Ben-Gurion University of the Negev

Ben-Gurion University of the Negev
The Jacob Blaustein Institutes for Desert Research
The Albert Katz International School for Desert Studies
Anthocyanins in Pistacia leaf at early vegetation and
senescence
Thesis submitted in partial fulfillment of the requirements for the degree of
"Master of Science"
By Artur Ghazaryan
21 October, 2010

Ben-Gurion University of the Negev
The Jacob Blaustein Institutes for Desert Research
The Albert Katz International School for Desert Studies
Anthocyanins in Pistacia leaf at early vegetation and
senescence
Thesis submitted in partial fulfillment of the requirements for the degree of
"Master of Science"
By Artur Ghazaryan
Under the Supervision of Prof. Avi Golan-Goldhirsh and Dr. Micha Guy
Department of Agriculture and Biotechnology for Sustainable Development
Author's Signature …………….……………………… Date …………….
Approved by the Supervisor…………….…………….. Date …………….
Approved by the Supervisor…………….…………….. Date …………….
Approved by the Director of the School …………… Date ………….…

Abstract
I
Anthocyanins in Pistacia leaf at early vegetation and senescence
Artur Ghazaryan
Thesis submitted in partial fulfillment of the requirements for the degree of
"Master of Science"
Ben-Gurion University of the Negev
The Jacob Blaustein Institutes for Desert Research
The Albert Katz International School for Desert Studies
(2010)
Twice a year, leaves of Pistacia turn into bright red, in early spring up to mid-summer
in new leaf growth and in autumn before leaf fall. Red coloration of the leaves and
stems is mainly due to anthocyanins that belong to the flavonoids. Anthocyanin content
and distribution pattern were determined in six Pistacia species: five deciduous (P.
chinensis, P. palaestina, P. khinjuk, P. atlantica, P. vera) and one evergreen (P.
lentiscus). These species are grown in the BIDR Pistacia germplasm collection plot
(http:/www.bgu.ac.il/pistacia), where they grow under similar environmental and soil
conditions. Flavonoids content and distribution was investigated in winter, spring and
autumn green and red leaves, as well as spring red stems. HPLC chromatograms
revealed seven major peaks that were annotated peaks #1 to 7. Authentic standards, MS
and NMR were used for quantification and identification of the peaks. Peak #2 was
identified as cyanidin-3-O-glucoside, peak #4 was identified as quercetin-3-O-
glucoside, peak #6 was identified as cyanidin and peak #7 as quercetin. Flavonoids
contents were higher in spring leaves compared to autumn leaves. Flavonoids contents
were not significantly different in green and red leaves of the same species and in the

II
same season, 3.26 ± 0.42; 3.60 ± 0.17 mg/gFW quercetin-3-O-glucoside, in P. chinensis
spring green and spring red leaves, respectively and 2.60 ± 0.46; 2.10 ± 0.43 mg/gFW,
in P. chinensis autumn green and autumn red leaves, respectively. Apparent color
difference between green and red leaves is due to the presence of other pigments, such
as chlorophyll and carotenoids that mask anthocyanin’s red color. Stem flavonoids
contents were much lower than in their corresponding leaves (4.33 ± 0.11 vs. 0.11 ±
0.01 mg/gFW quercetin-3-O-glucoside in P. palaestina). Based on quercetin-3-O-
glucoside content, P. palaestina is closer phylogenetically to P. chinensis and P.
lentiscus, P. khinjuk and P. atlantica group together. Distinct differences were detected
between spring juvenile and autumn senescent leaves in all parameters, flavonoids,
chlorophyll, carotenoids.
Newly growing branches, in spring or in autumn, start as red, then become green and
upon maturation turn brown-gray. Leaves masked by other leaves, were not red but
yellow. This is in agreement with light protection hypothesis of anthocyanins referred in
theeliterature.
Based on these observations, we proposed the "red cycle" model (Fig. 10). According to
the model, new growth of leaves or stems in early vegetation and photosynthetic
apparatus dismantlement and cellular break-down during leaf senescence cause
metabolic activity changes due to fluxes of metabolites and catabolites. Under these
conditions, anthocyanins plausible role can be protection of plant tissues from
environmental stresses and ROS.

Acknowledgement
III
I would like to thank Prof. Avi Golan-Goldhirsh and Dr. Micha Guy for their great
supervision and the excellent education.
My special thanks to Ahuva Vonshak for her technical assistance and advices in
laboratory.
I would like also to thank fellow researchers of the laboratory for cooperation and
wonderful friendship.
Special thanks to administrative staff Dorit Levin and Ilana Saller for making my life
bureaucracy-free.
Many thanks to my family. I miss you.
Many thanks to all the people in Israel I know! You made my life in this wonderful
country fruitful and enjoyable.

Table of Contents
IV
ABSTRACT .................................................................................................................I
ACKNOWLEDGEMENT .......................................................................................III
TABLE OF CONTENTS ......................................................................................... IV
LIST OF FIGURES ................................................................................................. IX
LIST OF TABLES .................................................................................................... X
LIST OF APPENDIX FIGURES .......................................................................... XII
LIST OF APPENDIX TABLES ............................................................................ XIII
ABBREVIATIONS ................................................................................................ XIV
1. INTRODUCTION ................................................................................................... 1
1.1. Red coloration of tree leaves............................................................................... 1
1.2. Anthocyanins ...................................................................................................... 2
1.2.1. Anthocyanin functions ................................................................................. 3
1.2.1.1. Anthocyanins as antioxidants ................................................................. 4
1.2.1.2. Photoprotection...................................................................................... 5
1.2.1.3. UV protection ........................................................................................ 8
1.2.1.4. Protection against herbivory................................................................... 9
1.2.1.5. Other functions ...................................................................................... 9
1.3. Pistacia tree ..................................................................................................... 10
1.4. Early vegetation ............................................................................................... 10
1.5. Leaf senescence ................................................................................................ 11
1.6. Flavonoids in Pistacia ...................................................................................... 11

V
1.7. Hypothesis and objectives ................................................................................. 13
2. MATERIAL AND METHODS ............................................................................ 14
2.1. Plant Material .................................................................................................. 14
2.2. Chlorophyll and carotenoids extraction and analysis ........................................ 14
2.3. Anthocyanin extraction ..................................................................................... 15
2.4. HPLC analysis of leaf and stem extract and anthocyanin separation ................ 15
2.4.1. Identification and quantification of the peaks ............................................. 15
2.5. MS analysis ...................................................................................................... 16
2.6. NMR analysis ................................................................................................... 17
2.7. Statistical analysis ............................................................................................ 17
3. RESULTS .............................................................................................................. 18
3.1. Red color phenomenon in Pistacia .................................................................... 18
3.2. Flavonoids pattern in Pistacia species .............................................................. 20
3.3. Identification and quantification of characteristic peaks ................................... 20
3.3.1. HPLC identification ................................................................................... 20
3.3.2. MS identification........................................................................................ 21
3.3.3. NMR identification .................................................................................... 21
3.4. Flavonoids content and pattern in leaves of various Pistacia species ................ 21
3.4.1. P. chinensis ................................................................................................ 21
3.4.1.1. Spring .................................................................................................. 21
3.4.1.2. Autumn................................................................................................ 24
3.4.2. P. palaestina .............................................................................................. 24

VI
3.4.2.1. Spring .................................................................................................. 24
3.4.2.2. Autumn................................................................................................ 25
3.4.3. P. khinjuk ................................................................................................... 26
3.4.3.1. Spring .................................................................................................. 26
3.4.3.2. Autumn................................................................................................ 28
3.4.4. P. atlantica ................................................................................................. 29
3.4.4.1. Spring .................................................................................................. 29
3.4.4.2. Autumn................................................................................................ 30
3.4.5. P.vera ........................................................................................................ 31
3.4.5.1. Spring .................................................................................................. 31
3.4.6. P. lentiscus ................................................................................................. 32
3.4.6.1. Cyprus ecotype .................................................................................... 32
3.4.6.1.1. Winter ........................................................................................... 32
3.4.6.1.2. Spring............................................................................................ 34
3.4.6.1.3. Autumn ......................................................................................... 34
3.4.6.2. Tunisian ecotype .................................................................................. 35
3.4.6.2.1. Winter ........................................................................................... 35
3.5. Flavonoids content in Pistacia stems ................................................................ 35
3.5.1. P. palaestina .............................................................................................. 35
3.5.2. P.vera ........................................................................................................ 36
3.5.3. P. chinensis ................................................................................................ 37
3.6. Chlorophyll and carotenoids content in Pistacia ............................................... 37
3.6.1. P. chinensis, P. palaestina and P. khinjuk ................................................... 37
3.6.1.1. Chlorophyll a/b ratio: ........................................................................... 39

VII
3.6.1.2. Spring and autumn total Chlorophyll ratios: ......................................... 39
3.6.1.3. Spring and autumn carotenoid contents ratios: ..................................... 40
3.6.2. P. atlantica ................................................................................................. 40
3.6.2.1. Chlorophyll a/b ratio: ........................................................................... 40
3.6.2.2. Spring and autumn total Chlorophyll ratios: ......................................... 41
3.6.2.3. Spring and autumn carotenoid contents ratios: ..................................... 41
3.6.3. P. vera ....................................................................................................... 41
3.6.3.1. Chlorophyll a/b ratio: ........................................................................... 41
3.6.3.2. Spring and autumn total Chlorophyll ratios: ......................................... 41
3.6.3.3. Spring and autumn carotenoid contents ratios: ..................................... 42
3.6.4. P. lentiscus ................................................................................................. 42
3.6.4.1. Cyprus ecotype .................................................................................... 42
3.6.4.1.1 Chlorophyll a/b ratio: ..................................................................... 42
3.6.4.1.2. Spring and autumn carotenoid contents ratios: ............................... 42
3.6.4.2. Tunisian ecotype .................................................................................. 42
3.6.4.2.1. Chlorophyll a/b ratio: ................................................................... 43
3.6.4.2.2. Spring and autumn total Chlorophyll ratios: ................................... 43
3.6.4.2.3. Spring and autumn carotenoid contents ratios: ............................... 43
3.6.5. Stems ......................................................................................................... 43
4. DISCUSSION ........................................................................................................ 45
4.1. Pistacia red coloration ..................................................................................... 46
4.1.1. Visual observation ...................................................................................... 46
4.2. Identification and quantification of characteristic peaks ................................... 48
4.3. Anthocyanin content variations ......................................................................... 49

VIII
4.3.1. Green vs. red comparison ........................................................................... 49
4.3.2. Spring vs. autumn comparison.................................................................... 51
4.4. Evolutionary relationships between Pistacia species ......................................... 52
4.5. Anthocyanin content in stems ............................................................................ 53
5. CONCLUSION ..................................................................................................... 54
6. REFERENCES...................................................................................................... 55
7. APPENDIX............................................................................................................ 64
7.1. Figures ............................................................................................................. 64
7.2. Tables ............................................................................................................... 69

List of Figures
IX
Fig. 1. Anthocyanidin chemical structure. Substitutions in R1-7 are hydroxyl, methoxyl,
carbohydrate ................................................................................................................. 3
Fig. 2. P. palaestina leaves in early spring (A); Newly developing male inflorescence
(circled) (B) ................................................................................................................ 18
Fig. 3. Pistacia chinensis leaves at senescence, in autumn (A); Leaf protection
(masking) by other leaves (circled) (B); Newly growing P. palaestina branch and leaves
(C); Red and turquoise fruits of Pistacia chinensis (D); P. chinensis leaves in autumn
(E); Yellow senescent leaves of P. chinensis (F) ......................................................... 19
Fig. 4. A schematic pattern of a representative HPLC chromatogram of Pistacia
flavonoids-enriched extracts showing the seven major characteristic peaks ................. 20
Fig. 5. Comparison of P. chinensis (A) and P. palaestina (B) HPLC chromatograms of
green and red leaf extracts at various seasons .............................................................. 22
Fig. 6. Comparison of P. khinjuk (A) and P. atlantica (B) HPLC chromatograms of
green and red leaf extracts at various seasons .............................................................. 27
Fig. 7. Comparison of P. vera HPLC chromatograms of flavonoids-enriched extract of
red and green leaves .................................................................................................... 31
Fig. 8. Comparison of P. lentiscus (Cyprus ecotype) HPLC chromatograms ............... 33
Fig. 9. Comparison of spring red stems HPLC chromatograms ................................... 36
Fig. 10. The "red cycle". A schematic model of reddening of young and senescent
leaves for protection of resettling/dedifferentiation of sink and source, respectively (in
some deciduous trees) ................................................................................................. 47

List of Tables
X
Table 1. Literature review of flavonoids identified in Pistacia .................................... 12
Table 2. Slope of the calibration curves of standards and their R-squared values ........ 16
Table 3. Flavonoids content and retention times in green and red leaves of P. chinensis.
Flavonoid content is expressed as mg/g fresh weight. The results are expressed as
mean±SD .................................................................................................................... 23
Table 4. Flavonoids content and retention times in green and red leaves of P.
palaestina. Flavonoid content is expressed as mg/g fresh weight. The results are
expressed as mean±SD ................................................................................................ 25
Table 5. Flavonoids content and retention times in green and red leaves of P. khinjuk.
Flavonoid content is expressed as mg/g fresh weight. The results are expressed as
mean±SD .................................................................................................................... 28
Table 6. Flavonoids content and retention times in green and red leaves of P. atlantica.
Flavonoid content is expressed as mg/g fresh weight. The results are expressed as
mean±SD .................................................................................................................... 30
Table 7. Flavonoids content and retention times in green and red leaves of P. vera.
Flavonoid content is expressed as mg/g fresh weight. The results are expressed as
mean±SD .................................................................................................................... 32
Table 8. Flavonoids content and retention times in green and red leaves of P. lentiscus.
Flavonoid content is expressed as mg/g fresh weight. The results are expressed as
mean±SD .................................................................................................................... 34
Table 9. Flavonoids content and retention times in red stems of P. palaestina, P. vera
and P. chinensis. Flavonoid content is expressed as mg/g fresh weight. The results are
expressed as mean±SD ................................................................................................ 37

Table 10. Chlorophyll and carotenoid contents in green and red Pistacia leaves and
stems. Chlorophyll and carotenoid contents are expressed as µg/g fresh weight. The
XI
results are expressed as mean±SD ............................................................................... 38
Table 11. Green vs. red comparison of Pistacia leaves flavonoids content harvested in
spring and in autumn ................................................................................................... 49
Table 12. Comparison of quercetin-3-O-glucoside leaf content between Pistacia species
................................................................................................................................... 53

List of Appendix Figures
XII
Fig. 1. Standard calibration curve of cyanidin-3-O-glucoside based on HPLC
chromatograms ........................................................................................................... 64
Fig. 2. Standard calibration curve of quercetin-3-O-glucoside based on HPLC
chromatograms ........................................................................................................... 64
Fig. 3. Standard calibration curve of cyanidin based on HPLC chromatograms ........... 65
Fig. 4. Standard calibration curve of quercetin based on HPLC chromatograms .......... 65
Fig. 5. MS spectrum of peak #2 .................................................................................. 66
Fig. 6. MS spectrum of peak #4 .................................................................................. 66
Fig. 7. MS spectrum of peak #6. (A) RT: 7.01-7.07, (B) RT: 7.33-7.42 ....................... 67
Fig. 8. NMR spectrum of peak #4 ............................................................................... 68
Fig. 9. NMR spectrum of peak #6 ............................................................................... 68

List of Appendix Tables
XIII
Table 1a. Identified characteristic flavonoids peaks in chromatograms of spring
harvested green Pistacia leaves ................................................................................... 69
Table 1b. Identified characteristic flavonoids peaks in chromatograms of spring
harvested red Pistacia leaves ...................................................................................... 69
Table 1c. Identified characteristic flavonoids peaks in chromatograms of autumn
harvested green Pistacia leaves ................................................................................... 69
Table 1d. Identified characteristic flavonoids peaks in chromatograms of autumn
harvested red Pistacia leaves ...................................................................................... 70
Table 1e. Identified characteristic flavonoids peaks in chromatograms of winter
harvested P. lentiscus leaves ....................................................................................... 70
Table 1f. Identified characteristic flavonoids peaks in chromatograms of spring
harvested red Pistacia stems ....................................................................................... 70
Table 2. Ratios of the combined relative areas of the characteristic flavonoids peaks to
the total peaks areas in chromatograms of green and red Pistacia leaves and stems.
Leaves were harvested in winter, spring and autumn. Stems were harvested in spring.
The results are the means of three independent experiments ±SD ................................ 71
Table 3. Retention time, MS fragmentation pattern and NMR chemical shifts of
identified peaks ........................................................................................................... 72

Abbreviations
C - Carbon
R - Residue, functional group
UV - ultraviolet
ROS - reactive oxygen species
CO2 - carbon dioxide
PS - photosystem
nm - nanometer
µm - micrometer
DNA - Deoxyribonucleic acid
RAPD - Random Amplification of Polymorphic
DNA
AFLP - Amplified Fragment Length
Polymorphism
o C - centigrade
mg - milligram
chl. - chlorophyll
carot. - carotenoid
OD - Optical Density
mL - milliliter
HCl - hydrochloric acid
HPLC - High-performance liquid
chromatography
RP - reverse phase
H2O - water
HCOOH - formic acid
CH3CN - acetonitrile
min. - minute
µL - microliter
gFW - gram fresh weight
MS - Mass Spectrometry
XIV
µm - micrometer
mm - millimeter
1H NMR - proton Nuclear Magnetic Resonance
MHz - Megahertz
SD - standard deviation
Fig. - Figure
P - P value
Spp. - species
RT - retention time
MW - molecular weight
δ - delta

1. Introduction
1.1. Red coloration of tree leaves
1
There are very few trees possessing constantly red leaves, with redness covering either
the whole leaf surface or appearing in patches, as in some tropical understory trees.
Transient redness, however, is much more common. Broadly we can distinguish
between developmentally and environmentally determined appearances of redness. In
the first case, young leaves of some trees are red but gradually turn to green upon
maturation (Karageorgou and Manetas, 2006); or, mature green leaves can become red
during senescence (Wheldale, 1916; Sanger, 1971; Chang et al., 1989; King, 1997;
Kozlowski and Pallardy, 1997). On the other hand, red color accumulation can be
induced in mature green leaves by various biotic or abiotic agents like wounding (Bopp,
1959; Costa-Arbulu et al., 2001; Stone et al., 2001), pathogen attack (Hipskind et al.,
1996), nutrient deficiency (Atkinson, 1973; Hodges and Nozzolillo, 1996; Kumar and
Sharma, 1999), UV-B radiation (Lindoo and Caldwell, 1978; Mendez et al., 1999) and
high light in combination with low temperatures (Christie et al., 1994; Close et al.,
2001; Krol et al., 1995; Hughes et al., 2005).
Autumn colors are due mainly to carotenoids (yellow-orange) and anthocyanins (red-
purple). In some species, such as Caryophyllaceae, red color is also due to betalains
(Clement and Mabry, 1996). Although carotenoids are present all year round in the
leaves, they are masked in mature leaves by the green color of chlorophyll. In autumn
they become visible because of the breakdown of chlorophyll into colorless metabolites,
but there is no evidence for a de novo synthesis of carotenoids (Tanaka et al., 2008).
Anthocyanins, by contrast, are newly generated in autumn, shortly before leaf fall
(Matile et al., 1992; King, 1997; Kozlowski and Pallardy, 1997; Matile, 2000). Thus,
red is produced actively in autumn and is not simply a side effect of leaf senescence. As

2
mentioned above anthocyanins (red color) are also produced in a variety of cases that
are not associated with senescence, for example in winter evergreens and in spring
young leaves.
More than a century the phenomenon of the red color remains mystery despite much
research about the biochemistry and physiology of this color change. Several
hypotheses have been suggested to explain possible role of red color. Among the roles
of red color suggested by scientists are protective functions against excess light
(sunscreen function), UV radiation, reactive oxygen species (antioxidant function),
water stress (osmoregulation), nutrient deficiency, pathogen attack, wounding,
herbivory etc. In spite of numerous functions, the general physiological and ecological
significance for the presence of anthocyanins (red color) in vegetative organs is still
very obscure.
1.2. Anthocyanins
Anthocyanins are a group of water-soluble flavonoids (glycosides of phenolic aglycons
with a flavan C6-C3-C6 skeleton). Anthocyanins are produced in the cytoplasm and
then transported into the vacuole (Harborne, 1988; Marrs et al., 1995; Shirley, 1996).
Chemically, they are glycosides of polyhydroxyl and polymethoxyl derivatives of 2-
phenylbenzopyrylium or flavylium salts. Individual anthocyanins differ in the number
of hydroxyl groups; the degree of methylation; the nature, number and location of
sugars attached to the molecule (Cao et al., 2001) (Fig.1).
Anthocyanin molecule consists of two parts: aglycon and glycosylated moiety, which in
turn can be acylated. The number of known anthocyanin aglycon structures is few. Only
18 have been reported (Harborne and Williams, 2001) while six of them (pelargonidin,
delphinidin, cyanidin, peonidin, petunidin, malvidin) have a wide distribution. Yet,
glycosylation at various positions with various sugars and acylation with various

Fig. 1. Anthocyanidin chemical structure. Substitutions in R1-7 are hydroxyl, methoxyl,
carbohydrate
3
phenolics result in a plethora of different anthocyanin structures. The major anthocyanin
found in leaves is the red cyanidin-3-glycoside (Harborne, 1976). Hence in seeking an
answer for the presence of anthocyanins we should not only ask "why leaves are
sometimes red" (Gould et al., 1995) but "why anthocyanic leaves are almost always
red" as well (Manetas, 2006).
1.2.1. Anthocyanin function
Red coloration of the leaves is due mainly to anthocyanins, which are synthesized de
novo during early vegetation in spring and leaf senescence in autumn (Feild et al.,
2001). What is the purpose of synthesis of a red pigment in leaves that soon will be shed
in autumn or turn to green upon maturation in spring? The explanation for the function
of red pigments (exclusively anthocyanins) in the leaves at early vegetation and
senescence could be plant interaction with biotic and abiotic factors (Archetti et al.,
2009). It is worthwhile to mention that all hypotheses suggested are defense based
mechanisms. Several possible functions have been suggested for the presence of
anthocyanins in vegetative parts of the plants, such as leaves and stems. The possible
abiotic functions are photoprotection (against excess high light and UV radiation),
protection against nutrient deficiency, reactive oxygen species (ROS), water stress etc.

4
(Lee, 2002a; Archetti, 2009). Many additional possible functions have been proposed
that rely on an interaction between plants and animals (biotic factors) (Archetti, 2009):
coevolution, fruit flag, direct defense, camouflage, anticamouflage and tritrophic
mutualism.
1.2.1.1. Anthocyanins as antioxidants
Anthocyanins have been shown to function as in vivo antioxidants, effectively
neutralizing various ROS (Lee and Gould, 2002; Nagata et al., 2003; Kytridis and
Manetas, 2006). Hydrogen peroxide is the most probable target for neutralization by
anthocyanins, because it is the only ROS known to be able to penetrate both
chloroplasts, where ROS are produced, and vacuole, where anthocyanins are stored
(Ougham et al., 2005). Experiments using leaf extracts have, however, yielded
conflicting results. Neil et al. (2002a) reported that leaf extracts from red varieties of
Elatostema rugosum had higher antioxidant capacity compared to green morphs and
anthocyanins contributed to that capacity more than the other low molecular weight
antioxidants. This, however, was not the case in Quintinia serrata, where both red and
green leaf extracts displayed the same antioxidative capacities (Neil et al., 2002b).
A critical point concerning antioxidative function of anthocyanins is their cellular
localization. One could argue that effective in vivo antioxidants should reside as close as
possible to the source of oxy-radical production. However, in leaves, the illuminated
chloroplasts of the mesophyll cells are the main source of ROS (Asada, 2000), while
anthocyanins are located in the upper and/or lower epidermis (Lee and Collins, 2001).
Moreover, the intracellular location of anthocyanins is the vacuole, although colorless
tautomers appear in the cytoplasm during the transit time between their biosynthesis and
transport to the vacuole (Hrazdina et al., 1978). As much as is known, there is no report
for the presence of anthocyanins in chloroplasts (Manetas, 2006). Flavonoids and

5
simple phenolics are also potential oxy-radical scavengers. Although slightly less
effective than anthocyanins (Bors et al., 1994), their concentration in leaves is at least
an order of magnitude higher than that of anthocyanins. (Grace et al., 1998; Jaakola et
al., 2004; Woodall and Stewart, 1998). Flavonoids reside in the central vacuole (Hutzler
et al., 1998), yet their presence in chloroplasts has also been documented (Saunders and
McClure, 1976). In addition, anthocyanins comprise the final steps of a biosynthetic
route yielding also flavonoids and simple phenolics (Saito and Yamasaki, 2002). Hence,
high levels of all these constituents may co-occur as a result of activation of the
pathway at an early stage (Close et al., 2001; Dominy and Lucas, 2004; Jaakola et al.,
2004; Karageorgou and Manetas, 2006; Lee and Lowry, 1980). Activation of the
phenylpropanoid pathway and thus increased synthesis of phenolics and flavonoids is a
common response to environmental stress (Dixon and Paiva, 1995). Based on plant
economics the "Optimal Defense theory" (Feeny, 1976; McKey, 1979; Rhoades and
Gates, 1976) and the "Growth-Differentiation Balance hypothesis" (Herms and Mattson,
1992) suggest that there is a trade-off between growth and defense of the plant.
Accordingly, phenylpropanoids (including anthocyanins and phenolics), needed for
plant defense, and proteins, needed for plant growth, compete for a common precursor,
phenylalanine. Thus, when growth is fast (available resources), the level of phenolics or
anthocyanins will be low. In contrast, under stressful conditions, such as, high light
radiation, low temperature, water-nutrient deficiency, growth is slowed, and defense
costs are high, as indicated by higher level of phenolics and anthocyanin.
1.2.1.2. Photoprotection
The idea behind the leaf photoprotection hypothesis dates back to the late 19 th century
(Kerner von Marilaum, 1897). This theory however was revived and proposed in its
present form only recently (Gould et al., 1995; Hoch et al., 2001; Feiled et al., 2001).

6
According to the revived hypothesis, anthocyanins function to relieve photo-oxidative
stress. Photoprotective role can be fulfilled in two ways: either by simply screening
visible radiation or/and by quenching oxy-radicals through the powerful antioxidant
capacity of anthocyanins (Wang et al., 1997). The above arguments are also valid for
young and senescing leaves. In young leaves, the ability to absorb photons and evolve
oxygen is developed before the full sufficiency of the CO2 reduction system (Ireland et
al., 1985; Miranda et al., 1981). This may explain their vulnerability to photoinhibition,
in spite of the higher pools in the xanthophyll cycle pigments (Barker et al., 1997;
Krause et al., 1995). Senescence is a programmed developmental stage. In trees, during
the senescence leaf metabolism is channeled towards the need of nutrient resorption
(mainly nitrogen) to persistent storage compartments like twigs and stems. It facilitates
rapid remobilization and used during the next growth season (Millard and Thomson,
1989). The risk of photo-oxidative damage is especially high in autumn, because: (1)
low temperatures reduce carbon fixation capacity, (2) increased light intensity owing to
a thinned canopy, affecting shade-adapted, understory trees, and (3) decreased self-
shading by chlorophyll as its breakdown occurs (Ougham et al., 2008). In plants, ROS
and photo-oxidative damage occurs under high light intensities when the rate of photons
absorption, and the concomitant photosynthetic electron flow, exceeds the capacity of
CO2 assimilation. Under these conditions, over-reduction of the electron transport chain
cause inactivation of PSII and the inhibition of photosynthesis. As a result, oxygen is
reduced by PSI to generate superoxide and hydrogen peroxide (Apel and Hirt 2004;
Mittler, 2002). The ability of anthocyanins to absorb light has been demonstrated
(Sarma and Sharma, 1999). The question is whether this contributes to photoprotection?
Recent studies support for a photoprotective function of anthocyanins in senescing
leaves (Feild et al., 2001), young leaves (Manetas et al., 2002) and evergreens (Hughes
et al., 2005; Hughes et al., 2007). Hughes et al.(2005) determined the maximal

7
photosystem II (PSII) efficiency of red and green leaves of the evergreen herb Galax
urceolata that were irradiated with wavelengths that are poorly or strongly absorbed by
anthocyanins. Their results suggest that anthocyanins function as light attenuators and
red leaves are less light stressed than non-red leaves. In contrast, other studies, failed to
support a photoprotective effect of anthocyanins, neither in senescing leaves (Lee et al.,
2003), young leaves (Manetas et al., 2003; Karageorgou and Manetas, 2006) nor mature
leaves (Kyparissis et al., 2007; Burger and Edwards, 1996; Hormaetxe et al., 2005;
Esteban et al., 2008). Ideally, the absorption spectrum of a photoprotector should match
that of its photosensitizer. However, leaf anthocyanins being almost always red
(Harborne, 1976) absorb maximally at 520-540 nm, i.e. at the green spectral region
where the probability of photon capture by chlorophylls is minimal. Although
anthocyanins absorb in the blue, the absorption coefficient is roughly 3-fold lower
compared to their green maximum. Taken together, it seems that the optical properties
of anthocyanins may not be designed for photoprotection. Green light seems to be
photosynthetically important deep within the leaf, yet deeply located chloroplasts are
self-shaded by overlying chlorophyll. For example, the contribution of red and blue
light to CO2 fixation in a relatively thick (750 µm) spinach leaf is greater than that of
green light at the first 150 µm of depth (Sun et al., 1998). This situation, however, is
reversed after ca. 250 µm and up to the lower epidermis (Sun et al., 1998).
Finally, anthocyanins are localized in the vacuoles (Harborne, 1988; Marrs et al., 1995;
Shirley, 1996), and are thus spatially separated from the chloroplasts and their
photosynthetic centers, the main sites of ROS production. This means that there is very
low chance of anthocyanin and ROS to interact. These findings do not support a
photoprotection function of anthocyanins.

1.2.1.3. UV protection
8
Anthocyanins generally have two absorption maxima, one in the UV (between 270-290
nm) and the other in the visible spectrum (500-550 nm) (Markham, 1982). Acylation of
anthocyanins with aromatic organic acids increases their UV absorbance by addition of
another absorption maximum in the 310-320 nm. The strong UV absorption of
anthocyanins has led to the hypothesis that they may protect leaves from the harmful
effects of UV radiation (Lee and Lowry, 1980; Coley and Kursar, 1996). Several studies
have supported this hypothesis: a positive correlation exists between leaf anthocyanin
content and UV-B radiation (Alexieva et al., 2001; Brandt et al., 1995; Lindoo and
Caldwell, 1978). Stapleton and Walbot (1994) demonstrated that the DNA in Zea mays
plants that contain flavonoids (primarily anthocyanins) was protected from damage
caused by UV radiation relative to the DNA in plants that were genetically deficient in
these compounds. It has also been reported that red Coleus varieties are less damaged
by both UV-B and UV-C radiation, when compared to green varieties (Burger and
Edwards, 1996). Therefore, anthocyanins have been implicated in UV-B protection (Lee
and Lowry, 1980). Moreover, some anthocyanin biosynthesis genes, i.e. chalcone
synthase (CHS) and xavanone 3-hydroxylase (F3H) are induced only upon UV-A
exposure (Zhou et al., 2007). However, a closer look at the anthocyanin spectral
absorbing properties and their contribution to total leaf UV-B absorbing capacity
renders their putative protection against UV-B radiation questionable. Molar absorption
coefficients for anthocyanins in the UV-B (280-320 nm) are ca. 3-5 times lower as
compared to corresponding values at their visible maxima (Giusti et al., 1999;
Harborne, 1976). Glycosylation per se does not appreciably alter the spectral
absorbance profile. However, acylation of anthocyanins by phenolic acids increases
UV-B absorptivity due to superimposed absorbance of the phenolic ring, resulting in
roughly equivalent absorption in both the visible and UV-B bands (Giusti et al., 1999;

9
Harborne, 1976). Yet, although molar absorptivities of acylated anthocyanins and the
rest of simpler phenolics and flavonoids in the UV-B band are comparable, the
concentrations of anthocyanins are always a small fraction (ca. 1-1.5 %) of the total
phenolic pool (Grace et al., 1998; Jaakola et al., 2004; Woodall and Stewart, 1998).
Accordingly, the contribution of anthocyanins to the total leaf UV-B absorbing capacity
is negligible (Woodall and Stewart, 1998).
1.2.1.4. Protection against herbivory
According to the “Coevolution Hypothesis“(Archetti, 2009), red coloration is a signal to
insects that the tree is not a suitable host for insects. Insect herbivores use leaves either
for direct consumption or for future use by their offspring (oviposition). Most folivorous
insects seem to possess three types of light receptor with sensitivity maxima at 350, 440
and 540 nm (Kelber, 2001; Kelber et al., 2003). These light receptors however, exhibit
decreasing sensitivities at the two margins, up to 300 and 620 nm, respectively (Kelber
et al., 2003). Accordingly, most insects can see UV-A, blue, green and may not see
human red. Experiments done suggest that red leaves are less suffering by insects, than
green leaves (Manetas, 2006; Karageorgou and Manetas, 2006). Recently, a new
hypothesis was suggested by Lev-Yadun et al (2004). According to him, a red color
could undermine the insect camouflage. Most folivorous insects are greenish and, being
on a green leaf, they probably escape predator attention. On a red leaf, however, they
become more conspicuous and thus an easy target to predators.
1.2.1.5. Other functions
Osmoregulation: It was suggested that anthocyanins could function as osmoregulators
to help decrease leaf osmotic potential, thereby contributing to drought tolerance during
senescence (Chalker-Scott, 2002). However, there is no strong evidence for a such

00
osmoregulatory function and it seems unlikely as anthocyanins contribute

1.5. Leaf senescence
11
Leaf senescence is a coordinated process in which deciduous plants get ready for winter
and recycle some of their valuable mineral nutrients (Killingbeck, 1996; Buchanan-
Wollaston, 1997; Thomas, 1997; Matile et al., 1999; Matile, 2000; Quirino et al., 2000).
The highly coordinated processes of leaf senescence (Smart, 1994; Quirino et al., 2000)
and chlorophyll degradation (Matile et al., 1999; Thomas, 2001) appear to increase the
resorption of nutrients, particularly nitrogen, back into woody plant tissues (Peri et al.
1999; Lee, 2002b). Hoch et al. (2001) argue that in plants anthocyanin accumulation
during leaf senescence functions to protect the photosynthetic machinery from
photoinhibition. A similar role of anthocyanin in photoprotection of chloroplasts during
leaf senescence of red-osier dogwood (Cornus stolonifera) was suggested by Feild et
al., 2001. In their study the authors provide evidence that the anthocyanins protect
chlorophyll by optical masking and thus reduce the danger of photooxidation to
senescing leaf cells. This photoprotection by anthocyanins would facilitate a more
efficient removal of nitrogen from mesophyll cells (where anthocyanins accumulate)
back into woody stems (Lee, 2002b)
1.6. Flavonoids in Pistacia
Currently, more than 20 flavonoids have been identified in Pistacia (leaves, fruits).
These flavonoids belong to anthocyanins, anthocyanidins, flavonols, isoflavonols,
which are represented in both aglycon and glycosilated forms (Table 1).

12
Table 1. Literature review of flavonoids identified in Pistacia
Flavonoid name Mol. formula Molar mass,
g/mol
Species Reference
Apigenin C15H10O5 270.24 P. terebinthus Topcu et al.,
2007
Cyanidin 3-Oarabinoside
C20H19O10 + 419.36 P. lentiscus Longo et al.,
2007
Cyanidin-3-Ogalactoside
C21H21O11 + 449.39 P. vera Seeram et al.,
2006
Cyanidin 3-O-glucoside C21H21O11 + 449.39 P. lentiscus Romani et al.,
2002
Daidzein C15H10O4 254.23 P. vera Ballistreri et al.,
2009
Daidzein-7-O-glucoside C21H20O9 416.38 P. vera Ballistreri et al.,
2009
Delphinidin 3-Oglucoside
C21H21O12 + 465.38 P. lentiscus Romani et al.,
2002
Dihydroquercetin
3,4-dihydro-4-(4'hydroxyphenyl)-7hydroxycoumarin
C15H12O7
C30H22O8
304.25
510.50
P. chinensis
P. chinensis
Keys et al., 1976
Nishimura et al.,
2000
Distylin
Eriodictyol
C15H12O7
C15H12O6
304.25
288.25
P. chinensis
P. vera
Keys et al., 1976
Ballistreri et al.,
2009
Fisetin
Genistein
Genistein-7-O-glucoside
C15H10O6
C15H10O5
C21H20O10
286.24
270.24
432.37
P. chinensis
P. lentiscus
P. vera
Keys et al., 1976
Vaya et al., 2006
Ballistreri et al.,
2009
6'-hydroxyhypolaetin-3'methyl
ether
C16H12O8 332.25 P. terebinthus Topcu et al.,
2007
Isoscutellarein 8-Oglucoside
C21H20O11 448.38 P. terebinthus Topcu et al.,
2007
Kaempferol-3-Oglucoside
C21H20O11 448.37 P. atlantica
P. chinensis
P. lentiscus
Kawashty et al.,
2000
Luteolin
Luteolin-7-O-glucoside
C15H10O6
C21H20O11
286.24
448.38
P. lentiscus
P. terebinthus
Vaya et al., 2006
Topcu et al.,
2007
Myricetin-3-Ogalactoside
C21H20O13 480.38 P. khinjuk Kawashty et al.,
2000
Myricetin-3-O-glucoside C21H20O13 480.38 P. khinjuk
P. lentiscus
Kawashty et al.,
2000
Myricetin-3-Orutinoside
C27H30O17 626.52 P. khinjuk Kawashty et al.,
2000
Naringenin
Quercetin
C15H12O5
C15H10O7
272.26
302.24
P. chinensis
P. terebinthus
Keys et al., 1976
Topcu et al.,
2007
Quercetin-3-galactoside C21H20O12 464.37 P. atlantica Kawashty et al.,
2000
Quercetin-3-O-glucoside C21H20O12 464.37 P. atlantica
P. chinensis
P. khinjuk
P. lentiscus
Kawashty et al.,
2000
Quercetin-3-Oglucoside-7-Ogalactoside
C27H30O17 626.52 P. atlantica Kawashty et al.,
2000
Quercetagetin-3-methyl
ether 7-O-glucoside
C22H22O13 494.40 P. terebinthus Topcu et al.,
2007
Quercetin-3-Orutinoside
C27H30O16 610.52 P. atlantica
P. khinjuk
P. lentiscus
Kawashty et al.,
2000

1.7. Hypothesis and objectives
Hypothesis:
13
The underlying hypothesis of this research work is that red coloration of Pistacia leaf,
stem and fruit represent developmental and biosynthetic changes associated with new
growth in spring and reprogramming of cellular activities during senescence in autumn.
It is hypothesized that the red color is derived mostly from anthocyanidins and their
derivatives.
Specifically, anthocyanin content in the leaves of Pistacia varies depending on leaf
developmental stage (spring, autumn): it is high in early growth and at senescence.
Objective:
To characterize and quantitate the various anthocyanidin derivatives in the leaves of six
Pistacia species during spring and autumn.

2. Material and Methods
2.1. Plant Material
14
Six Pistacia species were used as the experimental system: P. chinensis, P. palaestina,
P. khinjuk, P. atlantica, P. vera and P. lentiscus. All the studied species were grown at
BIDR Pistacia germplasm collection plot (http://www.bgu.ac.il/pistacia), where they
grow under practically identical environmental and soil conditions (Golan-Goldhirsh
and Kostiukovsky, 1998),
Red and green leaves were collected during autumn and spring and were stored at -
80 o C. Red stems were collected during spring and stored at -80 o C.
It should be noted that leaves of P. lentiscus (Cyprus ecotype, green only) were
harvested in autumn, winter and spring while those of the Tunisian ecotype (red only)
were harvested only in winter. Leaves of P. vera were harvested only in spring.
2.2. Chlorophyll and carotenoids extraction and analysis
The contents of chlorophylls (a and b) and of carotenoids were determined
spectrophotometrically. Leaves and stems of Pistacia (ca. 20 mg) were incubated in
100% methanol (1.5 mL) overnight, the methanol fraction was collected and analysed
spectrophotometrically. The pigment content (chlorophyll and carotenoid) was
calculated according to the following formula (Lichtenthaler, 1987):
Chla = 16.72 (OD665-OD750)-9.16 (OD652-OD750)
Chlb = 34.09 (OD652-OD750)-15.28 (OD665-OD750)
Carot. = {1000 (OD470-OD750)-1.63 Chla- 104.96 Chlb}/221

2.3. Anthocyanin extraction
15
Leaf and stem samples (200 mg fresh weight) of the selected Pistacia species were
ground in a mortar with a pestle in the presence of liquid nitrogen. Extraction solvent
was acidic methanol (Vanderauwera et al., 2005), 1.5 mL solution of 1% HCl in
methanol, and 1 mL of distilled water. Two mL of the extract were taken for further
analysis and centrifuged 2 times (13,000 rpm, 5 min, and room temperature) and 1.4 mL
supernatant was withdrawn. Chlorophyll was separated from the anthocyanins by back
extraction with 0.5 mL chloroform (Vanderauwera et al., 2005).
2.4. HPLC analysis of leaf and stem extract and anthocyanin separation
Anthocyanin separation was done using High Performance Liquid Chromatography
(HPLC, Merck & Hitachi Model D-7000) equipped with a UV Detector (L-4000A) and
the system was controlled by D7000 HSM Software. The samples were separated on a
reverse phase column, LiChrospher 100 RP-18 (5µm) (250x4mm). The binary mobile
phase consisted of solvents A, 90% H2O, 10% HCOOH (v/v) and B, 90% CH3CN, 10%
HCOOH (v/v). Typical running time was 45 min. The gradient was 0-2 min, 10% B; 2-
7 min, 10-20% B; 7-12 min, 20-30% B; 12-32 min, 30-70% B; 32-35 min, 70-100% B;
35-40 min, 100% B. Solvent flow rate was 1 mL/min, and the sample injection volume
was 10 µL. Detection was done at 325 nm (Jaiswal et al., 2010).
2.4.1. Identification and quantification of the peaks
To identify characteristic peaks, candidate flavonoid standards were purchased from
Sigma Aldrich (HPLC grade, >95% purity). The following standards were tested:
cyanidin chloride (Catalog #79457-1MG-F), cyanidin-3-O-glucoside chloride (Catalog
#52976-1MG-F), quercetin dihidrate (Catalog #Q0125). Quercetin-3-O-glucoside was
bought from Indofine Chem. Comp. (Hillsborough, NJ, USA, Catalog #020074). Stock

16
solutions (2 mg/mL) were made by dissolving the each standard in 0.5% HCl in
methanol (v/v) (cyanidin stock solution concentration was 5 mg/mL) and various
dilutions were analysed to establish standard curves, in order to relate peak area to the
amount of the flavonoid in the plant. The standard curves are shown in the Appendix.
The slope of each curve (Table 2) was used to calculate the actual concentration of the
corresponding compound in the plant in mg/g fresh weight (mg/gFW). Unidentified
peaks were expressed as cyanidin-3-O-glucoside chloride equivalent, using its standard
curve slope as conversion factor of peak area to mg/g fresh weight.
2.5. MS analysis
Table 2. Slope of the calibration curves of standards
and their R-squared values
Slope,
Compound
peak
area/µg
R 2
Cyanidin chloride 108673 0.9995
Cyanidin-3-O-glucoside chloride 144673 0.9996
Quercetin dihidrate 684903 0.9908
Quercetin-3-O-glucoside 578595 0.9977
Characteristic peaks of HPLC chromatograms were collected and identified by Mass
Spectroscopy (MS). All MS analyses were performed on a LCQ Fleet mass
spectrometer (Thermo Scientific) with an ESI source. Spectra were collected in positive
ion mode and analyzed by Xcalibur and Promass software (Thermo Scientific). For
LC/MS analyses, a Surveyor Plus HPLC System (Thermo Scientific) was used,
equipped with a Luna C18, 5 µm (150 × 4.6 mm) column. A flow rate of 0.5 mL/min,
using a mobile phase linear gradient of 0.1% aqueous formic acid (solvent A) and
CH3CN containing 0.1% formic acid (solvent B) was used.

2.6. NMR analysis
17
NMR spectra of purified compounds were determined using a Bruker Avance DPX200
(200 MHz) or Bruker Avance DPX500 (500 MHz) spectrometer.
2.7. Statistical analysis
All leaf and stem data are based on extracts of three different leaves, at any given
sampling point. Data are represented as a Mean value ± standard deviation (SD).
Student t-test and one-way ANOVA analyses were done to compare data, using
Statistica software.

3. Results
3.1. Red color phenomenon in Pistacia
18
During the lifecycle of Pistacia, leaves turn into bright red twice a year, in early spring
up to mid-summer in new leaf growth and in autumn before leaf fall (Figs. 2A and 3A).
During leaf formation in early spring, not only leaves but also newly formed
inflorescences are becoming bright red (Fig. 2B, circled), later on in summer leaves turn
green, male inflorescences reach anthesis within 2 to 3 weeks and senesce. Female
flowers set fruits, turn red and towards the end of summer, upon fruit maturation, in
fertile fruits, the pericarp turns turquoise-green (Fig. 3D). A similar reddening process
occurs in autumn, just before leaf senescence, however, the color change involves a
more complex variety of colors from light yellow to deep red, and the spectrum is quite
different in the various species (Fig. 3, A-F). Moreover, newly growing branches are
red (Fig. 3B) and later turn brown-gray. Leaves that had been shaded by neighboring
leaves, and by this had been protected against UV radiation, exhibit less redness and
were mostly yellowish (Fig. 3B, circled).
A
A B
B
Fig. 2. P. palaestina leaves in early spring (A); Newly developing male inflorescence (circled)
(B)

A
C
E
A
19
C D D
E F
F
Fig. 3. Pistacia chinensis leaves at senescence, in autumn (A); Leaf protection
(masking) by other leaves (circled) (B); Newly growing P. palaestina branch and leaves
(C); Red and turquoise fruits of Pistacia chinensis (D); P. chinensis leaves in autumn
(E); Yellow senescent leaves of P. chinensis (F)
B
B

3.2. Flavonoids pattern in Pistacia species
20
Flavonoids-enriched red and/or green leaf and stem extracts of six species of Pistacia
(that were harvested in autumn and spring) were subjected to HPLC analysis. HPLC
chromatograms of these extracts revealed up to seven major characteristic peaks, which
were annotated 1; 2; 3; 4; 5; 6 and 7, that appeared in the corresponding retention times
(minutes): 2, 7, 8, 10, 11, 13, 15 shown schematically in Fig. 4. Additionally, HPLC
analysis revealed about 30 minor peaks.
Fig. 4. A schematic pattern of a representative HPLC chromatogram of Pistacia flavonoidsenriched
extracts showing the seven major characteristic peaks
3.3. Identification and quantification of characteristic peaks
3.3.1. HPLC identification
Cyanidin, cyanidin-3-O-glucoside, quercetin and quercetin-3-O-glucoside were used to
identify characteristic peaks (Appendix, Figs. 1 to 4). Based on HPLC peaks retention
time, MS and NMR analyses the following compounds were identified: cyanidin-3-O-
glucoside corresponds to peak #2, quercetin-3-O-glucoside corresponds to peak #4,

21
cyanidin corresponds to peak #6 and quercetin to peak #7. The flavonoids content of
peaks' #2, 4, 6 and 7 (expressed as mg/gFW) was determined by using their appropriate
calibration curves (Appendix, Figs. 1 to 4). For the sake of comparison, in the case of
the unidentified peaks (#1, 3 and 5), their contents are expressed in cyanidin-3-O-
glucoside equivalents.
The flavonoids content of all the characteristic peaks, under all experimental conditions,
are given in Tables 3 to 9.
3.3.2. MS identification
Three characteristic peaks (peaks #2, 4 and 6) were purified by HPLC and subjected to
Mass Spectrometry analysis (Appendix, Figs. 5 to 8).
MS fragmentation pattern of characteristic peaks are given in Appendix, Table 3.
3.3.3. NMR identification
Three purified characteristic peaks (peaks #4 and 6) were analyzed by 1H NMR
(Appendix, Figs. 9 to 11). Table 3 in the Appendix shows specific chemical shifts (δ,
ppm) of the compounds.
3.4. Flavonoids content and pattern in leaves of various Pistacia species
3.4.1. P. chinensis
3.4.1.1. Spring
Chromatograms of flavonoid-enriched green and red leaf extracts of P. chinensis that
were harvested in spring revealed six and five major peaks, respectively (Fig. 5A,
Appendix, Tables 1a, 1b).
Green leaves: the highest flavonoid content was that of cyanidin, 12.66 ± 0.26 mg/gFW.
The flavonoid contents of peaks #1, 3 and 5 were 0.75 ± 0.15; 5.09 ± 0.43 and

A
22
A B
B
Fig. 5. Comparison of P. chinensis (A) and P. palaestina (B) HPLC chromatograms of green and red leaf extracts
at various seasons

23
4.20 ± 0.63 mg/gFW, respectively. Out of the six peaks, the content of peak #1 was the
lowest, 0.75 ± 0.15 mg/g FW, while those of peaks #3 and 5 did not differ significantly
(Table 3). Quercetin-3-O-glucoside content was 3.26 ± 0.42 mg/gFW, similar to that of
peak #5.
Table 3. Flavonoids content and retention times in green and red leaves of P. chinensis.
Flavonoid content is expressed as mg/g fresh weight. The results are expressed as
mean±SD
Peak # 1 2 3 4 5 6 7
Peak
identity
Retention
time
(min.)
Harvest
Spring
green
Spring
red
Autumn
green
Autumn
N.I.
Cyanidin-
3-Oglucoside
In spring red leaves, peaks #5 had the highest flavonoid content, 16.10 ± 1.98 mg/gFW.
The content of quercetin-3-O-glucoside did not differ statistically of that of cyanidin-3-
O-glucoside (4.84±2.22 mg/g FW) and was the lowest (Table 3). Peaks #1, cyanidin-3-
O-glucoside and 3 did not differ significantly of each other.
In the spring, as opposed to the autumn, different ratios of the combined relative areas
of the characteristic peaks (#; 1, 2, 3, 4, and 5) to the total peaks area were 0.74 ± 0.01
in green leaves and 0.52 ± 0.01 in red leaves (Appendix, Table 2).
N.I.
Quercetin-
3-Oglucoside
N.I. Cyanidin Quercetin
2.64±0.03 7.59±0.40 8.14±0.56 10.28±0.48 11.20±0.18 12.66±0.26 14.80±0.17
0.75±0.15
(a*)
6.45±0.24
(b)
5.09±0.43
(c)
3.26±0.42
(d)
4.20±0.63
(c,d)
11.20±0.61
(e)
-
6.57±0.48
(A)
4.84±2.22
(A,B)
7.73±1.05
(A)
3.60±0.17
(B)
16.10±1.98
(C)
- -
3.25±0.32
(k)
1.98±0.26
(l)
0.73±0.43
(m)
2.60±0.46
(k,l)
-
13.80±1.49
(n)
2.17±0.48
(l)
red
3.10±0.25
(K,M)
4.14±0.55
(K)
0.97±0.06
(L)
2.10±0.43
(M)
-
15.68±0.59
(N)
2.69±0.29
(M)
* For each harvest, means bearing the same letter are not significantly different according to t-test (P>0.05);
N.I. - not identified; Unidentified peaks, #1, 3 and 5 are expressed as cyanidin-3-O-glucoside equivalents;
Peaks #2, 4, 6 and 7 contents are expressed according to their own standard curves

3.4.1.2. Autumn
24
Chromatograms of flavonoid-enriched green and red leaf extracts of P. chinensis that
were harvested in autumn revealed six major peaks (Appendix, Tables 1c, 1d). In both
cases peak #5 was absent in the chromatogram (Fig. 5A).
In green leaves, the content of cyanidin was the highest, 13.80 ± 1.49 mg/gFW, while
that of peak #3 was the lowest, about 5.3 % of that of cyanidin (Table 3).
In red leaves, similar to green leaves, the content of cyanidin was the highest, 15.68 ±
0.59 mg/gFW and that of peak #3 was the lowest, 0.97 ± 0.06 mg/gFW (Table 3). It
should be noted that in green and red leaves similar ratios of the combined relative areas
of the characteristic peaks (#; 1, 2, 3, 4, 6 and 7) to the total peaks area were found 0.53
± 0.03 and 0.52 ± 0.03, respectively (Appendix, Table 2).
3.4.2. P. palaestina
HPLC analysis of flavonoid-enriched extracts of green and red leaves of P. palaestina
harvested in spring and autumn, revealed six major peaks (#; 1-6) while quercetin, that
was found in autumn leaves of P. chinensis, could not be detected (Fig. 5B, Appendix,
Tables 1, a-d).
3.4.2.1. Spring
In green leaf extracts of P. palaestina the flavonoid content of cyanidin-3-O-glucoside,
quercetin-3-O-glucoside and cyanidin did not differ significantly and were the highest,
5.61 ± 1.01, 3.86 ± 0.30, 4.62 ± 1.37, mg/gFW, respectively. Lowest, and similar,
flavonoid content were found in peaks #1 and 5, 1.39 ± 0.33 and 1.60 ± 0.51, mg/gFW,
respectively (Table 4).

25
Table 4. Flavonoids content and retention times in green and red leaves of P.
palaestina. Flavonoid content is expressed as mg/g fresh weight. The results
are expressed as mean±SD
Peak no. 1 2 3 4 5 6
Peak
identity
Retention
time
(min.)
Harvest
Spring
green
Spring
red
Autumn
green
Autumn
N.I.
Cyanidin-
3-Oglucoside
N.I.
Quercetin-
3-Oglucoside
N.I. Cyanidin
2.64±0.04 7.70±0.59 8.20±0.59 10.03±0.50 11.13±0.42 13.46±0.40
1.39±0.33 5.61±1.01 2.67±0.16 3.86±0.30 1.60±0.51 4.62±1.37
(a*) (b) (c) (b) (a,c) (b,c)
2.56±0.51 9.82±1.11 5.63±0.63 4.33±0.11 2.82±0.44 3.80±0.95
(A) (B) (C) (C) (A) (A,C)
1.95±0.45 1.87±0.32 0.93±0.28 1.51±0.21 1.06±0.17 3.56±1.11
(k) (k) (l) (k) (l)
(k)
2.23±0.48 4.69±2.06 2.66±0.53 2.21±0.40 4.06±1.47 7.68±5.30
red
(K) (K) (K) (K) (K) (K)
* For each harvest, means bearing the same letter are not significantly different according to
t-test (P>0.05); N.I. - not identified; Unidentified peaks, #1, 3 and 5 are expressed as
cyanidin-3-O-glucoside equivalents; Peaks #2, 4 and 6 contents are expressed according to
their own standard curves
In red leaves, the flavonoid content of cyanidin-3-O-glucoside was the highest, 9.82 ±
1.11 mg/gFW, while those of peaks #1 and 5 had the lowest amount, 2.56 ± 0.51 and
2.82 ± 0.44 mg/gFW, respectively (Table 4). The contents of peaks #3, quercetin-3-O-
glucoside and cyanidin did not differ significantly of each other (P>0.05) and was 5.63
± 0.63, 4.33 ± 0.11, 3.80 ± 0.95 mg/gFW, respectively.
Similar ratios of the combined relative peak areas of the characteristic peaks (#; 1, 2, 3,
4, 5 and 6) to the total peaks area were found in green and red leaves (0.77 ± 0.03 and
0.75 ± 0.02 mg/gFW, respectively) (Appendix, Table 2).
3.4.2.2. Autumn
In green leaves, the content of peaks #1, cyanidin-3-O-glucoside, quercetin-3-O-
glucoside and cyanidin did not differ significantly of each other and were the highest,
while those of peaks #3 and 5, 0.93 ± 0.28 and 1.06 ± 0.17, mg/gFW, respectively,
which were not significantly different (P>0.05) of each other, were the lowest amount
(Table 4).

26
In red leaves, the flavonoid content of all the peaks did not differ significantly of each
other. It should be noted that for some of the peaks very large standard deviations were
determined (peaks #5, cyanidin-3-O-glucoside and cyanidin) (Table 4).
Similar ratio of the combined relative peak areas of the characteristic peaks (#; 1, 2, 3,
4, 6) to the total peaks area were observed in extracts of green and red leaves, 0.69 ±
0.05 and 0.65 ± 0.04, respectively (Appendix, Table 2).
3.4.3. P. khinjuk
HPLC analysis of flavonoid-enriched extracts of green and red leaves of P. khinjuk
harvested both in spring and autumn, revealed six major peaks (#; 1-6) while quercetin,
that was found in P. chinensis, could not be detected (Fig. 6A, Appendix, Tables 1, a-d).
3.4.3.1. Spring
In green leaves the flavonoid content of cyanidin-3-O-glucoside was the highest (4.29 ±
0.36 mg/gFW), while that of peak #1 had the lowest content, 0.49 ± 0.08 mg/gFW. The
flavonoid contents of peaks #3 and quercetin-3-O-glucoside, 2.34 ± 0.28 and 2.30 ±
0.12 mg/gFW, respectively, were the second highest and did not differ significantly of
each other (P>0.05) (Table 5).
In red leaves, similarly to the green leaves, the content of cyanidin-3-O-glucoside was
the highest, 7.38 ± 0.37 mg/gFW, while that of peak #1 was the lowest, 0.85 ± 0.36
mg/gFW. Peaks #3 and 5 had the second highest flavonoid contents, 4.05 ± 0.23 and
4.00 ± 0.45, respectively and were not significantly different (P>0.05) of each other
(Table 5).

A
27
A B
B
Fig. 6. Comparison of P. khinjuk (A) and P. atlantica (B) HPLC chromatograms of green and red leaf
extracts at various seasons

28
Table 5. Flavonoids content and retention times in green and red leaves of P.
khinjuk. Flavonoid content is expressed as mg/g fresh weight. The results are
expressed as mean±SD
Peak no. 1 2 3 4 5 6
Peak
identity
Retention
time
(min.)
Harvest
Spring
green
Spring
red
Autumn
green
Autumn
N.I.
Cyanidin-
3-Oglucoside
N.I.
Quercetin-3-
O-glucoside
N.I. Cyanidin
2.65±0.01 7.97±0.19 8.52±0.19 10.33±0.12 11.39±0.06 13.42±0.04
0.49±0.08 4.29±0.36 2.34±0.28 2.30±0.12 1.74±0.06 1.14±0.03
(a*) (b) (c,d) (c)
(d)
(e)
0.85±0.36 7.38±0.37 4.05±0.23 2.84±0.09 4.00±0.45 2.28±0.15
(A) (B) (C) (D) (C) (E)
0.86±0.18 3.22±0.31 1.74±0.19 1.10±0.05 3.86±0.10 6.88±1.18
(k) (l) (m) (k)
(l)
(n)
1.29±0.14 4.84±0.35 3.34±0.44 1.13±0.03 5.40±0.46 6.97±2.46
red (K) (L) (M) (K) (L,M) (L)
* For each harvest, means bearing the same letter are not significantly different according to ttest
(P>0.05); N.I. - not identified; Unidentified peaks, #1, 3 and 5 are expressed as cyanidin-3-
O-glucoside equivalents; Peaks #2, 4 and 6 contents are expressed according to their own
standard curves
The ratios of the combined relative peak areas of the characteristic peaks (#; 1, 2, 3, 4, 5
and 6) to the total peaks area in green and red leaves extracts were 0.78 ± 0.01 and 0.68
± 0.03, respectively (Appendix, Table 2).
3.4.3.2. Autumn
In green leaves, the content of cyanidin was the highest, 6.88 ± 1.18 mg/gFW, while the
lowest contents were those of peaks #1 and quercetin-3-O-glucoside, 0.86 ± 0.18 and
1.10 ± 0.05 mg/gFW, respectively (Table 5). Flavonoid contents of cyanidin-3-O-
glucoside and peaks #5 did not differ significantly of each other (P>0.05) and had the
second highest flavonoid contents, 3.22 ± 0.31 and 3.86 ± 0.10 mg/gFW, respectively
(Table 5).
In red leaves, the flavonoid contents of cyanidin-3-O-glucoside, cyanidin and peak #5
were the highest and were not significantly different (P>0.05) of each other (Table 5).

29
The flavonoid contents of peaks #1 and quercetin-3-O-glucoside were the lowest, 1.29 ±
0.14 and 1.13 ± 0.03 mg/gFW, respectively.
The ratios of the combined relative peak areas of the characteristic peaks (#; 1, 2, 3, 4, 5
and 6) to the total peaks area in green and red leaves extracts were 0.59 ± 0.02 and 058
± 0.02, respectively (Appendix, Table 2).
3.4.4. P. atlantica
Chromatograms of flavonoid-enriched green and red leaf extracts of P. atlantica
harvested in spring and autumn revealed five and four major peaks, respectively. In
spring harvested green and red leaves, cyanidin (that was present in all other species)
and quercetin (which was present in P. chinensis) could not be detected (Fig. 6B,
Appendix, Tables 1, a,b). In autumn harvested green and red leaves, peaks #5, cyanidin,
and quercetin could not be detected (Fig. 6B, Appendix, Tables 1, c,d).
3.4.4.1. Spring
In green leaves, highest, and similar, flavonoid contents were detected in peaks #1, 3
and 5 whereas that of quercetin-3-O-glucoside was the lowest, 1.70 ± 0.45 mg/gFW
(Table 6).
In red leaves, the flavonoid content of peak #5 was the highest, 11.61 ± 2.12 mg/gFW.
Lowest flavonoid contents had cyanidin-3-O-glucoside, quercetin-3-O-glucoside and
peak #3 that were not statistically different of each other (Table 6).
The ratios of the combined relative peak areas of the characteristic peaks (#; 1, 2, 3, 4
and 5) to the total peaks area were about the same in green and red leaves, 0.55 ± 0.07
and 0.61 ± 0.01, respectively (Appendix, Table 2).

3.4.4.2. Autumn
00
In green leaves, the flavonoid content of peak #3 was the highest, 4.24 ± 0.26 mg/gFW,
while that of quercetin-3-O-glucoside was the lowest, 0.48 ± 0.01 mg/gFW (Table 6).
In red leaves, the flavonoid content of peak #3 was the highest, 3.97 ± 0.36 mg/gFW,
while those of cyanidin-3-O-glucoside and quercetin-3-O-glucoside were the lowest,
0.86 ± 0.19 and 0.68 ± 0.23 mg/gFW, respectively and did not differ significantly of
each other (P>0.05) (Table 6).
Table 6. Flavonoids content and retention times in green and red
leaves of P. atlantica. Flavonoid content is expressed as mg/g
fresh weight. The results are expressed as mean±SD
Peak no. 1 2 3 4 5
Peak
identity
Retention
time
(min.)
Harvest
Spring
green
Spring
red
Autumn
green
Autumn
N.I.
Cyanidin-
3-Oglucoside
N.I.
2.71±0.08 8.49±0.78 8.92±0.76
Quercetin-
3-Oglucoside
11.28±1.0
4
N.I.
12.28±0.0
9
5.68±0.69 3.43±0.52 5.45±1.04 1.70±0.45 5.09±1.80
(a*) (b,c) (a,b) (c) (a,c)
4.31±0.35 3.84±0.73 5.55±1.69 2.79±0.53 11.61±2.1
(A) (A,B) (A,B) (B) 2 (C)
2.91±0.47
(k)
1.06±0.01
(l)
4.24±0.26
(m)
0.48±0.01
(n)
-
red
2.53±0.17
(K)
0.86±0.19
(L)
3.97±0.36
(M)
0.68±0.23
(L)
-
* For each harvest, means bearing the same letter are not significantly
different according to t-test (P>0.05); N.I. - not identified; Unidentified
peaks, #1, 3 and 5 are expressed as cyanidin-3-O-glucoside equivalents;
Peaks #2 and 4 contents are expressed according to their own standard curves
The ratios of the combined relative peak areas of the characteristic peaks (#; 1, 2, 3, 4
and 5) to the total peaks area were 0.31 ± 0.02 in green leaves and 0.21 ± 0.01 in red
leaves (Appendix, Table 2).

3.4.5. P.vera
3.4.5.1. Spring
31
Leaves of this species were harvested only in spring. HPLC analysis of flavonoid-
enriched extracts revealed six characteristic peaks (peak #; 1, 2, 3, 4, 5 and 6) (Fig. 7,
Appendix, Tables 1, a,b).
Fig. 7. Comparison of P. vera HPLC chromatograms of flavonoidsenriched
extract of red and green leaves
In green and red leaves, the flavonoid content of cyanidin-3-O-glucoside was the
highest, 5.02 ± 0.08 and 7.59 ± 0.53 mg/gFW, respectively, while that of peak #1 was
the lowest, 0.52 ± 0.10 and 0.76 ± 0.20 mg/gFW, respectively (Table 7).

Table 7. Flavonoids content and retention times in green and red leaves of P.
vera. Flavonoid content is expressed as mg/g fresh weight. The results are
expressed as mean±SD
Peak no. 1 2 3 4 5 6
Peak
identity
Retention
time
(min.)
Harvest
N.I.
Cyanidin-
3-Oglucoside
N.I.
32
Quercetin-3-
O-glucoside
N.I. Cyanidin
2.67±0.01 7.97±0.42 8.50±0.42 10.33±0.25 11.32±0.19 13.37±0.21
0.52±0.10 5.02±0.08 2.28±0.16 1.29±0.08 2.34±0.32 2.23±0.38
Green
(a*) (b) (c) (d)
(c)
(c)
0.76±0.20 7.59±0.53 5.93±0.39 1.65±0.24 3.07±0.42 4.16±0.91
Red
(A) (B) (C) (D) (E) (C,E)
* For each harvest, means bearing the same letter are not significantly different according to t-test
(P>0.05); N.I. - not identified; Unidentified peaks, #1, 3 and 5 are expressed as cyanidin-3-Oglucoside
equivalents; Peaks #2, 4 and 6 contents are expressed according to their own standard
curves
The ratios of the combined relative peak areas of the characteristic peaks (#; 1, 2, 3, 4, 5
and 6) to the total peaks area were 0.64 ± 0.01 in green leaves and 0.58 ± 0.03 in red
leaves (Appendix, Table 2).
3.4.6. P. lentiscus
P.lentiscus is the only non-deciduous tree of the genus Pistacia that was examined in
this research and thus allowed us to collect leaf samples also in winter (February). It
should be noted that in winter the Cyprus ecotype stayed green, while the Tunisian
ecotype turned into bright red. HPLC analysis of flavonoid-enriched leaf extracts
revealed six characteristic peaks (peak #; 1, 2, 3, 4, 5 and 6), while quercetin that was
found in P. chinensis could not be detected (Fig. 8, Appendix, Tables 1, a,c,e).
3.4.6.1. Cyprus ecotype
3.4.6.1.1. Winter
In the Cyprus ecotype, highest, and statistically similar, flavonoid contents were those
of cyanidin-3-O-glucoside and peak #3, 5.81 ± 0.31 and 3.55 ± 1.43 mg/gFW,

33
Fig. 8. Comparison of P. lentiscus (Cyprus ecotype) HPLC
chromatograms
respectively. The flavonoid content of quercetin-3-O-glucoside, cyanidin, peaks #1 and
5 did not differ significantly (P>0.05) of each other and were the lowest (Table 8).
Similar ratios of the combined relative area of the characteristic peaks (#; 1, 2, 3, 4, 5
and 6) to the total peaks area were determined, about 0.75 ± 0.05 (Appendix, Table 2).

44
Table 8. Flavonoids content and retention times in green and red leaves of P.
lentiscus. Flavonoid content is expressed as mg/g fresh weight. The results are
expressed as mean±SD
Peak no. 1 2 3 4 5 6
Peak
identity
Retention
time
(min.)
Harvest
Winter
(Cyprus
ecotype)
Winter
(Tunisian
ecotype)
Spring
(Cyprus
ecotype)
Autumn
(Cyprus
ecotype)
N.I.
Cyanidin-
3-Oglucoside
N.I.
Quercetin-
3-Oglucoside
N.I. Cyanidin
2.62±0.03 7.32±1.55 7.71±1.55 9.51±1.40 10.88±0.86 12.86±0.80
1.82±0.58
(a*)
1.59±0.97
(A,B)
1.30±0.65
(k,m)
0.62±0.07
(K)
5.81±0.31
(b)
6.39±1.84
(B)
12.60±2.78
(l)
4.78±1.37
(L)
3.55±1.43
(a,b)
4.47±1.96
(B)
9.39±1.10
(l)
4.06±1.23
(L)
1.97±0.53
(a)
2.89±0.62
(A,B)
3.56±0.72
(m)
1.47±0.16
(L)
1.99±0.23
(a)
2.13±0.05
(A,B)
3.15±1.07
(m)
0.98±0.47
(K,L)
3.21±0.70
(a)
4.71±0.10
(B)
5.14±0.74
(m)
1.59±0.41
(K,L)
* For each harvest, means bearing the same letter are not significantly different according to ttest
(P>0.05); N.I. - not identified; Unidentified peaks, #1, 3 and 5 are expressed as cyanidin-3-
O-glucoside equivalents; Peaks #2, 4 and 6 contents are expressed according to their own
standard curves
3.4.6.1.2. Spring
Highest, and similar, flavonoid contents were those of cyanidin-3-O-glucoside and peak
#3, 12.60 ± 2.78 and 9.39 ± 1.10 mg/gFW, respectively, while those of peaks #1 and 5
were the lowest, 1.30 ± 0.65 and 3.15 ± 1.07 mg/gFW, respectively (Table 8).
Similar ratios of the combined relative areas of the characteristic peaks (#; 1, 2, 3, 4, 5
and 6) to the total peaks area of the Cyprus ecotype were determined, about 0.78 ± 0.03
(Appendix, Table 2).
3.4.6.1.3. Autumn
In the Cyprus ecotype, highest flavonoid contents were those of cyanidin-3-O-glucoside
and peak #3, 4.78 ± 1.37 and 4.06 ± 1.23 mg/gFW, respectively (Table 8). These peaks
did not differ significantly (P>0.05). In contrast, peaks #1 and 5 had the lowest
flavonoid contents, 0.62 ± 0.07 and 0.98 ± 0.47 mg/gFW, respectively (Table 8).

55
The ratios of the combined relative areas of the characteristic peaks (#; 1, 2, 3, 4, 5 and
6) to the total peaks area were about 0.65 ± 0.07 (Appendix, Table 2).
3.4.6.2. Tunisian ecotype
3.4.6.2.1. Winter
Highest flavonoid contents were those of cyanidin-3-O-glucoside, cyanidin and peak
#3, 6.39 ± 1.84, 4.47 ± 1.96 and 4.71 ± 0.10 mg/gFW, respectively, which were not
significantly different of each other (P>0.05) (Table 8). Lowest flavonoid contents
were detected in the rest of the peaks (quercetin-3-O-glucoside, peaks #1 and 5) that did
not differ significantly (P>0.05) of each other (Table 8).
The ratios of the combined relative areas of the characteristic peaks (#; 1, 2, 3, 4, 5 and
6) to the total peaks area of the Tunisian ecotype were about 0.67 ± 0.01 (Appendix,
Table 2).
3.5. Flavonoids content in Pistacia stems
Red stems of P. palaestina, P.vera and P. chinensis were harvested in the spring and
subjected to HPLC analysis. In P. palaestina and P. vera five characteristic peaks (#; 1,
2, 3, 4, and 5) were detected, while in P.chinensis only two peaks (#; 1 and 4) were
found (Fig. 9, Appendix, Table 1f).
3.5.1. P. palaestina
Highest flavonoid content was that of cyanidin-3-O-glucoside, 10.10 ± 1.51 mg/gFW,
while that of quercetin-3-O-glucoside was the lowest, 0.11 ± 0.01 mg/gFW (Table 9).
The ratios of the combined relative areas of the characteristic peaks (#; 1, 2, 3, 4 and 5)
to the total peaks area were about 0.76 ± 0.02 (Appendix, Table 2).

3.5.2. P.vera
66
Fig. 9. Comparison of spring red stems HPLC chromatograms
Similar to the stems of P. palaestina, highest flavonoid content had cyanidin-3-O-
glucoside, 1.82 ± 0.16 mg/gFW (Table 9). Peaks #1 and quercetin-3-O-glucoside, that
did not differ significantly (P>0.05), had the lowest amount, 0.21 ± 0.02 and 0.20 ± 0.03
mg/gFW, respectively (Table 9).
The ratios of the combined relative areas of the characteristic peaks (#; 1, 2, 3, 4 and 5)
to the total peaks area were about 0.50 ± 0.01 (Appendix, Table 2).

77
Table 9. Flavonoids content and retention times in red stems of P.
palaestina, P. vera and P. chinensis. Flavonoid content is expressed as
mg/g fresh weight. The results are expressed as mean±SD
Peak no. 1 2 3 4 5
Peak identity N.I.
Cyanidin-
3-Oglucoside
N.I.
Quercetin-3-
O-glucoside
Retention time
(min.)
Harvest
2.63±0.02 7.34±0.72 7.87±0.76 10.18±0.81 10.94±0.41
P. palaestina
0.92±0.14
(a*)
10.10±1.5
1 (b)
0.51±0.05
(c)
0.11±0.01
(d)
3.27±1.28
(a,c)
P. vera
0.21±0.02
(A)
1.82±0.16
(B)
1.03±0.12
(C)
0.20±0.03
(A)
1.13±0.19
(C)
P. chinensis
1.31±0.63
(k)
- -
1.71±0.79
(k)
-
* For each harvest, means bearing the same letter are not significantly different
according to t-test (P>0.05); N.I. - not identified; Unidentified peaks, #1, 3 and 5 are
expressed as cyanidin-3-O-glucoside equivalents; Peaks #2 and 4 contents are
expressed according to their own standard curves
3.5.3. P. chinensis
Only two flavonoids were detected in this species (quercetin-3-O-glucoside and peaks
#1) and their contents were not significantly different of each other, 1.31 ± 0.63 and
1.71 ± 0.79 mg/gFW, respectively, P>0.05 (Table 9).
The ratios of the combined relative areas of the characteristic peaks (quercetin-3-O-
glucoside and peak #1) to the total peaks area were 0.44 ± 0.09 (Appendix, Table 2).
3.6. Chlorophyll and carotenoids content in Pistacia
3.6.1. P. chinensis, P. palaestina and P. khinjuk
In three species, P. chinensis, P. palaestina and P. khinjuk, highest total chlorophyll and
carotenoids contents were found in spring harvested green leaves, about 1220-1575 and
117-262 µg/gFW, respectively whereas lowest contents of these pigments were found in
autumn harvested red leaves, about 21-28 and 42-87 µg/gFW, respectively (Table 10).
N.I.

38
Table 10. Chlorophyll and carotenoid contents in green and red Pistacia leaves and stems.
Chlorophyll and carotenoid contents are expressed as µg/g fresh weight. The results are
expressed as mean±SD
Spp.
(tissue)
P. chinensis
(leaves)
P. palaestina
(leaves)
P. khinjuk
(leaves)
P. atlantica
(leaves)
Sample Chl a Chl b
Chl
a/b
Chl a + Chl b Carotenoids
Sp.(G) 1195.7±93.9 378.5±31.2 3.2 1574.3±125.2 (a*) 261.9±10.0 (a)
Sp.(R) 227.1±13.2 55.61±7.8 4.1 282.7±21.0 (b) 121.8±3.6 (b)
Aut.(G) 255.6±13.7 53.8±12.5 4.8 309.4±26.0 (b) 117.6±13.6 (b)
Aut.(R) 10.1±10.1 18.11±9.0 0.6 28.2±19.0 (c) 87.4±31.1 (b)
Sp.(G) 785.9±283.5 237.4±115.8 3.3 1023.0±398.5 (a) 278.8±30.3 (a)
Sp.(R) 405.8±63.3 110.0±18.0 3.7 515.8±81.4 (a,b) 168.4±28.2 (b)
Aut.(G) 659.0±85.8 216.6±28.1 3.0 875.6±113.8 (a) 152.2±12.9 (b)
Aut.(R) 26.4±17.1 11.3±7.4 2.3 25.6±8.9 (c) 55.0±7.5 (c)
Sp.(G) 928.3±64.5 295.4±36.5 3.1 1223.7±98.7 (a) 227.8±20.2 (a)
Sp.(R) 434.0±73.1 127.9±15.5 3.4 561.9±88.2 (b) 133.8±17.7 (b)
Aut.(G) 244.4±23.6 79.2±6.5 3.1 323.7±29.9 (c) 100.4±1.7 (b)
Aut.(R) 15.0±5.9 6.4±2.3 2.3 21.5±7.8 (d) 41.9±3.5 (c)
Sp.(G) 586.5±11.9 140.5±14.4 4.2 727.0±23.7 (a)
224.1±37.5
(a,b)
Sp.(R) 627.9±152.6 154.7±24.8 4.1 755.8±127.8 (a) 212.1±6.5 (b)
Aut.(G) 1213.5±120.2 381.2±31.1 3.2 1594.7±139.4 (b) 299.1±26.7 (a)
Aut.(R) 124.4±32.0 44.0±3.6 2.8 168.4±34.1 (c) 90.5±11.8 (c)
P. vera Sp.(G) 660.1±96.5 202.8±14.9 3.3 862.9±109.8 (a) 173.3±21.7 (a)
(leaves) Sp.(R)
Sp.(G)
443.2±90.0 128.3±31.8 3.5 571.5±121.0 (b) 158.5±24.5 (a)
(Cyprus
ecotype)
Win.(G)
337.2±27.3 97.7±2.0 3.5 434.9±28.1 (a) 153.5±17.1 (a)
(Cyprus 251.3±72.2 93.4±16.1 2.7 394.4±23.2 (a) 131.5±18.6 (a)
P. lentiscus ecotype)
(leaves) Win.(R)
(Tunisian
ecotype)
Aut.(G)
136.3±98.7 51.5±37.1 2.6 127.1±68.0 (b) 76.6±15.8 (b)
(Cyprus
ecotype)
208.6±48.2 53.9±13.1 3.9 262.5±60.7 (b) 78.3±10.9 (b)
P. Palaestina
(stems)
100.2±15.8 44.1±6.9 2.3 139.5±20.7 (a) 34.7±6.4 (a)
P. vera
(stems)
Sp.(R) 137.9±16.2 53.2±4.5 2.6 187.0±18.9 (a) 41.5±3.0 (a)
P. chinensis
(stems)
130.1±16.8 54.6±7.8 2.4 184.7±24.5 (a) 47.7±6.2 (a)
* For each Spp. (tissue) total chlorophyll and carotenoids means bearing the same letter are not significantly
different according to t-test (P>0.05); Abbreviations are: Sp., spring; Aut., autumn; Win., winter; G, green;
R, red

3.6.1.1. Chlorophyll a/b ratio:
39
In autumn and spring harvested green leaves, high chlorophyll a/b ratios were found: in
P. palaestina, where it varied between 3.0 to 3.3, respectively, in P.khinjuk, 3.1 and in
P. chinensis, where it varied between 3.2 and 4.8 (calculated from data presented in
Table 10).
Lower chlorophyll a/b ratios were determined in autumn harvested red leaves, in P.
palaestina and P. khinjuk it was 2.3 and 0.6 in P. chinensis. In contrast, in red leaves
that were harvested in spring, an increase in the chlorophyll a/b ratio was found. In P.
palaestina, it increased by 11% to 3.7 and in P. khinjuk by 8% to 3.7. More profound
change was found in P.chinensis, where it increased by 29% to 4.4 (calculated from
data presented in Table 10).
3.6.1.2. Spring and autumn total Chlorophyll ratios:
P. chinensis: The ratio between total chlorophyll in spring harvested green leaves to that
of spring harvested red leaves was 5.6, while this ratio for autumn harvested leaves was
11.0 (calculated from data presented in Table 10).
P. palaestina: In the ratio between total chlorophyll in spring harvested green leaves to
that of spring harvested red leaves was lower, 2.0, than that determined in P. chinensis
however, this ratio for autumn harvested leaves had increased considerably to 34.2, as
compared to P. chinensis (calculated from data presented in Table 10).
P. khinjuk: The ratio between total chlorophyll in spring harvested green leaves to that
of spring harvested red leaves was similar to that of P. palaestina, 2.2, while this ratio
for autumn harvested leaves was 15.1 (calculated from data presented in Table 10).

00
3.6.1.3. Spring and autumn carotenoid contents ratios:
P. chinensis: The ratio between carotenoid content in spring harvested green leaves to
that of spring harvested red leaves was 2.1 and 1.3 for autumn harvested leaves
(calculated from data presented in Table 10).
P. palaestina: The ratio between carotenoids contents in spring harvested green leaves
to that of spring harvested red leaves was 1.67 and was lower than that of P. chinensis
while this ratio for autumn harvested leaves was 2.8 and was higher than that of P.
chinensis (calculated from data presented in Table 10).
P. khinjuk: The ratios between carotenoids contents in spring and autumn harvested
green leaves to that of spring harvested red leaves were 1.7 and 2.4, respectively, and
were similar to those of P. palaestina.
3.6.2. P. atlantica
Highest total chlorophyll and carotenoid contents were detected in autumn green leaves,
1595 and 299 µg/gFW, respectively, while the lowest contents of these pigments were
those of autumn red leaves, 168 and 90 µg/gFW, respectively (Table 10). No
significant difference (P>0.05) was found between the chlorophyll contents of spring
harvested green and red leaves and between the carotenoid contents of these leaves that
were harvested in spring.
3.6.2.1. Chlorophyll a/b ratio:
Higher chlorophyll a/b ratio was found in autumn green leaves compared to autumn red
leaves (Table 10). Similar chlorophyll a/b ratios were determined in spring harvested
green and red leaves, 4.2 and 4.1, respectively. It should be noted that these ratios are
higher, than those of autumn harvested leaves, by 31% and 46%, respectively.

41
3.6.2.2. Spring and autumn total Chlorophyll ratios:
In P. atlantica, the ratio between total chlorophyll in spring harvested green leaves to
that of spring harvested red leaves was 0.96, while this ratio for autumn harvested
leaves was 9.5 (calculated from data presented in Table 10).
3.6.2.3. Spring and autumn carotenoid contents ratios:
The ratio between carotenoids contents in spring harvested green leaves to that of spring
harvested red leaves was 1.05, this ratio however, increased three folds to 3.3 for
autumn harvested leaves and was close to that found in P. palaestina (calculated from
data presented in Table 10).
3.6.3. P. vera
Total chlorophyll content in spring green leaves, 863 µg/gFW, was higher than in spring
red leaves by about 33.8%. Similar carotenoid contents were found in spring green and
spring red leaves (Table 10).
3.6.3.1. Chlorophyll a/b ratio:
Chlorophyll a/b ratios in spring green and red harvested leaves were 3.3 and 3.5,
respectively. These values are close to those of P. palaestina.
3.6.3.2. Spring and autumn total Chlorophyll ratios:
The ratio between the total chlorophyll in spring harvested green leaves to that of spring
harvested red leaves was 1.5.

42
3.6.3.3. Spring and autumn carotenoid contents ratios:
The ratio between carotenoid content in spring harvested green leaves to that of spring
harvested red leaves was 1.1, similar to that of P. atlantica.
3.6.4. P. lentiscus
3.6.4.1. Cyprus ecotype
In the Cyprus ecotype of this species (green leaves only) highest total chlorophyll
contents were found in winter and spring harvested leaves, 435 and 394 µg/gFW,
respectively. It should be noted that these values did not differ significantly (P>0.05).
The total chlorophyll content of autumn leaves,127 µg/gFW, was by 33% and by 40%
lower than those of the winter and spring leaves, respectively.
3.6.4.1.1 Chlorophyll a/b ratio:
The chlorophyll a/b ratios of autumn and spring leaves were 3.9 and 3.5, respectively,
while a lower ratio, 2.7, was found in winter leaves (Table 10).
3.6.4.1.2. Spring and autumn carotenoid contents ratios:
The carotenoid contents were determined in winter and spring leaves. Spring and winter
cartonoid contents, 153.5 and 131.5 µg/gFW, respectively, did not differ significantly
(P>0.05). The total carotenoid content of autumn leaves, 78.3 µg/gFW, was by 40% and
by 49% lower than those of the winter and spring leaves, respectively (Table 10).
3.6.4.2. Tunisian ecotype
The chlorophyll of the Tunisian ecotype of P. lentiscus (red leaves only) was
determined only in the winter. The total chlorophyll content, 127.1 µg/gFW, was by
68% than that of the winter Cyprus ecotype.

3.6.4.2.1. Chlorophyll a/b ratio:
33
The chlorophyll a/b ratio of the Tunisian ecotype was 2.6, similar to that of the winter
Cyprus ecotype 2.7, however, the contents of chlorophyll a and b of this ecotype were
about half of those determined in the leaves of the Cyprus winter ecotype (Table 10).
3.6.4.2.2. Spring and autumn total Chlorophyll ratios:
The ratio between total chlorophyll in winter harvested green leaves (Cyprus ecotype)
to that of winter harvested red leaves (Tunisian ecotype) was 3.1.
3.6.4.2.3. Spring and autumn carotenoid contents ratios:
The cartenoid content of the red leaves was 76.6 µg/gFW. This value is lower by 42%
than that determined in the green leaves of the winter harvested Cyprus ecotype.
3.6.5. Stems
Chlorophyll and carotenoid contents of P. palaestina, P. vera and P. chinensis red
stems that were harvested in spring were determined.
The total chlorophyll contents, 139.5, 187.0 and 184.7 µg/gFW, respectively in these
stems were considerably lower than in the spring harvested red leaves of these species
by 63%, 67% and 35%, respectively. It should be noted however, that these chlorophyll
contents are not significantly different of each other (Table 10).
The chlorophyll a/b ratio in the stems of these species did not significantly (P>0.05)
differ among each other and were 2.3, 2.6 and 2.4, respectively. These values are
different from those determined in spring red leave of these species, 3.7, 3.5 and 4.1,
respectively.

44
The carotenoid contents of the stems, 34.7, 41.5 and 47.4 µg/gFW, respectively were
considerably lower than those determined in spring harvested red leaves by 80%, 74%
and 61%, respectively (Table 10).

4. Discussion
45
The Pistacia germplasm collection plot (http://www.bgu.ac.il/pistacia), located in
vicinity to the Desert Research Institutes at Sde Boker, provides a unique experimental
tool to study the response of the various Pistacia species under practically identical
environmental conditions. The Pistacia trees (P. chinensis, P. palaestina, P. khinjuk, P.
vera and P. lentiscus (Cyprus and Tunisian ecotypes) growing in the plot were
germinated from seeds that were collected at their natural habitat from all over the
world and thus represent diverse geographic and climate zones. In this research a
comprehensive seasonal comparison of flavonoids content in various Pistacia species is
reported for the first time. In most reported literature on flavonoids in plants in general
and in Pistacia in particular (Table 1), the focus is on this group of compounds in the
context of their natural product-medicinal and/or nutrition properties.
In response to environmental cues Pistacia leaves turn red twice a year, in the spring
and in the autumn, juvenile and senescence developmental stages, respectively. As all
the trees in the collection plot are growing under, practically, identical soil and climatic
conditions (light radiation, temperature, humidity, water supply, etc.) it was of interest
to study the biochemical changes that occur during the appearance of the red
pigmentation of the various Pistacia species.
The main objective of the research presented here was to elucidate the distribution
pattern of flavonoids in leaves and stems of different species of Pistacia tree. Autumn,
winter and spring samples were analyzed. It was hypothesized that anthocyanin content
in the leaves of Pistacia varies depending on leaf developmental stage (spring, autumn):
it is visually (Pics. 2 and 3) high in early growth and at senescence. Since the red color
of the trees is due to anthocyanins, research in the thesis includes anthocyanin

46
distribution pattern in the six Pistacia trees in different seasons, as well as identification
of the major anthocyanins.
Seven major peaks, out of a total of circa 30, were identified in chromatograms of
flavonoid-enriched extracts of six Pistacia species analyzed. The proportion of these
peaks was much more than 50% of the total peak area in most samples (Appendix,
Table 2). This highlights their potential major role in the red color phenomenon of the
leaves.
4.1. Pistacia red coloration
4.1.1. Visual observation
Red coloration of the leaves of deciduous trees attracted human curiosity for ages. Red
coloration of the leaves is due to anthocyanins, which are synthesized de novo during
early vegetation in spring and leaf senescence in autumn. The scientific literature
proposed several hypothetical functions for the red color in leaves, which all are
defense-based functions, such as protection against ROS, UV, excess high light
radiation and herbivory. It should be noted that most of the anthocyanins contained in
the leaves are red. Up to date there is no conclusive answer - why most of the leaf
anthocyanins are red?
Our visual observations indicate that newly growing branches, no matter in spring or in
autumn, are red at the beginning, then they become green and later brown-gray upon
maturation and secondary growth (Fig. 3C). During the early stages of vegetation,
young developing leaves are lacking secondary growth, hence are vulnerable to
different challenges. Thus, it might be a defense mechanism against them, such as high
level UV radiation. Moreover, during the winter, under low temperature and high
intensity of sunlight, the only evergreen species in our Pistacia germplasm, P. lentiscus
Cyprus ecotype stayed green, while the other ecotype called Tunisian turned into bright

47
red. Most probably this is related to light and temperature and genotypic factors rather
than herbivory problems.
Another observation that supports sunlight protection hypothesis is shown in Fig. 3B.
Here we can see that the leaves that were masked by other leaves were protected from
the sunlight and were not red but yellow. This means that there is no need 'to be red', if
the leaf is not under direct sunlight, alternatively this may reflect the need for sunlight
for synthesis of anthocyanins. So, one can conclude that anthocyanins biosynthesis is
related to sunlight.
Additionally, anthocyanins are the precursors of different phenolics and flavonoids
which are involved in ROS neutralization and UV protection. Hence, high levels of all
these constituents may co-occur as a result of activation of the pathway at an early stage
(Close et al., 2001; Dominy and Lucas, 2004; Jaakola et al., 2004; Karageorgou and
Manetas, 2006; Lee and Lowry, 1980).
Based on our visual observations and conclusions mentioned above, we propose the
"red cycle" model (Fig. 10).
Fig. 10. The "red cycle". A schematic model of reddening of young and senescent leaves for
protection of resettling/dedifferentiation of sink and source, respectively (in some deciduous
trees)

48
After winter dormancy deciduous trees enter into active growth, when new leaves and
stems are being formed and are protected by the red anthocyanins. This stage continues
up to mid-summer, when new growth stops. Newly growing centers, such as apical
meristems, serve as sink receiving nutrients and metabolites, mostly nitrogenous and
carbon-based, from root and bark. The flow of substances from source to sink, increases
metabolic activity of the sink. This may explain the accumulation of anthocyanins at the
sink fulfilling a protective role of newly developing meristems. In late spring and
summer, mature green leaves become the source for sinks of newly forming leaves,
which are also 'protected' by red color. During leaf senescence, senescing leaves serve
as a source, from where carbonaceous and nitrogenous compounds released during
cellular break-down and photosynthetic apparatus dismantlement, are mobilized to the
root and bark, which serve as sink (sink-source resettling). This process requires
resetting/reprogramming of metabolic activity of the senescing leaves (source, in this
case), which may require protection from radiation, explaining anthocyanins
accumulation in the senescing leaves.
4.2. Identification and quantification of characteristic peaks
HPLC separation on reverse phase column of the extracts yielded typical
chromatograms (Figs. 4 to 9). The reverse-phase separates the compounds according to
increasing hydrophobicity. The hydrophilic flavonoids eluted first and later on the
hydrophobic ones. Among the identified compounds, cyanidin-3-O-glucoside and
quercetin-3-O-glucoside had lower retention times (Tables 3 to 9) than that of cyanidin
and quercetin, which are the corresponding aglycons and are more hydrophobic
compared to their glycosylated counterparts. Identification of the various flavonoids
based on retention time of authentic compounds, UV absorption, NMR and MS data
confirm their identity (Appendix, Figs. 5 to 9, Table 3).

49
The amount of a separated compound on the chromatogram is proportional to its peak
area. Thus the identified peaks were quantified directly based on standard curves (Table
2 and Appendix, Figs. 1 to 4). The quantitative determination of the unidentified peaks
1, 3 and 5 was based on the standard curve of cyanidin-3-O-glucoside, therefore they
should be regarded as cyanidin-3-O-glucoside equivalents and not actual amount of
these compounds.
4.3. Variations in anthocyanin content of leaves
4.3.1. Green vs. red comparison
Anthocyanins are responsible for the red coloration in leaves. It was hypothesized that
anthocyanin content in the leaves should vary in green and red leaves. It was expected
to find high anthocyanin content in red leaves compared to green leaves.
However, evaluation of quercetin-3-O-glucoside (peak #4) content, showed no
significant difference (P>0.05) in red and green leaves of the same species and in the
same season in all cases, except in P. khinjuk in spring (Table 11).
Table 11. Green vs. red comparison of Pistacia leaves
flavonoids content harvested in spring and in autumn
Species Season
1 2 3
Peak #
4 5 6 7
P. chinensis
Spring
Autumn
-
+
+
-
-
+
+
+
-
ND
-
+
ND
+
P. palaestina
Spring
Autumn
-
+
-
+
-
-
+
+
-
+
+
+
ND
ND
P. khinjuk
Spring
Autumn
+
-
-
-
-
-
-
+
-
-
-
+
ND
ND
P. atlantica
Spring
Autumn
+
+
+
+
+
+
+
+
-
ND
ND
ND
ND
ND
P. vera
Spring
Autumn
+
ND
-
ND
-
ND
+
ND
+
ND
-
ND
ND
ND
Abbreviations: ND-not determined;
+ indicates that peak content is not significantly different between green and
red leaf in the given season (P>0.05);
- indicates that peak content is significantly different between green and
red leaf in the given season (P

50
The other peaks gave inconsistent results. Noteworthy is the insignificant differences
between green and red leaves of P. atlantica cyanidin-3-O-glucoside, quercetin-3-O-
glucoside, peaks #1 and 3 in spring and autumn. In P. chinensis, except cyanidin-3-O-
glucoside, autumn leaves had the same peak pattern, no matter of leaf color (green or
red).
Since P. lentiscus is the only evergreen species in our germplasm we collected samples
of P. lentiscus in winter too. We have examined two ecotypes of P. lentiscus: Cyprus
ecotype, which stayed green during the winter and Tunisian ecotype, whose leaves
turned into bright red color. Comparison of peaks of Cyprus ecotype (green leaves) and
Tunisian ecotype (red leaves) revealed that they are the same and do not differ
significantly of each other.
Concluding as mentioned above, that despite of apparent color difference between green
and red leaves, anthocyanin content is the same in these samples, at least for most of the
species and it is always the same for quercetin-3-O-glucoside (except in P. khinjuk in
spring), in agreement with Neil et al. (2002b), that in Quintinia serrata, both red and
green leaf extracts displayed the same antioxidative capacities.
Red color absence in green leaves might be due to the high quantity of other pigments,
such as chlorophyll and carotenoids that mask the red color (Table 10). Indeed,
chlorophyll and carotenoid content confirm that within the same season, green leaves
contain much higher, up to an order of magnitude more chlorophyll and carotenoids
than those of red leaves (Table 10).
The lower anthocyanins in autumn senescing leaves as compared to the juvenile spring
leaves is also reflected by the lower ratio of the major peaks out of the total peak area
(Appendix, Table 2). The increased ratio of small unidentified peaks may be related to
catabolism associated with senescence. This is in accordance with the decrease in the
ratio of chlorophyll a/b in autumn red leaves, indicative of changes in photosynthetic

51
apparatus. Also the decrease in carotenoids in autumn red leaves corroborate the trend
of catabolism during senescence. In P. chinensis, P. palaestina and P. khinjuk, the ratio
of total chlorophyll to carotenoids is reversed in autumn red leaves compared to spring
leaves (Table 10). This result may reflect the important role of carotenoids in protection
of the regulated catabolism during senescence (Matile et al., 1999).
4.3.2. Spring vs. autumn comparison
It was hypothesized that anthocyanin content in the leaves may vary in spring and
autumn leaves. However, in most cases spring leaves had significantly higher
concentration of anthocyanins than those of autumn leaves (Tables 3 to 9). Evaluation
of quercetin-3-O-glucoside content showed that it is higher in spring than in autumn
leaves, except P. chinensis green leaves (it was the same in spring and in autumn
leaves). The consistently increased ratio of the cyanidin-3-O-glucoside to its cyanidin
aglycon (the lowest in autumn green and the highest in spring red) (Tables 3 to 9), may
reflect high transglucosylation during spring. Alternatively, we may suggest faster rate
of catabolism of the anthocyanins during autumn. Cyanidin was not detectable in P.
chinensis (spring red), P. atlantica (all seasons) and in stems (Tables 3, 6 and 9,
respectively) suggestive of high transglucosylation activity in these cases. Romani et al.
(2002) reported that concentration of cyanidin-3-O-glucoside was the lowest (0.4 mg/g
dry weight) among 12 flavonoids identified in P. lentiscus. This value is about 20-fold
lower than that found in our work, comparing on a dry weight basis (assuming approx.
50% dry weight of leaves). These significantly different values can be related to
different ecotypes, season, age of the plants and growth conditions. It should be noted
that the content of the other flavonoids reported by Romani et al. (2002) ranged
between 0.8 to 26 mg/g dry weight.

52
Quercetin was identified only in autumn leaves of P. chinensis (Table 3). The only other
report of quercetin in Pistacia is in P. terebinthus, by Topcu et al. (2007). Worthwhile
noting that these species are phylogenetically closely related. The similarity between the
quercetin aglycon (peak #7) and its glucoside (peak #4) in green and red leaves in
autumn may suggest a different response of their metabolic pathways compared to the
changes in cyanidin (peak #6) and its glucoside (peak #2) to environmental cues. This
may be related to their different biosynthetic pathways, the first being a flavanone and
the later a flavanol. The absence of quercetin in all other species, as well as in spring
leaves of P. chinensis may suggest that the biosynthesis of this flavonoid is tilted
towards the glucoside on the expense of the aglycon. The quercetin-3-O-glucoside was
identified and quantified by us in the leaves and stems of all species tested (Tables 3 to
9). The only other report of this compound was by Kawashty et al. (2000), who
identified and qualitatively evaluated it in P. lentiscus, P. atlantica, P. chinensis and P.
khinjuk.
Quercetin being an antioxidant (Topcu et al. 2007), may function synergistically not
only as light protector but in protection against biotic and abiotic stresses.
Higher content of flavonoids in spring leaves compared to autumn leaves can be related
to intensive new growth and development in spring. At this developmental stage young
leaves are highly unprotected from UV radiation and light damage. Thus we may
suggest teleologically that there is a higher need for protection in spring compared to
autumn.
4.4. Evolutionary relationships between Pistacia species
Phylogenetic relationship among Pistacia species is based on genetic markers,
morphology of leaves and metabolite patterns (Golan-Goldhirsh et al., 2005 and
Barazani et al., 2003). Comparison of quercetin-3-O-glucoside content in different

53
Pistacia species (Table 12), reveals that, phylogeneticaly, P. palaestina is closer to P.
chinensis and P. lentiscus. P. khinjuk had similarity with P. atlantica, which was in
general agreement with the other phylogenetic criteria mentioned above.
Table 12. Comparison of quercetin-3-O-glucoside leaf content
between Pistacia species
Sample Sp.(G) Sp.(R) Aut.(G) Aut.(R)
P. chinensis vs. P. palaestina + - - +
P. chinensis vs. P. khinjuk - - - +
P. chinensis vs. P. atlantica - + - -
P. chinensis vs. P. vera - - ND ND
P. chinensis vs. P. lentiscus + ND - ND
P. palaestina vs. P. khinjuk - - + -
P. palaestina vs. P. atlantica - - - -
P. palaestina vs. P. vera - - ND ND
P. palaestina vs. P. lentiscus + ND + ND
P. khinjuk vs. P. atlantica + + - +
P. khinjuk vs. P. vera - - ND ND
P. khinjuk vs. P. lentiscus + ND - ND
P. atlantica vs. P. vera + - ND ND
P. atlantica vs. P. lentiscus - ND - ND
P. vera vs. P. lentiscus - ND ND ND
Abbreviations: Sp.-spring, Aut.-autumn, G-green, R-red, ND-not
determined;
+ indicates that quercetin-3-O-glucoside content is not significantly
different between species in the given season and in the given leaf color
(P>0.05);
- indicates that quercetin-3-O-glucoside content is significantly different
between species in the given season and in the given leaf color (P

5. Conclusion
Seven major flavonoids were detected in leaves and stems of six Pistacia species.
54
The following flavonoids were identified by comparison to authentic compounds, peak
retention time, MS and NMR analyses: cyanidin-3-O-glucoside, quercetin-3-O-
glucoside, cyanidin and quercetin,
1. Anthocyanin content is higher in spring than in autumn.
2. Anthocyanin content in green and red leaves of the same season and species does not
differ significantly.
3. Apparent color difference between green and red leaves is due to the presence of other
pigments, such as chlorophyll and carotenoids, which mask anthocyanins red color.
4. Based on quercetin-3-O-glucoside content, P. palaestina is closer phylogenetically to P.
chinensis and P. lentiscus, P. khinjuk and P. atlantica group together.
5. Distinct differences were detected between spring juvenile and autumn senescent leaves
in all parameters, flavonoids, chlorophyll, carotenoids.
6. Anthocyanins are hypothesized to be involved in photoprotection of newly growing
leaves and stems and senescing leaves.

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7. Appendix
7.1. Figures
Peak area (mAU, x10 6 )
64
Fig. 1. Standard calibration curve of cyanidin-3-O-glucoside based on HPLC
chromatograms
Peak area (mAU, x10 6 )
3.5
3
2.5
2
1.5
1
0.5
14
12
10
8
6
4
2
0
0
y = 144673x
R 2 = 0.9996
0 5 10 15 20 25
Injected amount (µg)
Fig. 2. Standard calibration curve of quercetin-3-O-glucoside based on HPLC
chromatograms
y = 578595x
R 2 = 0.9977
0 5 10 15 20 25
Injected amount (µg)

Peak area (mAU, x10 6 )
65
Fig. 3. Standard calibration curve of cyanidin based on HPLC chromatograms
Peak area (mAU, x10 6 )
6
5
4
3
2
1
0
14
12
10
8
6
4
2
0
y = 108673x
R 2 = 0.9995
0 10 20 30 40 50 60
Injected amount (µg)
y = 684903x
R 2 = 0.9908
0 5 10 15 20 25
Injected amount (µg)
Fig. 4. Standard calibration curve of quercetin based on HPLC chromatograms

Fig. 5. MS spectrum of peak #2
Fig. 6. MS spectrum of peak #4
66

B
A
67
Fig. 7. MS spectrum of peak #6. (A) RT: 7.01-7.07, (B) RT: 7.33-7.42

Fig. 8. NMR spectrum of peak #4
Fig. 9. NMR spectrum of peak #6
68

7.2. Tables
69
Table 1a. Identified characteristic flavonoids peaks in
chromatograms of spring harvested green Pistacia
leaves
Peak #
1 2 3 4 5 6 7
P. chinensis + + + + + + -
P. palaestina + + + + + + -
P. khinjuk + + + + + + -
P. atlantica + + + + + - -
P. vera + + + + + + -
P. lentiscus
+ + + + + + -
(Cyprus ecotype)
+ peak is present
- peak is absent
Table 1b. Identified characteristic flavonoids peaks in
chromatograms of spring harvested red Pistacia leaves
Peak #
1 2 3 4 5 6 7
P. chinensis + + + + + - -
P. palaestina + + + + + + -
P. khinjuk + + + + + + -
P. atlantica + + + + + - -
P. vera + + + + + + -
+ peak is present
- peak is absent
Table 1c. Identified characteristic flavonoids peaks in
chromatograms of autumn harvested green Pistacia
leaves
Peak #
1 2 3 4 5 6 7
P. chinensis + + + + - + +
P. palaestina + + + + + + -
P. khinjuk + + + + + + -
P. atlantica + + + + - - -
P. lentiscus
+ + + + + + -
(Cyprus ecotype)
+ peak is present
- peak is absent

70
Table 1d. Identified characteristic flavonoids peaks in
chromatograms of autumn harvested red Pistacia
leaves
Peak #
1 2 3 4 5 6 7
P. chinensis + + + + - + +
P. palaestina + + + + + + -
P. khinjuk + + + + + + -
P. atlantica + + + + - - -
+ peak is present
- peak is absent
Table 1e. Identified characteristic flavonoids peaks in
chromatograms of winter harvested P. lentiscus leaves
Peak #
1 2 3 4 5 6 7
Cyprus ecotype
(green leaves)
Tunisian ecotype
(red leaves)
+ peak is present
- peak is absent
+ + + + + + -
+ + + + + + -
Table 1f. Identified characteristic flavonoids peaks in
chromatograms of spring harvested red Pistacia stems
Peak #
1 2 3 4 5 6 7
P. palaestina + + + + + - -
P. vera + + + + + - -
P. chinensis + - - + - - -
+ peak is present
- peak is absent

71
Table 2. Ratios of the combined relative areas of the
characteristic flavonoids peaks to the total peaks areas
in chromatograms of green and red Pistacia leaves and
stems. Leaves were harvested in winter, spring and
autumn. Stems were harvested in spring. The results
are the means of three independent experiments ±SD
Spp. Sample R
P. chinensis
P. palaestina
P. khinjuk
P. atlantica
P. vera
P. lentiscus
Stems
Spring green 0.74 ± 0.01
Spring red 0.52 ± 0.01
Autumn green 0.53 ± 0.03
Autumn red 0.52 ± 0.03
Spring green 0.77 ± 0.03
Spring red 0.75 ± 0.02
Autumn green 0.69 ± 0.05
Autumn red 0.65 ± 0.04
Spring green 0.78 ± 0.01
Spring red 0.68 ± 0.03
Autumn green 0.59 ± 0.02
Autumn red 0.58 ± 0.02
Spring green 0.55 ± 0.07
Spring red 0.61 ± 0.01
Autumn green 0.31 ± 0.02
Autumn red 0.21 ± 0.01
Spring green 0.64 ± 0.01
Spring red 0.58 ± 0.03
Winter (Cyprus
ecotype)
0.75 ± 0.05
Winter (Tunisian
ecotype)
0.67 ± 0.01
Spring (Cyprus
ecotype)
0.78 ± 0.03
Autumn (Cyprus
ecotype)
0.65 ± 0.07
P. palaestina 0.76 ± 0.02
P. vera 0.50 ± 0.01
P. chinensis 0.44 ± 0.09

72
Table 3. Retention time, MS fragmentation pattern and NMR chemical shifts of
identified peaks
Retention
Peak
#
Peak
identity*
time, min.
(mean ± SD,
ca.)
Fragment, m/z
228.17, 239.25,
δ, ppm
2
Cyanidin-3-
O-glucoside
7.8±0.8
327.08, 459.33,
493.00, 515.17,
547.25, 592.75,
684.00
-
464.92, 507.17, 1.997
592.83, 610.08, 2.005, 2.016, 2.029, 2.030, 2.031,
684, 758
2.126, 2.145, 2.146, 2.151, 2.158,
2.166, 2.178, 2.200, 2.207, 2.220,
2.230, 2.233, 2.241, 2.243, 2.245
4
Quercetin-3-
O-glucoside
10.3±0.9
3.744, 3.752, 3.764, 3.772
4.147, 4.154, 4.161
5.360, 5.372
7.021, 7.022, 7.074, 7.077, 7.183,
7.185, 7.191, 7.198, 7.204, 7.222,
7.243, 7.259, 7.264, 7.363, 7.368,
7.372, 7.416, 7.421, 7.518, 7.523
8.041
448.92, 449.92, 0.923, 0.938
507.08, 600.92, 1.228, 1.243, 1.289, 1.981
6 Cyanidin 13.2±0.5 601.83, 602.83,
683.92, 685.00,
758.00
7 Quercetin 14.8±0.2 - -
* Cyanidin-3-O-glucoside, cyanidin and quercetin identification is based on retention time
correspondence to peaks # 2, 6 and 7, respectively and the literature referring their presence in
Pistacia leaves, according to MS data peak #2 is malvidin3-O-glucoside (or galactoside) with a
molecular ion MW=493 g/mol; quercetin-3-O-glucoside identification is based on retention time
correspondence to peak # 4, compound color, MS, NMR and the literature referring its presence in
Pistacia leaves