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Essential oil composition of five Artemisia (Compositae) species in regards to chemophenetics
Biochemical Systematics and Ecology 87 (2019) 103960
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Essential oil composition of five Artemisia (Compositae) species in regards to chemophenetics
T
Pedja Janaćkovića,∗, Nemanja Rajčevića, Milan Gavrilovića, Jelica Novakovića, Abdulhmid Giwelib, Danijela Steševićc, Petar D. Marina a
Department of Morphology and Systematics of Plants, University of Belgrade – Faculty of Biology, Belgrade, Studentski Trg 16, 11000, Belgrade, Serbia Department of Botany, University of Al - Gabel Al – Gharbe, Faculty of Science, Zintan, Libya c Department of Biology, University of Montenegro – Faculty of Natural Sciences and Mathematics, Podgorica, Montenegro Džordža Vašingtona bb, 20000, Podgorica, Montenegro b
ARTICLE INFO
ABSTRACT
Keywords: Artemisia Essential oil Chemophenetics
In this work we analysed the essential oils (EO) obtained by hydrodistillation from the aerial parts of five Artemisia species (A. arborescens L., A. campestris L., A. lobelii All., A. annua L. and A. absinthium L.) originated from Serbia, Montenegro, and Libya, by gas chromatography coupled with mass spectrometry. In total, 126 compounds were detected, and 120 were identified. Even though a high number of compounds were detected, each individual sample had only 25 to 50, attesting to a great diversity of compounds between taxa. Depending on the species and the locality (geographical origin), EO was dominated by either monoterpenes or sesquiterpenes, with β-pinene, chamazulene, germacrene D, camphor, pinocarvone and thuja-2,4(10)-diene being the dominant compounds. The chemophenetic value of the EO compositions was discussed in relation to the results of the multivariate statistical test, including the detailed survey of the available literature data.
1. Introduction In relation to the environment within which they grow, higher plants use a different mixture of specialized metabolites for adaptation (e.g., defence against herbivores or pathogenic microorganisms). This metabolic profile is genetically determined, and because of that, the similarity in their metabolite content is applicable in assessing the phylogenetic relationship of higher plants (Liu et al., 2017). Additionally, specialized metabolites have shown to be useful in finding cryptic species (Adams et al., 2005; Fujiwara et al., 2017), determination of genetic differentiation of populations (Adams et al., 2003) or even past hybridization events (Matsumoto et al., 2003). While this type of studies of plants is based on the presence or absence of selected specialized metabolites, they do not always include a metabolite content (Liu et al., 2017). In this regard, chemophenetic studies of plants have the main goal to describe the diversity of specialized metabolites in any given plant taxon. Thus, chemophenetic plant studies contribute to the phenetic description of plant taxa, similar to anatomical, morphological, and karyological approaches, which have already been recognized as major tools for establishing natural systems with the help of modern molecular methods (Zidorn, 2019). Artemisia L. (Artemisiinae - Anthemideae - Compositae), the largest ∗
genus of the tribe Anthemideae, comprises more than 500, mostly perennial species (Vallès et al., 2003; Vallès and Garnatje, 2005). It is a cosmopolitan genus containing wind-pollinated plants, mostly distributed in temperate areas of the northern hemisphere, colonising arid and semi-arid environments (steppes), with only a few representatives in the southern hemisphere (McArthur and Plummer, 1978; Pellicer et al., 2010). They are mostly medicinal plants, which are constantly the object of phytochemical and pharmaceutical investigations, mainly due to the large diversity of their chemicals in general and essential oil production, in particular (Abad et al., 2012). Regarding the phytochemistry of the genus Artemisia, mainly terpenoids, coumarins, flavonoids, caffeoylquinic acids, sterols, and acetylenes are present. Essential oils contain various volatile constituents, e.g. terpenes and phenolic-derived aromatic and aliphatic compounds. They are usually characterized by two-three dominant components at high concentrations (20–70%), compared to other compounds present in trace amounts (Abad et al., 2012). Essential oils (EO) have a broad range of bioactivity, due to the presence of diverse specialized metabolites which manifest their effects (e.g. chemical defence against diseases or predators) through different modes of action (Abad et al., 2012). Some of Artemisia species (e.g. A. absinthium L., A. annua L., A. afra Jacq. ex Willd., A. scoparia Waldst. & Kitam., A. maritima L.) are
Corresponding author. E-mail address: [email protected] (P. Janaćković).
https://doi.org/10.1016/j.bse.2019.103960 Received 14 August 2019; Received in revised form 29 October 2019; Accepted 2 November 2019 0305-1978/ © 2019 Elsevier Ltd. All rights reserved.
Biochemical Systematics and Ecology 87 (2019) 103960
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very rich in terpenoids (Bora and Sharma, 2011). Many Artemisia taxa possess strong and aromatic smell owing to high quantities of volatile terpenes, which occur mostly in leaves and flowers. The chemical composition of the essential oil of the genus Artemisia has been extensively investigated from around the world. Numerous studies have shown that Artemisia taxa exhibit significant intraspecific variability regarding the composition of their essential oil (Abad et al., 2012). The yield and composition of the essential oil of Artemisia species are influenced by the geographic region, harvesting season, plant part, fertilizer and pH of soils, drying conditions, extraction method, but mostly by its genotype (Abad et al., 2012). The taxa belonging to the genus Artemisia are characterized by a wide morphological and phytochemical variability associated with different geographical origins and genetics. Additionally, polyploidy is also common. Reported cytotypes differ in morpho-anatomy, fertility and phytochemical composition (Vallès et al., 2011). Thus, infrageneric taxonomy of the genus Artemisia is very complex and many attempts at infrageneric classification were made. This trend continues today (Vallès et al., 2003). Traditionally five major groups of the genus Artemisia are recognized as subgenera or sections (Absinthium, Artemisia, Dracunculus, Seriphidium, Tridentate) based mainly on the capitula type and fertility of florets (Torrell et al., 1999). Previous phylogenetic analyses on Artemisia s.str. using nuclear DNA ITS (Internal Transcribed Spacer) sequences support the monophyly of the genus and the monophyly of subgenera Dracunculus, Seriphidium, Tridentate, whereas Absinthium and Artemisia appear to be polyphyletic (Torrell et al., 1999; Vallès et al., 2003). Although essential oil of Artemisia species has been thoroughly investigated, our main goal was to examine the composition of the essential oil of five wild-growing Artemisia species (Artemisia arborescens (Vaill.) L., A. campestris L., A. lobelii All., A. annua L. and A. absinthium L.), and to evaluate, together with available literature data, their potential chemophenetic importance.
analysis. The extraction yield of oil was calculated according to the equation given: y = V/W x 100 where y is the oil yield (%, w/w), V is the mass of extracted plant oil (g) and W is the mass of dried plant material (g). 2.3. GC-FID and GC/MS analyses The GC-FID and GC/MS analyses were carried out with an Agilent 7890 A apparatus equipped with a 5975 C mass-selective detector (MSD), a flame ionization detector (FID), and an HP-5 MSI fused-silica cap (column length 30 m, diameter 0.25 mm, film thickness 0.25 mm). The oven temperature was programmed linearly, rising from 60° to 240° at 3°/min; the injector temperature was 220°; the detector temperature was 300°, and the transfer-line temperature was 240°. The carrier gas was He (1.0 mL/min at 210°, constant pressure mode) at an injection volume of 1 μL and a split ratio of 10: 1. Electron impact mass spectra (EI-MS; 70 eV) were acquired over the m/z range 40–550. Library search and mass spectral deconvolution and extraction were performed using the NIST AMDIS (automated mass spectral deconvolution and identification system) software, version 2.64.113.71, with the retention index (RI) calibration data analysis parameters set to the strong level and a 10% penalty for compounds without a RI. The RIs were experimentally determined using the standard method involving retention times (tR) of n-alkanes, which were injected after the essential oil under the same chromatographic conditions. The search was performed against our home-made library, containing 4972 spectra. The relative contents of identified compounds were computed from the GC peak areas. 2.4. Statistical analysis Statistical analysis was done on 21,248 numerical data. Standard statistics (mean, standard deviation, distribution) were used to study data prior to multivariate analyses: Principle Components Analysis (PCA), Discriminant Analysis (DA) and Hierarchical Cluster Analysis (HCA). All statistical analyses were performed using PAST 3.16. (Hammer et al., 2001).
2. Material and methods 2.1. Plant material Ten to fifteen individual plants (aerial parts) were collected in Serbia, Montenegro and Libya in full bloom in 2013, 2016 and 2017 (Table 1). Collected plant material was identified using floras of Serbia, Libya and Europe (Gajić, 1975; Jafri and El-Gadi, 1983; Tutin et al., 1976). Voucher specimens were deposited at the Herbarium (BEOU) of University of Belgrade – Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”.
3. Results and discussion 3.1. Artemisia EO composition and yield Yield and organoleptic characteristics of essential oils (EO) of studied Artemisia species are shown in Table 2. The conducted GC-FID and GC-MS analyses resulted in the detection of 126 compounds, making on average 97.8% of the total oil. In total, 120 compounds were identified in all samples, making up from 85.0% (AN) to 98.8% (LC) of the total oil. Even though a high number of compounds was detected, only 24 (AB) to 50 (CA2) compounds were found per individual sample, attesting to a great diversity of compounds in EO between taxa. All compounds are listed in Table 3. Depending on the species and locality, different terpene groups dominated EOs. Most of EOs were dominated by monoterpenes –
2.2. Isolation of essential oils Plant material was dried at room temperature and then chopped. Between 27 and 196 g of plant material was placed in a round-bottomed flask and 800 mL of distilled water was added. Hydrodistillation was performed for 3 h using the Clevenger-type apparatus, according to the procedure described in Ph. Eur. 6 (European Directorate for the Quality of Medicines, 2007). The obtained oils were stored at 4 °C before the GC Table 1 Composition of essential oils of the aerial parts of investigated Artemisia species. Species Artemisia Artemisia Artemisia Artemisia A. lobelii Artemisia Artemisia a
arborescens arborescens campestris campestris annua absinthium
Code
Country
Locality
Coordinates
Year
BEOUa
AR1 AR2 CA1 CA2 LC AN AB
Montenegro Montenegro Montenegro Libya Serbia Serbia Serbia
Budva Stari Ulcinj island Ćemovsko polje Zintan Zlatibor Belgrade Đerdap
N42.277 N41.993 N42.412 N31.959 N43.707 N44.798 N44.645
2016 2013 2016 2013 2013 2017 2016
17432 17433 17434 17435 17436 17437 17438
Voucher numbers in BEOU. 2
E18.839 E19.139 E19.250 E12.363 E19.540 E20.367 E22.547
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Table 2 Yield and organoleptic characteristics of essential oils of studied Artemisia species. Samplea
Dry plant material (g)
Obtained oil (g)
Yield (%, w/w)
Organoleptic characteristics
AR1 AR2 CA1 CA2 LC AN AB
94 94 94 196 100 27 150
0.026 0.214 0.046 0.353 0.295 0.044 0.233
0.027 0.227 0.048 0.180 0.295 0.162 0.155
ink blue, strong smell greenish-blue, strong smell bright yellow, strong smell transparent yellow, strong smell transparent yellow, strong smell golden yellow, strong smell dark brown, strong smell
a
For population details cf. Table 1.
absinthium, A. lobelii, A. campestris, A. annua and A. arborescens) show varying degree of variability within each species. A. absinthium show highest variability in the composition of EO, and differentiation of EO isolated from different plant organs. On the other hand, A. arborescens shows lowest variability of all studies species, with only three dominant components. Interestingly, A. annua samples of different geographic origin that were grown under same environmental conditions, show significant variability. This suggests stronger influence of genetic vs. environmental factors on the determination of EO composition. To test the chemophenetic significance of essential oil composition of Artemisia species, we also analysed all the available literature data, including present results. PCA (not shown here) showed a separation of all essential oils from different species based on one of the three most abundant components: β-pinene, β-thujone, and camphor. Based on the high abundances of β-pinene A. campestris, A. biennis Willd., A. dracunculus L., A. judaica, and A. pontica L. grouped together (Akrout et al., 2001, 2003; 2010, 2011; Chalchat et al., 2003; Derwich et al., 2009; Ghorab et al., 2013; Janaćković et al., 2015; Juteau et al., 2002b; Kazemi et al., 2009; Kordali et al., 2005; Lopes-Lutz et al., 2008). On the other hand, A. absinthium, A. vulgaris L. and A. herba-alba Asso., and most of the samples of A. arborescens were grouped together based on the high abundances of β-thujone, but also on the higher presence of chamazulene (Abderrahim et al., 2010; Akrout et al., 2010; Ariño et al., 1999; Basta et al., 2007; Blagojević et al., 2006; Derwich et al., 2009; Janaćković et al., 2015; Joshi, 2013; Judzentiene et al., 2012; Judzentiene and Mockutë, 2004; Juteau et al., 2003; Lopes-Lutz et al., 2008; Marongiu et al., 2006; Mihajilov-Krstev et al., 2014; Militello et al., 2011; Morteza-Semnani and Akbarzadeh, 2005; Ornano et al., 2013; Pino et al., 1997; Rezaeinodehi and Khangholi, 2008; Sharopov et al., 2011; Tucker et al., 1993; Younes, 2012). Remaining samples (A. annua, A. lobelii, A. scoparia Waldst. et Kit., A. ludoviciana Nutt., A frigida Willd., A. longifolia Nutt.) separated from all others, based on high levels of camphor (Bagchi et al., 2003; Ćavar et al., 2012; Goel et al., 2007; Holm et al., 1997; Juteau et al., 2002a; Khangholil and Rezaeinodehi, 2008; Lopes-Lutz et al., 2008; Morteza-Semnani and Akbarzadeh, 2005; Perazzo, 2003; Rasooli et al., 2003; Stojanovic et al., 2000; Tellez et al., 1999; Verdian-rizi et al., 2008). To test whether these differences were significant, discriminant analysis with 30 compounds that were present on average in mid-tohigh amounts (> 0.5%) was also done (Fig. 2). Species that were represented with multiple samples were assigned to different groups based on the species level. The rest of the species that were represented with only one sample were not assigned to a group but marked for the discriminant linear classifier and placed on a scatter plot after the discriminant analysis. Almost all of the species showed separation from another based on the EO composition. A. campestris and A. dracunculus grouped close to each other based on the higher abundances of βpinene. On the other hand, A. lobelii and A. annua grouped based on the higher abundances of camphor. Close to them, grouped A. scoparia, A. cana, A. frigida, A. longifolia, A. ludoviciana. Based on the higher abundances of β-thujone, A. herba-alba and A. absinthium were grouped close to each other, and close to them were A. pontica and A. judaica. A. arborescens separated from all the other on amounts of chamazulene.
monoterpene hydrocarbons in CA2, and oxygenated monoterpenes in AR2, LC and AN – 67.1, 75.4%, 61.4% and 56.3%, respectively. Sesquiterpene hydrocarbons were dominant in CA1 and AR1 – 65.5% and 61.4% respectively, while oxygenated sesquiterpenes were dominant only in A. absinthium EO (27.1%) from Belgrade (AB). The essential oil of A. arborescens from Budva (AR1) was characterized by an exceptionally high percentage of chamazulene (38.3%) and (E)-β-caryophyllene (15.6%), followed by camphene (7.1%) and α-pinene (5.7%). On the other hand, the essential oil composition of the population from Island of Stari Ulcinj (AR2) was characterized by an remarkably high percentage of camphor (39.5%) and β-thujone (28.6%), followed by p-cymene (4.6%), while chamazulene was found only in trace amounts. Essential oil of A. campestris from Podgorica (CA1) was characterized by an high percentage of germacrene D (24.2%), followed by bicyclogermacrene (14.7%), γ-himachalene (12.6%) and β-pinene (9.1%), while plants from Zintan (CA2) were characterized by a particularly high percentage of β-pinene (46.4%), followed by p-cymene (6.1%) and limonene (5.4%). The most dominant compound of A. lobeli from Zlatibor (LC) was camphor (41.9%), followed by 1,8-cineole (13.8%) and unidentified sesquiterpene (10.2%), while in oil of A. annua from Belgrade (AN) the most dominant was pinocarvone (29.4%), followed by artemisia ketone (19.2%) and caryophyllene oxide (5.9%). The most dominant compound of A. absinthium from Djerdap (AB) was thuja-2,4(10)-diene (15.9%) and caryophyllene oxide (14.9%), followed by β-thujone (12.8%). To assess the overall similarity of essential oil composition PCA was performed (Fig. 1). The first two PC axes account for 55.1% of the total variability. Based on camphor, chamazulene and α-pinene PCA showed the separation of all samples. Also, A. campestris from Libya and Serbia showed greater similarity between each other than two samples of A. arborescens from Montenegro, which is congruent with the available literature data on these two species (cf. Supplementary material). The literature data on ecological and biological significance of dominant compounds is scarce (Langenheim, 1994). However, available literature data testify of an array of biological activities of several compounds: i.e. (E)- β-caryophyllene and chamazulene show allelopathic effect (Sánchez-Muñoz et al., 2012; Solymosi, 2000), while germacrene D and camphor play role as an insect attractant (Arakaki et al., 2009; Røstelien et al., 2000). Hence, the essential oil composition can represent adaptive feature of studied species. Present results, show differences in dominant classes of compounds between species, and even within the same species from different geographic regions. These results are congruent with the literature data (Supplemental Table S1). However, since the present sample was rather small, detailed analysis using literature data was also performed. Pooled data (literature + present data) were analysed using multivariate statistical methods, to assess the variability within each of the analysed species. Results of intraspecific variability are presented in the Supplemental material. 3.2. Chemophenetics of Artemisia based on EO Essential oil compositon of the studied Artemisia species (A. 3
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Table 3 Composition of essential oils of the aerial parts of investigated Artemisia species. RI
Compounds
AR1
AR2
CA1
CA2
LC
AN
AB
878 926 926 934 951 952 974 978 995 996 996 1000 1008 1019 1023 1027 1031 1033 1040 1062 1062 1095 1106 1108 1100 1117 1121 1125 1127 1138 1139 1140 1145 1156 1160 1165 1177 1191 1192 1218 1243 1243 1337 1351 1374 1377 1386 1391 1393 1410 1421 1422 1430 1440 1441 1450 1454 1459 1462 1476 1478 1482 1487 1488 1494 1497 1497 1499 1500 1502 1503 1509 1510 1516
1,3-Dimethylbenzene Tricyclene α-Thujene α-Pinene Camphene Thuja-2,4(10)-diene Sabinene β-Pinene Myrcene p-mentha-1(7),8-diene Yomogi alcohol Mesitylene β-Phellandrene α-Terpinene o-Cymene p-Cymene Limonene 1,8-Cineole (Z)-β-Ocimene γ-Terpinene Artemisia ketone Terpinolene Linalool 2-Methylbutyl 2-methyl butyrate β-Thujone endo-Fenchol α-Thujone p-Menth-2-en-1-ol Chrysanthenone Nopinone iso-3-Thujanol trans-Pinocarveol Camphor endo-2-Methylbicyclo[3.3.1]nonane Pinocarvone Borneol Terpinen-4-ol α-Terpineol Myrtenol cis-3-Hexenyl valerate Isobornyl acetate Bornyl acetate δ-Elemene α-Longipinene Modheph-2-ene α-Copaene β-Bourbonene β-Cubebene β-Elemene α-Gurjunene (E)-β-Caryophyllene Lavandulylisobutanoate β-Copaene α-Guaiene Aromadendrene α-Himachalene α-Humulene (E)-β-Farnesene (Z)-Caryophyllene trans-Cadina-1(6),4-diene γ-Muurolene Germacrene D γ-Himachalene β-Selinene trans-Muurola-4(14),5-diene Valencene α-Selinene Pentadecane Bicyclogermacrene α-Cuprenene α-Muurolene Geranyl propionate (Z)-γ-Bisabolene γ-Cadinene
– – 0.49 5.68 7.08 – – 0.42 – – – – 0.64 1.47 – 0.84 3.16 – – 3.14 – 0.85 – – – – – – – – – – 6.44 – – – – – – – – – – – – 0.48 0.14 – – – 15.57 – – – – – 0.97 – – – 0.94 1.94 – 0.68 – 0.43 – – – – tr – – 0.94
– – 0.30 1.03 2.35 – – 0.08 – – – – – 0.40 – 4.58 – 0.73 – 0.77 – 0.20 1.08 0.36 1.52 – 28.59 0.33 – – tr – 39.46 – – 0.24 2.53 0.29 – – – – – – – 0.13 0.15 – tr – 0.46 – – – – – – – 0.07 – 0.17 – – 0.16 – – – – – – – – – –
– – – 1.12 0.47 – 0.25 9.14 1.05 – – – – – – 0.78 2.02 – 0.18 0.19 – – – – – – – – – – – – – – – – – – – – – 3.11 0.49 – – 0.65 – 0.15 0.17 0.12 1.62 – 0.22 0.29 – 0.55 0.46 3.43 0.34 – 0.43 24.15 12.62 – 0.27 – – – 14.74 0.76 – – 1.77 0.53
– – 0.17 6.77 0.34 – – 46.41 – 0.27 – – – 0.25 – 6.14 5.40 – – 0.90 – 0.41 0.15 – 0.73 0.20 tr – – 0.54 – 1.94 0.19 – 0.87 0.29 0.96 1.19 1.77 0.41 – – – – – 0.53 – – – – 0.20 – – – 0.60 – – – – 0.15 0.84 0.10 – – – 0.53 – – – – 0.32 – – 0.53
– 0.43 0.09 0.95 8.89 – – 0.77 – – – 0.25 – 0.11 – 0.87 – 13.80 – 0.20 – – – – – – – – 0.17 – – 0.19 41.94 – 0.63 3.38 0.49 – 0.80 – 0.39 – – 0.25 – – – – 0.35 – – – – – – – – – – – – – 0.19 0.30 – – 0.17 – – – – – – –
0.38 – – – 0.29 – – – – – 0.59 – – – – – – 4.72 – – 19.19 1.60 – – – – – – – – – – 1.34 – 29.40 1.68 – – – 1.09 – – – – 1.77 – – – – – 1.75 – – – – – – – – – – – 0.67 – 0.90 – – 0.53 – – – – – –
1.30 – 1.80 0.43 – 15.89 – – – – – – – 0.50 2.26 0.64 – – – 1.18 – – – – 0.93 – 12.83 – – – 1.10 – – 2.73 – – 5.51 – – – – – – – – – – – – – 0.81 0.68 – – – – – – – – – – – 5.66 2.01 – – – 0.95 – – 5.86 – –
(continued on next page) 4
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Table 3 (continued) RI
Compounds
AR1
AR2
CA1
CA2
LC
AN
AB
1519 1525 1526 1538 1540 1543 1545 1550 1565 1575 1578 1579 1581 1584 1592 1594 1608 1609 1610 1619 1623 1626 1629 1629 1632 1632 1632 1639 1641 1642 1643 1644 1650 1654 1654 1658 1662 1672 1677 1681 1681 1685 1687 1712 1718 1722 1730 1737 1743 1765 1771 2007
α-Muurolol allo-Aromadendrene epoxide β-Eudesmol α-Eudesmol Selin-11-en-4-alpha-ol Humalene-1,6-dien-3-ol 14-hydroxy-(Z)-caryophyllene C15H22O isomer Elemol acetate Helifolenol B C15H22O isomer Germacra-4(15),5,10(14)-trien-1-α-ol 10-Nor-calamenen-10-one C15H24O isomer Isobicyclogermacrenal Unknown #1b Chamazulene Unknown #2b Unknown #3b β-Costol α-Costol (E)-Nuciferylbutanoatea
Cadala-1(10),3,8-triene δ-Cadinene Myristicin trans-cadina-1,4-diene α-Cadinene C15H22O isomer α-Calacorene Elemol (E)-Nerolidol Nerylisovalerate syn-anti-antiHelifolen-12-al A[a] C15H24O isomer Humulane-1,6-dien-3-ol Caryophyllene oxide C15H22O isomer Salvial-4(14)-en-1-one Bornylangelate Geranylisovalerate Geranyl 2-methyl butanoate Junenol β-Cedrene epoxide trans-Isolongifolanone[a] α-Colacalene Selina-3,11-dien-6-alpha-ol γ-Eudesmol 10-epi-γ-Eudesmol cis-Cadin-4-en-7-ol C14H18 isomer Caryophylla-4(12),8(13)-dien-5-β-ol epi-α-Cadinol – – – – – – – – – – – – – – – – 38.25 – – – – 2.29
– 0.78 – – 0.19 – – – – – – – – 0.80 – – – – 0.30 0.31 – 0.61 – – – – – – – 1.04 – – 2.01 – – 0.16 – – – – – – – – – – 0.23 – – – – 3.42
– – 0.22 – – – – – – – – – – 2.54 – – – 0.68 – 0.15 – – – – – 0.43 – – – – 2.17 – – 1.53 – – – – – – – – 0.97 – – – – – – – – –
– 1.41 – 0.16 tr – 0.14 – 0.73 – – 5.32 0.53 0.97 0.31 0.16 – – 0.18 – – – – – – – – 0.84 – – – – 2.60 0.47 – – – – – – – – 0.31 – – – – – – – – –
– 1.89 – 0.12 tr – 0.14 0.12 0.56 – – 6.26 – 1.37 – 0.51 – 2.90 0.33 – – – 0.29 – 0.32 – – 0.35 – – – – 1.08 0.91 – – 1.14 – 0.39 0.18 – – – – – – – – – 0.69 0.65 –
– – – – – 4.20 – 2.56 – – 10.20 – – 0.40 – – – – – – – – – – 0.78 – – – – – – 0.70 – – 1.53 0.85 0.52 1.82 – – 0.98 2.07 – 6.15 0.74 1.02 – 1.32 0.44 – – –
– – – – – – – – – – – – – 5.93 – 0.50 0.42 – – – 2.37 – – 2.61 – – 0.65 – 1.88 – – – – – – 0.48 – – – – – – – – – – – – – – – –
1.54 – – – – – 5.10 – – 12.16 – – – 14.89 – – – – – – – – – – – – – – – –
Total monoterpenes Monoterpene hydrocarbons Monoterpenes oxygenated Total sesquiterpenes Sesquiterpene hydrocarbons Sesquiterpene oxygenated Other Unknown
30.20 23.76 6.44 64.46 61.40 3.06 0.06 2.29
85.16 9.79 75.37 7.28 1.47 5.82 0.49 3.81
15.18 15.18 0.00 78.38 65.51 12.87 3.94 0.00
76.03 67.13 8.90 22.06 6.03 16.03 0.75 0.00
73.96 12.56 61.40 24.05 1.26 22.79 0.78 0.00
58.20 1.88 56.32 24.69 5.08 19.61 3.27 8.94
43.06 22.69 20.37 43.12 16.07 27.05 11.05 0.00
TOTAL [%]: TOTAL (No):
97.01 31
96.75 40
97.50 45
98.84 50
98.79 35
98.70 35
97.24 24
a b
Tentatively identified by the combination of MS and RI. Mass spectra are shown in Appendix A (Supplemental Fig. S2).
Only essential oil of A. vulgaris was found close to this species. These groups are in concordance with the ITS phylogeny (Vallès et al., 2003), where A. dracunculus and A. campestris form a monophyletic group that is sister to other Artemisia species, which appears to be the case also with the essential oil composition (Fig. 2). A. absinthium and A. arborescens formed monophyletic group in the ITS analysis, and
this was also the case in the essential oil composition. Only discrepancy with the comparable ITS data is with A. herba-alba, but since there were only three samples available, this is somewhat to be expected with this type of data (essential oil composition). Combined ITS and ETS (External Transcribed Spacer) data Sanz et al. (2008) showed that all Artemisiinae genera constitute a monophyletic group. Also, they showed 5
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Fig. 1. Scatter plot of PCA analysis on the composition of the EO of studied species and localities. For population details cf. Table 1.
affiliation of the Artemisia species to the Artemisia vulgaris complex, North American endemic group, subgenus Seriphidium, Artemisia afra clade, Artemisia and Absinthium clade, Dracunculus clade and some informal groups and taxa. This appears to be the case with the essential oil composition, too (Fig. 2). The discrepancy is A. scoparia, which is positioned close to the North American group instead of in Dracunculus clade and A. herba-alba, which overlapped with Artemisia and Absinthium clade instead of subgenus Seriphidium. Further examination of EO of all other Artemisia species will certainly throw the light on concordance of EOs and molecular data. All this leads to the conclusion
that combining phytochemical/chemophenetic, anatomical, morphological and karyological analyses with modern molecular methods of other Artemisia taxa, will give a better infrageneric classification of this genus. 4. Conclusions Even though essential oil composition and biosynthetic pathways can vary to an extent depending on plant organ and some environmental factors, they are genetically determined and correspond to
Fig. 2. Scatter plot for discriminant analysis of 30 compounds present in the essential oil of different Artemisia species. The essential oil obtained from the leaves (●), areal parts (■) and leaves + flowers (◆). Lines show groups according to Sanz et al. (2008): Artemisia vulgaris complex, North American group, Seriphidium, Absinthium, Dracunculus. 6
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evolutionary trends. The qualitative and relative quantitative essential oil composition of the Artemisia species together with concordance with molecular data are applicable as a chemophenetic characters that may contribute to better understanding of systematics of Artemisia taxa.
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Essential oil composition of five Artemisia (Compositae) species in regards to chemophenetics
T
Pedja Janaćkovića,∗, Nemanja Rajčevića, Milan Gavrilovića, Jelica Novakovića, Abdulhmid Giwelib, Danijela Steševićc, Petar D. Marina a
Department of Morphology and Systematics of Plants, University of Belgrade – Faculty of Biology, Belgrade, Studentski Trg 16, 11000, Belgrade, Serbia Department of Botany, University of Al - Gabel Al – Gharbe, Faculty of Science, Zintan, Libya c Department of Biology, University of Montenegro – Faculty of Natural Sciences and Mathematics, Podgorica, Montenegro Džordža Vašingtona bb, 20000, Podgorica, Montenegro b
ARTICLE INFO
ABSTRACT
Keywords: Artemisia Essential oil Chemophenetics
In this work we analysed the essential oils (EO) obtained by hydrodistillation from the aerial parts of five Artemisia species (A. arborescens L., A. campestris L., A. lobelii All., A. annua L. and A. absinthium L.) originated from Serbia, Montenegro, and Libya, by gas chromatography coupled with mass spectrometry. In total, 126 compounds were detected, and 120 were identified. Even though a high number of compounds were detected, each individual sample had only 25 to 50, attesting to a great diversity of compounds between taxa. Depending on the species and the locality (geographical origin), EO was dominated by either monoterpenes or sesquiterpenes, with β-pinene, chamazulene, germacrene D, camphor, pinocarvone and thuja-2,4(10)-diene being the dominant compounds. The chemophenetic value of the EO compositions was discussed in relation to the results of the multivariate statistical test, including the detailed survey of the available literature data.
1. Introduction In relation to the environment within which they grow, higher plants use a different mixture of specialized metabolites for adaptation (e.g., defence against herbivores or pathogenic microorganisms). This metabolic profile is genetically determined, and because of that, the similarity in their metabolite content is applicable in assessing the phylogenetic relationship of higher plants (Liu et al., 2017). Additionally, specialized metabolites have shown to be useful in finding cryptic species (Adams et al., 2005; Fujiwara et al., 2017), determination of genetic differentiation of populations (Adams et al., 2003) or even past hybridization events (Matsumoto et al., 2003). While this type of studies of plants is based on the presence or absence of selected specialized metabolites, they do not always include a metabolite content (Liu et al., 2017). In this regard, chemophenetic studies of plants have the main goal to describe the diversity of specialized metabolites in any given plant taxon. Thus, chemophenetic plant studies contribute to the phenetic description of plant taxa, similar to anatomical, morphological, and karyological approaches, which have already been recognized as major tools for establishing natural systems with the help of modern molecular methods (Zidorn, 2019). Artemisia L. (Artemisiinae - Anthemideae - Compositae), the largest ∗
genus of the tribe Anthemideae, comprises more than 500, mostly perennial species (Vallès et al., 2003; Vallès and Garnatje, 2005). It is a cosmopolitan genus containing wind-pollinated plants, mostly distributed in temperate areas of the northern hemisphere, colonising arid and semi-arid environments (steppes), with only a few representatives in the southern hemisphere (McArthur and Plummer, 1978; Pellicer et al., 2010). They are mostly medicinal plants, which are constantly the object of phytochemical and pharmaceutical investigations, mainly due to the large diversity of their chemicals in general and essential oil production, in particular (Abad et al., 2012). Regarding the phytochemistry of the genus Artemisia, mainly terpenoids, coumarins, flavonoids, caffeoylquinic acids, sterols, and acetylenes are present. Essential oils contain various volatile constituents, e.g. terpenes and phenolic-derived aromatic and aliphatic compounds. They are usually characterized by two-three dominant components at high concentrations (20–70%), compared to other compounds present in trace amounts (Abad et al., 2012). Essential oils (EO) have a broad range of bioactivity, due to the presence of diverse specialized metabolites which manifest their effects (e.g. chemical defence against diseases or predators) through different modes of action (Abad et al., 2012). Some of Artemisia species (e.g. A. absinthium L., A. annua L., A. afra Jacq. ex Willd., A. scoparia Waldst. & Kitam., A. maritima L.) are
Corresponding author. E-mail address: [email protected] (P. Janaćković).
https://doi.org/10.1016/j.bse.2019.103960 Received 14 August 2019; Received in revised form 29 October 2019; Accepted 2 November 2019 0305-1978/ © 2019 Elsevier Ltd. All rights reserved.
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very rich in terpenoids (Bora and Sharma, 2011). Many Artemisia taxa possess strong and aromatic smell owing to high quantities of volatile terpenes, which occur mostly in leaves and flowers. The chemical composition of the essential oil of the genus Artemisia has been extensively investigated from around the world. Numerous studies have shown that Artemisia taxa exhibit significant intraspecific variability regarding the composition of their essential oil (Abad et al., 2012). The yield and composition of the essential oil of Artemisia species are influenced by the geographic region, harvesting season, plant part, fertilizer and pH of soils, drying conditions, extraction method, but mostly by its genotype (Abad et al., 2012). The taxa belonging to the genus Artemisia are characterized by a wide morphological and phytochemical variability associated with different geographical origins and genetics. Additionally, polyploidy is also common. Reported cytotypes differ in morpho-anatomy, fertility and phytochemical composition (Vallès et al., 2011). Thus, infrageneric taxonomy of the genus Artemisia is very complex and many attempts at infrageneric classification were made. This trend continues today (Vallès et al., 2003). Traditionally five major groups of the genus Artemisia are recognized as subgenera or sections (Absinthium, Artemisia, Dracunculus, Seriphidium, Tridentate) based mainly on the capitula type and fertility of florets (Torrell et al., 1999). Previous phylogenetic analyses on Artemisia s.str. using nuclear DNA ITS (Internal Transcribed Spacer) sequences support the monophyly of the genus and the monophyly of subgenera Dracunculus, Seriphidium, Tridentate, whereas Absinthium and Artemisia appear to be polyphyletic (Torrell et al., 1999; Vallès et al., 2003). Although essential oil of Artemisia species has been thoroughly investigated, our main goal was to examine the composition of the essential oil of five wild-growing Artemisia species (Artemisia arborescens (Vaill.) L., A. campestris L., A. lobelii All., A. annua L. and A. absinthium L.), and to evaluate, together with available literature data, their potential chemophenetic importance.
analysis. The extraction yield of oil was calculated according to the equation given: y = V/W x 100 where y is the oil yield (%, w/w), V is the mass of extracted plant oil (g) and W is the mass of dried plant material (g). 2.3. GC-FID and GC/MS analyses The GC-FID and GC/MS analyses were carried out with an Agilent 7890 A apparatus equipped with a 5975 C mass-selective detector (MSD), a flame ionization detector (FID), and an HP-5 MSI fused-silica cap (column length 30 m, diameter 0.25 mm, film thickness 0.25 mm). The oven temperature was programmed linearly, rising from 60° to 240° at 3°/min; the injector temperature was 220°; the detector temperature was 300°, and the transfer-line temperature was 240°. The carrier gas was He (1.0 mL/min at 210°, constant pressure mode) at an injection volume of 1 μL and a split ratio of 10: 1. Electron impact mass spectra (EI-MS; 70 eV) were acquired over the m/z range 40–550. Library search and mass spectral deconvolution and extraction were performed using the NIST AMDIS (automated mass spectral deconvolution and identification system) software, version 2.64.113.71, with the retention index (RI) calibration data analysis parameters set to the strong level and a 10% penalty for compounds without a RI. The RIs were experimentally determined using the standard method involving retention times (tR) of n-alkanes, which were injected after the essential oil under the same chromatographic conditions. The search was performed against our home-made library, containing 4972 spectra. The relative contents of identified compounds were computed from the GC peak areas. 2.4. Statistical analysis Statistical analysis was done on 21,248 numerical data. Standard statistics (mean, standard deviation, distribution) were used to study data prior to multivariate analyses: Principle Components Analysis (PCA), Discriminant Analysis (DA) and Hierarchical Cluster Analysis (HCA). All statistical analyses were performed using PAST 3.16. (Hammer et al., 2001).
2. Material and methods 2.1. Plant material Ten to fifteen individual plants (aerial parts) were collected in Serbia, Montenegro and Libya in full bloom in 2013, 2016 and 2017 (Table 1). Collected plant material was identified using floras of Serbia, Libya and Europe (Gajić, 1975; Jafri and El-Gadi, 1983; Tutin et al., 1976). Voucher specimens were deposited at the Herbarium (BEOU) of University of Belgrade – Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”.
3. Results and discussion 3.1. Artemisia EO composition and yield Yield and organoleptic characteristics of essential oils (EO) of studied Artemisia species are shown in Table 2. The conducted GC-FID and GC-MS analyses resulted in the detection of 126 compounds, making on average 97.8% of the total oil. In total, 120 compounds were identified in all samples, making up from 85.0% (AN) to 98.8% (LC) of the total oil. Even though a high number of compounds was detected, only 24 (AB) to 50 (CA2) compounds were found per individual sample, attesting to a great diversity of compounds in EO between taxa. All compounds are listed in Table 3. Depending on the species and locality, different terpene groups dominated EOs. Most of EOs were dominated by monoterpenes –
2.2. Isolation of essential oils Plant material was dried at room temperature and then chopped. Between 27 and 196 g of plant material was placed in a round-bottomed flask and 800 mL of distilled water was added. Hydrodistillation was performed for 3 h using the Clevenger-type apparatus, according to the procedure described in Ph. Eur. 6 (European Directorate for the Quality of Medicines, 2007). The obtained oils were stored at 4 °C before the GC Table 1 Composition of essential oils of the aerial parts of investigated Artemisia species. Species Artemisia Artemisia Artemisia Artemisia A. lobelii Artemisia Artemisia a
arborescens arborescens campestris campestris annua absinthium
Code
Country
Locality
Coordinates
Year
BEOUa
AR1 AR2 CA1 CA2 LC AN AB
Montenegro Montenegro Montenegro Libya Serbia Serbia Serbia
Budva Stari Ulcinj island Ćemovsko polje Zintan Zlatibor Belgrade Đerdap
N42.277 N41.993 N42.412 N31.959 N43.707 N44.798 N44.645
2016 2013 2016 2013 2013 2017 2016
17432 17433 17434 17435 17436 17437 17438
Voucher numbers in BEOU. 2
E18.839 E19.139 E19.250 E12.363 E19.540 E20.367 E22.547
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Table 2 Yield and organoleptic characteristics of essential oils of studied Artemisia species. Samplea
Dry plant material (g)
Obtained oil (g)
Yield (%, w/w)
Organoleptic characteristics
AR1 AR2 CA1 CA2 LC AN AB
94 94 94 196 100 27 150
0.026 0.214 0.046 0.353 0.295 0.044 0.233
0.027 0.227 0.048 0.180 0.295 0.162 0.155
ink blue, strong smell greenish-blue, strong smell bright yellow, strong smell transparent yellow, strong smell transparent yellow, strong smell golden yellow, strong smell dark brown, strong smell
a
For population details cf. Table 1.
absinthium, A. lobelii, A. campestris, A. annua and A. arborescens) show varying degree of variability within each species. A. absinthium show highest variability in the composition of EO, and differentiation of EO isolated from different plant organs. On the other hand, A. arborescens shows lowest variability of all studies species, with only three dominant components. Interestingly, A. annua samples of different geographic origin that were grown under same environmental conditions, show significant variability. This suggests stronger influence of genetic vs. environmental factors on the determination of EO composition. To test the chemophenetic significance of essential oil composition of Artemisia species, we also analysed all the available literature data, including present results. PCA (not shown here) showed a separation of all essential oils from different species based on one of the three most abundant components: β-pinene, β-thujone, and camphor. Based on the high abundances of β-pinene A. campestris, A. biennis Willd., A. dracunculus L., A. judaica, and A. pontica L. grouped together (Akrout et al., 2001, 2003; 2010, 2011; Chalchat et al., 2003; Derwich et al., 2009; Ghorab et al., 2013; Janaćković et al., 2015; Juteau et al., 2002b; Kazemi et al., 2009; Kordali et al., 2005; Lopes-Lutz et al., 2008). On the other hand, A. absinthium, A. vulgaris L. and A. herba-alba Asso., and most of the samples of A. arborescens were grouped together based on the high abundances of β-thujone, but also on the higher presence of chamazulene (Abderrahim et al., 2010; Akrout et al., 2010; Ariño et al., 1999; Basta et al., 2007; Blagojević et al., 2006; Derwich et al., 2009; Janaćković et al., 2015; Joshi, 2013; Judzentiene et al., 2012; Judzentiene and Mockutë, 2004; Juteau et al., 2003; Lopes-Lutz et al., 2008; Marongiu et al., 2006; Mihajilov-Krstev et al., 2014; Militello et al., 2011; Morteza-Semnani and Akbarzadeh, 2005; Ornano et al., 2013; Pino et al., 1997; Rezaeinodehi and Khangholi, 2008; Sharopov et al., 2011; Tucker et al., 1993; Younes, 2012). Remaining samples (A. annua, A. lobelii, A. scoparia Waldst. et Kit., A. ludoviciana Nutt., A frigida Willd., A. longifolia Nutt.) separated from all others, based on high levels of camphor (Bagchi et al., 2003; Ćavar et al., 2012; Goel et al., 2007; Holm et al., 1997; Juteau et al., 2002a; Khangholil and Rezaeinodehi, 2008; Lopes-Lutz et al., 2008; Morteza-Semnani and Akbarzadeh, 2005; Perazzo, 2003; Rasooli et al., 2003; Stojanovic et al., 2000; Tellez et al., 1999; Verdian-rizi et al., 2008). To test whether these differences were significant, discriminant analysis with 30 compounds that were present on average in mid-tohigh amounts (> 0.5%) was also done (Fig. 2). Species that were represented with multiple samples were assigned to different groups based on the species level. The rest of the species that were represented with only one sample were not assigned to a group but marked for the discriminant linear classifier and placed on a scatter plot after the discriminant analysis. Almost all of the species showed separation from another based on the EO composition. A. campestris and A. dracunculus grouped close to each other based on the higher abundances of βpinene. On the other hand, A. lobelii and A. annua grouped based on the higher abundances of camphor. Close to them, grouped A. scoparia, A. cana, A. frigida, A. longifolia, A. ludoviciana. Based on the higher abundances of β-thujone, A. herba-alba and A. absinthium were grouped close to each other, and close to them were A. pontica and A. judaica. A. arborescens separated from all the other on amounts of chamazulene.
monoterpene hydrocarbons in CA2, and oxygenated monoterpenes in AR2, LC and AN – 67.1, 75.4%, 61.4% and 56.3%, respectively. Sesquiterpene hydrocarbons were dominant in CA1 and AR1 – 65.5% and 61.4% respectively, while oxygenated sesquiterpenes were dominant only in A. absinthium EO (27.1%) from Belgrade (AB). The essential oil of A. arborescens from Budva (AR1) was characterized by an exceptionally high percentage of chamazulene (38.3%) and (E)-β-caryophyllene (15.6%), followed by camphene (7.1%) and α-pinene (5.7%). On the other hand, the essential oil composition of the population from Island of Stari Ulcinj (AR2) was characterized by an remarkably high percentage of camphor (39.5%) and β-thujone (28.6%), followed by p-cymene (4.6%), while chamazulene was found only in trace amounts. Essential oil of A. campestris from Podgorica (CA1) was characterized by an high percentage of germacrene D (24.2%), followed by bicyclogermacrene (14.7%), γ-himachalene (12.6%) and β-pinene (9.1%), while plants from Zintan (CA2) were characterized by a particularly high percentage of β-pinene (46.4%), followed by p-cymene (6.1%) and limonene (5.4%). The most dominant compound of A. lobeli from Zlatibor (LC) was camphor (41.9%), followed by 1,8-cineole (13.8%) and unidentified sesquiterpene (10.2%), while in oil of A. annua from Belgrade (AN) the most dominant was pinocarvone (29.4%), followed by artemisia ketone (19.2%) and caryophyllene oxide (5.9%). The most dominant compound of A. absinthium from Djerdap (AB) was thuja-2,4(10)-diene (15.9%) and caryophyllene oxide (14.9%), followed by β-thujone (12.8%). To assess the overall similarity of essential oil composition PCA was performed (Fig. 1). The first two PC axes account for 55.1% of the total variability. Based on camphor, chamazulene and α-pinene PCA showed the separation of all samples. Also, A. campestris from Libya and Serbia showed greater similarity between each other than two samples of A. arborescens from Montenegro, which is congruent with the available literature data on these two species (cf. Supplementary material). The literature data on ecological and biological significance of dominant compounds is scarce (Langenheim, 1994). However, available literature data testify of an array of biological activities of several compounds: i.e. (E)- β-caryophyllene and chamazulene show allelopathic effect (Sánchez-Muñoz et al., 2012; Solymosi, 2000), while germacrene D and camphor play role as an insect attractant (Arakaki et al., 2009; Røstelien et al., 2000). Hence, the essential oil composition can represent adaptive feature of studied species. Present results, show differences in dominant classes of compounds between species, and even within the same species from different geographic regions. These results are congruent with the literature data (Supplemental Table S1). However, since the present sample was rather small, detailed analysis using literature data was also performed. Pooled data (literature + present data) were analysed using multivariate statistical methods, to assess the variability within each of the analysed species. Results of intraspecific variability are presented in the Supplemental material. 3.2. Chemophenetics of Artemisia based on EO Essential oil compositon of the studied Artemisia species (A. 3
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Table 3 Composition of essential oils of the aerial parts of investigated Artemisia species. RI
Compounds
AR1
AR2
CA1
CA2
LC
AN
AB
878 926 926 934 951 952 974 978 995 996 996 1000 1008 1019 1023 1027 1031 1033 1040 1062 1062 1095 1106 1108 1100 1117 1121 1125 1127 1138 1139 1140 1145 1156 1160 1165 1177 1191 1192 1218 1243 1243 1337 1351 1374 1377 1386 1391 1393 1410 1421 1422 1430 1440 1441 1450 1454 1459 1462 1476 1478 1482 1487 1488 1494 1497 1497 1499 1500 1502 1503 1509 1510 1516
1,3-Dimethylbenzene Tricyclene α-Thujene α-Pinene Camphene Thuja-2,4(10)-diene Sabinene β-Pinene Myrcene p-mentha-1(7),8-diene Yomogi alcohol Mesitylene β-Phellandrene α-Terpinene o-Cymene p-Cymene Limonene 1,8-Cineole (Z)-β-Ocimene γ-Terpinene Artemisia ketone Terpinolene Linalool 2-Methylbutyl 2-methyl butyrate β-Thujone endo-Fenchol α-Thujone p-Menth-2-en-1-ol Chrysanthenone Nopinone iso-3-Thujanol trans-Pinocarveol Camphor endo-2-Methylbicyclo[3.3.1]nonane Pinocarvone Borneol Terpinen-4-ol α-Terpineol Myrtenol cis-3-Hexenyl valerate Isobornyl acetate Bornyl acetate δ-Elemene α-Longipinene Modheph-2-ene α-Copaene β-Bourbonene β-Cubebene β-Elemene α-Gurjunene (E)-β-Caryophyllene Lavandulylisobutanoate β-Copaene α-Guaiene Aromadendrene α-Himachalene α-Humulene (E)-β-Farnesene (Z)-Caryophyllene trans-Cadina-1(6),4-diene γ-Muurolene Germacrene D γ-Himachalene β-Selinene trans-Muurola-4(14),5-diene Valencene α-Selinene Pentadecane Bicyclogermacrene α-Cuprenene α-Muurolene Geranyl propionate (Z)-γ-Bisabolene γ-Cadinene
– – 0.49 5.68 7.08 – – 0.42 – – – – 0.64 1.47 – 0.84 3.16 – – 3.14 – 0.85 – – – – – – – – – – 6.44 – – – – – – – – – – – – 0.48 0.14 – – – 15.57 – – – – – 0.97 – – – 0.94 1.94 – 0.68 – 0.43 – – – – tr – – 0.94
– – 0.30 1.03 2.35 – – 0.08 – – – – – 0.40 – 4.58 – 0.73 – 0.77 – 0.20 1.08 0.36 1.52 – 28.59 0.33 – – tr – 39.46 – – 0.24 2.53 0.29 – – – – – – – 0.13 0.15 – tr – 0.46 – – – – – – – 0.07 – 0.17 – – 0.16 – – – – – – – – – –
– – – 1.12 0.47 – 0.25 9.14 1.05 – – – – – – 0.78 2.02 – 0.18 0.19 – – – – – – – – – – – – – – – – – – – – – 3.11 0.49 – – 0.65 – 0.15 0.17 0.12 1.62 – 0.22 0.29 – 0.55 0.46 3.43 0.34 – 0.43 24.15 12.62 – 0.27 – – – 14.74 0.76 – – 1.77 0.53
– – 0.17 6.77 0.34 – – 46.41 – 0.27 – – – 0.25 – 6.14 5.40 – – 0.90 – 0.41 0.15 – 0.73 0.20 tr – – 0.54 – 1.94 0.19 – 0.87 0.29 0.96 1.19 1.77 0.41 – – – – – 0.53 – – – – 0.20 – – – 0.60 – – – – 0.15 0.84 0.10 – – – 0.53 – – – – 0.32 – – 0.53
– 0.43 0.09 0.95 8.89 – – 0.77 – – – 0.25 – 0.11 – 0.87 – 13.80 – 0.20 – – – – – – – – 0.17 – – 0.19 41.94 – 0.63 3.38 0.49 – 0.80 – 0.39 – – 0.25 – – – – 0.35 – – – – – – – – – – – – – 0.19 0.30 – – 0.17 – – – – – – –
0.38 – – – 0.29 – – – – – 0.59 – – – – – – 4.72 – – 19.19 1.60 – – – – – – – – – – 1.34 – 29.40 1.68 – – – 1.09 – – – – 1.77 – – – – – 1.75 – – – – – – – – – – – 0.67 – 0.90 – – 0.53 – – – – – –
1.30 – 1.80 0.43 – 15.89 – – – – – – – 0.50 2.26 0.64 – – – 1.18 – – – – 0.93 – 12.83 – – – 1.10 – – 2.73 – – 5.51 – – – – – – – – – – – – – 0.81 0.68 – – – – – – – – – – – 5.66 2.01 – – – 0.95 – – 5.86 – –
(continued on next page) 4
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Table 3 (continued) RI
Compounds
AR1
AR2
CA1
CA2
LC
AN
AB
1519 1525 1526 1538 1540 1543 1545 1550 1565 1575 1578 1579 1581 1584 1592 1594 1608 1609 1610 1619 1623 1626 1629 1629 1632 1632 1632 1639 1641 1642 1643 1644 1650 1654 1654 1658 1662 1672 1677 1681 1681 1685 1687 1712 1718 1722 1730 1737 1743 1765 1771 2007
α-Muurolol allo-Aromadendrene epoxide β-Eudesmol α-Eudesmol Selin-11-en-4-alpha-ol Humalene-1,6-dien-3-ol 14-hydroxy-(Z)-caryophyllene C15H22O isomer Elemol acetate Helifolenol B C15H22O isomer Germacra-4(15),5,10(14)-trien-1-α-ol 10-Nor-calamenen-10-one C15H24O isomer Isobicyclogermacrenal Unknown #1b Chamazulene Unknown #2b Unknown #3b β-Costol α-Costol (E)-Nuciferylbutanoatea
Cadala-1(10),3,8-triene δ-Cadinene Myristicin trans-cadina-1,4-diene α-Cadinene C15H22O isomer α-Calacorene Elemol (E)-Nerolidol Nerylisovalerate syn-anti-antiHelifolen-12-al A[a] C15H24O isomer Humulane-1,6-dien-3-ol Caryophyllene oxide C15H22O isomer Salvial-4(14)-en-1-one Bornylangelate Geranylisovalerate Geranyl 2-methyl butanoate Junenol β-Cedrene epoxide trans-Isolongifolanone[a] α-Colacalene Selina-3,11-dien-6-alpha-ol γ-Eudesmol 10-epi-γ-Eudesmol cis-Cadin-4-en-7-ol C14H18 isomer Caryophylla-4(12),8(13)-dien-5-β-ol epi-α-Cadinol – – – – – – – – – – – – – – – – 38.25 – – – – 2.29
– 0.78 – – 0.19 – – – – – – – – 0.80 – – – – 0.30 0.31 – 0.61 – – – – – – – 1.04 – – 2.01 – – 0.16 – – – – – – – – – – 0.23 – – – – 3.42
– – 0.22 – – – – – – – – – – 2.54 – – – 0.68 – 0.15 – – – – – 0.43 – – – – 2.17 – – 1.53 – – – – – – – – 0.97 – – – – – – – – –
– 1.41 – 0.16 tr – 0.14 – 0.73 – – 5.32 0.53 0.97 0.31 0.16 – – 0.18 – – – – – – – – 0.84 – – – – 2.60 0.47 – – – – – – – – 0.31 – – – – – – – – –
– 1.89 – 0.12 tr – 0.14 0.12 0.56 – – 6.26 – 1.37 – 0.51 – 2.90 0.33 – – – 0.29 – 0.32 – – 0.35 – – – – 1.08 0.91 – – 1.14 – 0.39 0.18 – – – – – – – – – 0.69 0.65 –
– – – – – 4.20 – 2.56 – – 10.20 – – 0.40 – – – – – – – – – – 0.78 – – – – – – 0.70 – – 1.53 0.85 0.52 1.82 – – 0.98 2.07 – 6.15 0.74 1.02 – 1.32 0.44 – – –
– – – – – – – – – – – – – 5.93 – 0.50 0.42 – – – 2.37 – – 2.61 – – 0.65 – 1.88 – – – – – – 0.48 – – – – – – – – – – – – – – – –
1.54 – – – – – 5.10 – – 12.16 – – – 14.89 – – – – – – – – – – – – – – – –
Total monoterpenes Monoterpene hydrocarbons Monoterpenes oxygenated Total sesquiterpenes Sesquiterpene hydrocarbons Sesquiterpene oxygenated Other Unknown
30.20 23.76 6.44 64.46 61.40 3.06 0.06 2.29
85.16 9.79 75.37 7.28 1.47 5.82 0.49 3.81
15.18 15.18 0.00 78.38 65.51 12.87 3.94 0.00
76.03 67.13 8.90 22.06 6.03 16.03 0.75 0.00
73.96 12.56 61.40 24.05 1.26 22.79 0.78 0.00
58.20 1.88 56.32 24.69 5.08 19.61 3.27 8.94
43.06 22.69 20.37 43.12 16.07 27.05 11.05 0.00
TOTAL [%]: TOTAL (No):
97.01 31
96.75 40
97.50 45
98.84 50
98.79 35
98.70 35
97.24 24
a b
Tentatively identified by the combination of MS and RI. Mass spectra are shown in Appendix A (Supplemental Fig. S2).
Only essential oil of A. vulgaris was found close to this species. These groups are in concordance with the ITS phylogeny (Vallès et al., 2003), where A. dracunculus and A. campestris form a monophyletic group that is sister to other Artemisia species, which appears to be the case also with the essential oil composition (Fig. 2). A. absinthium and A. arborescens formed monophyletic group in the ITS analysis, and
this was also the case in the essential oil composition. Only discrepancy with the comparable ITS data is with A. herba-alba, but since there were only three samples available, this is somewhat to be expected with this type of data (essential oil composition). Combined ITS and ETS (External Transcribed Spacer) data Sanz et al. (2008) showed that all Artemisiinae genera constitute a monophyletic group. Also, they showed 5
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Fig. 1. Scatter plot of PCA analysis on the composition of the EO of studied species and localities. For population details cf. Table 1.
affiliation of the Artemisia species to the Artemisia vulgaris complex, North American endemic group, subgenus Seriphidium, Artemisia afra clade, Artemisia and Absinthium clade, Dracunculus clade and some informal groups and taxa. This appears to be the case with the essential oil composition, too (Fig. 2). The discrepancy is A. scoparia, which is positioned close to the North American group instead of in Dracunculus clade and A. herba-alba, which overlapped with Artemisia and Absinthium clade instead of subgenus Seriphidium. Further examination of EO of all other Artemisia species will certainly throw the light on concordance of EOs and molecular data. All this leads to the conclusion
that combining phytochemical/chemophenetic, anatomical, morphological and karyological analyses with modern molecular methods of other Artemisia taxa, will give a better infrageneric classification of this genus. 4. Conclusions Even though essential oil composition and biosynthetic pathways can vary to an extent depending on plant organ and some environmental factors, they are genetically determined and correspond to
Fig. 2. Scatter plot for discriminant analysis of 30 compounds present in the essential oil of different Artemisia species. The essential oil obtained from the leaves (●), areal parts (■) and leaves + flowers (◆). Lines show groups according to Sanz et al. (2008): Artemisia vulgaris complex, North American group, Seriphidium, Absinthium, Dracunculus. 6
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evolutionary trends. The qualitative and relative quantitative essential oil composition of the Artemisia species together with concordance with molecular data are applicable as a chemophenetic characters that may contribute to better understanding of systematics of Artemisia taxa.
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