Hydroxytyrosol Lipophilic Analogues: Synthesis, Radical Scavenging Activity and Human Cell Oxidative Damage Protection
Rosa Chillemi, Sebastiano Sciuto, Carmela Spatafora and Corrado Tringali
33 All rights of reproduction in any form reserved.
Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, 95125 Catania, Italy
12Olives and Olive Oil in Health and Disease Prevention.ISBN: 978-0-12-374420-3
135.1 IntroductIon
As detailed in other parts of this volume, a variety of stud-ies indicate that the regular dietary consumption of extra virgin olive oil has a protective effect towards coronary heart diseases (Keys, 1995; Tripoli et al., 2005) and breast cancer (Martin-Moreno et al., 1994). Many researches, ori-ented at identifying the main protective agents of olive oil, have pointed to the antioxidative phenolic constituents (Owen et al., 2000; Leenen et al., 2002; Kok and Kromhout, 2004). The olive oil phenols include tyrosol (1, 4-hydroxyphene-thyl alcohol), hydroxytyrosol (2, reported also as 3,4- dihydroxyphenethyl alcohol, 3,4-DHPEA; in the following indicated with the acronym HT) (Tuck and Hayball, 2002) and their secoiridoids and conjugate forms: oleuropein (3), ligstroside (4), verbascoside (5). Further conjugates of HT identified in Olea europaea are demethyloleuropein (6), oleuropein aglycon (7), 2-(3,4-dihydroxyphenyl)ethyl ester of elenolic acid dialdehyde (3,4-DHPEA-EDA, 8), hydroxytyrosol 4--glucoside (9) and oleuroside (10) (Fernandez-Bolanos et al., 2008). Hydroxytyrosol is incorpo-rated in the aglycon of oleuropein and other conjugates and is released by hydrolysis during olive storage and pressing (Brenes et al., 2001).
Among these phenols, hydroxytyrosol is recognized as the main antioxidant and protective principle of virgin olive oil (Fernandez-Bolanos et al., 2008) and is also considered an important anticancer component of this Mediterranean condiment (Manna et al., 1997; Fabiani et al., 2002). In fact, many literature data indicate the potent in vitro anti-oxidant activity of HT (Saija et al., 1998; Stupans et al., 2002; Roche et al., 2005) in agreement with theoretical predictions on ortho-diphenols (Goupy et al., 2003). In addition, 2 has been proven to prevent oxidative damage in human erythrocytes (Manna et al., 1999). Further studies
suggest that 2 has a high oral bioavailability and is easily absorbed, unlike oleuropein (Manna et al., 2000; Tuck et al., 2001; Tuck and Hayball, 2002).
Some natural lipophilic analogues of HT are known: hydroxytyrosol acetate (11) is present in olive oil in a per-centage around 10% of HT, which has been reported to be in the range 1.5–15 mg kg1 oil (Tuck and Hayball, 2002). The homovanillic alcohol (12) has been identified as a lipo-philic human metabolite of HT and is commercially avail-able; it has been reported as a radical scavenger comparable to HT and reputed by some authors to contribute to the ben-eficial properties exerted by olive oil (Tuck et al., 2002).
Some studies on olive phenols (Manna et al., 1997; Paiva-Martins et al., 2003; Morellò et al., 2005) have high-lighted the importance of the lipophilic character of the antioxidant with reference to the dispersion medium (bulk oil, emulsions), to the cell uptake and membrane crossing, and to the substrate to be protected (LDL or other cellular constituents). In addition to these data which are of par-ticular interest in the biomedical field, the agro-industry is undergoing new pressures due to the widespread concern about the use of synthetic additives and antioxidants (such as BHT, 13) and to a growing demand for safer foods and beverages. This has led to a drive, by the industries of the agro-alimentary sector, to search for new antioxidants and other nutritional supplements of natural origin or obtained by simple modification of natural products. In fact, oxida-tion is one of the main causes of food deterioration, espe-cially in oils and fats. Moreover, oxidized lipids, when absorbed in mammals, are incorporated into lipoproteins and may contribute to atherosclerosis (Parthasarathy et al., 1992; Regnstrom et al., 1992). Thus, the lipophilic HT analogues may be useful in the formulation of ‘beneficial foods’ for the prevention and treatment of chronic patholo-gies associated with reactive radical damage or as additives
SectIon | III Tyrosol and Hydroxytyrosol1234
OH
OH
R R
OH
O
OO
O
O
OH
OOH
OHOH
MeO
OH
OHO
OH
O OOH
O
O
OH
OH
OH
O
OH
OH
OH
OH
O
OO
O
O
OH
OOH
OHOH
OH
OH
OH
O
OO
O
OH
MeO
OH
OH
O
OO
O
O
OH
O
OH
OHOH
OHOH OH
OH
O
OO
O
O
OH
OOH
OHOH
MeO
1 R = H 2 R = OH
3 R = OH 4 R = H
5 6
87
019
FIgure 135.1
for preserving foods from oxidation processes. On this basis, we compiled this chapter focusing on hydroxytyro-sol lipophilic analogues, and in particular on their synthe-sis, their radical scavenging activities and other protective properties. Due to space constraints, we reviewed only the recent literature and have included almost exclusively those synthetic analogues which have been submitted to antioxi-dant activity assays.
135.2 LIpophILIc hydroxytyroSoL anaLogueS
The most common methodology to convert HT (2) into more lipophilic analogues is the esterification of the pri-mary alcoholic group without affecting the catechol moi-ety, which is known to be essential for the antioxidant and protective effects of 2. The chemical methods for selective
esterification of alcoholic groups have been paralleled, in recent years, by enzymatic methods, which avoid the use of toxic reagents and allow mild reaction conditions. Thus, Buisman et al. (1998) screened seven lipases in view of enzymatic esterification of 2. In a preliminary test, the
authors observed low reaction rates except for lipase B from Candida antarctica (CAL-B). In a subsequent series of experiments 2 and 3,5-di-t-butyl-4-hydroxybenzyl alco-hol (3,5-DB-4-HBA, 14), whose antioxidant properties have been previously reported ( Papadopoulos and Boskou, 1991; Baldioli et al., 1996), were esterified according to Scheme 135.1. The 3,5-DB-4-HBA octanoate (15) was obtained with 93% yield within 5 h, whereas the HT octanoate (16) was obtained with 65% yield in 20 h.
Both esters were evaluated for antioxidant perform-ance by measurement of OSI in refined sunflower oil, in comparison with BHT (13) and 14. The authors observed that the addition of the octanoate esters increases the oxida-tion induction time of sunflower oil. Nevertheless, the highest antioxidant activity is observed for 14 followed by BHT, whereas the lowest antioxidant activity is obtained for 15. The analogue 16 resulted a less effective antioxi-dant than HT.
As cited above, hydroxytyrosol acetate (11) is a natu-ral constituent of olive oil, but it has also been obtained by synthesis. Gordon et al. (2001) prepared 11 from 2 by a series of chemical conversions, namely protection of the catechol moiety with benzyl bromide, followed by acetyl-ation with acetic acid and subsequent deprotection through
catalytic hydrogenation. The antioxidant activity of 11 was assessed in comparison with that of other olive oil components, namely hydroxytyrosol (2), oleuropein (3), 3,4-DHPEA-EA (17) and -tocopherol (18) by scaveng-ing of DPPH• radicals as well as by measurement of the oxidative stability of bulk olive oil and oil-in-water emul-sions. In the DPPH• scavenging test, the concentration required for 50% reduction in DPPH• radical concentration in 15 min was determined (EC50, reported as ratio moles of antioxidant/moles of DPPH•). At 15 min, the scaveng-ing activity decreased in the order: 2 (EC50 0.19) 18 (EC50 0.25), 11 (EC50 0.26). The antioxidant effect of 2 and 11 in bulk oil was evaluated in comparison with 18. The parameters examined were the peroxide value (PV) for primary oxidation products, and the p-anisidine value (AV) for secondary oxidation products; both values showed that the antioxidant activity decreased in the order: 2 11 18 control.
These results are in agreement with the polar paradox: that is, more polar antioxidants are more effective in less polar media (Porter, 1993; Frankel et al., 1994). When the study was carried out in oil-in-water emulsions (olive oil stripped of natural phenolic compounds and tocophe-rols), oxidation was faster than in bulk oil, and the order
SectIon | III Tyrosol and Hydroxytyrosol1236
blood cells (RBC) (see Figures 135.6A and B).
of antioxidant activity was different, following the order: 18 11 2 control. In conclusion, 2 and 11 have a comparable antioxidant activity with 11 being less effec-tive in oil, but slightly more effective in emulsion. In a fur-ther paper by the same group (Paiva-Martins et al., 2003), the antioxidant activity of 2, 3, 8, 11, and 17 in a soybean phospholipid liposome system was studied, in comparison with 18 and the water-soluble tocopherol analogue, Trolox. Lipid peroxidation, initiated by AAPH, was determined spectrophotometrically. The end of the lag phase was defined as the point where the slope rapidly increased. The radical scavenging activity of the phenols was evaluated by the ABTS assay (Re et al., 1999), that is, measuring their ability to trap the stable free radical ABTS•. The above-cited phenols showed comparable lipid antioxidant activity with a duration of the lag phase almost twice that of 18 and better than Trolox. Synergistic effects (11–20% increase in lag phase) were observed in the antioxidant activity of combinations of 18 with olive oil phenols both with and without ascorbic acid. Localization of compounds in lipo-somes was studied by fluorescence anisotropy of probes and fluorescence quenching: these showed that the olive oil phenols did not penetrate into the membrane, but their effectiveness as antioxidants showed they were associated with the surface of the phospholipid bilayer.
Torres de Pinedo et al. (2005) reported an efficient enzy-matic synthesis of a series of long-chain phenolic esters with the antioxidant ortho-dihydroxy moiety, although they did not carry out any biological or chemical assay on their prod-ucts. Two of these products were HT esters bearing polyun-saturated acyl chains, namely HT all-Z eicosapentaenoate (19) and HT all-Z docosahexaenoate (20). We consider these products worthy of citation here, in view of the well-known antioxidative properties of polyunsaturated fatty acids which in these esters are coupled to the properties of the catechol moiety. According to Scheme 135.2, they carried out the reaction in the presence of Candida antarctica lipase (CAL) and obtained the esters in good yields and short reaction times.
The same authors (Torres de Pinedo et al., 2007) have more recently reported the synthesis of a series of phenolic
fatty acid esters and their evaluation as lipophilic antioxi-dants in an oil matrix. The HT analogues 2124, the latter being a methylated HT derivative, were prepared by enzy-matic acylation of the corresponding phenolic alcohols, using lipase from CAL-B. The HT palmitate 21, the HT stearate 22, the HT oleate 23 and the 3-hydroxy-4-methoxyphenethyl palmitate 24 were obtained as pure compounds, in good to excellent yields. The two oleoyl derivatives 25 and 26 were instead prepared by chemical acylation of the phenolic alco-hol with oleic acid, using EDCI as activating reagent. These two compounds were obtained as a 1:1 unseparated mixture in lower yield (65%).
The radical scavenging activity of all compounds was evaluated through the above-cited ABTS assay. Their effi-cacy as food antioxidants was evaluated in refined olive oil using the Rancimat method (Matthaus, 1996), by measuring the IT, that is the time taken until a rapid oxidation of the oil spiked with each antioxidant is observed. The palmitates 21 and 24 showed better scavenging capacities than the positive controls, -tocopherol and ascorbyl palmitate. Actually, the authors report as ‘surprising’ the result obtained for com-pound 24, lacking the ortho-phenolic function. Moreover, all the catecholic analogues 21–23 showed higher IT in the Rancimat test than did the control antioxidants and com-pound 24. The type of fatty acid (palmitate, stearate or oleate) acylating the phenolic alcohols determined negligi-ble differences in ITs. As expected, the mixture of acylated phenols 25 and 26 was a less effective antioxidant than the analogues bearing the ortho-dihydroxy moiety.
The protective effect of HT lipophilic analogues against oxidative stress in human cells was recently studied by Manna et al. (2005). Two chemically stable HT analogues were prepared by standard chemical conversions, namely the triacetate 27 and the diacetate 28. As expected, the acetylated analogues were devoid of any chemical antioxi-dative property, determined by FRAP assay (Benzie and Strain, 1999). Conversely, both acetyl derivatives, at micro-molar concentrations, equally protected against oxidative damages induced by t-BHP in human colon carcinoma Caco-2 cells (see Figures 135.5A and B) and human red
The HT triacetate 27 has been the subject of a furtherpaper by the same authors (Capasso et al., 2006). The prepa-ration of 27 (overall yield of 35.6%) from olive mill waste-water (OMWW) organic extract is reported, based on a
R1
R3
R2
R
O
O
OH
( )n
O
O
O
O
O
23 R1 = R2 = OH, R3 = OH
25 R1 = OH, R2 = OH, R3 =
( )7 ( )7
( )7 ( )7
21 R = OH n = 14
22 R = OH n = 16
mild treatment with an acetylating mixture (HClO4-SiO2 and acetic anhydride). This stable form of HT may be use-ful as a bioantioxidant, to be converted into the active form by intracellular esterases.
More recently, Trujillo et al. (2006) evaluated some lipophilic hydroxytyrosyl esters as antioxidants in lipid matrices and biological systems. The esters were obtained by a procedure under patent (Alcudia et al., 2004): to a solution of HT in ethyl or methyl ester (acetate, butyrate, palmitate, stearate, oleate or linoleate) p-TSA was added, and the mixture was stirred for 24 h. After standard work-up, the HT esters 11, 16, 21, 23, 2931 were obtained in 62–86% yields.
The authors evaluated the antioxidant activities of these lipophilic HT analogues in comparison with those of HT, 13 and 18 in both glyceridic matrix and biological systems. A lipid glyceridic matrix obtained from virgin olive oil was spiked with the antioxidant under test and subjected to
O26 R1 = OH, R2 = R3= OH ( )7 ( )724 R = OCH3 n = 14FIgure 135.4
accelerated oxidation in a Rancimat apparatus operating at 90 °C. The IT was measured as the time required to have a sharp increase of a conductivity measurement, and was reported versus the concentration C (mmol kg1) of each antioxidant (see Figure 135.8). HT and its analogues 11, 16, 21, 23, 2931 showed comparable antioxidant activi-ties, which resulted higher than those of 13 or 18. In a sec-ond experiment, the capacity of esters of hydroxytyrosol to protect proteins and lipids against oxidation caused by peroxyl radicals was measured. A brain homogenate model was used, because brain tissue is very vulnerable to oxida-tive damage due to its relative lack of antioxidant enzymes.
The protective effect of the above-cited esters against the damage caused by CH has been evaluated by measuring the MDA, the most abundant aldehyde produced as product of lipid peroxidation when lipid hydroperoxides break down in biological systems (Esterbauer and Cheeseman, 1990), and carbonyl group content, because of the introduction of carbonyl groups into proteins by oxidative mechanisms. The results
have been compared with those for 2, 13 and 18. The capacity of HT and HT acetate (11), palmitate (21), and oleate (23) to prevent lipid peroxidation was similar to that of 18, but lower than that of 13. Interestingly, the hydroxytyrosyl linoleate (31) showed greater activity than the other esters.
A novel methodology of derivatization has been applied by Bernini et al. (2007) to a series of phenolic alcohols, including HT. These authors prepared the carboxymethyl-ated HT analogue 32 by an eco-friendly, chemoselective and efficient method, employing dimethyl carbonate in the presence of DBU or sulfuric acid as catalysts (see Scheme 135.3). After work-up, the product was obtained in quan-titative yield. The antioxidant activity of 32 was investi-gated using the DPPH• radical scavenging test. The results showed that this new compound has an antioxidant activity (EC50 0.11 mol L1 antioxidant mol1 L DPPH•) similar to that reported for HT (Tuck et al., 2002).
A paper of Rietjens et al. (2007) is focused on the con-troversies concerning the antioxidant potential of the olive oil antioxidants, and in particular HT; this study includes tyrosol (1) and homovanillic alcohol (12): the authors observed that both these compounds, lacking a free cat-echol moiety, resulted less potent radical scavengers with respect to HT, although 12 was a relatively good scavenger of ONOOH and OH•.
Recently, some of us (Grasso et al., 2007) have car-ried out a study on a series of HT lipophilic analogues, to evaluate the possible effects of an enhanced lipophilicity on the antioxidative properties of HT and 12. An environ-mentally friendly and chemoselective enzymatic method was preferred to the previously reported chemical conver-sions. Tyrosol, more stable than hydroxytyrosol, was used as a model for a preliminary acetylation screening with 12 different lipases, employing vinyl acetate as reagent and t-butyl methyl ether as solvent, on the basis of previous satisfactory results (Nicolosi et al., 2002). The best results were obtained with Mucor miehei and Candida antarctica (CAL) lipases, this latter being less expensive and conse-quently employed on a preparative scale.
Ten lipophilic esters were prepared by acylation at the alcoholic function of 2 (11, 22, 29, 33, 34) and 12 (35–39) employing vinyl acetate, propionate, butyrate, decanoate and stearate. CAL and the acyl donor were added to a
solution of the substrate (2 or 12) in tert-butyl methyl ether and the mixture was shaken at 40 °C: HT acetate (11), HT propionate (33) and HT butyrate (29) were obtained after 35 min with 95–96% yield; HT decanoate (34) was obtained in 93% yield after 75 min, whereas HT stearate (22) was obtained in 92% yield after 3 h. Analogously, 12 afforded with comparable yields the corresponding acetate (35), pro-pionate (36), butyrate (37), decanoate (38) and stearate (39) with reaction times in the range 60 min–4 h. Compounds 11, 22, 29, 33, 34, 35–39 were submitted to the DPPH assay and compared with 1, 2 and 12 (see Table 135.1). As expected, 2 proved a much more effective scavenger than 1. Compound 12 resulted clearly less active than 2, in contrast with previous literature data (Tuck et al., 2002). The SC50 values of the two groups of analogues related to 2 and 12 are respectively in the range 20.5–24.8 and 40.5–44.6 M. These data confirmed that the anti-radical activity of HT is not notably influenced by the presence and length of an acyl chain at the alcoholic function, and that the homova-nillic system has lower radical scavenging activity with respect to the HT system. Compounds 2, 12 and their lipophilic analogues were also subjected to the atypical Comet test (Collins, 2004) on whole blood cells in order to evaluate both their possible basal DNA damaging proper-ties and their capacity to counteract the H2O2 caused oxi-dative stress. The results were expressed as TDNA or tail intensity, considering this value indicative of the number of DNA breaks.
Figure 135.10A reports the TDNA values obtained by treatment of whole cells with compounds 1, 2 and 12 and the HT analogues 11, 22, 29, 33 and 34, compared with the val-ues obtained for untreated control cells (C). Figure 135.10B reports the TDNA values obtained when these compounds were added before the H2O2 insult in comparison with the data obtained for solely H2O2 treated cells (H2O2). The data in Figure 135.10A show that 1 and 12 have a signifi-cant basal DNA-damaging effect with respect to the con-trol values, whereas no significant damage is caused by 2. Interestingly, HT acetate (11) and HT propionate (33) show
OH
OH
O R
O
OH
O R
O
OMe
33 R = C2H5
34 R = C9H19
35 R = CH3
36 R = C2H5
37 R = C3H7
38 R = C9H19
39 R = C17H35
FIgure 135.9
a minimal DNA-damaging activity, while the damage sig-nificantly increases for compounds 22, 29 and 34 bear-ing respectively stearoyl, butyryl and decanoyl acyl chain. Figure 135.10B clearly shows that 2 is highly protective towards the oxidative induced DNA damage in whole blood cells, differently from 1 and 12. Among the lipophilic analogues, 11 and 33 are comparable to 2 in counteract-ing oxidative stress, but the protective effect progressively decreases in the order 29 22 ≈ 34.
In separate experiments, the reference compounds 1, 2 and 12 and the homovanillic alcohol analogues 35–39 were examined and the TDNA values thus obtained are reported in Figures 135.11A and B. The experiment on basal DNA damage confirms that 2 does not cause any significant dam-age at basal level, whereas 12 and its lipophilic analogues 35–39 are comparable in causing a basal damage. The experiment reported in Figure 135.11B confirms the good
Table 135.1 DPPH• scavenging activity.
Compounds SC50 (M)a SD
2 33.2 5.6
3 24.6 6.5
4 46.1 3.4
5 21.9 1.4
6 22.9 5.1
7 24.7 1.0
8 24.8 10.2
9 20.5 6.6
10 42.9 5.8
11 44.6 1.8
12 41.8 2.2
13 44.5 4.2
14 40.5 1.8
Note the correspondence with our text: 2 1; 3 2; 4 12; 5 11; 6 33; 7 29; 8 34, 9 22; 10 35; 11 36; 12 37; 13 38 and 14 39.Reprinted from, S. Grasso, L. Siracusa, C. Spatafora, M. Renis, C. Tringali. Hydroxytyrosol lipophilic analogues: enzymatic synthesis, radical scavenging activity and DNA oxidative damage protection. Bioorganic Chemistry 35: 137–152. Copyright (2007), with permission from Elsevier.aSC50 (M), Scavenging capacity: phenol concentration, expressed in M, able to quench 50% of DPPH radicals in a 92 M solution (mAU 1, solvent: cyclohexane). Each reported value is the mean of three separate measurements.
SectIon | III Tyrosol and Hydroxytyrosol1240
protective properties elicited by 2 versus H2O2-induced DNA damage, as well as the poor protection given by 12. The lipophilic analogues of the latter show slightly higher TDNA values than that of 12. In this work, we determined the log P value for all compounds. The results of the Comet test show a good protective effect for 2 and its analogues 11 and 33, with log P 1.20, a moderate effect for 29 (log P 1.77) and negligible protection for 34 and 22 (log P 5), these latter exerting a significant DNA damage in basal condi-tions. Thus, it appears that a longer acyl chain than C4 and a higher log P than 2 cause the loss of the protective properties of HT. In conclusion, the lipophilic analogues 11, 22, 29, 33
100
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10
0C 2
*
3
*
4 5 6 7 8 9
*
**
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NA
= %
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ragm
ente
d D
NA
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90
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0H2O2B
A
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NA
FIgure 135.10 Atypical alkaline COMET assay. *Significantly differ-ent from control untreated whole blood cells (p 0.001). °Significantly different from H2O2 alone treated cells (p 0.001). The results are reported as TDNA % of fragmented DNA. (A) Basal DNA damage: whole blood cells were treated for 20 min at 37 °C with the tested com-pounds at the concentration 50 M. (B) Oxidative DNA damage protec-tion: whole blood cells were firstly treated for 20 min at 37 °C like (A) and second, after a wash with PBS 1X, for 20 min at 37 °C with H2O2 (200 M). Each experiment, performed in duplicate, was repeated three times and the mean ± SEM for each value was calculated. Note the corre-spondence with our text: 2 1; 3 2; 4 12; 5 11; 6 33; 7 29; 8 34 and 9 22. Reprinted from, S. Grasso, L. Siracusa, C. Spatafora, M. Renis, C. Tringali. Hydroxytyrosol lipophilic analogues: enzymatic synthesis, radical scavenging activity and DNA oxidative damage protec-tion. Bioorganic Chemistry 35: 137–152. Copyright (2007), with permis-sion from Elsevier.
and 34 may be profitably used, in principle, as antioxidants in bulk lipids or emulsions; 11 and 33, in particular, could be further evaluated for possible applications in pharmaceutical, nutritional or cosmetic fields. The experiments carried out on compound 12 and its analogues 35–39 gave a different result, indicating that the homovanillic system has poor antioxidant activity, without correlation with increased lipophilicity.
A very recent study of Fabiani et al. (2008) also deals with the preventive properties of HT and other related olive phenolics against the oxidative DNA damage in human peripheral blood mononuclear cells (PBMC) and includes the effects towards leukaemia HL60 cells. HT and a complex
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0C 2
* * * * * * *
3 4 10 11 12 13 14
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NA
= %
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ente
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NA
(A)
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0H2O2(B) 2 3 4 10 11 12 13 14
TD
NA
= %
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ente
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NA
FIgure 135.11 Atypical alkaline COMET. *Significantly different from control untreated whole blood cells (p 0.001). °Significantly different from H2O2 alone treated cells (p 0.001). The results are reported as TDNA % of fragmented DNA. (A) Basal DNA damage: whole blood cells were treated for 20 min at 37 °C with the tested compounds at the concentration 50 M. (B) Oxidative DNA damage protection: blood cells were firstly treated for 20 min at 37 °C like (A) and second, after a wash with PBS 1X, for 20 min at 37 °C with H2O2 (200 M). Each experiment, performed in duplicate, was repeated three times and the mean ± SEM for each value was calculated. Note the correspondence with our text: 2 1; 3 2; 4 12; 10 35; 11 36; 12 37; 13 38 and 14 39. Reprinted from S. Grasso, L. Siracusa, C. Spatafora, M. Renis, C. Tringali. Hydroxytyrosol lipophilic analogues: enzymatic synthesis, radical scavenging activity and DNA oxidative damage protection. Bioorganic Chemistry 35: 137–152. Copyright (2007), with permission from Elsevier.
mixture of phenols obtained from virgin olive oil and olive mill wastewater reduced the H2O2-induced DNA damage at concentrations as low as 1 M, as determined by Comet assay. The protection by HT was 93% in HL60 and 89% in PBMC at 10 M. A comparable protective effect was observed for 8 on both cultured cells. Other phenolic puri-fied compounds, among them 1, 3 and 5, showed a lower protective effect (range of protection 25–75%).
In an interesting and recent study by Palozza et al. (2008) a group of novel -tocopherol analogues are reported, some of them being also HT lipophilic analogues, namely compounds 40–42. The authors designed novel chromanyl derivatives sharing a chromanyl head with -tocopherol but differing in the side chain. In particular, compounds 40–42 included the further antioxidant moiety of HT or its nitrogenated analogue, dopamine.
The protective effect of these compounds towards free-radical-induced oxidative stress was investigated in isolated membranes as well as in intact cells. Tocopherol analogues were added to rat liver microsomes and to RAT-1 fibrob-lasts and were exposed to the pro-oxidant action of free radical sources including AAPH, t-BOOH, and H2O2. The antioxidant efficiency was evaluated by measuring several parameters of oxidative stress, including MDA and conju-gated diene formation, ROS production, cell viability, and expression of heat-shock proteins (hsp70, hsp90) and was compared with that of natural -tocopherol and -tocot-rienol. The results clearly demonstrate that all synthesized compounds were active in: (i) inhibiting lipid peroxidation in microsomes and (ii) preventing H2O2-induced ROS pro-duction, cell damage, and heat-shock protein expression in immortalized RAT-1 fibroblasts. The concomitant pres-ence of a chromanyl head and an additional catechol moiety markedly increased the antioxidant potency of the molecule. In particular, 40 and 41, resulting from the molecular combi-nation of Trolox with HT and dopamine, respectively, were much more potent than -tocopherol, -tocotrienol, and the other synthetic compounds.
A very recent addition to the above-cited literature reports the isolation and characterization of a new HT
secoiridoid derivative from Olea europaea leaves, namely the bis methylacetal of oleuropein aglycone 43 (Paiva-Martins and Pinto, 2008). The radical scavenging activity of this new compound, evaluated by DPPH• assay, resulted much higher than those of 8 or 18. The authors observed an easy conversion of 43 into 8 in acidic aqueous media, and consequently call to attention the need for a careful identi-fication of compounds by HPLC-MS, usually performed in acidic conditions.
Summary poIntS
l More than 40 hydroxytyrosol (HT) lipophilic analogues are reported.
l Selective chemical and enzymatic methods for the preparation of HT lipophilic analogues are described.
l Analogues including an ortho-diphenol moiety are normally more effective antioxidants than those with a single free phenolic function.
l The HT metabolite homovanillic alcohol and its lipophilic analogues are poor antioxidants and protec-tive agents with respect to HT and the majority of its lipophilic analogues.
l The majority of the lipophilic HT analogues showed very good radical scavenging activities and/or effec-tively inhibited lipid oxidations. In some cases, the HT analogues were more effective than other widely employed antioxidants, such as -tocopherol or BHT.
l Some of the reported lipophilic HT analogues were also demonstrated to be protective towards oxidative
OH
OOH O
O
OMe
OMe
43
FIgure 135.13
SectIon | III Tyrosol and Hydroxytyrosol1242
damage to human cells, and in particular towards H2O2-induced DNA damage.
l In a study of H2O2-induced DNA damage, the HT esters with a longer acyl chain than C4 and a higher log P than 2 showed low protective properties and caused significant DNA basal damage.
l The conjugation with a chromanyl head markedly increased the antioxidant potency of HT.
reFerenceS
Alcudia, F., Cert, A., Espartero, J. L., Mateos, R., and Trujillo, M., 2004. Method of preparing hydroxytyrosol esters, esters thus obtained and use of same. PCT WO 2004/005237.
Baldioli, M., Servili, M., Perretti, G., Montedoro, G.F., 1996. Antioxidant activity of tocopherols and phenolic compounds of virgin olive oil. J. Am. Oil Chem. Soc. 73, 1589–1593.
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