Chemical Characteristics and Anticancer Activity of Essential Oil from Arnica Montana L. Rhizomes and Roots

Arnica montana L. is a medicinal plant with diverse biological activities commonly used in pharmacy and cosmetics. The attributes of A. montana are mainly related to the concentration and chemical composition of essential oils (EOs). Therefore, the objective of this study was to characterize the chemical composition of EOs derived from A. montana rhizomes and roots taking into account the age of the plants and to investigate the effect of the analyzed EOs on induction of apoptosis, necrosis, and autophagy in human glioblastoma multiforme T98G and anaplastic astrocytoma MOGGCCM cell lines. Rhizomes and roots of mountain arnica were harvested at the end of the third and fourth vegetation periods. The chemical composition of essential oils was determined with the GC–MS technique. Among the 37 components of the essential oil of A. montana, 2,5-dimethoxy-p-cymene (46.47%–60.31%), 2,6-diisopropylanisole (14.48%–23.10%), thymol methyl ether (5.31%–17.79%), p-methoxyheptanophenone (5.07%–9.65%), and α-isocomene (0.68%–2.87%), were detected in the rhizomes and roots of the three-year-old plants and in the rhizomes and roots of the four-year-old plants. The plant part (rhizome, root) and plant age can be determinants of the essential oil composition and, consequently, their biological activity. The induction of apoptosis (but not autophagy nor necrosis) at a level of 28.5%–32.3% is a promising result, for which 2,5-dimethoxy-p-cymene, 2,6-diisopropylanisole, thymol methyl ether, and p-methoxyheptanophenone are probably mainly responsible. The present study is the first report on the anticancer activities of essential oils from A. montana rhizomes and roots.


Introduction
Natural products and their derivatives are increasingly desirable sources of novel therapeutic molecules worldwide [1]. Researchers' attention is focused on the chemical characteristic of essential oils (Eos) derived from plants and the wide range of their biological properties, including antioxidant, The parts of Arnica plants contain a wide range of secondary metabolites. Essential oils are located in flower heads, rhizomes, roots, and achenes [9,10,15,21,26,40]. The investigations of EOs contained in the flower heads of different European A. montana populations have shown differences in the chemical profile of EOs. The EOs obtained from plants cultivated in Poland were characterized by dominance of β-caryophyllene, caryophyllene oxide, decanal, germacrene D, and farnesyl acetate [9,21]. As reported by Ristic et al. [40], β-caryophyllene, germacrene D, and decanal dominated in the EO of A. montana grown on Tara Mountain in Serbia. In turn, in a study from Lithuania, Judžentienė and Būdienė [15] reported the presence of (2E, 6E)-farnesol, (2Z, 6E)-farnesyl acetate, (Z, Z)-geranyl linalool, β-caryophyllene, and caryophyllene oxide; however, the two main EO compounds were not identified. Pljevljakušić [17] demonstrated that 2,5-dimethoxy-p-cymene, thymol methyl ether, p-methoxyheptanophenone, and 2,6-diisopropylanisole were the main constituents of oils from rhizomes and roots of two-year-old and three-year-old plants growing on Tara Mountain (subalpine climate) in Serbia. Weremczuk-Jeżyna [26] showed that EOs were dominated by 10-isobutyryloxy-8,9-didehydro-thymol isobutyrate and 10-isobutyryloxy-8,9-didehydro-thymol methyl ether. In turn, Danila et al. [41] reported 35 compounds in EOs of A. montana rhizomes and roots collected from wild populations in the Romanian East-Carpathians, with the following major constituents: 3-t-butyl-1,2-dimethoxybenzene, thymol, tetradecane, non-3-en-2-one, and thymol methyl ether. However, there is no information on the biological activity of these oils. Recently, another new source of EOs from Arnica montana L. achenes and their chemical characteristics have been reported [10]. This study is the first report on the anticancer activities of essential oil from A. montana achenes and generally the only example of studies of the biological activity of Arnica EOs so far. The main components in the EOs from the Arnica achenes included 2,5-dimethoxy-p-cymene, cumene, thymol methyl ether, 2,6-diisopropylanisole, decanal, and 1,2,2,3-tetramethylcyclopent-3-enol. Such a wide range of different components of Arnica EOs implies a very broad spectrum of the possible use of these chemically different secondary metabolites.
Studies conducted lately have shown that EOs from achenes exert an anticancer effect by induction of cell death in anaplastic astrocytoma and glioblastoma multiforme cells [10]. This facilitates the use of this secondary metabolite in further studies that will contribute to development of glioma therapy in the future. Moreover, the concentration of EOs in achenes and in flower heads is similar; however, rhizomes and roots, which can contain even up to 4% of EOs in dry mass with a varied chemical composition, are a richer source of EO [17]. Therefore, the objective of this study was: (i) to characterize the chemical composition of EOs derived from A. montana rhizomes and roots taking into account the age of the plants, and (ii) to investigate the effect of the analyzed EOs on induction of apoptosis, necrosis, and autophagy in human glioblastoma multiforme T98G and anaplastic astrocytoma MOGGCCM cell lines.

Differentiation of the EO Content
The principal correspondence analysis (PCA) of the 37 chemical variables from the studied samples revealed two principal components with a greater effect on the chemical composition of EO, but only four main components are presented in Figure 3. The PCA analysis showed distribution of the samples along the first axis. The two PCA axes account for 98.93% of the total variance, with 92.77% of the total variance explained by the first axis; therefore, two principal components are

Differentiation of the EO Content
The principal correspondence analysis (PCA) of the 37 chemical variables from the studied samples revealed two principal components with a greater effect on the chemical composition of EO, but only four main components are presented in Figure 3. The PCA analysis showed distribution of the samples along the first axis. The two PCA axes account for 98.93% of the total variance, with 92.77% of the total variance explained by the first axis; therefore, two principal components are sufficient to describe the presented samples (Table 2). Axis 1 is the linear combination of the studied oils, which better summarizes the variations in the original data matrix in a single number, whereas Axis Molecules 2020, 25, 1284 6 of 21 2 better summarizes the remaining information. The dimensionality of the data was therefore reduced from 37 variables to two uncorrelated components with 5% loss of variation.
The principal correspondence analysis (PCA) of the 37 chemical variables from the studied samples revealed two principal components with a greater effect on the chemical composition of EO, but only four main components are presented in Figure 3. The PCA analysis showed distribution of the samples along the first axis. The two PCA axes account for 98.93% of the total variance, with 92.77% of the total variance explained by the first axis; therefore, two principal components are sufficient to describe the presented samples (Table 2). Axis 1 is the linear combination of the studied oils, which better summarizes the variations in the original data matrix in a single number, whereas Axis 2 better summarizes the remaining information. The dimensionality of the data was therefore reduced from 37 variables to two uncorrelated components with 5% loss of variation.
The chemical variables are represented as a function of both Axis 1 and Axis 2. 2,5-dimethoxyp-cymene, thymol methyl ether, 2,6-diisopropylanisole, and p-methoxyheptanophenone are the main factors determining the chemical differentiation of EOs ( Figure 3, Table 2). Axis 1 shows a high positive correlation with 2,5-dimethoxy-p-cymene and a negative correlation with thymol methyl ether; therefore, this principal component separates samples with high content of 2,5-dimethoxy-p-cymene and low content of thymol methyl ether (EO-RO3, EO-RO4, EO-RH4) on the right side and samples with low content of 2,5-dimethoxy-p-cymene and high content of thymol methyl ether (EO-RH3) on the left side of the ordination space. In turn, Axis 2 of PCA shows a high positive correlation with 2,6-diisopropylanisole and a negative correlation with p-methoxyheptanophenone and α-isocomene. This principal component separates samples situated in the upper part of the ordination space with high content of 2,6-diisopropylanisole and samples located in the lower part of the ordination space with high content of p-methoxyheptanophenone and α-isocomene.

Anticancer Activity
To estimate the sensitivity of T98G and MOGGCCM cells to the treatment with the EOs derived from Arnica rhizomes and roots, a staining method with dyes specific for apoptosis, necrosis, and autophagy; i.e., Hoechst 33342, propidium iodide, and acridine orange, respectively, was employed (Figures 4 and 5). The multi-way ANOVA results showed a statistically significant main effect of the plant age (A) and the EO concentration (C); however, there was no statistically significant effect of the plant part (R) on apoptosis, necrosis, and autophagy of the T98G cells (Table 3). In the case of this cell line, there was a statistically significant effect of the R× A, R × C, A × C, and R × A × C interactions on apoptosis, a significant effect of the A × C interaction on necrosis, and a significant effect of the R × C, A × C, and R × A × C interactions on autophagy. Different results were obtained in the case of the MOGGCCM cells. The essential oils did not cause autophagy; however, all analyzed factors and their interactions were found to have a statistically significant effect on the level of apoptosis, but only the EO concentration had an effect on necrosis (Table 3). Table 3. Effect of the main factors and their interactions on the level of apoptosis, necrosis, and autophagy. Result of multi-way analysis of variance (ANOVA). T98G-glioblastoma multiforme cell line, MOGGCCM-anaplastic astrocytoma cell line.

Cell Line T98G MOGGCCM
Factor Apoptosis Necrosis Autophagy Apoptosis Necrosis The microscopic observations demonstrated that EO-RH3 added to the T98G culture medium had a considerable effect on induction of cell death (Figure 4, EO-RH3). A significant 15.7% increase in the number of apoptotic cells was induced by EO-RH3 at the concentration of 0.5 µL/mL (Figure 4, EO-RH3). The increase in the EO concentration to 1 µL/mL resulted in a further increase in the level of apoptosis to 32.3% and simultaneously initiated necrosis and autophagy at a level of 2.0% and 2.3%, respectively. The EO concentration of 2 µL/mL caused a decrease in the percentage of apoptotic cells (13.3%) to the level characteristic for the concentration of 0.5 µL/mL. Moreover, although apoptosis occurred at the EO concentration of 2 µL/mL, necrosis (34.7%) and autophagy (1.0%) were initiated.
The application of EO-RH4 to the T98G culture medium (Figure 4, EO-RH4) caused a similar response (Figure 4, EO-RH3). A significant 12.0% increase in the number of apoptotic cells induced by EO-RH4 was observed at the concentration of 0.5 µL/mL. A further increase in the EO-RH4 concentration to 1 µL ml also resulted in a ca. 19.7% increase in the level of apoptosis; however, this value was statistically significantly lower than the level of apoptosis caused by EO-RH3 at the concentration of 1 µL/mL (Figure 4, EO-RH3). In turn, the concentration of 2 µL/mL caused a gradual decrease in the percentage of apoptotic cells to 14.7% to the level characteristic for the concentration of 0.5 µL/mL. Besides apoptosis, EO-RH4 initiated necrosis at a level of 12.3% and autophagy at a level of 6.3%. The application of 0.5 μL/mL of EO-RO3 to the T98G culture medium had a considerable effect on cell death, namely, there was a significant 3.5% increase in the number of apoptotic cells ( Figure  4, EO-RO3). A further increase in the EO-RO3 concentration also resulted in a statistically significant increase in the level of apoptosis to 28.5%, whereas necrosis was initiated at a level of 1.0% only. In The application of 0.5 μL/mL of EO-RO4 to the MOGGCCM culture medium had a considerable effect on cell death, namely a significant 6.3% increase in the number of apoptotic cells was observed The application of 0.5 µL/mL of EO-RO3 to the T98G culture medium had a considerable effect on cell death, namely, there was a significant 3.5% increase in the number of apoptotic cells (Figure 4, EO-RO3). A further increase in the EO-RO3 concentration also resulted in a statistically significant increase in the level of apoptosis to 28.5%, whereas necrosis was initiated at a level of 1.0% only. In turn, the EO-RO3 concentration of 2 µL/mL caused a decrease in the percentage of apoptotic cells (25.5%), which was not statistically significant. Although apoptosis occurred at this EO-RO3 concentration, necrosis and autophagy were initiated at a level of 26.5% and 2%, respectively.
Our experiments showed a significant 12.7% increase in the number of apoptotic cells upon the addition of EO-RO4 at the concentration of 0.5 µL/mL (Figure 4 The application of 0.5 µL/mL of EO-RO4 to the MOGGCCM culture medium had a considerable effect on cell death, namely a significant 6.3% increase in the number of apoptotic cells was observed ( Figure 5, EO-RO4). A further increase in the EO-RO4 concentration to 1 µL/mL also resulted in a statistically significant increase in the level of apoptosis to 29.0%, whereas necrosis was initiated at a level of 1.0% only. In turn, the EO-RO4 concentration of 2 µL/mL did not cause a significant change in the percentage of apoptotic cells (27.0%). Although apoptosis occurred at this EO-RO4 concentration, necrosis was initiated at a level of 23.7%. Additionally, it is worth mentioning that none of the EOs studied caused autophagy in the MOGGCCM culture medium.
To sum up, EO-RH3, EO-RH4, and EO-RO4 at the concentration of 1 µL/mL have the most promising influence on apoptosis of the T98G and MOGGCCM cell lines with a low level of necrosis at the same time.
The IC 50 analysis based on the microscopic observations and colorimetric assay revealed that the MOGGCCM cells were more sensitive to initiation of cell death after the treatment with the essential oils, in comparison to the T98G cell line (Tables 4 and 5). The analyzed essential oils had no effect on cell death initiation in normal fibroblasts ( Figure 6).
As shown by multiyear observations of Arnica individuals conducted in field conditions [25], A. montana can produce clumping ramets which develop from the short rhizomes of the spreading ramets. In the central part of the four-year-old genets, accumulation of necromass and no production of new ramets were detected, which was a symptom of the onset of genet division. Therefore, the lower EO concentration in the rhizomes of the older Arnica plants can be an effect of genet senescence and greater abundance of dead fragments in the raw material. However, the genet division and genet senescence do not explain the lower concentration of EO in the roots of the older Arnica plants.
Secondary metabolism is often found to be unique to an organism or is an expression of the individuality of a species [47,48]. Secondary metabolites are produced as a result of organism adaptation to its surrounding environment or are produced to act as a possible defense mechanism against predators and assist in the survival of the organism [48,49]. Biosynthesis of EOs is not only controlled genetically, but is also strongly affected by various biotic and abiotic stresses [50]. Therefore, the EO composition is determined by growth conditions, climate, altitude, soil type, agricultural methods and practices, developmental stage, plant part, and harvesting time [51]. Altitude is one of the abiotic stresses associated with differences in a number of environmental factors such as air temperature, precipitation, light intensity, wind exposure, UV-B radiation, ozone density, and oxidizing air pollutants [52]. A consequence of the altitude differentiation is the variable chemical composition of EOs [53,54]. The presented literature data on the concentration and chemical composition of EO in rhizomes and roots, in comparison with the results of our research, originated from studies carried out in natural habitats and from different field experiments conducted in different regions of Europe, from lowland to mountain regions [17,26,41]. The sources of the plant material differed as well. Therefore, many factors, e.g., edaphic and climatic determinants, have an influence on plant growth and production of primary and secondary metabolites. Probably, climatic conditions are the main determinants of the EO concentration in mountain Arnica rhizomes and roots and the chemical composition of EO.
Gliomas are very aggressive brain tumors with very high resistance to chemotherapy [39]; therefore, studies of the effectiveness of different substances in elimination of human glioma cells through apoptosis and autophagy are necessary [35][36][37][38]. Jakubowicz-Gil et al. [35][36][37][38] made an attempt to use single natural chemicals, such as quercetin, or combinations with temozolomide [35][36][37] and sorafenib [38], which were shown as potent apoptosis inducers in T98G and MOGGCCM cells. Conventional cancer therapies cause serious side effects [28]. However, many alternative treatments and therapies based on plants have been explored in the recent years, which is especially important for patients that do not tolerate extreme side effects [46]. Plants have been analyzed to identify their anticancer properties and characterized chemically to reveal the presence of many bioactive compounds [29,30]. The effect of EOs with a specific chemical composition has been reported to vary depending on the features of particular cancer types. Ribeiro et al. [55] investigated the cytotoxic potential of L. gracilis EOs against tumor cell lines. The results showed that the oils inhibited human melanoma (MDA-MB-435) and colon carcinoma (HCT-8) cell lines. Lima et al. [56] indicated that the cytotoxicity of EOs from L. lucidus against liver carcinoma and breast cancer cell lines was stronger than against other cell lines. da Silva et al. [57] showed varied cytotoxic activity of Piper aleyreanum oils on colon and melanoma cell lines. Similarly, Yu et al. [58] demonstrated various levels of cytotoxic activity of P. klotzschianum oils inhibiting human hepatocellular carcinoma, human promyelocytic leukemia, and murine melanoma cell lines. A broad cytotoxicity spectrum was indicated in the case of essential oil of P. cernuum against human melanoma, human cervical tumor, and human glioblastoma cells [59,60]. Considering the anticancer activity of the EOs described in the present study and the level of apoptosis caused by EOs extracted from the underground parts of A. montana (Figures 4 and 5), this plant species can be included in the medicinal plant group with high anticancer potential.
Apoptosis is one of the main mechanisms by which chemotherapeutic agents induce cell death in cancer cells [61]. The increase in the EO-RH3, EO-RH4, EO-RO3, EO-RO4 concentration to 1 µL/mL resulted in an increase in the level of apoptosis to 18.0%, 21.0%, 28.7%, and 29% in the MOGGCCM cell line, respectively (Figure 4). Similar results were obtained in the case of the T98G cell line, namely, the increase in the EO-RH3, EO-RH4, EO-RO3, EO-RO4 concentration to 1 µL/mL resulted in an increase in the level of apoptosis to 32.3%, 19.7%, 28.5%, and 32.3%, respectively ( Figure 5), which suggests that the essential oil is a promising natural anticancer product. In the world literature, there is another report of the effect of A. montana essential oil on the induction of apoptosis of glioma T98G and MOGGCCM cell lines [10]; hence, this species can be regarded as an important source of anticancer secondary metabolites. This is an important finding, especially in the light of recent investigations showing that gliomas naturally resist apoptosis [62].
Exploration of natural plant products as an option to find new chemicals as anticancer agents is one of the fastest growing areas of research, and the available studies indicate EOs from various plant species and their constituents as anticancer agents [63]. A recent study has shown that EOs can be a potential alternative for glioblastoma treatment [64][65][66]. Quassinti et al. [65] showed that EO of Hypericum hircinum had antiproliferative activity on human glioblastoma tumor cells T98G. Detoni et al. [64] demonstrated that EO of Zanthoxylum tinguassuiba could be a potential alternative for gliobastoma treatment. As reported by Bayala et al. [66], essential oil of Ageratum conyzoides shows the highest antitumor activity on SF-763 cells, while the SF-767 glioblastoma cell line was the most sensitive to EOs of Ocimum basilicum and Lippia multiflora. In the literature, there are examples of research on the effects of EOs or their main ingredients on glioma cells. Thymol, i.e., the main component of many aromatic and medicinal plants has been shown to have a stimulating effect on apoptosis and an inhibitory effect on cell growth in DBTRG-05MG human glioblastoma [63]. α-Bisabolol, which is a natural compound strongly inducing apoptosis in glioma cells [2], or β-Elemene, i.e., a natural plant drug obtained from Curcuma wenyujin inducing apoptosis in glioblastoma cells, exert promising anticancer effects against a broad spectrum of tumors [32,67]. Another example of molecules characterized by an anticancer effect on human glioblastoma cell line U87MG is aloe emodin, i.e., an anthraquinone compound present in the leaves of Aloe arborescens, and hispolon, which is a polyphenolic compound isolated from Phellinus linteus [68]. The study presented in this paper is the first attempt to search for phytochemicals contained in Arnica rhizomes and roots characterized by antiglioma activity. Our earlier study showed anticancer activity of EOs from mountain Arnica achenes on the T98G and MOGGCCM cell lines [10]. Puzzling is fact that the EOs from achenes have a similar chemical composition to that from rhizomes and roots, with 2.5-dimethoxy-p-cymene, thymol methyl ether, and 2,6-diisopropylanisole as the main ingredients.
The components of the EOs present in the analyzed Arnica rhizomes and roots determine the biological activity of formulations derived from these plant raw materials. 2,5-dimethoxy-p-cymene, thymol methyl ether, p-methoxyheptanophenone, and 2,6-diisopropylanisole are the main components of the EOs in the underground parts of mountain Arnica. In view of continuation of the study, it is highly promising that the medicinal plant A. triplinervis from the family Asteraceae has recently been identified for the first time as a new source of antiviral phytocompounds targeting the entry of viral pathogens to cells, with 2.5-dimethoxy-p-cymene as the main component of EO extracted from leaves [46]. The study on the use of 2.5-dimethoxy-p-cymene has demonstrated that this molecule isolated from A. triplinervis EO is a potent inhibitor of ZIKV infection in human cells. Moreover, assessment of the biosafety of 2.5-dimethoxy-p-cymene extracted from A. triplinervis has shown that injection of this molecule in zebrafish does not lead to any signs of stress and does not affect fish survival, demonstrating the absence of acute toxicity of this compound [46]. However, different results were obtained by Unnikrishnan et al. [45]. Namely, the essential oils from Eupatorium triplinerve stems and leaves were found to have a significant antimicrobial effect, compared to that of 2.5-dimethoxy-p-cymene. Hence, the researchers recapitulated that the antimicrobial effect of the EO could be attributed to its minor components [45]. In turn, thymol methyl ether is the major constituent of EO of Crithmum maritimum [69], exhibits considerable activity mostly against Gram+ bacteria [70], and is one of the main ingredients of EO in the aerial parts of Baccharis grisebachii characterized by antimicrobial properties [71]. This molecule is also one of the major constituents of EO isolated from aerial parts of several Thymus species [72,73]. EO of T. numidicus is characterized by insecticidal properties [73], whereas EO isolated from aerial parts of T. fontanesii showed strong in vitro growth inhibition activity against Gram-bacteria and antifungal activity [72].
Plant-derived compounds have a high impact as therapeutic agents, both alone and in combination with conventional drugs [74]. The literature shows many examples of the potential synergistic, additive, or even antagonistic biological effects of combinations of individual EO compounds applied at different concentrations [74][75][76]. Generally, the biological activity of an EO is related to its chemical composition and the major functional groups of compounds, and the components at high concentrations play a major role in the biological effect of EOs. However, less abundant compounds may also be important, as various molecules can act synergistically with the major compounds [77]. Our results showed no negative effect of EOs on the normal cell line ( Table 2). This is very important in the context of our further studies of the anticancer and antiglioma activity of EORH and EORO. Therefore, in the continuation of the studies of the antiglioma activity, not only the dominant EO ingredient 2.5-dimethoxy-p-cymene but also less abundant compounds (alone and in combination) will be taken into account. The mechanisms of action of these main bioactive phytochemical compounds contained in the studied EO (2,5-dimethoxy-p-cymene, thymol methyl ether, p-methoxyheptanophenone, 2,6-diisopropylanisole) are unknown, similarly as the metabolic pathways responsible for biosynthesis of these molecules.
It is obvious that experiment conducted on pure secondary metabolites will bring more valuable and precise information about anticancer activity of studied extracts. Therefore, more detailed experiments toward estimating the precise molecular mechanism leading to cell death upon EOs and purified compounds will be continued in the near future. Given our previous experience in studying isolated flavonoids (quercetin) and coumarins (osthole, imperatorin), the research will be continued in accordance to anti-migratory potential, the level of typical for cell death marker proteins and related genes expression. We will also try to combine isolated compounds with cytostatic drugs used in anticancer therapy to assess their usefulness in combination therapy. Because such experiments were not undertaken yet, we hope to receive some new and valuable information with practical potential. Therefore, further studies on the anticancer effect of Arnica EOs and the main EO components (individually and in combination) against deadly gliomas will be an attempt at elucidation of the molecular anticancer mechanisms of this interesting constituent of A. montana EO.

Collection of Raw Material
Mountain Arnica montana subsp. montana plants were harvested at the end of the third and fourth vegetation periods from two experimental fields (3-year-old and 4-year-old plantation) at the University of Life Sciences in Lublin, eastern Poland. Standard fertilization was used: nitrogen (40.0 kg ha −1 ), phosphorus (24.0 kg ha −1 ), and potassium (66.4 kg ha −1 ) were applied every year. After harvesting randomly selected 3-year-old and 4-year-old Arnica plants, their underground parts were carefully washed with tap water and finally with deionized water. After washing, the rhizomes and roots were separated from underground organs and subsequently air-dried and crushed.

Assay of the Essential Oil Content
Twenty grams of crushed rhizomes and roots (mean particle size-0.5 mm) of mountain Arnica were submitted to water-distillation in a Deryng apparatus with 500 mL water for 3 h according to the Polish Pharmacopoeia VI [78]. The method of indirect distillation was applied. The essential oils were collected over water, separated, dried over anhydrous sodium sulphate, and stored in the dark at 4 • C prior to GC-MS analysis. The analysis was carried out in four repetitions. The moisture content of the mountain Arnica rhizomes and roots (9.62%) was determined by heating 10 g of the raw material at a temperature of 105 • C in an air oven until three consecutive constant weights were recorded using an electronic balance measuring with an accuracy of 0.001 g. The essential oil content was calculated to absolute dry weight.

GC-MS Analysis
The chromatographic analysis was performed according to procedures described previously [9,21]. The analysis was performed in triplicate. The essential oils were analyzed using a Varian 4000 GC-MS/MS system (Varian, Palo Alto, California, CA, USA). The compounds were separated on a 30 m × 0.25 mm × 0.25 µm VF-5 ms column (Varian, Palo Alto, California, CA, USA). The column temperature was increased from 50 to 250 • C at a rate of 4 • C/min; injector temperature 250 • C; split ratio 1:50; injection volume 5 µL. The MS parameters were as follows: EI mode, with ionization voltage 70 eV, ion source temperature, 200 • C; scan range, 40-870 Da.

Qualitative and Quantitative Analysis
The qualitative analysis was carried out on the basis of MS spectra, which were compared with the spectra from the NIST library [79] and with data available in the literature [43,80]. The identity of the compounds was confirmed by their retention indices [42] taken from the literature [43,80] and our data for standards described previously [9,21]. The quantitative analysis was performed with the internal standard addition method (alkanes C 12 and C 19 ) according to procedures described previously [81]. . The cultures were kept at 37 • C in a humidified atmosphere of 95% air and 5% CO 2 . Primary cultures of human skin fibroblast were prepared according to a method described previously [82].

Neutral Red Staining
To confirm the ID50 values (µL/mL) obtained from the microscopic observation, a colorimetric assay with the Neutral Red Assay Kit-Cell Viability/Cytotoxicity (ab234039, Abcam) was used. The principle of this assay is based on the detection of viable cells via the uptake of the neutral red dye. Viable cells can take up neutral red via active transport, whereas non-viable cells cannot take up this chromophore. The neutral red uptake assay was performed according to manufacturer's protocol. OD 540 nm was measured with a BioTek 800 TS microplate reader. The experiment was performed in triplicate.

Statistical Analysis
One-way and multi-way analysis of variance (ANOVA) and subsequent Tukey's tests were used. The differences were considered significant at p < 0.05. The statistical analyses were carried out using the Statistica 6.0 software (Stat. Soft, Inc., Kraków, Polska). Principal component analysis (PCA) was applied to explain the relationships between oil components and to show variability factors. Prior to the PCA, the data on all 37 components of EO were centered. The analyses were carried out using the statistical package (MVSP) program version 3.1. [83].

Conclusions
Among the 37 components of the essential oil of A. montana, 2,5-dimethoxy-p-cymene (46.47%, 56.42%, 60.31%, 60.23%), 2,6-diisopropylanisole (14.48%, 19.78%, 17.41%, 23.10%), thymol methyl ether (17.79%, 9.18%, 5.31%, 3.87%), p-methoxyheptanophenone (9.65%, 6.19%, 7.02%, 5.07%), and α-isocomene (1.05%, 0.68%, 2.87%, 1.36%), were detected in the rhizomes and roots of the 3-year-old plants and in the rhizomes and roots of the 4-year-old plants. Their quality and chemical composition are different from those of essential oils from flower heads, achenes, and rhizomes and roots of Arnica populations in different regions of Europe. Essential oils exert an anticancer effect by induction of anaplastic astrocytoma and glioblastoma multiforme cell death. Given the activity expressed by induction of apoptosis at a level of 28.5%-32.3%, the substance can be used in subsequent studies focused on the development of glioma therapy in the future. The plant part (rhizome, root) and plant age can be determinants of the concentration and essential oil composition and, consequently, their biological activity. The knowledge and information obtained in this study indicate a need for further research on the anticancer effect of EO components (individually and in combination) on the T98G and MOGGCCM cell lines and elucidation of the molecular anticancer mechanisms of this interesting constituent of A. montana EOs constituting a potential source of natural anticancer compounds.