**3.1.2 Biological activity**

#### **3.1.2.1 Antioxidant activity**

The antioxidant activity of hydroxytyrosyl esters has been measured with different methods, including DPPH (1,1-diphenyl-2-picrylhydrazyl radical), ABTS (2,2'-azino-bis(3 etilbenzotiazolin-6-sulfonic acid), FRAP (ferric reducing antioxidant power) and Rancimat (Mateos et al., 2008; Gordon et al., 2001; Bouallagui et al., 2011b). The Rancimat test is a method commonly used to evaluate the antioxidant power in lipophilic food matrices, such as oils and fats, while the ABTS and FRAP assays are used for the evaluation of antioxidant activity in hydrophilic medium; the ABTS assay evaluating the radical-scavenging capacity, and the FRAP method determining the reducing activity.

The Rancimat test revealed a lower activity for ester derivatives compared to HT, in agreement with the so-called polar paradox, according to which hydrophilic antioxidants are more effective in less polar media, such as bulk oils, whereas lipophilic antioxidants are more effective in relatively more polar media, such as in oil-in-water emulsions or liposomes (Frankel et al., 1994; Shahidi & Zhong, 2011).

The use of enzymes, like lipases, as catalysts in non-aqueous solvents to prepare lipophilic derivatives directly from HT has been widely described in the last few years (Grasso et al., 2007; Torres de Pinedo et al., 2005; Mateos et al., 2008; Buisman et al., 1998). This procedure

The esterication of phenols with carboxylic fatty acids and lipases as biocatalysts was firstly investigated by Buisman et al., (1998), using hydroxytyrosol, octanoic acid in hexane, and immobilized lipases from *Candida antartica* (CAL-B). Furthermore, a strong dependence of the yield on the solvent used was observed; so, in diethyl ether a conversion of 85% was obtained within 15 hours (35 ºC), while conversions of roughly 20% were found in the case of solvents like chloroform, dichloromethane or THF. Yields of 70–80% were observed using

Different enzymes have been tested on hydroxytyrosol (Grasso et al., 2007) including lipases from *A. niger, C. cylindracea, M. javanicus, P. cepacia, M. miehei, C. viscosum, P. uorescens, R. arrhizus, R. niveus, C. antarctica*, porcine pancreas and wheat germ, using vinyl acetate as reagent and *tert*-butyl methyl ether as solvent. The best results were obtained with *C. antarctica* in terms of short reaction time, chemioselective conversion and good yield. *C. antarctica* lipase (CAL) was selected for acylation of hydroxytyrosol and homovanillic alcohol with vinyl esters of different acyl chains on a preparative scale, as shown in Table 1. The use of *C. antarctica* with increasing alkyl chain length required longer reaction times. The homovanillyl alcohol and its esters were found to exhibit scarce effectiveness both as

Transesterification of HT with ethyl saturated, mono- and poly-unsaturated fatty acid esters, catalized by Novozym 435 (immobilized *C. antarctica* lipase B), in vacuum under solventless conditions, has been successfully developed (Torres de Pinedo et al. 2005). This procedure gave hydroxytyrosyl esters in 59-98% yield for the saturated fatty acid esters, and

The antioxidant activity of hydroxytyrosyl esters has been measured with different methods, including DPPH (1,1-diphenyl-2-picrylhydrazyl radical), ABTS (2,2'-azino-bis(3 etilbenzotiazolin-6-sulfonic acid), FRAP (ferric reducing antioxidant power) and Rancimat (Mateos et al., 2008; Gordon et al., 2001; Bouallagui et al., 2011b). The Rancimat test is a method commonly used to evaluate the antioxidant power in lipophilic food matrices, such as oils and fats, while the ABTS and FRAP assays are used for the evaluation of antioxidant activity in hydrophilic medium; the ABTS assay evaluating the radical-scavenging capacity,

The Rancimat test revealed a lower activity for ester derivatives compared to HT, in agreement with the so-called polar paradox, according to which hydrophilic antioxidants are more effective in less polar media, such as bulk oils, whereas lipophilic antioxidants are more effective in relatively more polar media, such as in oil-in-water emulsions or

avoids the use of toxic reagents and allows mild reaction conditions.

32-97% yield for the mono- and poly-unsaturated fatty acid esters.

and the FRAP method determining the reducing activity.

liposomes (Frankel et al., 1994; Shahidi & Zhong, 2011).

*n*-pentane and *n*-hexane, in spite of the poor solubility of HT in such solvents.

**3.1.1.6 Lipase-catalyzed transesterification** 

radical scavengers and antioxidant agents.

**3.1.2 Biological activity 3.1.2.1 Antioxidant activity** 

Phenol: acylating agent 1:20, *C. antarctica* lipase*, t*-BuOMe, 40 ºC

Table 1. Enzymatic esterification of HT and homovanillyl alcohol (Grasso et al., 2007)

The order of the scavenging activities toward the ABTS radical was hydroxytyrosyl esters ≥ -tocopherol > hydroxytyrosol > tyrosyl >tyrosyl esters BHT. In a similar trend, comparison of FRAP values obtained for the free hydroxytyrosol and tyrosol with the corresponding esters revealed that while hydroxytyrosyl esters showed a significantly higher reducing activity than their precursor, all the tyrosyl esters showed a lower antioxidant activity than that of tyrosol. The same conclusion was obtained from DPPH assay of the radical scavenging activity (Grasso et al., 2007).

In connection with the size of the acyl chain, the reported literature seems to conclude that the antioxidant capacity of hydroxytyrosyl esters is better for medium-sized (C4C9) alkyl chains in comparison with HT, whereas further elongation of the acyl chain does not improve the antioxidant activity. This confirms that antioxidant capacity does not depend

Biological Properties of Hydroxytyrosol and Its Derivatives 385

The ability of hydroxytyrosol and its esters to prevent iron-induced oxidative stress has been studied on human cervical cells (HeLa cells) by the TBARS protocol (Bouallagui et al., 2011b). Pre-incubation of HeLa cells in the presence of 100 μM phenolic compounds led to a significant improvement of the oxidative status. In fact, thiobarbituric acid-reactive substance (TBARS) production was decreased by 30%, 36% and 38% with hydroxytyrosol,

The study of the metabolism of hydroxytyrosol, tyrosol, and hydroxytyrosyl acetate has been carried out using human hepatoma cells (HepG2) as a model system of the human liver (Mateos et al., 2005). The results showed extensive uptake and metabolism of hydroxytyrosol and scarce metabolism of tyrosol, while hydroxytyrosyl acetate showed an interesting behavior, with formation of deacetylated hydroxytyrosol after only 2 h. Because hydroxytyrosyl acetate was stable in the culture medium, the hydroxytyrosol detected in the

Neuroprotection exerted by HT derivatives has been investigated in rat brain slices subjected to hypoxia-reoxygenation, both *in vitro* and after oral administration (González-Correa et al., 2008). This study was carried out to confirm to the previously demonstrated neuroprotective effects of virgin olive oil in rats (González-Correa et al., 2007). Although the studies gave positive results in the neuroprotective activity of both HT and hydroxytyrosyl

Hydroxytyrosyl alkyl ethers have been obtained (Madrona et al., 2009) in a three-step procedure starting from hydroxytyrosol isolated from olive oil waste waters (Scheme 8). This procedure requires first the selective protection of the aromatic hydroxyl groups *via* benzylation with benzyl bromine in the presence of K2CO3, and then the addition of an alkyl iodide under basic conditions, and the subsequent deprotection by catalytic hydrogenation

The yield for the alkylation step varies depending on the length of the alkyl chain; as depicted in Scheme 8, the yields decrease as the length of the alkyl chain increases, due to the reduced solubility of the corresponding long chain alkyl iodides in the solvent (DMSO).

Fig. 3. Hydroxytyrosol lipophilic analogues

**3.1.2.5 Transport, absorption and metabolism** 

hydroxytyrosyl acetate and hydroxytyrosyl oleate, respectively.

acetate, mechanisms that underlie this effect are still unknown.

**3.2 Lipophilic hydroxytyrosyl alkyl ethers** 

(Pd/C) to obtain the corresponding ethers.

**3.2.1 Synthetic approaches** 

extracellular medium should be attributed to the action of the hepatic cells. **3.1.2.6 Neuroprotective effect of hydroxytyrosyl and hydroxytyrosol acetate** 

**3.1.2.4 Prevention of oxidative stress** 

only on lipophilicity. A possible explanation could be related to the fact that the conformational freedom of the ester chain increases with the acyl chain length, and this could result in folded structures in which catechol hydroxyls are shielded (Tofani et al., 2010; Pereira-Caro et al., 2009; Medina et al., 2009).

This antioxidant activity has also been proved in biological assays, in order to check the ability of hydroxytyrosyl esters to protect proteins and lipids against oxidation caused by peroxyl radicals, using a brain homogenate as an *ex vivo* model (Trujillo et al., 2006) and cumene hydroperoxide to induce oxidation. The results obtained showed a protective effect in these systems, which was more effective in preventing the generation of carbonyl groups in proteins than the generation of malondialdehyde in lipid; hydroxytyrosyl linoleate showed the greatest activity. This fact proves that the introduction of a lipophilic chain in the hydroxytyrosol molecule increases both protein and lipid protection.

Dichlorodihydrofluorescein (DCF) fluorometric assay on whole cells, carried out to check the antioxidant activity of a large serie of hydroxytyrosyl esters (Tofani et al., 2010) on rat muscle cells, showed that hydroxytyrosol esters had a better antioxidant activity compared to HT due to the better penetration into the cells of the lipophilic derivatives.

Hydroxytyrosol fatty acid esters have shown a nonlinear tendency in antioxidant capacity in fish oil-in-water emulsions (Lucas et al., 2010), where a maximum of antioxidant efficiency appeared for hydroxytyrosol octanoate in a study of hydrosytyrosyl esters with alkyl chains varying from C2 to C18. These results seem to be in disagreement with the antioxidant polar paradox.

#### **3.1.2.2 Cardiovasvular diseases**

Platelet aggregation is considered one of the main events in arterial thrombosis; therefore aggregation prevention is a major goal of cardiovascular research. It has been proved that hydroxytyrosol acetate inhibits platelet aggregation induced by ADP, collagen or arachidonic acid and stimulates nitric oxide production, more efficiently than hydroxytyrosol, and as effectively as acetylsalicylic acid; the latter is the most widely used drug in the world to prevent ischaemic cardiovascular diseases because of its antiplatelet aggregating action. This conclusion has been achieved *in vivo* in a study of oral administration of this ester to rats (González-Correa et al., 2008b), and *in vitro* in both human whole blood and platelet-rich plasma (González-Correa et al., 2009).

#### **3.1.2.3 DNA damage oxidative protection**

The atypical Comet test on whole blood cells has been applied to several hydroxytyrosyl esters to check their capacity to counteract the oxidative stress caused by H2O2 and the basal DNA damage.

The results obtained show that antidamaging properties on DNA of HT acetate and propanoate are comparable to those of HT, whereas the protective effect progressively decreases in the order butanoate < decanoate estearate (Fig. 3). This behavior was not observed for the lipophilic analogues of homovanillyl alcohol which appear to be scarcely protective, indicating that *o*-diphenols are more effective antioxidants than simple phenols (Grasso et al., 2007).

Fig. 3. Hydroxytyrosol lipophilic analogues

#### **3.1.2.4 Prevention of oxidative stress**

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

only on lipophilicity. A possible explanation could be related to the fact that the conformational freedom of the ester chain increases with the acyl chain length, and this could result in folded structures in which catechol hydroxyls are shielded (Tofani et al.,

This antioxidant activity has also been proved in biological assays, in order to check the ability of hydroxytyrosyl esters to protect proteins and lipids against oxidation caused by peroxyl radicals, using a brain homogenate as an *ex vivo* model (Trujillo et al., 2006) and cumene hydroperoxide to induce oxidation. The results obtained showed a protective effect in these systems, which was more effective in preventing the generation of carbonyl groups in proteins than the generation of malondialdehyde in lipid; hydroxytyrosyl linoleate showed the greatest activity. This fact proves that the introduction of a lipophilic chain in

Dichlorodihydrofluorescein (DCF) fluorometric assay on whole cells, carried out to check the antioxidant activity of a large serie of hydroxytyrosyl esters (Tofani et al., 2010) on rat muscle cells, showed that hydroxytyrosol esters had a better antioxidant activity compared

Hydroxytyrosol fatty acid esters have shown a nonlinear tendency in antioxidant capacity in fish oil-in-water emulsions (Lucas et al., 2010), where a maximum of antioxidant efficiency appeared for hydroxytyrosol octanoate in a study of hydrosytyrosyl esters with alkyl chains varying from C2 to C18. These results seem to be in disagreement with the antioxidant polar

Platelet aggregation is considered one of the main events in arterial thrombosis; therefore aggregation prevention is a major goal of cardiovascular research. It has been proved that hydroxytyrosol acetate inhibits platelet aggregation induced by ADP, collagen or arachidonic acid and stimulates nitric oxide production, more efficiently than hydroxytyrosol, and as effectively as acetylsalicylic acid; the latter is the most widely used drug in the world to prevent ischaemic cardiovascular diseases because of its antiplatelet aggregating action. This conclusion has been achieved *in vivo* in a study of oral administration of this ester to rats (González-Correa et al., 2008b), and *in vitro* in both

The atypical Comet test on whole blood cells has been applied to several hydroxytyrosyl esters to check their capacity to counteract the oxidative stress caused by H2O2 and the basal

The results obtained show that antidamaging properties on DNA of HT acetate and propanoate are comparable to those of HT, whereas the protective effect progressively decreases in the order butanoate < decanoate estearate (Fig. 3). This behavior was not observed for the lipophilic analogues of homovanillyl alcohol which appear to be scarcely protective, indicating that *o*-diphenols are more effective antioxidants than simple phenols

the hydroxytyrosol molecule increases both protein and lipid protection.

to HT due to the better penetration into the cells of the lipophilic derivatives.

human whole blood and platelet-rich plasma (González-Correa et al., 2009).

2010; Pereira-Caro et al., 2009; Medina et al., 2009).

paradox.

DNA damage.

(Grasso et al., 2007).

**3.1.2.2 Cardiovasvular diseases** 

**3.1.2.3 DNA damage oxidative protection** 

The ability of hydroxytyrosol and its esters to prevent iron-induced oxidative stress has been studied on human cervical cells (HeLa cells) by the TBARS protocol (Bouallagui et al., 2011b). Pre-incubation of HeLa cells in the presence of 100 μM phenolic compounds led to a significant improvement of the oxidative status. In fact, thiobarbituric acid-reactive substance (TBARS) production was decreased by 30%, 36% and 38% with hydroxytyrosol, hydroxytyrosyl acetate and hydroxytyrosyl oleate, respectively.

#### **3.1.2.5 Transport, absorption and metabolism**

The study of the metabolism of hydroxytyrosol, tyrosol, and hydroxytyrosyl acetate has been carried out using human hepatoma cells (HepG2) as a model system of the human liver (Mateos et al., 2005). The results showed extensive uptake and metabolism of hydroxytyrosol and scarce metabolism of tyrosol, while hydroxytyrosyl acetate showed an interesting behavior, with formation of deacetylated hydroxytyrosol after only 2 h. Because hydroxytyrosyl acetate was stable in the culture medium, the hydroxytyrosol detected in the extracellular medium should be attributed to the action of the hepatic cells.

#### **3.1.2.6 Neuroprotective effect of hydroxytyrosyl and hydroxytyrosol acetate**

Neuroprotection exerted by HT derivatives has been investigated in rat brain slices subjected to hypoxia-reoxygenation, both *in vitro* and after oral administration (González-Correa et al., 2008). This study was carried out to confirm to the previously demonstrated neuroprotective effects of virgin olive oil in rats (González-Correa et al., 2007). Although the studies gave positive results in the neuroprotective activity of both HT and hydroxytyrosyl acetate, mechanisms that underlie this effect are still unknown.

#### **3.2 Lipophilic hydroxytyrosyl alkyl ethers**
