ANTICANCER COMPOUNDS FROM MEDICINAL PLANTS BIOLOGICA NYSSANA 6 (2)  December 2015: 75-80 Radojković-Kostevski, I. et al.  Variations in the headspace volatile profiles… 75 Original Article Received: 11 October 2015 Revised: 12 November 2015 Accepted: 20 December 2015 Variations in the headspace volatile profiles of three different Achillea coarctata Poir. (Asteraceae) populations Ivana Radojković-Kostevski1, Goran Petrović1, Gordana Stojanović1, Jelena Stamenković1, Bojan Zlatković2 1University of Niš, Faculty of Sciences and Mathematics, Department of Chemistry, Višegradska 33, 18000 Niš, Serbia 2University of Niš, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Višegradska 33, 18000 Niš, Serbia * E-mail: peca@pmf.ni.ac.rs Abstract: Radojković-Kostevski, I., Petrović, G., Stojanović, G., Stamenković, J., Zlatković, B.: Variations in the headspace volatile profiles of three different Achillea coarctata Poir. (Asteraceae) populations. Biologica Nyssana, 6 (2), December 2015: 75-80. This study presents a detailed compositional analysis of six Achillea coarctata Poir. samples obtained by static headspace method and interrelationships based on the volatiles profiles from different plant parts, three different populations and geological substrates, using multivariate statistical analysis. The most dominant components were mutual for aerial vegetative plant parts and inflorescences collected at the same locality. Main compounds differed in percentages for two localities (values in parenthesis refer to aerial plant parts and inflorescences, respectively): 1) 1,8-cineole (40.7%; 39.9%), β-pinene (29.6%; 36.4%) and α-pinene (7.2%; 3.3%); 2) 1,8-cineole (51.8%; 53.3%), β-pinene (18.0%; 28.2%) and α-pinene (5.6%; 4.5%). the most abundant constituents identified in third locality were 1,8-cineole (37.2%; 35.6%), β-pinene (18.6%; 11.7%) and o- cymene (11.6%; 11,7%). Samples collected on different geological substrates are qualitative and quantitative various according to agglomerative hierarchical clustering analysis and can be grouped in two clades and two subclades. Key words: Achillea coarctata Poir., headspace, volatile profiles, statistical analysis Apstrakt: Radojković-Kostevski, I., Petrović, G., Stojanović, G., Stamenković, J., Zlatković, B.: Razlike headspace profila isparljivih komponenti tri različite populacije Achillea coarctata Poir. (Asteraceae). Biologica Nyssana, 6 (2), December 2015: 75-80. U ovom radu su predstavljeni rezultati dobijeni ispitivanjem hemijskog sastava lako isparljivih komponenti iz šest uzoraka biljke Achillea coarctata Poir. headspace statičkom metodom, i ispitana je zavisnost varijacije hemijskih profila od ispitivanih delova biljke i geološke podloge, korišćenjem multivarijantne statističke analize. Uzorci nadzemnog dela biljke i cveta prikupljani na jednom staništu sadržali su iste glavne komponente. Rezultati su pokazali da je razlika između dva lokaliteta u sadržaju istih glavnih komponenti, čiji su procenti prikazani u zagradama za nadzemni deo biljke i cvet: 1) 1,8-cineol (40.7%; 39.9%), β-pinen (29.6%; 6 (2) • December 2015: 75-80 BIOLOGICA NYSSANA 6 (2)  December 2015: 75-80 Radojković-Kostevski, I. et al.  Variations in the headspace volatile profiles… 76 36.4%) i α-pinen (7.2%; 3.3%); 2) 1,8-cineol (51.8%; 53.3%), β-pinen (18.0%; 28.2%) i α-pinen (5.6%; 4.5%). Kod trećeg staništa najdominantnije su bile sledeće komponente: 1,8-cineol (37.2%; 35.6%), β-pinen (18.6%; 11.7%) i o-cimen (11.6%; 11.7%), za nadzemni deo biljke odnosno cvet. Prema rezultatima AHC analize, uzorci prikupljeni sa različitih podloga se kvalitativno i kvantitativno razlikuju i grupisani su u dva klastera i dva subklastera. Key words: Achillea coarctata Poir., headspace, profili isparljivih komponenti, statistička analiza Introduction The genus Achillea L. (Asteraceae) is comprised of about 115 species found in the Northern Hemisphere, mostly in the Euro-Asian continent that are commonly known as yarrows (B e n e d e k et al., 2008; N e me t h & B e r n a t h , 2008; R a d u l o v i ć et al., 2010). The Achillea L. species belong to the oldest medicinal plants that are used both for pharmaceutical purposes and in folk medicine. Achillea species are diuretic, emmenagogue agents, used for healing wounds, curing stomachache and diarrhea, with antichloristic, antispasmodic, antiseptic and infection preventing properties. They have also been used to reduce sweating and to stop bleeding (A l s o h a i l i & A l - f a w w a z , 2014). The Achillea genus has a wide distributional range, and the differences in oil composition may be affected by different environmental factors such as plant genetic type, seasonality, and developmental stage, because it is a chemically polymorphic and perennial plant. Terpenoids (1,8-cineole, camphor, borneol, pinenes, artemisia ketone, santolina alcohol, farnesane, caryophyllene and its oxides, cubebene, germacrenes, eudesmol, α-bisabolol and oxides, farnesene, γ-gurjunene, γ-muurolene and chamazulene) are the principle components of Achillea essential oils (M o t a v a l i z a d e h k a k h k y et al., 2013). A. coarctata is a perennial herb with yellow ligules growing in dry hillsides and sandy oils, with the range restricted to the Balcan Peninsula, the South Ukraine and the Asia Minor (G a j i ć , 1975; S i m i ć et al., 1999; T z a k o u et al., 2009). As far as we know, there are three reports about chemical composition of A. coarctata essential oil. Different components are reported as the main constituents of the oil among these three studies. First known analysis represents caryophyllene oxide, 1,8-cineole and trans-linalool oxide as dominant constituents (S i m i ć et al., 1999). Tzakou found 1,8-cineole, camphor and borneol, while Toker reported 1,8- cineole, camphor and viridiflorol to be major components of the essential oil (T z a k o u et al., 2009; T o k e r et al., 2003). The aims of this study were to perform a detailed compositional analysis of six A. coarctata samples obtained by static headspace method and to establish interrelationships based on the volatiles profiles from different plant parts and different populations using multivariate statistical analysis. Material and methods Plant material The plant material (flowering stage) was collected at three different locations in Serbia (Rujan mountain; Preševo (Mitrovac); Pčinja valley (Trgovište)), in June 2014. The plant materials were identified by Bojan Zlatković and the voucher specimens were deposited in the Herbarium Moesiacum Niš (HMN), Department of Biology and Ecology, Faculty of Science and Mathematics, University of Niš under the acquisition numbers 9367, 9368 and 9369. Types of geological substrates were identified according to Basic Geological Mapp 1:100.000 (K a r a j o v a n o v i ć & H r i s t o v , 1976; B a b o v i ć & C v e t k o v i ć , 1977). Sample preparation 300 mg of milled fresh plant material was put into 20 mL HS vial and soaked with 2 mL of distilled water. The sample was heated at 80 °C for 20 minutes with the next mixing program: shaking for 5 seconds, pause for 2 seconds. 500 μL of vapor generated from the aerial parts was drawn out from the vial using a gas-tight syringe (90 °C) and injected directly in the chromatographic column via a transfer line (75 °C). GC and GC/MS analysis The samples were analyzed by a 7890/7000B GC/MS/MS triple quadrupole system in MS1 scan mode (Agilent Technologies, USA) equipped with a Combi PAL sampler and Headspace for G6501B/G6509B. The fused silica capillary column HP-5MS (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. The carrier gas was helium with a flow of 1.0 mL/min. 500 μL of HS vapor was injected via a transfer line (75 °C). Post run: back flash for 1.89 min, at 280 °C, with helium pressure of 50 psi. MS conditions were as follows: BIOLOGICA NYSSANA 6 (2)  December 2015: 75-80 Radojković-Kostevski, I. et al.  Variations in the headspace volatile profiles… 77 ionization voltage of 70 eV, acquisition mass range 50-650, scan time 0.32 s. GC analysis was carried out under the same experimental conditions using the same column as described for the GC/MS. The percentage composition of the samples was computed from the GC peak areas without any corrections. Identification of volatile compounds HS volatiles were identified by comparison of their linear retention indices (relative to C8-C32 n-alkanes on the HP-5MS column) with literature values and their MS with those of authentic standards, as well as those from 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 essential oils by the application of the AMDIS software (Automated Mass Spectral Deconvolution and Identification System, Ver. 2.1, DTRA/NIST, 2011). Some components were identified by co-injection of pure substances. Multivariate Statistical Analysis The contents of the components of the A. coarctata headspace volatiles obtained in this study were analyzed by agglomerative hierarchical cluster analysis (AHC). The AHC was performed with Euclidean distances as metric and using single linkage method as aggregation criterion using the “Statistica, version 8.1” software. Results and discussion Plant materials were collected at three different locations - Rujan mountain (samples RN, RC), Preševo (Mitrovac) (samples PN, PC) and Pčinja valley (Trgovište) (samples TN, TC). Each location has different geological substrate type: 1) serpentine (Preševo), 2) silicate (Rujan mountain), 3) conglomerates and molasse (Pčinja valley). Compositions of aerial plant parts and inflorescences headspace volatiles of A. coarctata from three different localities obtained by GC and GC/MS, are presented in Tab. 1. Number of identified compounds in RN, TN, PN, RC, TC and PC samples were 38, 36, 33, 34, 28 and 32, respectively (representing 98.9% (RN); 98.1% (TN); 98.3% (PN); 98.2% (RC); 98.8% (RC) and 98.6% (TC) of total HS volatiles. The most dominant components were mutual for aerial plant parts and inflorescences collected at the same locality. Main compounds found in RN and RC samples were 1,8-cineole (40.7%; 39.9%), β- pinene (29.6%; 36.4%) and α-pinene (7.2%; 3.3%). The same components were found as the most dominant in samples TN and TC, with following percentage: 1,8-cineole (51.8%; 53.3%), β-pinene (18.0%; 28.2%) and α-pinene (5.6%; 4.5%), for TN and TC respectively. The most abundant constituents identified in PN and PC samples were 1,8-cineole (37.2%; 35.6%), β-pinene (18.6%; 11.7%) and o- cymene (11.6%; 11.7%). As far as we know, there is no any published data on headspace volatiles composition of Achillea coarctata. Three papers revealed no different compounds as the most abundant in obtained essential oils compositions: (1) caryophyllene oxide, 1,8-cineole and trans-linalool oxide; (2) 1,8-cineole, camphor and borneol and (3) 1,8-cineole, camphor and viridiflorol, respectively (S i m i ć et al., 1999; T z a k o u et al., 2009; T o k e r et al., 2003). Content of monoterpenoids, which are more volatile compounds, is higher in headspace volatiles then in essential oil, while sesquiterpenoids, which have relatively high retention times, were only found in traces or not found at all. The results of the AHC analysis are depicted in Fig. 1. Table 1 lists the identified constituents with their contents of the six A. coarctata headspace volatile samples included in the AHC. The dendrogram depicted in Fig. 1, obtained as the result of the AHC, indicates the existence of two statistically different classes of samples (C1–C2). It is obvious that the chemical compositions of the aerial parts and inflorescences of the samples collected at the same locations are almost identical and they are grouped in the same stocks since they have the same main components that differ only in percentages in different parts of plants. Samples PC and PN are separated from the rest of the A. coarctata samples and constituted the first clade (C1). The samples TC, TN, RC and RN composed the second clade (C2). There was further subdivision within clade C2, into two more subclades consisted of the samples collected from the same sites. The second clade is more homogeneous than the first one and it is flatter on the dendrogram. Samples TC, TN, RC and RN were characterized by high contents of same compounds (1,8-cineole, β-pinene and α-pinene), reason for their strong association (same clade). Greater dissimilarity level (1.6) observed in this analysis separated samples PC and PN in clade C1 from the rest of the samples considering that the most abundant constituents identified in these samples were 1,8-Cineole, β-Pinene and o-Cymene. Since each population has different geological substrate: serpentine (samples PC and PN, Preševo), conglomerates and molasses (samples TC and TN, Pčinja valley) and biotite gneiss (samples RC and RN, Rujan mountain) it can be concluded that, with respect to the sample origin, different substrates produce various chemotypes. Chemical compositions BIOLOGICA NYSSANA 6 (2)  December 2015: 75-80 Radojković, I. et al.  Variations in the headspace volatile profiles… 78 Table 1. Chemical composition of the A. coarctata volatiles achieved by GC and GC/MS Relative amount % RIref RIexp Compound RN TN PN RC TC PC Class 801 802 Hexanal 0.1 0.3 0.2 tr 0.4 tr O 846 851 2(E)-Hexenal 0.2 1.2 0.4 0.3 0.3 tr O 850 854 3(Z)-Hexenol 0.2 0.7 0.3 - - - O 859 865 2(Z)-Hexenol 0.1 0.2 0.8 - - - O 863 867 n-Hexanol 0.8 1.1 2.1 0.2 0.2 0.3 O 921 923 Tricyclene 0.2 tr 0.2 0.2 tr 0.2 M 924 928 α-Thujene 0.1 tr 0.3 0.1 0.1 0.3 M 932 935 α-Pinene 7.2 5.6 11.5 3.3 4.5 4.7 M 946 951 Camphene 3.5 1.6 3.2 3.0 1.7 3.1 M 952 962 Benzaldehyde 0.1 0.5 tr 0.2 0.2 0.1 O 969 976 Sabinene 0.7 1.0 2.8 1.5 1.5 3.5 M 974 979 β-Pinene 29.6 18 18.6 36.4 28.2 25.7 M 988 992 Myrcene 0.2 - - 0.3 - - M 988 994 Dehydro-1,8-Cineole - 0.1 tr - 0.1 tr MO 1014 1019 α-Terpinene 0.6 0.9 1.3 0.7 0.7 2.8 M 1022 1027 o-Cymene 0.5 0.9 11.6 0.2 0.4 11.7 M 1024 1031 Limonene 0.2 0.2 0.5 0.2 tr 0.5 M 1026 1034 1,8-Cineole 40.7 51.8 37.2 39.9 53.3 35.6 MO 1054 1060 γ-Terpinene 1.0 2.3 0.9 1.1 1.2 1.2 M 1065 1069 cis-Sabinene hydrate 0.6 0.6 - - - - MO 1086 1092 Terpinolene 0.2 0.4 0.2 0.2 0.2 0.2 M 1095 1100 Linalool - - - 0.3 0.4 0.4 MO 1107* 1108 6-Ethenyldihydro-2,2,6- trimethyl-2H-pyran-3(4H)-one 3.7 1.9 0.5 3.1 0.9 1.6 O 1122 1128 α-Campholenal - tr tr - - - MO 1135 1142 trans-Pinocarveol - 0.1 - - - - MO 1141 1148 Camphor 3.7 1.5 1.6 1.9 1.1 1.6 MO 1160 1166 Pinocarvone tr 0.1 tr tr tr tr MO 1165 1169 Borneol 0.2 0.2 0.3 0.3 0.3 0.4 MO 1170 1176 cis-Linalool oxide (pyranoid) 0.1 - - - - - MO 1174 1180 Terpinen-4-ol 0.6 1.2 0.4 0.6 0.7 0.6 MO 1186 1193 α-Terpineol 0.8 0.9 0.5 0.9 0.8 0.6 MO 1195 1199 Myrtenal tr 0.3 - 0.1 0.1 tr MO 1288 1291 Lavandulyl acetate - 0.1 0.2 0.1 - 0.4 MO 1374 1381 α-Copaene 0.2 0.9 0.4 0.6 0.2 0.3 S 1417 1427 (E)-Caryophyllene 0.4 0.9 1.7 0.4 0.8 1.8 S 1454 1458 (E)-β-Farnesene tr 0.1 - - - - S 1458 1466 allo-Aromadendrene 0.7 - 0.2 0.8 - 0.4 S 1474 1482 10-epi-β-Acoradiene 0.2 - - 0.2 - - S 1484 1488 Germacrene D 0.7 2.3 0.3 0.6 0.5 0.2 S 1500 1503 Bicyclogermacrene 0.1 0.1 tr - - - S 1513 1519 γ-Cadinene 0.1 - - 0.1 - - S 1522 1529 δ-Cadinene tr 0.1 0.1 0.1 - 0.2 S 1582 1592 Caryophyllene oxide - - - - - 0.2 SO 1635 1642 cis-Cadin-4-en-7-ol 0.6 - - 0.3 - - SO Total 98.9 98.1 98.3 98.2 98.8 98.6 Monoterpenoids 90.7 87.8 91.3 91.3 95.3 93.5 Hydrocarbons(M) 44.0 30.9 51.1 47.2 38.5 53.9 Oxygenated(MO) 46.7 56.9 40.2 44.1 56.8 39.6 Sesquiterpenoids 3.0 4.4 2.7 3.1 1.5 3.1 Hydrocarbons (S) 2.4 4.4 2.7 2.8 1.5 2.9 Oxygenated (SO) 0.6 0 0 0.3 0 0.2 Others (O) 5.2 5.9 4.3 3.8 2.0 2.0 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-alaknes; (*): identified by NIST Chemistry WebBook Retention indices; tr: traces (<0.1%); (-): not detectid. Samples RN, TN, PN- aerial plant parts (collected at Rujan mountain, Pčinje valley (Trgovište) and Preševo (Mitrovac), respectively); samples RC,TC, PC- inflorescences (collected at Rujan mountain, Pčinje valley (Trgovište) and Preševo (Mitrovac), respectively). BIOLOGICA NYSSANA 6 (2)  December 2015: 75-80 Radojković-Kostevski, I. et al.  Variations in the headspace volatile profiles… 79 Fig. 1. Dendrogram obtained by agglomerative hierarchical clustering (constituent contents used as cases) of samples collected on biotite gneiss and conglomerates and molasses do not differ to a great extent while the sample from serpentine is slightly different. It has been already noticed that the deficiency of water and indispensable mineral elements result in numerous structural and functional adaptations of plants species that grow on a serpentine substrate (S t e v a n o v i ć et al., 2003). 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