Evaluation of the chemical composition of essential oils with respect to the maturity of flower heads of Arnica montana L. and Arnica chamissonis Less. cultivated for industry

Industrial Crops and Products 76 (2015) 857–865

Contents lists available at ScienceDirect

Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop

Evaluation of the chemical composition of essential oils with respect to the maturity of flower heads of Arnica montana L. and Arnica chamissonis Less. cultivated for industry Radosław Kowalski a,∗ , Danuta Sugier b , Piotr Sugier c , Barbara Kołodziej b a

Department of Analysis and Evaluation of Food Quality, University of Life Sciences in Lublin, 8 Skromna Street, 20-704 Lublin, Poland Department of Industrial and Medicinal Plants, University of Life Sciences in Lublin, 15 Akademicka Street, 20-950 Lublin, Poland c Department of Ecology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, 19 Akademicka Street, 20-033 Lublin, Poland b

a r t i c l e

i n f o

Article history: Received 24 April 2015 Received in revised form 19 June 2015 Accepted 16 July 2015 Keywords: Arnica montana Arnica chamissonis GC/MS Oil composition E-caryophyllene Alpha-pinene Isopropyl hexadecanoate Farnesyl acetate Alpha-cis-bergamotene

a b s t r a c t The aim of the studies was to evaluate the chemical composition of essential oils extracted from different mature flower heads of Arnica montana L. and Arnica chamissonis Less. cultivated for industry. The impact of different stages of flower development on the quantity and chemical composition of volatile oils of Arnica montana L. and Arnica chamissonis Less. in eastern Poland were studied. The flower heads were harvested in four different development phases and the chemical composition of essential oils was investigated by GC/MS. Fifty compounds in the case of A. montana and 62 components in the case of A. chamissonis constitued over 90% of the total oil content. In the case of the two plant species studied, the flower head maturity determined the quantity and chemical composition of oils. The content of volatile oils in flower heads of A. montana was in the range from 0.158% to 0.195% and in A. chamissonis from 0.137% to 0.194%. The highest content of volatile oils in the flower heads of the two plant species studied was noted in the full flowering phase, when ligulate florets were opened, and up to half of the disc of tubular florets were opened. Differences in the volatile oil content were noted between the species studied only in the stage of yellow buds, whereas in the other stages the contents were similar. E-caryophyllene, alpha-pinene, isopropyl hexadecanoate, farnesyl acetate, alpha-cis-bergamotene, and decanal are the main components that determined the oil chemical differentiation. Generally, higher amounts of E-caryophyllene, farnesyl acetate, and germacrene D were detected in the flower heads of A. montana in relation to A. chamissonis. Among the main volatile oil components, isopropyl hexadecanoate and alpha-pinene were present only in the flower heads of A. chamissonis, while alpha-cis-bergamotene was only noted in A. montana. The investigations of A. montana volatile oils in the flower heads have shown the existence the differences in the chemical profile in relation to other European populations of this species. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The genus Arnica L. comprises ca. 32 species predominantly confined to the boreal and montane region of the northern hemisphere (Maguire, 1943). Mountain arnica (Arnica montana L.) is a herbaceous perennial herb and a medicinal plant widely used as a herbal remedy and occurring throughout the entire range of the species, from South Norway and Latvia southwards to the Apennines and the South Carpathians (Hultén and Fries, 1986). It is mainly found in grasslands and shrublands as well as alpine mountain

∗ Corresponding author. Tel.: +48 81 462 33 32. E-mail address: [email protected] (R. Kowalski). http://dx.doi.org/10.1016/j.indcrop.2015.07.029 0926-6690/© 2015 Elsevier B.V. All rights reserved.

environments, but it also grows in dry pine forests, meadows on siliceous soils, marginal parts of spruce forests, open forest edges, mowing pastures, and margins of peatlands (Luijten et al., 1996; Kahmen and Poschlod, 2000; Maurice et al., 2012; Wołkowycki, 2012). Eutrophication, habitat fragmentation, agricultural intensification, and collection for medicinal purposes have led to a rapid decline in the mountain arnica in many European countries; therefore, this species is a rare plant under strict protection and is included in the IUCN Red List of Threatened Species (Falniowski et al., 2012) and in the Red Data Books and Red Data Lists of many ˛ European countries (Korneck et al., 1996; Zarzycki and Szelag, 2006). Arnica montana is a medicinally important plant species widely applied in pharmaceutical and cosmetic industry (Ganzera et al.,

858

R. Kowalski et al. / Industrial Crops and Products 76 (2015) 857–865

2008; Merfort, 2010). Arnica is a source of sesquiterpene lactones, flavonoids, essential oils, terpenoids, and phenolic acids (Willuhn et al., 1995; Willuhn, 1998; Merfort and Wendisch, 1988; ˙ et al., 2011; Gawlik-Dziki Ganzera et al., 2008; Weremczuk-Jezyna et al., 2009, 2011; Sugier, 2013), and exhibits antiseptic, antiinflammatory, antiradical, antibacterial, antisclerotic, antifungal, and antioxidant activities (Ganzera et al., 2008; Gawlik-Dziki et al., 2009, 2011; Sugier and Gawlik-Dziki, 2009; Saluk-Juszczak et al., 2010; Gaspar et al., 2014). Cultivation of other Arnica species with a similar pharmacological effect is one of the different approaches to meet the industrial demand for Arnica and guarantee the supply of a standardised plant drug or of its active substances (Nichterlein, 1995). One of them is Arnica chamissonis Less., a species extending from the Alaskan Archipelago south to the San Bernardino Mountains in California and in the southern Rocky Mountains into New Mexico (Maguire, 1943). This is an attractive plant species for the pharmaceutical industry, as it contains numerous pharmacologically active substances and is characterised by similarity of pharmacological effects to A. montana (Willuhn et al., 1983; Leven and Willuhn, 1987; Nichterlein, 1995). Moreover, A. chamissonis is a good source of bioactive compounds (Leven and Willuhn, 1987; Merfort and Wendisch, 1987; Roki et al., 2001; Schmidt et al., 2006; Gawlik-Dziki et al., 2009), a valuable source of herbal raw material and a pharmaceutical substitute for the endangered mountain arnica (Willuhn, 1998; Cassell et al., 1999; Sugier and Gawlik-Dziki, 2009; Gawlik-Dziki et al., 2011). A. chamissonis extracts exhibit potent antiinflammatory and antiradical activity and possesses high antioxidant abilities that might be helpful in preventing or slowing the progress of free radical-dependent diseases (Cassell et al., 1999; Gawlik-Dziki et al., 2009). Additionally, in comparison to Arnica montana, it is easier to cultivate due to its low ecological demand (Cassell et al., 1999; Nowak, 2002), which is important in the aspect of raw material production and use thereof in pharmaceutical and cosmetics industry. In recent years, considerable attention of researchers is focused on factors determining the quantity and quality of mountain arnica raw material, whereas studies of A. chamissonis as a viable substitute for A. montana in herbal medicine are scarce. The content of secondary metabolites in mountain arnica varies between the parts of the plant, temperature during growth, type of vegetation, and differences in natural habitats, altitude, and climatic factors (Spitaler et al., 2008; Albert et al., 2009; Perry et al., 2009; Cornu et al., 2010; Seemann et al., 2010). The aromatic and curative properties of arnica are mainly related to the contents and chemical composition of essential oil. This substance may undergo quantitative and qualitative changes during the vegetative period (Willuhn, 1972). High content of essential oil and their chemical composition in rhizomes and roots have been previously reported (Willuhn, 1972; ˙ Rohloff, 2003; Weremczuk-Jezyna et al., 2006, 2011; Pljevljakuˇsic´ et al., 2012). There are only a few reports describing the constituents of essential oils in A. montana underground parts. The investigations of A. montana volatile oils in the flower heads of different European populations of this species have shown differences in the chemical profile of A. montana oils (Rohloff, 2003; ¯ ˙ 2009). The composiRistic´ et al., 2007; Judˇzentiene˙ and Budien e, tion of essential oil in flowers, leaves, and roots of Arnica montana differed (Rohloff, 2003); however, the variation of volatile oil content of Arnica spp. in relation to the maturity of flower heads has not been well investigated. Therefore the objective of this study was: (i) to characterise the chemical oil profile in A. montana and A. chamissonis cultivated in eastern Poland, (ii) to evaluate the influence of different stages of flower development on the quantity, and (iii) the chemical composition of essential oil in the flower heads. Different stages of development of A. montana flowers showed highly significant differences in the concentration of total sesquiterpene lactones and quercetin (Douglas et al., 2004;

Sugier, 2013). The knowledge of the variation in the content of volatile oils related to the flowering phase can help in choosing optimised harvesting time and ensure the required quality and quantity of raw material. This aspect is very important from the point of view of the use of these two very important herbal plant species for the pharmaceutical and cosmetic industry. 2. Material and methods 2.1. Experimental site conditions The experiment (2008–2009) was performed on experimental fields at the University of Life Sciences in Lublin located in the eastern part of Poland, 51◦ 33◦ N; 22◦ 44◦ E on grey-brown podsolic soil with a granulometric composition of heavy loamy sand. In order to determine the edaphic conditions, before plantation establishment, topsoil samples were randomly selected from the depth of 15 cm. The soil was characterised by an average content of organic matter (1.41%), average phosphorus (55.4 mg P kg−1 , PN-R-04023: 1996), low potassium (64.9 mg K kg−1 , PN-R-04022: 1996 + Az1:2002), very low magnesium (12.5 mg mg kg−1 , PN-R04020: 1994 + Az1:2004), and acidic reaction (pH in 1 mol dm−3 KCl 5.38, PN-ISO 10390: 1997). Phosphorus-potassium fertilisation was applied every year in the following doses: 24.0 kg P and 66.4 kg K per hectare, whereas 40.0 kg of nitrogen was applied in two equal doses: in the spring, before the beginning of vegetation, and after collection of flowers. During the vegetation, the plants were weeded three times (by hand), and mechanical cultivation of inter rows was ensured. In the present study, seeds (achenes) for sowing were collected from 15 individuals of A. montana and 15 individuals of A. chamissonis at the end of June 2007, from the collection of the Department of Industrial and Medicinal Plants of the University of Life Sciences in Lublin. The experimental design was a randomised complete block with four replications (plots of 10 m−2 ). Arnica spp. plantations were established by seedlings obtained from seedbed transplanting in the second decade of May 2008 at a 40 cm × 20 cm distance. In the first year of vegetation, the mountain arnica did not bloom; thus, the raw material for the chemical analyses was taken from 2-year-old individuals of A. montana and A. chamissonis. Collection of inflorescences was carried out depending on the stage of flower head maturity. Four categories were distinguished: FM1 – stage of yellow buds (ligulate florets not opened, tubular florets closed), FM2 – beginning of flowering (flower heads fully emerged, ligulate florets opened, tubular florets opened in 1–2 rows of flower heads). FM3 – full flowering (flower heads fully emerged, ligulate florets opened, tubular florets opened up to half of the disc), FM4 – end of flowering (ligulate florets dried out, all tubular florets opened). Inflorescences were dried in a drying chamber at 40 ◦ C immediately after the harvest. 2.2. Qualitative and quantitative analysis of essential oil 2.2.1. Assay of the essential oil content The content of essential oil in Arnica spp. inflorescences was assayed with the method of distillation. The distillation was conducted in conformance with the Polish Pharmacopoeia VI (2002). The method of indirect distillation was applied (with xylene), while the distillation time was 3 hours. 2.2.2. GC analysis 2.2.2.1. GC/MS. ITMS Varian 4000 GC–MS/MS (Varian, USA) equipped with a CP-8410 auto-injector and a 30 m × 0.25 mm i.d. VF-5ms column (Varian, USA), film thickness 0.25 ␮m, was used; carrier gas He at a rate of 0.5 ml/min; injector and detector temperature 250 ◦ C and 200 ◦ C, respectively; split ratio 1:50; injection

R. Kowalski et al. / Industrial Crops and Products 76 (2015) 857–865

volume 5 ␮l. A temperature gradient was applied (50 ◦ C for 1 min, then incremented by 4 ◦ C/min to 250 ◦ C, then held at 250 ◦ C for 10 min); ionisation energy 70 eV; mass range 40–870 Da; scan time 0.80 s. 2.2.2.2. GC/FID. GC Varian 3800 (Varian, USA) equipped with a CP-8410 auto-injector and a 30 m × 0.25 mm DB-5 column (J&W Scientific, USA), film thickness 0.25 ␮m, carrier gas helium 0.5 ml/min, injector and detector FID temperatures 260 ◦ C; split ratio 1:100; injection volume 5 ␮l. A temperature gradient was applied (50 ◦ C for 1 min, then incremented by 4 ◦ C/min to 250 ◦ C, 250 ◦ C for 10 min). 2.2.3. Qualitative analysis The qualitative analysis was carried out on the basis of MS spectra, which were compared with the spectra of the NIST library (NIST, 2005) and with data available in the literature (Joulain and König, 1998; Adams, 2001). The identity of the compounds was confirmed by their retention indices (Van Den Dool and Kratz, 1963) taken from the literature (Joulain and König, 1998; Adams, 2001) and our own data for standards (alphapinene, Sigma–Aldrich, USA; p-cymene, Sigma–Aldrich, USA; limonene, Sigma–Aldrich, USA; linalool, Sigma–Aldrich, Spain; menthone, Sigma–Aldrich, Switzerland; menthol, Sigma–Aldrich, Switzerland; decanal, Sigma–Aldrich, USA; thymol, Sigma–Aldrich Chemic GmbH, Germany; E-caryophyllene, Sigma–Aldrich, Spain; alpha-humulene, Sigma–Aldrich Chemic GmbH, Germany; germacrene D, Sigma–Aldrich, Spain, spathulenol, Sigma–Aldrich, Spain; caryophyllene oxide, Sigma–Aldrich, Switzerland; alpha-bisabolol, Sigma–Aldrich, USA). 2.2.4. Quantitative analysis The quantitative analysis was performed by means of internal standard addition method (alkanes C12 and C19 ) according to procedures described previously (Kowalski and Wawrzykowski, 2009). Essential oil was diluted 1000 times using n-hexane to achieve 1 ml volume, then 1 mg of C12 and 1 mg C19 were added to the diluted oil. Samples prepared in this way were subjected to GC–MS and GC–FID determinations. The quantitative analysis was performed on the basis of calibration curves plotted to find the dependence between the ratio of the peak area for the analyte to the area for the internal standard (Aanalyte :Ai.s. ) vs. the analyte concentration (Canalyte ) for alpha-pinene, p-cymene, limonene, linalool, menthone, menthol, decanal, thymol, E-caryophyllene, alphahumulene, germacrene D, spathulenol, caryophyllene oxide, and alpha-bisabolol, in an appropriate concentration range (Kowalski and Wawrzykowski, 2009). The following alkanes were applied as internal standards: C12 (for compounds with a retention index <1300, alpha-pinene, linalool, p-cymene, menthone, menthol, decanal) and C19 (for compounds with a retention index >1300, E-caryophyllene, caryophyllene oxide, thymol, E-caryophyllene, alpha-humulene, germacrene D, spathulenol, caryophyllene oxide, alpha-bisabolol). The contents of the analysed substances were read from the calibration curves obtained, for which the data on the peak areas originated from the GC separations of Arnica oil components and internal standards. The final result took into account all dilutions during the whole analytical procedure. 2.3. Statistical analysis Prior to the analysis, the quantity data were tested for normality with the Shapiro-Wilk test. Variance heterogeneity was checked using Levene’s test. Data were normally distributed with homogeneous variances, therefore one-way analysis of variance (ANOVA) with subsequent Tukey tests were used. The results were expressed as means ± SD, and the differences were considered significant at

859

P < 0.05. The statistical analyses were carried out using the Statistica 6.0 software programme. The chemical composition of oils in the different phases of Arnica spp. head development were examined with a clustering analysis of unweighted pair group mean averages (UPGMA). Principal component analysis (PCA) was applied in order to describe the group of chemical data, to establish the relationships between the different chemical variables (oils content), and to detect the most important factors of variability. The data were clustered before the analysis. Both cluster and PCA analyses were conducted using the multivariate statistical package (MVSP) program version 3.1. (Kovach, 1999). 3. Results and discussion 3.1. Quantity of volatile oils The content of volatile oils in the flower heads of Arnica spp. harvested in different flower maturity stages is presented in Table 1. The flowering stages differed significantly in terms of the volatile oil content in the flower heads of the two species studied. The content of volatile oils in the flower heads of A. montana was in the range of 0.158% (FM1) and 0.195% (FM3). In the case of A. chamissonis, the content of volatile oils in the flower heads was from 0.137% (FM1) to 0.194% (FM3). Differences in the contents of volatile oils between the species studied were noted only in FM1, whereas in the other flower maturity stages they were similar (Table 1). The content of volatile oils in the flower heads of A. montana in FM3 (0.195%) was statistically higher in relation to FM1, FM2, and FM4, whereas the differences in the three stages mentioned were not statistically different. Similarly, in the case of A. chamissonis, the highest content of volatile oils in the flower heads was noted in FM3 (0.194%) and was significantly higher in relation to the other stages. However, the lowest content of volatile oils was recorded in FM1 and FM4. The values are substantially higher in relation to data from Serbia, where Ristic´ et al. (2007) report that the content of essential oil derived from two species, A. montana and A. chamissonis, ¯ was approximately 0.08% (v/w). Similarly, Judˇzentiene˙ and Budien e˙ (2009) in Lithuania obtained essential oil content below 0.1% (v/w). 3.2. Chemical composition of volatile oils Analysis of the chemical composition of the volatile oils revealed qualitative differences between the species studied (Table 3). Fifty compounds in the case of A. montana, and 62 components in the case of A. chamissonis constitued over 90% of the total oil content. The results of our studies indicated that volatile oils taken from flower heads of A. montana are characterised by a higher concentration of sesquiterpenes (ca. 56–71%) in relation to A. chamissonis. However, the proportion of monoterpenes in oils of A. chamissonis flower heads was almost 22% (FM1), whereas in A. montana–ca. 11% (FM1). Ristic´ et al. (2007) presented similar relationships. The dominant components detected in the flower heads of the two species studied included cumene (from 0.94% in the oil from A. chamissonis “FM4” to 1.99% in the oil from A. montana “FM1”), thuja2,4(10)-diene (from 0.87% in the oil from A. montana “C” to 2.25% in the oil from A. chamissonis “FM1”), nonanal (from 1.36% in the oil from A. chamissonis “FM1” to 2.73% in the oil from A. montana “FM1”), decanal (from 6.66% in the oil from A. montana “FM3” to 12.50% in the oil from A. chamissonis “FM2”), thymol (from 1.36% in the oil from A. chamissonis “FM1” to 2.73% in the oil from A. montana “FM1”), eugenol (from 0.10% in the oil from A. chamissonis “FM1” to 2.44% in the oil from A. montana “FM3”), dodecanal (from 0.54% in the oil from A. montana “FM3” to 2.25% in the oil from A. chamissonis

860

R. Kowalski et al. / Industrial Crops and Products 76 (2015) 857–865

Table 1 Volatile oil content in A. montana and A. chamissonis flower heads in relation to the flower maturity stage. FM1 – stage of yellow buds (ligulate florets not opened, tubular florets closed), FM2 – beginning of flowering (flower heads fully emerged, ligulate florets opened, tubular florets opened in 1–2 rows of flower heads). FM3 – full flowering (flower heads fully emerged, ligulate florets opened, up to half of the disc tubular florets opened), FM4 – end of flowering (ligulate florets dry out, all tubular florets opened). The values designated by the different small letters in the lines of the table are significantly different (p = 0.05). The values designated by the different capital letters in the columns of the table are significantly different (p = 0.05). (Tukey-test, p < 0.05).

A. montana A. chamissonis

FM1

FM2

FM3

FM4

0.158aA ± 0.009 0.137aB ± 0.010

0.172aA ± 0.014 0.159bA ± 0.011

0.195bA ± 0.012 0.194cA ± 0.013

0.162aA ± 0.016 0.149abA ± 0.011

“FM2”), E-caryophyllene (from 0.76% in the oil from A. chamissonis “FM1” to 17.98% in the oil from A. montana “FM3”), germacrene D (from 0.66% in the oil from A. chamissonis “FM1” to 3.96% in the oil from A. montana “FM2”), spathulenol (from 0.71% in the oil from A. montana “FM3” to 4.28% in the oil from A. chamissonis “FM4”), caryophyllene oxide (from 4.46% in the oil from A. montana “FM2” to 6.72% in the oil from A. montana “FM3”), humulene epoxide II (from 0.51% in the oil from A. montana “FM2” to 2.29% in the oil from A. chamissonis “FM4”), alpha-cadinol (from 1.65% in the oil from A. montana “FM2” to 2.77% in the oil from A. chamissonis “FM4”), pentadecanal (from 1.69% in the oil from A. montana “FM1” to 3.69% in the oil from A. chamissonis “FM4”), 2-hexyl-E-cinnamaldehyde (from 0.44% in the oil from A. montana “FM3” to 1.60% in the oil from A. chamissonis “FM2”), and farnesyl acetate (from 8.57% in the oil from A. chamissonis “FM4” to 16.14% in the oil from A. montana “FM1”). The other dominant components of the A. montana essential oil that were not detected in A. chamissonis included E-,E,Z-1,3,5,8-undecatetraene – up to 2.63%, alpha-cis-bergamotene – up to 7.10%, and alpha-trans-bergamotene – up to 3.46%. In turn, the main components of A. chamissonis oil that were not detected in A. montana included alpha-pinene – up to 15.23%, epoxy-alloalloaromadendrene – up to 1.90%, n-octadecane – up to 2.65%, and isopropyl hexadecanoate – up to 12.41%. Comparison of previous results obtained by Judˇzentiene˙ and ¯ Budien e˙ (2009) with the results of the present study shows that the quantitative composition of A. montana oils is similar, but there are considerable quantitative differences in the oils. In their investigations of volatile oils from A. montana and A. chamissonis, Ristic´ et al. (2007) found qualitative-quantitative differences between the two species, which was also confirmed in this research. As reported by Ristic´ et al. (2007), E-caryophyllene (31.55–34.59%) and germacrene D (12.47–16.30%) dominated in the A. montana oil, whereas in our investigations, the compounds reached 11.12–17.98% and ¯ e˙ (2009) 5.72–8.92%, respectively; in turn, Judˇzentiene˙ and Budien reported that E-caryophyllene accounted for 1.90% and germacrene D – 0.40%. Germacrene D had been previously identified as the major component of A. chamissonis oil with the content in the range from 18.03 to 38.32% as well as alpha-pinene present in the range of 6.58–19.14% (Ristic´ et al., 2007), while the proportions of these compounds in these investigations were from 3.28 to 5.70% and from 3.44 to 15.23%, respectively. 3.3. Differentiation of the content of volatile oils Cluster analysis of the chemical oil composition showed that two separate groups, A. montana and A. chamissonis samples, could be clearly distinguished among the samples studied (Fig. 1). The percent similarity of the two groups is ca. 50%; however, the similarity of the volatile oil composition is different between the two separated clusters. In the case of A. montana, the highest percent similarity (over 90%) of volatile oil composition was recorded between AM-FM3 and AM-FM4, and in the case of A. chamissonis between AC-FM2 and AC-FM4. In the case of the two herb species, the lowest similarity of the volatile oil composition was recorded between the first and other three stages of flower head maturity (Fig. 1). The presented results correspond to the data reported by

Ristic´ et al. (2007) from Tara Mountain, where similarity of the composition of essential oils extracted from flower heads at the beginning of flowering was lower than in the middle and end of flowering of the two herbal plant species. PCA of the six chemical variables from the samples studied showed that two principal components had a higher influence on the chemical composition (Fig. 2). The two PCA axes account for 93.0% of the total variance, with 85.5% of the total variance explained by the first one; therefore, two principal components are sufficient for description of the samples studied. Axis 1 is the linear combination of the oils studied that summarizes better the variations in the original data matrix in a single number, whereas Axis 2 summarizes the remaining information better. The dimensionality of the data was therefore reduced from 77 variables (all determined oil chemicals) to two uncorrelated components with 7% loss of variation. The chemical variables are represented as a function of both Axis 1 and Axis 2 (Fig. 2). E-caryophyllene, alpha-pinene, isopropyl hexadecanoate, farnesyl acetate, alpha-cis-bergamotene, and decanal are the main factors determining the oil chemical differentiation (Table 2). Axis 1 showed a high correlation with farnesyl acetate, alpha-cis-bergamotene, and E-caryophyllene (positive values) and with alpha-pinene and isopropyl hexadecanoate (negative values). This principal component separates samples with high farnesyl acetate, alpha-cis-bergamotene, and E-caryophyllene values (A. montana) from those with high alpha-pinene and isopropyl hexadecanoate values (A. chamissonis). Therefore, in the case of A. montana, the E-caryophyllene, alpha-cis-bergamotene, and farnesyl acetate values increased from AM-FM1 to AM-FM3 and decreased to AM-FM4, and adversely, the isopropyl hexadecanoate and pinene values decreased from AM-FM1 to AM-FM3 and decreased to AM-FM4. In the samples of A. chamissonis, E-caryophyllene, alpha-cis-bergamotene, and farnesyl acetate increased and isopropyl hexadecanoate and pinene decreased toward AC-FM1 < AC-FM3 < AC-FM2 < AC-FM4. Axis 2 of PCA showed a high positive correlation with decanal and negative with alpha-pinene (Tab. 2). This principal component separates samples with high decanal and low alpha-pinene values from those with high alpha-pinene and low decanal values. Therefore, in the case of A. montana, the P values increased from AM-FM1 to AM-FM3 and decreased to AM-FM4, and adversely, the decanal value decreased from AM-FM1 to AM-FM3 and increased to AM-FM4. In the samples of A. chamissonis, the decanal value increased toward AC-FM1 < AC-FM3 < AC-FM2 < AC-FM4, and the alpha-pinene value decreased. 3.4. Components of essential oils and their value The components of essential oil present in the analysed arnica species determine the biological activity of formulations derived from these plant raw materials. E-caryophyllene, alpha-pinene, isopropyl hexadecanoate, farnesyl acetate, alpha-cis-bergamotene, and decanal are the main components of essential oils in Arnica spp. As reported in previous papers, alpha-pinene exhibits a wide spectrum of bioactivity, i.e. antiinflammatory (Bae et al., 2012),

R. Kowalski et al. / Industrial Crops and Products 76 (2015) 857–865

861

Fig. 1. Cluster analysis of the chemical oil composition in A. montana (AM) and A. chamissonis (AC) in relation to the different flower maturity stages. FM1 – stage of yellow buds (ligulate florets not opened, tubular florets closed), FM2 – beginning of flowering (flower heads fully emerged, ligulate florets opened, tubular florets opened in 1–2 rows of flower heads). FM3 – full flowering (flower heads fully emerged, ligulate florets opened, up to half of the disc tubular florets opened), FM4 – end of flowering (ligulate florets dry out, all tubular florets opened).

Fig. 2. Principal component analysis (PCA) ordination diagram grouping flower heads of A. montana (AM) and A. chamissonis (AC)) with respect to the chemical composition of volatile oils (77 variables) in relation to the different flower maturity stages. FM1 – stage of yellow buds (ligulate florets not opened, tubular florets closed), FM2 – beginning of flowering (flower heads fully emerged, ligulate florets opened, tubular florets opened in 1–2 rows of flower heads). FM3 – full flowering (flower heads fully emerged, ligulate florets opened, up to half of the disc tubular florets opened), FM4 – end of flowering (ligulate florets dry out, all tubular florets opened).

Table 2 Results of the principal components analysis on the basis of chemical data; (a) eigenvalues and percentage of the variance explained by the first two PCA axes; (b) loading components for each environmental variable associated with the two axes. Chemical variables (a) Eigenvalues Percentage Cumulative percentage (b) Pinene Decanal Bergamotene Caryophyllene Farnesyl acetate Isopropyl hexadecanoate

Axis 1

Axis 2

136.368 85.493 85.493

12.041 7.549 93.042

−0.406 −0.054 0.262 0.641 0.239 −0.383

−0.741 0.449 −0.144 −0.384 −0.026 −0.100

antibacterial (Dorman et al., 2000), antioxidant (Wang et al., 2008; Aydin et al., 2013), anticancer (Wang et al., 2012), and antinociceptive (Him et al., 2008) activity. It is necessary to underline that, in the A. chamissonis flower heads, this substance constitutes from a few to several dozen percent, depending on the maturity of the flower heads (Table 2). Decanal exhibits antioxidant

(Liu et al., 2012a,b), antibacterial (Mahboubi and Feizabadi, 2009; Liu et al., 2012a,b), and antitumor (Liu et al., 2012a,b) activities and is a basic oil component of the species studied. Similarly, Ecaryophyllene is known to possess antiinflammatory (Tambe et al., 1996; Cho et al., 2007), antioxidant (Lourens et al., 2004; Singh et al., 2006), anticarcinogenic (Kubo et al., 1996), antibiotic (Alma et al., 2003; Lourens et al., 2004; Pichette et al., 2006), and local anaesthetic (Ghelardini et al., 2001) activities. Isopropyl hexadecanoate was previously detected as one of the major components in Matricaria recutita flowers (Jamalian et al., 2012), and the substance is used for production of cosmetics (emollient) and as an intermediate product for chemical synthesis. The high content of this compound in the A. chamissonis flower heads, especially in the FM3 phase, when the highest essential oil content was noted (Tables 1 and 2), make the plant species very interesting from the point of view of cosmetic industry. As an oxygenated sesquiterpene, farnesyl acetate can determine the antibacterial activity of essential oils (Pauli, 2001). For instance, oil derived from the overground parts of Mantisalca duriaei Briq. Et Cavill with farnesyl acetate as a dominant compound is characterised by interesting antibacterial activity (Boussaada et al., 2009), likewise the oil from Alpinia pahangensis Ridl rhizome, with farnesyl acetate as a major

862

Table 3 Volatile oil composition in A. montana and A. chamissonis in relation to the different stages of flower head maturity. Compounds

IR

Arnica montana FM1 [%]

932 939 956 975 983 992 994 1009 1010 1028 1032 1095 1099 1105 1128 1130 1146 1151 1162 1172 1178 1184 1200 1205 1212 1236 1246 1302 1312 1316 1333 1354 1364 1389 1395 1401 1410 1416 1422 1429 1440 1440 1449 1469 1487 1496

1.99 – 1.21 1.62 – – – – 0.46 0.30 0.31 0.30 0.44 2.73 0.10 – – – 1.76 0.82 2.63 2.32 – – 10.36 0.50 0.13 3.73 – 0.16 – – 1.43 – 0.39 0.09 0.15 0.85 4.40 11.12 – 2.04 0.37 1.42 – 5.75

FM3

FM2 ±SD ±0.220 ±0.092 ±0.080

±0.086 ±0.090 ±0.020 ±0.076 ±0.020 ±0.073 ±0.008

±0.080 ±0.009 ±0.082 ±0.020

±0.090 ±0.042 ±0.010 ±0.140 ±0.010

±0.158 ±0.102 ±0.016 ±0.019 ±0.156 ±0.350 ±0.080 ±0.024 ±0.031 ±0.003 ±0.125

[%]

±SD

[%]

0.97 – 0.41 0.97 – – – – 0.41 0.16 0.19 0.12 0.28 2.32 0.01 – – – 0.15 0.01 2.47 0.22 – – 9.56 0.73 0.17 3.96 – 0.16 – – 2.04 – 0.74 0.14 0.12 0.74 6.24 13.52 – 3.27 0.59 2.02 – 8.92

±0.177

1.02 – 0.19 0.87 – 0.00 0.00 0.00 0.27 0.01 0.16 0.10 0.44 1.58 0.10 – – – 0.32 0.15 1.75 0.28 – – 6.66 0.72 0.24 3.47 – 0.15 – – 2.44 – 0.47 0.14 0.21 0.54 7.10 17.98 – 3.46 0.56 1.83 – 7.92

±0.058 ±0.017

±0.026 ±0.003 ±0.016 ±0.008 ±0.005 ±0.091 ±0.000

±0.002 ±0.056 ±0.014

±0.030 ±0.126 ±0.006 ±0.174 ±0.003

±0.101 ±0.098 ±0.046 ±0.025 ±0.059 ±0.014 ±0.046 ±0.004 ±0.045 ±0.099 ±0.169

FM4 ±SD ±0.375 ±0.036 ±0.028

±0.065 ±0.000 ±0.002 ±0.001 ±0.000 ±0.030 ±0.016

±0.003 ±0.001 ±0.022 ±0.029

±0.181 ±0.020 ±0.092 ±0.083 ±0.003

±0.041 ±0.080 ±0.087 ±0.002 ±0.013 ±0.056 ±0.418 ±0.064 ±0.011 ±0.033 ±0.172

FC1

[%]

±SD

[%]

1.92 – 0.80 1.49 – 0.00 0.00 0.00 0.21 0.01 0.15 0.10 0.52 2.20 0.01 – – – 0.11 0.01 2.13 0.21 – – 7.88 0.41 0.01 3.06 – 0.17 – – 1.94 – 0.52 0.11 0.17 0.64 5.02 16.60 – 2.07 0.46 1.94 – 8.35

±0.148

1.00 15.23 – 2.25 1.40 0.53 0.83 0.37 – 0.46 0.50 0.10 0.17 1.36 – 0.28 0.26 0.16 0.28 0.14 – 0.35 0.01 0.09 8.15 – – 0.66 0.20 0.39 0.09 0.22 0.10 0.41 0.39 0.09 – 1.71 – 0.76 0.08 – 0.66 0.39 0.10 3.28

±0.013 ±0.119

±0.002 ±0.000 ±0.005 ±0.007 ±0.012 ±0.192 ±0.000

±0.050 ±0.000 ±0.073 ±0.033

±0.238 ±0.090 ±0.000 ±0.380 ±0.019

±0.148 ±0.029 ±0.023 ±0.014 ±0.032 ±0.203 ±0.593 ±0.027 ±0.029 ±0.053 ±0.038

FC2 ±SD ±0.013 ±0.235 ±0.259 ±0.157 ±0.055 ±0.107 ±0.049 ±0.017 ±0.109 ±0.033 ±0.152 ±0.011 ±0.007 ±0.007 ±0.030 ±0.047 ±0.120 ±0.011 ±0.000 ±0.037 ±0.168

±0.048 ±0.017 ±0.088 ±0.044 ±0.050 ±0.080 ±0.007 ±0.029 ±0.106 ±0.002 ±0.060 ±0.000 ±0.004 ±0.006 ±0.006 ±0.101

FC3

[%]

±SD

1.08 5.88 – 1.93 0.80 0.67 0.57 0.84 – 0.47 0.59 0.18 0.10 1.97 – 0.16 0.15 0.16 0.37 0.18 – 0.43 0.01 0.10 12.50 – – 0.79 0.11 0.69 0.34 0.44 0.88 0.60 0.37 0.10 – 2.25 – 1.17 0.14 – 0.59 0.58 0.23 5.16

±0.052 ±0.248 ±0.175 ±0.061 ±0.083 ±0.180 ±0.195 ±0.108 ±0.042 ±0.057 ±0.085 ±0.137 ±0.002 ±0.026 ±0.005 ±0.168 ±0.085 ±0.006 ±0.000 ±0.093 ±0.799

±0.016 ±0.011 ±0.025 ±0.065 ±0.002 ±0.012 ±0.039 ±0.042 ±0.069 ±0.032 ±0.017 ±0.002 ±0.006 ±0.007 ±0.003 ±0.147

[%] 0.95 7.98 – 1.40 0.76 0.48 0.59 0.66 – 0.26 0.24 0.14 0.08 1.49 – 0.22 0.30 0.30 0.15 0.13 – 0.19 0.01 0.11 8.13 – – 1.03 0.12 0.50 0.41 0.18 1.36 0.61 0.44 0.01 – 1.73 0.00 0.89 0.10 – 0.52 0.51 0.21 4.19

FC4 ±SD

[%]

±SD

±0.028 ±0.334

0.94 3.44 – 1.58 0.49 0.28 0.39 0.20 – 0.18 0.01 0.10 0.13 1.58 – 0.16 0.11 0.12 0.17 0.12 – 0.14 0.01 0.11 10.89 – – 1.07 0.01 0.59 0.18 0.23 0.72 0.93 0.69 0.17 – 2.24 – 1.30 0.14 – 0.70 0.70 0.20 5.70

±0.147 ±0.544

±0.436 ±0.311 ±0.286 ±0.210 ±0.314 ±0.200 ±0.139 ±0.010 ±0.000 ±0.078 ±0.009 ±0.012 ±0.012 ±0.027 ±0.135 ±0.003 ±0.000 ±0.061 ±0.060

±0.010 ±0.003 ±0.002 ±0.065 ±0.007 ±0.007 ±0.007 ±0.113 ±0.000 ±0.002 ±0.056 ±0.021 ±0.015 ±0.015 ±0.006 ±0.273

±0.400 ±0.237 ±0.242 ±0.122 ±0.049 ±0.002 ±0.000 ±0.001 ±0.001 ±0.108 ±0.003 ±0.006 ±0.024 ±0.008 ±0.025 ±0.035 ±0.000 ±0.013 ±0.133

±0.059 ±0.000 ±0.089 ±0.256 ±0.038 ±0.193 ±0.059 ±0.046 ±0.026 ±0.013 ±0.001 ±0.006 ±0.007 ±0.008 ±0.002 ±0.002

R. Kowalski et al. / Industrial Crops and Products 76 (2015) 857–865

Cumene Pinene Camphene Thuja-2,4(10)-diene Pinene Myrcene 2-Amilfuran Octanal Phellandrene Cymene Limonene n-Undecane Linalool Nonanal Menth-2-en-1-ol Campholenal Verbenol Verbenol Menthone Menthone 1,3,5,8-Undecatetraene Menthol n-Dodecane Myrtenal Decanal Thymol, methyl ether Carvacrol, methyl ether Thymol Carvacrol Undecanal Decadienal <2E,4E-> Longipinene Eugenol Maaliene Isocomene n-Tetradecane Methyl eugenol Dodecanal Bergamotene Caryophyllene Copaene Bergamotene Farnesene <(Z)-beta-> Humulene Amorphene Germacrene D

Arnica chamissonis

1507 1510 1511 1523 1527 1530 1535 1541 1554 1589 1594 1600 1603 1620 1623 1656 1659 1661 1671 1685 1688 1692 1699 1725 1759 1800 1844 1968 2000 2100 2131

– – 0.12 0.25 0.28 0.57 0.58 – 0.29 1.07 5.46 – 1.23 0.73 0.61 – 0.68 – 1.92 0.38 0.97 – 1.39 1.69 0.90 – 16.14 – – – 0.84 10.92 56.5 16.77 4.36 3.02 0.84 3.57

±0.002 ±0.026 ±0.042 ±0.023 ±0.059 ±0.007 ±0.012 ±0.335 ±0.062 ±0.026 ±0.026 ±0.334 ±0.032 ±0.026 ±0.019 ±0.049 ±0.068 ±0.000 ±0.290

±0.006

– – 0.19 0.30 0.47 1.01 0.94 – 0.42 1.06 4.46 – 1.68 0.68 0.51 – 1.42 – 1.65 0.34 0.80 – 1.16 1.92 1.29 – 13.48 – – – 0.65 4.97 63.47 15.68 4.86 2.73 0.65 3.68

±0.015 ±0.042 ±0.050 ±0.041 ±0.179 ±0.079 ±0.061 ±0.014 ±0.083 ±0.093 ±0.079 ±0.587 ±0.254 ±0.103 ±0.288 ±0.075 ±0.093 ±0.020 ±0.221

±0.051

– – 0.22 0.22 0.17 0.61 0.98 – 0.25 0.71 6.72 – 1.16 0.60 0.52 – 0.50 – 1.69 0.38 0.97 – 1.36 1.76 0.44 – 15.46 – – – 1.15 5.44 70.52 11.51 4.43 1.99 1.15 1.96

±0.002 ±0.027 ±0.002 ±0.175 ±0.009 ±0.280 ±0.007 ±0.199 ±0.063 ±0.001 ±0.005 ±0.070 ±0.053 ±0.041 ±0.164 ±0.150 ±0.187 ±0.010 ±0.330

±0.050

– – 0.17 0.28 0.25 1.01 0.57 – 0.44 1.01 6.17 – 1.61 0.82 0.68 – 0.71 – 2.41 0.53 1.35 – 1.30 2.15 0.45 – 13.82 – – – 2.04 5.63 66.28 14.14 3.48 2.34 2.04 3.08

±0.014 ±0.001 ±0.007 ±0.047 ±0.027 ±0.017 ±0.066 ±0.334 ±0.095 ±0.061 ±0.058 ±0.092 ±0.136 ±0.033 ±0.193 ±0.021 ±0.148 ±0.052 ±0.477

±0.123

0.13 0.16 – 0.51 0.22 0.34 – 0.15 0.00 3.97 5.83 0.24 0.82 0.84 2.09 0.22 0.01 1.90 2.09 – – 1.70 0.67 2.75 1.11 2.24 9.59 8.01 1.35 1.61 – 22.2 36.04 16.17 0.86 5.76 8.01 2.95

±0.003 ±0.014 ±0.010 ±0.001 ±0.008 ±0.004 ±0.090 ±0.042 ±0.000 ±0.013 ±0.005 ±0.024 ±0.009 ±0.000 ±0.002 ±0.012

±0.059 ±0.009 ±0.246 ±0.005 ±0.001 ±0.318 ±0.249 ±0.009 ±0.496

0.19 0.23 – 0.77 0.52 0.53 – 0.10 0.00 4.27 5.94 0.70 1.04 1.12 2.15 0.21 1.35 1.09 2.35 – – 1.37 0.67 2.91 1.60 2.65 8.57 5.67 0.99 1.46 – 12.87 38.32 23.39 0.9 6.27 5.67 4.6

±0.008 ±0.001 ±0.025 ±0.008 ±0.012 ±0.023 ±0.119 ±0.168 ±0.012 ±0.030 ±0.017 ±0.055 ±0.035 ±0.029 ±0.034 ±0.042

±0.041 ±0.010 ±0.670 ±0.019 ±0.033 ±0.166 ±0.187 ±0.164 ±0.411

0.15 0.24 – 0.63 0.33 0.49 – 0.22 0.00 4.08 5.63 0.57 0.92 1.31 2.11 0.19 1.36 0.98 2.59 – – 1.37 0.70 2.99 1.58 2.06 8.71 12.41 1.38 1.63 – 13.96 36.21 17.85 1.15 5.94 12.41 4.48

±0.015 ±0.021 ±0.042 ±0.034 ±0.036 ±0.045 ±0.015 ±0.020 ±0.033 ±0.148 ±0.036 ±0.038 ±0.103 ±0.015 ±0.135 ±0.047

±0.096 ±0.005 ±0.525 ±0.047 ±0.108 ±0.252 ±0.917 ±0.007 ±0.067

0.18 0.31 – 0.73 0.28 0.56 – 0.15 0.00 4.28 6.51 0.65 1.03 1.26 2.29 0.25 1.07 1.10 2.77 – – 1.52 0.82 3.69 1.28 2.39 10.85 6.66 1.24 1.42 – 7.76 43.31 21.36 1.08 6.15 6.66 3.68

±0.001 ±0.006 ±0.007 ±0.000 ±0.010 ±0.023 ±0.060 ±0.027 ±0.005 ±0.066 ±0.010 ±0.019 ±0.071 ±0.062 ±0.087 ±0.048

±0.062 ±0.019 ±0.021 ±0.076 ±0.114 ±0.102 ±0.101 ±0.081 ±0.100

R. Kowalski et al. / Industrial Crops and Products 76 (2015) 857–865

n-Pentadecane Cubebol Muurolene Tridecanal Cadinene Amorphene Sesquiphellandrene Nerolidol Sesquisabinene hydrate Spathulenol Caryophyllene oxide n-Heksadecane Salvial-4(14)-en-1-one Tetradecanal Humulene epoxide II Muurolol Amyl cinnamaldehyde Alloaromadendrene Cadinol Bisabolol Cadalene Valeranone Bisabolol Pentadecanal Cinnamaldehyde <2-hexyl-(E)-> n-Octadecane Farnesyl acetate Isopropyl hexadecanoate n-Eicosane n-Heneicosane Methyl linoleate Monoterpenes Sesquiterpenes Aliphatic aldehydes Phenols Aliphatic hydrocarbons Fatty acids and their derivatives Others

IR – retention indices (from temperature-programming, using definition of Van Den Dool and Kratz, 1963). FM1 – stage of yellow buds (ligulate florets not opened, tubular florets closed), FM2 – beginning of flowering (flower heads fully emerged, ligulate florets opened, tubular florets opened in 1–2 rows of flower heads). FM3 – full flowering (flower heads fully emerged, ligulate florets opened, up to half of the disc tubular florets opened), FM4 – end of flowering (ligulate florets dry out, all tubular florets opened).

863

864

R. Kowalski et al. / Industrial Crops and Products 76 (2015) 857–865

component as well (Awang et al., 2011). It is necessary underline that in the case the two Arnica spp. studied this substance constitutes from a few to several dozen percent, depending on the maturity of the flower heads (Table 2). Alpha-cis-bergamotene is the major component of oils that are characterised by antioxidant and antibacterial activity (Liu et al., 2012a,b; Senatore et al., 2003). Given the similarity of the chemical composition of the oils characteristic for both the arnica cultivars, it can be suggested that the analysed group of secondary metabolites should exhibit common biological properties, which is confirmed by the fact that A. montana and A. chamissonis flowers have been regarded as pharmaceutical raw material by the European Commission (). 4. Conclusions • In the case of the two plant species studied, the flower head maturity determined the quantity and chemical composition of oils. • The highest content of volatile oils in the flower heads of the two plant species studied was noted in the full flowering phase, when ligulate florets were opened, and up to half of the disc of tubular florets were opened. • Differences in the volatile oil content were noted between the species studied only in the stage of yellow buds, whereas in the other stages the contents were similar. • E-caryophyllene, alpha-pinene, isopropyl hexadecanoate, farnesyl acetate, alpha-cis-bergamotene, and decanal are the main components that determined the oil chemical differentiation. • The investigations of A. montana volatile oils in the flower heads have shown the existence the differences in the chemical profile in relation to other European populations of this species.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop.2015.07. 029 References Adams, R.P., 2001. Identification of Essential Oil Compounds by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured, Carol Stream, IL. Albert, A., Sareedenchai, V., Heller, W., Seidlitz, H.K., Zidorn, C., 2009. Temperature is the key to altitudinal variations of phenolics in Arnica montana L. cv. ARBO. Oecologia 169 (1), 1–8. Alma, M.H., Mavi, A., Yildirim, A., Digrak, M., Hirata, T., 2003. Screening chemical composition and in vitro antioxidant and anti-bacterial activities of the essential oils from Origanum syriacum L. growing in Turkey. Biological and Pharmaceutical Bulletin 26, 1725–1729. Awang, K., Ibrahim, H., Rosmy Syamsir, D., Mohtar, M., Mat Ali, R., Azah Mohamad Ali, N., 2011. Chemical constituents and antimicrobial activity of the leaf and rhizome oils of Alpinia pahangensis Ridl., an endemic wild ginger from Peninsular Malaysia. Chemistry and Biodiversity 8 (4), 668–673. Aydin, E.A., Türkez, H.B., Geyiko˘glu, F.A., 2013. Antioxidative, anticancer and genotoxic properties of ␣-pinene on N2a neuroblastoma cells. Biologia (Poland) 68 (5), 1004–1009. Bae, G.-S., Park, K.-C., Choi, S.B., Jo, I.-J., Choi, M.-O., Hong, S.-H., Song, K., Park, S.-J., 2012. Protective effects of alpha-pinene in mice with cerulein-induced acute pancreatitis. Life Sciences 91 (17–18), 866–871. Boussaada, O., Ammar, S., Mahjoub, M.A., Saidana, D., Chriaa, J., Chraif, I., Daami, M., Helal, A.N., Mighri, Z., 2009. Chemical composition and antimicrobial activity of volatile components from capitula, stems-leaves and aerial parts of Mantisalca duriaei Briq. et Cavill growing wild in Tunisia. Journal of Essential Oil Research 21 (2), 179–184. Cassell, A.C., Walsh, C., Belin, M., Cambornac, M., Rohit, J.R., Lubrano, C., 1999. Establishment of plantation from micropropagated Arnica chamissonsis a pharmaceutical substitute for the endangered A. montana. Plant Cell Tissue and Organ Culture 156, 139–144. Cho, J.A., Chang, H.J., Lee, S.-K., Kim, H.-J., Hwang, J.-K., Chun, H.S., 2007. Amelioration of dextran sulphate sodium-induced colitis in mice by oral administration of ␤-caryophyllene, a sesquiterpene. Life Sciences 80, 932–939.

Cornu, C., Joseph, P., Gaillard, S., Bauer, C., Vedrinne, C., Bissery, A., Melot, G., Bossard, N., Seeman, A., Wallner, T., Poschlod, P., Heilmann, J., 2010. Variations of sesquiterpene lactone contents in different Arnica montana populations: influence of ecological parameters. Planta Medica 76 (8), 837–842. Dorman, H.J.D., Figueiredo, A.C., Barroso, J.G., Deans, S.G., 2000. In vitro evaluation of antioxidant activity of essential oils and their components. Flavour and Fragrance Journal 15 (1), 12–16. Douglas, J.A., Smallfield, B.M., Burgess, E.J., Perry, N.B., Anderson, R.E., Douglas, M.H., Glennie, V.A., 2004. Sesquiterpene lactones in Arnica montana, a rapid analytical method and the effects of flower maturity and simulated mechanical harvesting on quality and yield. Planta Medica 70, 166–170. Falniowski, A., Bazos, I., Hodálová, I., Lansdown, R., Petrova, A., 2012. Arnica montana. In: IUCN Red List of Threatened Species, Version 2012.2. IUCN. Polish Pharmacopoeia VI. The Republic of Poland, The Minister of Health, Warsaw, 2002. Ganzera, M., Egger, C., Zidorn, C., Stuppner, H., 2008. Quantitative analysis of flavonoids and phenolic acids in Arnica montana L. by micellar electrokinetic capillary chromatography. Analytica Chimica Acta 614 (2), 196–200. Gaspar, A., Cracinescu, O., Trif, M., Moisei, M., Moldovan, L., 2014. Antioxidant and anti-inflammatory properties of active compounds from Arnica montana L. Romanian Biotechnological Letters 19 (3), 9353–9365. ´ Gawlik-Dziki, U., Swieca, M., Sugier, D., Cichocka, J., 2009. Seeds of Arnica montana and Arnica chamissonis as a potential source of natural antioxidants. Herba Polonica 55, 60–71. ´ Gawlik-Dziki, U., Swieca, M., Sugier, D., Cichocka, J., 2011. Comparison of in vitro lipoxygenase, xanthine oxidase inhibitory and antioxidant activity of Arnica montana and Arnica chamissonis tinctures. Acta Scientiarum Polonorum, Hortorum Cultus 10, 15–27. Ghelardini, C., Galeotti, N., Di Cesare, M.-L., Mazzanti, G., Barto-lini, A., 2001. Local anaesthetic activity of b-caryophyllene. Farmaco 56, 387–389. Him, A., Ozbek, H., Turel, I., Oner, A.C., 2008. Antinociceptive activity of alpha-pinene and fenchone. Pharmacologyonline 3, 363–369. Hultén, E., Fries, M., 1986. Atlas of North European Vascular Plants: North of the Tropic of Cancer, vol. 1. Koeltz Scientific Books, Königstein, Germany. Jamalian, A., Shams-Ghahfarokhi, M., Jaimand, K., Pashootan, N., Amani, A., RazzaghiAbyaneh, M., 2012. Chemical composition and antifungal activity of Matricaria recutita flower essential oil against medically important dermatophytes and soil-borne pathogens. Journal de Mycologie Medicale 22 (4), 308–315. Joulain, D., König, W.A., 1998. The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. E.B. Verlag, Hamburg. ˙ A., Budien ¯ ˙ J., 2009. Analysis of the chemical composition of fower Judˇzentiene, e, essential oils from Arnica montana of Lithuanian origin. Chemija 20 (3), 190–194. Kahmen, S., Poschlod, P., 2000. Population size, plant performance, and genetic variation in the rare plant Arnica montana L. in the Rhön, Germany. Basic and Applied Ecology 1 (1), 43–51. Korneck, D., Schnittler, M., Vollmer, I., 1996. Red list of pteridophyta and spermatophyta in Germany. In: Ludwig, G, Schnittler, M (Eds.), Red List of Endangered Plants in Germany. Schriftenreihe für Vegetationskunde, vol. 28. Bundesamt für Naturschutz, Bonn, Germany, pp. 21–187 (German). Kowalski, R., Wawrzykowski, J., 2009. Effect of ultrasound-assisted maceration on the quality of oil from the leaves of thyme Thymus vulgaris L. Flavour and Fragrance Journal 24, 69–74. Kovach, W., 1999. MVSP – A Multivariate Statistical Package for Windows, ver. 3.1. Kovach Computing Services. Pentraeth, Wales, UK, pp. 133p. Kubo, I., Chaudhuri, S.K., Kubo, Y., Sanchez, Y., Ogura, T., Saito, T., Ishikawa, H., 1996. Cytotoxic and antioxidative sesquiterpe-noids from Heterotheca inuloides. Planta Medica 62, 427–430. Liu, K., Chen, Q., Liu, Y., Zhou, X., Wang, X., 2012a. Isolation and Biological Activities of Decanal, Linalool, Valencene, and Octanal from Sweet Orange Oil. Journal of Food Science 77 (11), C1156–C1161. Liu, C.J., Zhang, S.Q., Zhang, J.S., Liang, Q., Li, D.S., 2012b. Chemical composition and antioxidant activity of essential oil from berries of Schisandra chinensis (Turcz.) Baill. Natural Product Research 26 (23), 2199–2203. Leven, W., Willuhn, G., 1987. Sesquiterpene lactones from Arnica chamissonis less.: VI. Identification and quantitative determination by high-performance liquid and gas chromatography. Journal of Chromatography A 410, 329–342. Lourens, A.C., Reddy, D., Baser, K.H., Viljoen, A.M., Van Vuuren, S.F., 2004. In vitro biological activity and essential oilcomposition of four indigenous South African Helichrysum species. Journal of Ethnopharmacology 95, 253–258. Luijten, S.H., Oostermeijer, J.G.B., van Leeuwen, N.C., Den Nijs, H.C.M., 1996. Reproductive success and clonal genetic structure of the rare Arnica montana (Compositae) in The Netherlands. Plant Systematics and Evolution 201 (1–4), 15–30. Maguire, B., 1943. A monograph of the genus Arnica (Senecioneae, Compositae). Brittonia 4, 386–510. Mahboubi, M., Feizabadi, M.M., 2009. Antimicrobial activity of Ducrosia anethifolia essential oil and main component, decanal against methicillin-resistant and methicillin-susceptible Staphylococcus aureus. Journal of Essential Oil-Bearing Plants 12 (5), 574–579. Maurice, T., Colling, G., Muller, S., Matthies, D., 2012. Habitat characteristics, stage structure and reproduction of colline and montane populations of the threatened species Arnica montana. Plant Ecology 213 (5), 831–842. Merfort, I., Wendisch, D., 1987. Flavonoid Glycosides from Arnica montana and Arnica chamissonis. Planta Medica 53 (5), 434–437. Merfort, I., Wendisch, D., 1988. Flavonoid glucuronides from the flowers of Arnica montana. Planta Medica 54, 247–250.

R. Kowalski et al. / Industrial Crops and Products 76 (2015) 857–865 Merfort, I., 2010. Arnika–Aktueller Stand hinsichtlich Wirksamkeit, Pharmakokinetik und Nebenwirkungen. Zeitschrift Phytother 31 (4), 188–192. Nichterlein, K., 1995. Arnica montana (Mountain Arnica): in vitro culture and the production of sesquiterpene lactones and other metabolites. Biotechnology in Agriculture and Forestry 33, 47–61. NIST/EPA/NIH., 2005. Mass Spectral Library with Search Program: Data Version: NIST 08, Software Version 2.0f. National Institute of Standards and Technology. ´ ´ Nowak, T., 2002. Lowland arnica–culivation in gostynskoleszczy nski region. Wiadomo´sci Zielarskie 1, 18–19. Pauli, A., 2001. Antimicrobial properties of essential oil constituents. International Journal of Aromatherapy 11 (3), 126–133. Perry, N.B., Burgess, E.J., Rodriguwz, M.A., Romero Franco, R., López Morquero, E., Smallfield, B.M., Joyce, N.I., Litrtlejohn, R.P., 2009. Sesquiterpene lactones in Arnica montana helenalin and dihydrohelenalin chemotypes in Spain. Planta Medica 75 (6), 660–666. Pichette, A., Larouche, P.-L., Lebrun, M., Legault, J., 2006. Composition and antibacterial activity of Abies balsamea essential oil. Phytotherapy Research 20, 371–373. ´ D., Ranˇcic, ´ D., Ristic, ´ M., Vujisic, ´ L., Radanovic, ´ D., Dajic-Stevanovi ´ ´ Pljevljakuˇsic, c, Z., 2012. Rhizome and root yield of the cultivated Arnica montana L., chemical composition and histochemical localization of essential oil. Industrial Crops and Products 39, 177–189. ´ M., Krivokuca-Djokic, D., Radanovic, ´ D., Nastovski, T., 2007. Essential oil of Ristic, Arnica montana and Arnica chamissonis. Hemijska industrija 61, 272–277. Rohloff, J., 2003. Cultivation of herbs and medicinal plants in Norway—essential oil production and quality control. In: PhD Thesis. Norwegian University of Science, Trondheim, Norway. ˇ ´ N., Savikin-Fodulovi ´ K., Krivokuca-Doki ´ ´ D., Ristic, ´ M., Grubiˇsic, ´ Roki, D., Menkovic, c, c, D., 2001. Flavonoids and essential oil in flower heads of introduced Arnica chamissonis. Journal of Herb, Spices and Medicinal Plants 8 (4), 19–27. Saluk-Juszczak, J., Pawlaczyk, I., Olas, B., Kolodziejczyk, J., Ponczek, M., Nowak, P., Tsirigotis-Woloszczak, M., Wachowicz, B., Gancarz, R., 2010. The effect of polyphenolic-polysaccharide conjugates from selected medicinal plants of Asteraceae family on the peroxynitrite-induced changes in blood platelet proteins. International Journal of Biological Macromolecules 47, 700–705. Schmidt, T.J., Stausberg, S., von Raison, J., Berner, M., Willuhn, G., 2006. Lignans from Arnica species. Natural Product Research 20, 443–453. Seemann, A., Wallner, T., Poschlod, P., Heilmann, J., 2010. Variation of sesquiterpene lactone contents in different Arnica montana populations: influence of ecological parameters. Planta Medica 76 (8), 837–842. Senatore, F., Lentini, F., Venza, F., Bruno, M., Napolitano, F., 2003. Composition and antibacterial activity of the essential oil of Anisochilus carnosus (Linn. fil.) Benth., a Tamil plant acclimatized in Sicily. Flavour and Fragrance Journal 18 (3), 202–204. Singh, G., Marimuthu, P., de Heluani, C.S., Catalan, C.A., 2006. Antioxidant and biocidal activities of Caru m nigrum (seed) essential oil, oleoresin, and their selected components. Journal of Agriculture Food Chemistry 54, 174–181.

865

Spitaler, R., Winkler, A., Lina, I., Yanar, S., Stuppner, H., Zidorn, C., 2008. Altitudinal variation on the phenolic contents in flowering heads of Arnica montana cv. ARBO: a 3-year comparison. Journal of Chemical Ecology 34 (3), 369–375. Sugier, D., Gawlik-Dziki, U., 2009. The influence of foliar fertilization on yielding and quality of mountain arnica (Arnica montana L.) and chamisso arnica (Arnica chamissonis var. foliosa). Annales UMCS Agricultura 64 (3), 129–139. Sugier, D., 2013. Yield and chemical composition of mountains arnica (Arnica montana L.) raw material in relation to the method of plantation establishment and the harvesting time of flower heads. Annales UMCS Agricultura 68 (3), 51–62. Tambe, Y., Tsujiuchi, H., Honda, G., Ikeshiro, Y., Tanaka, S., 1996. Gastric cytoprotection of the non steroidal anti-inflammatory sesquiterpene, ␤-caryophyllene. Planta Medica 62, 469–470. Van Den Dool, H., Kratz, D.J., 1963. A generalization of the retention index system including liner temperature programmed gas-liquid partition chromatography. Journal of Chromatography 11 (2), 463–467. Wang, W., Li, N., Luo, M., Zu, Y., Efferth, T., 2012. Antibacterial activity and anticancer activity of Rosmarinus officinalis L. essential oil compared to that of its main components. Molecules 17 (3), 2704–2713. Wang, W., Wu, N., Zu, Y.G., Fu, Y.J., 2008. Antioxidative activity of Rosmarinus officinalis L. essential oil compared to its main components. Food Chemistry 108 (3), 1019–1022. ˙ ´ Weremczuk-Jezyna, I., Kisiel, W., Wysokinska, H., 2006. Thymol derivatives from hairy roots of Arnica montana. Plant Cell Reports 25 (9), 993–996. ˙ ´ Weremczuk-Jezyna, L., Wysokinska, H., Kalemba, D., 2011. Constituents of the essential oil from hairy roots and plant roots of Arnica montana. Journal of Essential Oil Research 23 (1), 91–97. Willuhn, G., 1972. Untersuchungen über die Inhaltsstoffe von Arnica–Arten–VI. Charakterisierung und preparative Auftrennung der ätherischen Öle aus den Wurzeln, Rhizomen, Blättern und Blütenkörbchen verschiedener Arnica–Arten. Planta Medica 21 (4), 329–342. Willuhn, G., Kresken, J., Wendisch, D., 1983. Sesquiterpenelactone aus Arnica chamissonis III: 4-0-acetyl-6-desoxychamissonolid und 6-0-propionyl-11,13dihydrohelenalin. Planta Medica 47, 157–160. Willuhn, G., Merfort, I., Pasreiter, C.M., Schmidt, T.J., 1995. Chemistry and systematics of the genus Arnica. In: Hind, D.J.N., Jeffrey, C., Pope, G.W.(Hrsg.) (Eds.), Advances in Compositae Systematics. Royal Botanic Gardens, Kew, England. Willuhn, G., 1998. Arnika Flowers: Pharmacology, toxicology and analysis of the sesquiterpene lactones–their main active substances. In: Lawson, L.D., Bauer, R. (Eds.), Phytomedicines of Europe – Chemistry and Biological Activity. ACS Symposium Series 691. American Chemical Society, Washington DC. Wołkowycki, D., 2012. Mountain arnica. Arnica montana L. In: Perzanowska, J. (Ed.), ´ Warszawa, Poland, Monitoring of Plant Species. Methodical Guide, Part III. GIOS, p. 2012. ˛ Z., 2006. Red list of the vascular plants in Poland. In: Mirek, Z., Zarzycki, K., Szelag, ˛ Z. (Eds.), Red List of the Plants and Fungi in Zarzycki, K., Wojewoda, W., Szelag, Poland. Szafer Institute of Botany, Polish Academy of Sciences, Kraków, Poland.