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Chemical Composition and Allelopathic Potential of Essential Oils Obtained from Acacia cyanophylla Lii n dl . Cultivated in Tunisia by Asma El Ayeb-Zakhama a ), Lamia Sakka-Rouis a ), Afifa Bergaoui b ), Guido Flamini* c ), Hichem Ben Jannet b ), and Fethia Harzallah-Skhiri* a ) a
) Laboratory of Genetics Biodiversity and Valorisation of Bioresources (LR11ES41), Higher Institute of Biotechnology of Monastir, University of Monastir, Rue Tahar Haddad, 5000 Monastir, Tunisia (phone: þ 216-73-463711; 216-73-465404; e-mail: [email protected]) b ) Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity, Medicinal Chemistry and Natural Products, Faculty of Sciences of Monastir, University of Monastir, Tunisia c ) Dipartimento di Farmacia, University of Pisa, Via Bonanno 33, I-56126 Pisa (e-mail: [email protected])
Acacia cyanophylla Lindl. (Fabaceae), synonym Acacia saligna (Labill.) H. L.Wendl., native to West Australia and naturalized in North Africa and South Europe, was introduced in Tunisia for rangeland rehabilitation, particularly in the semiarid zones. In addition, this evergreen tree represents a potential forage resource, particularly during periods of drought. A. cyanophylla is abundant in Tunisia and some other Mediterranean countries. The chemical composition of the essential oils obtained by hydrodistillation from different plant parts, viz., roots, stems, phyllodes, flowers, and pods (fully mature fruits without seeds), was characterized for the first time here. According to GC-FID and GC/MS analyses, the principal compound in the phyllode and flower oils was dodecanoic acid (4), representing 22.8 and 66.5% of the total oil, respectively. Phenylethyl salicylate (8; 34.9%), heptyl valerate (3; 17.3%), and nonadecane (36%) were the main compounds in the root, stem, and pod oils, respectively. The phyllode and flower oils were very similar, containing almost the same compounds. Nevertheless, the phyllode oil differed from the flower oil for its higher contents of hexahydrofarnesyl acetone (6), linalool (1), pentadecanal, a-terpineol, and benzyl benzoate (5) and its lower content of 4. Principal component and hierarchical cluster analyses separated the five essential oils into four groups, each characterized by its main constituents. Furthermore, the allelopathic activity of each oil was evaluated using lettuce (Lactuca sativa L.) as a plant model. The phyllode, flower, and pod oils exhibited a strong allelopathic activity against lettuce.
Introduction. – Acacia Mill., belonging to the subfamily Mimosoideae, is the second largest genus in the family Fabaceae [1], with ca. 1350 species [2]. The Acacia species range in size from small shrubs to large trees and are ecologically important as ÐpioneerÏ species, because they rapidly establish cover after major natural disturbances [3]. Researchers showed that Acacia trees are known as a source of components with bioactive properties, suggesting a large inhibitory potential in this genus. The main use of Acacia species is as a fodder source and in soil stabilization, but it is also used in the traditional medicine for its hypoglycemic [4], antibacterial [5], and anti-inflammatory [6] activities. Other authors reported its spasmogenic and vasoconstrictor actions [7] and also its cytotoxic [8] and antioxidant [9] activities. Little is known about the volatile chemistry of most species of Acacia [10]. In fact, Seigler [10] reported on the phytochemistry of Acacia and mentioned only plant volatiles obtained from A. Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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farnesiana (L.) Willd. Floral volatiles have been described from A. farnesiana, A. berlandieri Benth., A. rigidula Benth. (all three native to North America) [11], A. praecox Griseb., A. caven (Molina) Molina, A. Aroma Hook & Arn. (all three from Argentina) [12] [13], and from A. karroo Hayne, an African species [14]. More recently, floral and leaf volatiles of Acacia cyclops A.Cunn. ex G.Don have been studied [15]. A. cyanophylla Lindl. (syn. A. saligna (Labill) H. L.Wendl) is an Australian tree introduced in Tunisia for the first time in 1930 for rangeland rehabilitation, particularly in the semiarid zones and is now extensively grown and cultivated throughout the territory [16]. A. cyanophylla plantations are located in the subhumid and semiarid bioclimatic stages, as the species is highly resistant to drought and salinity [17]. To the best of our knowledge, until now, the chemical composition of A. cyanophylla essential oil has not been reported. Hence, the chemical composition of the root, stem, phyllode, flower, and pod (fully mature fruit without seeds) essential oils (EOs) was characterized and their allelopathic potential evaluated. Results and Discussion. – Essential-Oil Yield. The mean oil yields for the five investigated plant organs of A. cyanophylla varied significantly from 0.01% (w/w) for the roots to 0.08% for the pods. Phyllodes, stems, and flowers afforded 0.03, 0.04, and 0.06% of essential oil, respectively. Essential-Oil Composition. Chromatographic analysis (GC-FID and GC/MS) of the EOs extracted from the different organs of A. cyanophylla collected in spring (roots, stems, phyllodes, and flowers) or in summer (pods), allowed the identification of 51 compounds (Table), representing 91.9 – 97.7% of the total oil composition. The chemical structures of some of the major compounds are shown in Fig. 1. Root-Oil Composition. In total, 14 compounds were identified in the root oil, representing 97.7% of the total volatiles (Table). The main constituent was phenylethyl salicylate (51 1), 8; 34.9%), followed by benzyl benzoate (43, 5; 32.4%), 2-phenylethyl benzoate (46, 7; 19.4%), and hexahydrofarnesyl acetone (45, 6; 4.4%). For all other compounds, lower contents were detected (0.2 – 1.5%). Seven out of the 14 compounds were exclusive to this oil. Stem-Oil Composition. As in the root oil, also in the stem oil 14 compounds were identified (Table). The major ones were heptyl valerate (22, 3; 17.3%), tetradecanoic acid (44; 15.0%), benzyl butyrate (17, 2; 14.2%), dodecanoic acid (syn. lauric acid, 32, 4; 9.4%), pentadecane (30; 6.7%), compound 6 (6.6%), and heptadecane (40, 5.7%). The remaining constituents were present at amounts varying from 1.9 to 3.4%. Seven of the 14 compounds were found only in the stem oil. Phyllode-Oil Composition. In the Table, it can be seen that the A. cyanophylla phyllode oil, with 21 compounds identified, was the richest in constituents. Compounds 4 (22.8%), 6 (12.1%), and 5 (11.5%) as well as linalool (3, 1; 9.4%), heptadecan-2-one (47; 8.8%), and pentadecanal (41, 4.7%) were the most important ones. Only six compounds were exclusive to this oil, even if detected at low amounts (1.1 – 1.7%). Flower-Oil Composition. Among its 16 constituents, compound 4 dominated the composition of this oil, with a content of 66.5%. Compound 6, heptadecan-2-one (47), 1)
Italic numbers in parentheses refer to the entries in the Table.
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Table. Chemical Composition of the Essential Oils ( EOs) Extracted from Different Organs of Acacia cyanophylla Lindl. Cultivated in Tunisia Entry Compound name and class
LRI a ) Content [%] b ) Root EO Stem EO Phyllode EO Flower EO Pod EO
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Octanol Undecane Linalool (1) Nonanal 4-Oxoisophorone a-Terpineol Decanal b-Cyclocitral Nerol Geraniol ( E )-Dec-2-enal Nonanoic acid Thymol Tridecane ( Z )-Tridec-2-ene ( E )-Tridec-2-ene Benzyl butyrate (2) 1,2-Dihydro-1,1,6-trimethylnaphthalene 2-Methylbutyl heptanoate Eugenol Decanoic acid Heptyl valerate (3) ( E )-Methyl cinnamate ( Z )-Jasmone a-Himachalene ( E )-Geranyl acetone ( E )-b-Ionone Tridecan-2-one Benzyl tiglate Pentadecane Tridecanal Dodecanoic acid (4) ( E )-Hex-3-enyl benzoate Hexadecane Tetradecanal Benzophenone cis-Methyldihydrojasmonate Heptadec-1-ene Pentadecan-2-one Heptadecane Pentadecanal Methyl tetradecanoate Benzyl benzoate (5) Tetradecanoic acid Hexahydrofarnesyl acetone (6) 2-Phenylethyl benzoate (7)
1071 1100 1101 1104 1144 1191 1206 1222 1229 1256 1263 1278 1292 1300 1305 1316 1346 1353
– c) – 0.9 – – 1.0 – – 0.2 1.0 – – – – – – – –
– 2.8 – 2.5 – – – – – – – – – 1.9 2.6 1.9 14.2 –
– – 9.4 1.0 – 3.0 1.3 – – 2.5 – – 1.5 – – – – –
– – 3.2 – – 1.4 – 0.3 – 0.9 0.3 – – – – – – 0.5
1355 1358 1373 1375 1380 1395 1450 1455 1487 1497 1498 1500 1509 1570 1585 1600 1612 1626 1655 1695 1698 1700 1716 1727 1762 1769 1844 1858
– – – – 0.4 – – – – – 0.2 – – – 0.3 1.5 – – – – – – – – 32.4 – 4.4 19.4
– – – 17.3 – – 1.9 – – – – 6.7 – 9.4 – – – – 3.4 – – 5.7 – – – 15.0 6.6 –
1.1 1.2 2.1 – – – – 2.1 1.1 – – 1.6 1.1 22.8 – – 1.2 1.7 – – 1.3 – 4.7 – 11.5 – 12.1 –
– – 0.5 – – – – 0.4 0.4 0.6 – – – 66.5 – – – – – – 0.8 – 1.3 – – 5.0 8.4 –
0.5 0.2 – 0.3 – – – – – 0.6 – – – – – – 0.4 – – 0.7 0.6 – 0.3 0.6 1.4 – – – 2.6 – 0.3 – – – 6.8 – 8.6 – 0.2 – – 23.3 –
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Table (cont.) Entry Compound name and class
LRI a ) Content [%] b ) Root EO Stem EO Phyllode EO Flower EO Pod EO
47 48 49 50 51
Heptadecan-2-one Nonadecane 2-Phenylethyl acetate Methyl hexadecanoate Phenylethyl salicylate (8)
1897 1900 1910 1928 1956
– – 0.7 0.4 34.9
– – – – –
8.8 – – – –
5.8 – – – –
36.0 – 13.3 –
Esters Carboxylic acids Hydrocarbons Apocarotenoids Oxygenated monoterpenes Aldehydes Others
88.7 – 1.5 4.4 3.1 – –
34.9 24.4 21.6 6.6 – 2.5 1.9
12.6 24.9 1.6 23.9 18.1 9.3 2.7
– 72.0 0.5 15.6 6.3 1.6 0.3
14.6 3.2 51.7 25.0 1.7 – 0.5
Total [%]
97.7
91.9
93.1
96.3
96.7
a
) LRI: Linear retention index determined relative to the tR of a series of n-alkanes (C9 – C28 ) on a HP-5 capillary column. b ) Content determined on a HP-5 capillary column. c ) –: Not detected.
Fig. 1. Chemical structures of some major compounds identified in the essential oils of Acacia cyanophylla cultivated in Tunisia. 1, Linalool; 2, benzyl butyrate; 3, heptyl valerate; 4, dodecanoic acid; 5, benzyl benzoate; 6, hexahydrofarnesyl acetone; 7, 2-phenylethyl benzoate; 8, phenylethyl salicylate.
and tetradecanoic acid (44) were other important components (8.4, 5.8, and 5.0%, resp.). Three compounds detected in small amounts (0.3 – 0.5%) were exclusive to this oil. Pod-Oil Composition. Altogether, 18 compounds were identified in the pod EO. Among the main constituents were two compounds exclusive to this oil, viz., nonadecane (48; 36.0%) and heptadec-1-ene (38, 6.8%). Compound 6, another main compound of the pod oil, had the highest content (23.3%) in this oil as compared to the other EOs. Methyl hexadecanoate (50; 13.3%) and heptadecane (40; 8.6%) were other
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important constituents of this EO. Among the minor constituents, five were characteristic for the pod oil. Variation of the Chemical Compound Classes within the EOs of the Different Plant Parts. The chemical classes representing the essential-oil constituents are reported in the Table. Their contents varied significantly within the EOs of the different plant parts. The main chemical class was composed of the esters, characterizing both the root and stem EOs (88.7 and 34.9%, resp.). In total, 13 esters were detected, eight in the root EO, three in the stem EO, two in the phyllode EO, and four in the pod EO. Among them, compounds 2, 3, 5, 7, and 8 should be mentioned. No compound belonging to the class of the esters was found in the flower oil. In the phyllode and flower EOs, the class of the carboxylic acids predominated (24.9 and 72.0%, resp.). The stem EO also showed a high content of carboxylic acids (24.4%). This compound class was particularly represented by compound 4 and tetradecanoic acid (44). No carboxylic acids were found in the root oil. The third major class was that of the hydrocarbons (0.5 – 51.7%), represented by ten compounds, of which only one was detected in the root, phyllode, and flower EOs, 6 in the stem EO, and 4 in the pod EO. Among them, the hydrocarbon nonadecane (48) reached the highest percentage (36.0%) and characterized the pod EO. The class of the apocarotenoids reached its maximum contents in the phyllode, flower, and pod EOs (23.9, 15.6, and 25.0%, resp.), and it was represented in particular by compound 6. Oxygenated monoterpenes (1.7 – 18.1%) were mainly represented by compound 1 in the phyllode EO (9.4%). No compound belonging to this class was present in the stem oil. The contents of the aldehyde class varied from 0.0 to 9.3%. No aldehydes were detected in the root and pod oils, while pentadecanal (41) reached its highest content in the phyllode oil. Comparing the present results with those reported for the EOs isolated from A. cyclops, A. farnesiana, A. berlandieri, A. rigidula, A. praecox, and A. caven flowers, many differences were found. The oils of these species were characterized by completely different major compounds, and dodecanoic acid (4), the major compound of the flower EO of A. cyanophylla (66.5%; Table), was not detected at all in these EOs. Indeed, the flower EO of A. cyclops contained (Z)-hex-3-en-1-yl acetate (24.1%), 4-oxoisophorone (23.7%), (Z)-b-ocimene (10.6%), heptadecane (8.3%), and nonadecane (5.7%) as major components [15]. A. farnesiana flower oil contained methyl salicylate (47.5%), anisaldehyde (17.3%), geraniol (9.8%), and benzaldehyde (6%) as main compounds [11]. The major components of the A. rigidula EO were panisaldehyde (5.5%) and jasmone (2%) [11], and those of A. berlandieri were linalool oxide B (1.1%) and octanol (1%) [11]. For the A. praecox EO, the major components were linalool (1; 13.5%), undecane (13.3%), eugenol (10.5%), decane (10.5%), and oct1-en-3-ol (9.1%) [12]. Finally, nonadecane (15.4%), heneicosane (13.5%), tricosane (7.7%), pentacosane (6.6%), kaurene (6.6%), and (E,E)-farnesyl acetate (6.2%) were reported as main constituents of the A. caven flower oil [18]. Some minor compounds, such as geraniol, b-ionone, b-cyclocitral, geranyl acetone, a-terpineol, and compound 1, were found similarly in the EOs of A. farnesiana, A. berlandieri, and A. rigidula. For the A. praecox oil, only 1 was a shared compound with the present flower EO. The chemical composition of the leaf EOs of A. tortilis (Forsk.) Hayne and A. cyclops was different from that of the EOs investigated here. In fact, for the A. tor-
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tilis leaf oil, the major components were a-humulene (12.0%), a-cadinol (10.6%), nerolidol (9.9%), g-cadinene (7.4%), and (E)-oct-2-enal (6.0%) [19]. The A. cyclops leaf oil was characterized by high relative contents of (Z)-b-ocimene (70.7%) [15]. On the other hand, in the stem-bark oil of A. nilotica (L.) Delile, menthol (34.9%) and limonene (15.3%) were identified as major compounds [20]. For A. albida, in the essential oil obtained from the same plant part, the only compound detected in high amounts was a-pinene (18.6%) [20]. To the best of our knowledge, the essential oils isolated from roots, stems, and pods of Acacia species have never been analyzed previously. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA). For the PCA and the HCA, 18 essential-oil components having average contents of at least 3.0% of the total oil composition in at last one plant organ were selected. The PCA horizontal axis explained 37.34% of the total variance and the vertical axis a further 31.99% (Fig. 2). The PCA highlighted the main compounds that characterized each EO. The HCA based on the Euclidean distances between groups indicated three groups of EOs (Groups A, B, and C), identified by their main components, with a
Fig. 2. Plot obtained by principal component analysis of the 18 main compounds of the five plant organ essential oils of Acacia cyanophylla cultivated in Tunisia. &, Essential oils obtained from five different plant tissues; ^, essential-oil components having average contents of at least 3.0% of the total oil composition in at least one plant tissue; 2-Hep ¼ heptadecan-2-one.
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Fig. 3. Dendrogram obtained by cluster analysis based on the Euclidean distances between groups of the five plant organ essential oils of Acacia cyanophylla cultivated in Tunisia. The principal compounds that characterize each essential oil (EO) are indicated by their entries listed in the Table.
dissimilarity > 15 (Fig. 3). With a dissimilarity > 3, Group C was further divided into two Subgroups, C1 and C2 . Group A, formed by the stem EO, was characterized by compound 3 (22, 17.3%), tetradecanoic acid (44, 15.0%), compound 2 (17, 14.0%), pentadecane (30, 6.7%), and cis-methyldihydrojasmonate (37, 3.4%). Group B, represented by the pod EO, was characterized by nonadecane (48, 36.0%), compound 6 (45, 23.3%), methyl hexadecanoate (50, 13.3%), and heptadec-1-ene (38, 6.8%). Moreover, the stem and pod EOs both contained heptadecane (40) in appreciable amounts. Subgroup C1 comprised the sole root EO, characterized by its peculiar richness in compounds 8 (51, 34.9%), 5 (43, 32.4%), and 7 (46, 19.4%). The phyllode and flower EOs composing Subgroup C2 had a very similar oil composition and were thus closely correlated. They shared six compounds, namely, compounds 6 (45), 4 (32), and 1 (3), heptadecan-2-one (47), pentadecanal (41), and a-terpineol (6). The phyllode EO differed quantitatively from the flower one because of its higher contents in compounds 1 and 6, pentadecanal, and a-terpineol. It had compound 5 in common with the root EO. Contrariwise, the flower EO was differenced by its high content of compound 4 and the absence of compound 2. It had high contents of tetradecanoic acid in common with the stem EO. Phytotoxic Potential of the Essential Oils. The allelopathic effect of the EOs isolated from the different organs of A. cyanophylla on lettuce (Lactuca sativa L.)-seed germination and seedling early growth varied according to the plant organ and the concentration of the oils (Fig. 4). In the conducted contact tests, the germination of lettuce seeds was totally inhibited by the flower and phyllode oils at the highest dose of 1 mg/ml. The pod oil tested at the same concentration showed a much lower germination-inhibition effect ( ¢ 20%), and, on the contrary, no inhibition was detected in the contact tests with the stem and root oils. When tested at concentrations of 0.4 and 0.04 mg/ml, the flower oil affected the germination of lettuce seeds to a lower extent (¢50.0% and ¢ 55.0%, resp.) than at 1 mg/ml. The root, stem, phyllode, and pod oils showed no or only a very low germination-inhibition activity (0.0% to ¢ 5.0%). None of the oils was effective when tested at the lowest concentration of 0.004 mg/ml. The oils of all A. cyanophylla organs investigated inhibited the early growth of lettuce (Fig. 4). A dose-dependent inhibitory effect was observed on the elongation of the lettuce roots and shoots. Hence, the root and shoot elongation seemed to be more affected by the oils than the seed germination. Indeed, the inhibition of the shoot
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Fig. 4. Allelopathic effects of four increasing concentrations of the essential oils extracted from different plant organs (roots, stems, phyllodes, flowers, and pods) of Acacia cyanophylla from Tunisia on a) the seed germination, b) the shoot elongation, and c) the root elongation of Lactuca sativa seedlings. For experimental details, cf. Exper. Part. Values (bars) with different letters differ significantly by DuncanÏs multiple range test at p < 0.05.
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elongation varied from ¢ 4.2 to ¢ 100.0% and that of the root elongation from ¢ 10.0 to ¢ 100.0%. The highest inhibition (100%) was observed for the flower and phyllode EOs at the highest dose of 1 mg/ml. At the same concentration, the elongation of the two organs was also considerably inhibited by the pod essential oil ( ¢ 80.5 %). The root and stem oils at 1 mg/ml were less active, with inhibition percentages of ¢ 41.1 and ¢ 34.8%, respectively, for the shoot elongation and ¢ 24.2 and ¢ 36.8%, respectively, for the root elongation. When the oils were tested at 0.4, 0.04, and 0.004 mg/ml, the percentages of inhibition never exceeded 50.0%, except for the flower EO at 0.4 mg/ml (56.8 and 54.2% for the shoot and root elongation, resp.). Many studies demonstrated the phytotoxicity of essential oils [21 – 26]. In this study, the most phytotoxic A. cyanophylla oils were those isolated from the flowers, phyllodes, and pods. The flower and phyllode EOs showed the highest contents of compound 4 (32), which has previously been reported to have allelopathic activity [27 – 29]. Furthermore, Magalh¼es et al. [27] described 4 as an allelochemical inhibiting the growth of lettuce. The pod oil contained the highest contents of nonadecane (48, 36.0%) and compound 6 (45, 23.3%). Razavi and Nejad-Ebrahimi [22], studying Ecballium elaterium (L.) A.Rich., also identified 6 in its leaf oil and reported about its phytotoxic activity. The flower, phyllode, and pod oils contained a complexity of different allelochemicals, which may operate in synergy and contribute to their allelopathic effect. These findings indicate the possibility to use allelopathic oils or compounds for the biological control of weeds. Indeed, these biologically active essential oils obtained from natural source may be developed to obtain commercial herbicides that might be more environment-friendly than synthetic herbicides. Conclusions. – It was demonstrated that the A. cyanophylla oils, especially those obtained from phyllodes, flowers, and pods, inhibited or reduced the germination and/ or growth of lettuce. To the best of our knowledge, this is the first study reporting the composition and allelopathic activity of the EOs obtained from different fresh plant organs of A. cyanophylla. This species might be a good raw source of allelopathic compounds that could be developed as herbicides. In nature, the plant material falls to the ground and forms the litter under and around the tree. The phytotoxic compounds resulting from the decomposing material might be transmitted to neighboring species. A. cyanophylla has been introduced and planted in poor and degraded soils in Tunisia, hence, it was considered as an introduced tree in this ecosystem. Allelochemicals were released into the environment of this tree and should modify the growing sites by affecting the development of the plant diversity and the number of species. Although the evidence is strong that some kind of allelopathy is the principal agent of growth inhibition, there is a pressing need to investigate for resource competition and allelopathy interactions in the communities under field conditions. From the ecological point of view, those findings are of prime importance, and it would be interesting to study the role played by allelopathy in those communities. Along with laboratory experiments, field experiments are needed to study the interactions among various physical, chemical, and biological properties of the soil. The consideration of allelopathy in an integrated community and ecosystem context requires the recognition of the large number of different processes that can be affected
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by the same chemical or its derivatives. Understanding the biochemical characteristics of an ecosystem and the efficiency of allelochemicals needs a study associating chemical ecology, biotic and abiotic environmental conditions, and interactions between species (noxious, introduced, or native ones) with biogeography and evolutionary-biology history. This should be kept in mind, if we are interested to demonstrate that toxic compounds induce or not evolutionary changes in natural field settings. Experimental Part Plant Material. Roots, stems, phyllodes, and flowers of A. cyanophylla were collected during the blooming stage in spring (March, 2012), and fully mature fruits (pods) were harvested in summer (June, 2012) from ca. 20-year-old trees cultivated in the area of Monastir (latitude 35846’0’’ N, longitude 10859’0’’ E, coastal region, east of Tunisia, subhumid climate). Voucher specimens (No. Acy20 – 24) were deposited with the Herbarium of the Laboratory of Genetic Biodiversity and Valorization of Bioressources, High Institute of Biotechnology of Monastir, Tunisia. The fresh plant material was separated into phyllodes, stems, flowers, roots, and pods without seeds. Essential-Oil Extraction. For each extraction, 3 100 g of fresh plant material cut into small pieces were subjected to hydrodistillation for 4 h using a Clevenger-type apparatus. The obtained oils were dried (anh. Na2SO4 ) and weighed. The calculated essential-oil yield was expressed in % (w/w) of the fresh material. All oils were stored in sealed glass vials at 48 until analysis. All extractions were done in triplicate. GC Analysis. The GC analyses were carried out with a Hewlett-Packard 5890 apparatus equipped with a flame ionization detector (FID) and a HP-5 cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm). The oven temp. was programmed isothermal at 508 for 1 min, rising from 50 to 2808 at 58/min, and held isothermal at 2808 for 3 min; injector temp., 2508; detector temp., 2808; carrier gas, N2 (1.2 ml/ min). The injected volume was 1 ml (10% essential oil in hexane). Linear retention indices (LRIs) were determined rel. to the retention times (tR ) of a series of n-alkanes (C9 – C28 ). GC/MS Analysis. The essential oils were analyzed with a Varian CP-3800 apparatus equipped with a Varian Saturn 2000 ion trap mass-selective detector and a HP-5 cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm). The oven temp. was programmed rising from 60 to 2408 at 38/min; injector and transfer-line temp., 220 and 2408, resp.; carrier gas, He (1 ml/min); ionization voltage, 70 eV; injection volume, 0.2 ml (10% essential oil in hexane). Compound Identification. The identification of the constituents was based on the comparison of i) their tR with those of authentic samples and ii) their LRIs and mass spectra with those listed in the commercial libraries NIST 98 and ADAMS and in a home-made mass-spectral library, built up from pure substances and components of known oils, and with literature data [30] [31]. Moreover, the molecular weights of all the identified compounds were confirmed by GC/CI-MS, using MeOH as CI ionizing gas. Phytotoxicity Assay. The inhibition potential of the essential oils obtained from the five plant tissues of A. cyanophylla on the seed germination and the shoot and root elongation of lettuce (Lactuca sativa L.) seedlings was investigated. Lettuce seeds were purchased from a local seed shop, sterilized for 5 min with NaClO (1%), and then rinsed with dist. H2O [32] [33]. Three replicates, each comprising 20 seeds, were prepared for the contact tests with each of the five oils, using sterile Petri dishes (90 mm diameter) lined with double-sterile filter paper (Whatman No. 2). The oils were dispersed as an emulsion in dist. H2O using Tween 20. Four doses of the oils (0.004, 0.04, 0.4 and 1 mg/ml) were obtained by dilution of the emulsion in deionized H2O. The dishes were then moistened with 5 ml of A. cyanophylla oils at different concentrations or with 5 ml dist. H2O, used as a negative control. Thereafter, the dishes were sealed with ParafilmÔ, to prevent losses of moisture and to avoid contamination, and placed in a growth chamber to allow germination in the dark at an average temp. of 23 28 for 7 d. A seed was considered germinated when the protrusion of the radicle became evident [32]. The experimental design was a randomized block with three replicates for each treatment and control. After 7 d, the germination percentage was determined. Then, the seedlings of L. sativa were collected, and the shoot and the root lengths were measured, to evaluate the allelopathic activity of the
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oils. The inhibitory or stimulatory effects were calculated using Eqn. 1, which was slightly modified from Chung et al. [34]. Inhibition (¢)/stimulation (þ) [%] ¼ (EOe ¢ Ce)/Ce 100
(1)
where EOe (essential-oil effect) is the parameter measured in presence of A cyanophylla essential oil and Ce (control effect) the parameter measured in presence of dist. H2O. Statistical Analysis. Data from the experiments were subjected to analysis of variance (ANOVA) using SPSS 13.0 for Windows (SPSS Inc. Chicago, IL, USA). The inhibition data relative to the pffiffiffi germination and plant development were transformed using arcsin square-root (arcsin x ) transformation before ANOVA. Means were separated at the 5% significance level by a least significant difference test (DuncanÏs multiple range test). To evaluate whether the essential-oil constituents are useful in reflecting the chemical relationships between organs, 18 compounds detected in the oil samples at an average concentrations of at least 3% of the total oil composition in one plant organ were subjected to PCA and HCA using SPSS 13.0 for Windows.
REFERENCES [1] E. L. Rice, in ÐChemically Mediated Interactions between Plants and Other OrganismsÏ, Eds. G. A. Cooper-Driver, T. Swain, E. E. Conn, Plenum Press, New York, 1985, pp. 81 – 105. [2] R. K. Brummitt, Taxon 2004, 53, 826. [3] P. R. Lister, P. Holford, T. Haigh, D. A. Morrison, in ÐProgress in New CropsÏ, Ed. J. Janick, ASHS Press, Alexandria, VA, 1996, pp. 228 – 236. [4] A. Wadood, N. Wadood, S. A. W. Shah, J. Pak. Med. Assoc. 1989, 39, 208. [5] S. A. Sotohy, A. N. Sayed, M. M. Ahmed, DTW, Dtsch. Tierrztl. Wochenschr. 1997, 104, 432. [6] A. A. Dafallah, Z. Al-Mustafa, Am. J. Chin. Med. 1996, 24, 263. [7] S. Amos, P. A. Akah, C. J. Odukwe, K. S. Gamaniel, Phytother. Res. 1999, 13, 683. [8] Y. Tezuka, K. Honda, A. H. Banskota, M. M. Thet, S. Kadota, J. Nat. Prod. 2000, 63, 1658. [9] S. T. Chang, J. H. Wu, S. Y. Wang, P. L. Kang, N. S. Yang, L. F. Shyur, J. Agric. Food Chem. 2001, 49, 3420. [10] D. S. Seigler, Biochem. Syst. Ecol. 2003, 31, 845. [11] R. A. Flath, T. R. Mon, G. Lorenz, C. J. Whitten, J. W. Mackley, J. Agric. Food Chem. 1983, 31, 1167. [12] J. A. Zygadlo, A. L. Lamarque, D. M. Maestri, A. Velasco Negueruela, M. J. Perez Alonso, M. C. Garcia Vallejo, Flavour Fragrance J. 1996, 11, 3. [13] A. L. Lamarque, D. M. Maestri, J. A. Zygadlo, N. R. Grosso, Flavour Fragrance J. 1998, 13, 266. [14] R. Kaiser, Rev. Ital. EPPOS 1997, 18, 18. [15] M. J. Kotze, A. Jîrgens, S. D. Johnson, J. H. Hoffmann, S. Afr. J. Bot. 2010, 76, 701. [16] Banque mondiale, ÐUne Strat¦gie pour le D¦veloppement des Parcours en Zones Arides et SemiArides, Annexe III, Rapport TechniqueÏ, Tunisie, 1995, p. 162. [17] National Academy of Sciences (NAS), ÐFirewood Crops: Shrub and Tree Species for Energy ProductionÏ, National Academy of Sciences, Washington, D.C., 1980, p. 237. [18] R. A. Malizia, D. A. Cardell, J. S. Molli, R. J. Grau, J. Essent. Oil Res. 2002, 14, 2. [19] I. A. Ogunwandea, T. Matsuib, K. Matsumotob, M. Shimodac, D. Kubmarawad, J. Essent. Oil Res. 2008, 20, 116. [20] A. O. Ogunbinu, S. Okeniyi, G. Flamini, P. L. Cioni, I. A. Ogunwande, I. T. Babalola, J. Essent. Oil Res. 2010, 22, 540. [21] M. Verdeguer, M. A. Blazquez, H. Boira, Biochem. Syst. Ecol. 2009, 37, 362. [22] S. M. Razavi, S. Nejad-Ebrahimi, Nat. Prod. Res. 2010, 24, 1704. [23] D. R. Batish, H. P. Singh, M. Kaur, R. K. Kohli, S. Singh, Biochem. Syst. Ecol. 2012, 41, 104. [24] C. Wright, B. K. Chhetri, W. N. Setzer, Am. J. Essent. Oils Nat. Prod. 2013, 1, 18. [25] S. V. Wyse, B. R. Burns, New Zeal. J. Ecol. 2013, 37, 178. [26] V. Rowshan, S. Karimi, Int. J. Agric. Res. Rev. 2013, 3, 788. [27] H. M. Magalh¼es, P. S. N. Lopes, F. O. Silv¦rio, H. F. J. Silva, Allelopathy J. 2012, 30, 49.
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[28] M. D. Wang, H. Chen, X. Liu, Y. Gao, K. Wu, X. Jia, Afr. J. Biotechnol. 2010, 9, 8672. [29] P. Liu, L. Jiang, S. Wan, J. Wei, S. Yu, L. Yang, M. Wang, J. Agric. Sci. Technol. 2009, 11, 107. [30] R. P. Adams, in ÐIdentification of Essential Oil Components by Gas Chromatography/Mass SpectrometryÏ, 4th edn., Allured Publishing Corporation, Carol Stream, IL, 2007, p. 804. [31] D. Joulain, W. A. Kçnig, ÐThe Atlas of Spectral Data of Sesquiterpene HydrocarbonsÏ, EB Verlag, Hamburg, 1998. [32] S. Mabrouk, K. Bel Hadj Salah, A. Elaissi, L. Jlaiel, H. Ben Jannet, M. Aouni, F. Harzallah-Skhiri, Chem. Biodiversity 2013, 10, 209. [33] N. Zahed, K. Hosni, N. Ben Brahim, M. Kallel, H. Sebei, Acta Physiol. Plant. 2010, 32, 1221. [34] I. M. Chung, J. K. Ahn, S. J. Yun, Crop. Prot. 2001, 20, 921. Received May 14, 2014
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Chemical Composition and Allelopathic Potential of Essential Oils Obtained from Acacia cyanophylla Lii n dl . Cultivated in Tunisia by Asma El Ayeb-Zakhama a ), Lamia Sakka-Rouis a ), Afifa Bergaoui b ), Guido Flamini* c ), Hichem Ben Jannet b ), and Fethia Harzallah-Skhiri* a ) a
) Laboratory of Genetics Biodiversity and Valorisation of Bioresources (LR11ES41), Higher Institute of Biotechnology of Monastir, University of Monastir, Rue Tahar Haddad, 5000 Monastir, Tunisia (phone: þ 216-73-463711; 216-73-465404; e-mail: [email protected]) b ) Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity, Medicinal Chemistry and Natural Products, Faculty of Sciences of Monastir, University of Monastir, Tunisia c ) Dipartimento di Farmacia, University of Pisa, Via Bonanno 33, I-56126 Pisa (e-mail: [email protected])
Acacia cyanophylla Lindl. (Fabaceae), synonym Acacia saligna (Labill.) H. L.Wendl., native to West Australia and naturalized in North Africa and South Europe, was introduced in Tunisia for rangeland rehabilitation, particularly in the semiarid zones. In addition, this evergreen tree represents a potential forage resource, particularly during periods of drought. A. cyanophylla is abundant in Tunisia and some other Mediterranean countries. The chemical composition of the essential oils obtained by hydrodistillation from different plant parts, viz., roots, stems, phyllodes, flowers, and pods (fully mature fruits without seeds), was characterized for the first time here. According to GC-FID and GC/MS analyses, the principal compound in the phyllode and flower oils was dodecanoic acid (4), representing 22.8 and 66.5% of the total oil, respectively. Phenylethyl salicylate (8; 34.9%), heptyl valerate (3; 17.3%), and nonadecane (36%) were the main compounds in the root, stem, and pod oils, respectively. The phyllode and flower oils were very similar, containing almost the same compounds. Nevertheless, the phyllode oil differed from the flower oil for its higher contents of hexahydrofarnesyl acetone (6), linalool (1), pentadecanal, a-terpineol, and benzyl benzoate (5) and its lower content of 4. Principal component and hierarchical cluster analyses separated the five essential oils into four groups, each characterized by its main constituents. Furthermore, the allelopathic activity of each oil was evaluated using lettuce (Lactuca sativa L.) as a plant model. The phyllode, flower, and pod oils exhibited a strong allelopathic activity against lettuce.
Introduction. – Acacia Mill., belonging to the subfamily Mimosoideae, is the second largest genus in the family Fabaceae [1], with ca. 1350 species [2]. The Acacia species range in size from small shrubs to large trees and are ecologically important as ÐpioneerÏ species, because they rapidly establish cover after major natural disturbances [3]. Researchers showed that Acacia trees are known as a source of components with bioactive properties, suggesting a large inhibitory potential in this genus. The main use of Acacia species is as a fodder source and in soil stabilization, but it is also used in the traditional medicine for its hypoglycemic [4], antibacterial [5], and anti-inflammatory [6] activities. Other authors reported its spasmogenic and vasoconstrictor actions [7] and also its cytotoxic [8] and antioxidant [9] activities. Little is known about the volatile chemistry of most species of Acacia [10]. In fact, Seigler [10] reported on the phytochemistry of Acacia and mentioned only plant volatiles obtained from A. Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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farnesiana (L.) Willd. Floral volatiles have been described from A. farnesiana, A. berlandieri Benth., A. rigidula Benth. (all three native to North America) [11], A. praecox Griseb., A. caven (Molina) Molina, A. Aroma Hook & Arn. (all three from Argentina) [12] [13], and from A. karroo Hayne, an African species [14]. More recently, floral and leaf volatiles of Acacia cyclops A.Cunn. ex G.Don have been studied [15]. A. cyanophylla Lindl. (syn. A. saligna (Labill) H. L.Wendl) is an Australian tree introduced in Tunisia for the first time in 1930 for rangeland rehabilitation, particularly in the semiarid zones and is now extensively grown and cultivated throughout the territory [16]. A. cyanophylla plantations are located in the subhumid and semiarid bioclimatic stages, as the species is highly resistant to drought and salinity [17]. To the best of our knowledge, until now, the chemical composition of A. cyanophylla essential oil has not been reported. Hence, the chemical composition of the root, stem, phyllode, flower, and pod (fully mature fruit without seeds) essential oils (EOs) was characterized and their allelopathic potential evaluated. Results and Discussion. – Essential-Oil Yield. The mean oil yields for the five investigated plant organs of A. cyanophylla varied significantly from 0.01% (w/w) for the roots to 0.08% for the pods. Phyllodes, stems, and flowers afforded 0.03, 0.04, and 0.06% of essential oil, respectively. Essential-Oil Composition. Chromatographic analysis (GC-FID and GC/MS) of the EOs extracted from the different organs of A. cyanophylla collected in spring (roots, stems, phyllodes, and flowers) or in summer (pods), allowed the identification of 51 compounds (Table), representing 91.9 – 97.7% of the total oil composition. The chemical structures of some of the major compounds are shown in Fig. 1. Root-Oil Composition. In total, 14 compounds were identified in the root oil, representing 97.7% of the total volatiles (Table). The main constituent was phenylethyl salicylate (51 1), 8; 34.9%), followed by benzyl benzoate (43, 5; 32.4%), 2-phenylethyl benzoate (46, 7; 19.4%), and hexahydrofarnesyl acetone (45, 6; 4.4%). For all other compounds, lower contents were detected (0.2 – 1.5%). Seven out of the 14 compounds were exclusive to this oil. Stem-Oil Composition. As in the root oil, also in the stem oil 14 compounds were identified (Table). The major ones were heptyl valerate (22, 3; 17.3%), tetradecanoic acid (44; 15.0%), benzyl butyrate (17, 2; 14.2%), dodecanoic acid (syn. lauric acid, 32, 4; 9.4%), pentadecane (30; 6.7%), compound 6 (6.6%), and heptadecane (40, 5.7%). The remaining constituents were present at amounts varying from 1.9 to 3.4%. Seven of the 14 compounds were found only in the stem oil. Phyllode-Oil Composition. In the Table, it can be seen that the A. cyanophylla phyllode oil, with 21 compounds identified, was the richest in constituents. Compounds 4 (22.8%), 6 (12.1%), and 5 (11.5%) as well as linalool (3, 1; 9.4%), heptadecan-2-one (47; 8.8%), and pentadecanal (41, 4.7%) were the most important ones. Only six compounds were exclusive to this oil, even if detected at low amounts (1.1 – 1.7%). Flower-Oil Composition. Among its 16 constituents, compound 4 dominated the composition of this oil, with a content of 66.5%. Compound 6, heptadecan-2-one (47), 1)
Italic numbers in parentheses refer to the entries in the Table.
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Table. Chemical Composition of the Essential Oils ( EOs) Extracted from Different Organs of Acacia cyanophylla Lindl. Cultivated in Tunisia Entry Compound name and class
LRI a ) Content [%] b ) Root EO Stem EO Phyllode EO Flower EO Pod EO
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Octanol Undecane Linalool (1) Nonanal 4-Oxoisophorone a-Terpineol Decanal b-Cyclocitral Nerol Geraniol ( E )-Dec-2-enal Nonanoic acid Thymol Tridecane ( Z )-Tridec-2-ene ( E )-Tridec-2-ene Benzyl butyrate (2) 1,2-Dihydro-1,1,6-trimethylnaphthalene 2-Methylbutyl heptanoate Eugenol Decanoic acid Heptyl valerate (3) ( E )-Methyl cinnamate ( Z )-Jasmone a-Himachalene ( E )-Geranyl acetone ( E )-b-Ionone Tridecan-2-one Benzyl tiglate Pentadecane Tridecanal Dodecanoic acid (4) ( E )-Hex-3-enyl benzoate Hexadecane Tetradecanal Benzophenone cis-Methyldihydrojasmonate Heptadec-1-ene Pentadecan-2-one Heptadecane Pentadecanal Methyl tetradecanoate Benzyl benzoate (5) Tetradecanoic acid Hexahydrofarnesyl acetone (6) 2-Phenylethyl benzoate (7)
1071 1100 1101 1104 1144 1191 1206 1222 1229 1256 1263 1278 1292 1300 1305 1316 1346 1353
– c) – 0.9 – – 1.0 – – 0.2 1.0 – – – – – – – –
– 2.8 – 2.5 – – – – – – – – – 1.9 2.6 1.9 14.2 –
– – 9.4 1.0 – 3.0 1.3 – – 2.5 – – 1.5 – – – – –
– – 3.2 – – 1.4 – 0.3 – 0.9 0.3 – – – – – – 0.5
1355 1358 1373 1375 1380 1395 1450 1455 1487 1497 1498 1500 1509 1570 1585 1600 1612 1626 1655 1695 1698 1700 1716 1727 1762 1769 1844 1858
– – – – 0.4 – – – – – 0.2 – – – 0.3 1.5 – – – – – – – – 32.4 – 4.4 19.4
– – – 17.3 – – 1.9 – – – – 6.7 – 9.4 – – – – 3.4 – – 5.7 – – – 15.0 6.6 –
1.1 1.2 2.1 – – – – 2.1 1.1 – – 1.6 1.1 22.8 – – 1.2 1.7 – – 1.3 – 4.7 – 11.5 – 12.1 –
– – 0.5 – – – – 0.4 0.4 0.6 – – – 66.5 – – – – – – 0.8 – 1.3 – – 5.0 8.4 –
0.5 0.2 – 0.3 – – – – – 0.6 – – – – – – 0.4 – – 0.7 0.6 – 0.3 0.6 1.4 – – – 2.6 – 0.3 – – – 6.8 – 8.6 – 0.2 – – 23.3 –
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Table (cont.) Entry Compound name and class
LRI a ) Content [%] b ) Root EO Stem EO Phyllode EO Flower EO Pod EO
47 48 49 50 51
Heptadecan-2-one Nonadecane 2-Phenylethyl acetate Methyl hexadecanoate Phenylethyl salicylate (8)
1897 1900 1910 1928 1956
– – 0.7 0.4 34.9
– – – – –
8.8 – – – –
5.8 – – – –
36.0 – 13.3 –
Esters Carboxylic acids Hydrocarbons Apocarotenoids Oxygenated monoterpenes Aldehydes Others
88.7 – 1.5 4.4 3.1 – –
34.9 24.4 21.6 6.6 – 2.5 1.9
12.6 24.9 1.6 23.9 18.1 9.3 2.7
– 72.0 0.5 15.6 6.3 1.6 0.3
14.6 3.2 51.7 25.0 1.7 – 0.5
Total [%]
97.7
91.9
93.1
96.3
96.7
a
) LRI: Linear retention index determined relative to the tR of a series of n-alkanes (C9 – C28 ) on a HP-5 capillary column. b ) Content determined on a HP-5 capillary column. c ) –: Not detected.
Fig. 1. Chemical structures of some major compounds identified in the essential oils of Acacia cyanophylla cultivated in Tunisia. 1, Linalool; 2, benzyl butyrate; 3, heptyl valerate; 4, dodecanoic acid; 5, benzyl benzoate; 6, hexahydrofarnesyl acetone; 7, 2-phenylethyl benzoate; 8, phenylethyl salicylate.
and tetradecanoic acid (44) were other important components (8.4, 5.8, and 5.0%, resp.). Three compounds detected in small amounts (0.3 – 0.5%) were exclusive to this oil. Pod-Oil Composition. Altogether, 18 compounds were identified in the pod EO. Among the main constituents were two compounds exclusive to this oil, viz., nonadecane (48; 36.0%) and heptadec-1-ene (38, 6.8%). Compound 6, another main compound of the pod oil, had the highest content (23.3%) in this oil as compared to the other EOs. Methyl hexadecanoate (50; 13.3%) and heptadecane (40; 8.6%) were other
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important constituents of this EO. Among the minor constituents, five were characteristic for the pod oil. Variation of the Chemical Compound Classes within the EOs of the Different Plant Parts. The chemical classes representing the essential-oil constituents are reported in the Table. Their contents varied significantly within the EOs of the different plant parts. The main chemical class was composed of the esters, characterizing both the root and stem EOs (88.7 and 34.9%, resp.). In total, 13 esters were detected, eight in the root EO, three in the stem EO, two in the phyllode EO, and four in the pod EO. Among them, compounds 2, 3, 5, 7, and 8 should be mentioned. No compound belonging to the class of the esters was found in the flower oil. In the phyllode and flower EOs, the class of the carboxylic acids predominated (24.9 and 72.0%, resp.). The stem EO also showed a high content of carboxylic acids (24.4%). This compound class was particularly represented by compound 4 and tetradecanoic acid (44). No carboxylic acids were found in the root oil. The third major class was that of the hydrocarbons (0.5 – 51.7%), represented by ten compounds, of which only one was detected in the root, phyllode, and flower EOs, 6 in the stem EO, and 4 in the pod EO. Among them, the hydrocarbon nonadecane (48) reached the highest percentage (36.0%) and characterized the pod EO. The class of the apocarotenoids reached its maximum contents in the phyllode, flower, and pod EOs (23.9, 15.6, and 25.0%, resp.), and it was represented in particular by compound 6. Oxygenated monoterpenes (1.7 – 18.1%) were mainly represented by compound 1 in the phyllode EO (9.4%). No compound belonging to this class was present in the stem oil. The contents of the aldehyde class varied from 0.0 to 9.3%. No aldehydes were detected in the root and pod oils, while pentadecanal (41) reached its highest content in the phyllode oil. Comparing the present results with those reported for the EOs isolated from A. cyclops, A. farnesiana, A. berlandieri, A. rigidula, A. praecox, and A. caven flowers, many differences were found. The oils of these species were characterized by completely different major compounds, and dodecanoic acid (4), the major compound of the flower EO of A. cyanophylla (66.5%; Table), was not detected at all in these EOs. Indeed, the flower EO of A. cyclops contained (Z)-hex-3-en-1-yl acetate (24.1%), 4-oxoisophorone (23.7%), (Z)-b-ocimene (10.6%), heptadecane (8.3%), and nonadecane (5.7%) as major components [15]. A. farnesiana flower oil contained methyl salicylate (47.5%), anisaldehyde (17.3%), geraniol (9.8%), and benzaldehyde (6%) as main compounds [11]. The major components of the A. rigidula EO were panisaldehyde (5.5%) and jasmone (2%) [11], and those of A. berlandieri were linalool oxide B (1.1%) and octanol (1%) [11]. For the A. praecox EO, the major components were linalool (1; 13.5%), undecane (13.3%), eugenol (10.5%), decane (10.5%), and oct1-en-3-ol (9.1%) [12]. Finally, nonadecane (15.4%), heneicosane (13.5%), tricosane (7.7%), pentacosane (6.6%), kaurene (6.6%), and (E,E)-farnesyl acetate (6.2%) were reported as main constituents of the A. caven flower oil [18]. Some minor compounds, such as geraniol, b-ionone, b-cyclocitral, geranyl acetone, a-terpineol, and compound 1, were found similarly in the EOs of A. farnesiana, A. berlandieri, and A. rigidula. For the A. praecox oil, only 1 was a shared compound with the present flower EO. The chemical composition of the leaf EOs of A. tortilis (Forsk.) Hayne and A. cyclops was different from that of the EOs investigated here. In fact, for the A. tor-
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tilis leaf oil, the major components were a-humulene (12.0%), a-cadinol (10.6%), nerolidol (9.9%), g-cadinene (7.4%), and (E)-oct-2-enal (6.0%) [19]. The A. cyclops leaf oil was characterized by high relative contents of (Z)-b-ocimene (70.7%) [15]. On the other hand, in the stem-bark oil of A. nilotica (L.) Delile, menthol (34.9%) and limonene (15.3%) were identified as major compounds [20]. For A. albida, in the essential oil obtained from the same plant part, the only compound detected in high amounts was a-pinene (18.6%) [20]. To the best of our knowledge, the essential oils isolated from roots, stems, and pods of Acacia species have never been analyzed previously. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA). For the PCA and the HCA, 18 essential-oil components having average contents of at least 3.0% of the total oil composition in at last one plant organ were selected. The PCA horizontal axis explained 37.34% of the total variance and the vertical axis a further 31.99% (Fig. 2). The PCA highlighted the main compounds that characterized each EO. The HCA based on the Euclidean distances between groups indicated three groups of EOs (Groups A, B, and C), identified by their main components, with a
Fig. 2. Plot obtained by principal component analysis of the 18 main compounds of the five plant organ essential oils of Acacia cyanophylla cultivated in Tunisia. &, Essential oils obtained from five different plant tissues; ^, essential-oil components having average contents of at least 3.0% of the total oil composition in at least one plant tissue; 2-Hep ¼ heptadecan-2-one.
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Fig. 3. Dendrogram obtained by cluster analysis based on the Euclidean distances between groups of the five plant organ essential oils of Acacia cyanophylla cultivated in Tunisia. The principal compounds that characterize each essential oil (EO) are indicated by their entries listed in the Table.
dissimilarity > 15 (Fig. 3). With a dissimilarity > 3, Group C was further divided into two Subgroups, C1 and C2 . Group A, formed by the stem EO, was characterized by compound 3 (22, 17.3%), tetradecanoic acid (44, 15.0%), compound 2 (17, 14.0%), pentadecane (30, 6.7%), and cis-methyldihydrojasmonate (37, 3.4%). Group B, represented by the pod EO, was characterized by nonadecane (48, 36.0%), compound 6 (45, 23.3%), methyl hexadecanoate (50, 13.3%), and heptadec-1-ene (38, 6.8%). Moreover, the stem and pod EOs both contained heptadecane (40) in appreciable amounts. Subgroup C1 comprised the sole root EO, characterized by its peculiar richness in compounds 8 (51, 34.9%), 5 (43, 32.4%), and 7 (46, 19.4%). The phyllode and flower EOs composing Subgroup C2 had a very similar oil composition and were thus closely correlated. They shared six compounds, namely, compounds 6 (45), 4 (32), and 1 (3), heptadecan-2-one (47), pentadecanal (41), and a-terpineol (6). The phyllode EO differed quantitatively from the flower one because of its higher contents in compounds 1 and 6, pentadecanal, and a-terpineol. It had compound 5 in common with the root EO. Contrariwise, the flower EO was differenced by its high content of compound 4 and the absence of compound 2. It had high contents of tetradecanoic acid in common with the stem EO. Phytotoxic Potential of the Essential Oils. The allelopathic effect of the EOs isolated from the different organs of A. cyanophylla on lettuce (Lactuca sativa L.)-seed germination and seedling early growth varied according to the plant organ and the concentration of the oils (Fig. 4). In the conducted contact tests, the germination of lettuce seeds was totally inhibited by the flower and phyllode oils at the highest dose of 1 mg/ml. The pod oil tested at the same concentration showed a much lower germination-inhibition effect ( ¢ 20%), and, on the contrary, no inhibition was detected in the contact tests with the stem and root oils. When tested at concentrations of 0.4 and 0.04 mg/ml, the flower oil affected the germination of lettuce seeds to a lower extent (¢50.0% and ¢ 55.0%, resp.) than at 1 mg/ml. The root, stem, phyllode, and pod oils showed no or only a very low germination-inhibition activity (0.0% to ¢ 5.0%). None of the oils was effective when tested at the lowest concentration of 0.004 mg/ml. The oils of all A. cyanophylla organs investigated inhibited the early growth of lettuce (Fig. 4). A dose-dependent inhibitory effect was observed on the elongation of the lettuce roots and shoots. Hence, the root and shoot elongation seemed to be more affected by the oils than the seed germination. Indeed, the inhibition of the shoot
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Fig. 4. Allelopathic effects of four increasing concentrations of the essential oils extracted from different plant organs (roots, stems, phyllodes, flowers, and pods) of Acacia cyanophylla from Tunisia on a) the seed germination, b) the shoot elongation, and c) the root elongation of Lactuca sativa seedlings. For experimental details, cf. Exper. Part. Values (bars) with different letters differ significantly by DuncanÏs multiple range test at p < 0.05.
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elongation varied from ¢ 4.2 to ¢ 100.0% and that of the root elongation from ¢ 10.0 to ¢ 100.0%. The highest inhibition (100%) was observed for the flower and phyllode EOs at the highest dose of 1 mg/ml. At the same concentration, the elongation of the two organs was also considerably inhibited by the pod essential oil ( ¢ 80.5 %). The root and stem oils at 1 mg/ml were less active, with inhibition percentages of ¢ 41.1 and ¢ 34.8%, respectively, for the shoot elongation and ¢ 24.2 and ¢ 36.8%, respectively, for the root elongation. When the oils were tested at 0.4, 0.04, and 0.004 mg/ml, the percentages of inhibition never exceeded 50.0%, except for the flower EO at 0.4 mg/ml (56.8 and 54.2% for the shoot and root elongation, resp.). Many studies demonstrated the phytotoxicity of essential oils [21 – 26]. In this study, the most phytotoxic A. cyanophylla oils were those isolated from the flowers, phyllodes, and pods. The flower and phyllode EOs showed the highest contents of compound 4 (32), which has previously been reported to have allelopathic activity [27 – 29]. Furthermore, Magalh¼es et al. [27] described 4 as an allelochemical inhibiting the growth of lettuce. The pod oil contained the highest contents of nonadecane (48, 36.0%) and compound 6 (45, 23.3%). Razavi and Nejad-Ebrahimi [22], studying Ecballium elaterium (L.) A.Rich., also identified 6 in its leaf oil and reported about its phytotoxic activity. The flower, phyllode, and pod oils contained a complexity of different allelochemicals, which may operate in synergy and contribute to their allelopathic effect. These findings indicate the possibility to use allelopathic oils or compounds for the biological control of weeds. Indeed, these biologically active essential oils obtained from natural source may be developed to obtain commercial herbicides that might be more environment-friendly than synthetic herbicides. Conclusions. – It was demonstrated that the A. cyanophylla oils, especially those obtained from phyllodes, flowers, and pods, inhibited or reduced the germination and/ or growth of lettuce. To the best of our knowledge, this is the first study reporting the composition and allelopathic activity of the EOs obtained from different fresh plant organs of A. cyanophylla. This species might be a good raw source of allelopathic compounds that could be developed as herbicides. In nature, the plant material falls to the ground and forms the litter under and around the tree. The phytotoxic compounds resulting from the decomposing material might be transmitted to neighboring species. A. cyanophylla has been introduced and planted in poor and degraded soils in Tunisia, hence, it was considered as an introduced tree in this ecosystem. Allelochemicals were released into the environment of this tree and should modify the growing sites by affecting the development of the plant diversity and the number of species. Although the evidence is strong that some kind of allelopathy is the principal agent of growth inhibition, there is a pressing need to investigate for resource competition and allelopathy interactions in the communities under field conditions. From the ecological point of view, those findings are of prime importance, and it would be interesting to study the role played by allelopathy in those communities. Along with laboratory experiments, field experiments are needed to study the interactions among various physical, chemical, and biological properties of the soil. The consideration of allelopathy in an integrated community and ecosystem context requires the recognition of the large number of different processes that can be affected
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by the same chemical or its derivatives. Understanding the biochemical characteristics of an ecosystem and the efficiency of allelochemicals needs a study associating chemical ecology, biotic and abiotic environmental conditions, and interactions between species (noxious, introduced, or native ones) with biogeography and evolutionary-biology history. This should be kept in mind, if we are interested to demonstrate that toxic compounds induce or not evolutionary changes in natural field settings. Experimental Part Plant Material. Roots, stems, phyllodes, and flowers of A. cyanophylla were collected during the blooming stage in spring (March, 2012), and fully mature fruits (pods) were harvested in summer (June, 2012) from ca. 20-year-old trees cultivated in the area of Monastir (latitude 35846’0’’ N, longitude 10859’0’’ E, coastal region, east of Tunisia, subhumid climate). Voucher specimens (No. Acy20 – 24) were deposited with the Herbarium of the Laboratory of Genetic Biodiversity and Valorization of Bioressources, High Institute of Biotechnology of Monastir, Tunisia. The fresh plant material was separated into phyllodes, stems, flowers, roots, and pods without seeds. Essential-Oil Extraction. For each extraction, 3 100 g of fresh plant material cut into small pieces were subjected to hydrodistillation for 4 h using a Clevenger-type apparatus. The obtained oils were dried (anh. Na2SO4 ) and weighed. The calculated essential-oil yield was expressed in % (w/w) of the fresh material. All oils were stored in sealed glass vials at 48 until analysis. All extractions were done in triplicate. GC Analysis. The GC analyses were carried out with a Hewlett-Packard 5890 apparatus equipped with a flame ionization detector (FID) and a HP-5 cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm). The oven temp. was programmed isothermal at 508 for 1 min, rising from 50 to 2808 at 58/min, and held isothermal at 2808 for 3 min; injector temp., 2508; detector temp., 2808; carrier gas, N2 (1.2 ml/ min). The injected volume was 1 ml (10% essential oil in hexane). Linear retention indices (LRIs) were determined rel. to the retention times (tR ) of a series of n-alkanes (C9 – C28 ). GC/MS Analysis. The essential oils were analyzed with a Varian CP-3800 apparatus equipped with a Varian Saturn 2000 ion trap mass-selective detector and a HP-5 cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm). The oven temp. was programmed rising from 60 to 2408 at 38/min; injector and transfer-line temp., 220 and 2408, resp.; carrier gas, He (1 ml/min); ionization voltage, 70 eV; injection volume, 0.2 ml (10% essential oil in hexane). Compound Identification. The identification of the constituents was based on the comparison of i) their tR with those of authentic samples and ii) their LRIs and mass spectra with those listed in the commercial libraries NIST 98 and ADAMS and in a home-made mass-spectral library, built up from pure substances and components of known oils, and with literature data [30] [31]. Moreover, the molecular weights of all the identified compounds were confirmed by GC/CI-MS, using MeOH as CI ionizing gas. Phytotoxicity Assay. The inhibition potential of the essential oils obtained from the five plant tissues of A. cyanophylla on the seed germination and the shoot and root elongation of lettuce (Lactuca sativa L.) seedlings was investigated. Lettuce seeds were purchased from a local seed shop, sterilized for 5 min with NaClO (1%), and then rinsed with dist. H2O [32] [33]. Three replicates, each comprising 20 seeds, were prepared for the contact tests with each of the five oils, using sterile Petri dishes (90 mm diameter) lined with double-sterile filter paper (Whatman No. 2). The oils were dispersed as an emulsion in dist. H2O using Tween 20. Four doses of the oils (0.004, 0.04, 0.4 and 1 mg/ml) were obtained by dilution of the emulsion in deionized H2O. The dishes were then moistened with 5 ml of A. cyanophylla oils at different concentrations or with 5 ml dist. H2O, used as a negative control. Thereafter, the dishes were sealed with ParafilmÔ, to prevent losses of moisture and to avoid contamination, and placed in a growth chamber to allow germination in the dark at an average temp. of 23 28 for 7 d. A seed was considered germinated when the protrusion of the radicle became evident [32]. The experimental design was a randomized block with three replicates for each treatment and control. After 7 d, the germination percentage was determined. Then, the seedlings of L. sativa were collected, and the shoot and the root lengths were measured, to evaluate the allelopathic activity of the
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oils. The inhibitory or stimulatory effects were calculated using Eqn. 1, which was slightly modified from Chung et al. [34]. Inhibition (¢)/stimulation (þ) [%] ¼ (EOe ¢ Ce)/Ce 100
(1)
where EOe (essential-oil effect) is the parameter measured in presence of A cyanophylla essential oil and Ce (control effect) the parameter measured in presence of dist. H2O. Statistical Analysis. Data from the experiments were subjected to analysis of variance (ANOVA) using SPSS 13.0 for Windows (SPSS Inc. Chicago, IL, USA). The inhibition data relative to the pffiffiffi germination and plant development were transformed using arcsin square-root (arcsin x ) transformation before ANOVA. Means were separated at the 5% significance level by a least significant difference test (DuncanÏs multiple range test). To evaluate whether the essential-oil constituents are useful in reflecting the chemical relationships between organs, 18 compounds detected in the oil samples at an average concentrations of at least 3% of the total oil composition in one plant organ were subjected to PCA and HCA using SPSS 13.0 for Windows.
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