PHYTOCHEMICAL ANALYSIS

Phytochem. Anal. 13, 79–86 (2002)

DOI: 10.1002/pca.627

Identification and Quantification of Galloyl

Derivatives, Flavonoid Glycosides and
Anthocyanins in Leaves of Pistacia lentiscus L.

A. Romani,1 P. Pinelli,1 C. Galardi,1 N. Mulinacci1 and M. Tattini2*

1
Dipartimento di Scienze Farmaceutiche, Università degli Studi, Via G. Capponi 9, I-50121, Firenze, Italy
2
Istituto sulla Propagazione delle Specie Legnose, Consiglio Nazionale delle Ricerche, Via Ponte di Formicola 76, I-50018, Scandicci,
Firenze, Italy

Separation, identification and quantification of polyphenols was carried out on leaves of Pistacia lentiscus L.,
an evergreen member of the family Anacardiaceae, using semi-preparative HPLC, HPLC-photodiode array
detection and HPLC-MS analysis, together with 1H- and 13C NMR. Three major classes of secondary
metabolites were detected: (i) gallic acid and galloyl derivatives of both glucose and quinic acid; (ii) flavonol
glycosides, i.e. myricetin and quercetin glycosides; and (iii) anthocyanins, namely delphinidin 3-O-glucoside
and cyanidin 3-O-glucoside. Low amounts of catechin were also detected. The concentration of galloyl
derivatives was extremely high, representing 5.3% of the leaf dry weight, and appreciable amounts of
myricetin derivatives were also detected (1.5% on a dry weight basis). These findings may be useful in
establishing a relationship between the chemical composition of the leaf extract and the previously reported
biological activity of P. lentiscus, and may also assign a new potential role of P. lentiscus tissue extracts in
human health care. Copyright # 2002 John Wiley & Sons, Ltd.
Keywords: 1H-NMR; 13C-NMR; HPLC-PAD; HPLC-MS; 3,5-O-digalloyl quinic acid; 3,4,5-O-trigalloyl quinic acid;
myricetin glucuronide; delphinidin 3-O-glucoside.

INTRODUCTION in animal cell metabolism in an attempt to relate both in

The ecological role of several evergreen sclerophyll
species that have typically developed under a Mediterra-
nean climate has been reconsidered recently (Naveh,
1995). Pistacia lentiscus L., an evergreen member of the
family Anacardiaceae (Brosse, 1979), plays a key role in
the maintenance of ecosystems that are at incipient risk of
desertification in view of its exceptionally high resistance
to both drought and excess light stress (Margaris, 1983;
Naveh, 1995; Castro-Diez et al., 1998; Filella et al., 1998).
It has previously been suggested that plant species
have developed a very efficient secondary metabolism to
cope with severe stress conditions such as those typical of
the Mediterranean climate (Halbrock and Scheel, 1989;
Dixon and Paiva, 1995; Shirley, 1996). As a conse-
quence, such species are generally very rich in poly-
phenols which, in addition to protecting plant cells from
the detrimental effects of short-wave (UV-B and UV-A)
radiation from the sun, may also counter the negative
effects of reactive oxygen species (free radicals) on cell
metabolism (Husain et al., 1987; Vogelman, 1993;
Hanasaki et al., 1994).
Recently, the role of naturally occurring polyphenols,
particularly flavonoids, and gallate esters of both quinic
acid and catechin, has also been extensively investigated
* Correspondence to: M. Tattini, Istituto sulla Propagazione delle Specie
Legnose, Consiglio Nazionale delle Ricerche, Via Ponte di Formicola 76, I-
50018, Scandicci, Firenze, Italy.
Email: tattini@ipsl.fi.cnr.it
Contract/grant sponsor: Eu Environment and Climate Research and
Development Program within the Project “Modelling Mediterranean
Vegetation Dyanamics”; Contract/grant number: ENV4-CT97-0680.
vivo and in vitro biological activity of secondary
metabolites with their chemical structure (Nishizawa et
al., 1989; Sichel et al., 1991; Vlietinck et al., 1998;
Qiong et al., 1999). However, most of the experiments
have been conducted on metabolites isolated from “crop”
species, whereas few data concern the so-called “wild-
neglected” species (Kim et al., 1994; Manthey and
Buslig, 1998).
P. lentiscus has long been used in the ethno- or
popular-medicine of Mediterranean people (El-Kamali
and Khalid, 1996; Amico and Sorce, 1997). Aerial parts
of P. lentiscus have a hypotensive action, possibly related
to a polymeric procyanidin fraction, and an interesting
anti-bacterial activity which has been correlated to an
unspecified phenolic fraction (Bonsignore et al., 1998;
Sanz et al., 1992, 1998). Furthermore, P. lentiscus is well
known for the synthesis of the essential oils of the mastic
gum resin (Tassou and Nychas, 1995; Al-Said et al.,
1986). Chemotaxonomic studies, conducted on some
members of the Anacardiaceae distributed in India,
suggested the ubiquitous presence of myricetin and gallic
acid in the leaf tissue; other members were also rich in
proanthocyanins and flavonols such as quercetin and
kaempferol (Umadevi et al., 1988). However, to the best
of our knowledge no specific study has been carried out to
elucidate the polyphenol composition of leaves of P.
lentiscus.
It is suggested that there is an urgent need for an in-
depth phytochemical analysis in order to identify relevant
classes of secondary compounds that may help in a better
understanding of (i) environment-induced changes in
secondary metabolism as a part of the evolutionary
process of selected plant species (Harborne, 1993;

Copyright # 2002 John Wiley & Sons, Ltd. Received 29 December 2000
Accepted 21 June 2001
80 A. ROMANI ET AL.
Copper-Driver and Bhattacharya, 1998), and (ii) action
mechanisms of tissue preparations and extracts in an
attempt to relate the chemical composition of tissues to
specific biological/pharmacological activities (Larson,
1995; Miller et al., 1996; Sawa et al., 1999). As a
consequence, the ecological and biological role of the
“wild-neglected” species typically distributed in Medi-
terranean-type ecosystems would be completely eluci-
dated. It was decided, therefore, to conduct an experiment
aimed at identifying and quantifying different classes of
secondary metabolites in leaves of P. lentiscus by using
several chromatographic techniques, including semi-
preparative HPLC, HPLC-diode array detection (PAD)
and HPLC-MS, as well as NMR analysis.
EXPERIMENTAL
Plant material and sample preparation. Fully-ex-
panded leaves were collected from adult plants of
Pistacia lentiscus L. growing in Southern Tuscany (42°
46'N, 10° 53'E) at the end of June and September 1999,
respectively. The authenticity of the plant material was
verified by the Department of Botany of the University of
Florence and a voucher sample is deposited at the Istituto
salla Propagazione delle Specie Legnose. Fully-devel-
oped leaves were collected from both the apical (leaves
were coloured slightly red) and basal portions of the
current-year shoot at midday and combined into an
individual sample. Three replicate samples were prepared
at each harvesting date. Leaf lamina was rapidly frozen in
liquid nitrogen and stored at 80°C before proceeding
with the analysis.
Extraction, purification and isolation of individual
metabolites. The extraction and purification procedures
were similar to those previously reported for analogous
tissues of Myrtus communis (Romani et al., 1999). In brief,
freeze-dried tissue (1–2 g) was extracted with 4  80 mL
of 70% ethanol adjusted to pH 2.0 with formic acid to
avoid degradation of the anthocyanins. The raw ethanol
solution was then evaporated to dryness under vacuum
(Rotavapor 144 R; Büchi, Flawil, Switzerland) at room
temperature and dissolved in water at pH 2 (with formic
acid) to a final volume of 100 mL. The solution was then
defatted with 4  70 mL of n-hexane and evaporated to
dryness under reduced pressure at room temperature, and
finally redissolved in pH 2 water to a final volume of
10 mL. The aqueous solution was deposited on a 20 mL
Extrelut1 (Merck, Darmstadt, Germany) cartridge and,
after 20 min from deposition, eluted with (i) 200 mL n-
hexane until the eluate was colourless, (ii) 200 mL of ethyl
acetate, and (iii) 200 mL of methanol at pH 2 (with formic
acid). The ethyl acetate and methanol fractions were
evaporated to dryness at room temperature, diluted with
1 mL of pH 2 water: methanol:acetonitrile (20:60:20,
v/v/v) and used for the separation, identification and
quantification of secondary metabolites.
The isolation of individual polyphenols was carried out
using 150 g of freeze-dried leaf tissue which was extracted
with 4  250 mL 70% ethanol adjusted to pH 2 (with
formic acid). The raw ethanolic extract was partitioned
with 4  300 mL of n-hexane, evaporated to dryness at
room temperature under reduced pressure, rinsed with
100 mL of pH 2 water and further extracted with
Copyright # 2002 John Wiley & Sons, Ltd.
4  50 mL ethyl acetate. The ethyl acetate fraction was
then reduced to dryness under vacuum at room tempera-
ture, redissolved in 20 mL of ethanol adjusted to pH 2 with
formic acid, and analysed by semi-preparative HPLC.
Identification and quantification of individual poly-
phenols. The identification of individual metabolites was
carried out using their retention times, and both spectro-
scopic and spectrometric data. In a few cases, molecular
characterisation of isolated compounds was performed by
using 1H- and 13C-NMR. Authentic standards of gallic
acid, (‡)-catechin, myricetin 3-O-rhamnoside, quercetin
3-O-rhamnoside, delphinidin 3-O-glucoside and cyanidin
3-O-glucoside were purchased from Extrasynthése
(Lyon, France). 5-O-galloyl-quinic acid (familiar name,
theogallin) and monogalloyl glucose were extracted and
isolated from a commercial extract of green tea (Green-
select2; Indena, Milano, Italy). Myricetin 3-O-rutinoside
was extracted and isolated from undeveloped leaves of
Ribes nigrum as previously reported (Rolland et al.,
1977).
The quantitative determination of individual polyphe-
nols was performed directly by HPLC-PAD using a five-
point regression curve (r2  0.99) operating in the range
0–30 mg. Calibration of galloyl and myricetin derivatives
was performed at 280 and 350 nm using gallic acid and
myricetin 3-O-rhamnoside as reference compounds,
respectively. The actual amounts of both galloyl and
myricetin derivatives in leaves of P. lentiscus were
calculated after applying corrections for changes in the
molecular weight. Finally, authentic standards were used
for the calibration curves of (‡)-catechin, quercetin 3-O-
rhamnoside, delphinidin 3-O-glucoside and cyanidin 3-
O-glucoside, which were performed at 280, 350 and
520 nm for catechin, the flavonol glycoside and the
anthocyanins, respectively.
Analytical techniques and equipment. HPLC-PAD
analyses were performed using an Hewlett Packard (Palo
Alto, CA, USA) model HP 1090L liquid chromatograph
equipped with a diode array detector and managed by an
HP 9000 workstation. Analytical conditions were the
same as previously applied to the separation and
identification of polyphenols in corresponding tissues of
Phillyrea latifolia (Romani et al., 1996). Anthocyanins
were separated using an Aquapore RP300 (Brownlee
Laboratories, Santa Clara, CA, USA) column (250 
4.6 mm i.d.; 7 mm) operating at 26°C equipped with an
Aquapore RP300 precolumn (30  4.6 mm i.d.). The
mobile phase was (A) water:formic acid (95:5), (B)
water:acetonitrile:methanol:formic acid (50:22.5:22.5:5),
and (C) methanol. A three-step linear solvent gradient
system was used starting from 0% B and increasing to
70% B during a 70 min period. The amount of B reached
9% between 0 and 11 min, and then increased to 55%
between 21 and 60 min, and finally attained 70% between
64 and 70 min. The contribution of C was 2% during the
entire run, and the flow rate was 1 mL/min.
Semi-preparative HPLC analysis was performed using
a Perkin Elmer (Norwalk, CT, USA) series 250 LC
equipped with a LC 95 UV–vis detector managed by
Turbochrom 4 software (PE Nelson, San Jose, CA, USA).
The mobile phase was water at pH 3.2 (with formic
acid):acetonitrile at a flow rate of 5.0 mL/min following
the 106 min solvent gradient system previously reported
(Romani et al., 1996). The column was a Merck
Phytochem. Anal. 13: 79–86 (2002)
 GALLOYL DERIVATIVES AND FLAVONOIDS IN PISTACIA LENTISCUS 81

Figure 1. Chromatographic pro®le of the ethyl acetate extract of leaves of P. lentiscus acquired by HPLC-PAD detected at the
relative maxima of absorbance of galloyl derivatives (280 nm) and ¯avonols (350 nm), respectively. Key to peak identities: 1,
monogalloyl glucose; 2, gallic acid; 3, 5-O-galloyl quinic acid; 4, 3,5-O-digalloyl quinic acid; 5, (‡)-catechin; 6, 3,4,5-O-trigalloyl
quinic acid; 7, myricetin glucuronide; 8, myricetin 3-O-rutinoside; 9, myricetin 3-O-rhamnoside; and 10, quercetin 3-O-
rhamnoside. [A LiChrosorb RP18 column was used at 26°C and eluted with water at pH 3.2 (with formic acid): acetonitrile at a ¯ow
rate of 1.0 mL/min following the seven-step linear gradient system from 100% to 55% water in 67 min as previously reported by
Romani et al. (1996).]

LiChrosphere RP18 (250  10 mm i.d.; 10 mm) equipped
with a LiChrosphere RP18 pre-column (10  10 mm i.d.)
and maintained at 26°C.
HPLC-MS analysis was performed using the 67 min
elution program previously described (Romani et al.,
1999). The HPLC-DAD equipment detailed above was
interfaced with an Hewlett Packard HP 1100 MSD API-
electrospray operating in the negative ion mode under the
following conditions: gas temperature, 350°C; nitrogen
flow rate, 10.0 L/min; nebulizer pressure, 40 psi;
quadrupole temperature, 40°C; capillary voltage,
3500 V; fragmentation voltage, 80 to 180 eV. MS were
recorded in the range 0–1000 amu.
The 1H-NMR and COSY spectra were recorded at
300K on a Bruker (Rheinstetten, Germany) Advance-600
spectrometer operating at 600.13 MHz using the 5 mm
inverse probe equipped with z-shielded gradient. Sam-
ples were dissolved in deuterated methanol and the
solvent signal was used for spectral calibration (1H:
3.39 ppm). Data were processed by means of an AV600
computer using a Win-NMR software version 2.5 (Bruker).
The COSY experiments were performed using the internal
pulse sequence for two-dimensional homonuclear shift
correlation, using gradient pulses for selection, type COSY
magnitude 90°. 13C-NMR and 13C-NMR DEPT spectra
were recorded at 300K on a Bruker AC 200 operating at
50.1 MHz. The solvent and spectral calibration (13C:
49.9 ppm) were as for the 1H-NMR experiments.
RESULTS AND DISCUSSION
Extraction and purification methods
In Figs 1 and 2, HPLC-PAD chromatograms of the ethyl
acetate and methanol fractions, respectively, of the leaf
extract of Pistacia lentiscus, recorded at 280 and 350 nm,
are reported. The data suggest that both fractions need to
be analysed when performing a qualitative and quanti-
tative analysis of polyphenols in leaves of P. lentiscus. In
fact, HPLC-PAD analysis of the ethyl acetate fraction
was crucial in identifying and quantifying 3,4,5-O-
trigalloyl quinic acid and quercetin 3-O-rhamnoside
(Fig. 1), whereas monogalloyl-glucose and 5-O-galloyl
quinic acid (theogallin) were only detected in the
methanol fraction (Fig. 2). Galloyl and myricetin
derivatives were, however, detected in appreciable
amounts in both fractions. Consequently the quantifica-
tion of even a specific class of secondary metabolites
cannot be performed by analysing an individual fraction.
The results also indicate the presence of tannins with high
molecular weights in the leaves of P. lentiscus (Fig. 2;
peaks having retention times 60–90 min), as has been
previously reported (Sanz et al., 1992; Gravano et al.,
2000).
Identification and quantitative determination of
individual polyphenols
Typically, the identities of individual polyphenols were
ascertained using data from HPLC-PAD, HPLC-MS and
NMR analyses, by comparison and combination of
retention times, UV–vis, and both MS and NMR spectra.
Gallic acid, monogalloyl glucose, and 5-O-galloyl quinic
acid were identified by comparison of their retention
times, UV–vis and MS with those of authentic standards
or pure compounds, the latter isolated from a commercial
extract of green tea. Other galloyl derivatives were
completely identified using HPLC-MS and NMR spectral
data.
In Fig. 3 the MS of digalloyl and trigalloyl quinic acid

Copyright # 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 79–86 (2002)
82 A. ROMANI ET AL.
Figure 2. Chromatographic pro®le of the methanol extract of leaves of P. lentiscus acquired by HPLC-DAD at 280 and 350 nm,
respectively. Analytical conditions as reported in Fig. 1.
derivatives are reported. The MS of the former consisted
of four major peaks at m/z 495, 343, 191 and 169,
corresponding to the quasi-molecular ion [M-H] , the
fragments after two successive losses of gallic acid ([M-
152-H] and [M-304-H] ), and the fragment of gallic
acid, respectively. Analogously, the peaks at m/z 647,
495, 343 and 169 were consistent with a trigalloyl
substitution of the quinic acid core. The definitive
molecular characterization of the galloyl quinic acid
derivatives was carried out by comparison of 1H-NMR
spectral data (Table 1) with those previously reported by
Pauli et al. (1998) for caffeoyl derivatives of quinic acid.
In detail, the presence of two galloyl moieties was
revealed by the signals at 7.12 and 7.30 ppm (singlets),
each integrating for two protons. The chemical shift, both
the splitting and J-values of the seven protons of the
quinic acid core, were measured by means of COSY and
selective decoupling experiments. The chemical shifts of
protons 3 and 5 were in agreement with an esterification
of the hydroxyl groups linked to carbons 3 and 5 as also
confirmed by 13C spectral data (Table 1) and led to the
unambiguous elucidation of the structure as 3,5-O-
digalloyl quinic acid. The identification of 3,4,5-O-
trigalloyl quinic acid was performed in a similar manner
(Table 1).
The presence of a wide range of gallate esters of quinic
acid has been previously reported in the bark of Quercus
stenophylla and in the galls of Guiera senegalensis,
although quantitative determination of individual com-
pounds has not been reported (Nishimura et al., 1984;
Bouchet et al., 1998a). It has been shown (Umadevi et al.,
1988) that the family Anacardiaceae is characterized by
the occurrence of both gallic acid and myricetin
derivatives. The present data agree with these findings
and at the same time give a more in-depth description of
the galloyl derivatives present in the leaves of P.
lentiscus.
Copyright # 2002 John Wiley & Sons, Ltd.
The other relevant class of polyphenols in the leaf
extract of Pistacia lentiscus (i.e. flavonol glycosides,
mostly myricetin derivatives and small amounts of
quercetin-3-O-rhamnoside) was identified by combina-
tion of retention time, UV–vis and MS. In Fig. 4, the MS
of a myricetin–glucuronide is reported. The most relevant
peaks, recorded at m/z 493 and 317, corresponded to the
quasi-molecular ion [M-H] and to the fragment after the
loss of glucuronic acid [M-176-H] . The peak at m/z 987
corresponded to the dimer of myricetin glucuronide. The
chemical structure of this myricetin derivative has still
not been fully elucidated, although glycosides of
myricetin in most plant species are in position 3
(Harborne, 1994). Myricetin 3-O-b-D-glucuronide has
been previously detected in different tissue extracts of
Potentilla anserina and Epilobium angustifolium (Hier-
mann et al., 1991; Kombal and Glasl, 1995).
Two other myricetin derivatives were detected in the
leaf tissue of P. lentiscus, namely myricetin 3-O-
rhamnoside and myricetin 3-O-rutinoside. Identification
was performed by comparison of UV–vis and MS of leaf
extracts of P. lentiscus with those of standard or pure
compounds, myricetin 3-O-rutinoside being extracted
and isolated from developing leaves of Ribes nigrum
(Rolland et al., 1977). In leaves of other members of the
Anacardiaceae family, namely Buchanania lanzan and
Rhus parviflora, myricetin 3'-rhamnoside-3-galactoside
and myricetin 3-O-rhamnoside have been previously
identified (Nair et al., 1977; Arya et al., 1992).
Catechin, quercetin 3-O-rhamnoside, and both delphi-
nidin 3-O-glucoside and cyanidin 3-O-glucoside were
identified by comparison of retention times and UV–vis
spectra of the leaf extract with those of authentic
standards. Derivatives of quercetin have been previously
reported to be largely distributed in species of Anacar-
diaceae (Umadevi et al., 1988; Kombal and Glasl, 1995)
and delphinidin is closely related in structure to myricetin
Phytochem. Anal. 13: 79–86 (2002)
 Table 1. 1H-NMR, COSY, 13C-NMR and DEPT spectral data for 3,5 di-O-galloyl quinic acid and 3,4,5, tri-O-galloyl quinic acid isolated from leaves of Pistacia lentiscus L.a

Copyright # 2002 John Wiley & Sons, Ltd.

R1 R2 R3
1 3,5-di-O-galloyl quinic acid galloyl H galloyl
2 3,4,5-di-O-galloyl quinic acid galloyl galloyl galloyl

1 H Ð Ð 2ax 2eq 3 4 5 6ax 6eq Ð G1 G2 G3 G4 G5 G6

ppm; Ð Ð 2.40 2.22 5.55 4.08 5.51 2.26 2.34 Ð Ð 7.12, 7.20 Ð Ð Ð 7.12, 7.20
splitting; dd ddd ddd dd ddd dd ddd s s
J* (Hz) 14.9, 7.1 14.9, 3.9, 3.2, 7.1, 7.2, 3.2 7.2, 7.4, 13.8, 7.4 13.8, 4.1,
1.4 3.9 4.1 1.4
C COOH 1 2 Ð 3 4 5 6 Ð C=O G1 G2 G3 G4 G5 G6
ppm 177.7 74.8 37.7* Ð 72.8# 70.7 72.5# 36.0* Ð 168.1, ;121.6, 110.3, 146.4 139.8, 146.4 110.3, 110.5
167.7 121.9 110.5 139.9
2 H Ð Ð 2ax 2eq 3 4 5 6ax 6eq Ð G1 G2 G3 G4 G5 G6
ppm; Ð Ð 2.40 2.22 5.51 5.21 5.63 2.26 2.34 Ð Ð 7.04, 7.07, Ð Ð Ð 7.04, 7.07,
7.14 7.14
splitting; dd ddd ddd dd ddd dd ddd s s
J* (Hz) 14.9, 7.0 14.9, 3.9, 3.1, 7.0, 9.0, 3.1 9.0, 7.3, 13.8, 7.3 13.8, 4.0,
1.4 4.0 4.0 1.4
C COOH 1 2 Ð 3 4 5 6 Ð C=O G1 G2 G3 G4 G5 G6
ppm 178.0 74.7 38.3* Ð 70.0# 72.6 72.1# 36.5* Ð 168.2, 120.8, 110.3, 146.4 140.1, 146.4 110.3, 110.5
167.8, 121.1 110.5 140.3
168.0
1,2 COSY Ð Ð 2eq 3 2ax, 3, 6eq 2ax 2eq 4 3, 5 4, 6ax 6eq 5, 6eq 2eq 5, 6ax Ð Ð Ð Ð Ð Ð Ð
GALLOYL DERIVATIVES AND FLAVONOIDS IN PISTACIA LENTISCUS

DEPT C C CH2 Ð CH CH CH CH2 Ð C C CH C C C CH

a 1
H-NMR and COSY spectra were recorded at 300K on a Bruker Advance-600 spectrometer operating at 600.13 MHz. 13C-NMR and DEPT spectra were recorded at 300K on a Bruker
AC spectrometer operating at 50.1 MHz. The J couplings were measured directly or by simpli®cation of the signals after selective decouplings. The symbols
# and * are interchangeable in the same column.

Phytochem. Anal. 13: 79–86 (2002)

83
84 A. ROMANI ET AL.

Figure 3. Negative ion MS of 3,5-O-digalloyl and 3,4,5-O-trigalloyl quinic acids acquired during API-electrospray HPLC-MS
analysis (for analytical protocol see Experimental section).

(Griffiths and Smith, 1972). Catechin and its galloyl
derivatives, in addition to appreciable amounts of
myricetin and quercetin glycosides, have been previously
found in the leaves of M. communis, an evergreen
member of the Myrtaceae, that occurs together with P.
lentiscus in most arid zones of the southern Mediterra-
nean basin. In our experiment, kaempferol glycosides
were not detected in the leaves of P. lentiscus, this
species differing from other members of Anacardiaceae
(Umadevi et al., 1988).
Polyphenol composition of leaves of Pistacia
lentiscus
weight. It should be pointed out that the leaves analysed
were from plants of P. lentiscus growing under a daily
light irradiance of approximately 30 mmol/m2 photon
flux density. The content of both hydrolysable tannins
and flavonol glycosides may markedly increase in leaves
of P. lentiscus plants adapted to full solar radiation (daily
light irradiance >60 mmol/m2) as commonly occurs in
several plant species exposed to excess light (Lavola,
1998; Tattini et al., 2000).
Galloyl derivatives accounted for more than 70% of
the total polyphenols present as determined by HPLC-
DAD analysis (Table 2), and they may also serve for the
synthesis of high molecular weight tannins (Hergert,

The polyphenol composition of leaves of P. lentiscus

appears of great interest from both an ecological and Table 2. Relative amounts of galloyl derivatives, flavonoid
biological point of view (Table 2). The total amount of glycosides and anthocyanins found in leaves of
polyphenols is high, representing 7.5% of leaf dry Pistacia lentiscus La

Concentrationc
b
Polyphenol (mg/g dry weight)

Monogalloyl glucose (1) 2.8  0.15

Gallic acid (2) 3.7  0.56
5-O-Galloyl quinic acid (3) 9.6  2.25
3,5-O-Digalloyl quinic acid (4) 26.8  4.67
(‡)-Catechin (5) 1.8  0.98
3,4,5-O-trigalloyl quinic acid (6) 10.3  2.45
Myricetin glucuronide (7) 3.9  0.65
Myricetin 3-O-rutinoside (8) 4.5  0.18
Myricetin 3-O-rhamnoside (9) 6.8  1.04
Quercetin 3-O-rhamnoside (10) 3.7  0.52
Delphinidin 3-O-glucoside 0.8  0.22
Cyanidin 3-O-glucoside 0.4  0.12
a
Leaves were collected from both the apical and the basal
portion of the current-year shoot from three plants at the
end of June and at the end of September.
b
Numbers in brackets refer to the peak number shown in
Figs 1 and 2 (except for anthocyanins, where chromato-
Figure 4. Negative ion MS of a myricetin glucuronide grams are not given).
acquired during API-electrospray HPLC-MS analysis. The c
Quantitative determination was performed using a ®ve
chemical structure of myricetin 3-O-glucuronide has been point regression curve (r2 > 0.99) obtained from authentic
reported for convenience (for analytical protocol see Experi- standards and isolated compounds. Data are means  stan-
mental section). dard deviation (n = 6).

Copyright # 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 79–86 (2002)
 GALLOYL DERIVATIVES AND FLAVONOIDS IN PISTACIA LENTISCUS
1989) when plants are faced with conditions of water
deficit and wind stress (Bussotti et al., 1998). Synthesis of
both low and high molecular weight hydrolysable tannins
may also contribute significantly to the usage of captured
photosynthetic energy under excess light conditions
(Lewis and Yamamoto, 1989).
The large presence of myricetin derivatives in leaves
of P. lentiscus (20% of total polyphenol content; Table 2)
may also have an interesting ecological significance, as
primitive characters (low degree of evolution) are often
associated with angiosperm groups containing myricetin
(Giannasi, 1988). Anthocyanins frequently occur in
young leaves of many plant species and may protect
photosystem II from solar radiation as they absorb at
wavelengths typical of chlorophyll b (Gould et al., 1995).
In leaves of P. lentiscus, which are not protected by
specific functional organs (glandular trichomes or leaf
hairs) usually found in species adapted to arid environ-
ments (Tattini et al., 2000), anthocyanins may play a key
role in the integrated mechanisms of adaptation to a
combination of excess light and drought conditions
(Gould et al., 1995).
The concentration of gallate esters of quinic acid in
leaves of P. lentiscus is high enough (approximately
4.7% on dry weight basis; Table 2) reasonably to suggest
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