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Article

Stages of Development and Solvents Determine the Anticancer Potential of Mountain Arnica (Arnica montana L.) Inflorescence Extracts

by
Piotr Sugier
1,*,
Joanna Jakubowicz-Gil
2,
Danuta Sugier
3,
Łukasz Sęczyk
3,
Adrian Zając
2,
Mateusz Pięt
4 and
Roman Paduch
4,5
1
Department of Botany, Mycology and Ecology, Institute of Biological Sciences, Maria Curie-Skłodowska University, 19 Akademicka Street, 20-033 Lublin, Poland
2
Department of Functional Anatomy and Cytobiology, Institute of Biological Sciences, Maria Curie-Skłodowska University, 19 Akademicka Street, 20-033 Lublin, Poland
3
Department of Industrial and Medicinal Plants, University of Life Sciences in Lublin, 15 Akademicka Street, 20-950 Lublin, Poland
4
Department of Virology and Immunology, Institute of Biological Sciences, 19 Akademicka Street, 20-033 Lublin, Poland
5
Department of General and Pediatric Ophthalmology, Medical University of Lublin, 1 Chmielna Street, 20-079 Lublin, Poland
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(24), 12976; https://doi.org/10.3390/app132412976
Submission received: 21 October 2023 / Revised: 26 November 2023 / Accepted: 1 December 2023 / Published: 5 December 2023
(This article belongs to the Special Issue Natural and Synthetic Pharmaceutical Ingredients: Physico-Chemical Characterization and Drug Development)
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Abstract

:
In recent years, new sources of secondary metabolites (SMs) in medicinal plants have been identified, and the introduction of these plants into field conditions has been carried out to obtain chemically diverse standardized raw material (RM). An example is mountain arnica Arnica montana L., one of Europe’s endemic endangered medicinal plant species, commonly used in pharmacy, cosmetics, and medicine. Its inflorescences (Arnicae flos) are characterized by anti-inflammatory, antiradical, antioxidant, antibacterial, antifungal, and antitumor properties. The main goals of the present research included: (i) characterization of the chemical composition of the inflorescences of A. montana harvested in different development stages; and (ii) presentation of the role of the development stage and different extraction methods in the antitumor activity of extracts through analyses of apoptosis, autophagy, and necrosis induction in human cervical carcinoma HeLa, human colon carcinoma HT29, and human colon metastatic carcinoma SW620 cell lines. The development stage was found to modify the composition of pharmacologically active substances, e.g., sesquiterpene lactones (SLs), flavonoids (Fs), and essential oil (EO), in arnica inflorescences. The content of Fs and EO increased during flowering to the full flowering phase; however, the highest content of SLs was noted in the full flowering phase and at the end of flowering. More promising results, i.e., a relatively high level of apoptosis and a low level of necrosis induced by the arnica extracts, were demonstrated in the HeLa cell line (full flowering; concentration: 0.5 µL/mL), the HT29 cell line (beginning of flowering; concentration: 0.5 µL/mL), and the SW620 cell line (stage of yellow buds; concentration: 1 µL/mL). This extremely valuable medicinal plant species provides a very broad range of RMs (e.g., inflorescences, rhizomes, roots, achenes, and all plant); therefore, attention should be paid to the more frequent use of water as a solvent in studies on the biological activity of mountain arnica extracts.

1. Introduction

As a source of extremely interesting secondary metabolites (SMs) characterized by antioxidant and anticancer activity, medicinal plants are of great interest to researchers around the world [1,2,3,4]. In recent years, new sources of SM in medicinal plants have been identified and investigated [5,6], and the introduction of these plants to cultivation has been carried out to obtain standardized raw material (RM) [7,8,9]. An example is mountain arnica Arnica montana L. from the Asteraceae family, which has recently gained great levels of interest from researchers [10,11,12]. It is worth noting that mountain arnica, which has been considered a valuable source of RM for the industry for many decades, is also a protected species in Europe, and its populations are endangered [13]. A. montana is an endemic mountain plant species in Europe [14,15,16]. Therefore, the introduction of this plant to field cultivation primarily reduces the pressure of the acquisition of RM from natural habitats, and, secondly, facilitates the application of cultivation factors that contribute to the generation of high yields of active substances and the production of more chemically diverse herbal RM [7,8,9].
The yield of SMs in the RM of cultivated medicinal plants, their composition, and their chemical differentiation depend on factors such as soil quality [7,9], soil and foliar fertilization [3,6,17,18], seasonal variations [19,20], plant density [2], and weather conditions [21,22]. In addition to environmental factors that determine the chemical profiles of medicinal plants, other important factors include the propagule type during introduction [9], the stage of development [19], morphological features, and plant organs [23,24]. In this way, the quantity and quality of the RM can be modified based on the knowledge of the biology and ecology of the species and with the use of agricultural tools. In the case of Arnica spp., different plant parts can be used, i.e., flower heads, rhizomes, or all underground parts of plants characterized by varied contents and different chemical compositions of such SMs as total phenolics, flavonoids (Fs), phenolic acids, sesquiterpene lactones (SLs), and essential oils (EOs) [23,24] with their broad biological activity [4,11,25,26,27,28]. Analyses of individual parts of the plant revealed the highest antiradical activity of A. montana flower head tincture. Flower heads, herbs, and rhizomes exhibited high chelating power and their ability to inhibit lipid peroxidation. Essential oils from A. montana rhizomes, roots, and achenes possess anticancer activity [23,24]. Other factors include the plant’s age, harvesting term, and stage of development, especially in the case of flowers or flower heads harvested as a raw material [9]. Although many studies have been conducted to characterize mountain arnica raw material, the knowledge regarding the use of water as a solvent or biological feature–solvent interactions are still insufficient [7,28,29,30,31]. Therefore, the results presented in this paper fill this gap, and are a subsequent stage towards determining the potential of this species in the study of biological properties and the practical use of this knowledge.
There are available results presenting the impact of nitrogen fertilization on A. montana extracts and their antioxidant and anticancer activity; however, the stages of development as a factor modifying the quality of extracts and antitumor activity have not been investigated so far. The present study is the first report on the anticancer activity of A. montana water and ethanol extracts, with emphasis on the role of the development stage. The use of various extractants can reveal the additional anticancer potential of arnica inflorescences. Such an approach, namely the use of new extraction methods or the simultaneous use of different extraction methods, has indicated greater opportunities for the evaluation of the quality of extracts and their various antioxidant and biological activities [3,4,32]. The RM collected in different stages of flowering and the use of different extractants can reveal the antitumor potential of mountain arnica inflorescences. Therefore, the objective of the present study was: (i) to characterize the chemical composition of A. montana inflorescences as pharmacopeial material obtained in different plant development stages; and (ii) to show the influence of the development stages and solvent types on the anticancer activity of arnica extracts.

2. Materials and Methods

2.1. Study Site

The field studies were conducted in a plantation site in Eastern Poland (N 51°31′25″; E 22°45′04″). All details of the field experiment were described in a previous publication [22]. The following stages of development constituted the first main experimental factor: YB—yellow bud; BF—beginning of flowering; FF—full flowering; and EF—end of flowering. After each harvest, the fresh weight of the inflorescence was determined. Next, the RM was dried in a drying room at 40 °C. After drying, the samples were weighed to determine the dry mass (DM) of the RM; next, chemical analyses were performed.

2.2. Chemical Analyses

2.2.1. Sesquiterpene Lactones

Sesquiterpene lactones (SLs) were determined using the chromatographic method specified in the Polish Pharmacopoeia VIII [33]. A total of 1 g of dried and milled Arnicae anthodium raw material was weighed on a laboratory scale and transferred to a 250 mL round-bottom flask. Subsequently, 50 mL of a methanol and water mixture in a 1:1 ratio was added. The contents of the flask were heated under a reflux in a water bath at 50–60 °C for 30 min with frequent shaking. A cotton wool pad was used to filter the mixture into a 500 mL round-bottom flask. Next, the filter residue and cotton wool pad were flooded with a methanol–water mixture and heated under the same conditions for 30 min. The reaction mixture was filtered into a 500 mL round-bottom flask, and both filtrates were combined. The extract was supplemented with an internal standard solution (3 mL) and evaporated to an approx. 18 mL volume under reduced pressure. A methanolic solution of α-santonin (over 99% purity; Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 0.001 g/mL was used as an internal standard. The concentrated extract was placed on a chromatographic column packed with approx. 15 g of diatomaceous earth (Sigma-Aldrich, St. Louis, MO, USA). The solution was left on the column for approx. 20 min. Next, sesquiterpene lactones were eluted from 200 mL of a mixture of ethyl acetate and methylene chloride (equal volumes). The eluate was transferred into a 500 mL round-bottom flask and evaporated to dryness under reduced pressure using a vacuum evaporator. The dry residue was dissolved in 3 mL of 50% HPLC-grade methanol, and the solution was purified on a Captiva column filled with 0.2 μm grain-size polypropylene under reduced pressure. Subsequently, the sample was employed for HPLC analysis performed on a Varian ProStar apparatus equipped with a UV–Vis detector model 210. The results were presented as the % of the sum of sesquiterpene lactones expressed as dihydrohelenalin tiglinate equivalents.

2.2.2. Flavonoids

The spectrophotometric method described by the Polish Pharmacopoeia VIII was used for the determination of the flavonoid content [33]. A total of 0.5 g of ground raw material was placed in a 250 mL round-bottom flask. Next, 20 mL of acetone, 2 mL of 25% hydrochloric acid, and 1 mL of a 0.5% urotropine (methenamine) solution were added, and the mixture was kept under a reflux in a MML 547 water bath (AJL Electronic, Gloucester, UK) for 30 min. The hydrolyzate was filtered through a cotton wool filter into a 100 mL volumetric flask. The precipitate and cotton wool were transferred into a 250 mL round-bottom flask, flooded with 20 mL of acetone, and boiled again for 10 min. The extract was filtered into the same volumetric flask and supplemented with acetone to the mark. Next, 20 mL of the acetone extract was placed in a pear-shaped funnel, following which 15 mL of distilled water was added, and the mixture was shaken. Subsequently, ethyl acetate (15 and 10 mL portions) was added with shaking each time. Following the separation of the water–acetone and acetate layers, the lower layer was poured out, and the upper (acetate) layer was collected for further analysis. The acetate layer was placed in a 50 mL volumetric flask and supplemented with ethyl acetate to the mark. To prepare solutions for the analyses, 10 mL of the acetate extract was transferred into a 25 mL volumetric flask. A 1% aluminum chloride solution (2 mL) was added and supplemented with a mixture of methanol and acetic acid (ratio: 19:1) to the required volume. The comparative solution was prepared analogously but with no aluminum chloride solution. The solutions were left to stand for 45 min, and their absorbance was measured at 425 nm on a HITACHI U-2900 spectrophotometer. The content of flavonoids (Fs) was expressed as quercetin equivalents.

2.2.3. Essential Oil

The content of essential oils (EOs) was determined using the hydrodistillation method proposed in the Polish Pharmacopoeia VIII [33]. All chemical analyses were carried out in triplicate.

2.3. Extraction Procedure

To study the anticancer activity, ethanolic and water extracts of A. montana inflorescences were prepared. Thus, 250 mg of plant material (dried inflorescences were ground to powder) was triple extracted using 5 mL of the solvent (99% ethanol or water) in a laboratory shaker for 30 min and then centrifuged.

2.4. Cells, Culture Conditions, and Drug Treatment

This study employed the following human carcinoma cell lines: cervical HeLa B (ECACC No. 85060701); colon metastatic SW620 (ATCC No. CCL-227); and colon HT29 (ATCC No. HTB-38). The cells were cultured in RPMI 1640 medium (GIBCO BRL, Washington, DC, USA) supplemented with 5% fetal bovine serum (FBS) (GIBCO BRL) (v/v) and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL, and amphotericin B 0.25 µg/mL) in 8-well Lab-Tek Chamber slides. Cells at a density of 1 × 106 cells/mL were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The cancer cells were exposed to the ethanolic and water extracts of A. montana (final concentrations of 1, 2, and 5 µL/mL) dissolved in the culture medium for 24 h.

2.5. Detection of Apoptosis, Autophagy, and Necrosis

Apoptosis, autophagy, and necrosis were identified through microscopic observations after staining with Hoechst 33342 (Sigma), acridine orange (Sigma), and propidium iodide (Sigma) fluorochromes, respectively, as described previously [34].

2.6. Statistical Analysis

After testing the data for normality and homoscedasticity, two-way analysis of variance (ANOVA) was carried out, followed by Tukey’s test. The results were presented as mean values and standard deviations, and the differences were considered significant at p < 0.05. The statistical analyses were performed using Statistica 6.0 software (Stat. Soft, Inc., Krakow, Poland). Principal component analysis (PCA) was performed to explain the relationships between the parameters and to determine variability factors. The data were centered and log-transformed prior to the PCA. The analyses were carried out in MVSP program version 3.1 [35].

3. Results

3.1. Chemical Characteristics of Arnicae flos

A significant impact of the development stages on the content of all the SMs analyzed in the study was clearly visible. The highest concentrations of flavonoids (Fs) (0.69%) and essential oils (EOs) (0.29%) were noted during the full flowering stage (FF), whereas their lowest levels were recorded at the end of flowering (EF) (Table 1). The content of sesquiterpene lactones (SLs) increased during flowering. The highest SL content was noted in the FF and EF phases and was similar—1.35% and 1.36%, respectively.
Figure 1 shows the PCA results prepared on the basis of the concentrations of SLs, Fs, and EOs in the A. montana inflorescences depending on the stages of development. The first two PCA axes explain 99.8% of the variability, and show the presence of two main gradients. Fs and EOs are positively correlated with axis 1, whereas SLs are positively correlated with axis 2 (Figure 1). The variation in chemistry was clearly determined via the stages of development. While the contents of Fs and EOs increased until the full flowering stage and then decreased, the SL content increased until the end of flowering.

3.2. Anticancer Activity

Our analyses demonstrate an influence of the extraction method, extract concentration (EC), and DS × C and S × C interactions on apoptosis and necrosis in the HeLa cell line (Table 2). In the case of the HT29 cell line, there was a significant impact of the stages of development, extraction method, EC, and DS × C and S × C interactions on apoptosis induction. A significant impact of all the factors and their interactions on cell apoptosis in the SW620 line was observed. A significant impact of all the factors on necrosis in all the cell lines was also observed. The analyzed extracts had no impact on the initiation of cell death in normal fibroblasts. What was also interesting was that the studied substances had no impact on autophagy initiation in all the studied cell lines; therefore, these data were not presented.
Our experiments demonstrated that the ethanol extract prepared from inflorescences in the stage of yellow buds (EYB) and the water extract prepared from inflorescences in the stage of yellow buds (WYB) added to the HeLa culture medium had a considerable impact on the induction of cell death (Figure 2). A significant increase in the number of apoptotic cells exposed to the former (8.3%) and latter (9.0%) extract was observed already at the EC of 0.5 µL/mL. The increase in the EC to 1 µL/mL caused a significant increase in the level of apoptosis to 27.0% in the EYB variant and to 17.3% in the WYB treatment, and contributed to a significant increase in the necrosis level to 52.7% and 19.7%, respectively.
The application of the ethanol extract prepared from inflorescences harvested before flowering (EBF), in full flowering (EFF), and at the end of flowering (EEF), and the water extract prepared from inflorescences collected before flowering (WBF) and at the end of flowering (WEF), to the HeLa cell line caused a similar response—an increase in the apoptosis level. At the EC of 0.5 µL/mL, the level of apoptosis varied between 6% and 7% in the ethanol extracts and between 6.3% and 13% in the water extracts, with a very low level of necrosis. The increase in the EC to 1 µL/mL significantly increased the level of apoptosis to 28.7–35.0% (ethanol extracts) and to 24.3–26.7% (water extracts). However, it should be emphasized that this was accompanied by a high level of necrosis: 48–52% in the ethanol extract variant, and 20–22.3% in the treatment with the water extracts. The impact of the extractant on the induction of HeLa cell death was only noted at the EC of 0.5 µL/mL of extracts from inflorescences collected in the FF phase. A statistically significantly higher level of apoptosis was noted after the addition of the water extract (13%) versus the ethanol extract (6%) to the HeLa culture medium (Figure 2).
The PCA ordination of the apoptosis and necrosis of the HeLa cell line induced by the ethanol and water extracts at the EC of 0.5 µL/mL and the chemical composition of A. montana raw material are presented in Figure 3. The first two axes explained 77.7% of the variability. Apoptosis, necrosis, Fs, and EOs were positively correlated with axis 1, whereas SLs were positively correlated with axis 2. Axis 1 showed an increase in the level of apoptosis and in the concentrations of Fs and EOs in the RM. Probably, the concentrations of these SMs determine the number of apoptotic HeLa cells. Simultaneously, on the right site of the ordination space, there is a clearly visible group of extracts characterized by the highest impact on the apoptosis of the HeLa cell line—WFF, WBF, EBF, and EFF. The mountain arnica extracts added to the HT29 cells had a visible impact on the induction of cell death (Figure 4). A considerable increase in the quantity of apoptosis was caused by EYB and WYB at the EC of 0.5 µL/mL. The increase in the EC resulted in a continued increase in the apoptosis to 9.0% and 20% in the EYB and WYB variants, respectively, and simultaneously initiated necrosis at a level of 11% and 8.7%, respectively.
The applications of EBF, WBF, EFF, and WFF to the HT29 culture medium also caused an increase in the apoptosis level. At the EC of 0.5 µL/mL, the level of apoptosis was between 8.7% and 13.0% in the ethanol extracts and between 8.7% and 14.3% in the water extracts, with a very low necrosis rate (<2% in the alcohol extracts and <1% in the water extracts). The increase in the EC to 1 µL/mL significantly increased the level of apoptosis to 17.7–19.0% (ethanol extracts) and to 23.0–26.0% (water extracts). However, it should be emphasized that this was accompanied by a high level of necrosis: 32.3–52.3% in the treatment with the ethanol extracts, and 27.0–35.0% in the water extract variant. In the case of the EEF and WEF extracts applied at the EC of 1 µL/mL, the level of apoptosis was 4.7% and 9.0%, respectively, at a low level of necrosis. In the other EEF and WEF treatments, the values of apoptosis and necrosis were low.
The impact of the solvent type on the induction of cell death was clearly visible. The level of apoptosis induced by WYB (20.0%) and WFF (26.0%) at the EC of 1 µL/mL was clearly higher than in the EYB (9.0%) and EFF (19.0%) variants. A significant 14.3% increase in the apoptosis of HT29 induced by WBF was registered at the EC of 0.5 µL/mL. This value was significantly higher than that obtained with the EBF variant (13.0%).
The results of the PCA analysis of the HT29 cell line (Figure 5) were similar to those revealed by the analysis of the HeLa cell line (the percentage of variability explained by axis 1 was 58.1 and by axis 2 was 26.4). Apoptosis, Fs, and EOs were positively correlated with axis 1, whereas SLs and necrosis were positively and negatively correlated with axis 2, respectively. Axis 1 showed an increase in the level of apoptosis and in the concentration of Fs and EOs in the RM. Probably, the concentrations of these SMs determine the number of apoptotic HT29 cells. Simultaneously, on the right site of the ordination space, there was a clearly visible group of extracts characterized by the highest impact on the apoptosis of the HT29 cell line—WBF, WFF, EBF, and EFF.
We observed a 17.7% increase in the level of apoptosis in SW620 under the impact of WYB at the EC of 1 µL/mL and at a low value of necrosis (1.2%) (Figure 6). A continued increase in the concentration of WYB resulted in a reduced level of apoptosis and an increase in necrosis. A similar reaction was registered following the applications of WBF, WFF, and WEF, namely a decrease or maintenance of a similar level of apoptosis, and a drastic increase in the level of necrosis at the EC of 2.5 µL/mL. The positive response of the SW620 cell line was only observed after the application of the water extracts. Under the impact of the ethanolic extracts, no apoptosis was observed in the majority of treatments, or the apoptosis rate was very low with a simultaneous maximum level of necrosis.
The results of the PCA analysis of the apoptosis and necrosis in the SW620 cell line exposed to the ethanol and water extracts at the EC of 1.0 µL/mL and the chemical characteristics of mountain arnica raw material are presented in Figure 7. The first two PCA axes explained 71.7% of the variability—43.6% and 28.1%, respectively. Necrosis was positively correlated with the first axis, apoptosis was positively correlated with the second axis, and Fs and EOs were positively correlated with both axes. In the upper area of the PCA, there was a group of extracts characterized by the highest impact on the apoptosis of the SW620 cell line—WYB, WBF, WFF, and EBF, and simultaneously extracts prepared from RM rich in Fs and EOs.

4. Discussion

4.1. Chemical Characteristics of Raw Material

The inflorescences of mountain arnica are a very rich source of SLs and contain higher amounts of this metabolite than the level of approximately 0.1% detected in whole A. montana plants [11]. The SL content was within the ranges presented by other studies: Aiello et al. [7]—from 0.45% to 1.51%, Dall’Acqua et al. [36]—from 0.54% to 1.50%, and Seemann et al. [37]—from 0.40% to 1.55%, and definitely higher than those reported by Douglas et al. [38]—from 0.66% to 0.94%. Compared to the standards (minimum: 0.40%) presented in the Polish Pharmacopoeia VIII, the contents of these compounds were two-fold higher in the RM collected in the yellow bud stage and over three-fold higher in the material harvested in full flowering and at the end of flowering [33]. Our previous research revealed that nitrogen fertilization was a significant factor increasing the SL content in inflorescences [3]. The stages of development clearly modified the concentration of SLs in the inflorescences (Table 1), and the highest SL content was observed under full flowering (1.35%) and at the end of flowering (1.36%). A similar response of different plant species was reported earlier [38]. In a study on the localization of sesquiterpene lactone biosynthesis in mountain arnica flowers, Parafiniuk et al. [12] showed that the lactone concentration increased from the top of the corolla to the ovary of single florets, with the pappus calyx as a source of their production. Therefore, the highest content of SLs is exhibited by A. montana inflorescences at the end of flowering and possibly by the achene pappus, where the compounds may serve as protection against diseases [39].
The stages of development also clearly influenced the content of flavonoids in the arnica inflorescences (Table 1). Their contents ranged from 0.43% DM at the end of flowering to 0.69% under full flowering, which confirms earlier findings. The data presented in the literature have also indicated that flavonoid compounds and SLs in Arnicae anthodium can be modified using different cultivation models [9] and plant development stages [38]. The present study has confirmed that the highest content of these metabolites is stored in inflorescences under the full flowering stage.
The EO quantity in RM is significantly dependent on the level of inflorescence maturity, along with nitrogen fertilization [3,38]. The present results showed that the stages of development significantly modified the EO content in arnica inflorescences as well. The EO concentration in the arnica inflorescences analyzed in our study ranged from 0.19% to 0.29% (Table 1). The content of this SM in the inflorescences was higher than that reported from Serbia [40] and Lithuania [41]. Analyses of its concentration in different plant parts showed higher EO content than in A. montana achenes [23], but lower than in underground parts of plants cultivated in Poland [24] and Serbia [42].

4.2. Anticancer Activity

The quantity and quality of the RM of medicinal plants can be modified based on the knowledge of the biology and ecology of the plant species and with the use of agricultural tools [7,8,9]. In field conditions, the chemical profile of Arnica spp. can be determined via the propagule type during introduction [9], soil quality [7,9], soil and foliar fertilization [3,18], morphological features, and plant organs [23,24], and may be responsible for the biological activity of plant material [23,24,25,26,27]. There are reports available in the literature on the apoptotic activity of mountain arnica extracts against melanoma WM-266–4 cells [4] and cytotoxic properties against colorectal cancer HCT-116 cells [43]. The present study has shown that the stage of plant development, the solvent type, and their interactions can modify the chemical composition of extracts and their antitumor activity against human carcinoma (HeLa), colon (HT29), and colon metastatic SW620 cell lines. Our earlier studies revealed that nitrogen fertilization, solvent type, and their interaction had an impact on apoptosis in the same cell lines, which confirms the relevance of research on the biological activity of extracts with the use of the abovementioned factors, including the more common use of water as an extractant [3]. The present research has indicated that the collection of flower heads during full flowering guarantees the production of RM, and their extracts have been characterized by the highest proapoptotic activity.
A. montana is a pharmacopeial plant species; therefore, extraction in the majority of studies is carried out with the method described in pharmacopeias [40], where ethanol is used as a solvent. Moreover, eluents such as 80% methanol [10,44], 41% ethanol [34], and 95% ethanol [45] can be used during extraction. Recently, Žitek et al. [4] conducted research to achieve a high yield and content of active ingredients via supercritical CO2 extraction. However, the use of water as an extractant has been investigated sporadically [3]. Hence, there is poor information of the biological activity of arnica water extracts and the importance of hydrophilic SMs. Moreover, the use of various eluents, including ethanol and water, gives the opportunity to demonstrate the proapoptotic activity of arnica extracts.
In the literature, it has been suggested that the plant species variety and extractant polarity affect the extractability of flavonoids or polyphenols [45,46]. In our study, the extracts were not comprehensively characterized chemically; hence, we are unable to explain the effect of particular molecules on the anticancer activity. Nevertheless, the strict dependence between the F content in the RM and the apoptosis level in all the studied lines suggested that these SMs may serve a crucial function in anticancer activity. Moreover, the highest impact of the extracts on apoptosis in the analyzed cell lines was visible when water was used as a solvent. It cannot be excluded that hydrophilic compounds, as in the case of other medicinal plants [32], may have an impact on biological activity. The A. montana inflorescence is characterized by high concentrations of quercetin, and the antitumor activity of this metabolite has been presented many times [47,48,49,50,51]. Therefore, the role of this component and of other hydrophilic constituents in the biological activity of aqueous extracts is very probable. However, this requires further research, including the identification of SMs in mountain arnica extracts and observations of the biological model’s response.

5. Conclusions

The development stage is a crucial factor that determines the composition of SMs in A. montana inflorescences and the antitumor activity of the analyzed extracts. The development stage can modify the composition of SMs, such as SLs, Fs, and EOs in arnica RM. The contents of Fs and EOs increased during flowering to the full flowering phase; however, the highest concentration of SLs was noted under full flowering and at the end of flowering. RM harvested in different development stages varies in their composition of SMs, and collecting this plant material at an appropriate stage of development can serve as a modifying factor of the chemical composition of arnica inflorescences. The development stage and different extraction methods had an impact on apoptosis and necrosis in the human cervical carcinoma HeLa, human colon carcinoma HT29, and human colon metastatic carcinoma SW620 cell lines. More promising results, i.e., a relatively high level of apoptosis and a low level of necrosis induced by the arnica extracts, were demonstrated in the HeLa cell line (full flowering; concentration: 0.5 µL/mL), the HT29 cell line (beginning of flowering; concentration: 0.5 µL/mL), and the SW620 cell line (stage of yellow buds; concentration: 1 µL/mL). The present study is the first report on the anticancer activity of A. montana water and ethanol extracts, with emphasis on the role of the development stage. Nevertheless, further studies are required to explore their potential for future medicinal purposes. In further studies, other factors and their interactions modifying the quality of Arnicae flos should be analyzed, such as interactions of abiotic and biological factors increasing the SM yield and modification of the chemical composition of RM and, consequently, the biological activity of extracts. From the point of view of anticancer activity, water was a more effective solvent than ethanol. A. montana provides a very broad range of RM (e.g., inflorescences, rhizomes, roots, achenes, and all plant); therefore, attention should be paid to the water as an eluent in research on the biological activity of mountain arnica.

Author Contributions

Conceptualization, P.S., D.S. and J.J.-G.; methodology, P.S., J.J.-G., D.S., Ł.S., A.Z., M.P. and R.P.; formal analysis, P.S., D.S., J.J.-G.; investigation, D.S., P.S., J.J.-G., A.Z., M.P. and R.P.; resources, P.S., D.S. and J.J.-G.; data curation, D.S., P.S. and J.J.-G.; writing—original draft preparation, D.S., P.S., J.J.-G., A.Z., M.P., R.P. and Ł.S.; writing—review and editing, D.S., P.S., J.J.-G., A.Z., M.P., R.P. and Ł.S.; visualization, P.S., J.J.-G. and D.S. All authors have read and agreed to the published version of the manuscript.

Funding

The research was financially supported by the Polish Ministry of Science and Higher Education.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to copyright protection.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Applsci 13 12976 g001
Figure 1. PCA analysis of the content of SMs in A. montana inflorescences. SLs—sesquiterpene lactones; Fs—flavonoids; and EO—essential oil. YB—stage of yellow buds; BF—beginning of flowering; FF—full flowering; and EF—end of flowering.
Figure 1. PCA analysis of the content of SMs in A. montana inflorescences. SLs—sesquiterpene lactones; Fs—flavonoids; and EO—essential oil. YB—stage of yellow buds; BF—beginning of flowering; FF—full flowering; and EF—end of flowering.
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Figure 2. Apoptosis and necrosis induction in human cervical carcinoma HeLa cells treated with the ethanol extract (A) and water extract (B) (concentration: 0, 0.5, and 1 μL/mL) from the inflorescences of A. montana plants. Explanations have been detailed in Figure 1. Yellow color—apoptosis; orange color—necrosis. Explanations have been detailed in Figure 1. Values designated by different letters are significantly different (p < 0.05). *—statistically significant difference (p < 0.05) between values of apoptosis induced by ethanol and water extracts from plants in different development stages.
Figure 2. Apoptosis and necrosis induction in human cervical carcinoma HeLa cells treated with the ethanol extract (A) and water extract (B) (concentration: 0, 0.5, and 1 μL/mL) from the inflorescences of A. montana plants. Explanations have been detailed in Figure 1. Yellow color—apoptosis; orange color—necrosis. Explanations have been detailed in Figure 1. Values designated by different letters are significantly different (p < 0.05). *—statistically significant difference (p < 0.05) between values of apoptosis induced by ethanol and water extracts from plants in different development stages.
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Figure 3. PCA analysis of the apoptosis (%) and necrosis (%) of the HeLa cell line induced by the ethanol and water extracts at the concentration of 0.5 µL/mL, and the chemical characteristics of A. montana raw material. SLs—sesquiterpene lactones; Fs—flavonoids; EO—essential oil; EYB—ethanol extract prepared from inflorescences in the stage of yellow buds; EBF—ethanol extract prepared from inflorescences at the beginning of flowering; EFF—ethanol extract prepared from inflorescences in full flowering; EEF—ethanol extract prepared from inflorescences at the end of flowering; WYB—water extract prepared from inflorescences in the stage of yellow buds; WBF—water extract prepared from inflorescences at the beginning of flowering; WFF—water extract prepared from inflorescences in full flowering; and WEF—water extract prepared from inflorescences at the end of flowering.
Figure 3. PCA analysis of the apoptosis (%) and necrosis (%) of the HeLa cell line induced by the ethanol and water extracts at the concentration of 0.5 µL/mL, and the chemical characteristics of A. montana raw material. SLs—sesquiterpene lactones; Fs—flavonoids; EO—essential oil; EYB—ethanol extract prepared from inflorescences in the stage of yellow buds; EBF—ethanol extract prepared from inflorescences at the beginning of flowering; EFF—ethanol extract prepared from inflorescences in full flowering; EEF—ethanol extract prepared from inflorescences at the end of flowering; WYB—water extract prepared from inflorescences in the stage of yellow buds; WBF—water extract prepared from inflorescences at the beginning of flowering; WFF—water extract prepared from inflorescences in full flowering; and WEF—water extract prepared from inflorescences at the end of flowering.
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Figure 4. Apoptosis and necrosis induction in human colon carcinoma HT29 cells treated with the ethanol extract (A) and water extract (B) (concentration: 0, 0.5, and 1 μL/mL) from the inflorescences of A. montana plants. Explanations have been detailed in Figure 1. Yellow color—apoptosis; Orange color—necrosis. Values designated by different letters are significantly different (p < 0.05). *—statistically significant difference (p < 0.05) between values of apoptosis induced by ethanol and water extracts from plants in different development stages.
Figure 4. Apoptosis and necrosis induction in human colon carcinoma HT29 cells treated with the ethanol extract (A) and water extract (B) (concentration: 0, 0.5, and 1 μL/mL) from the inflorescences of A. montana plants. Explanations have been detailed in Figure 1. Yellow color—apoptosis; Orange color—necrosis. Values designated by different letters are significantly different (p < 0.05). *—statistically significant difference (p < 0.05) between values of apoptosis induced by ethanol and water extracts from plants in different development stages.
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Figure 5. PCA analysis of the apoptosis (%) and necrosis (%) of the HT29 cell line induced by the ethanol and water extracts at the concentration of 0.5 µL/mL, and the chemical characteristics of A. montana raw material. SLs—sesquiterpene lactones; Fs—flavonoids, EO—essential oil; EYB—ethanol extract prepared from inflorescences in the stage of yellow buds; EBF—ethanol extract prepared from inflorescences at the beginning of flowering; EFF—ethanol extract prepared from inflorescences in full flowering; EEF—ethanol extract prepared from inflorescences at the end of flowering; WYB—water extract prepared from inflorescences in the stage of yellow buds; WBF—water extract prepared from inflorescences at the beginning of flowering; WFF—water extract prepared from inflorescences in full flowering; and WEF—water extract prepared from inflorescences at the end of flowering.
Figure 5. PCA analysis of the apoptosis (%) and necrosis (%) of the HT29 cell line induced by the ethanol and water extracts at the concentration of 0.5 µL/mL, and the chemical characteristics of A. montana raw material. SLs—sesquiterpene lactones; Fs—flavonoids, EO—essential oil; EYB—ethanol extract prepared from inflorescences in the stage of yellow buds; EBF—ethanol extract prepared from inflorescences at the beginning of flowering; EFF—ethanol extract prepared from inflorescences in full flowering; EEF—ethanol extract prepared from inflorescences at the end of flowering; WYB—water extract prepared from inflorescences in the stage of yellow buds; WBF—water extract prepared from inflorescences at the beginning of flowering; WFF—water extract prepared from inflorescences in full flowering; and WEF—water extract prepared from inflorescences at the end of flowering.
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Figure 6. Apoptosis and necrosis induction in human colon metastatic carcinoma SW620 cells treated with the ethanol extract (A) and water extract (B) (concentration: 0, 1.0, and 2.5 μL/mL) from the inflorescences of A. montana plants. Explanations have been detailed in Figure 1. Yellow color—apoptosis; Orange color—necrosis. Values designated by different letters are significantly different (p < 0.05). *—statistically significant difference (p < 0.05) between values of apoptosis induced by the ethanol and water extracts from plants in different development stages.
Figure 6. Apoptosis and necrosis induction in human colon metastatic carcinoma SW620 cells treated with the ethanol extract (A) and water extract (B) (concentration: 0, 1.0, and 2.5 μL/mL) from the inflorescences of A. montana plants. Explanations have been detailed in Figure 1. Yellow color—apoptosis; Orange color—necrosis. Values designated by different letters are significantly different (p < 0.05). *—statistically significant difference (p < 0.05) between values of apoptosis induced by the ethanol and water extracts from plants in different development stages.
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Figure 7. PCA analysis of the apoptosis (%) and necrosis (%) of the SW620 cell line induced by the ethanol and water extracts at the concentration of 1 µL/mL, and the chemical characteristics of A. montana raw material. SLs—sesquiterpene lactones; Fs—flavonoids; EO—essential oil; EYB—ethanol extract prepared from inflorescences in the stage of yellow buds; EBF—ethanol extract prepared from inflorescences at the beginning of flowering; EFF—ethanol extract prepared from inflorescences in full flowering; EEF—ethanol extract prepared from inflorescences at the end of flowering; WYB—water extract prepared from inflorescences in the stage of yellow buds; WBF—water extract prepared from inflorescences at the beginning of flowering; WFF—water extract prepared from inflorescences in full flowering; and WEF—water extract prepared from inflorescences at the end of flowering.
Figure 7. PCA analysis of the apoptosis (%) and necrosis (%) of the SW620 cell line induced by the ethanol and water extracts at the concentration of 1 µL/mL, and the chemical characteristics of A. montana raw material. SLs—sesquiterpene lactones; Fs—flavonoids; EO—essential oil; EYB—ethanol extract prepared from inflorescences in the stage of yellow buds; EBF—ethanol extract prepared from inflorescences at the beginning of flowering; EFF—ethanol extract prepared from inflorescences in full flowering; EEF—ethanol extract prepared from inflorescences at the end of flowering; WYB—water extract prepared from inflorescences in the stage of yellow buds; WBF—water extract prepared from inflorescences at the beginning of flowering; WFF—water extract prepared from inflorescences in full flowering; and WEF—water extract prepared from inflorescences at the end of flowering.
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Table 1. Content of SLs, Fs, and EOs in A. montana inflorescences (% of DM) depending on the stages of development; values designated by different letters are significantly different (p < 0.05).
Table 1. Content of SLs, Fs, and EOs in A. montana inflorescences (% of DM) depending on the stages of development; values designated by different letters are significantly different (p < 0.05).
Stages of DevelopmentSLsFsEOs
Yellow buds1.036 a ± 0.0230.496 b ± 0.0080.256 b ± 0.003
Beginning of flowering1.225 b ± 0.0210.615 c ± 0.0050.288 c ± 0.002
Full flowering1.353 c ± 0.0190.689 d ± 0.0130.293 d ± 0.001
End of flowering1.362 c ± 0.0230.422 a ± 0.0060.194 a ± 0.001
Table 2. Effect of the main factors (development stage (DS), solvent (S), and concentration (C)) and their interactions on the levels of apoptosis (%) and necrosis (%). HeLa—cervical carcinoma; HT29—colon carcinoma; and SW620—colon metastatic carcinoma.
Table 2. Effect of the main factors (development stage (DS), solvent (S), and concentration (C)) and their interactions on the levels of apoptosis (%) and necrosis (%). HeLa—cervical carcinoma; HT29—colon carcinoma; and SW620—colon metastatic carcinoma.
Cell LineHeLaHT29SW620
ApoptosisNecrosisApoptosisNecrosisApoptosisNecrosis
DSF = 1.74F = 4.07F = 78.60F = 182.01F = 6.52F = 66.80
p = 0.171p < 0.05p < 0.001p < 0.001p < 0.001p < 0.001
SF = 4.83F = 106.91F = 38.97F = 18.98F = 107.43F = 1130.70
p < 0.05p < 0.001p < 0.001p < 0.001p < 0.001p < 0.001
CF = 404.93F = 763.89F = 403.73F = 816.05F = 25.86F = 1315.42
p < 0.001p < 0.001p < 0.001p < 0.001p < 0.001p < 0.001
DS × SF = 0.84F = 4.38F = 2.50F = 7.95F = 7.93F = 89.80
p = 0.479p < 0.01p = 0.071p < 0.001p < 0.001p < 0.001
DS × CF = 3.31F = 5.09F = 25.47F = 189.35F = 7.30F = 85.84
p < 0.01p < 0.001p < 0.001p < 0.001p < 0.001p < 0.001
S × CF = 14.65F = 110.20F = 22.57F = 20.84F = 27.34F = 299.36
p < 0.001p < 0.001p < 0.001p < 0.001p < 0.001p < 0.001
DS × S × CF = 0.70F = 3.13F = 1.04F = 7.36F = 8.34F = 71.19
p = 0.654p < 0.05p = 0.413p < 0.001p < 0.001p < 0.001
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Sugier, P.; Jakubowicz-Gil, J.; Sugier, D.; Sęczyk, Ł.; Zając, A.; Pięt, M.; Paduch, R. Stages of Development and Solvents Determine the Anticancer Potential of Mountain Arnica (Arnica montana L.) Inflorescence Extracts. Appl. Sci. 2023, 13, 12976. https://doi.org/10.3390/app132412976

AMA Style

Sugier P, Jakubowicz-Gil J, Sugier D, Sęczyk Ł, Zając A, Pięt M, Paduch R. Stages of Development and Solvents Determine the Anticancer Potential of Mountain Arnica (Arnica montana L.) Inflorescence Extracts. Applied Sciences. 2023; 13(24):12976. https://doi.org/10.3390/app132412976

Chicago/Turabian Style

Sugier, Piotr, Joanna Jakubowicz-Gil, Danuta Sugier, Łukasz Sęczyk, Adrian Zając, Mateusz Pięt, and Roman Paduch. 2023. "Stages of Development and Solvents Determine the Anticancer Potential of Mountain Arnica (Arnica montana L.) Inflorescence Extracts" Applied Sciences 13, no. 24: 12976. https://doi.org/10.3390/app132412976

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