Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium

Phytochemistry xxx (2016) 1e10

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Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium Nicolas Baert*, Jorma Kim, Maarit Karonen, Juha-Pekka Salminen Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, Turku, FI-20014, Finland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 August 2016 Received in revised form 21 October 2016 Accepted 8 November 2016 Available online xxx

Rosebay willowherb (Epilobium angustifolium) contains large amounts of polyphenolic compounds, including tellimagrandin I-based oligomeric ellagitannins (ETs). The aim of this study was to assess the interpopulational and inter-organ variability of the polyphenol fingerprint of E. angustifolium. Seven ETs, 11 flavonol glycosides and neochlorogenic acid were quantified by UHPLC-DAD-ESI-QqQ-MS in the leaves, flowers and stem parts of plants from 10 populations. Total polyphenol content of leaves and flowers ranged from 150 to 200 mg/g dry wt, of which 90% was constituted by dimeric to heptameric ETs. Flowers contained, on average, 10% more oenothein B (dimeric ET) and 2 times less oenothein A (trimeric ET) than leaves. Tetrameric and pentameric ETs exhibited rather similar levels in leaves and flowers whereas hexameric and heptameric were 3e4 times more abundant in flowers than in leaves. Quercetin3-O-rhamnoside, myricetin-3-O-rhamnoside and kaempferol-3-O-rhamnoside were specific to flower tissue and were absent from leaves. The inflorescence stem showed the highest content in total polyphenols with an average of 250 mg/g dry wt and contained remarkably large amounts of oenothein B and A. Polyphenol content steadily decreased along the inflorescence stem and reached its lowest level in the vegetative part of the stem. The interpopulational variability of most polyphenols was within a two- to threefold range across the 10 sampled populations. Myricetin-3-O-glucoside and myricetin-3-O-glucuronide, however, showed a more population-specific distribution with concentrations varying from 0 to 2.3 mg/g dry wt. Finally, this study showed that the levels of oenothein B and A in the plant are not interdependent but that their relative abundance is constant within a population. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Epilobium angustifolium Onagraceae Population study Oligomeric ellagitannins Flavonol glycosides Oxidised oenothein A

1. Introduction Rosebay willowherb (Epilobium angustifolium L., family: Onagraceae) is used in traditional medicine for the treatment of several ailments such as benign prostate hyperplasia, diarrhoea, skin burns, mouth ulcers and persistent coughs (Granica et al., 2014). Extracts of the plant possess antioxidant, anti-inflammatory, anti-proliferative, immunomodulatory and antimicrobial properties in vitro
(Kiss et al., 2006; Kosalec et al., 2013; Ramstead et al., 2012; Stajner et al., 2007). The chemical composition of Epilobium sp. extracts has been extensively studied and was found to be rich in polyphenolic  th et al., 2009). Epicompounds (Granica et al., 2014; Hevesi To lobium angustifolium notably contains large amounts of the macrocyclic ellagitannin (ET) oenothein B, which represents about 6e8% of the dry mass of leaves and flowers (Granica et al., 2012;

* Corresponding author. E-mail address: nicolas.baert@utu.fi (N. Baert).

Shikov et al., 2010). It has been recently discovered that the plant also contains larger oligomeric ETs, at least up to heptamer, and that this series of tellimagrandin I (TI)-based ETs represents ca. 15% of the dry weight of leaves and flowers (Baert et al., 2015). In addition to its benefits on human health, rosebay willowherb may promote animal welfare. In the last few decades there has been a growing interest in the utilisation of tannin-rich forages in ruminant nutrition. Tannin-containing fodders can improve the utilisation of dietary proteins, promote animal growth rate, enhance animal resistance to intestinal parasites and reduce the environmental impact of the livestock industry by decreasing methane and nitrous oxide emissions from ruminants (Bodas et al., 2012; Hoste et al., 2015; Min et al., 2003). Recent in vitro and in vivo studies also suggest that flavonoids could increase the utilisation of dietary energy and decrease enteric methane formation in ruminants (Chen et al., 2015; Kim et al., 2015; Oskoueian et al., 2013). The mechanisms of action of polyphenols in the digestive tract of ruminants are not yet fully understood but recent studies clearly indicate that even small variations in the chemical structure can

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Please cite this article in press as: Baert, N., et al., Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.11.003

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N. Baert et al. / Phytochemistry xxx (2016) 1e10

have significant consequences on the activity (Baert et al., 2016; € m et al., 2016; Karonen et al., 2015). Despite the strong inEngstro terest for the polyphenols of willowherb, very little is known about their actual distribution in the plant or their inter-individual variability. In this study we measured the variability of the polyphenol fingerprint of E. angustifolium across 10 populations as well as the distribution of the same polyphenols in various organs of the plant. Additionally, we assessed the intra- and inter-organ correlations that link these polyphenols in order to gain some insight into the biochemistry of the plant. Inter-population and inter-organ distribution of some polyphenols of rosebay willowherb have been previously investigated (Averett et al., 1979; Baert et al., 2015; Jürgenson et al., 2012), however, this study is the very first to report compound-specific, quantitative variations of 19 polyphenols, including oligomeric ETs, in E. angustifolium.

have been labelled as quercetin-3-O-(600 -galloyl)-galactoside and quercetin-3-O-(galloyl)-hexoside, respectively. Total polyphenols in leaves and flowers ranged from 151 to 206 mg/g dry wt and total flavonoid content varied from 5.8 to 16.6 mg/g dry wt. These numbers are consistent with two recent studies which reported total phenolic content of 90.5e144.5 and 97.6e135.2 mg/g dry wt and total flavonoid content of 6.7e22.9 and 10.7e17.3 mg/g dry wt in aerial parts of E. angustifolium (Ka
skoniene_ et al., 2015; Monschein et al., 2015). In our experiment, oligomeric ellagitannins constituted about 90% (w/w) of total polyphenols in all analysed plant tissues. Oenothein B was by far the most abundant compound, accounting for 40e60% (w/w) of the total amount of polyphenols. Its concentration in leaves and flowers varied from 55 to 115 mg/g dry wt.

2. Results and discussion

Samples analysed by UHPLC-DAD-ESI-QqQ-MS showed a peak at 2.74 min corresponding to an m/z value of 1173.5. Samples were then analysed by UPLC-DAD-ESI-Orbitrap-MS in order to get high resolution mass spectra for this unknown compound. Analyses by Orbitrap-MS revealed a doubly charged pseudo-molecular ion at m/z 1182.098 ([M2H]2) and a sodium adduct at m/z 1193.089 ([M3H þ Na]2). An ion at m/z 1173.093 (which had been observed by ESI-QqQ-MS) was also visible and corresponded to a water loss ([M2HH2O]2). MS2 analyses showed fragments at m/z 935.078, 765.059, 615.064, 300.999 and 275.020 that are characteristic of TI-based oligomeric ETs (Baert et al., 2015). The unknown compound thus matched the description of an oxidised derivative of oenothein A previously found in evening primrose (Oenothera biennis) which, like E. angustifolium, is a member of the Onagraceae family (McArt et al., 2013). In that study, authors found that herbivory damage on leaves of evening primrose induced a 75% increase (P ¼ 0.006) in the concentration of oxidised oenothein

2.1. Polyphenols of Epilobium angustifolium From our sample pool we quantified 19 polyphenols, of which 18 had been previously identified in E. angustifolium and one (compound 2) had never been reported in any Epilobium species (Fig. 1 and Table 1). Similarly to what was reported by Stolarczyk et al. (2013) and Monschein et al. (2015), we observed the presence of two quercetin glycosides (compounds 11 and 12) with an m/z value of 615 in the extracts of E. angustifolium. Stolarczyk et al. identified the flavonol with the shorter retention time by NMR as quercetin-3-O-(600 -galloyl)-galactoside but did not identify the exact sugar moiety of the second one. Because their LC conditions were very similar to what we used in this study, we can safely assume that the two compounds follow the same order of elution. Consequently, 11 and 12

2.2. Oxidised oenothein A

Fig. 1. UV chromatograms of Epilobium angustifolium leaf extracts (A and B) and flower extracts (C and D) at 280 and 349 nm.

Please cite this article in press as: Baert, N., et al., Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.11.003

N. Baert et al. / Phytochemistry xxx (2016) 1e10

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Table 1 Retention time, UV absorption maxima and MS/MS data of the main polyphenols found in Epilobium angustifolium. Peak #

RT (min)

UV Absorption maxima (nm)

[MeH] (m/z)

Main product ions (m/z)

Identification

References

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

2.48 2.74 2.94; 3,23a 3.33; 3.50a 3.58e3.79 3.81e4.02 4.05e4.24 4.12e4.30 3.63 3.66 3.83 3.89 3.98 4.01 4.06 4.31 4.38 4.43 4.82

245, 224, 222, 227, 225, 223, 223, 223, 259, 259, 264, 266, 258, 265, 255, 253, 265, 255, 263,

353 1182.1b 783.1b 1175.1b 1044.4c 1305.8c 1567.1c 1828.5c 479 493 615 615 463 463 477 433 461 447 431

191 765, 765, 765, 301, 301, 301, 301, 316, 316, 300, 300, 316, 300, 300, 300, 284, 300, 284,

Neochlorogenic acid Unknown (oxidised oenothein A derivative) Oenothein B Oenothein A Tetrameric tellimagrandin I Pentameric tellimagrandin I Hexameric tellimagrandin I Heptameric tellimagrandin I Myricetin-3-O-glucoside Myricetin-3-O-glucuronide Quercetin-3-O-(600 -galloyl)-galactoside Quercetin-3-O-galloylhexoside Myricetin-3-O-rhamnoside Quercetin-3-O-galactoside Quercetin-3-O-glucuronide Quercetin-3-O-arabinoside Kaempferol-3-O-glucuronide Quercetin-3-O-rhamnoside Kaempferol-3-O-rhamnoside

Stolarczyk et al., 2013 McArt et al., 2013 Kiss et al., 2004; Baert et al., 2015 Ducrey et al., 1997; Baert et al., 2015 Baert et al., 2015 Baert et al., 2015 Baert et al., 2015 Baert et al., 2015 th et al., 2009 Ducrey et al., 1995; Hevesi To Hiermann et al., 1991; Hiermann 1995 Ducrey et al., 1995; Stolarczyk et al., 2013 th et al., 2009; Stolarczyk et al., 2013 Hevesi To th et al., 2009 Ducrey et al., 1995; Hevesi To Ducrey et al., 1995; Stolarczyk et al., 2013 th et al., 2009 Ducrey et al., 1995; Hevesi To th et al., 2009 Ducrey et al., 1995; Hevesi To th et al., 2009 Ducrey et al., 1995; Hevesi To th et al., 2009 Ducrey et al., 1995; Hevesi To th et al., 2009 Ducrey et al., 1995; Hevesi To

a b c

324 259sh 262 261 266 267 269 269 355 353 352 352 350 353 353 354 349 349 344

301 301 301 275 275 275 275 317 317 301 301 317 301 301 301 285 301 285

Isomer peak. [M2H]2. [M3H]3.

A in flower buds. These results imply thatdat least in O. biennisdthe production of this compound is part of a defence strategy against insect herbivores. Whether this is also the case for E. angustifolium remains to be demonstrated. The mass of this ET is 2366 Da which is 14 Da more than oenothein A (2352 Da) and putatively comes from the oxidation of a hexahydroxydiphenoyl (HHDP) group of oenothein A into a dehydrohexahydroxydiphenoyl (DHHDP) group (þ16 Da) along with the formation of an HHDP group from 2 free galloyl moieties (2 Da). The hypothesis of a DHHDP group is consistent with the fact the molecule elutes earlier than oenothein A because the DHHDP group is more hydrophilic than the HHDP group. Additionally, the presence of two OH-groups on the same carbon atom of the DHHDP group would explain why the molecule easily undergoes water fragmentation (Moilanen et al., 2013). Finally, unlike all the oligomeric ETs from willowherb, the UV spectrum of the oxidised oenothein A did not feature a clear valley around 250 nm, thus suggesting a higher HHDP to galloyl ratio in the molecule, which supports our hypothesis (Moilanen et al., 2013; Salminen et al., 2011). The complete structure elucidation of the molecule would require its isolation followed by NMR analyses of hydrolysis and derivatization product. 2.3. Inter-organ distribution 2.3.1. Leaves vs. flowers The polyphenol fingerprints of leaves and flowers were quantitatively dominated by ETs, and particularly by oenothein B (3) and A (4) (Fig. 1). Those two compounds, however, were distributed differently in leaves and flowers: oenothein B (3) was 10% more abundant in flowers than in leaves whereas the level of oenothein A (4) was, on average, two times higher in leaves than in flowers. Across all the sampled plants, the concentrations of large oligomeric ETs (compounds 5 to 8) were, on average, higher in flowers than in leaves. This trend was particularly marked for 7 and 8, which often showed three to four times higher concentrations in flowers compared to leaves. Compound 2 was generally more abundant in flowers (average ¼ 8.9 m/g dry wt) than in leaves (average ¼ 6.9 mg/g dry wt) but the highest level recorded was 13.0 m/g dry wt in the leaves of population #2 (Table 2).

Neochlorogenic acid (5-caffeoylquinic acid) (1) and quercetin-3O-glucuronide (15) also showed a very specific distribution across the plant organs (Fig. 2). Concentrations of 1 and 15 in leaves were, on average, three times and two times higher, respectively, than in flowers (Table 2). Quercetin-3-O-glucuronide (15) is a major compound in E. angustifolium and has been previously identified, with kaempferol-3-O-glucuronide (17), as a chemotaxonomic marker of  th et al., 2009). Comthat species (Ducrey et al., 1995; Hevesi To pound 17, however, was present at similar levels in leaves and flowers (Table 2). Additionally, leaves were the only organs containing myricetin-3-O-glucoside (9), myricetin-3-O-glucuronide (10) and quercetin-3-O-arabinoside (16). The most striking characteristic of flowers, on the other hand, was the presence of myricetin-3-O-rhamnoside (13), quercetin-3-O-rhamnoside (18) and kaempferol-3-O-rhamnoside (19). Apart from very small amounts of 18 being present in the topmost part of the stems, these three flavonol rhamnosides are unique to flower tissue. Furthermore, they represent, together, 70e85% of the total flavonoid content of flowers. These results could support the hypotheses that certain flavonol glycosides are involved in the visual attractiveness of flowers towards insect pollinators or affect the fertility of the pollen (Falcone Ferreyra et al., 2012; Gronquist et al., 2001; Thompson et al., 1972). A study of the precise spatial distribution of 13, 18 and 19 in willowherb flowers may permit us to understand the roles of these flavonols in the plant. Interestingly, these three compounds had been previously reported in aerial parts of the plant only (Ducrey et al., 1995; Stolarczyk et al., 2013). We thus provide the first time evidence that they are in fact absent from the leaves of E. angustifolium. 2.3.2. Open flowers vs. flower buds The differences between flower buds and open flowers were investigated in 10 individual plants from one population. In the studied population, open flowers and flower buds showed very similar levels for all the quantified compounds, with the sole exception of quercetin-3-O-hexoside (14) which was not detected in open flowers (Fig. 2 and Table 3). It should be noted, nevertheless, that 14 was present in open flowers of 8 out of the 10 sampled populations, albeit in small quantities (Table 2). This result indicates that the amounts of ellagitannins and flavonol glycosides in

Please cite this article in press as: Baert, N., et al., Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.11.003

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Compound

#

Willowherb populations 1

2

3

4

5

6

7

8

9

10

Flowers Neochlorogenic acid Oxidised oenothein A Oenothein B Oenothein A Tetramer Pentamer Hexamer Heptamer Myricetin-3-O-Glc Myricetin-3-O-GlcA Quercetin-3-O-(600 -galloyl)-Gal Quercetin-3-O-(galloyl)-hexoside Myricetin-3-O-Rha Quercetin-3-O-Gal Quercetin-3-O-GlcA Quercetin-3-O-Ara Kaempferol-3-O-GlcA Quercetin-3-O-Rha Kaempferol-3-O-Rha Leaves Neochlorogenic acid Oxidised oenothein A Oenothein B Oenothein A Tetramer Pentamer Hexamer Heptamer Myricetin-3-O-Glc Myricetin-3-O-GlcA Quercetin-3-O-(600 -galloyl)-Gal Quercetin-3-O-(galloyl)-hexoside Myricetin-3-O-Rha Quercetin-3-O-Gal Quercetin-3-O-GlcA Quercetin-3-O-Ara Kaempferol-3-O-GlcA Quercetin-3-O-Rha Kaempferol-3-O-Rha

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0.69 ± 0.02e 7.57 ± 0.16bc 94.72 ± 2.63abcd 16.37 ± 0.33d 11.46 ± 0.82c 10.09 ± 0.28c 1.39 ± 0.13b 1.13 ± 0.11b nq nq 0.03 ± 0.01ab 0.16 ± 0.01c 1.45 ± 0.07a 0.37 ± 0.02a 2.72 ± 0.08ab nq 0.54 ± 0.02cd 6.34 ± 0.25a 4.22 ± 0.17d 3.54 ± 0.11d 4.25 ± 0.11c 91.35 ± 2.70bc 30.85 ± 0.80d 9.64 ± 0.69e 8.15 ± 0.11d 0.50 ± 0.03d 0.29 ± 0.06d 0.38 ± 0.15b 0.29 ± 0.14b 0.34 ± 0.02b 0.10 ± 0.01d nq 1.48 ± 0.10a 4.11 ± 0.15cd 0.67 ± 0.04ab 0.62 ± 0.02def nq nq

0.89 ± 0.07de 7.71 ± 0.41bc 87.89 ± 5.02bcde 23.77 ± 1.41abc 17.23 ± 0.81bc 12.39 ± 0.44bc 2.21 ± 0.31ab 1.71 ± 0.35ab nq nq nq 0.12 ± 0.01d 1.11 ± 0.03bc 0.24 ± 0.01bc 1.98 ± 0.04cd nq 1.15 ± 0.04b 3.42 ± 0.08ef 5.87 ± 0.12abc 5.30 ± 0.13ab 13.03 ± 1.53a 61.45 ± 3.20de 43.64 ± 3.22bc 21.14 ± 1.51bc 11.49 ± 0.45bc 0.93 ± 0.10bcd 0.88 ± 0.13bcd 0.83 ± 0.14b 0.70 ± 0.15b 0.25 ± 0.03bc 0.09 ± 0.01d nq 1.19 ± 0.06a 4.90 ± 0.11bcd 0.56 ± 0.02ab 1.05 ± 0.06bc nq nq

1.24 ± 0.02bc 6.60 ± 0.10c 67.12 ± 0.87f 26.15 ± 0.57a 17.93 ± 1.22abc 12.76 ± 0.49abc 2.28 ± 0.30ab 1.98 ± 0.27ab nq nq 0.02 ± 0.01b 0.12 ± 0.01d 1.34 ± 0.03abc 0.24 ± 0.01bc 2.83 ± 0.05ab nq 0.51 ± 0.01d 4.40 ± 0.06cd 5.45 ± 0.11bc 5.39 ± 0.13ab 5.32 ± 0.17c 67.08 ± 1.89de 56.19 ± 1.64a 16.49 ± 1.15cde 9.53 ± 0.28cd 0.70 ± 0.06cd 0.51 ± 0.05cd 2.31 ± 0.21a 1.61 ± 0.09a 0.20 ± 0.01cd 0.07 ± 0.01d nq 0.80 ± 0.05b 6.19 ± 0.18a 0.71 ± 0.03a 0.28 ± 0.01f nq nq

0.87 ± 0.01e 11.88 ± 0.87a 99.04 ± 3.35abc 22.68 ± 1.14abc 21.82 ± 1.31ab 13.08 ± 0.47abc 2.47 ± 0.24ab 1.95 ± 0.24ab nq nq 0.08 ± 0.01a 0.28 ± 0.01a 1.06 ± 0.10cd 0.22 ± 0.02bc 2.08 ± 0.04c nq 0.68 ± 0.06cd 3.79 ± 0.05de 5.46 ± 0.32bc 5.78 ± 0.22a 6.55 ± 0.42bc 73.85 ± 3.69cd 48.44 ± 1.91ab 22.56 ± 1.44abc 11.46 ± 0.33bc 1.00 ± 0.10bcd 0.81 ± 0.08bcd nq 0.17 ± 0.10b 0.56 ± 0.04a 0.21 ± 0.01b nq 0.54 ± 0.06bcd 4.73 ± 0.13bcd 0.66 ± 0.09ab 0.89 ± 0.14cd nq nq

1.82 ± 0.04a 10.93 ± 0.38a 71.64 ± 2.02ef 19.73 ± 0.30cd 17.83 ± 0.78bc 12.82 ± 0.28abc 2.28 ± 0.21ab 2.13 ± 0.25ab nq nq nq 0.15 ± 0.01cd 1.10 ± 0.03c 0.19 ± 0.01bc 2.82 ± 0.06ab nq 1.77 ± 0.01a 4.12 ± 0.05d 6.36 ± 0.19ab 4.64 ± 0.07bc 7.71 ± 0.45bc 55.21 ± 1.94e 35.53 ± 1.36cd 24.22 ± 1.55abc 13.28 ± 0.49ab 1.25 ± 0.10abc 1.40 ± 0.15ab nq nq 0.10 ± 0.01d 0.11 ± 0.01cd nq 0.29 ± 0.01def 5.17 ± 0.11abc 0.45 ± 0.01bcd 1.75 ± 0.08a nq nq

1.11 ± 0.04cd 7.15 ± 0.22c 83.37 ± 1.54cdef 25.12 ± 0.80a 24.50 ± 1.40ab 15.51 ± 0.60a 3.42 ± 0.28a 2.58 ± 0.23ab nq nq nq 0.20 ± 0.01b 1.45 ± 0.06a nq 1.61 ± 0.04d nq 0.63 ± 0.02cd 5.09 ± 0.10b 6.76 ± 0.26a 4.71 ± 0.10bc 6.13 ± 0.35bc 66.09 ± 1.70de 43.18 ± 1.22bc 18.97 ± 1.32cd 9.99 ± 0.34cd 0.89 ± 0.12cd 0.64 ± 0.07cd nq nq 0.10 ± 0.01d 0.21 ± 0.01b nq 0.10 ± 0.01ef 5.23 ± 0.10ab 0.16 ± 0.01e 0.41 ± 0.03ef nq nq

1.16 ± 0.01bc 9.69 ± 0.18ab 102.27 ± 1.93ab 24.44 ± 0.27ab 24.78 ± 1.75ab 14.89 ± 0.69ab 3.05 ± 0.36a 2.28 ± 0.28ab nq nq nq 0.25 ± 0.01a 1.40 ± 0.03ab nq 1.80 ± 0.04cd nq 1.17 ± 0.03b 2.82 ± 0.06f 5.94 ± 0.16abc 4.00 ± 0.05cd 4.20 ± 0.09c 95.30 ± 3.12b 47.95 ± 1.20ab 30.33 ± 1.49a 14.82 ± 0.52a 1.80 ± 0.13a 1.67 ± 0.16a nq nq 0.18 ± 0.01cd 0.29 ± 0.01a nq 0.02 ± 0.01f 3.85 ± 0.04d 0.22 ± 0.01de 1.28 ± 0.03b nq nq

1.30 ± 0.02bc 9.63 ± 0.38ab 98.99 ± 2.74abc 23.23 ± 0.37abc 25.55 ± 1.59a 15.33 ± 0.61ab 3.09 ± 0.25a 2.72 ± 0.27a nq nq 0.04 ± 0.01ab 0.22 ± 0.01b 0.78 ± 0.02d 0.15 ± 0.01c 2.52 ± 0.08b nq 0.71 ± 0.02c 2.91 ± 0.06f 5.17 ± 0.19cd 4.65 ± 0.10bc 6.10 ± 0.42bc 72.79 ± 1.45de 44.67 ± 0.93bc 26.87 ± 0.87ab 14.33 ± 0.26a 1.52 ± 0.11ab 1.39 ± 0.12ab nq 0.17 ± 0.11b 0.29 ± 0.01bc 0.21 ± 0.01b nq 0.49 ± 0.02bcd 4.88 ± 0.13bcd 0.44 ± 0.02bcd 0.45 ± 0.03ef nq nq

1.35 ± 0.04b 7.07 ± 0.23c 79.35 ± 2.80def 20.19 ± 0.36bcd 19.43 ± 1.04ab 13.41 ± 0.42ab 2.06 ± 0.22ab 2.00 ± 0.23ab nq nq 0.02 ± 0.01b 0.12 ± 0.01d 0.76 ± 0.02d 0.38 ± 0.05a 3.08 ± 0.11a nq 1.20 ± 0.02b 4.33 ± 0.07cd 6.30 ± 0.14abc 5.71 ± 0.15a 5.99 ± 0.42c 94.20 ± 2.70b 39.22 ± 0.96bcd 20.00 ± 1.13bcd 11.64 ± 0.34bc 1.25 ± 0.10abc 1.08 ± 0.10abc nq 0.16 ± 0.10b 0.19 ± 0.03cd 0.11 ± 0.01cd nq 0.44 ± 0.08cde 5.63 ± 0.42ab 0.52 ± 0.06abc 0.70 ± 0.02cde nq nq

1.15 ± 0.04bc 11.34 ± 0.41a 112.16 ± 3.31a 16.08 ± 0.17d 18.55 ± 1.15abc 13.34 ± 0.48ab 2.57 ± 0.20ab 2.31 ± 0.20ab nq nq 0.04 ± 0.01ab 0.16 ± 0.01c 1.50 ± 0.03a 0.28 ± 0.01ab 2.62 ± 0.08b nq 0.65 ± 0.01cd 4.88 ± 0.07bc 5.12 ± 0.13cd 4.48 ± 0.07c 9.79 ± 0.50ab 115.58 ± 4.60a 39.66 ± 0.58bcd 13.28 ± 0.97de 9.19 ± 0.27d 0.84 ± 0.04cd 0.51 ± 0.05cd 0.49 ± 0.16b 0.68 ± 0.14b 0.21 ± 0.01cd 0.15 ± 0.01c nq 0.73 ± 0.04bc 5.36 ± 0.11ab 0.29 ± 0.01cde 1.06 ± 0.02bc nq nq

aef

Means within a row with different superscript letters differ (P < 0.05). nq: below limit of quantification.

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Please cite this article in press as: Baert, N., et al., Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.11.003

Table 2 Inter-population variations of the concentration of the main polyphenols in the leaves and flowers of Epilobium angustifolium. Results are expressed as mean ± SEM (n ¼ 10).

N. Baert et al. / Phytochemistry xxx (2016) 1e10

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Fig. 2. Matrices of Pearson product-moment correlation coefficients between the concentrations of the main polyphenols of Epilobium angustifolium within leaves (A) and flowers (B). A blank square means a non-significant correlation (P ¼ 0.05, n ¼ 100).

flowers do not significantly change during the process of blooming. However, we did not examine anthocyanin levels which have been shown to vary during flower development (Justesen et al., 1997; Schmitzer et al., 2009). It is also possible that the developmental difference between the flower buds and the open flowers that we collected was not large enough to visualise differences in the polyphenol fingerprint. Repeated sampling throughout the entire flowering season would provide a more comprehensive understanding of the fluctuations of polyphenols that occur in the flowers of rosebay willowherb.

2.3.3. Stem parts Of all analysed tissues, stem A showed the largest content in total polyphenols with an average of 252 mg/g dry wt, of which 90% was made by oligomeric ETs 2, 3 and 4. In comparison, within the same population, leaf and flower tissues contained 157 and 178 mg/ g dry wt, respectively. Moreover, stem A contained the highest levels of oenothein A and B compared to all other organs (P < 0.05), showing concentration that were twice as much as those found in leaves (for oenothein B) and flowers (for oenothein A) within the same population (Table 3). The polyphenol composition of stem A featured characteristics from both leaves and flowers (Table 3). Like leaves, it did not contain the flavonol rhamnosides that are characteristic of flowers (except very small amount of 18). The levels of neochlorogenic acid (1) and quercetin-3-O-glucuronide (15) in stem A, however, were identical to those found in flowers (Table 3). Furthermore, we observed a declining gradient in polyphenol content along the stem. The concentration of polyphenols quickly decreased along the top half of the stem and remained relatively

constant throughout the bottom half, with stem parts C, D and E showing similar fingerprints (Fig. 2). This result indicates that, unlike the vegetative stem, the inflorescence shoot accumulates relatively high levels of polyphenols at its apex and, more specifically, high levels of oenothein B and A (Table 3). This could be part of a defence strategy aimed at protecting floral meristems from pathogens and insect herbivores in order to increase the reproductive success of the plant. There is, indeed, strong evidence that ETs are involved in the defence mechanisms of plants against insects and pathogens (Barbehenn and Constabel, 2011). Increased production of oenothein A, in particular, was found to be an adaptive trait favoured by natural selection in Oenothera biennis (Agrawal et al., 2012; Johnson et al., 2009).

2.4. Intra- and inter-organ correlations In addition to measuring polyphenol concentration in various organs, we examined the relationships that existed between those compounds within the leaves, within the flowers and between leaves and flowers of each individual plant. Concentrations of tetra- to heptameric ET strongly correlated with each other in leaves and flowers (0.72  r  0.94), meaning that high levels of tetramer were always associated with high levels of pentamer, hexamer, heptamer and reciprocally (Fig. 3). This observation seems to indicate that the biosynthesis of tetra- to heptameric ET could be the work of a single enzyme which elongates oligomers larger than dimer. The concentrations of oenothein B (3) and A (4), however, did not correlate with each other, thus suggesting that they are produced by two different enzymes.

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Table 3 Inter-organ distribution of the main polyphenols of Epilobium angustifolium. Results are expressed as mean ± SEM (n ¼ 10). Compound

#

Flowers

Flower buds

Leaves

Stem A

Stem B

Stem C

Stem D

Stem E

Neochlorogenic acid Oxidised oenothein A Oenothein B Oenothein A Tetramer Pentamer Hexamer Heptamer Myricetin-3-O-Glc Myricetin-3-O-GlcA Quercetin-3-O-(600 -galloyl)-Gal Quercetin-3-O-(galloyl)-hexoside Myricetin-3-O-Rha Quercetin-3-O-Gal Quercetin-3-O-GlcA Quercetin-3-O-Ara Kaempferol-3-O-GlcA Quercetin-3-O-Rha Kaempferol-3-O-Rha

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.11 ± 0.04b 7.15 ± 0.22a 83.37 ± 1.54b 25.12 ± 0.80c 24.5 ± 1.40a 15.51 ± 0.60a 3.42 ± 0.28a 2.58 ± 0.23b nq nq nq 0.20 ± 0.01a 1.45 ± 0.06a nq 1.61 ± 0.04b nq 0.63 ± 0.02a 5.09 ± 0.10a 6.76 ± 0.26b

1.10 ± 0.04b 7.38 ± 0.51a 75.44 ± 2.83bc 22.83 ± 0.30c 21.29 ± 1.07ab 13.99 ± 0.47ab 3.02 ± 0.37ab 2.15 ± 0.28b nq nq nq 0.18 ± 0.01a 1.24 ± 0.04b 0.81 ± 0.09a 1.49 ± 0.06b nq 0.64 ± 0.03a 5.16 ± 0.07a 7.91 ± 0.22a

4.71 ± 0.10a 6.13 ± 0.35ab 66.09 ± 1.70c 43.18 ± 1.22b 18.97 ± 1.32b 9.99 ± 0.34c 0.89 ± 0.1cd 0.64 ± 0.07a nq nq 0.10 ± 0.01 0.21 ± 0.01a nq 0.10 ± 0.01b 5.23 ± 0.10a 0.16 ± 0.01 0.41 ± 0.03b nq nq

1.36 ± 0.07b 7.65 ± 0.74a 148.28 ± 4.35a 52.14 ± 1.64a 25.62 ± 1.36a 12.36 ± 0.38b 2.00 ± 0.24bc 1.26 ± 0.16a nq nq nq 0.14 ± 0.01b nq nq 1.48 ± 0.07b nq 0.10 ± 0.01c 0.13 ± 0.01b nq

0.47 ± 0.02c 7.16 ± 0.51a 51.32 ± 1.68d 24.32 ± 0.97c 7.63 ± 0.38c 1.37 ± 0.06d 0.71 ± 0.07d 0.41 ± 0.05a nq nq nq nq nq nq 0.81 ± 0.05c nq nq nq nq

0.29 ± 0.01c 4.46 ± 0.32b 26.61 ± 0.70e 16.34 ± 0.41d 2.44 ± 0.16cd 0.55 ± 0.03d 0.17 ± 0.02d nq nq nq nq nq nq nq 0.34 ± 0.01d nq nq nq nq

0.24 ± 0.01c 3.88 ± 0.20b 21.00 ± 0.87e 13.49 ± 0.55d 1.54 ± 0.14cd 0.43 ± 0.03d 0.13 ± 0.03d nq nq nq nq nq nq nq 0.26 ± 0.02d nq nq nq nq

0.02 ± 0.02c 4.17 ± 0.21b 17.01 ± 1.00e 11.5 ± 0.31d 1.10 ± 0.06cd 0.31 ± 0.01d 0.03 ± 0.02d nq nq nq nq nq nq nq 0.09 ± 0.03d nq nq nq nq

aee Means within a row with different superscript letters differ (P < 0.05). nq: below limit of quantification.

Additionally, we observed that certain populations of willowherb tended to produce more oligomeric ETs at the expense of the main quercetin glycosides (14, 15, 16 and 18) and reciprocally (Fig. 2). Populations #1 and #7 clearly illustrate this trend by standing at both ends of the spectrum. Flowers of population #1 exhibited the lowest concentration of large ETs (sum of compounds 4 to 8) with an intra-population average of 40.4 mg/g whereas the sum of quercetin glycosides (14 þ 15 þ 18) coincidentally showed the highest value with an intra-population average of 9.4 mg/g. A similar but less pronounced trend was also observed with the leaves of that population. Conversely, the sum of ETs 4 to 8 in leaves of population #7 ranked the highest with an intra-population

average of 96.6 mg/g and the sum of quercetin glycosides (14 þ 15 þ 16) showed the lowest intra-population average with only 4.1 mg/g. Those correlations are likely the result of a metabolic trade-off whereby some plants invest more carbon and energy toward the production of either large oligomeric ETs or quercetin glycosides. Such trade-off could stem from the regulation of the synthesis of gallic acid. The biosynthesis of gallic acid competes with the formation of shikimic acid which is a precursor of p-coumaroyl-CoA, an essential building block of the carbon skeleton of flavonoids (Dewick and Haslam, 1969; Falcone Ferreyra et al., 2012; Ossipov et al., 2003). Therefore, an upregulation of the biosynthesis of

Fig. 3. Principal Component Analysis biplot showing the score of each plant sample on PC1 and PC2, and the loadings of the dependent variables on PC1 and PC2.

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N. Baert et al. / Phytochemistry xxx (2016) 1e10

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gallic aciddnecessary for the production of ETsdwould induce a decreased production of p-coumaroyl-CoA, ultimately resulting in a decreased production of some flavonoids. The observation of inter-organ correlations shows that the negative association between ETs and quercetin is not limited to single organs but is visible across the entire plant. This was particularly clear with the concentration of 18 in flowers which exhibited a strong negative relationship (0.75  r  0.61) with the concentration of ETs 4 to 8 in leaves. The trade-off between ETs and quercetin glycosides observed in the 10 sampled populations may stem from various origins. It could be the result of different genotypes or environmental factors, or simply due to differences in the maturity of plants. Multiple sampling throughout the growing season would help us understand the dynamic of the production of polyphenols and may give a better view of the interdependency relationships linking those compounds.

2.5. Inter-population variations For most polyphenols, the interpopulational variation was moderate as their concentrations varied only two- to threefold across the 10 populations. A few polyphenols, however, exhibited a more prominent contrast. Myricetin-3-O-glucoside (9) and myricetin-3-O-glucuronide (10) showed the largest inter-population fluctuations. Compound 9 varied from being below the limit of quantification in 6 out of 10 populations to being the second most abundant flavonol glycoside in the leaves of population #3, reaching the average concentration of 2.31 mg/g dry wt (Table 2). Interestingly, the concentrations of 9 and 10 showed one of the strongest positive association among all polyphenols (r ¼ 0.89), thus highlighting their common biosynthetic origin (Fig. 3). Quercetin-3-O-galactoside (14) was undetected in flowers of populations #6 and #7 but showed relatively similar levels in all other populations, ranging from 0.15 to 0.38 mg/ g dry wt. Quercetin-3-O-(600 -galloyl)-galactoside (11) was present at very low concentration in the leaves of 6 populations but was not quantifiable in the other 4 populations (Table 2). Its concentration in flowers, on the other hand, ranged from 0.10 to 0.56 mg/g dry wt. However, we observed no relationship between the concentration of 11 in leaves and its concentration in flowers (Fig. 4). Finally, hexamer and heptamer varied by up to 260% and 470% respectively, whereas all other ETs remained within a threefold range of variation. The ratio of oenothein A/oenothein B (OA/OB) in leaves and in flowers varied substantially between populations but was very consistent within each population (Table 4). We thus observed contrasting phenotypes in the relative proportion of those two molecules. Populations #1 and #10, for instance, are characterized by low OA/OB ratios (0.17 and 0.14, respectively in flowers) whereas, at the other end of the spectrum, population #3 shows a high OA/OB ratio (0.39 in flowers). All the other populations exhibit intermediate values. These patterns resemble those observed earlier with Oenothera biennis, in which extreme genotypes can be classified as either oenothein B- or oenothein A-dominant and other genotypes are found with intermediate proportions of these major metabolites (Agrawal et al., 2012; Johnson et al., 2009). In contrast to O. biennis, we did not witness any oenothein A-dominant individuals in E. angustifolium in this study or in our earlier studies where we accumulated E. angustifolium tissues for ellagi€ m et al., 2016; tannin purification (Baert et al., 2015; Engstro Karonen et al., 2015; Moilanen et al., 2015; Moilanen and Salminen, 2008). These results show that even within a small sampling area of 4 km2 we can observe clear, contrasting phenotypes regarding the

Fig. 4. Matrix of Pearson product-moment correlation coefficients between the concentrations of the main polyphenols in the leaves and flowers of Epilobium angustifolium. A blank square means a Pearson's correlation coefficient jrj < 0.1 or a nonsignificant correlation (P ¼ 0.05, n ¼ 100).

production of the two major water-soluble polyphenolic constituents of E. angustifolium. Analyses of populations over a wider geographic area may give some insight about the roles of certain polyphenols in the fitness of the plant and the environmental factors that drive the natural selection of these molecules. A recent study showed that O. biennis populations can be characterized by their oenothein A to B ratio and that this pattern is driven also by latitude: populations growing at higher latitudes produced more oenothein B and less oenothein A (Anstett et al., 2015). Such latitudinal patterns are yet to be studied with E. angustifolium, since our plants grew at high latitudes and were characterized by higher oenothein B and lower oenothein A levels similarly to O. biennis. These patterns, however, are very likely to affect the biological activity of E. angustifolium and its potential to

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N. Baert et al. / Phytochemistry xxx (2016) 1e10

Table 4 Ratio of oenothein A/oenothein B in 10 populations of Epilobium angustifolium. Results are expressed as mean (SD). OA/OB ratio in Flowers Leaves

Willowherb populations #1

#2 f

0.17 (<0.01) 0.34e (0.03)

#3 bcd

0.27 (<0.01) 0.70b (0.07)

#4 a

0.39 (0.02) 0.84a (0.04)

#5 e

0.23 (0.01) 0.67b (0.12)

#6 bc

0.28 (0.01) 0.65b (0.05)

#7 b

0.30 (0.03) 0.65b (0.04)

#8 cde

0.24 (0.02) 0.50cd (0.02)

#9 de

0.24 (0.02) 0.61bc (0.04)

#10 cde

0.26 (0.03) 0.42de (0.03)

0.14f (0.01) 0.34e (0.04)

aef

Means within a row with a different superscript letter differ (P < 0.05).

be used, for example, as an active feed additive for ruminants. It has been indeed shown that trimeric and tetrameric TI are more potent than oenothein B in protein-binding activity (Karonen et al., 2015) and in inhibiting the activity of methanogenic bacteria found in the rumen of cows (Baert et al., 2016). Therefore, oenothein A-rich populations and plant parts should be favoured in order to maximize these types of activities. Regarding the anthelmintic activity, however, the proportions of different oligomeric ETs seems less € m et al. relevant than their total concentration as shown by Engstro (2016). Nevertheless, for each of the aforementioned activity, the inflorescence shoot is predictably the most bioactive part of E. angustifolium with the highest levels of trimer, tetramer and total ETs. 3. Conclusion The main result of this study is the inter-organ distribution pattern of the polyphenols of E. angustifolium. The most distinct feature in this pattern concerned the distribution of flavonol rhamnosides which were the most abundant flavonol glycosides in flowers but were absent from leaves. Additionally, we found that the apex of the inflorescence shoot exhibited the highest content in total polyphenols and was particularly rich in oenothein A and B. Furthermore, the concentration of all polyphenols decreased along the inflorescence shoot and reached a minimum in the vegetative part of the stem. The study of inter-population variations in polyphenol content revealed that populations of E. angustifolium can be characterized by the relative abundance of oenothein A over oenothein B in the whole plant. We also showed that myricetin-3O-glucoside, myricetin-3-O-glucuronide, quercetin-3-O-(600 -galloyl)-galactoside and quercetin-3-O-galactoside were specific to certain populations. 4. Experimental 4.1. General experimental procedures Plant samples were analysed by UHPLC-DAD-ESI-QqQ-MS with an Acquity™ UPLC (Waters Corp., Milford, MA, USA) coupled with a XEVO™ TQ triple-quadrupole mass spectrometer (Waters Corp., Milford, MA, USA). The column was an Acquity UPLC BEH Phenyl 1.7 mm, 2.1  100 mm (Waters Corp., Wexford, Ireland). Elution was carried out with a binary solvent system consisting of acetonitrile (A) and 0.1% aqueous formic acid (B) at a constant flow rate of 0.5 ml min1. Elution pattern was the following: 0e0.5 min: 0.1% A; 0.5e5.0 min: 0.1e30% A (linear gradient); 5.0e5.1 min: 30e90% A (linear gradient); 5-1e7.1 min: 90% A (washing); 7.1e7.2 min: 90e0.1% A (linear gradient); 7.2e8.5 min: 0.1% A (stabilisation). UVeVis (190e500 nm) and MS data (m/z 100 to 2000) were recorded from 0 to 6 min. The electrospray was set on negative mode. Capillary voltage was 3.4 kV; desolvation temperature: 650  C; source temperature: 150  C; desolvation gas and cone gas (N2) flow rate: 1000 and 100 l h1, respectively; collision gas: argon. Identification of the oxidised oenothein A derivative was carried out by UHPLC-DAD-ESI-Orbitrap-MS. The UHPLC unit, DAD, column

and elution gradient were identical to those described above. The mass spectrometer was a Thermo Scientific™ Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer with an ESI source set on negative mode. Spray voltage 3.0 kV; capillary temperature 380  C; S-lens RF level 60. Full MS scan and MS2 of the main ions were recorded using the Full MS/dd-MS2 (Top5) procedure of the Thermo Q Exactive software. 4.2. Plant material Samples of Epilobium angustifolium L. (Onagraceae) were collected from 10 distinct populations located around the Aura River in Turku (Southwest Finland) in July 2012. The sampled populations were located at the following GPS coordinates (latitude; longitude): #1 (60.479329; 22.287653), #2 (60.455512; 22282835), #3 (60.458527; 22.290842), #4 (60.460387; 22.292889), #5 (60.462790; 22.284113), #6 (60.460737; 22.299457), #7 (60.461479; 22.277219), #8 (60.465219; 22.284897), #9 (60.466325; 22.302436), #10 (60.467136; 22.305477). From each population, 10 individual plants were harvested. Quickly after collection, samples were brought back to the laboratory. For the study of inter-population variations, 5 leaves and 5 flowers were cut off each individual plant from populations #1 to #10. For the study of polyphenol distribution across organs we utilised plants from population #6 only and those individuals were subjected to further sampling. Additionally to leaves and flowers, we also collected 5 flower buds and 5 pieces of stem. Stem pieces were 5 cm long and were collected at 5 different heights along the stem. They were labelled with letters “A” to “E”. Stem A was taken from the apex of the plant. Stem B corresponded to the bottom of the inflorescence. Stem C, D and E corresponded to the vegetative part of the stem; they were equally spaced from each other and spanned from the middle of the stem to its very bottom. Every pieces of stem were thoroughly cleared of petioles and pedicels. Before extraction, all plant samples were frozen, lyophilised and ground to a fine homogeneous powder with a ball-mill. 4.3. Statistical calculations Principal component analysis was performed to visualise the inter-organ distribution of polyphenols across 8 different plant parts from 10 individuals belonging to the same population. Pearson product-moment correlation matrices were computed in order to observe potential relationships between polyphenols within leaves, within flowers and between leaves and flowers within the same plant. Statistical evaluation of the quantitation data was performed by one-way ANOVA followed by Scheffe's method of pairwise comparison of means. All statistical calculations were carried out using the software R (R Core Team, 2016). 4.4. Sample preparation Samples were prepared for quantitative analysis according to a previously described protocol (Baert et al., 2015). Shortly, 10 mg of ground lyophilised plant samples were extracted twice with

Please cite this article in press as: Baert, N., et al., Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.11.003

N. Baert et al. / Phytochemistry xxx (2016) 1e10

1400 mL acetone/water (4:1 v/v). The acetone was evaporated from the combined extracts and the remaining water was removed by lyophilisation. The samples were then dissolved in 500 mL of ultrapure water, vortexed and filtered (0.2 mm, PTFE). An additional 1:31 dilution of the extract was prepared for each sample. Both extracts (diluted and non-diluted) were analysed by UHPLC-DADESI-QqQ-MS. The non-diluted extracts were used to quantify hexameric and heptameric ETs, the diluted extracts were used to quantify all the other compounds. 4.5. Compound identification The quantified polyphenols were identified by comparing retention time, UV absorption maxima, m/z values of pseudomolecular ion and fragments with previously published data. Quercetin-3-O-galactoside and 5-caffeoylquinic acid were identified by comparison with commercial standards (see Table 1). 4.6. Quantitative analyses Tetrameric to heptameric tellimagrandin I-based oligomeric ETs (compounds 5 to 8) were quantified by UHPLC-ESI-QqQ-MS, using MRM (Multiple Reaction Monitoring) transitions that had been developed and optimised in a previous work (Baert et al., 2015). Oenothein B, oenothein A and compound 2 were quantified by UHPLC-DAD at 280 nm. The concentrations of all the oligomeric ETs (compounds 3 to 8) were calculated using the purified molecules as external calibration standards. The concentration of compound 2 was expressed as oenothein A equivalent. The quantitative measurements of flavonoid glycosides and 5-O-caffeoylquinic acid were done by UHPLC-DAD at 349 nm. Myricetin-3-O-rhamnoside, kaempferol-3-O-glucoside, quercetin-3-O-glucoside and chlorogenic acid were used as external calibration standards for the quantification of myricetin glycosides, kaempferol glycosides, quercetin glycosides and 5-O-caffeoylquinic acid, respectively. 4.7. Solvents and chemicals Technical grade acetone from VWR (Haasrode, Belgium) was used for the extractions. LC-MS CHROMASOLV acetonitrile (SigmaAldrich, Steinheim, Germany) was used for UHPLC-DAD-ESI-QqQMS analyses. Water was purified with a Millipore Synergy water purification system from Merck KGaA (Darmstadt, Germany). Formic acid 98e100% was from Sigma-Aldrich (Seelze, Germany). Myricetin-3-O-rhamnoside, kaempferol-3-O-glucoside, quercetin3-O-galactoside and quercetin-3-O-glucoside were purchased from ExtraSynthese (Genay, France). Chlorogenic acid was purchased from Biopurify Phytochemicals Ltd (Chengdu, China). Acknowledgements These investigations were supported by the European Commission (PITN-GA-2011-289377, “LegumePlus” project) and by the Academy of Finland (Grant no. 258992 to J.-P.S. and 251388 to M.K.) and the Turku University Foundation (Grant to N.B.). The Strategic Research Grant (Ecological Interactions) enabled the use of the UPLC-MS/MS instrument. References Agrawal, A.A., Hastings, A.P., Johnson, M.T.J., Maron, J.L., Salminen, J.-P., 2012. Insect herbivores drive real-time ecological and evolutionary change in plant populations. Sci. (80-) 338, 113e116. http://dx.doi.org/10.1126/science.1225977. Anstett, D.N., Ahern, J.R., Glinos, J., Nawar, N., Salminen, J.-P., Johnson, M.T.J., 2015. Can genetically based clines in plant defence explain greater herbivory at higher latitudes? Ecol. Lett. 18, 1376e1386. http://dx.doi.org/10.1111/ele.12532.

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Please cite this article in press as: Baert, N., et al., Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.11.003