3390 J. Agric. Food Chem., Vol. 55, No. 9, 2007
Gambacorta et al.
byproduct. This can produce either transacetalization at the
glycoside moiety or transesterification processes at the ester
bond.
LITERATURE CITED
(
1) See, for example: (a) Silva, S.; Gomes, L.; Leit a˜ o, F.; Coelho,
A. V.; Boas, L. V. Phenolic compounds and antioxidant activity
of Olea europaea L. fruits and leaves. Food Sci. Technol. Int.
Therefore, oleuropein 2 was acetalized (Scheme 5) under the
same conditions described for the preparation of compound 6.
The only difference was the use of a Dean-Stark apparatus in
which the condensation funnel was filled with 4 Å molecular
sieves, to selectively remove methanol (24) distilled as azeotrope
with CHCl3 (28). This setup minimizes the methanol-induced
transesterification and transacetalization processes. The reaction
progress was followed by HPLC without isolating any inter-
mediates, and the process was carried on until a steady
chromatographic profile was obtained. Results are reported in
Figure 3.
2
006, 12 (5), 385-396. (b) O’Dowd, Y.; Driss, F.; Dang,
P. M.; Elbim, C.; Gougerot-Pocidalo, M.; Pasquier, C., et al.
Antioxidant effect of hydroxytyrosol, a polyphenol from olive
oil: scavenging of hydrogen peroxide but not superoxide anion
produced by human neutrophils. Biochem. Pharmacol. 2004, 68
(10), 2003-2008. (c) Mateos, R.; Dom ´ı nguez, M. M.; Espartero,
J. L.; Cert, A. Antioxidant effect of phenolic compounds,
R-tocopherol, and other minor components in virgin olive oil.
J. Agric. Food Chem. 2003, 51, 7170-7175. (d) Owen, R. W.;
Haubner, R.; Mier, W.; Giacosa, A.; Hull, W. E.; Spiegelhalder,
B.; Bartsch, H. The isolation, structural elucidation and antioxi-
dant potential of the major phenolic compounds in brined olive
drupes. Food Chem. Toxicol. 2003, 41, 703-717.
As expected, oleuropein 2 (Figure 3A, tR ) 9.42 min) quickly
reacted to afford a complex mixture with a lower polarity. The
retention times of main peaks ranged between 13 and 17 min
(2) (a) Panizzi, L.; Scarpati, M. L.; Oriente, G. Costituzione
dell’oleuropeina glucoside amaro e ad azione ipotensiva dell’ulivo.
Gazz. Chim. Ital. 1960, 90, 1449-1485. (b) Esti, M.; Cinquanta,
L.; La Notte, E. Phenolic compounds in different olive varieties.
J. Agric. Food Chem. 1998, 46, 32-35. (c) Ryan, D.; Antolovich,
M.; Prenzler, P.; Robards, K.; Lavee, S. Biotrasformations in
phenolic compounds in Olea europaea L.. Sci. Hortic. 2002,
(Figure 3B). These peaks are probably related to acetalization
and/or polyacetalization processes of the glucose moiety in 2.
However, prolonged reaction times (3 h, Figure 3C) resulted
in a change of the chromatographic profile in favor of products
with higher retention times (20-21 min), likely derived from
exhaustive acetalization. Further heating of the mixture did not
affect the chromatographic profile, and the crude mixture was
directly saponified to afford acetonide 3 together with a small
amount (8%) of an unknown compound (Figure 3D, tR ) 17.72
min). This was tentatively identified as the isochromane
acetonide 7 on the bases of the similarity between the GC-MS
spectra of 7 (see Materials and Methods) and 4. Compound 7
was probably derived from acetalization of the isochromane 4,
produced in turn by cyclization of hydroxytyrosol 1 (see
Scheme 2). The latter compound is the expected product of
oleuropein 2 transesterification in the presence of methanol (see
Scheme 4). After chromatographic purification over silica, the
pure acetonide 3 was obtained in high yield (76% referred to
9
2, 147-176.
(
3) Lo Scalzo, R.; Scarpati, M. L.; Verzegnassi, B.; Vita, G. Olea
europaea chemicals repellent to Dacus oleae females. J. Chem.
Ecol. 1994, 20, 1813-1823.
(4) Le Tutour, B.; Guedon, D. Antioxidative activities of Olea
europaea leaves and related phenolic compounds. Phytochemistry
1
992, 31, 1173-1178.
(
5) (a) Montedoro, G.; Servili, N.; Baldioli, M.; Miniati, E. Simple
and hydrolyzable phenolic compounds in virgin olive oil. 1. Their
extraction, separation, and quantitative and semiquantitative
evaluation by HPLC. J. Agric. Food. Chem. 1992, 40, 1571-
1
576. (b) Owen, R. W.; Giacosa, A.; Hull, W. E.; Haubner, R.;
Spiegelhalder, B.; Bartsch, H. The antioxidant/anticancer po-
tential of phenolic compounds isolated from olive oil. Eur. J.
Cancer 2000, 36, 1235-1247.
2
) and quantitatively converted into hydroxytyrosol 1 as
described above.
(6) (a) Brenes, M.; Rejano, L.; Garcia, P.; Sanchez, A. H.; Garrido,
A. A highly convenient synthesis of hydroxytyrosol and its
recovery from agricultural waste waters. J. Agric. Food Chem.
To the best of our knowledge, this is the first high-yielding
procedure to produce stabilized acetonide 3 from the abundant
1
999, 47, 1745-1748. (b) Romero, C.; Brenes, M.; Yousfi, K.;
(4) and easily extractable (2) natural glycoside oleuropein.
Garcia, P.; Garcia, A.; Garrido, A. Effect of cultivar and
processing method on the contents of polyphenols in table olives.
J. Agric. Food Chem. 2004, 52, 479-484.
Because deprotection affords pure hydroxytyrosol in high yield,
the overall procedure can be regarded as a suitable alternative
of previous methods that access 1 from 2 by chemical or
enzymatic processes (20-22).
In conclusion, we have found that the natural and unstable
antioxidant hydroxytyrosol 1 can be stabilized if the catechol
function is protected as acetonide 3. This novel compound is
highly stable in air and under light, over silica, and in a wide
range of basic and acidic conditions. It can be purified and stored
long-term. In addition, we have reported an easy and mild
method for deprotection so that the acetonide 3 can be regarded
as a stable source of hydroxytyrosol 1.
(
7) Briante, R.; La Cara, F.; Tonziello, M. P.; Febbraio, F.; Nucci,
R. Antioxidant activity of the main bioactive derivatives from
oleuropein hydrolysis by hyperthermophilic â-glycosidase.
J. Agric. Food Chem. 2001, 49, 3198-3203.
(
8) Frankel, E. N.; Huang, S.; Kanner, J.; German, J. B. Interfacial
phenomena in the evaluation of antioxidants: bulk oils Vs
emulsions. J. Agric. Food Chem. 1994, 42, 1054-1059.
9) Gordon, M. H.; Pavia-Martins, F.; Almeida, M. Antioxidant
activity of hydroxytyrosol acetate compared with that of other
olive oil polyphenols. J. Agric. Food Chem. 2001, 49, 2480-
(
2
485.
However, the protection cannot be introduced directly;
therefore, 3 must be prepared either by a high-yielding two-
step process or by chemical elaboration of the natural and
abundant glycoside oleuropein 2. In view of both the high
amount of oleuropein in olive leaves and the easy extraction,
this procedure constitutes a new interesting route to antioxi-
dant 1.
(10) Mir o´ -Casas, E.; Covas, M.; Fit o´ , M.; Farr e´ -Albadalejo, M.;
Marrugat, J.; de la Torre, R. Tyrosol and hydroxytyrosol are
absorbed from moderate and sustained doses of virgin olive oil
in humans. Eur. J. Clin. Nutr. 2003, 57 (1), 186-190.
(11) Tuck, K. L.; Hayball, P. L. Major phenolic compounds in olive
oil: metabolism and health effects. J. Nutr. Biochem. 2002, 13,
6
36-644.
(
12) Manna, C.; Della Ragione, F.; Cucciolla, V.; Borriello, A.;
D’Angelo, S.; Galletti, P.; Zappia, V. Biological effects of
hydroxytyrosol, a polyphenol from olive oil endowed with
antioxidant activity. AdV. Exp. Med. Biol. 1999, 472, 115-
130.
As a final consideration, the high stability of 3 can also be
regarded to cause trouble as it is aimed to deprotect the catechol
function under very mild physiological conditions (derma or
gastric pH). Further work is in progress to answer this question.