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Chemical and biomolecular analyses to discriminate three taxa of Pistacia genus from Sardinia Island (Italy) and their antifungal activity
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[A. Marengo, A.Piras, D. Falconieri, S. Porcedda, P. Caboni, P. Cortis, C. Foddis, C.
Loi, M.J. Gonçalves, L. Salgueiro, A. Maxia, Chemical and biomolecular analyses to
discriminate three taxa of Pistacia genus from Sardinia Island (Italy) and their
antifungal activity, Natural Product Research,
DOI:10.1080/14786419.2017.1378211]
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1Arianna Marengoa, Alessandra Pirasb, Danilo Falconiericd, Silvia Porceddab, Pierluigi
2
Cabonia, Pierluigi Cortisa, Caterina Foddisc, Claudia Loia, Maria José Gonçalvese, Lígia
3
Salgueiroe, Andrea Maxiaa*
4 5
Affiliations
6 a
Department of Life and Environmental Sciences, Botany section, University of Cagliari, Viale 7
Sant’Ignazio da Laconi 13, 09123 Cagliari, Italy; 8
b
Department of Chemical and Geological Sciences, University of Cagliari, Cittadella Universitaria, 9
SP 8, Monserrato – Sestu km 0.700, 09042, Monserrato (CA), Italy; 10
c
Co.S.Me.Se – Consorzio per lo Studio dei Metaboliti Secondari, Viale Sant’Ignazio, 13, 09123 11
Cagliari, Italy. 12
d
Istituto Tecnico Industriale M. Giua, via Montecassino, 09134 Cagliari, Italy 13
e
CNC.IBILI, Faculty of Pharmacy, University of Coimbra, Azinhaga de S. Comba 3000-354 14
Coimbra, Portugal 15
16
*Corresponding author: a.maxia@unica.it 17
Chemical and biomolecular analyses to discriminate three taxa of Pistacia genus
19from Sardinia Island (Italy) and their antifungal activity
2021
Abstract
22
This work reports the results and the comparison concerning the chemical and biomolecular 23
analyses and the antifungal activity of three wild Pistacia species (Anacardiaceae) from Sardinia. 24
Volatile oils from leaves and twigs of Pistacia x saportae, P.lentiscus and P. terebinthus 25
were characterized using GC-FID and GC-MS techniques and tested against some fungal strains. 26
Two DNA nuclear regions (ITS and 5S-rRNA-NTS) were amplified through PCR technique and 27
sequenced. 28
The three Pistacia have similar chemical profile, although there are some important 29
quantitative differences. 30
The analysis of ITS and 5S-rRNA-NTS regions, reveals a species-specific nucleotide 31
variation among the three taxa. This method could emerge as a powerful tool for the species 32
identification, especially because the discrimination of these three taxa appears difficult for non-33
expert botanists. 34
Concerning the anti-fungal activity, P.lentiscus and P. x saportae show the highest activity 35
against Cryptococcus neoformans, with a MIC value of 0.32 μL/mL. 36
37
Key Words: Pistacia genus, essential oil, ITS, 5S-rRNA-NTS, antifungal activity
38 39
1. Introduction
40
The genus Pistacia belongs to the Anacardiaceae family. It contains about 12 taxa, shrubs or trees 41
with resinous bark, which are distributed from the Mediterranean area to the Eastern Asia, Southern 42
USA and Mexico (Yi et al. 2008; Bozorgi et al. 2013; Xie et al. 2014). 43
It is represented in Sardinia Island (Italy) by three taxa: P. lentiscus L., P. terebinthus L. and 44
their hybrid P. x saportae Burnat (Pignatti 1982; Arista et al. 1990). In P. lentiscus and P. 45
terebinthus macroscopic morphological differences are noticeable. On the other hand, their hybrid, 46
P. x saportae, reports characteristics of both species, for this reason the discrimination of these 47
species appear to be difficult for non-expert botanists (Tutin et al. 1968; Arista et al. 1990; Werner 48
et al. 2001). 49
In the Mediterranean area and in Sardinia, - characterized by a well-established traditional 50
culture - these species are traditionally used, for the treatment of several ailments. Moreover, also 51
food uses are reported and Pistacia essential oils are used in cosmetic, pharmaceutical and food 52
industries (Ballero et al. 2001; Atzei 2003; Loi et al. 2004; Lancioni et al. 2007; Maxia et al. 2008; 53
Aissi et al. 2016). To the authors’ knowledge, no traditional uses of P. x saportae are reported. 54
This work reports the results and the comparison concerning the chemical and biomolecular 55
analyses and the antifungal activity of the three Pistacia species collected from Sardinia. 56
The phytochemical data and antifungal activity of the essential oil of P. terebinthus from 57
Sardinia has been already published by Piras et al. (Piras et al. 2017). In this study P. lentiscus and 58
P. x saportae samples were collected in the same period and from the same locality and then 59
analysed in the exact same approach operated by Piras et al. (2017) for the analyses of P. 60
terebinthus. We aim at evaluating and comparing the chemical data and antifungal activity of these 61
three species from Sardinia. 62
P. lentiscus and P. terebinthus are traditionally used also in the treatment of mycosis. In 63
spite of that, there are very few works focusing on oil activities on phyto-pathogenic fungi (Duru et 64
al. 2003; Ismail et al. 2013; Piras et al. 2017; Rauf et al. 2017). In return, several phytochemical 65
studies indicating hydrocarbon and oxygenated monoterpenes as the major chemical constituents in 66
Pistacia essential oils (Castola et al. 2000; Duru et al. 2003; Amhamdi et al. 2009; Bozorgi et al. 67
2013; Ismail et al. 2013; Aissi et al. 2016; Pulaj et al. 2016; Rauf et al. 2017), are performed. Few 68
studies of these species from Sardinia were executed, and in particular P. x saportae essential oil 69
was here analysed for the first time (Picci et al. 1987; Congiu et al. 2002; Usai et al. 2006; Barra et 70
al. 2007; Maxia et al. 2011; Foddai et al. 2015; Piras et al. 2017). 71
As far as we know, it is difficult to find a molecular marker specific for P. x saportae 72
(Werner et al. 2001; Yi et al. 2008; Xie et al. 2014). Here, the nucleotide composition of the nuclear 73
ribosomal DNA (nrDNA) internal transcribed spacer (ITS) gene and the 5S-rRNA gene spacer 74
region (5S-rRNA-NTS) were investigated in order to search for biomolecular markers of the three 75
analysed taxa. Until now ITS sequences of all the three taxa are deposited in GenBank (NCBI), 76
none of them are from Sardinia. In this work, P. lentiscus, P. terebinthus and P. x saportae 5S-77
rRNA-NTS has been sequenced for the first time. 78
The aim of this work is to characterize and discriminate the three above-mentioned Pistacia 79
taxa from Sardinia and to evaluate and compare the potential antifungal activity of their essential 80
oils. This could be useful in the species identification, taking into account that sometimes in plants, 81
hybrid identification is difficult. Even if morphological differences are detectable, for non-expert 82
botanist it is difficult to distinguish the three taxa, so these data can be an additional tool to 83
discriminate closely related species. Additionally, it could be particularly useful to authenticate 84
dried and processed herbal materials, when the morphological parameters cannot be evaluated 85
(Arista et al. 1990; Galimberti et al. 2013). 86
87
2. Results and discussion
88 89
2.1. Essential oils composition
90
Essential oil yields (expressed as mass percentages, on dry basis) of P. lentiscus L., P. terebinthus 91
and P. x saportae are, respectively, 0.33 %, 0.25 % and 0.27 %. A total of 30 components, 92
accounting for (97.9 98.8) % of the oil composition (Table S1), were identified in the essential 93
oils isolated from leaves and twigs of the three taxa. 94
The oils contained very high amounts of hydrocarbon monoterpenes (63.0 90.9) %. 95
Differently from the other two oils, P. lentiscus oil also contained relatively high amounts of 96
oxygenated monoterpenes (29.3 %). 97
As can be seen from Table S1, the constituents of the three oils were the same; however, 98
some important quantitative differences were present. The dominant components in P. lentiscus oil 99
were: terpinen-4-ol (25.2 %), -phellandrene (11.9 %), -phellandrene (10.2 %) and -terpinene 100
(10.1 %). 101
P. terebinthus and P. x saportae oil samples were found to be rich in -pinene (35 and 102
30.3%). Other main constituents in the P. x saportae essential oil were (Z)--ocimene (26.7 %) and 103
(E)--ocimene (11.1 %); whereas the second major constituent in the P. terebinthus oil was 104
terpinolene (35.2 %). 105
These data are confirmed by Principal Component Analysis (PCA), results are reported in 106
Fig.S1 caption in Supplementary material section. 107
Our results are partially in accordance with literature data on Sardinian P. lentiscus and P. 108
terebinthus essential oils. Most of the identified compounds have been already reported in the two 109
species from Sardinia (Picci et al. 1987; Congiu et al. 2002; Usai et al. 2006; Barra et al. 2007; 110
Maxia et al. 2011). 111
In a study by Barra et al. (2007), P. lentiscus samples, collected in different seasons and 112
from different localities in Sardinia, were analysed. It emerged that, α-pinene, β-myrcene, p-cymene 113
and terpinen-4-ol were the main compounds found in all the samples. Nevertheless, differences in 114
the concentration of single compounds of the essential oil are noticeable, both among P. lentiscus 115
single plants of the same area and between samples collected in different period from different 116
stations. The higher terpinen-4-ol concentration detected in this work is in accordance with results 117
of samples collected in May (Barra et al. 2007). The P. lentiscus sample analysed in this work is 118
characterized by low level of α-pinene, which is usually predominant in the essential oil of this 119
species. Similar results are obtained from a sample from Orroli (CA, Sardinia), collected in May (α-120
pinene: 8.8 %) (Barra et al. 2007). Another sample collected in Costa Rey (CA, Sardinia), is 121
characterized by a very low level of α-pinene (0.2 %), while the predominant components are β-122
pinene (18.7 %), β-phellandrene (12.6 %) and β-caryophyllene (13.2 %) (Congiu et al. 2002). The 123
high percentage of terpin-4-ol and the lower content of α-pinene are supported by Picci et al. (1987) 124
in a sample from Sardinia. 125
The analysis of P. terebinthus from Calagonone (NU, Sardinia), by Usai et al. (2006), 126
confirms α-pinene, β-pinene and β-phellandrene as major compounds of the essential oil obtained 127
from the leaves and the twigs. However, terpinolene, the predominant compound of the oil analysed 128
in this work (33.8 %), is present in very low amount in the Calagonone sample (1.3 % in leaves and 129
1.7 % in twigs). 130
Several works on P. lentiscus and P. terebinthus harvested in different sites in the 131
Mediterranean area are present (Castola et al. 2000; Duru et al. 2003; Vidrich et al. 2004; Kivçak & 132
Akay 2005; Amhamdi et al. 2009; Said et al. 2011; Ismail et al. 2013; Negro et al. 2014; Aissi et al. 133
2016; Pulaj et al. 2016). It is interesting to notice the variability of the essential oil chemical 134
composition. 135
To the best of our knowledge, this is the first time that the essential oil chemical 136
composition of P. x saportae has been investigated. 137
138
2.2. Sequences information and cluster analysis
139
The nucleotide composition of ITS and 5S-rRNA-NTS regions was determined for each individuals 140
of each Pistacia taxa. ITS gene sequence contains approximately 710-723bp, the total length of the 141
5S-rRNA gene spacer region is 413-426 bp. The 5S-rRNA-NTS region exhibits a lower conserved 142
sites percentage (86.7 % vs. 90.8 %) and a higher variables sites percentage (13.3 % vs. 9.1 %), 143
compared to ITS. 144
P. lentiscus and P. x saportae ITS sequences are very similar, there are only few nucleotide 145
variations at the far ends. It is possible to find some species-specific nucleotide variations in P. 146
terebinthus ITS sequence, compared to the other two taxa. 147
5S-rRNA-NTS sequences of each individuals were obtained. Unfortunately, it was more 148
difficult to obtain good quality sequences for P. x saportae and the unclear nucleotides were 149
replaced with an “N”. From the alignment, it is possible to notice some nucleotide variations among 150
the three taxa. Additionally, the comparison of 5S-rRNA-NTS regions reveals the presence of gaps 151
in some sequences. For example, P. lentiscus presents a gap between positions 134/135 bp and 152
240/241 bp. Moreover, P. terebinthus is characterized by a gap between positions 279/280 bp. P. 153
lentiscus and P. x saportae both show a gap, respectively, between positions 353/354 and 349/350. 154
The Neighbor Joining (NJ) and the Unweighted Pair Group Method with Arithmetic Mean 155
(UPGMA) trees were built both from the ITS and the 5S-rRNA-NTS sequences alignment (Figure 156
1, A, B, C and D). From the alignment of the individuals sequences of each taxa it is possible to 157
notice the presence of few intra-specific nucleotide variation (data not shown). Additionally, for 158
both regions, NJ and UPGMA trees determine the formation of two main clusters. One containing 159
all the individuals belonging to P. lentiscus and P. x saportae and one containing only the 160
individuals belonging to P. terebinthus, for ITS (Figure 1, A and B). One containing all the 161
individuals belonging to P. lentiscus and P. terebinthus and one containing only the individuals 162
belonging to P. x saportae, for 5S-rRNA-NTS (Fig. 1, C and D). Moreover, Fig. 1 A and 1 B show 163
that P. lentiscus and P. x saportae cluster into two different groups, from Fig. 1 C and 1 D it is 164
possible to notice that P. lentiscus and P. terebinthus individuals are grouped into two clusters, 165
according to taxa origin. 166
The three Pistacia 5S-rRNA-NTS regions were here sequenced for the first time. On the 167
other hand, the obtained ITS sequences of P. terebinthus are in agreement with those deposited in 168
GenBank (EF193086); only one nucleotide variation is present (position 138). Few variation in the 169
nucleotide composition among the deposited P. lentiscus and P. x saportae ITS sequences are 170
present, this suggest that ITS sequence is less stable for these taxa (Yi et al. 2008; Xie et al. 2014). 171
According to the obtained results, 5S-rRNA-NTS region shows a higher nucleotide 172
composition variation among the three taxa, compared to the ITS sequence. These results are 173
confirmed by the hierarchical clustering analysis. In fact, the dendrograms suggest that the ITS 174
region is able to discriminate P. terebinthus from the other two taxa. P. lentiscus and P. x saportae 175
ITS sequences are very similar, with only few nucleotide variations at the far ends, so, in this case 176
ITS gene can’t be considered a good molecular maker to discriminate between these two taxa. 177
This is partially in accordance with previous phylogenetic works, that, in most cases P. x 178
saportae, clustered together with P. lentiscus (Yi et al. 2008; Xie et al. 2014). The difficulty to find 179
a molecular marker specific for P. x saportae is due to the similarity of its sequences to the other 180
Pistacia sequences (Yi et al. 2008; Xie et al. 2014). 181
The nucleotide variability of 5S-rRNA-NTS region among the three taxa may be useful for 182
their discrimination. It is possible to notice some nucleotide variations able to make a 183
discrimination among the three taxa. This result is confirmed by the hierarchical clustering analysis. 184
This gene could be used as a molecular marker for the discrimination of P. lentiscus, P. terebinthus 185
and their hybrid P. x saportae. 186
[Figure 1 near here] 187
188
2.3. Antifungal activity
189
In addition to results obtained for P. terebinthus from Piras et al., the antifungal activity of P. 190
lentiscus and P. x saportae essential oils from Sardinia is presented in Table S2. The activity against 191
T. verrucosum was also evaluated. The highest antifungal activity was observed against 192
Cryptococcus neoformans with MIC value of 0.32 μL/mL for both P. lentiscus and P. x saportae. 193
For dermatophyte strains MIC values varied from (1.25 to 2.5) µL/mL, while for Candida strains 194
the oils showed low activity, particularly those of Pistacia terebinthus, with MICs values of 5 195
μL/mL. For most yeasts and dermatophytes, MIC was equivalent to MLC suggesting a fungicidal 196
effect of the oils. 197
Antifungal activity of Pistacia lentiscus, P. terebinthus, P. vera essential oils was previously 198
reported against eight phyto-pathogenic fungi (Ismail et al. 2013). The authors reported that the 199
essential oils showed significant inhibition of fungal growth; this study also indicated that the 200
antifungal activity is variable depending of the fungal strain and the tested oils. 201
Moreover, the antifungal activity of P. lentiscus and P. terebinthus essential oils against 202
some agricultural pathogens (Pythium ultimum, Rhizoctonia solani, Aspergillus flavus) has been 203
documented (Duru et al. 2003; Kordali et al. 2003; Barra et al. 2007). 204
Antimicrobial activity of Pistacia vera was also reported against Gram negative and Gram 205
positive bacteria and Candida albicans (Alma et al. 2004). The oil showed variable levels of 206
inhibition with good activity against C. albicans. 207
In what concern P. lentiscus and P. x saportae it is the first time that the antifungal activity 208
of these oils was evaluated against yeast and filamentous fungi. Our results on P. lentiscus and P. x 209
saportae showed the highest antifungal activity against Cryptococcus neoformans, which is cause, 210
together with C. gattii, of Crytococcosis, of invasive fungal infection (Brizendine et al. 2011). 211
The activity of P. lentiscus may be due to the contribution of its main compound, terpinen-212
4-ol, which is a terpene alcohol that exhibits in vitro antifungal properties against Cryptococcus 213
neoformans, Candida spp., Malassezia spp., Rhodotorula spp., Trichosporon spp., Aspergillus 214
spp., Penicillium spp., and dermatophytes (Hammer et al., 2003, 2004; Carson et al., 2006; Morcia 215
et al., 2012). In what concerns P. x saportae the antifungal activity can be attributed to the main 216
compounds, α-pinene and (Z)-β-ocimene. Different studies have shown effective antifungal 217
activity to these compounds (Zuzarte et al., 2011; Gonçalves et al., 2012). 218
219
3. Conclusions
220
In conclusion, the chemical analysis shows a quantitative variation of some compounds, among the 221
three taxa. Since no marker compounds have been found, the chemical analysis is not sufficient for 222
an easy discrimination of the three taxa. 223
The performed biomolecular analysis can be a powerful tool for the species identification. 224
Our results suggest that 5S-rRNA-NTS region presents an higher variability compared to ITS gene, 225
so it can be considered a good molecular marker for the three taxa. However, a combination of the 226
two sequences could discriminate the three taxa in an easier way, also because more comparable 227
ITS sequences are present in the database. The three taxa 5S-rRNA-NTS region has been here 228
amplified and sequenced for the first time. Further studies should be focused on the 5S-rRNA-NTS 229
sequencing of other Pistacia species in order to improve the sequences database. 230
Finally, the anti-fungal activity investigation highlights that P. lentiscus and P. x saportae 231
oils from Sardinia Island (Italy) revealed suitability to be incorporated in pharmaceutical 232
formulations for the prevention and treatment of fungal infections, particularly cryptococcosis. 233
These findings also justify the traditional use of Pistacia species as antiseptic. 234
Supplementary material:
The experimental section, including Tables and Figures, can be accessed as supplementary material. 235
236
Acknowledgments:
237
The authors wish to thank Co.S.Me.Se for financial support. 238
239
Conflict of interest:
240
The authors declare no conflict of interest. 241
242
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Figures captions:
Figure 1. Hierarchical cluster analysis of individuals ITS and 5S-rRNA-NTS sequences belonging to P. lentiscus, P. terebinthus, P. x saportae (numbers at the node indicate bootstrap values). Joining tree (A) and UPGMA tree (B) built with individuals ITS sequences. Neighbor-Joining tree (C) and UPGMA tree (D) built with individuals 5S-rRNA-NTS sequences.
SUPPLEMENTARY MATERIAL
Chemical and biomolecular analyses to discriminate three taxa of Pistacia genus
from Sardinia Island (Italy) and their antifungal activity
Arianna Marengoa, Alessandra Pirasb, Danilo Falconiericd, Silvia Porceddab, Pierluigi Cabonia, Pierluigi Cortisa, Caterina Foddisc, Claudia Loia, Maria José Gonçalvese, Lígia Salgueiroe, Andrea Maxiaa*
Affiliations
a
Department of Life and Environmental Sciences, Botany section, University of Cagliari, Viale Sant’Ignazio da Laconi 13, 09123 Cagliari, Italy;
b
Department of Chemical and Geological Sciences, University of Cagliari, Cittadella Universitaria, SP 8, Monserrato – Sestu km 0.700, 09042, Monserrato (CA), Italy;
c
Co.S.Me.Se – Consorzio per lo Studio dei Metaboliti Secondari, Viale Sant’Ignazio, 13, 09123 Cagliari, Italy.
d
Istituto Tecnico Industriale M. Giua, via Montecassino, 09134 Cagliari, Italy e
CNC.IBILI, Faculty of Pharmacy, University of Coimbra, Azinhaga de S. Comba 3000-354 Coimbra, Portugal
Abstract
This work reports the results and the comparison concerning the chemical and biomolecular analyses and the antifungal activity of three wild Pistacia species (Anacardiaceae)from Sardinia.
Volatile oils from leaves and twigs of Pistacia x saportae, P.lentiscus and P. terebinthus
were characterized using GC-FID and GC-MS techniques and tested against some fungal strains. Two DNA nuclear regions (ITS and 5S-rRNA-NTS) were amplified through PCR-based technique and sequenced.
The three Pistacia have similar chemical profile, although there are some important quantitative differences.
The analysis of ITS and 5S-rRNA-NTS regions, reveals a species-specific nucleotide variation among the three taxa. This method could emerge as a powerful tool for the species identification, especially because the discrimination of these three taxa appears difficult for non-expert botanists.
Concerning the anti-fungal activity, P.lentiscus and P. x saportae show the highest activity against Cryptococcus neoformans, with a MIC value of 0.32 μL/mL.
Experimental section
Plant material
Aerial parts of four individuals of each wild Pistacia taxa were collected from Baunei, Sardinia, in May 2015 and voucher specimen for each taxa was deposited at the Herbarium of Cagliari, University of Cagliari, Italy (no. 280 for P. lentiscus, no. 279a for P. terebinthus and no. 279b for P. x saportae). The fresh materials were air-dried in an oven (BINDER FP 115) at 40 °C, under forced ventilation for 48 h. A part of plant materials was used for the biomolecular analysis and another part was subjected to steam distillation in order to obtain the essential oil (EO).
Essential oil isolation and analysis
Isolation of essential oils by hydrodistillation was performed, as reported by Piras et al. (2017), from leaves and twigs, in a Clevenger-type apparatus for 3h. All essential oils were stored at 4 °C until use.
The samples were analyzed by using a gas chromatograph equipped with a flame ionization detector (GC-FID) to obtain the quantitative composition and by gas chromatography coupled to mass spectrometry (GC-MS) for constituents identification, according to Zuzarte et. al, 2013. Quantitative analyses of the extracts were performed using a gas chromatograph (Agilent 7890A, Palo Alto, CA, USA), equipped with a 30 m × 0.25 mm i.d. with 0.25 µm stationary film thickness DB-5 capillary column (Agilent J&W) and a FID. The following temperature program was used: from 60 ºC to 246 ºC at a rate of 3 ºC min-1 and then held at 246 ºC for 20 min (total analysis time 82 min). Carrier gas helium (purity ≥ 99.9999 % – Air Liquide Italy); flow rate, 1.0 mL min-1
; injector temperature, 250 ºC and detector temperature, 300 ºC were the other operating conditions. Injection of 1 μL of diluted sample (1:100 in hexane, w/w/) was performed with 1:10 split ratio, using an autosampler (Agilent, Model 7683B).
GC-MS analyses were carried out using a gas chromatograph (Agilent 6890N) equipped with a 30 m 0.25 mm i.d. with 0.25 µm stationary film thickness HP-5ms capillary column (Agilent J&W) coupled with a mass selective detector having an electron ionization device, EI, and a quadrupole analyzer (Agilent 5973). The temperature program and the chromatographic operating conditions (except detector) were the same used for GC-FID. The MS conditions were as follows: MS transfer line temperature 240 °C; EI ion source temperature, 200 °C with ionization energy of 70 eV; quadrupole temperature 150 °C; scan rate, 3.2 scan s-1 at m/z scan range, (30 to 480). To handle and process chromatograms and mass spectra was used the software MSD ChemStation (Agilent, rev. E.01.00.237).
Constituents of the samples were identified by comparing: mass spectra fragmentation patterns with those of a computer library (NIST/EPA/NIH Mass spectral library 2005; Adams 2007), and linear retention indices (RI), based on a homologous series of C8-C26 n-alkanes, with those reported in literature (Adams 2007). Relative amounts of individual components were calculated based on GC peak areas without FID response factor correction. Three replicates were performed for each sample. The average of these three values and the standard deviation were determined for each compound identified.
Genomic DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from ten milligrams of dried ground leaves by using the Eurogold Plant DNA Mini Kit (Euroclone, Pero, Italy). Approximately 20 ng of isolated genomic DNA was used as a template for PCR amplification.
The ITS region was amplified with forward primer ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and reverse primer ITS4 (5’- TCCTCCGCTTATTGATATGC-3’) (White et al. 1990).
The 5S-rRNA-NTS region was amplified with forward primer 5S-P1
(5’-GTGCTTGGGCGAGAGTAGTA-3’) and reverse primer 5S-P2
(5’-TTAGTGCTGGTATGATCGCA-3’) (Rubiolo et al. 2009; Gnavi et al. 2010).
PCR reaction mixture contained: 2.5 μL of 10× PCR buffer (Thermo-Scientific, Waltham, MA USA), 0.2 mMdeoxynucleoside triphosphates (dNTPs), 20 pmol of forward and reverse primers, and 0.5 U of Taq DNA polymerase (Thermo-Scientific, Waltham, MA USA), in a final volume of 25 μL. PCRs were carried out in a T-Gradient Thermalcycler (Biorad, Hercules, CA, USA). Cycling conditions consisted of an initial 4 min at 94 °C, followed by 30 s of denaturing at 94 °C, 45 s of annealing at 53 °C for ITS primers and 52 °C for 5S-rRNA-NTS primers and 45 s of elongation at 72 °C, repeated for 35 cycles with 10 min of final extension at 72 °C. PCR products were separated by 1.5 % agarose gel electrophoresis and visualised by ethidium bromide staining under UV. PCR products were purified following the manufacturer’s protocol (Agencourt® AMPure® kit) and employed as a template for sequencing. The amplicons were sequenced in both directions in a ABI 3730 XL automated sequencer, with the primers used for amplification (IGA Technology Services, Udine, Italy). Obtained sequences were deposited in NCBI (National Center for Biotechnology Information) database(Coordinators 2017) (TableS3).
Statistical elaboration
Statistical analysis were performed using SPSS 15.0 (IBM Corporation) software.
Chemical data were processed through Principal Component Analysis (PCA), in order to reduce the multivariate space in which objects were distributed, the chromatographic area percentages of the 30 compounds identified by GC-MS have been used as variables.
Gene sequences were analysed and aligned with MEGA7 software (ClustalW) using default parameters to evaluate the integrity of each sequence. A multiple sequences alignment was performed using, respectively, ClustalW with Gap Opening and Gap Extension Cost values of 15
trees were calculated and built using the MEGA7 software with the Bootstrap method (1000 replicates) and K2P model, considering gaps in the Partial Deletion option.
Fungal organisms
The yeasts used to evaluate the antifungal activity of the essential oils were Candida spp. clinical strains: Candida guilliermondii MAT23 and C. krusei H9, isolated from recurrent cases of vulvovaginal candidiasis; three American Type Culture Collection (ATCC) reference strains (C. albicans ATCC 10231, C. tropicalis ATCC 13803, and C. parapsilosis ATCC 90018) and one strain of Cryptococcus neoformans (Colección Española de Cultivos Tipo, CECT, C. neoformans type strain-1078).
Concerning the moulds, seven dermatophyte strains, three isolated from nails and skin (Epidermophyton floccosum FF9, Microsporum canis FF1, and Trichophyton mentagrophytes FF7) and four from CECT collection (M. gypseum 2908, T. mentagrophytes var. interdigitale 2958, T. verrucosum 2992, and T. rubrum 2794), were tested.
The strains were stored in Sabouraud dextrose broth (BioMérieux) with 20 % glycerol at – 80 ºC. Previously to each test, all the strains were subcultured in Sabouraud dextrose agar (SDA; Becton-Dickinson), to certify optimal growth and purity.
Antifungal activity
The antifungal activity was evaluated according to Piras et al. (Piras et al. 2017). Minimum inhibitory concentrations (MICs) of the essential oils, were determined by broth macrodilution methods based on the Clinical and Laboratory Standards Institute (CLSI) reference protocols M27-A3, S3 (CLSI 2008a), and M38-A2 (CLSI 2008b), for yeasts and filamentous fungi, respectively.
Two-fold serial dilutions of the essential oils, ranging from 0.16 to 10 μL/mL, were prepared in DMSO (Sigma) with a maximum concentration of 1 % (v/v). The test tubes were incubated aerobically at 35 ºC for 48 h or 72 h (Candida spp./Cryptococcus neoformans) and at 30
ºC for seven days (dermatophytes). MIC was considered the lowest concentration resulting in 100 % growth inhibition. In addition, a reference antifungal compound, fluconazole (Pfizer), was used to monitor the sensitivity of the tested microorganisms. Growth and sterility controls were performed in RPMI (Roswell Park Memorial Institute) medium.
The minimum lethal concentrations (MLCs) were determined after incubation time (48 h for Candida spp., 72 h for Cryptococcus neoformans and seven days for dermatophytes), by removing 20 μL from all tubes deprived of visible growth to SDA plates. The MLC was defined as the lowest concentration showing a total absence of growth on SDA plates, resulting from the subculture of MIC plates. All determinations were performed in duplicate and three independent experiments were run with concordant results. A range of values is presented when different results were obtained.
References
CLSI. 2008a. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approv Stand third ed, M27-A3, Wayne, PA Clin Lab Stand Institute.
CLSI. 2008b. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approv Stand third ed, M27-A3, Wayne, PA.
Coordinators NR. 2017. Database Resources of the National Center for Biotechnology Information.
Nucleic Acids Res. 45:D12–D17.
https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkw1071
Gnavi G, Bertea CM, Maffei ME. 2010. PCR , sequencing and PCR –RFLP of the 5S-rRNA-NTS region as a tool for the DNA fingerprinting of medicinal and aromatic plants. Flavour Fragr J. 25:132–137.
Piras A, Marzouki H, Maxia A, Marengo A, Falconieri D, Gonçalves MJ, Cavaleiro C, Piras A, Marzouki H, Maxia A, Marengo A. 2017. Chemical characterisation and biological activity of leaf essential oils obtained from Pistacia terebinthus growing wild in Tunisia and Sardinia Island. Nat
Prod Res. 6419. http://dx.doi.org/10.1080/14786419.2017.1289204
Rubiolo P, Matteodo M, Bicchi C, Gnavi G, Bertea C, Maffei M. 2009. Chemical and Biomolecular Characterization of Artemisia umbelliformis Lam., an Important Ingredient of the Alpine Liqueur “ Genepi#.” J Agric Food Chem. 57:3436–3443.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc A Guid to Methods Appl. 3:315–322.
Table S1. Retention time (TR), linear retention index (RI) and chromatographic area percentages of compounds identified in leaf essential oil of P. lentiscus and P. terebinthus and P. x saportae. Data related to P. terebinthus come from the article by Piras et al. (Piras et al. 2017).
Compounds listed in order to their elution on the HP-5 column. Mean and standard deviation of 3 samples. Standard deviation were recorded, values were insignificant and omitted from the Table to avoid congestion; tr: trace, i.e.,
percentage lower than 0.1 %.
TR RI
P. lentiscus P. terebinthus P. x saportae Compound
4.963 925 0.2 0.4 1.3 tricyclene 5.050 929 0.4 - tr -thujene 5.230 937 7.6 35.0 30.3 -pinene 5.593 952 1.0 2.4 5.1 camphene 6.222 976 6.3 tr tr sabinene 6.323 979 0.9 4.5 3.3 -pinene 6.686 992 2.3 1.3 1.1 myrcene 7.000 1002 - 0.5 tr meta-mentha-1(7),8-diene 7.106 1006 11.9 0.5 0.1 -phellandrene 7.473 1018 7.9 3.6 0.5 -terpinene 7.490 1019 - 2.1 tr 1-para-menthene 7.717 1026 1.2 tr 0.2 ortho-cymene 7.871 1031 10.2 4.5 2.9 -phellandrene 8.199 1041 - 0.2 26.7 (Z)--ocimene 8.526 1051 - 0.3 11.1 (E)--ocimene 8.863 1061 10.1 0.4 0.7 -terpinene 9.934 1089 3.0 35.2 1.6 terpinolene 13.348 1178 25.2 0.3 0.9 terpinen-4-ol 13.895 1190 2.7 2.3 1.7 -terpineol 17.859 1285 1.4 1.4 1.4 bornyl acetate 23.368 1418 1.3 0.7 4.4 (E)-caryophyllene 24.709 1452 tr 0.3 0.5 -humulene 25.645 1475 tr tr 0.5 -muurolene 25.847 1480 1.7 1.4 1.1 germacrene D 26.411 1493 tr - 0.7 -selinene 26.620 1498 0.4 tr 0.4 -muurolene 26.992 1508 0.5 tr tr germacrene A 27.552 1523 1.2 0.6 1.6 -cadinene 31.988 1640 0.7 tr 0.3 epi--cadinol 32.439 1652 0.7 tr tr -cadinol Total identified 98.8 97.9 98.4 Hydrocarbon monoterpenes, HM 63.0 90.9 84.9 Oxygenated monoterpenes, OM 29.3 4.0 4.0 Hydrocarbon sesquiterpenes, HS 5.1 3.0 9.2 Oxygenated sesquiterpenes, OS 1.4 tr 0.3
Table S2. Antifungal activity (MIC and MLC) of Pistacia spp. and of fluconazole for yeasts and
dermatophyte. Data related to P. terebinthus come from the article by Piras et al. (Piras et al. 2017). Strains Pistacia lentiscus Pistacia terebinthus Pistacia x saportae Fluconazole
MIC / (μL/mL) MLC / (μL/mL) MIC / (μL/mL) (Piras et al. 2017) MLC / (μL/mL) (Piras et al. 2017) MIC / (μL/mL) MLC / (μL/mL) MIC / (μg/mL) MLC / (μg/mL)
Candida albicans ATCC
10231 2.5 2.5 5 5 2.5 5 1 >128 Candida tropicalis ATCC 13803 2.5 2.5-5 5 5 5 5 4 >128 Candida krusei H9 2.5 2.5 5 5 2.5 2.5 64 64-128 Candida guillermondii MAT23 2.5 2.5 5 5 2.5 2.5 8 8 Candida parapsilosis ATCC 90018 2.5 5 5 5 5 5 <1 <1 Cryptococcus neoformans CECT 1078 0.32 0.64 1.25 2.5 0.32 0.64-1.25 16 128 T. mentagrophytes FF7 1.25 1.25 2.5 2.5 1.25-2.5 2.5 16-32 32-64 T. mentagrophytes var. interdigitale CECT 2958 2.5 5 2.5 5 2.5 2.5 128 ≥128 Trichophyton rubrum CECT 2794 1.25 1.25 1.25 1.25 1.25 1.25 16 64 T. verrucosum CECT 2992 2.5 2.5 2.5 5 2.5 2.5 128 >128 Microsporum canis FF1 1.25 2.5 1.25 1.25 1.25 1.25 128 128 M. gypseum CECT 2905 2.5 2.5 2.5 2.5 1.25 1.25 128 >128 Epidermophyton floccosum FF9 1.25 1.25 1.25 1.25 1.25 1.25 16 16
Table S3. GeneBank accession numbers for each sequence obtained for each plant sample.
Species ITS 5s-rRNA_NTS
P. lentiscus 1 KY549572 KY563233
P. lentiscus 2 KY549573 KY563234
P. lentiscus 3 KY549574 KY563235
P. lentiscus 4 KY549575 KY563236
P. terebinthus 1 KY549576 KY563237
P. terebinthus 2 KY549577 KY563238
P. terebinthus 4 KY549579 KY563240
P. x saportae 1 KY549568 KY563229
P. x saportae 2 KY549569 KY563230
P. x saportae 3 KY549570 KY563231
Fig. S1. PCA analysis of the three Pistacia taxa, the chromatographic area percentages of the 30
compounds identified by GC-MS in their essential oils have been used as variables. (A) Score plot of the three Pistacia taxa. (B) Loading plot of the variables.
The PCA statistical analysis was performed in order to try to differentiate the three Pistacia samples and to search for discriminating variables.
As shown in Fig.A the first component (PC1), which explains the 64.6% of the variation, separates P. x saportae and P. terebinthus samples from P. lentiscus. Additionally, P. x saportae and P. terebinthus are discriminated by the second component (PC2, 35.4% of the variation explained). Fig. B reports the influence of the compounds identified in the essential oils in the distribution of the samples in the loading plot. It is noteworthy that, as reported in section 2.1 (Results and discussion-Essential oils composition), among the compounds that positively explain PC1, α-phellandrene, β-phellandrene and terpinen-4-ol are present in an higher amount in P. lentiscus. At the same time, α-pinene and α-humulene are present in a comparable amount in P. x saportae and P. terebinthus and are negatively correlated with PC1. Additionally, it is possible to observe a positive correlation of (Z)-β-ocimene and (E)-β-ocimene and a negative correlation of terpinolene with PC2. These compounds are more abundant in P. saportae and P. terebinthus, respectively. This analysis gives information on the most representative compounds in each sample. However, since only one data per taxa is available and the three Pistacia have similar chemical profile it is difficult to discriminate them by only evaluating their essential oils compositions.
