Petrović, G., Stamenković, J., Stojanović, G., Zlatković, B., Jovanović, O.: Essential oil analysis of different plant parts of Geocaryum cynapioides (Guss.) L. Engstrand. Biologica Nyssana, 9 (1). September, 2018: 31-35. BIOLOGICA NYSSANA 9 (1) ⚫ September 2018: 31-35 Petrović et al. ⚫ Essential oil analysis of different plant parts of Geocaryum… 31 Original Article Received: 29 January 2018 Revised: 29 Mart 2018 Accepted: 27 June 2018 Essential oil analysis of different plant parts of Geocaryum cynapioides (Guss.) L. Engstrand Goran Petrović1, Jelena Stamenković1, Gordana Stojanović1, Bojan Zlatković2, Olga Jovanović1 1University of Niš, Faculty of Science and Mathematics, Department of Chemistry, Višegradska 33, 18000 Niš, Serbia 2University of Niš, Faculty of Science and Mathematics, Department of Biology and Ecology, Višegradska 33, 18000 Niš, Serbia * E-mail: jelena.stamenkovic@pmf.edu.rs Abstract: Petrović, G., Stamenković, J., Stojanović, G., Zlatković, B., Jovanović, O.: Essential oil analysis of different plant parts of Geocaryum cynapioides (Guss.) L. Engstrand. Biologica Nyssana, 9 (1). September, 2018: 31-35. Phytochemical analysis by GC and GC/MS of the essential oil samples, obtained from fresh aerial parts, shoots and inflorescences of Geocaryum cynapioides (Guss.) L. Engstrand, allowed the identification of 55 components in total, comprising 99.5%, 99.7% and 99.0% of the oils compositions, respectively. Regarding the aerial parts essential oil, the major of 52 identified volatile compounds were (E)-β-Farnesene (66.6%), 7- epi-cis-Sesquisabinene hydrate (17.4%) and (E,E)-α-Farnesene (3.7%). The same compounds were dominant among the 48 components detected in the shoots and 42 volatiles in inflorescences oil, but in different proportions. Hydrocarbon sesquiterpenes had the highest contribution in all investigated Geocaryum cynapioides essential oils, with a share of about three quarters of all, followed by one quarter of oxygenated sesquiterpenes, while the monoterpenoid and nonterpenoid compounds were detected only in trace amounts. Key words: Geocaryum cynapioides, essential oil, phytochemical analysis, GC/MS Apstrakt: Petrović, G., Stamenković, J., Stojanović, G., Zlatković, B., Jovanović, O.: Analiza etarskih ulja iz različitih biljnih delova vrste Geocaryum cynapioides (Guss.) L. Engstrand. Biologica Nyssana, 9 (1). Septembar, 2018: 31-35. Fitohemijskom analizom uzoraka etarskih ulja izolovanih iz svežeg biljnog materijala različitih delova (nadzemnog dela, stabljike i cvasti) biljne vrste Geocaryum cynapioides (Guss.) L. Engstrand, pomoću GC i GC/MS metode, identifikovano je ukupno 55 komponenti, što predstavlja 99,5% nadzemnog dela, 99,7% stabljike i 99,0% cvasti. U etarskom ulju izolovanom iz svežeg nadzemnog dela biljke identifikovane su ukupno 52 komponente među kojima su (E)-β-Farnezen (66,6%), 7-epi-cis-Seskvisabinen hidrat (17,4%) i (E,E)-α-Farnezen (3,7%) bile najzastupljenije. Iste te komponente su bile dominantne i u etarskim uljima 9 (1) • September 2018: 31-35 DOI: 10.5281/zenodo.1470846 BIOLOGICA NYSSANA 9 (1) ⚫ September 2018: 31-35 Petrović et al. ⚫ Essential oil analysis of different plant parts of Geocaryum… 32 izolovanim iz stabljike i cvasti ali u različitim procentnim udelima. U svim ispitivanim uzorcima tri četvrtine ukupnog sastava etarskog ulja činili su ugljovodonični seskviterpeni, zatim oksigenovani seskviterpeni koji su činili jednu četvrtinu, dok su monoterpenoidi i neterpenoidne komponente detektovani samo u tragovima. Ključne reči: Geocaryum cynapioides, etarsko ulje, fitohemijska analiza, GC/MS Introduction The genus Geocaryum L. (Apiaceae) is small genus, comprised of only 8 accepted species (The Plant list, 2012). It belongs to the tribe Scandiceae, subtribe Scandicinae (Spalik & Downie, 2001) with 11 another larger genera like Chaerophyllum, Anthriscus, Osmorhiza etc. Geocaryum cynapioides (Guss.) L. Engstrand is endemic species which inhabits pastures and meadows of the high mountains in southern parts of the Balkan Peninsula and central and southern parts of Italy (Ball, 1968). It is a perennial herb, 45 cm tall, with flexuous or curved internodes and undivided uppermost cauline leaves, linear or filiform lamina up to 20 mm, flowering from May to July. According to the Plant list database, G. cynapioides has as many as 18 synonymous names classifying it in other genera such as Bunium, Carum or even Chaerophyllum. Downie (Downie et al., 2010) finally hemotaxonomically defined its position within the tribe Scandiceae on the basis of his investigation of nuclear ribosomal DNA. But, it is very unusual that there are no any previous data about the chemical composition of essential oils, as well as about the extracts of species belonging to this genus, and their biological activities. As far as we know, there is only one report on chemical composition of G. cynapioides essential oil (Radulović et al., 2008) which relates to the plant as a whole, including underground tubers, for which it is literally known that they might be completely different in chemical composition. The aim of this study was to perform a detailed phytochemical analysis of Geocaryum cynapioides (Guss.) L. Engstrand essential oils obtained from different plant parts. Material and methods Plant material The plant material (flowering stage) was collected at Vlasina Lake plateau, Serbia, in June 2017 and was identified by one of the authors Bojan Zlatković. The voucher specimen was deposited in the Herbarium Moesiacum Niš (HMN), Department of Biology and Ecology, Faculty of Science and Mathematics, University of Niš under the acquisition number 13317. Sample preparation The essential oils samples were prepared by hydrodistillation of fresh chopped whole aerial plant parts (194 g), shoots (158 g) and inflorescences (54 g), for 2.5 hours using Clevenger type apparatus, according to the method recommended in British Pharmacopoeia (British Pharmacopoeia, 1988). The essential oils were extracted with n-hexane, dried over anhydrous sodium sulfate and evaporated. The yields of the oils were 0.06%, 0.05% and 0.08%, based on the weight of fresh plant, respectively. Samples were re-dissolved in n-hexane to obtain the desired optimal concentrations and stored at -20 °C in the dark until analyzed. - Hexane - Analytical reagent grade, Fisher Chemical, UK; - Na2SO4 - Pro analysis grade, Sigma-Aldrich, Germany. GC and GC/MS analysis The sample of essential oil (20 mg/ml) was analyzed by a 7890/7000B GC/MS/MS triple quadrupole system in MS1 scan mode (Agilent Technologies, USA) equipped with a Combi PAL sampler. The fused silica capillary column HP-5 MS (5% phenylmethylsiloxane, 30 m x 0.25 mm, film thickness 0.25 μm) was used. The injector and interface operated at 250 and 300 °C, respectively. Temperature program: from 50 to 290 °C at a heating rate of 4 °C min-1. The carrier gas was helium with a flow of 1.0 mL min-1. One microliter of the oil solution in hexane was injected (1:100, split ratio 40:1). Post run: back flash for 1.89 minutes, at 280 °C, with helium pressure of 50 psi. MS conditions were as follows: ionization voltage of 70 eV, acquisition mass range 40-440, scan time 0.32 seconds. GC-FID analysis was carried out under the same experimental conditions using the same column as described for the GC/MS. The percentage composition of the sample was computed as an average of the GC peak areas obtained in triplicate, without any corrections. Identification of volatile compounds EO constituents were identified by comparison of their linear retention indices relative to C8-C32 alkanes on the HP-5 MS column (Van Den Dool & Kratz, 1963), with literature values and their MS with BIOLOGICA NYSSANA 9 (1) ⚫ September 2018: 31-35 Petrović et al. ⚫ Essential oil analysis of different plant parts of Geocaryum… 33 Table 1. Chemical compositions of the G. cynapioides essential oils obtained by GC/MS Relative amount % RIexp RIref Compound SO IO AO Class 998 988 Myrcene tr tr tr M 1034 1024 Limonene tr tr tr M 1042 1032 (Z)-β-Ocimene 1.5 0.2 1.0 M 1052 1044 (E)-β-Ocimene 0.6 0.1 0.4 M 1083 1088* 1-Nonen-3-ol 0.1 - tr O 1091 1086 Terpinolene 0.2 - 0.1 M 1101 1095 Linalool tr tr - MO 1100 1100 Undecane - tr - O 1106 1100 n-Nonanal tr 0.1 tr O 1147 1144 2(Z)-Nonen-1-al 0.1 - tr O 1161 1157 2(E)-Nonen-1-al tr - tr O 1287 1283 Isobornyl acetate tr - tr MO 1291 1288 Lavandulyl acetate - tr tr MO 1299 1300 Tridecane tr - tr O 1278 1374 α-Copaene tr tr tr S 1383 1379 Geranyl acetate 0.1 - tr MO 1388 1387 β-Bourbonene 0.1 - tr S 1394 1389 β-Elemene 0.1 - tr S 1406 1405 Sesquithujene 0.1 0.1 tr S 1416 1410 α-Cedrene tr tr tr S 1423 1417 (E)-Caryophyllene 0.8 1.6 1.0 S 1429 1429* Himachala-2,4-diene 0.2 0.1 0.1 S 1432 1430 β-Copaene tr . tr S 1437 1432 α-trans-Bergamotene tr tr tr S 1444 1440 (Z)-β-Farnesene tr tr tr S 1461 1454 (E)-β-Farnesene 61.0 72.0 66.6 S 1481 1481 γ-Curcumene - 0.1 0.1 S 1485 1484 Germacrene D 2.7 3.0 2.8 S 1494 1491* (Z,E)-α-Farnesene 1.8 1.2 1.5 S 1500 1500 Bicyclogermacrene 0.6 0.2 0.4 S 1503 1506 (Z)-α-Bisabolene 0.1 tr 0.1 S 1508 1505 (E,E)-α-Farnesene 4.4 2.6 3.7 S 1513 1514 β-Curcumene 0.5 0.2 0.4 S 1525 1521 β-Sesquiphellandrene tr tr tr S 1525 1522 δ-Cadinene 0.2 tr 0.1 S 1533 1529 (E)-γ-Bisabolene tr tr tr S 1555 1542 cis-Sesquisabinene hydrate 2.1 1.4 1.7 SO 1564 1561 (E)-Nerolidol 0.4 0.3 0.3 SO 1579 1574 Germacrene D-4-ol 0.1 - tr SO 1583 1577 trans-Sesquisabinene hydrate 0.4 0.1 0.1 SO 1593 1586* 7-epi-cis-Sesquisabinene hydrate 19.3 13.1 17.4 SO 1611 1611 Tetradecanal tr - tr O 1634 1632 α-Acorenol 0.6 0.3 0.4 SO 1651 1636 β-Acorenol 0.2 0.1 0.1 SO 1672 1670 epi-β-Bisabolol 0.7 0.4 0.5 SO 1685 1683 epi-α-Bisabolol 0.2 - 0.1 SO 1698 1699 β-Sinensal tr tr tr SO 1900 1900 Nonadecane - 0.1 tr O 2000 2000 Eicosane - tr tr O 2100 2100 Heneicosane - 0.1 tr O 2200 2200 Docosane - tr - O BIOLOGICA NYSSANA 9 (1) ⚫ September 2018: 31-35 Petrović et al. ⚫ Essential oil analysis of different plant parts of Geocaryum… 34 those of authentic standards, as well as those from Adams (Adams, 2007), Wiley 6, NIST11, Agilent Mass Hunter Workstation B.06.00 software and a homemade MS library with the spectra corresponding to pure substances and components of known EOs by the application of the AMDIS software (Automated Mass Spectral Deconvolution and Identification System, Ver. 2.1, DTRA/NIST, 2011). Results and discussion Chemical compositions of the aerial plant parts, shoots and inflorescences essential oils of Geocaryum cynapioides, obtained by GC and GC/MS analyses, are presented in Tab. 1. Number of identified compounds in aerial parts (AO), inflorescences (IO) and shoots (SO) samples were 52, 42 and 48 respectively, representing 99.5%, 99.0% and 99.7% of total volatiles. The composition of the essential oils of the shoots and the oil obtained from inflorescences was found to be quite comparable. Hydrocarbon sesquiterpenes had the highest contribution in all investigated G. cynapioides essential oils, with a share of about three quarters of all, followed by one quarter of oxygenated sesquiterpenes. The shoot essential oil had a little bit lower content of hydrocarbon sesquiterpenes compared to the inflorescences oil, 72.6% and 81.1%, respectively. In both essential oils this class of compounds predominated over the oxygenated sesquiterpenes. A reverse distribution was observed for the oxygenated sesquiterpenes which are prevalent in shoots essential oil over the inflorescences oil (24.0% vs. 15.7%, respectively). Hydrocarbon monoterpenes were present in negligible amounts in both oils, while the oxygenated monterpenes were not detected at all. Obtained results indicate that the composition of the G. cynapioides aerial parts essential oil represents the average value of the contents of essential oils derived from inflorescences and shoots essential oils. Phytochemical analysis by GC and GC/MS of the essential oil samples, obtained from fresh aerial parts, shoots and inflorescences of G. cynapioides, allowed the identification of 55 components in total. Regarding the aerial parts essential oil, the major of 52 identified volatile compounds were (E)-β- Famesene (66.6%), 7-epi-cis-Sesquisabinene hydrate (17.4%) and (E,E)-α-Farnesene (3.7%). The same compounds were dominant among the 48 components detected in the shoots and 42 volatiles in inflorescences oil, but in different proportions. As we already shown, chemical composition of inflorescences could be pretty similar (Kostevski et al., 2016) or essentially various (Petrović et al., 2018; Stamenković et al., 2015) in comparison to the composition of the shoots essential oil. On the other hand, the composition of the volatile components obtained from all the above-ground parts of the plant and root is always completely different (Petrović et al., 2017; Stamenković et al. 2015). Compared with the previously published results of G. cynapioides essential oil (Radulović et al., 2008), certain differences in chemical compositions of aerial parts essential oils can be observed. That can be explained by the fact that sample A consisted of fresh aerial parts (inflorescences, stems and leaves) and underground tubers, while sample B was composed of stems, leaves and umbels full of ripe fruit (fruiting stage). Sample B differs significantly both by the number of identified components and by the relative content of compound classes, especially in relation to monoterpenoids content (hydrocarbon monoterpenes Relative amount % RIexp RIref Compound SO IO AO Class 2300 2300 Tricosane 0.1 0.6 0.2 O 2500 2500 Pentacosane tr 0.4 0.1 O 2700 2700 Heptacosane 0.1 0.2 0.1 O 2900 2900 Nonacosane 0.3 0.4 0.2 O Total 99.7 99.0 99.5 Monoterpene hydrocarbons - M 2.3 0.3 1.5 Monoterpene oxygenated - MO 0.1 - - Sesquiterpene hydrocarbons - S 72.6 81.1 76.8 Sesquiterpene oxygenated - SO 24.0 15.7 20.6 Others - O 0.7 1.9 0.6 Compounds are listed in order of elution from a HP-5 MS column; RIref: Literature Retention indices; RIexp: Experimental Retention indices relative to C8-C32 n-alkanes; (*): identified by NIST Chemistry WebBook Retention indices; tr: traces (<0.1%); (-): not detected. Essential oils: SO - shoots; IO - inflorescences; AO - aerial plant parts. BIOLOGICA NYSSANA 9 (1) ⚫ September 2018: 31-35 Petrović et al. ⚫ Essential oil analysis of different plant parts of Geocaryum… 35 6.5% and oxygenated monterpenes 4.9%). However, the main difference that can be noticed is that one of the main components identified in our investigation, 7-epi-cis-Sesquisabinene hydrate, was not found at all. The probable reason is the similarity of the mass spectra and the proximity of the retention indices of this compound and trans-Sesquisabinene hydrate, which was found in a significant amount, which led to a misidentification. According to the results of this study, the essential oil of G. cynapioides consisted mainly of sesquiterpene compounds, implying that there are great differences in composition of investigated essential oil compared to other reports about the Scandicinae species` essential oils. Considering the fact that Geocaryum genus is not investigated at all, more studies are needed regarding essential oil constituents of its species. Obtained data could be helpful in future research to clarify the chemotaxonomic relationships between the genera within the tribe Scandiceae and subtribe Scandicinae. Acknowledgements. The authors are grateful to the Ministry of Education, Science and Technological Development for financial support through the grant within frame of basic research, No 172047. References Adams, R. P. 2007: Identification of essential oil components by gas chromatography and mass spectrometry (4th ed.). Carol Stream: Allured Publishing Corporation. Ball, P. W. 1968: Huetia P. W. Boiss. In: T. G. Tutin, V. H. Heywood, N. A. Burges, D. M. Moore, D. H., Valentine, S. M. Walters, D. A. Webb (eds.), Flora europaea 2 (pp. 330). Cambridge: Cambridge University Press. Downie, S. R., Katz-Downie, D. S., Spalik, K. 2000: A phylogeny of Apiaceae tribe Scandiceae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences. American Journal of Botany, 87: 76-95. 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