**3.3 Hydroxytyrosol-derived isochromans**

386 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

Scheme 8. Synthesis of hydroxytyrosyl alkyl ethers by alkylation with alkyl iodides

steric effect of the hydrocarbon chains.

**3.2.2 Biological activity** 

other xenobiotics.

The oxidative stability of lipid matrix in the presence of these compounds, measured by the Rancimat method, has shown that these derivatives retain the high protective capacity of free hydroxytyrosol and similar induction times, having higher induction times than butylhydroxytoluene (BHT) and α-tocopherol (Madrona et al., 2009). These results are in agreement with those obtained in the case of hydroxytyrosyl esters, covered in the previous section (Mateos et al., 2008). The antioxidant activity has been checked by the DPPH, FRAP and ABTS assays in a hydrophilic medium (Pereira-Caro et al., 2009). The antioxidant activity of the lipophilic hydroxytyrosyl ethers was slightly lower in bulk oils and higher in hydrophilic media in comparison with their reference HT, supporting the polar paradox. The length of the alkyl chain did have a positive influence in hydrophilic medium for ethers with a short alkyl chain (methyl, ethyl, propyl), while ethers with longer alkyl chains (from butyl to octadecyl) maintained or decreased their antioxidant activity, probably due to the

In order to evaluate the safety and potential biological activity of these ethers, studies of their transport, absorption and metabolism in cellular and animal models have been developed (Pereira-Caro et al., 2010a, 2010b) using a human hepatoma cell line (HepG2) as a model system of the human liver and human enterocyte-like Caco-2/TC7 cells, which are commonly used to characterize the intestinal absorption of a range of drugs, nutrients, and

The results showed a direct relationship between the lipophilic nature of each compound and the level of metabolization; as an example, hydroxytyrosyl butyl ether biotransformation was complete after 18 h, whereas small amounts of the others remained after the same time. Furthermore, an intestinal absorption increase was observed from methyl to *n*-butyl ethers.

Protective effects against oxidative stress have also been studied (Pereira-Caro et al., 2011) using HepG2 cells, the ones previously employed to assess the metabolism of the synthesized HT ethers. The results obtained show the potential to prevent cell damage induced by *tert*-butyl hydroperoxide (*t*-BuOOH) and the ability to maintain unaltered cellular redox status, partially after 2 hours of pretreatment and almost completely after 20 hours. These results are in accordance with those obtained with hydroxytyrosol (Martín et al., 2010), but they also show the relevance of the role of the lipophilic character of the Isochroman fragment is a ubiquitous scaffold that can be found in natural products, drugs and agrochemicals (Larghi & Kaufman, 2006). Access to dihydroxyisochromans derived from HT can be achieved by using the oxa-Pictet-Spengler reaction, by reaction of arylethanols with aldehydes, ketones or masked-carbonyl derivatives (Guiso et al. 2001). The reaction is highly regioselective, as intramolecular cyclization takes place mainly in the less hindered position, as it can be deduced from its reaction mechanism, shown in Scheme 9. Two of the synthetized isochromans (Fig. 4) have been detected in olive oil (Bianco et al., 2001).

Fig. 4. Isochromans naturally present in olive oil

Scheme 9. Synthesis of hydroxytyrosol isochroman derivatives

Hydroxytyrosol isochroman derivatives shown in Fig. 4 were effective free radical scavengers able to inhibit platelet aggregation and thromboxane release (Togna, 2003).

#### **3.4 Hydroxytyrosol glucoronide derivatives**

One of the major metabolic pathways found *in vivo* for dietary phenolic compounds such as hydroxytyrosol is *O*-conjugation via glucuronidation and sulfation. Therefore, it is of interest to study these metabolites and their biological activities.

Biological Properties of Hydroxytyrosol and Its Derivatives 389

The synthesis of 2-arylhydroxytyrosols from 2-halohydroxytyrosol derivatives has been described (Bernini et al., 2008a). The reaction of the corresponding 2-chloro precursors via Suzuki-Miyaura cross-coupling reaction with arylboronic acids containing electrondonating, electron-withdrawing, as well as *ortho* substituents, yielded this family of

The complexation of hydroxytyrosol with commercially-available β-cyclodextrin (β-CD) (López-García et al., 2010; Rescifina et al. 2010) and hydroxypropyl-β-cyclodextrin (HP-β-CD) (López-García et al., 2010) in aqueous solutions has been studied. The stoichiometries, the association constants and the geometry of the complexes have been determined by NMR techniques. The stoichiometries of both complexes are 1:1 and the association constants are 93±7 M-1 for HT/β-CD complex and 43±1 M-1 for HT/HP-β-CD complex (López-García et al., 2010). In both cases, the insertion of the catechol moiety took place by directing the hydroxyalkyl chain to the primary rim. The postulated geometry of the 1:1 HT/-CD

H-5

7

OH

OH

OH

OH

H-3

OH 14

Fig. 6. Postulated geometry of the 1:1 HT/-CD inclusion complex

**3.6 Arylhydroxytyrosol derivatives** 

compounds in high to excellent yields (Scheme 11).

Scheme 11. Synthesis of 2-arylhydroxytyrosol derivatives

inclusion complex is depicted in Fig. 6.

**3.7 Complexation of hydroxytyrosol with β-cyclodextrins** 

Biocatalyzed syntheses of hydroxytyrosol and other phenolic glucuronides have been developed using porcine liver microsomes (Khymenets et al., 2006, 2010). This type of glucuronides has also been synthesised stereoselectively (Lucas et al., 2009) in the phenolic or aliphatic hydroxyl groups using efficient chemical method from *O*-partially protected hydroxytyrosol and glucuronosyl trichloroacetimidate donors (Scheme 10).

The antioxidant activities of hydroxytyrosol conjugates have been evaluated, concluding that none of these glucuronides displayed significant antioxidant activities at the concentration tested (Khymenets et al., 2010).

Scheme 10. Synthesis of hydroxytyrosol glucuronides

#### **3.5 Hydroxytyrosol glucosides**

The three isomers of hydroxytyrosol -D-glucopyranosides (Fig.5) have been reported to be present in olives (Bianco et al., 1998). The 4-glucoside (Romero et al., 2004) and the 1 glucosides (Medina et al., 2007) have been found in table olive brines, and have been analysed as antimicrobial compounds against *Lactobacillus pentosus* with negative results. It has been recently shown that hydroxytyrosol 4-glucoside was the main phenolic compound in the aqueous phase of fresh alpeorujo, followed by hydroxytyrosol, and hydroxytyrosol 1 glucoside.

Fig. 5. The three isomers of hydroxytyrosol

#### **3.6 Arylhydroxytyrosol derivatives**

388 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

Biocatalyzed syntheses of hydroxytyrosol and other phenolic glucuronides have been developed using porcine liver microsomes (Khymenets et al., 2006, 2010). This type of glucuronides has also been synthesised stereoselectively (Lucas et al., 2009) in the phenolic or aliphatic hydroxyl groups using efficient chemical method from *O*-partially protected

The antioxidant activities of hydroxytyrosol conjugates have been evaluated, concluding that none of these glucuronides displayed significant antioxidant activities at the

The three isomers of hydroxytyrosol -D-glucopyranosides (Fig.5) have been reported to be present in olives (Bianco et al., 1998). The 4-glucoside (Romero et al., 2004) and the 1 glucosides (Medina et al., 2007) have been found in table olive brines, and have been analysed as antimicrobial compounds against *Lactobacillus pentosus* with negative results. It has been recently shown that hydroxytyrosol 4-glucoside was the main phenolic compound in the aqueous phase of fresh alpeorujo, followed by hydroxytyrosol, and hydroxytyrosol 1-

hydroxytyrosol and glucuronosyl trichloroacetimidate donors (Scheme 10).

concentration tested (Khymenets et al., 2010).

Scheme 10. Synthesis of hydroxytyrosol glucuronides

Fig. 5. The three isomers of hydroxytyrosol

**3.5 Hydroxytyrosol glucosides** 

glucoside.

The synthesis of 2-arylhydroxytyrosols from 2-halohydroxytyrosol derivatives has been described (Bernini et al., 2008a). The reaction of the corresponding 2-chloro precursors via Suzuki-Miyaura cross-coupling reaction with arylboronic acids containing electrondonating, electron-withdrawing, as well as *ortho* substituents, yielded this family of compounds in high to excellent yields (Scheme 11).

Scheme 11. Synthesis of 2-arylhydroxytyrosol derivatives

#### **3.7 Complexation of hydroxytyrosol with β-cyclodextrins**

The complexation of hydroxytyrosol with commercially-available β-cyclodextrin (β-CD) (López-García et al., 2010; Rescifina et al. 2010) and hydroxypropyl-β-cyclodextrin (HP-β-CD) (López-García et al., 2010) in aqueous solutions has been studied. The stoichiometries, the association constants and the geometry of the complexes have been determined by NMR techniques. The stoichiometries of both complexes are 1:1 and the association constants are 93±7 M-1 for HT/β-CD complex and 43±1 M-1 for HT/HP-β-CD complex (López-García et al., 2010). In both cases, the insertion of the catechol moiety took place by directing the hydroxyalkyl chain to the primary rim. The postulated geometry of the 1:1 HT/-CD inclusion complex is depicted in Fig. 6.

Fig. 6. Postulated geometry of the 1:1 HT/-CD inclusion complex

Biological Properties of Hydroxytyrosol and Its Derivatives 391

Appendino G, Minassi A, Daddario N, Bianchi F, & Tron GC. (2002) Esterification of

Aruoma O, Deiane M, Jenner A, Halliwell B, Kaur M, Banni S, et al. (1998) Effect of

Bernini R, Mincione E, Barontini M, & Crisante F. (2007) Procedimento per la preparazione

Bernini R, Cacchi S, Fabrizi G, & Filisti E. (2008a) 2-Arylhydroxytyrosol derivatives via

Bernini R, Mincione E, Barontini M, & Crisante F. (2008b) Convenient synthesis of

Bernini R, Mincione E, Barontini M, & Crisante, F. (2008c) Method for preparing hydroxytyrosol and hydroxytyrosol derivatives. PCT/IB2008/000598. Bianco A, Mazzei RA, Melchioni C, Romeo G, Scarpati ML, Soriero A, et al. (1998)

Bianco, A., Coccioli, F., Guiso, M. & Marra, C. (2001) The occurrence in olive oil of a new class of phenolic compounds: hydroxy-isochromans. Food Chem. 77: 405–411. Biesalski HK. (2007) Polyphenols and inflammation: basic interactions. Curr Opin Clin Nutr

Bisignano G, Tomaino A, Lo Cascio R, Crisafi G, Uccella N, & Saija A. (1999) On the in-vitro

Bouallagui Z, Han J, Isoda H, & Sayadi S. (2011a) Hydroxytyrosol rich extract from olive

Bouallagui Z, Bouaziz M, Lassoued S, Engasser JM, Ghoul M, & Sayadi S. (2011b)

Buisman GJH, van Helteren CTW, Kramer GFH, Veldsink JW, Derksen JTP, & Cuperus FP.

Bulotta S, Corradino R, Celano M, D'Agostino M, Maiuolo J, Oliverio M, et al. (2011)

semisynthetic peracetylated derivatives. Food Chem 127: 1609–1614. Burke TR Jr, Fesen MR, Mazumder A, Wang J, Carothers AM, Grunberger D, et al. (1995) Hydroxylated aromatic inhibitors of HIV-1 integrase. J Med Chem 38: 4171–4178. Carluccio MA, Ancora MA, Massaro M, Carluccio M, Scoditti E, Distante A, et al. (2007)

lipophilic antioxidants. Biotechnol Lett 20: 131–136.

Am J Physiol Heart Circ Physiol 293: 2344–2354.

low-density lipoprotein oxidation. J Agric Food Chem 46: 5181–5187. Balsano C & Alisi A. (2009) Antioxidant effects of natural bioactive compounds. Curr Pharm

hydroxytyrosol found in extra virgin olive oil on oxidative DNA damage and on

di derivati dell'idrossitirosolo e di idrossitirosolo via demetilazione ossidativa

hydroxytyrosol and its lipophilic derivatives from tyrosol or homovanillyl alcohol.

Microcomponents of olive oil III. Glucosides of 2(3,4-dihydroxy-phenyl)ethanol.

antimicrobial activity of oleuropein and hydroxytyrosol. J Pharm Pharmacol 51:

leaves modulates cell cycle progression in MCF-7 human breast cancer cells. Food

Hydroxytyrosol acyl esters: Biosynthesis and Activities. Appl Biochem Biotechnol

(1998) Enzymatic esterifications of functionalized phenols for the synthesis of

Antiproliferative and antioxidant effects on breast cancer cells of oleuropein and its

Homocysteine induces VCAM-1 gene expression through NF-kB and NAD(P)H oxidasa activation: protective role of Mediterranean diet polyphenolic antioxidants.

phenolic acids and alcohols. Org Lett 4: 3839–3841.

Suzuki-Miyaura cross-coupling. Org Lett, 10: 3457–3460.

Des 15: 3063-3073.

MI2007 A001110.

J Agric Food Chem 56: 8897–8904.

Food Chem 63, 461–464.

Metab Care 10: 724–728.

Chem Toxicol 49: 179–184.

971–974.

163: 592–599.

Moreover, the antioxidant activity of encapsulated HT, together with the photoprotection effect of β-CD on HT, has been evaluated by scavenging of the stable DPPH radical. It has been proven that β-Cyclodextrin acts as a secondary antioxidant and provides a moderate improvement of the radical scavenging activity of HT measured by the DPPH assay.

β-Cyclodextrin exerts a strong photoprotection of HT upon UV irradiation, which could be deduced from the EC50 values (Table 2). For equimolecular mixtures of HT and -CD at 1.2 mM, the observed degradation after 24 h and 48 h is similar to the degradation found for HT at the same concentration and time (entries 4 and 5) showing no protection at 24 h and only a slight protection after 48 h. However, using 1:4 mixtures of HT (1.2 mM) and -CD (4.8 mM), a remarkable reduction of the degradation rate was observed when compared with pure HT. In this way, the complexation of HT with cyclodextrins might enhance stability, improve its performance as antioxidant and extend its storage life (López-García et al., 2010).


Table 2. Effect of the encapsulation of HT on its photostability
