Qualitative and quantitative analyses of bioactive compounds from ex vitro Chamaenerion angustifolium (L.) (Epilobium augustifolium) herb in different harvest times

Industrial Crops & Products 123 (2018) 208–220

Contents lists available at ScienceDirect

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

Qualitative and quantitative analyses of bioactive compounds from ex vitro Chamaenerion angustifolium (L.) (Epilobium augustifolium) herb in different harvest times

T

⁎

Gryszczyńska Agnieszkaa, , Dreger Mariolaa, Piasecka Annab,c, Kachlicki Piotrb, Witaszak Nataliab, Sawikowska Anetab,d, Ożarowski Marcina,e, Opala Bognaa, Łowicki Zdzisława, Pietrowiak Aureliaa, Miklaś Magdalenaa, Mikołajczak Przemysław Łukasza,f, Wielgus Karolinaa a

Institute of Natural Fibres and Medicinal Plants, Wojska Polskiego 71b, 60-630 Poznań, Poland Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, Poznań, Poland Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, Poznań, Poland d Department of Mathematical and Statistical Methods, Poznań University of Life Sciences, Wojska Polskiego 28, Poznań, Poland e Department of Pharmaceutical Botany and Plant Biotechnology, Poznań University of Medical Sciences, Św. Marii Magdaleny 14, 61-861 Poznań, Poland f Department of Pharmacology, Poznań University of Medical Sciences, Rokietnicka, 5a, 60-806 Poznań, Poland b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Chamaenerion angustifolium (L.) HPLC-DAD-MSn UPLC-PDA-MS/MS UPLC-MS/MS Flavonoids Polyphenolic acids

The rosebay willowherb (Chamaenerion angustifolium (L.) Scop. syn. Epilobium angustifolium L.) from Onagraceae family is a valuable medicinal plant. The aim of our research was to measure the effect of different harvest times on the profile and concentration of oenothein B, polyphenols, flavonoids and sterols after ex vitro Chamaenerion angustifolium multiplication. For this purpose, we compared phytochemical properties of the herb in two ex vitro lines: German (DE) and Polish (PL), collected in two different harvest times: during and after the flowering period – in June and in September respectively. The qualitative and quantitative analyses were conducted using advanced LC–MS systems, including: HPLC-DAD-MSn, UPLC-PDA-MS/MS, HPLC-DAD, UPLC–MS/MS and the spectrophotometric methods. In the course of the qualitative analysis of herb samples, 45 phenolic metabolites were identified, from which gallic acid, oenothein B and quercetin 3-O-arabinoside were the principal compounds. In both lines, the quantitative determination with the use of two chromatographic methods showed the highest concentration of oenothein B, β−sitosterol and ellagic acid, whereas in the analysis with the spectrophotometric method, it was the total polyphenols content that predominated (expressed as gallic acid). In most cases, the flowering period was a better source of active compounds. The chemical characteristics of ex vitro raw material is similar to the field crop plants profile, confirming the positive application of the ex vitro cultivation method.

1. Introduction Epilobium augustifolium (rosebay willowherb) is one of the Onagraceae family (Stolarczyk et al., 2013). In recent years this species is also named Chamaenerion angustifolium (L.). The plant is very popular all over the world and it is one of the most common among over 200 species (Schepetkin et al., 2009). There are 26 species in Europe (Granica et al., 2014), including 14 species in Poland (Kujawski et al., 2010a). Epilobium can be split into two groups depending on different size of flowers: the small ones (E. parviflorum, E. montanum and E. roseum) and the large ones (E. angustifolium and E. hirsutum) (Stolarczyk et al., 2013). The healing properties of this plant are used in traditional

⁎

Corresponding author. E-mail address: [email protected] (G. Agnieszka).

https://doi.org/10.1016/j.indcrop.2018.06.010 Received 11 February 2018; Received in revised form 31 May 2018; Accepted 4 June 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

medicine, for example in China and America (Stolarczyk et al., 2013). Willowherb is characterized by photoprotective, anti-inflammatory and antitumoral effects according to the in vitro studies (Baert et al., 2015). There are many papers describing the influence of Epilobium extracts on the hormone-dependent prostate cancer cell apoptosis (Stolarczyk et al., 2013), on the estrogen receptor alpha and beta expression in the in vivo model (Kujawski et al., 2010b), as well as on the activity in PC-3 cells (Kiss et al., 2006). The Epilobium herb is a rich source of bioactive compounds which belong to the polyphenolic and flavonoid groups (Bazylko et al., 2007). The herb also contains steroids, teriterpens and fatty acids (Granica et al., 2014). These include the following compounds: kaemperol,

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

clear identification as PL (Institute of Natural Fibres & Medicinal Plants) and DE (Rieger-Hofmann® GmbH). The seeds were surfacesterilized by soaking in 70% ethanol (1 min) and then in ACE® solution (2:1) with a drop of Tween 20 (3–4 min), and afterwards they were washed three times in sterile water. The seeds were germinated on solid MS medium (Murashige and Skoog, 1962) without PGR (Plant Growth Regulators). After five weeks, the seedlings were cut for root explants (1.0 cm) and transferred to the induction medium according to Turker (Turker et al., 2008) with slight modifications: MS medium was supplemented with BAP (0.1 mg/l), IAA (0.5 mg/l), vitamin C (0.1 g/l) and casein hydrolysate (0.5 g/l), sucrose (30.0 g/l) and agar (8.0 g/l). Multishoots were regularly subcultured on the fresh induction medium every four weeks. The regenerated shoots were individually separated and transferred into Magenta vessels (300 ml, Sigma Aldrich) with rooting medium: ½ full strength MS medium with IAA (0.5 mg/l) and vitamin C (0.1 g/l). All cultures were incubated at a temperature of 23 °C ( ± 2 °C) under 16/8 h photoperiod (cool-white fluorescent lights 30 μmol m−2s−1). The pH media were adjusted to 5.7 before autoclaving (0.1 MPa, 121 °C, 20 min). The rooted plants were transferred into the soil substrate and perlite (Kekkila Paperpot) and acclimatized in closed tunnels in the greenhouse conditions (21 °C) for two weeks. The plants were hardening in the open tunnels for two weeks in a temperature of 16 °C, and for another two weeks in the field conditions. The plant material (underground parts), derived from PL and DE marked plants, was collected and dried at 35 °C – 40 °C, separately in June and September 2015. Voucher specimens were identified by the authors, checked by dr Artur Adamczak (Institute Natural Fibres and Medicinal Plants, Poznań, Poland) and deposited in the Institute Natural Fibres and Medicinal Plants (Poznań, Poland).

quercetin, myricetin and their monoglycosidic derivatives. Moreover, there are: ellagic acid, valoneic acid dilactone, chlorogenic acid, gallic acid, protocatechuic acid, cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid (Granica et al., 2014). The presence of flavonoids, especially the quercetin glucuronide, which is a marking substance for Epilobium augustifolium (Bazylko et al., 2007), differs from other Epilobium species, in which myrcetin rhamnoside is the main flavonoid compound (Kiss et al., 2011). One of the most characteristic compounds of willowherb is oenothein B, which belongs to tannins (Baert et al., 2015), and more specifically to the macrocyclic ellagitannin with a dimeric structure (Ramstead et al., 2012). There are some opinions that this compound is responsible for the activity of Epilobium augustifolium extracts (Granica et al., 2014; Bazylko et al., 2007). Some studies have shown that oenothein B decreases the growth of several tumors in vivo and produces the macrophages activation (Ramstead et al., 2012; Miyamoto et al., 1993). This compound can activate monocytes/macrophages and neutrophils causing an increase of intercellular Ca+2 flow (Schepetkin et al., 2009). Many researchers have conducted numerous experiments on other properties of oenothein B, which are juxtaposed by Granica et al. They mention, for example, in vivo and in vitro experiments in which anti-HIV, anti-inflammatory, anti-prostate hyperplasia and immunomodulatory activities of oenothein B have been tested (Granica et al., 2014). The consumption of medicinal plants and the demand for raw material is growing throughout the world. More than 50% of plants are harvested from the wild (Traffic International, Therapy for Medicinal Plants, 2015). Unfortunately, it involves some problems, such as: misidentification of botanical origin, variable content of active compounds, or the presence of toxins and contaminants. On the other hand, the cultivation under controlled conditions can improve the yield of active compounds and the production stability, providing high quality and homogenous raw material. In vitro cultures and micropropagation techniques offer the opportunity to significantly shorten the process of reproduction, and allow us to achieve a large scale multiplication of clonally identical plants. Micropropagation is defined as aseptic asexual plant propagation under controlled conditions of light, nutrients and temperature (Máthé et al., 2015). It gives many unique advantages over conventional propagation methods, such as: high efficiency, rapid multiplication of valuable genotypes, production of disease-free plants. Moreover, there is lack of environmental restrictions (seasonal and climatic conditions, pathogen and pest expositions, etc.). The standardization and quality control of raw material is essential for therapeutic efficacy and a safe application of herbal drugs, as well as to fulfill the demand for high quality plant material by pharmaceutical companies, the phytochemical studies including a wide range of analyses are needed for a better assessment. The aim of our research was to measure the effect of different harvest times on the profile and concentration of oenothein B, polyphenols, flavonoids and sterols after ex vitro Chamaenerion angustifolium multiplication. A detailed description of the results concerning micropropagation was recently reported by Dreger et al. (Dreger et al., 2016). In this study, some modern analytical techniques, such as HPLC-DAD-MSn and UPLC-PDA-MS/MS, were used to verify the quality of raw material, whereas in order to perform the qualitative and quantitative analyses HPLC-DAD and UPLC-MS/MS methods were applied.

2.2. Qualitative and quantitative analyses 2.2.1. LC–MS qualitative procedure Approximately 100 mg of herb was placed in Eppendorf tubes with 2 ml of 80% methanol, next 5 μl of apigenin IS1 (c = 1.0 mg/ml, DMSO solution) was added. After that, the sample was homogenized using a ball mill (MM 400, Retsch, Germany), subsequently it was placed in an ultrasonic bath for 20 min and next centrifuged (11,000g) for 10 min. The resulting supernatant was passed through the 0.45 μm syringe filters GHP Acrodisc 13 (Waters, USA) and subjected to HPLC-MS analysis. Prior to the UPLC-MS analysis, 200 μl of the obtained sample had been diluted with 800 μl of 80% methanol. The HPLC and UPLC analyses were performed and the MSn (up to the MS5) and a high-resolution MS/MS spectra were recorded in the negative and the positive ion modes using the previously published approach (Ożarowski et al., 2017; Piasecka et al., 2017). The individual compounds were identified via comparison of the exact molecular masses, mass spectra and retention times to those of the standard compounds, databases available online (PubChem, ChEBI, Metlin and KNApSAck) and literature data (Baert et al., 2015; Karonen et al., 2010). Chromatographic data from the UPLC-MS/MS system were exported in ASCII format and served for mathematical baseline removal for the best visualization of chromatograms as it was described in details by Piasecka et al. Piasecka et al., 2017). The baselines from the raw chromatographic UPLC-UV data were removed by the rollingBall method using package baseline in R. The peaks were detected in individual chromatograms using profiles of the second derivative smoothed by a cubic smoothing spline with the smooth.spline function in R. Some detected individual peaks were considered to be irrelevant and were removed from the subsequent analysis (peaks characterized either by a length of less than 10 retention time points or by a low intensity value within the peak after two differentiations). A Venn diagram was implemented in R script using the venn function in the gplots package. The bar chart that shows the area of identified peaks was performed

2. Material and methods 2.1. Sample plant preparation procedures The C. angustifolium seeds were obtained from Rieger-Hofmann® GmbH (Germany) and from Garden of Medicinal Plants in Plewiska (Institute of Natural Fibres & Medicinal Plants, Poland), and they were used for induction of in vitro cultures and micropropagation. The seeds and seedlings were treated separately in the same way and marked for 209

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

using the ggplot function in the R ggplot2 package.

Table 1 Polyphenolic acids and flavonoids with the fragmentation of parent ions and obtained retention times.

2.3. Quantitative procedure 2.3.1. Analysis of sterols Approximately 1.0 g of herb was placed in a 25 ml flask, then 20 ml of methanol was added, and the mixture was extracted in an ultrasonic bath for 60 min. The sample was cooled down, filled with methanol up to 25 ml, and the solution was passed through the 0.45 μm filters. A chromatography separation was obtained on the Zorbax Eclipse XDB-C8 column, 150 mm × 4.6 mm × 5 μm (Agilent, USA). The mobile phase was as follows: water:H3PO4:tetrahydrofurane (80:15:8 V/V/V). The β-sitosterol, stigmosterol and campesterol were detected at λ = 208 nm. The peaks were identified comparing the retention time and UV-VIS spectra with those of the standard solution.

Compounds

MRM [Da]

Retention time [min]

quercetin-D3 (IS2) caffeic acid gallic acid p-coumaric acid ellagic acid rutin quercetin myricetin

303.9 179.0 168.9 162.9 300.9 609.0 300.9 316.9

3.05 2.60 1.00 2.90 2.80 2.90 3.05 3.00

> > > > > > > >

150.9 134.0 124.9 118.0 144.9 299.9 150.9 150.8

was 0.15 ml/min. The assay was performed in a gradient elution procedure: 0.0 min – 95% of phase B, 1 min – 95% of phase B, 1.5 min – 50% of phase B, 2.5 min – 25% of phase B, 4 min – 0% of phase B, 6 min – 0% of phase B, 9 min – 90% of phase B, 10 min – 95% of phase B. The column temperature was 25 °C; the ion source temperature was 100 °C; the desolvation temperature was 325 °C. The sample injection had 7 μl of volume, the desolvation gas flow rate was 500 l/h. The analysis was performed in negative ion charge, using multiple reaction monitoring (MRM) for qualitative and quantitative analyses. The quercetin-D3 was used as an internal standard (IS2) and the value of 303.9 > 150.9 Da was characteristic for its fragmentation. Table 1 shows the fragmentation and retention times for all the above-mentioned substances.

2.3.2. Analysis of oenothein B In this research we used the adapted extraction method of oenothein B, which was developed by Bazylko et al. (Bazylko et al., 2007). Approximately 0.5 g of herb was placed in a 100 ml volumetric flask, 70 ml of water was added to the sample and the mixture was extracted in an ultrasonic bath for 60 min in 40 °C. The sample was cooled down and 10.0 ml of acetonitrile was added. Later, the sample was filled up with water and the solution was passed through the 0.45 μm filters. The detection method of oenothein B was adapted to this study (Kiss et al., 2011). LiChrospher 100 RP-18e, 250 mm × 4 mm × 5 μm (Merck, Germany) was used as a stationary phase. The mixture of 2 mobile phases eluted the above compound: 2.5% CH3COOH (phase A) and 2.5% CH3COOH: acetonitrile (2:8 V/V) (phase B), which were used in the gradient separation procedure. The separation was performed in the following conditions: 0 min – 7% phase B, 30 min – 20% phase B, 60 min – 40% phase B. The column temperature was 25 °C, the flow rate was 1.0 ml/min, the detection was at λ = 263 nm. The peaks were identified comparing the retention time and UV–VIS spectra with those of the standard solution.

2.3.4. Total flavonoids The Polish Pharmacopoeia extraction method of total flavonoids determination was used to prepare the sample (Polish Pharmacopoeia VI, 2002). 2.3.5. Total polyphenols Approximately 100.0 mg of herb was placed in a round-bottomed flask and 10 ml of water was added. The sample was heated under the reflux condenser for 30 min. The solution was cooled down and filtrated through a filter into the 50 ml volumetric flask. The filter was returned into the round-bottomed flask and extracted with 10 ml of water. The solution was cooled down again, filtrated through the filter, and all combined filtrates were filled up to 50 ml with water. The test solution was prepared in a tube filled with 7.0 ml water, 0.5 ml Follins`s reagent and 0.5 ml of the sample. After 3 min, 2.0 ml of 20% sodium carbonate was added and the sample was placed in a 25 °C water bath for 1 h. Immediately after that, the absorbance of test solution was measured at λ = 760 nm for comparison with the compensation liquid. The compensation liquid was prepared in the same way but without the sample.

2.3.3. Analysis of flavonoids and polyphenolic acids with UPLC–MS/MS method 2.3.3.1. Sample preparation procedure – determination of flavonoids and ellagic acid. The extraction method developed by Hevesi Tóth et al. (Hevesi Tóth et al., 2009) was adapted in our laboratory and modified. Approximately 2.0 g of herb was placed in a 100 ml round-bottomed flask and extracted with 80% acetone under the reflux condenser for 2 h. After cooling down and filtrating, the solution was evaporated to dryness. The dry residue was dissolved with 20 ml of methanol. Next, 1.5 ml of methanolic solution was dissolved with 3.5 ml of 2.5% acetic acid, and 1.0 ml of this solution was applied on SPE column (C18, 500 mg, 3 ml), which had previously been activated with 3 ml of methanol and 3 ml of 2.5% acetic acid. The compounds were eluted with 2.5 ml of methanol: 2.5% acetic acid mixture (70:30, V/V) and 1.0 ml of methanol. Finally, 49 μl of IS2 (quercetin-D3 c = 0.103 mg/ ml) was added to the sample and the solution was filled up with methanol to a volume of 5.0 ml. The resulting solution was passed through the 0.2 μm filters.

2.3.6. Total tannins The European Pharmacopoeia extraction method of total tannins was used to prepare the sample (European Pharmacopoeia 8, 2013). 2.4. Standards solutions The internal standard of apigenine in qualitative analysis was prepared in dimethylsulfoxide and its concentration was c = 1.0 mg/ml. The calibration curves of oenothein B, kaemposterol, stigmosterol and β-sitosterol were prepared from the methanolic solution. Approximately 1.00 mg of the reference substance was weighed in a 10 ml volumetric flask, it was dissolved in 8.0 ml methanol and the solution was filled up to 10 ml with the same solvent (stock solution). Other concentrations of compounds were prepared by mixing the above (except oenothein B) and filling them up with methanol to 2.0 ml. The concentration range was 100–1000 ng/ml (n = 5). The concentration of all stock solutions used in the quantitative analysis of flavonoids and polyphenolic acids was c = 0.1 mg/ml. The exact amount 1.00 mg of the standard substance was weighed in a 10 ml

2.3.3.2. Sample preparation procedure – determination of caffeic, gallic and p-coumaric acids. Approximately 250.0 mg of herb was placed in a 10 ml volumetric flask and 9 ml of methanol was added, next the sample was sonificated for 30 min. After cooling down and adding IS2 (98 μl of 0.13 mg/ml), the sample was filled up with methanol to a volume of 10.0 ml and then filtrated through the 0.2 μm filters. The separation of flavonoids and polyphenolic acids (see: sample preparation 2.3.3.1 and 2.3.3.2) was performed on an Acquity UPLC BEH C18 column, 50 mm × 2.1 mm × 1.7 μm (Waters, USA). The mobile phases were as follows: acetonitrile: methanol 80:20 (V/V) (phase A), 0.1% (V/V) formic acid solution in water (phase B). The flow rate 210

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

volumetric flask and dissolved in 8.0 ml methanol, the solution was filled up to 10 ml with the same solvent (stock solution). Next, 1.0 ml of the stock solution was transferred to a 10 ml volumetric flask and filled up with methanol (working solution at concentration 0.01 mg/ml). The number of dilutions of each analyte was made in order to obtain a standard curve. The solution mixture was prepared by taking an adequate volume of the working solution and filling it up to 2.0 ml. In this way, the dilutions performed were in the range 100–1000 ng/ml (n = 5).

Thanks to a high-resolution MS, accurate mass measurements made it possible to distinguish isobaric flavonoids from the substituents with the same nominal masses. We confirmed that the losses of fragment 176.0321 from the [M−H]− ions of compounds 25, 30 and 38 referred to chemical formula C6H8O6 adequate to glucuronic acid, whereas in compound 45 the [M–176.0472]− ion referred to the loss of C10H8O3 adequate to ferulic acid (Fig. 2A and B). A similar calculation with the error lower than 2 ppm enabled us to differentiate rhamnose from pcoumaric acid (146.05814 for C6H10O4 and 146.03644 for C9H6O2, respectively) in compounds 39 and 47. Compounds 37 and 43 had glucose and caffeic acid in their structures, as a result both gave fragments with the same nominal masses (162 amu) in a low-resolution MS. The analysis in HR MS enabled us to differentiate both substituents by the exact mass measurement (162.05334 for C6H10O5 and 162.03156 for C9H6O3, respectively) (Fig. 2C). The MS2 spectra of compounds 37 and 43 revealed a creation of the [M - C9H6O3]− product ion corresponding to the loss of caffeic acid followed by the rupture of Ohexoside (fragment 162.0533 amu) that allowed us to annotate these compound as myricetin and quercetin caffeoylhexosides. To our knowledge, flavonols glycoconjugates acylated with hydroxycinnamic acids: p-coumaric, caffeic and ferulic acids were detected for the first time in the Epilobium species. The CID of the negative ions of compounds 16, 24, 25, 32, 33, 35, 37, 40 and 45 led to the formation of highly abundant [Aglycone – H]−% radical-ions resulting from the homolytic cleavage of the bond between glycan and aglycone (for a review of this phenomenon see (Kachlicki et al., 2016)) (Fig. 2C). The predominance of these ions has proved to be diagnostic for the presence of 3-O-glycosyl substitution, thus the mentioned flavonols were assigned as 3-O-glycoconjugates. On the other hand, compounds 21, 26, 27, 36, 38, 39, 42, 43 and 47 shared common fragmentation in the negative ionization leading to the formation of [Aglycone - H]−% ions, which indicated that most likely Oglycosylation occurred in position 7C of the respective aglycones. The presence of galloyl substituents in 7C of the aglycone was also confirmed by measuring the exact masses of metabolites using MS. This trihydroxybenzoic acid occurred in conjugates with sugars and phenolic acids (1, 2, 3, 5, 8, 10, 12, 18, 22 and 23). Thanks to the high-resolution MS, three isomers of digalloylglucose (3, 8 and 10) could be easily identified. Nevertheless, it was difficult to establish whether the acid and sugar were bound via glycosidic or ester bond because no additional fragments were observed (Parveen et al., 2013). Thus, the differentiation between ester and O-glycosidic bond needed further analysis in modified CID conditions (Wojakowska et al., 2013). For instance, the fragmentation of compounds 3, 8 and 10 led to [M152.01093]− and [M-170.02130]− ions corresponding to the losses of the first unit of the gallic acid either as C7H4O4 or C7H6O5 fragments, respectively. The subsequent detachment of 162.05411 amu (calculated for hexose) resulted in obtaining the main product ion at 169.01321 m/ z adequate for the second gallic acid. Thus, 3, 8 and 10 were annotated as digalloylglucose (A). In addition, two quercetin derivatives acylated with gallic acid (metabolites 26 and 34 were detected in Epilobium on the basis of HR MS for the first time (Fig. 3B). Similarly to flavonols acylated with hydroxycinnamic acid (37, 39, 43 and 47), first order fragmentation of 26 resulted in the main [M-152.01093]− product ion that indicated the galloyl substituent. The detachment of glucose from aglycone was observed in the next step of fragmentation. Mass spectra obtained in the MSn mode in both positive and negative ionization revealed minor ions that indicated the presence of galloyl-glucose structure in 26. In the positive ionization, the minor ion at m/z = 315 corresponded to the conjugated form of galloyglucose and the detachment of quercetin as a neutral fragment. Other minor ions at m/z = 345 and 411 corresponded to the loss of galloylglucose fragments. This fragmentation allowed the annotation of 26 as galloyglucoside of quercetin. Nevertheless, detailed structure elucidations require further thorough studies and can only be achieved by NMR analysis.

2.5. Statistical analysis The data were expressed as mean ± SD and the statistical comparison of results was carried out using ANOVA followed by the Tukeypost hoc test for independent samples. P values of 0.05 or less were considered as statistically significant. 3. Results and discussion In our research we compared 2 lines of in vitro plants, which were transferred to the soil and then collected in different growth phases. The qualitative and quantitative analyses of herbs helped us to choose the raw material with a higher concentration of bioactive compounds. We believe that the preparation of dry extracts from such material will allow the future development of new herbal products. The qualitative analysis was an important part of the experiment. Since the information describing chemical composition of ex vitro plants from C. augustifolium was not available, we performed the qualitative analyses in all sample lines. To recognize the compound composition of samples, the vast advanced research was carried out in Institute of Plant Genetics, Polish Academy of Sciences in Poznań, Poland. The qualitative analysis was mainly focused on the identification of glycosides presence in the herb. To our knowledge, so far not many studies on secondary metabolites of Epilobium species have been conducted using mass spectrometry, thus the herb from this plant material was subjected to a careful examination by two complementary LC–MS systems. The fragmentation schemes generated by the ion trap mass spectrometer served to identify particular substituents in these structures. The identification correctness was confirmed by a high-resolution mass spectrometer. Forty-seven phenolic metabolites were identified in the methanolic extracts of Epilobium plants. All phenolic and flavonoid compounds are shown in Table 2. The predominant identified compounds were bioactive esters of gallic acid, which had a maximum absorption around 270 nm, but glycosides of flavones were also detected, especially flavonols and hydroxycinnamic acid derivatives with a maximum absorption around 330 nm. Therefore, the annotation of metabolites in all samples required chromatographic analysis at both 270 nm and 330 nm (Fig. 1A and B). The chromatogram at 270 nm revealed that peak 17 had two apices, which can indicate two not fully separated isomers of the compound. Even if the LC–MS data did not confirm the prevalence of two isomers in the peak, we postulate an unusual behaviour of 17 in the given chromatographic separation conditions. The principal metabolites in the analysed samples were gallic acid, oenothein B (metabolite 15) and quercetin glucuronide (30), which composed the parent compound – ellagotanin. Glycosides of 3-methylmyricetin, named annulatin, (41, 44 and 46) as well as kaempferol (35, 38, 39, 40 and 42) were identified for the first time in the genus, while myricetin (16, 21, 24, 25, 37), quercetin (26, 27, 30, 33, 34, 36, 43, 45, 47) and 3-methoxy-quercetin glycoconjugates (32) are already well known phytochemicals of this species (Nakanishi et al., 2007). In the negative modes [M-162-H]−; [M-146-H]−; and [M-132-H]− ions indicated the loss of an O-hexose (glucose or galactose, impossible to distinguish by MS), O-deoxyhexose (rhamnose) and O-pentose (arabinose or xylose), respectively. The derivatives of hydroxycinnamic acids (6, 8, 9, 11, 13 and 14) were identified in a lower amount. 211

3.1

3.9

4.8

6.2

6.5

6.6

8.7

9.5 9.7

9.7

9.6

2

3

4

5

6

7

8

9 10

11

12

212

7.01

19.9

19.8

16

17

7.55

7.81

7.9

8.54

8.69 8.83

9.17

9.26

9.39

18

19

20

21

22 23

24

25

26

20.0

5.90

15

6.9

5.49

5.39

5.33

4.70

4.29 4.65

4.25

4.32

4.30

3.82

3.54

3.33

3.27

2.90

LC-HRMS RT [min]

14

10.4

3.0

1

13

LC–MS RT [min]

no

myricetin-3-O-glucoside glucuronide quercetin-7-O-[galloyl]glucoside

myricetin-3-O-arabinoside

digallic acid galloylconiferolglucose

myricetin 7-O-glucoside

ellagitanin tetramer

gallic acid derivative

digalloylrhamnoside

oenothein A

myricetin 3-O-rhamnoside

oenothein B

caffeoylquinic acid izomer

3-feruloylquinic acid

gemin D isomer

5-coumaroylquinic acid

5-caffeoylquinic acid digalloylglucose izomer

digalloyl caffeic acid

syringoylquinic acid

4-caffeoylquinic acid

4-glucosyloxy-3-hydroxybenzoic acid galloylshikimic acid

digalloylglucose

B-glucogallin

gemin D

metabolite identification

633.0735

C27H22O8

321.1555 493.0624 449.0713 655.1308 615.1017

C14H26O8 C21H18O14 C20H18O12 C31H28O16 C28H24O16

1044.76233c

C136H94O88 479.0827

415.1961

C20H32O9

C21H20O13

467.0816

C20H20O15

1175.60938b

337.0941

C16H18O8

C102H71O66

353.0873 483.0783

C16H18O9 C20H20O14

463.0515

483.0775

C20H20O14

C20H16O13

371.0985

C16H20O10

783.06927a

353.0873

C16H18O9

C68H46O44

325.056

C14H14O9

353.0868

315.0725

C13H16O9

C16H18O9

483.0780

C20H20O14

367.1035

331.0672

C13H16O10

C17H20O9

633.0734

measured

615.1027

655.1305

449.0725

321.1544 493.0610

479.0831

1044.7621

415.1968

467.0826

1175.6094

463.0513

783.0675

353.0873

367.1035

633.0733

337.0929

353.0880 483.0780

483.0783

371.0984

353.0879

325.0563

315.0722

483.0783

331.0671

633.0733

calculated

exact mass of [M−H]−

C27H22O18

chemical formula

−2.7379

−1.5982

655, 493, 479, 330, 316, 244, 179 615, 463, 301, 271, 255, 169, 107

449, 316, 287, 179, 151

−3.3380 −2.7241

0.5198

321, 179, 164, 159 793, 359, 317, 271

−0.7945

415, 301, 179 935, 765, 633, 597, 450, 300, 275, 249, 169 479, 331, 317, 271, 179

0.1910

−1.6158

−2.0582

−0.0360

0.4047

2.198

935, 765, 698, 633, 615, 597, 301, 275 463, 316, 317, 287, 271, 242, 179 935, 785, 765, 727, 633, 569, 450, 335, 300, 291, 275, 249 467, 423, 315, 169

353, 191, 179, 173, 135

−1.4032

0.0810

633, 465, 301, 275, 231,185 367, 193, 173, 134

483, 331, 313, 271, 211, 169 353, 191, 127, 109, 85 483, 331, 314, 271, 211, 169 337, 191, 163, 119

353, 191, 173, 135, 127, 93 371, 191, 179, 153

325, 169, 125

315, 153, 109

633, 463, 331, 301, 275, 257, 249 331, 271, 241, 211, 169, 125 483, 331, 313, 193, 169

Fragmentation in negative ion mode

0.2361

0.6480

1.9676 0.5159

1.7143

0.2971

1.7083

1.8221

7.0823

−0.0455

0.3001

0.0433

Δ ppm

*

*

*

* *

*

*

*

*

*

*

*

*

* *

*

*

*

*

*

*

Line PL June

Table 2 Characterization of phenolic compounds and flavonoids in different plants of Epilobium by HPLC–MSn and UPLC-Q-Exactive MS/MS.

*

*

*

* *

*

*

*

*

*

*

*

*

*

* *

*

*

*

*

*

*

*

Line PL September

*

*

*

* *

*

*

*

*

*

*

*

*

*

*

* *

*

*

*

*

*

*

*

Line DE June

*

*

*

* *

*

*

*

*

*

*

*

*

*

*

*

* *

*

*

*

*

*

*

*

*

Line DE September

355

369

358

269, 345 273 268

264

269

267

269, 345 264

264

292

324sh

273

273

292 267

273

299 sh 324 275

271

273

278

272

λ max [nm]

431

75,820

16129729

5,352,000

16174339

9799386

75500

12310830

75491

460899

(Hofmann et al., 2016) (Hofmann et al., 2016) (Baert et al., 2015) (Kachlicki et al., 2008) PubChem (Hofmann et al., 2016) (Kachlicki et al., 2008) (Nakanishi et al., 2007) (Kachlicki et al., 2008)

(Taniguchi et al., 2002) (Hofmann et al., 2016) (Hofmann et al., 2016) (Hofmann et al., 2016) (Hofmann et al., 2016) (Piasecka et al., 2015) (Hofmann et al., 2016) (Hofmann et al., 2016) std (Hofmann et al., 2016) (Piasecka et al., 2015) (Taniguchi et al., 2002) (Piasecka et al., 2015) (Clifford et al., 2008) (Baert et al., 2015) (Kachlicki et al., 2008) (Baert et al., 2015)

ref

3

3

3

a 3

3

3

3

2

3

2

2

2

3

2

1 2

2

3

d

2

3

2

3

3

IL

(continued on next page)

C00035947

20,020,654**

C00002652*

C00037173*

ID

G. Agnieszka et al.

Industrial Crops & Products 123 (2018) 208–220

213

23.1

23.8

24.3

24.2

25.0

22.8

25.3

30.3

26.5

30.4

29.5

30.0

32

33

34

35

36

37

38

39

40

41

42

43

12.46

12.38

11.87

11.66

11.20

11.27

11.06

10.89

10.80

11.14

10.78

10.74

10.21

10.22

quercetin-7-O-pcoumaroylglucoside

quercetin-3-Oferuloylglucoside annulatin O-glucoside

quercetin-7-Ocaffeoylglucoside annulatin O-rhamnoside

kaempferol 7-O-rhamnoside

annulatin O-arabinoside

kaempferol 7-O-pcoumaroylglucoside kaempferol 3-O-arabinoside

myricetin-3-O-caffeoylglucoside kaempferol 7-O-glucuronide

quercetin 7-O-rhamnoside

quercetin-O-[digalloyl]glucoside kaempferol 3-O-glucoside

3-O-methylquercetin Oarabinoside quercetin 3-O-arabinoside

[2M-H]-477

[2M-H]-463 1,2,3,4,6-O-galloylglucoside quercetin glucuronide

quercetin 7-O-glucoside

metabolite identification

433.0776 767.1096 447.0933 447.0934 641.1154 461.0728 593.1303 417.0829 463.0882 431.0984 625.1199 477.1025 639.1359 493.09723 609.1251

C35H28O20 C21H20O11 C21H20O11 C30H26O16 C21H18O12 C30H26O13 C20H18O10 C21H20O12 C21H20O10 C30H26O15 C22H22O12 C31H28O15 C22H21O13 C30H26O14

955.1428

C42H36O26

C20H18O11

477.0676

C21H18O13

447.0980

927.1843 939.1118

C42H46O24 C41H32O26

C21H20O11

463.0886

measured

609.1250

493.0982

639.1355

477.1033

625.1199

431.0984

463.0884

417.0827

593.1301

461.0725

641.1148

447.0933

447.0933

767.1101

433.0777

447.0992

955.1422

477.0675

927.1837 939.1109

463.0882

calculated

exact mass of [M−H]−

C21H20O12

chemical formula

767, 615, 463, 301, 273

−0.7275

0.1354

−1.998

0.5023

−1.5967

0.0655

609, 463, 447, 301, 285, 254, 221, 177

625, 463, 301, 273, 179, 151 477, 331, 272, 257, 169, 152 639, 477, 463, 300, 271, 255, 179, 151 493, 331

431, 285, 255, 194, 163

−0.0274

0.5199

0.5439

593, 447, 285, 257, 241, 195, 151 417, 371, 327, 284, 255, 227, 151 463, 331, 300, 191

447, 301, 271, 255, 179, 151, 107 641, 479, 316, 317, 299, 288, 271, 214 461, 285, 199, 163, 141

0.4149

0.5765

0.9510

0.2219

0.0171

0.0756

447, 285, 255, 227, 179

447, 343, 327, 315, 284, 255, 227, 151 433, 300, 271, 255, 179

−2.6756

0.6472

477, 301, 258, 228, 179, 151 955, 601, 477, 301, 151

927, 797, 463, 301, 107 939, 769, 617, 465

463, 301, 271, 179, 151

Fragmentation in negative ion mode

0.2782

0.8998 0.9066

0.7835

Δ ppm

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

* *

*

Line PL June

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

* *

*

Line PL September

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

* *

*

Line DE June

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

* *

*

Line DE September

sh

sh

sh

sh

279 sh 349 287 sh 313

298

264 sh 348

348

295 356 296 356 298 361 298 348 348

276

357

λ max [nm]

26367429

5,481,882 *** 24,773,541

44,258,923 ***

44,259,226 ***

5282102***

16736526

5274585

58735

28529

ID

(Howard and Mabry, 1970) (Kachlicki et al., 2008) (Piasecka et al., 2015) (Kachlicki et al., 2008) (Kachlicki et al., 2008) (Piasecka et al., 2015) (Nakanishi et al., 2007) (Piasecka et al., 2015) (Kachlicki et al., 2008) (Howard and Mabry, 1970) (Kachlicki et al., 2008) (Piasecka et al., 2015) (Howard and Mabry, 1970) (Piasecka et al., 2015) (Howard and Mabry, 1970) (Piasecka et al., 2015)

(Hofmann et al., 2016) (Nakanishi et al., 2007)

(Kachlicki et al., 2008)

ref

3

3

3

3

3

3

3

3

3

3

3

3

2

3

3

3

2

2

3

IL

b-measurement of [M-2 H]2− ion ; c-measurement of [M-3 H]3–;d-measurement of [M-4 H]4−. ID – identification number in followed data base: * – Knapsack; ** – ChemSpider; *** PubChem. Ref – references. IL – metabolite identification level according to Metabolomics Standards Initiative recommendation (Sumner et al., 2007). The levels include: 1) Identified compounds, 2) Putatively annotated compounds without chemical reference standards, based upon physicochemical properties and spectral similarity with public spectral libraries, 3) Putatively characterized compound classes based upon characteristic physicochemical properties of a chemical class of compounds, or by spectral similarity to known compounds of a chemical class, 4) Unknown compounds – although unidentified or unclassified, these metabolites can still be differentiated and quantified based upon spectral data. std – identification on the basis of standard compound fragmentation.

12.72

47

33.7

12.71

46

12.69

22.2

31

33.4

22.1

30

9.68 10.55

45

20.6 21.7

28 29

9.54

12.51

20.5

27

LC-HRMS RT [min]

44

LC–MS RT [min]

no

Table 2 (continued)

G. Agnieszka et al.

Industrial Crops & Products 123 (2018) 208–220

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

Fig. 1. Chromatograms of secondary metabolites profile of Chamaenerion Line DE from September recorded at two complementary wavelengths: A) 270 nm and B) 330 nm.

molecular [M-2H]2– and [M-3H]3– ions detected in compounds 17, and 20, respectively, indicated macrocyclic ellagitanins from tri- to tetrameric structures with a similar fragmentation pattern as described by Baert et al. (Baert et al., 2015). Therefore, metabolite 17 was assumed to be oenothein A and metabolite 20 was assumed to be ellagitanin tetramer. The quality profile of secondary metabolites from plant materials, differentiated by origin and time of sampling, had a high resemblance, as shown in Fig. 5. Among 47 identified peaks, 37 were common in all samples. Only 2 peaks were specific to line PL and 5 to line DE. One PL peak was characteristic of the June line, whereas the September line had 5 common metabolites – both in PL and DE. However, some significant differences were observed in the content of particular metabolites. The content of oenothein B (15), a metabolite identified in the highest peak, detected in line PL slightly decreased in the September

Gallic acid was also observed in relatively high molecular weight compounds ellagitanins (15, 17 and 20) that quantitatively dominated the secondary metabolites of studied extracts. Gallic acid as part of the main metabolites could have a significant influence on the bioactivity of studied samples. The identification of ellagitanins was done mainly by measurement of pseudo-molecular ions and their fragmentation. The main [M-2 H]2− ion of compound 15 and its fragmentation indicated ions at m/z 783.06927, 765.0508 and 633.0738, which were described as characteristic for tellimagrandin I based oligomers with m-DOG type bonds between two galloyl groups attached to each other by CeC bonds [5, 34]. In addition, ellagitannins can be characterized by the presence of m/z = 301 corresponding to [ellagic acid-H]-. The theoretical chemical formula for [M-2 H]2− ion of 15 C68H46O44 was adequate to the dimers of tellimagrandin I called oenothein B (Węglarz et al., 2011) (Fig. 4). Other multicharged pseudo-

214

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

Fig. 2. Mass spectra recorded in LC-HR-MS in negative ion mode showing differences in fragmentation of compounds with substituents with the same nominal masses: A) quercetin glucuronide (30), B) quercetin-3-O-feruloylglucoside (45), C) myricetin-3-O-caffeoyl-glucoside (37).

215

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

Fig. 3. Mass spectra recorded in LC-HR-MS in negative ion mode showing metabolites with gallic acid A) digalloylglucose (3) and B) quercetin-7-O-[galloyl]glucoside (26).

comparison with the concentration measured in the samples from June (line DE: 2.82 ± 0.03 mg/100 g and Line PL: 3.37 ± 0.15 mg/100 g, respectively) (Table 3). Moreover, the concentration of this compound differed statistically between these two lines, and in line PL the content of oenothein B was higher than in the line DE. Since 1997 many researchers have analysed the presence and concentration of oenothein B in different parts of the Epilobium species. Its content depends on the particular species, the time of herb harvesting (Granica et al., 2014) and the part of plant analysed. Beart et al. (Baert et al., 2015) compared the content of many compounds from this group that create the oligomeric structures of ellagitannin. They investigated 3 parts of E. augustifolium: flowers, leaves and stems. The richest source of oenothein B were flowers (approximately 80 mg/g dry mass), then

harvest, whereas in line DE an increase of content was observed in the same time of harvest. As a result, compound 15 was present in a higher amount in line PL in June and in line DE in September. The content of typical groups of compounds is essential for phytochemical characteristics of the plants. The concentrations of selected groups and chosen compounds are shown in Tables 3–5. The most representative substance in the willowherb is oenothein B. This ellagitannin is very important and it is responsible for the activity of this species (Granica et al., 2014). In our study, we analysed the samples of herb collected during flowering (June) and after a flowering period (September). In two samples of the herb harvested in September, the content of oenothein B was statistically higher (line DE:3.12 ± 0.02 mg/100 g and line PL:3.56 ± 0.04 mg/100 g, respectively) in 216

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

Fig. 4. Mass spectra recorded in LC-HR-MS in negative ion mode showing fragmentation of oenothein B (15) and postulated scheme of fragmentation.

et al., 2012). The content range of ellagitannin in our experiment, based on ex vitro plants, was on the similar level as in the above-mentioned studies that analysed field crop plants, and our results were within the range of 2.818–3.368 g/100 g. However, many scientists were focused in particular on checking the properties of dry extracts, especially water extracts, but not raw material. In these extracts, the content of ellagitannin is much higher than in the raw plant material and it reaches even 15% (Baert et al., 2015). From the sterols content analysis, it can be assumed that the main sterol present in every line of the herb and in two harvest times was βsitosterol (Table 3). In both lines, the herb after flowering (September) was a richer source of this compound and its content was on a similar level (line DE: 118.81 ± 1.26 mg/100 g and line PL: 115.33 ± 4.10 mg/100 g). On the contrary, a higher concentration of stigmosterol was obtained in June, comparing to September, in both lines (line DE: 7.95 ± 0.94 and line PL: 8.39 ± 1.52 mg/100 g). The highest statistically significant difference of the content between both lines was observed in the campesterol case. In line DE the concentrations of this compound were only 13.28 ± 2.64 and 12.25 ± 1.02 mg/100 g, in June and September, respectively, whereas in line PL the content was about 3–4 fold higher (48.318 ± 6.40 and 38.53 ± 6.78 mg/100 g). Moreover, it should be stressed that during the flowering period (June), the level of campesterol was higher than in

Fig. 5. Venn diagram showing similarities of secondary metabolites profiles in all studies samples: 1 – Line PL from July, 2 – Line PL from September, 3 – Line DE from July, 4 – Line DE from September.

leaves (approximately 60 mg/g dry mass) (Baert et al., 2015). Granica et al. compared a range of oenothein B in the herb from different species of Epilobium, i.e. C. augustifolium, and they confirmed the 41.77 ± 1.47 mg/g concentration of oenothein B in the plant (Granica

Table 3 Concentration of oenothein B and sterols in line DE and line PL harvested in two periods. Sample

Line DE Line PL

Concentration

June September June September

Oenothein B [g/100 g]

Campesterol [mg/100 g]

Stigmasterol [mg/100 g]

β-sitosterol [mg/100 g]

2.82 3.12 3.37 3.56

13.28 12.35 48.32 38.53

7.95 5.72 8.39 6.25

84.49 ± 5.85 118.81 ± 1.26# 94.95 ± 1.52 115.33 ± 4.10#

± ± ± ±

0.03 0.02# 0.15* 0.04*#

± ± ± ±

2.64 1.02 6.40* 6.78*#

n = 3. Values are expressed as mean ± SD. Line DE – from Rieger-Hofmann® GmbH, Germany. Line PL – from Garden in the Institute of Natural Fibres & Medicinal Plants, Poland. * – Statistical significance vs. proper DE line, p < 0.05. # –Statistical significance vs. proper June group, p < 0.05. 217

± ± ± ±

0.94 1.26# 1.52 1.78#

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

Table 4 Concentration of flavonoids and polyphenolic acids measured with UPLC-MS/MS method. Compound

Line DE

Line PL

June

September

June

September

0.03 ± 0.00# 1.82 ± 0.22# N/D 123.31 ± 5.93# 8.92 ± 0.49 5.10 ± 0.15# N/D

0.24 ± 0.01* 3.91 ± 0.02* N/D 189.15 ± 2.36* 2.15 ± 0.12* 11.47 ± 0.25 N/D

0.27 ± 0.02*# 7.04 ± 0.09*# N/D 120.10 ± 7.01# 1.01 ± 0.05* 4.57 ± 0.32# N/D

concentration [mg/100 g] caffeic acid gallic acid p-coumaric acid ellagic acid quercetin rutin myricetin

0.12 ± 0.01 1.22 ± 0.05 N/D 102.38 ± 1.09 10.96 ± 0.49 11.95 ± 0.12 N/D

n = 3. Values are expressed as mean ± SD. Line DE – from Rieger-Hofmann® GmbH, Germany. Line PL – from Garden in the Institute of Natural Fibres &Medicinal Plants, Poland. * – Statistical significance vs. proper DE line, p < 0.05. # – Statistical significance vs. proper June group, p < 0.05. N/D – not detected.

period, the content of this compound was slightly higher, although the differences did not reach statistical significance. On the contrary, in the analysis of rutin concentration, it was found that the lines did not differ in the periods (June: line DE – 11.95 ± 0.12 mg/100 g, line PL – 11.47 ± 0.25 mg/100 g; Septmeber: line DE – 5.10 ± 0.15 mg/100 g, line PL – 4.57 ± 0.32 mg/100 g), whereas during the flowering period the content of this compound was more than 2 fold higher in every line. In addition to the determination of flavonoids, we analysed the concentration of some polyphenolic acids (Table 4). The presence of caffeic acid, gallic acid, ellagic acid and p-coumaric acid was checked in all samples. The main detected acid in the samples was ellagic acid (102.38 ± 1.09–189.15 ± 2.36 mg/100 g), which reached the highest concentration in line PL (June). In line DE, a better source of this compound was the sample from herb collected in September (123.31 ± 5.93 mg/100 g). Analysing the results for gallic acid level, it was found that samples from the period after flowering showed a higher concentration of this acid (line DE: 1.82 ± 0.22 mg/100 g; line PL: 7.04 ± 0.09 mg/100 g). However, line PL had much higher content of gallic acid in both periods comparing to line DE. A caffeic acid occurred in both harvest periods but in lower concentrations in comparison to ellagic and gallic acids. Moreover, in all samples the concentration did not exceed 0.3 mg/100 g. It should be stressed that none of the samples was a source of p-coumaric acid. Our results can be compared with the studies of Maruška et al. (Maruška et al., 2014). The authors of this paper analysed the plants collected in Kaunas Botanical Garden, Lithuania in terms of hyperoside,

the September samples (Table 3). Many papers give information about the presence of sterols, but few contain raw data about their concentrations. For example, in Pelcʼs et al. work (Pelc et al., 2005) two species of Epilobium were analysed, but not the E. augustifolium species. They determined 5 sterols: cholesterol, brassicasterol, campesterol, stigmosterol and β-sitosterol in seed oil, therefore, a simple comparison of our results is not possible. On the contrary, Węglarz et al. (Węglarz et al., 2011) published a work which describes the concentrations of β-sitosterol, campesterol and βsitosterol D-glucoside in 20 wild populations of E. augustifolium collected in southern Poland while blooming, and the herb was the raw material for further analysis. In almost every sample analysed by Węglarz et al. (Węglarz et al., 2011), the major sterol was β-sitosterol (85.80–171.18 mg/100 g), similarly to the results in our experiment (Table 3). However, in 6 populations a dominant sterol was campesterol (24.24–334.49 mg/100 g), whereas in our ex vitro lines the level of this compound was lower (12.35–48.32 mg/100 g). The stigmasterol content in the 20 wild herbs was 4.90–36.69 mg/100 g (Węglarz et al., 2011), while in our samples the concentration of this compound was within the range 5.718–8.387 mg/100 g. In order to determine the flavonoids and ellagic acid content, we used a modified method developed by Hevesi Tóth et al. (Hevesi Tóth et al., 2009). In our studies, line DE was a better source of quercetin comparing to line PL in both harvested periods (10.96 ± 0.49 and 8.92 ± 0.49 mg/100 g vs. 2.15 ± 0.12 and 1.01 ± 0.05 mg/100 g, in June and September, respectively) (Table 4). During the flowering

Table 5 Concentration of the bioactive groups of compounds detected by UV–vis methods. Total flavonoids expressed as hyperoside

Samples

Total flavonoids expressed as quercetin

Total tannins expressed as pyrogallol

Total polyphenols expressed as gallic acid

0.73 0.41 0.72 0.56

3.73 4.22 5.86 3.69

8.82 6.56 8.51 5.46

concentration [g/100 g] Line DE Line PL

June September June September

1.04 0.58 1.04 0.80

± ± ± ±

0.03 0.01# 0.02 0.01*#

± ± ± ±

0.02 0.01# 0.02 0.01*#

n = 3. Values are expressed as mean ± SD. Line DE – from Rieger-Hofmann® GmbH, Germany. Line PL – from Garden in the Institute of Natural Fibres &Medicinal Plants, Poland. * – Statistical significance vs. proper DE line, p < 0.05. # – Statistical significance vs. proper June group, p < 0.05. 218

± ± ± ±

0.11 0.09# 0.24* 0.01*#

± ± ± ±

0.31 0.43# 0.71 0.40*#

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

pharmaceuticals, for example, it will be a source for new drugs against benign prostatic hypertrophy.

myricetin, quercetin, quercetirin and kaempferol content. Different parts of E. augustifoliumi were analysed in different vegetation phases. The highest amount of quercetin was detected during the bud period (0.07 mg/g), like in our experiment during flowering (June). However, it should be emphasized that Maruška et al. (Maruška et al., 2014) in their research identified the presence of myricetin, which in our experiment was not detected. The concentration of main phytochemical groups, expressed as total flavonoids, tannins and polyphenols, measured by spectrophotometric methods are shown in Table 5. In all cases, a higher concentration of bioactive compounds was detected during the flowering period, except for the total tannins content for line DE. The total flavonoids content expressed as hyperoside during the flowering period was presented on the same level in both lines (line DE: 1.04 ± 0.03 g/100 g, line PL: 1.04 ± 0.02 g/100 g). A similar relationship was observed for total flavonoids content expressed as quercetin, however, the obtained values were lower. Analysing the content of total polyphenols expressed as gallic acid, it was observed that during the flowering period, the obtained values were at the same level in both lines (line DE: 8.82 ± 0.31 g/100 g, line PL: 8.51 ± 0.71 g/100 g). On the other hand, in the samples from September the amounts of polyphenols were lower than in June, but the values in line DE were statistically significant comparing to PL line after the flowering period (6.56 ± 0.43 and 5.46 ± 0.40 mg/100 g, respectively). Our values are generally in line with the amounts obtained by other authors, given that the typical concentration of total flavonoids in herb from E. augustifolium is between 1–2%, whereas the content of tannins is about 12% (Monschein et al., 2015). For example Monschein et al. (Monschein et al., 2015) collected the samples in Austria and analysed them during flowering period taking into consideration the harvesting impact dependent on the high or low altitude. They confirmed the presence of polyphenols (expressed as gallic acid) in the range 97.6–135.2 mg/g (Monschein et al., 2015). In the experiment conducted by Kaškonienė et al. (Kaškoniene et al., 2016) in the samples, collected in different places and vegetation periods in Lithuania, the total polyphenolic compounds were in the range 90.5–144.5 mg/g and the total flavonoids contents (expressed as rutin) were 6.7–22.9 mg/g. Meanwhile, Kiss et al. (Kiss et al., 2011) measured 27.0–85.0 mg/g polyphenols (expressed as chlorogenic acid) and 0.1–2.4 mg/g flavonoids (expressed as rutin) (Kiss et al., 2011, Kaškoniene et al., 2016). In another Kaškonienė`s et al. paper, the authors performed the analysis of fresh and dried plants focused on the total polyphenols and flavonoids content. They found out that in dried plants, the concentration of total polyphenols was in the range 70.6–126.8 mg/g and total flavonoids (expressed as rutin) were 14.3–41.0 mg/g (Kaškoniene et al., 2015). Jürgenson et al. (Jürgenson et al., 2012) compared the concentration of total polyphenols, total flavonoids and total tannins in different parts of E. augustifolium, in different periods and vegetable states. They mentioned that the concentrations of the above compounds in June were as follows: 66.7 mg/g, 54.0 mg/g, 0.21 mg/g in stems; 32.2 mg/g, 186.4 mg/g and 1.88 mg/g in leaves, respectively. In September, the research results concerning stems was: 55.9 mg/g, 60.4 mg/g and 0.19 mg/g, respectively (Jürgenson et al., 2012).

Acknowledgement The work was supported by the Polish National Centre for Research and Development grant no. PBS2/A8/23/2013. References Baert, N., Karonen, M., Salminen, J.P., 2015. Isolation, characterization and quantification of the main oligomericmacrocyclic ellagitannins in Epilobium angustifolium by ultra-high performance chromatography with diode array detection and electrospray tandem mass spectrometry. J. Chromatogr. A 1419, 26–36. Bazylko, A., Kiss, A.K., Kowalski, J., 2007. High-performance thin-layer chromatography method for quantitative determination of oenothein B and quercetin glucuronide in aqueous extract of Epilobium angustifolium herb. J. Chromatogr. A 1173, 146–150. Clifford, M.N., Kirkpatrick, J., Kuhnert, N., Roozendaal, H., Salgado, P.R., 2008. LC–MSn analysis of the cis isomers of chlorogenic acids. Food Chem. 106 (1), 379–385. Dreger, M., Wegenke, J., Makowiecka, J., Michalik, T., Wielgus, K., 2016. Application of multi-shoots cultures in micropropagation of willow herb (Chamaenerion angustifolium (L.) Scop.). Herba Pol. 62, 28–39. European Pharmacopoeia 8, 2013. European Directorate for the Quality of Medicines, Strasburg. Granica, S., Bazylko, A., Kiss, A.K., 2012. Determination of macrocyclic ellagitannin oenothein B in plant materials by HPLC-DAD-MS: method development and validation. Phytochem. Anal. 23 (6), 582–587. Granica, S., Piwowarski, J.P., Czerwińska, M.E., Kiss, A.K., 2014. Phytochemistry, pharmacology and traditional uses of different Epilobium species (Onagraceae): a review. J. Ethnopharm. 156, 316–346. Hevesi Tóth, B., Blazics, B., Kéry, A., 2009. Polyphenol composition and antioxidant capacity of Epilobium species. J. Pharm. Biomed. Anal. 49, 26–31. Hofmann, T., Nebehaj, E., Albert, L., 2016. Antioxidant properties and detailed polyphenol profiling of European hornbeam (Carpinus betulus L.) Leaves by multiple antioxidant capacity assays and high-performance liquid chromatography/multistage electrospray mass spectrometry. Ind. Crops Prod. 87, 340–349. Howard, G., Mabry, T.J., 1970. Myricetin 3-O-methyl ether 3,-O-glucoside, the major flavonoid of Oenothera speciosa (Onagraceae). Phytochemistry 9 (11), 2413–2414. Jürgenson, S., Matto, V., Raal, A., 2012. Vegetational variation of phenolic compounds in Epilobium angustifolium. Nat. Prod. Res. 26 (20), 1951–1953. Kachlicki, P., Einhorn, J., Muth, D., Kerhoas, L., Stobiecki, M., 2008. Evaluation of glycosylation and malonylation patterns in flavonoid glycosides during LC/MS/MS metabolite profiling. J. Mass Spectrom. 43, 572–586. Kachlicki, P., Piasecka, A., Marczak, Ł., Stobiecki, M., 2016. Structural characterization of flavonoid glycoconjugates and their derivatives with mass spectrometric techniques. Molecules 21 (11), 1494. Karonen, M., Parker, J., Agrawal, A., Salminen, J.-P., 2010. First evidence of hexameric and heptameric ellagitannins in plants detected by liquid chromatography/electrospray ionisation mass spectrometry. Rapid Commun. Mass Spectrom. 24, 3151–3156. Kaškoniene, V., Stankevičius, M., Drevinskas, T., Akuneca, I., Kaškonas, P., Bimbiraite -Surviliene, K., Maruška, A., Ragažinskiene, O., Kornyšova, O., Briedis, V., Ugenskiene, R., 2015. Evaluation of phytochemical composition of fresh and dried raw material of introduced Chamerion angustifolium L. using chromatographic, spectrophotometric and chemometric techniques. Phytochemistry 115, 184–193. Kaškoniene, V., Maruška, A., Akuņeca, I., Stankevičius, M., Ragažinskienė, O., Bartkuvienė, V., Kornyšova, O., Briedis, V., Ugenskienė, R., 2016. Screening of antioxidant activity and volatile compounds composition of Chamerion angustifolium (L.) Holub ecotypes grown in Lithuania. Nat. Prod. Res. 30 (12), 1373–1381. Kiss, A.K., Kowalski, J., Melzig, M.F., 2006. Induction of neutral endopeptidase activity in PC-3 cells by an aqueous extract of Epilobium angustifolium L. and oenothein B. Phytomedicine 13 (4), 284–289. Kiss, A.K., Bazylko, A., Filipek, A., Granica, S., Jaszewska, E., Kiarszys, U., Kośmider, A., Piwowarski, J., 2011. Oenothein B’s contribution to the anti-inflammatory and antioxidant activity of Epilobium sp. Phytomedicine 18, 557–560. Kujawski, R., Bogacz, A., Derebecka-Hołysz, N., Cichocka, J., Kujawski, J., Mikołajczak, P.Ł., Bobkiewicz-Kozłowska, T., Grześkowiak, E., Krajewska-Patan, A., Czerny, B., Mrozikiewicz, P.M., 2010a. Rośliny lecznicze z rodzaju Epilobium – działanie biologiczne i farmakologiczne. Herba Pol. 56 (1), 66–82. Kujawski, R., Mrozikiewicz, P.M., Bogacz, A., Cichocka, J., Mikołajczak, P.Ł., Czerny, B., Bobkiewicz-Kozłowska, T., Grześkowiak, E., 2010b. Wpływ standaryzowanego wyciągu z Epilobium angustifolium na ekspresji mRNA receptórow estrogenowych alpha i beta w modelu in vivo. [Influence of standardized extract of Epilobium angustifolium on estrogen receptor alpha and beta expression in in vivo model]. Ginekol. Pol. 81, 600–605. Maruška, A., Ragažinskienė, O., Vyšniauskas, O., Kaškonienė, V., Bartkuvienė, V., Kornyšova, O., Briedis, V., Ramanauskienė, K., 2014. Flavonoids of willow herb (Chamerion angustifolium (L.) Holub) and their radical scavenging activity during vegetation. Adv. Med. Sci. 59, 136–141. Máthé, Á., Hassan, F., Kader, A.A., 2015. In vitro micropropagation of medicinal and aromatic plants. In: Máthe, A. (Ed.), Medicinal and Aromatic Plants of the World, and Aromatic Plants of the World 1. Springer Science+Business Media, Dordrecht, pp. 305–336. http://dx.doi.org/10.1007/978-94-017-9810-5_15. Miyamoto, K., Nomura, M., Sasakura, M., Matsui, E., Koshiura, R., Murayama, T.,

4. Conclusions As a result of our experiment, we succeeded in receiving the in vitro plants of Chamaenerion angustifolium (L.). To our knowledge, there have not been similar results before. The chemical characteristics of ex vitro raw material, qualitatively and quantitatively analysed for the content of main active compounds, including oenothein B, sterols, flavonoids and polyphenolic acids, is similar to the chemical characteristics of the field crop plants. In conclusion, the application of micropropagation cultivation method allowed us to elaborate the controlled growing of willowherb. This may enable us to obtain the standardized, safe and homogenous material, which can be used to produce new 219

Industrial Crops & Products 123 (2018) 208–220

G. Agnieszka et al.

Polish Pharmacopoeia VI, 2002. Polskie Towarzystwo Farmaceutyczne. Warszawa. . Ramstead, A.G., Schepetkin, I.A., Quinn, M.T., Jutila, M.A., 2012. oenothein B, a cyclic dimeric ellagitannin isolated from Epilobium angustifolium. PLoS 7 (11), e50546. Schepetkin, I.A., Kirpotina, L.N., Jakiw, L., Khlebnikov, A.I., Blaskovich, Ch.L., Jutila, M.A., Quinn, M.T., 2009. Immunomodulatory activity of oenothein B isolated from Epilobium angustifolium. J. Immunol. 183, 6754–6766. Stolarczyk, M., Naruszewicz, M., Kiss, A.K., 2013. Extracts from Epilobium sp. herbs induce apoptosis in human hormone-dependent prostate cancer cells by activating the mitochondrial pathway. J. Pharm. Pharm. 65, 1044–1054. Sumner, L.W., Amberg, A., Barett, D., Bealle, M.H., Beger, R., Daykin, C.A., Fan, T.W.-M., Fiehn, O., Goodacre, R., Griffin, J.L., Hankemeier, T., Hardy, N., Harnly, J., Higashi, R., Kopka, J., Lane, A.N., Lindon, J.C., Mariott, P., Nicholls, A.W., Reilly, M.D., Thaden, J.J., Viant, M.R., 2007. Proposed minimum reporting standards for chemical analysis. Metabolomics 3, 211–221. Taniguchi, S., Imayoshi, Y., Yabu-uchi, R., Ito, H., Hatano, T., Yoshida, T., 2002. A macrocyclic ellagitannin trimer, oenotherin T1, from Oenothera species. Phytochemistry 59, 191–195. Traffic International, Therapy for Medicinal Plants http://www.traffic.org/home/2008/ 5/19/therapy-for-medicinal-plants.html. 2015 (Accessed 29 June 2015). Turker, A.U., Mutlu, E.C., Yıldırım, A.B., 2008. Efficient in vitro regeneration of fireweed, a medicinal plant. Acta Physiol. Plant. 30, 421–426. Węglarz, Z., Kosakowska, O., Pelc, M., Geszprych, A., Przybył, J.L., Bączek, K., 2011. Intraspecific variability of fireweed (Chamaenerion angustifolium /L./ Scop.) and evening primrose (Oenothera biennis L.) in respect of sterol content. Herba Pol. 57 (2), 7–15. Wojakowska, A., Piasecka, A., García-López, P.M., Zamora-Natera, F., Krajewski, P., Marczak, Ł., Kachlicki, P., Stobiecki, M., 2013. Structural analysis and profiling of phenolic secondary metabolites of Mexican lupine species using LC-MS techniques. Phytochemistry 92, 71–86.

Furukawa, T., Hatano, T., Yoshida, T., Okuda, T., 1993. Antitumor activity of oenothein B, a unique macrocyclic ellagitannin. Jpn. J. Cancer Res. 84, 99–103. Monschein, M., Jaindl, K., Buzimkić, S., Bucar, F., 2015. Content of phenolic compounds in wild populations of Epilobium angustifolium growing at different altitudes. Pharm. Biol. 53 (11), 1576–1582. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco culture. Physiol. Plant. 15, 473–497. Nakanishi, T., Inatomi, Y., Murata, H., Ishida, S.S., Fujino, Y., Miura, K., Yasuno, Y., Inada, A., Lang, F.A., Murata, J., 2007. Triterpenes and flavonols glucuronides from Oenothera cheiranthifolia. Chem. Pharm. Bull. 55, 334. Ożarowski, M., Piasecka, A., Gryszczyńska, A., Sawikowska, A., Pietrowiak, A., Opala, B., Mikołajczak, P.Ł., Kujawski, R., Kachlicki, P., Buchwald, W., Seremak-Mrozikiewicz, A., 2017. Determination of phenolic compounds and diterpenes in roots of Salvia miltiorrhiza and Salvia przewalskii by two LC–MS tools: Multi-stage and a high-resolution tandem mass spectrometry with assessment of antioxidant capacity. Phytochem. Lett. 20, 331–338. Parveen, I., Wilson, T., Donnison, I.S., Cookson, A.R., Hauck, B., Threadgill, M.D., 2013. Potential sources of high value chemicals from leaves, stems and flowers of Miscanthus sinensis “Goliath” and Miscanthus sacchariflorus. Phytochemistry 92, 160–167. Pelc, M., Kosakowska, O., Węglarz, Z., Przybył, J., Geszprych, A., 2005. Sterols and fatty acids in the seeds of evening primrose (Oenothera sp.) and willow herb (Epilobium sp.). Herba Pol. 51 (3/4), 20–24. Piasecka, A., Sawikowska, A., Krajewski, P., Kachlicki, P., 2015. Combined mass spectrometric and chromatographic methods for in-depth analysis of phenolic secondary metabolites in barley leaves. J. Mass Spectrom. 50, 513–532. Piasecka, A., Sawikowska, A., Kuczyńska, A., Ogrodowicz, P., Mikołajczak, K., Krystkowiak, K., Gudyś, K., Guzy-Wróbelska, J., Krajewski, P., Kachlicki, P., 2017. Drought related secondary metabolites of barley (Hordeum vulgare L.) leaves and their mQTLs. Plant. J. 89 (5), 898–913. http://dx.doi.org/10.1111/tpj.13430.

220