Abstract There is increasing interest in olive phenolic

compounds because of their biological properties as well

as their contribution to the colour, taste and shelf life of
olive products. Phenolic compounds in natural black
Spanish olives are characterised by HPLC-UV and
HPLC-MS. Hydroxytyrosol-4--D-glucoside has been
identified as the major phenolic compound in natural
black olives. A qualitative and quantitative study of
phenolic substances such as hydroxytyrosol, salidroside,
tyrosol, verbascoside, luteolin-7-glucoside, rutin, deme-
thyloleuropein and oleuropein has been carried out.
Traces of other phenolic compounds such as catechol,
vanillic acid, apigenin-7-rutinoside and ligustroside have
also been found by mass spectroscopy. Secoiridoid agly-
cons, the main phenolic compounds in olive oil, were not
detected in olive flesh except the dialdehydic form of
elenolic acid linked to hydroxytyrosol (HyEDA), partic-
ularly in the Arbequina olive variety and only traces
were detected in other varieties. Among anthocyanins,
cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside
were the only anthocyanin compounds detected in all the
varieties studied. The UV and MS spectra of several un-
known phenolic compounds have also been described.
Keywords Phenolic compounds
Hydroxytyrosol-4--D-glucoside Anthocyanins
Natural black olives Mass spectrometry
Introduction
Natural black olives are the raw material for a number of
products, particularly olive oil and table olives (naturally
black olives), and contain a high proportion of phenolic
compounds. Olive polyphenols comprise a major group
of secondary metabolites present in olive oil, pomace,
vegetation water, packed olives and fermentation brines.
They also play an important role in the chemical, orga-
noleptic and nutritional properties of olive products.
The first complete study on the polyphenols in olive
fruit was carried out by Vzquez et al. [1], and new tech-
nologies have recently been applied as HPLC-UV, CC,
RMN [2, 3] and LC-MS [3, 4] to make a better qualita-
tive and quantitative determination of olive polyphenols.
Currently, the development of new methodologies has
been necessary for obtaining a phenol extract; the appli-
cation of solid phase extraction (SPE) [5] and liquid-liq-
uid extraction (L-L) [2] in the separation of phenolic
compounds from olive fruit are the protocols most used.
Oleuropein, the main component that produces bitter-
ness in olives, decreases during the last maturation step
of the fruit [6], while elenolic acid glucoside and deme-
thyloleuropein increase [7]. In fact, the latter compound
has been proposed as a variety marker by Esti et al. [8],
because this polyphenol was found only in some Italian
varieties. In addition, other polyphenols were found in
olive fruit with a different ripeness index, such as
hydroxytyrosol, tyrosol, vanillic acid, caffeic acid,
p-coumaric acid and verbascoside [4, 9]. However,
hydroxytyrosol-4--D-glucoside, also reported in olive
flesh [10, 11], has recently been proposed as the main
polyphenol compound in naturally black olives [12].
Besides these compounds, other natural phenols, also
identified in olives, are flavonol compounds such as rutin,
luteolin-7-glucoside and apigenin-7-glucoside, and several
anthocyanin pigments [13, 14]. The latter are responsible
for the purple colour of natural black olives and a qualita-
tive study of these pigments was carried out by Vzquez
and Maestro [15, 16], reporting that cyanidin-3-O-gluco-
side and cyanidin-3-O-rutinoside were the main anthocya-
nins in natural black olive fruits. Luh and Mahecha [17]
detected other minor anthocyanin pigments in Manzanillo
olives such as peonidin-3-glucoside acylated with p-cou-
maric acid, cyanindin-3-glucoside acylated with p-cou-
maric acid and cyanidin-3-O-rutinoside acylated with
caffeic acid. Delphinidin-3-rhamnoglucoside was also
detected in olives for the first time by Tanchev et al. [18].
C. Romero (

) P. Garca M. Brenes A. Garca A. Garrido

Food Biotechnology Department, Instituto de la Grasa (CSIC),
Avda. Padre Garcia Tejero 4, 41012-Sevilla, Spain
e-mail: crb@cica.es
Eur Food Res Technol (2002) 215:489496
DOI 10.1007/s00217-002-0619-6
ORI GI NAL PAP ER
Concepcin Romero Pedro Garca Manuel Brenes
Aranzazu Garca Antonio Garrido
Phenolic compounds in natural black Spanish olive varieties
Received: 10 July 2002 / Published online: 19 October 2002
Springer-Verlag 2002
The phenolic fraction of olive pulp is complex, and
some peaks remain unidentified. A comparison between
different Italian varieties showed significant quantitative
differences in some polyphenols, such as verbascoside
and oleuropein derivatives [19]. Besides, when the phe-
nolic composition of different varieties from several
countries (Spain, Italy, France and Portugal) was studied
[11], the data were quantitatively dissimilar between the
varieties for the same phenol.
Spain is the major producer of olive oil and table
olives in the world and extensive information is not avail-
able about the phenolic fraction of Spanish olive variet-
ies. Therefore, the purpose of this investigation was to
determine the phenolic fraction of different natural black
Spanish olive varieties and to obtain a better knowledge
of the quali-quantitative profiles of these compounds.
Materials and methods
Olives
Assays were run with Gordal, Manzanilla, Picual, Hojiblanca,
Lechn, Arbequina and Cacerea varieties of olive fruits, which
were collected by hand in several Spanish provinces: Cabra
(Crdoba), Arahal (Sevilla), Paradas (Sevilla) Casariche (Sevilla),
Hinojos (Huelva) and Plasencia (Cceres).
Ripeness Index (r.i.)
It was calculated using a subjective evaluation colour of the skin
and flesh proposed by Uceda and Fras [20].
Superficial colour analyses
Colorimetric measurements on olives were performed using a
BYK-Gardner Model 9000 Color-view spectrophotometer,
equipped with computer software to calculate the CIE L* (light-
ness), a* (redness) and b* (yellowness) parameters. Interference
by stray light was minimised by covering samples with a box
which had a matt black interior. The data of each measurement are
the average of 20 olives.
Extraction of phenolic compounds
The olive fruits were frozen in liquid nitrogen and crushed in a
mortar after removal of the stone, yielding an uniform powder.
Non-anthocyanin compounds. The phenolic extracts of olive
fruits were obtained using the methodology proposed by Romero
et al. [12]. The proposed method consisted of extracting the phe-
nolic compounds with a solution of methanol:water plus 100 ppm
of sodium salt of diethyldithio-carbamic acid. A C
18
cartridge
was used to purify the phenolic extract and syringic acid was
used as internal standard.
Anthocyanin compounds. The anthocyanin extracts of olive fruits
was obtained by following the procedure described in Fig. 1. The
proposed method consisted of extracting the phenolic compounds
with a solution of methanol:hydrochloric acid. A washing step
with hexane was required to remove the fat from the extract.
HPLC analysis of phenolic compounds
Non-anthocyanin compounds. The HPLC system consisted of
a Waters 2690 Alliance with a pump, a column heater and autosam-
pler modules included, the detection being carried out with a Waters
996 photodiode array detector. The system was controlled with Mil-
lennium
32
software (Waters Inc., Milford, MA). A 25 cm4.6 mm
i.d., 5-m Lichrospher 100 (Merck, Darmstadt, Germany) column
was used. Separation was achieved by linear gradient elution using
an initial composition of 90% water (pH 2.5 adjusted with 0.15%
phosphoric acid) and 10% methanol. The concentration of the latter
solvent was increased to 30% in 10 min and maintained for 20 min.
Subsequently, the methanol percentage was raised to 40% in
10 min, maintained for 5 min. Finally, the methanol percentage for
the last three steps was increased to 60, 70 and 100% in 5-min peri-
ods. Initial conditions were reached in 15 min. An injection volume
of 20 l, a flow rate of 1 ml/min and a temperature of 35 C were
used. Chromatograms were recorded at 280 nm.
Anthocyanin compounds. The HPLC method used was based on
that proposed by Romero and Bakker [21]. The HPLC system
consisted of a Waters 717 plus autosampler (20 l), a Waters 600
E pump, a Waters column heater module (40 C), and a Waters
996 photodiode array detector operated with Millenium 2010 soft-
ware (Waters Inc., Milford, MA). A 25 cm4.6 mm i.d., 5-m Ex-
trasil ODS-2 (Technokroma, Barcelona, Spain) column was used
and the elution conditions were as follows: flow rate=1 ml/min;
solvent A, water with 1% perchloric acid, solvent B, methanol.
The mobile phase consisted initially of 80% of A; using a linear
gradient, the concentration of methanol was increased to 50% over
35 min, to 98% at 40 min, held for 2 min at 98% of B to wash the
column, and then returned to the initial conditions (20% of B) for
10 min. Chromatograms were recorded at 520 nm.
Reference compounds
The evaluation of each non-anthocyanin compound was
performed using a regression curve in triplicate of three different
concentrations (r
2
0.99), using siringic acid (0.19 mmol/l) as
internal standard. Oleuropein, apigenin-7-rutinoside and luteolin-7-
O-glucoside were purchased from Extrasynthese S. A. (Lyon Nord,
490
Fig. 1 Flowchart of the extraction and separation of anthocyanin
compounds from olives
Genay, France); catechol, tyrosol, vanillic acid and rutin were
provided by Sigma Chemical Co. (St. Louis, MO); verbascoside
was isolated by analytical HPLC and its absorbance was measured
at 330 nm in a 10-mm quartz cell, and the concentration was calcu-
lated using the molar absorptivity value of 111 [22]; dialdehydic
form of elenolic acid linked to hydroxytyrosol (HyEDA), hydroxy-
tyrosol and hydroxytyrosol-4--D-glucoside were obtained by pre-
parative HPLC as described elsewhere [12, 23]. The evaluation of
each anthocyanin compounds was performed using a four-point re-
gression curve (r
2
0.998) obtained using the available standards;
cyanindin-3-O-glucoside and cyanidin-3-O-rutinoside were pur-
chased from Extrasynthese S. A. (Lyon Nord, Genay, France).
HPLC-MS analysis of phenolic compounds
Sample extracts were analysed using a HPLC system consisting of
a Waters 600 E pump, a Waters column heater module (35 C), a
Waters 996 photodiode array detector and a ZMD4 mass spectro-
photometer (Waters Inc., Mildford, MA) equipped with an electro-
spray ionisation ion source (ESI). The system was controlled with
MassLynk NT 3.4 software (Waters Inc., Milford, MA). A
25 cm4.6 mm i.d., 5-m Spherisorb ODS-2 (Waters Inc.,
Milford, MA) column and an injection volume of 20 l were used.
Separation was achieved by gradient elution (see above, HPLC
analysis of non-anthocyanin compounds). The ionspray mass
spectra in the negative-ion mode were obtained under the following
conditions: capillary voltage, 3 kV; cone voltage, 20 and 70 V; ex-
tractor voltage, 12 V; desolvation temperature, 250 C and source
temperature, 80 C. A constant flow of 1 ml/min was used for each
analysis, with an approximately 4:1 split ratio (UV:MS detectors).
Results and discussion
The ripeness index (r.i.) and the colour parameters of the
olive fruits are given in Table 1. Olives of different vari-
eties with a similar ripeness index were studied. By way
of an exception, olives with two values of r.i. were con-
sidered for the Manzanilla and Hojiblanca varieties. The
correlation coefficients between the r.i. and the colour
values were very poor: r.i. vs L* (r
2
=0.488), r.i. vs a*
(r
2
=0.441) and r.i. vs b* (r
2
=0.031); data that do not
agree with those reported by Esti et al. [8]. We must bear
in mind that the r.i. is a subjective value of the external
and internal degree of the purple colour in the olive
fruits, whereas the CIELAB parameters are exclusively
an objective value of the external colour. In general, the
lightness was very similar for all varieties (22 units);
the highest value of L* (32.17) corresponded to the Ar-
bequina variety. Fruits of this variety showed the highest
values of yellowness (1.88) and redness (8.99).
491
Table 1 Ripeness Index and
CIE L* a* b* parameters of
Spanish olives
Cultivars Ripeness Index
a
L* a* b*
Gordal 3.19 22.3 (1.26)
b
7.82 (1.01) 0.23 (0.45)
Picual 3.00 20.13 (0.26) 3.02 (0.84) 1.34 (0.07)
Arbequina 2.91 32.17 (3.00) 8.99 (0.65) 1.88 (0.46)
Lechn 3.00 24.33 (1.68) 4.26 (0.51) 0.21 (0.49)
Cacerea 3.07 23.23 (0.13) 6.12 (0.30) 0.08 (0.12)
Manzanilla (a) 4.04 20.28 (0.90) 3.87 (0.52) 0.39 (0.37)
Manzanilla (b) 4.94 20.03 (0.33) 3.12 (0.27) 0.68 (0.06)
Hojiblanca (a) 2.86 22.66 (0.89) 5.77 (1.10) 1.08 (0.20)
Hojiblanca (b) 3.53 21.37 (0.43) 0.29 (0.10) 0.72 (0.14)
a
Ripeness Index= (in
i
)/100;
i=number of the group,
n
i
=number of olives in it
b
Standard deviation
Figure 2 shows the HPLC chromatogram of phenolic
compounds in Hojiblanca olives. A similar chromato-
graphic profile was obtained for the rest of the varieties
studied although peaks response varied. Among the
peaks, hydroxytyrosol-4--D-glucoside (peak 1) showed
the highest response to the UV detector; it was the main
polyphenol found in all the varieties studied. In fact,
hydroxytyrosol-4--D-glucoside has recently been
proposed as a very important phenolic compound in nat-
ural black olives and derived products [12].
Hydroxytyrosol, tyrosol, verbascoside, luteolin-7-
glucoside and oleuropein (peaks 2, 5, 8, 9 and 12 respec-
tively) were present in all varieties and they have also
been reported in several Italian varieties [8, 11]. Vanillic
acid (peak 6) and rutin (peak 11) were not detected in the
Gordal and Arbequina, and rutin was not present in the
Manzanilla varieties. Demethyloleuropein (peak 7) was
mainly found in the Arbequina variety, while, traces of
apigenin-7-rutinoside (peak 13) and ligustroside (peak
14) have been detected by mass spectroscopy in all the
natural black olive varieties studied.
Peak 3 was identified as catechol by means of its
retention time and confirmed by comparison of the char-
acteristic UV and mass spectra of a standard with those
obtained from peak 3. This is the first time that this phe-
nol has been described in natural black olive pulp and it
was found in the Lechn, Manzanilla and Hojiblanca
varieties. Likewise, peak 4 was assigned to salidroside
(tyrosol glucoside), which has been reported in pulp ol-
ive before [19, 24], and particularly in olive seeds [25].
The dialdehydic form of elenolic acid linked to hy-
droxytyrosol (HyEDA) was identified as peak 10. This
compound has been found in olive oil [23, 26] and dur-
ing this study it was mainly found in Arbequina olives.
Other varieties also showed traces of this secoiridoid
aglycon.
Peaks a, b, c, d, e, f and g correspond to
unknown phenolic compounds. Peak a was only found
in the Hojiblanca olives, and its UV spectrum was simi-
lar to that of oleuropein with a maximum at 228.5 nm.
The mass spectrum of this compound displayed a major
signal at m/z 701, when operating the mass spectrometer
at cone voltage of 20 V with an ESI probe in negative
mode. Significant fragments at m/z 153 and 123 were
also obtained when operating at cone voltage of 70 V
(data not shown). The latter signals correspond to the
molecular ion [M

] and a fragment of hydroxytyrosol,

respectively. Thus, this compound identified as peak a
could be a hydroxytyrosol derivative. Similar com-
pounds, with a high molecular mass, have previously
been reported in olive leaf extracts [27].
Moreover, peaks d and f were only found in the
Arbequina olives. While peak d showed a UV spec-
trum with two maxima at 210 and 238 nm, peak f did
not have a well-defined spectrum. The mass spectra of
these compounds displayed major signals at m/z 673 and
467 for peak d and f respectively. At high cone volt-
age (70 V) the fragments were 119, 121, 139, 165, 209
and 389 for peak d" and 70, 95 and 166 for peak f. In
fact, these data only allow us to make a qualitative
description of these compounds but we cannot confirm
any structure for them.
The anthocyanin composition of natural black
Spanish olives (Fig. 2) was very simple; two pigments
only were detected; cyanidin-3-O-glucoside and cyani-
din-3-O-rutinoside (peaks 15 and 16 respectively). In
general, these compounds have been identified as the
main coloured pigments in several natural black olive
varieties by different authors [13, 15, 16]. Other antho-
cyanin pigments such as cyanidin-3-O-rutinoside acyla-
ted with caffeic acid, peonidin-3-glucoside acylated with
p-coumaric acid and cyanidin-3-O-glucoside acylated
with p-coumaric acid have also been reported [16, 17,
18] but we have not found them in the Spanish varieties,
even using HPLC-MS detection.
Oleuropein is considered as the main phenolic com-
pound in olive fruits, its content diminishing with olive
maturation [6]. Nevertheless, when we evaluated natural
black olives, hydroxytyrosol-4--D-glucoside (HyGlu)
was the most important phenol found. Gordal, Hoji-
blanca and Lechn varieties with a similar ripeness index
showed the highest level of this glucoside, about
6570 mmol per kilogram of dry weight (Fig. 3). It was
also observed for the Manzanilla variety that the level of
this phenol increased with maturation, which confirms
previous data [12].
492
Fig. 3 Concentration of hydroxytyrosol (Hy), hydroxytyrosol-4-
-D-glucoside (HyGlu), tyrosol (Ty) and salidroside (TyGlu) com-
pounds (mmoles per kilogram dry weight) in seven natural black
Spanish olive varieties. Salidroside was quantified as tyrosol. Va-
riety identification: (G) Gordal, (P) Picual, (A) Arbequina, (L) Le-
chn, (C) Cacerea, (M(a)) Manzanilla (a), (M(b)) Manzanilla (b),
(Hj(a)) Hojiblanca (a), (Hj(b)) Hojiblanca (b)
Fig. 2 HPLC chromatograms of the phenolic compounds in natu-
ral black olives of Hojiblanca variety: A non-anthocyanin com-
pounds (see materials and methods), determination at 280 nm: (1)
hydroxytyrosol-4--D-glucoside, (2) hydroxytyrosol, (3) catechol,
(4) salidroside, (5) tyrosol, (I.S.) internal standard (siringic acid),
(6) vanillic acid, (7) demethyloleuropein, (a) unknown, RT 24.01,
(8) verbascoside, (9) luteolin-7-glucoside, (10) dialdehydic form
of elenolic acid linked to hydroxytyrosol (HyEDA), (11) rutin,
(12) oleuropein, (13) apigenin-7-rutinoside, (b) unknown RT
35.53, (14) ligustroside, (c) unknown RT 41.25, (d) unknown RT
42.45, (e) unknown RT 43.73, (f) unknown RT 49.85, (g) un-
known RT 52.01; B anthocyanin compounds (see materials and
methods), determination at 520 nm: (15) cyanidin-3-O-glucoside,
(16) cyanidin-3-O-rutinoside
Apart from the glucoside of hydroxytyrosol, impor-
tant levels of another phenols were found in the natural
black Spanish olives studied. Oleuropein (548 mmol/kg
dry weight) (Table 2), hydroxytyrosol (417 mmol/kg
dry weight), tyrosol (26 mmol/kg dry weight) and sal-
idroside (26 mmol/kg dry weight) (Fig. 3). Salidroside
is a glucoside of tyrosol (TyGlu) that was present in all
varieties and, in general, its concentration was higher
than that of tyrosol, except for the Cacerea variety,
whose behaviour was diametrically opposed. Tyrosol
was not detected in some varieties, such as Arbequina
and Manzanilla (b).
The importance of these glucosides (hydroxytyrosol-
4--D-glucoside and salidroside) has not been considered
until now. These natural black olives are the raw material
for olive oil production and natural black table olives;
the latter product goes through a process of fermentation
[28]. Therefore, we must keep in mind that both phenolic
compounds may signify (i) an important source of glu-
cose, which could make the fermentation process easier,
and (ii) a source of high amounts of simple phenols,
principally, hydroxytyrosol, which has shown antimicro-
bial, hypoglycemic, hypolipidemic and hypocholesterol
properties [29, 30, 31].
Only a few other phenols were present in a quantifi-
able amount in olives (Table 2): verbascoside
(0.030.23 mmol/kg dry weight), luteolin-7-glucoside
(0.292.12 mmol/kg dry weight) and rutin
(0.170.73 mmol/kg dry weight). Moreover, other phe-
nols such as catechol, vanillic acid, apigenin-7-rutino-
side and ligustroside were only found in trace levels by
mass spectrometry. The identification of these phenols
was possible based on its UV and mass spectra data,
which were compared with the respective standards.
HPLC-MS with an ESI probe in negative mode and a
cone voltage of 20 V was used, and extract ions [M

] at
m/z 109, 167, 577 and 523 were obtained, which corre-
spond to catechol, vanillic acid, apigenin-7-rutinoside
and ligustroside respectively.
Arbequina is the only variety that displayed a signifi-
cant level of demethyloleuropein (38.82 mmol/kg dry
weight) and HyEDA (19.19 mmol/kg dry weight). In ad-
dition, both phenolic compounds could be detected in the
Lechn, Cacerea, Manzanilla and Hojiblanca varieties,
but in trace levels only. Demethyloleuropein is a well-
known phenol and it increases with the maturation
degree of olive pulp [7], but, HyEDA has only been
identified before in olive oil [23, 26], another product for
which natural black olives are the raw material.
Figure 4 shows the mass spectra of unknown peaks
named b, c, e and g, when the mass spectrome-
ter was operated at cone voltage of 20 V with an ESI
probe in negative mode. The mass spectrum of peak b
revealed a molecular ion [M

] at m/z 551, while if a cone

voltage of 70 V was used, the fragments obtained were
mainly at m/z 161, 133, 179, 221 and 285 (Fig. 5). The
signal at m/z 179 could be assigned to the molecular ion
of caffeic acid and the UV-spectrum of peak b was
similar to that of caffeic acid, with maxima at 219 and
329 nm (Fig. 4). Then, peak b could be tentatively as-
signed to a caffeic acid derivative.
Moreover, mass spectrum of peak c showed a molec-
ular ion [M

] at m/z 535 (Fig. 4) and its fragments obtained

with cone voltage of 70 V were 69, 117, 145, 163 and 205
(Fig. 5), where 163 could correspond to the presence of
p-coumaric acid. This hypothesis is corroborated for the
typical UV-spectrum of p-coumaric acid with two maxima
(228.5 and 310.1), similar to those obtained for the UV-
spectrum of peak c (Fig. 6). Thus, the peak c can be
also tentatively assigned to a p-coumaric acid derivative.
Finally, two more unknown peaks could be qualita-
tively described, peak e, with a UV-spectrum with four
493
Table 2 Other phenolic compounds (mmoles per kilogram dry weight) in seven natural black Spanish olive varieties by HPLC
n
a
Compound Gordal Picual Arbequina Lechn Cacerea Manzanilla Manzanilla Hojiblanca Hojiblanca
(a) (b) (a) (b)
3 Catechol nd nd nd nd
6 Vanillic acid nd nd nd nd nd nd nd
7 Demethyloleuropein
b
38.82 nd nd nd nd nd nd
(2.34)
8 Verbascoside 0.05
c
0.04 0.15 0.37 0.04 0.03 0.23 0.17 0.16
(0.00)
d
(0.00) (0.01) (0.02) (0.00) (0.04) (0.04) (0.01) (0.04)
9 Luteolin-7-glucoside 0.25 0.84 1.41 0.20 1.05 0.94 0.29 2.12 1.86
(0.04) (0.04) (0.08) (0.02) (0.01) (0.13) (0.10) (0.07) (0.13)
10 HyEDA
e
19.19 nd nd nd nd nd nd
(1.47)
11 Rutin nd 0.27 0.73 0.17 0.16
(0.02) (0.08) (0.02) (0.04)
12 Oleuropein 8.34 20.49 4.61 26.51 8.97 48.06 14.80 19.55 13.06
(1.20) (0.90) (0.31) (1.77) (0.34) (6.07) (2.36) (0.56) (4.98)
13 Apigenin-7-rutinoside nd nd nd nd nd nd nd nd nd
14 Ligustroside nd nd nd nd nd nd nd nd nd
a
The number is the peak number shown in the chromatogram of
Fig. 2
b
The demethyloleuropein has been quantified as oleuropein
c
Results are mean values of three independent determinations
d
Standard deviation
e
The dialdehydic form of elenolic acid linked to hydroxytyrosol
494
Fig. 4 Negative ion mass spec-
tra (ESI) of b, c, e and
g compounds obtained under
cone voltage of 20 V
Fig. 5 Negative ion mass spec-
tra (ESI) of b, c, e and
g compounds obtained under
cone voltage of 70 V
maxima at 203, undefined, 287 and 333 nm (Fig. 6),
which has an aspect very similar to vanillic acid; howev-
er, the maxima were not coincident. If we pay attention
to the signals obtained for the mass spectrum at cone
voltage of 20 V, apparently this peak e could be a mix-
ture of two compounds, whose molecular ions were 770
and 925 amu (atomic mass units) (Fig. 4). The mass
spectrum at a higher voltage (70 V) showed several sig-
nals, mainly 135, 161, 179, 377, 539, 621 and 654, that
may be the fragments of both compounds included in
this peak e.
The fourth unknown peak (g), that is important in
relation to its high response to the HPLC chromatogram
and its presence in all varieties, did not show a clear UV-
spectrum (Fig. 6) with three maxima (210, 239, unde-
fined nm). Its mass spectrum showed a molecular ion
mass at m/z 686 (Fig. 4) and the fragments obtained with
a cone voltage of 70 V (Fig. 5) were assigned to the
signals at m/z 89, 119, 149, 179, 223 and 403.
These four peaks are a constant in all HPLC chro-
matograms for all the olive pulps studied, but only a
qualitative characterisation of them has been possible
until now.
As regards anthocyanins, these pigments are the char-
acteristic phenolic compounds of natural black olives.
They are responsible for the purple colour, which is an
important organoleptic parameter for table olives. The
amount of these compounds increased with maturation,
as clearly shown in Fig. 7, when comparing their level in
the Manzanilla and Hojiblanca varieties with two differ-
ent ripeness indices. The Manzanilla variety showed an
increase (2.68 mmol/kg dry weight) in the cyanidin-3-O-
rutinoside level as a consequence of the maturation rise.
However, the total amount of anthocyanin in Picual
olives was too low (0.06 mmol/kg dry weight) and some
varieties such as Lechn and Cacerea are particularly
rich in cyanidin-3-O-glucoside (0.20 mmol/kg dry
495
Fig. 6 UV spectra of b, c,
e and g compounds
Fig. 7 Concentration of anthocyanin compounds (mmoles per ki-
logram dry weight) in seven natural black Spanish olive varieties.
See Fig. 3 for variety identification
weight). Gordal, Arbequina, Manzanilla and Hojiblanca
olives with a similar ripeness index had an analogous an-
thocyanin composition, and values of 0.150.20 mmol/kg
dry weight were found for cyanidin-3-O-rutinoside and
0.010.11 mmol/kg dry weight for cyanidin-3-O-gluco-
side.
Acknowledgement This research work was supported by the
CICYT (Spanish Government), under the project AGL-2000
1539-CO201.
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