of HT to male and female Sprague Dawley rats promoted the glucuronidation pathway (25-30%) over the sulfated pathway (14%) (Kotronoulas et al. 2013).In contrast, at 100 mg/kg dose, the sulfation pathway was the most predominant (75%).Therefore, these results showed that the dose of phenolic compounds ingested seems to determine in large extent their metabolic disposal (Kotronoulas et al. 2013).Likewise, other factors may also contribute to these differences such as gender or individual microbiota as already mentioned.For example, in a study by Domínguez-Perles et al. (2017), female rats have shown higher excretion of HT metabolites than males (1.4-3.9-fold) and the second metabolite by abundance in urine in male rats treated with 1 mg/kg of DOPAC or HT was Tyr, with a clear microbiota origin.Moreover, this study reported a recovery of the compounds from 9.7% to 154% in males and from 22.1% to 241% in female animals suggesting an interference by the metabolism of dopamine neurotransmitters (Figure 4) that also produce T, HT, HVA, and homovanillic acid (77) (Galmés et al. 2021).
Although the structure of metabolites derived from HT and T found in biological fluids are well defined, the same cannot be said about the structure of secoiridoids metabolites and only more recently their structures have been, at least partially, clarified (Figures 7 and 8).The first human intervention studies have failed to clearly identify oleuropeidine, oleacein or oleocanthal in either plasma or urine following VOO ingestion (Miró-Casas, Covas, Farre, et al. 2003;Miró-Casas, Covas, Fitó, et al. 2003;Vissers et al. 2002), while oleacein has only been measured in the plasma of one subject (out of five) in a study performed by Suarez et al. (Suárez et al., 2009).The inability to detect oleacein and oleuropeidine (or their glucuronides) in biological fluids are more likely to be because they are not the major bioavailable forms in vivo.Moreover, the high number of possible structural isomers and stereoisomers of the several possible metabolites formed (Figures 7-9) makes these reach very low concentrations in body fluids and tissues, being only detected/quantified by the more recent UHPLC-MS technics.
Oleuropein has shown low absorption kinetics in vitro.This low absorption can be explained by its hydrophilic character, requiring alternative uptake mechanisms from those of HT and/or the need for microbiota activity to be absorbed (Corona et al. 2006;López de las Hazas et al. 2016;Vissers et al. 2002).Despite this, in vivo studies endorse the distribution of oleuropein derivatives to different tissues and organs of animals after ingestion of olive pomace (Serra et al. 2012).Moreover, it was found that after supplementation to rats with 5 mg of oleuropein/kg/day for 21 days (López de las Hazas et al. 2016), a much higher concentration (up to 50-fold higher) in urine of homavanillic acid, HT sulfate and homovanillic acid sulfate was achieved when compared with the concentrations for these metabolites found after hydroxytyrosol supplementation or phenolic extract supplementation.In humans (de Bock et al. 2013), conjugated metabolites of hydroxytyrosol were the primary metabolites recovered in plasma and urine after oleuropein rich (>80%) leaf extract ingestion.It was also found a wide inter-individual variation and a gender effect on the bioavailability of oleuropein rich extract, with males displaying greater plasma area under the curve for conjugated HT (11,600 vs. 2550 ng/mL; p = 0.048).Then, oleuropein can be hydrolyzed and converted to HT by phase I metabolism reactions in the body, causing increases in the HT metabolites bioavailability, at the expense of oleuropein (de Bock et al. 2013;García-Villalba et al. 2010;Khymenets et al. 2010;Miró-Casas, Covas, Farre, et al. 2003;Rubió, Macià, et al. 2012;Suárez et al. 2011).In contrast, oleuropein and ligstroside aglycone derivatives, which are structurally related to oleuropein and ligstroside, have shown to be quickly up taken and, at least, partially hydrolyzed to HT by carboxylesterases, in particular by CES2 present in the small intestine in both humans and rats, leading to a rise in the HT concentration in plasma, intestinal lumen and several tissues such as stomach, small intestine and liver (López-Yerena, Pérez, et al. 2021;López-Yerena, Vallverdú-Queralt, Jáuregui, et al. 2021;López-Yerena, Vallverdú-Queralt, Lamuela-Raventós, et al. 2021;López de las Hazas et al. 2016).Therefore, after consumption of VOO, the concentration of HT and its metabolites rises more than expected if taken into consideration only the HT existent in the oil, and it is proportional to the total phenol content and not to the HT content of the oil (López de las Hazas et al. 2016;Vissers et al. 2002).
A human study (García-Villalba et al. 2010), concerning the exploratory analysis of human urine by LC-ESI-TOF MS after intake of a high phenol rich olive oil showed the presence of compounds with a molecular ions compatible with reduced forms of oleacein (101) and oleuropeidine ( 122), together with their O-methyl conjugates (90, 123) (Figures 7 and 9).It was also detected secoiridoids in the hydrated form (compounds 92-95, 111-114), probably due to the addition of water molecules from the aqueous fluids and extraction solutions to the carbonyl groups in a similar fashion to what occurs when oleacein is dissolved in water.In fact, according to the results obtained in vitro, hydrated metabolites should be present in a higher concentration than non-hydrated forms, although the equilibrium of this reaction may be influenced by the interaction of carbonyl groups with proteins or by media pH.Although in this study most of the metabolites found were phase I metabolites or methylconjugates, glucuronides of these phase I metabolites were also identified, HT-EDA + CH 2 +Glu and HT-EA + CH 2 +Glu (91 and 121), together with the glucuronides of oleacein (98, 99, HT-EDA + Glu) and oleuropeidine (119, 120, HT-EA + Glu).Moreover, a glucuronide of a reduced form of oleuropeidine (124, 125, HT-EA + H 2 +Glu) were found in important concentrations.A further class of secoiridoid metabolites was also identified, having these secoiridoid metabolite molecules an increase in molecular ion mass of 16, suggesting, according to the authors, the existence of an extra hydroxyl group in these secoiridoid metabolites.Latter, López-Yerena et al. (2020) suggested that this increase in the mass of 16 units could be probably due to the occurrence of oleocanthal and oleuropeidine oxidation by aldehyde dehydrogenases, with the production of oleocanthalic (28) and oleaceinic (27) acids and its metabolite derivatives (Figure 7).Silva et al. (2018) published data concerning the presence of secoiridoid metabolites, not only in urine but also in human plasma, after consumption of 50 mL of VOO containing 51, 203, 30, and 10 mg/kg of oleacein, ligstrosidine, oleocanthal and oleuropeidine respectively.These authors reported the presence of unchanged oleacein in plasma in low concentration and considerable amounts of glucuronides of the reduced forms of the above secoiridoids, before and after O-methylation, in agreement with the metabolization pathways previously reported by Garcia-Villalba et al (García-Villalba et al. 2010).Also detected were secoiridoid glucuronides in the hydrated form and secoiridoid metabolite molecules with a molecular mass increase of more 16, confirming previous works (García-Villalba et al. 2010).However, although similar metabolites were found in both studies, the importance of each metabolite for the pool of all secoiridoid metabolites in urine were somewhat contradictory as the concentration of phase I metabolites in the Garcia-Villalba et al study were found to be much higher than in the Silva et al study, where the most important metabolites found were glucuronides.This is of great importance as the bioactivity of VOO phenolic compounds will depend on the metabolic pathways they will suffer in the body.As already mention, food matrix, the composition of the extracts, dose of phenolic compounds ingested, gender, or individual microbiota seems to determine in large extent their metabolic disposal and maybe the reason for these contradictory reports (Lenaerts et al. 2007;Lozano-Castellón et al. 2020;Zhu et al. 2017).Moreover, the use of a mixture of phenolic compounds may increase the uncertainty in the tentative identification of some secoiridoid metabolites by MS technics as some oleuropein and ligstroside derivatives (García-Villalba et al. 2010) may have the same molecular ion.The presence of several stereoisomers of different metabolites also increases the difficulty in the tentative identification of compounds by MS.Both García-Villalba et al. (2010) and Silva et al. (2018) were able to detect, in phenolic extracts from biological fluids, several chromatographic picks with close retention times with the same molecular ion and similar mass spectra pattern, indicating the presence of these stereoisomers.In fact, apart from the possibility of enantiomeric isomers, there are the possible presence of diastereoisomers due, not only to the presence of double bounds, but also to the substitution of the pyranic ring in some of the metabolites (Figures 7 and 9).
Preliminary studies into the ability of oleuropeidine and oleacein, as pure compounds, to cross the small intestine were investigated using a human Caco-2 cell model (Pinto et al. 2011).Analysis of the apical side (AP) and basolateral (BL) side medium showed enterocyte-mediated transfer of both oleuropeidine and oleacein over a 2 h period from the apical side to the basolateral side, in a time dependent and concentration dependent manner.HT was also detected on both the AP and BA sides, whereas HVA was only detected on the AP side of the Caco 2 cells.
Although the Caco-2 cell model is generally considered to be a suitable model for intestinal mediated first-pass metabolism, this cell system may not wholly reflect physiological conditions in vivo as several enzymes involved in metabolism are overexpressed relative to the human intestinal epithelial cells.On the other hand, uridine-diphospho-glucuronosyl transferase (Pinto et al. 2011), catechol-O-methyl transferase, aldolase and retinal dehydrogenase (Lenaerts et al. 2007) are under expressed in these cells, resulting in an incomplete picture of the actual pattern of oleacein and oleuropeidine absorption and metabolism.To address this problem, an experiment was performed using isolated, perfused rat intestinal model, which possess a full metabolic capacity for up to two hours post-isolation (Pinto et al. 2011).Following the perfusion of the ileum with oleuropeidine solution, HPLC-MS detected in the serosal fluid HVA, hydroxytyrosol glucuronide (81, 82, HT-Glu), and homovanillic alcohol glucuronide (72, HVA-Glu).In addition, it was also detected the E-and Z-isomers of oleuropeidine, its glucuronides and, as the major bioavailable forms, two glucuronides of each reduced forms of the Eand Z-isomers of oleuropeidine (124, 125, HT-EA + H 2 +Glu, Figure 9).These results indicate that during transfer across the ileum, oleuropeidine isomers undergo both a two-electron reduction to yield HT-EA + H 2 and monoglucuronidation at two positions (positions 3′ or 4′, Figure 9).In fact, the carbonyl group may suffer the action of NADPH-dependent aldo-keto reductases (Jez et al. 1997) that are widely distributed in mammals and are capable of catalyzing the reduction of a variety of carbonyl-containing compounds.These enzymes are responsible, for example, for the reduction of retinal to retinol in the human small intestine (Crosas et al. 2003) as well as the reduction of various other molecules, including carbohydrates, aliphatic and aromatic aldehydes and steroids.
Similar observations were made following the perfusion of the ileum with oleacein solution (Pinto et al. 2011), with the presence in the serosal fluid of HVA, HT glucuronide, HVA glucuronide being detected.However, again, the major metabolites were glucuronides of the reduced forms of oleacein (107, 108, HT-EDA + H 2 +Glu, Figure 7).Neither oleacein nor its reduced forms were detected in the serosal fluid by HPLC-MS.
In these studies, it was not possible to confirm which of the carbonyl functional groups of oleacein had undergone reduction since reduction at either carbonyls would yield MS similar fragmentation patterns (López-Yerena, Pérez, et al. 2021;Pinto et al. 2011).At first sight, the oxidation of the conjugated carbonyl could make more sense as this moiety is quite similar to the one found in the retinal molecule.However, in the case of oleuropeidine, where there is no conjugated carbonyl group, this reduction was also observed.Moreover, it has never been reported in the several studies (García-Villalba et al. 2010;López-Yerena, Pérez, et al. 2021;López-Yerena, Vallverdú-Queralt, Lamuela-Raventós, et al. 2021;Silva et al. 2018) performed with oleacein the presence of the hydrated form of these reduced molecules, what may indicate that the non-conjugated carbonyl is the one that suffers reduction.Nevertheless, it was also found that the hydroxyl group formed by a carbonyl reduction can actually add to the non-reduced carbonyl group, forming a pyrane like ring, similar to the one found in oleuropeidine (Paiva-Martins et al. 2015) (102, 103, Figure 7).This will enable further addition of water molecule to this metabolite.
Perfusion of the jejunum (Pinto et al. 2011) with both oleacein and oleiropeidine also resulted in their extensive reduction and glucuronidation in a similar manner to that seen in the ileum.However, these metabolites were transferred to a greater extent in the jejunum compared to the ileum, in particular in the case of oleuropeidine metabolites that were found in four times higher concentration in the jejunum serosal fluid than in the ileum serosal fluid.Transfer of compounds and appearance of metabolites reached a maximum after about one hour, reflecting an initial slow uptake of components into cells, followed by their enzymatic reduction and metabolism.There were also notable differences in the patterns of metabolism occurring between the jejunum and ileum, with ileum appearing to display a lower reductase and glucuronidase activity than the jejunum (Pinto et al. 2011).Nevertheless, when considering the whole small intestine, glucuronides of reduced forms of oleacein and oleuropeidine were the major intestinal metabolites, concerning 40-60% of the total metabolites entering the portal blood (Pinto et al. 2011).
Using a single-pass intestinal perfusion (SPIP) rat model, oleacein was assayed at 0.15 mg/mL (468 µM) (López-Yerena, Pérez, et al. 2021).Simultaneous luminal blood sampling and analysis of the intestinal fluid and mesenteric blood by LC-ESI-LTQ-Orbitrap-MS was then performed.This study confirmed that oleacein was mostly metabolized by phase I reactions, undergoing hydrolysis, reduction and oxidation, and showing metabolite levels much higher in the plasma than in the lumen.According to these results, oleacein is well absorbed in the intestine, with an intestinal permeability similar to that of the highly permeable model drug naproxen (López-Yerena, Pérez, et al. 2021).A large number of metabolites were identified, and their relatively high abundance indicate an important intestinal first-pass metabolism of oleacein during absorption.The most abundant metabolites found in plasma and lumen in similar concentrations (Figure 7) were hydroxytyrosol and possibly oleaceinic acid (27, HT-EDA + O), followed by the reduced and hydrated forms of oleacein, HT-EDA + H 2 (101) and HT-EDA + H 2 O (93), respectively.Glucuronides of the reduced form of oleacein (107, 108, HT-EDA + H 2 +Glu), oleacein hydrate (95/96, HT-EDA + H 2 O + Glu), O-methyl conjugated oleacein (91, HT-EDA + CH 2 +Glu), and O-methyl conjugated oleaceinic acid (HT-EDA + CH 2 +O + Glu) were also detected but in much lower concentrations probably as a consequence of the lack of hepatic metabolism that was not evaluated in this study.These metabolites were also found in the ileum tissue samples, in particular HT, the reduced form of oleacein ( 101) and oleacein hydrate (93) (Figure 7).Although HT was the more concentrated compound found in plasma, intestinal lumen and ileum tissue, the pool of secoiridoid metabolites accounted for more than 60% of all identified metabolites.
The distribution of oleacein and its metabolites in rat plasma and tissues (stomach, intestine, liver, kidney, spleen, lungs, heart, brain, thyroid and skin) 1, 2 and 4.5 h after acute intake of a refined VOO containing 0.3 mg/mL of oleacein was also investigated by LC-ESI-LTQ-Orbitrap-MS (López-Yerena, Vallverdú-Queralt, Lamuela-Raventós, et al. 2021).In this study, a quite different metabolite pattern was observed when compared with the intestinal perfusion (SPIP) rat model (López-Yerena, Pérez, et al. 2021).In contrast with this study, where the possible oleaceinic acid (27, DHPEA-EDA + O) was found as the most concentrated metabolite in plasma and ileum tissue, this metabolite was not found either in rat plasma or in any of the analyzed tissues.In this case, after 2 hours of oleacein intake, the most abundant secoiridoid metabolites found in plasma were the methyl conjugate of oleacein (90, HT-EDA + CH 2 ), representing 25% of the total metabolites identified (p < 0.05), followed by the glucuronide of oleacein hydrate (95, 96, HT-EDA + H 2 O).These secoiridoid metabolites were not identified in plasma in the intestinal perfusion (SPIP) rat model study.Nevertheless, in both studies HT was one of the most important metabolites, representing in this study around 20% of the total quantified metabolites.Some of these contradictory results may be caused by the different dosage, different route and vehicle of administration, which can bypass or trigger the activation of some metabolic pathways, and by the absence or presence of organs and tissues leading to different metabolic disposal.
The metabolite content of several organ tissues showed quite important differences between them (López-Yerena, Vallverdú-Queralt, Lamuela-Raventós, et al. 2021).In general, the maximum concentration of metabolites in stomach and intestine was observed after one hour of intake.However, in other tissues, except for kidneys and skin, where the maximum concentration was achieved at 4.5 h, the maximum concentration was achieved after 2 h, with a significant decrease 4.5 hours after intake.Oleacein was found in stomach (C max = 8.7 µmol/kg), small intestine (C max = 2.2 µmol/kg), liver (C max = 0.28 µmol/kg) and heart tissues (C max = 0.45 µmol/ kg), but not in any other tissues, in an important concentration after 1 h of intake.This concentration in the case of the heart tissue was kept stable during at least 4.5 h after the oral intake.Although, in the vitro studies, the concentrations detected in the heart were very low compared with the concentrations considered necessary for bioactivity (1-10 µM) (Czerwińska et al., 2012;Fernandes et al. 2020;Paiva-Martins et al. 2013, 2015;Segade et al. 2016) this is the first demonstration that oleacein is able to reach the heart, where it may exert a beneficial effect.Moreover, the stable concentration for oleacein in the heart tissue over the 4.5 hours rises the possibility of accumulation in this tissue upon sustained intake.Other secoiridoid metabolites such as the glucuronide of the oleacein hydrate (95/96, HT-EDA + H 2 O + Glu), the methyl conjugated oleacein (90, HT-EDA + CH 2 ) and the reduced form of oleacein (101, HT-EDA + H 2 ) were also found in the heart tissues in important concentrations, in particular after 2 h of intake.On the other hand, the methyl conjugated oleacein (90, HT-EDA + CH 2 ) was the most concentrated secoiridoid metabolite found not only in the liver (C max = 5.79 µmol/kg) but also in brain tissue (C max =0.52 µmol/kg).The reduced form of oleacein (101, HT-EDA + H 2 ) was the most important metabolite in the intestine tissue (C max = 5,71 µmol/kg), confirming previous results in perfused intestine experiments (Pinto et al. 2011), and spleen (C max = 0.68 µmol/ kg).In Table 2, we can observe the metabolites and their order of concentration in the different tissues.
Most of the metabolites found in tissues, except for kidney tissue, were mainly as non-glucuronide conjugated Table 2. oleacein and its metabolites found in plasma and in different rat tissues after oral ingestion of oleacein (Ht-eda) at 2.5 mg/kg.numbers represent the order of concentration found in the tissue, being 1 de most concentrated metabolite.nd, not detected.adapted from reference (lópez-Yerena, vallverdú-Queralt, lamuela-raventós, et al. 2021).For metabolite identification, please see Figure 7. metabolites.This is of great importance as the bioactivity of VOO phenolic compounds seems to decrease drastically with glucuronide and sulfate conjugation (Fernandes et al. 2020;Khymenets et al. 2010;Paiva-Martins et al. 2013).On the other hand, oleacein, phase I metabolites and methyl conjugates seems to have important bioactivity in preventing oxidative stress, as vasorelaxant and as anti-inflammatory at quite low concentrations (Czerwińska, Kiss, and Naruszewicz 2012;Fernandes et al. 2020;Paiva-Martins et al. 2013, 2015;Segade et al. 2016).The liver, heart, spleen, and lungs were the target tissues where the secoiridoid metabolites were detected in larger concentrations, which can explain the health benefits attributed to EVOO consumption as HT does not seem to reach any of these tissues.

Metabolite
The absorption and metabolism of oleocanthal was also studied using a single-pass intestinal perfusion (SPIP) rat model similar to that used for oleacein (López-Yerena et al. 2020).Samples were analyzed by UHPLC-MS-MS for the presence of oleocanthal and its metabolites.In contrast with oleacein, oleocanthal was poorly absorbed in the intestine, as indicated by the low effective permeability coefficient (2.23 ± 3.16 × 10 −5 cm/s) and apparent permeability coefficient (4.12 ± 2.33 × 10 −6 cm/s) when compared to the highly permeable reference compound levofloxacin (López-Yerena et al. 2020).The hydrated form of oleocanthal (92, T-EDA + H 2 O) was the most concentrated secoiridoid found in the intestine lumen and its concentration increased almost linearly over the 60 minutes of experiment (Figure 7).Oleocanthal was mostly metabolized by phase I metabolism, undergoing reduction probably catalyzed by NADPH-dependent aldo-keto reductases (AKR) located in the small intestine epithelium with the formation of the reduced form of oleocanthal (100, T-EDA + H 2 ).Furthermore, no other metabolites were detected in the intestinal lumen.Thus, reduction seems, indeed, to be an important phase I metabolic route for secoiridoids.In plasma, the most important secoiridoid found was, again, the hydrated form of oleocantal (92, T-EDA + H 2 O), representing up to 65% of all secoiridoids, followed by its glucuronide (94, up to 25% of all secoiridoids).The oleocanthal hydrate has also been identified in plasma after VOO phenolic consumption (Silva et al. 2018).
An oxidation product of oleocanthal (T-EDA + O), obtained probably by the action of the enterocyte cytochromes P450 enzymes was detected.In this case, the possibility of a further aromatic hydroxylation by Cytochrome P450 enzymes, instead of the carbonyl group oxidation, with the formation of oleacein instead of ligstrosidic acid, needs to be considered (Figure 8).Actually, according to the literature, the fragmentation pattern of this metabolite by MS is compatible with the presence of oleacein, since the main fragments observed had m/z = 153 and 183, corresponding to hydroxytyrosol and the dialdehydic form of elenolic acid, respectively.Moreover, the presence of glucuronides of a possible methylated conjugate of the oxidative form of oleocanthal (T-EDA + O+CH 2 +Glu = HT-EDA + CH 2 +Glu) indicates that this metabolite seems to bear a catecholic structure.In addition, the presence of hydrates of the oxidized form eliminates the hypothesis of non-conjugated carbonyl group oxidation.A previous study (García-Villalba et al. 2010) has identified this metabolite in human urine, although the authors raised doubt about whether it was a oleocanthal metabolite or a hydroxytyrosol derivative such as oleacein (Figure 8).
A further glucuronide, of the reduced oleocanthal (106, T-EDA + H 2 +Glu), were also detected in plasma.Interestingly, tyrosol was not detected in none of the samples.Both results are in accordance with previous results obtained by Silva et al (Silva et al. 2018) which found, after VOO intake, only oleocanthal hydrate and glucuronides of reduced oleocanthal, both in plasma and urine.
Recently, the first study concerning the distribution of olecanthal and its metabolites in rat plasma and tissues (stomach, intestine, liver, kidney, spleen, lungs, heart, brain, thyroid and skin) after 1, 2 and 4.5 h of acute intake of a refined olive oil containing 0.3 mg/mL of oleocanthal has also been performed by LC-ESI-LTQ-Orbitrap-MS (López-Yerena, Vallverdú-Queralt, Lamuela-Raventós, et al. 2021).In general, and in a similar fashion to oleacein, the maximum concentration of metabolites in stomach and intestine was observed after one hour of intake.However, in other tissues except for kidneys and skin, where the maximum concentration was achieved at 4.5 h, the maximum concentration was achieved at 2 h after intake with a significant decrease after 4.5 h.It was observed, again, a quite different metabolite pattern when compared with the intestinal perfusion (SPIP) rat model study (López-Yerena et al. 2020).In contrast with this study, where the oleocanthal hydrate (92, T-EDA + H 2 O) was found as the most concentrated secoiridoid in plasma and ileum lumen, this secoiridoid was not the most important compound found neither in plasma nor in any other analyzed tissue (Table 3).In this study, the oxidation of oleocanthal, with the probable production of oleacein (T-EDA + O = HT-EDA), seems to be the most important metabolic pathway for this compound in vivo.This metabolite was the most concentrated both in stomach (C max = 12.5 µM/kg) and intestine (C max = 4.8 µM/kg).The probable hydrate (T-EDA + O+H 2 O = HT-EDA + H 2 O) in the lungs (C max = 6.3 µM/kg) or the methyl conjugate of this metabolite (T-EDA-CH 2 +O = HT-EDA + CH 2 ) were then the most concentrated metabolites (Table 3) found not only in plasma (6 µM/kg) but also in most of the analyzed tissues, such as liver (31 µM/kg), spleen (15 µM/kg), brain (C max = 10 µM/kg) and heart tissues (C max = 7.5 µM/kg).Although the glucuronides of the oxidized form of oleocanthal (T-EDA + O+Glu = HT-EDA + Glu) were detected in several tissues in relatively low concentration, the glucuronide of the oleocanthal hydrate (94, T-EDA + H 2 O + Glu) was the most important glucuronide metabolite in the kidney (C max = 16.5 µM/kg), liver (C max = 4 µM/kg) and plasma (C max = 3.2 µM/kg).Tyrosol was only found in stomach and intestine tissues but not in plasma or other organs probably because of its metabolization by enzymes of the gastrointestinal mucosal epithelium before entering systemic circulation.Moreover, T metabolites were not detected, possibly because they were at a very low concentration.
Since olecanthal oxidation seems to be the most important metabolic pathway for this compound in vivo, future confirmation of oleacein as one of the most important metabolites of oleocanthal, will be of foremost importance in understanding the VOO phenols bioactivity.If oleacein and its metabolites can arise in the body from olecanthal intake, this will influence the health benefits attributed to oleocanthal consumption as oleacein being a catecholic, presents a much higher radical scavenging capacity than oleocanthal.It is also important to point out, that the concentration found for these metabolites in tissues, even for the ones with the lowest concentration, were one order of magnitude higher than the ones found in the oleacein study using similar in vivo system and conditions (López-Yerena, Vallverdú-Queralt, Lamuela-Raventós, et al. 2021).Furthermore, the majority of metabolites found were not as a glucuronide or sulfate conjugate.These results are somewhat surprising as oleocanthal showed to be poorly absorbed when evaluated by the single-pass intestinal perfusion (SPIP) rat model (López-Yerena et al. 2020).

Conclusion
Most bioavailability studies conducted so far agree that plasma and tissue concentrations of VOO polyphenol metabolites is often higher than the concentration reached by the ingested parent compound.Therefore, these metabolites are likely to significantly contribute to the beneficial bioactivity correlated to the regular consumption of olive polyphenols.However, the data available, in particular related with secoiridoid metabolism, is still quite scarce and some of the metabolites do not have their structures well defined.Moreover, too little is known about interaction of parental phenols and its metabolites with plasma and cell membrane proteins or their stability during analytical procedures, what may underestimate their concentration in the body.Therefore, very little is still known about the underling mechanisms of the in vivo bioactivity of VOO polyphenols and of which compounds have a major contribution for the health effects of VOO consumption.This present review is intended to summarize what is currently known about the major VOO polyphenols biochemical transformations occurring in the human body as their chemistry is fundamental in understanding and evaluating the bioavalability and health benefits associated with VOO consumption in future research works.
Table 3. oleocanthal and its metabolites found in plasma and in different rat tissues after oral ingestion of oleocanthal (t-eda) at 2.5 mg/kg.numbers represent the order of concentration found in the tissue, being 1 de most concentrated metabolite.nd, not detected.adapted from reference (lópez-Yerena, vallverdú-Queralt, Jáuregui, et al. 2021).For metabolite identification, please see Figure 7

Figure 1 .
Figure 1.structures of the most important olive oil phenolic secoiridoids.

Figure 2 .
Figure 2. structures of olive oil secoiridoid phenolic compounds and possible biochemical transformations pathways of oleuropein and ligstroside aglycones in olives, during olive oil extraction and during olive oil phenolic compounds extraction.
; Dean et al. 2004; Jan, Ho et al. 2009; Jansen et al. 2005; Knust et al. 2006; Liu, Saarinen, and Thompson 2006; ) and are widely distributed throughout the organism(D' Angelo et al. 2001; Weinbrenner  et al. 2004).Pharmacokinetic analysis in rats indicate extensive and fast uptake of these compounds and distribution of their metabolites by different organs including skeletal muscle, kidneys, liver, lungs, heart(D' Angelo et al. 2001) and brain(D' Angelo et al. 2001;López de las Hazas et al. 2018).The knowledge of their metabolic pathways in the body is very relevant during ADME in humans, as the un-metabolized forms are most of the time undetectable in

Table 1 .
Main phenols present in virgin olive oil.
and text.