Identification and occurrence of phenylethanoid and iridoid glycosides in six Polish broomrapes (Orobanche spp. and Phelipanche spp., Orobanchaceae)

Phytochemistry 170 (2020) 112189

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Identification and occurrence of phenylethanoid and iridoid glycosides in six Polish broomrapes (Orobanche spp. and Phelipanche spp., Orobanchaceae)

T

Dariusz Jedrejeka,∗, Sylwia Paweleca, Renata Piwowarczykb, Łukasz Pecioa, Anna Stochmala a b

Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, 24-100, Puławy, Poland Department of Botany, Institute of Biology, Jan Kochanowski University, 25-406, Kielce, Poland

ARTICLE INFO

ABSTRACT

Keywords: Broomrape Orobanche Phelipanche Phenylethanoid glycosides Iridoids NMR HR-QTOF/MS

There are about 200 holoparasitic broomrapes (Orobanchaceae) known worldwide, however, only several species have been so far investigated phytochemically. Among Orobanche s.l. are both rare and endangered species, as well as onerous crop pests. This study aims to give a phytochemical description, both qualitative and quantitative, of six broomrape species (Orobanche and Phelipanche taxa) growing in Poland, including species that have not been tested in detail (O. caryophyllacea, O. lutea, O. picridis, and P. arenaria). Sixteen metabolites, including 14 phenylethanoid glycosides (PhGs) and 2 iridoid glycosides (IrGs), were isolated and identified using NMR spectroscopy and hydrolysis, revealing the presence of two previously undescribed PhGs in P. ramosa, named ramoside A and 2′-acetylramoside A. In addition, in the example of O. caryophyllacea, we have reported as the first occurrence of IrGs in broomrapes. Concentrations of phenylethanoids, the main constituents of broomrapes, in the studied plant material (flowering shoots with haustoria) were determined using the UHPLCPDA method. It was found that P. ramosa has been the richest source of PhGs. In addition, the differences between broomrapes have been visualized using principal component and cluster analysis. The results of the antiradical DPPH test of 13 PhGs confirmed previous findings on the relation of the antioxidant potential with the structure of phenolic moieties – phenolic acid and phenylethanoid unit.

1. Introduction The broomrape is a general name for parasitic herbaceous plants of the Orobanche L. sensu lato genus (Orobanchaceae family). There are about 200 species of broomrapes known worldwide but these plants are mainly found in the temperate climate zone of the northern hemisphere, with the greatest density in Mediterranean Basin, western and central Asia (Pusch and Günther, 2009; Piwowarczyk et al., 2019). In Poland, the genus comprises 17 native species, which are mostly rare and endangered, such as Orobanche coerulescens Stephan ex Willd., and O. picridis F.W. Schultz, nevertheless, the occurrence of serious agricultural pest, Phelipanche ramosa (L.) Pomel has also been confirmed. The area of their greatest concentration in Poland includes the belt of the Polish Uplands (Piwowarczyk et al., 2011). Broomrapes are non-photosynthetic (chlorophyll-lacking) root holoparasites that are totally dependent on their host plants. The number of preferred hosts is variable, and parasitic species can be divided into three groups: monophagous, oligophagous (dominating) and polyphagous (Piwowarczyk et al., 2019). All broomrapes have a specially adapted intrusive organ, haustorium, that functions as a bridge

∗

connecting the parasite with its host, and allows the uptake of water and essential nutrients (Heide-Jorgensen, 2008). In addition, other molecules, such as nucleic acids, proteins, and bioactive specialized metabolites, can be transported both ways with this channel. For example, there are few reports on the sequestration of phytochemicals, such as alkaloids, by Orobanche spp. from their host species (Scharenberg and Zidorn, 2018). Broomrapes spend most of their life cycle attached to the host plant and hidden underground and become visible above the surface of the soil only at the time of flowering, in the form of short, scaly stems ended with yellow, purple, white or bluish flowers. The identification of parasites within the Orobanche taxon, due to a small number of morphological characteristics (significant reduction of vegetative organs) and high morphological variability, poses many difficulties (Paran et al., 1997). The taxonomy and phylogeny of the Orobanche genus are still discussed contentiously. At the beginning of the 21st century, on the basis of combined results of morphological, micro-morphological and molecular analyses, the Orobanche genus was divided into two separate genera, Orobanche and Phelipanche (Schneeweiss, 2007; Joel, 2009). Therefore, some species of the old Orobanche genus have been

Corresponding author. E-mail address: [email protected] (D. Jedrejek).

https://doi.org/10.1016/j.phytochem.2019.112189 Received 11 July 2019; Received in revised form 5 October 2019; Accepted 26 October 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

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transferred to the newly created Phelipanche genus, an example of which are Phelipanche ramosa (L.) Pomel and P. aegyptiaca (Pers.) Pomel, formerly described as O. ramosa L. and O. aegyptiaca Pers. The chemotaxonomic analyses in combination with statistics (multivariate methods) have been found as a good auxiliary tool in the case of difficulty in distinguishing and classification of plant taxa using standard botanical methods (Martucci et al., 2014). An important factor in plant chemotaxonomy studies is the occurrence of taxon-specific chemical markers, such as phytochemicals, present only in representatives of a given family/genus/species. The Orobanchaceae family is known for the richness of metabolites from the group of phenylethanoid glycosides (PhGs) (Scharenberg and Zidorn, 2018). Within the Orobanchaceae, the Cistanche species, due to the extensive use of Herba Cistanche in Chinese traditional medicine, especially in the treatment of genitourinary system deficiencies, have been best recognized for their phytochemical composition. To date, more than 35 different PhGs have been isolated and identified in Cistanche plants, together with some other minor constituents, including benzyl glycosides, iridoids, and lignans (Jiang and Tu, 2009). In turn, among about 200 Orobanche and Phelipanche species, only several have been so far investigated phytochemically, including O. cernua Loefling, O. coerulescens, and P. ramosa, however, their qualitative chemical composition proved to be similar to Cistanche spp. (PhGs as major components, and other minor accompanying compounds, such as flavones (Qu et al., 2015) or terpenes, have been described) (Scharenberg and Zidorn, 2018). Extracts from either Cistanche or broomrape plants, as well as some phenylethanoid glycosides isolated from them (such as acteoside, echinacoside and crenatoside), were positively tested for a variety of biological activities including antioxidant, radical scavenging, anti-inflammatory, and memory-enhancing (Scharenberg and Zidorn, 2018), cytotoxic against cancer cell lines (Qu et al., 2016), and antibacterial and antifungal (Genovese et al., 2019). Additionally, as recently reported by Renna et al. (2018), stems of Orobanche crenata Forssk. (crenate broomrape) – noxious weed of the legume crops, serve as a culinary delicacy in the Puglia region (southern Italy). The nutritional and culinary properties of crenate broomrape have been compared to asparagus, while the parasite has possessed several times higher antioxidant activity; thus, the broomrape plant has potential as a novel functional food. Nevertheless, despite the confirmed presence of phenylethanoid glycosides in O. crenata (Scharenberg and Zidorn, 2018), Renna et al. (2018) did not mention which ingredients of this plant are responsible for the high anti-oxidative action demonstrated in their research. The aim of the phytochemical study was to perform a qualitative and quantitative investigation of six broomrape species growing in Poland, including plants that have not been tested in detail: Orobanche caryophyllacea Sm., O. lutea Baumg., O. picridis F.W. Schultz and Phelipanche arenaria Pomel. The results of the quantitative content of phenylethanoid glycosides, main constituents of all studied broomrapes, were used to compare the tested plants using chemometric methods (principal component analysis and cluster analysis). In addition, isolated PhG compounds were tested for antioxidant activity using DPPH method.

time). As can be seen in Fig. 1, the phytochemical profiles of broomrapes belonging to the genus Orobanche (Fig. 1A and B) were different compared to both Phelipanche spp, which were also significantly different from each other (Fig. 1C and D). Additionally, the species O. caryophyllacea, due to the exclusive presence of two compounds tentatively identified as iridoid glycosides (compd. 1 and 2, Fig. 1A), could be distinguished from other three species of the genus Orobanche (O. coerulescens, O. lutea and O. picridis; Fig. 1B), which, in turn, showed far-reaching phytochemical similarity, confirmed further by the results of quantitative and chemometric analyses. Due to the lack or incompleteness of literature data on some broomrape species, including O. caryophyllacea, O. lutea, O. picridis and P. arenaria, and some difficulties in identifying phenylethanoid glycosides (the presence of isobaric compounds with similar spectroscopic properties) a decision was made to isolate the main plant specialized metabolites and their subsequent identification based on spectroscopic and chemical methods. From the 18 major constituents of the set of studied broomrapes, we were able to isolate 16 compounds (1–2, 4–15, 17–18; according to Table 1) by the use of the multistep purification process, and they were further identified on the basis of NMR spectroscopy, LC-MS analyses of products of their hydrolysis, and literature data. While compounds 3 and 16 (Table 1) could not be isolated and were tentatively described on the basis of UHPLC-HR-MS/MS and literature data. Summarizing, two undescribed (10 and 17, Table 1) and sixteen already known natural products (1–9, 11–16, and 18; Table 1) were identified in the set of studied broomrape species grown in Poland. The structures of all detected phytochemicals are shown in Fig. 2. Among already described compounds were 14 PhGs: echinacoside (3) (Becker et al., 1982), arenarioside (4) (Andary et al., 1985a), acteoside (syn. verbascoside) (5) (Andary et al., 1982), tubuloside A (6) (Kobayashi et al., 1987), poliumoside (7) (Andary et al., 1985b), isoacteoside (8) (Kobayashi et al., 1987), crenatoside (syn. orobanchoside) (9) (Afifi et al., 1993), 3-O-methylpoliumoside (11) (Mostafa et al., 2007), pheliposide (12) (Andary et al., 1985a), leucosceptoside A (13) (Miyase et al., 1982), 2′-O-acetylacteoside (14) (Kobayashi et al., 1987), 2′-O-acetylpoliumoside (syn. brandioside) (15) (Lahloub et al., 1991), 2′-O-acetylisoacteoside (syn. tubuloside B) (16) (Kobayashi et al., 1987) and wiedemannioside D (18) (Abougazar et al., 2003), and 2 iridoid glycosides: asperulosidic acid (1) (Wang et al., 1999) and asperuloside (2) (Inouye et al., 1969) (Fig. 2). The 1H and 13C NMR data of known isolated compounds (1–2, 4–9, 11–15, and 18) are presented in the Supplementary Materials (Tables 1S–4S). Additionally, some general conclusions can be drawn regarding the MS (ESIneg) fragmentation of phenylethanoid glycosides (Table 1): for all described PhG compounds, the most intense (100%) fragment ion represented hydroxycinnamic acid moiety (m/z 161 [CA-H-H2O]– or m/z 175 [FA-HH2O]– or m/z 145 [p-CoA-H-H2O]–), and the second most intense (about 20%) fragment ion in MS/MS spectra of most PhGs was a product of detachment of a phenolic acid fragment ([phenolic acid-H2O]– for nonacetylated PhGs or [phenolic acid-H2O+42(acetyl)]– for acetylated PhGs) from a deprotonated ion [M-H]–. Compound 10 was a previously undescribed phenylethanoid glycoside isolated from P. ramosa, and received a trivial name ramoside A. The negative- and positive-ion HR-QTOF-MS spectra of 10 showed deprotonated molecule at m/z 753.2593 [M-H]– and adduct ions at m/z 772.3013 [M+NH4]+ and 755.2756 [M+H]+, respectively, and its molecular formula was determined as C35H46O18. In the MS/MS spectrum of ion peak at m/z 755.2756 the following fragment ions were observed: m/z 609.2171 [M+H-146(deoxyhexosyl)]+, m/z 463.1587 [M+H-292(deoxyhexosyl-deoxyhexosyl)]+, m/z 455.1539 [M+Hm/z 309.0957 [M+H300(deoxyhexosyl-hydroxytyrosol)]+, 446(deoxyhexosyl-deoxyhexosyl-hydroxytyrosol)]+, m/z 147.0434 [pCoA+H–H2O]+. The 13C-NMR of compound 10 showed 32 signals that were classified as two CH3, three CH2, 21 CH, and six quaternary carbon atoms (Table 2). The 1H-NMR spectrum of 10 demonstrated signals

2. Results and discussion Preliminary UHPLC-PDA-HR-MS/MS analyses of the extracts from the whole plants of six broomrape species, including Orobanche caryophyllacea, O. coerulescens, O. lutea (two different host plants), O. picridis, Phelipanche arenaria, and P. ramosa (two different host plants), revealed the presence of several metabolites tentatively identified as phenylethanoid glycosides (PhGs), which are known constituents of Cistanche and Orobanche plants. In Fig. 1, a UHPLC-PDA (max plot) chromatograms of the studied holoparasites are presented, and the identified compounds were marked with numbers that are in accordance with Table 1 (numbers were assigned according to retention 2

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Fig. 1. Representative UHPLC-PDA chromatograms of extracts from six studied broomrape species: Orobanche caryophyllacea (A); O. coerulescens, O. lutea (parasitizing on Medicago falcata and M. sativa) and O. picridis (B, all three species showed far-reaching phytochemical similarity); Phelipanche arenaria (C), and P. ramosa (parasitizing on tobacco and tomato plants) (D).

corresponding to: hydroxytyrosol moiety – two methylenes [δH 2.79, 2.82 (1H each, H2-7), and 3.73, 3.99 (1H each, H2-8)], and ortho- and meta-coupled ABC-type aromatic protons [δH 6.57 (1H, H-2), 6.69 (1H, H-3), and 6.70 (1H, H-6); β-glucopyranosyl unit [δH 4.37 (1H, H-1′)]; two α-rhamnopyranosyl units [δH 5.19 (1H, H-1″), and 4.63 (1H, H1‴)]; and a trans-p-coumaroyl moiety – a trans-olefin protons [δH 6.34 (1H, H-8‴′), and 7.67 (1H, H-7‴′)], and AA’XX’-type aromatic protons [δH 6.81, 7.47 (2H each, H-3‴′,5‴′ and 2‴′, 6‴′) (Table 2). Connection locations between sugar and acyl moieties in 10 were revealed by an HMBC spectrum, which showed correlations between the following proton-carbon pairs: H-1′(Glc) and C-8 (hydroxytyrosol, δC 72.4), H-4′ [Glc, δH 5.00 (1H)] and the p-coumaroyl carbonyl carbon (δC 168.0, C9‴′), H-1′′ (Rha) and C-3′ (Glc, δC 81.6), H-1‴ (Rha) and C-6′ (Glc, δC 67.6). The α/β-orientation of anomeric protons was based on measurements of direct 1JHC coupling constants using F2-coupled perfect CLIP-HSQC spectra (Castañar et al., 2015) – 159 Hz for H-1′-C-1′ of Glc, 171 Hz for H-1″-C-1″ of Rha and 169 for H-1‴-C-1‴ of Rha (Bubb, 2003). Finally, acid hydrolysis of 10 and subsequent UHPLC-PDA-MS/ MS analysis of derivatization reaction products allowed to identify Dglucose and L-rhamnose. Therefore, the structure of ramoside A was elucidated to be 2-(3,4-dihydroxyphenyl)ethyl O-α-L-rhamnopyranosyl-(1 → 3)-[α-L-rhamnopyranosyl-(1 → 6)]-4-O-trans-p-coumaroylβ-D-glucopyranoside (10) (Fig. 2). Compound 17, isolated from P. ramosa, was an acylated derivative of ramoside A (10). The negative- and positive-ion HR-QTOF-MS spectra of 17 showed deprotonated molecule at m/z 795.2695 [M-H]– and adduct ions at m/z 814.3121 [M+NH4]+ and 797.2832 [M+H]+, respectively, and its molecular formula was determined as C37H48O19. The 1H and 13CNMR spectra of 17 were very similar to those of 10, except for additional signals due to the presence of an acetyl group (Table 2). Its location has been set at C-2′ based on HMBC correlation between H-2′ [Glc, δH 4.87 (1H)] and the acetyl carbonyl carbon (δC 171.4) (Table 2). Key 2D-NMR

(COSY, HMBC and NOESY) correlations for compound 17 are presented in Fig. 3. Acid hydrolysis of 17 and subsequent UHPLC-PDA-MS/MS analysis of derivatization reaction products, as for compound 10, allowed to identify D-glucose and L-rhamnose. Therefore, the structure of 2′-O-acetylramoside A was elucidated to be 2-(3,4-dihydroxyphenyl)ethyl O-α-Lrhamnopyranosyl-(1 → 3)-[α-L-rhamnopyranosyl-(1 → 6)]-2-O-acetyl-4-Otrans-p-coumaroyl-β-D-glucopyranoside (17) (Fig. 2). Summarizing, to the best of our knowledge, three broomrape species i.e. O. caryophyllacea, O. lutea, and O. picridis have never been the subject of detailed phytochemical study, therefore, we are the first who described the occurrence of PhGs and iridoids (O. caryophyllacea) in these species. Moreover, although iridoid glycosides (IrGs) are known constituents (genuine or sequestered from host plant) of the holoparasitic Cistanche spp. (Orobanchaceae) (Jiang and Tu, 2009), they have never been reported in Orobanche and Phelipanche genera. However, the sequestration of IrGs, including asperulosidic acid and asperuloside, by the hemiparasite Euphrasia stricta J.F.Lehm (Orobanchaceae, formerly Scrophulariaceae) from its host Galium verum L. (Rubiaceae) was previously described (Rasmussen et al., 2006). In the other three broomrape species i.e. O. coerulescens, P. arenaria and P. ramosa, which have been already examined for phytochemicals in a few studies, we have also reported some metabolites for the first time: O. coerulescens – leucosceptoside A (13); P. arenaria – echinacoside (3), acteoside (5), tubuloside A (6), isoacteoside (8), leucosceptoside A (13), and 2′-Oacetyloacteoside (14); P. ramosa – isoacteoside (8), 3-O-methylpoliumoside (11), leucosceptoside A (13), and wiedemannioside D (18). Phenylethanoid glycosides represent the largest group of natural products identified in parasitic plants of the family Orobanchaceae. Due to the fact that PhG compounds are usually not found in host species, they are considered as specialized metabolites synthesized by the parasites, as opposed to compounds sequestered from the hosts (alkaloids, flavonoids, and polyacetylenes) (Scharenberg and Zidorn, 2018). 3

4

12.4

12.8 12.8

13.8 14.0 15.6 15.9

15.9

Ramoside Aa,b

3-O-Methylpoliumoside

Pheliposidea Leucosceptoside Aa

2′-O-Acetylacteosidea 2′-O-Acetylpoliumosidea 2′-O-Acetylisoacteoside 2′-O-Acetylramoside Aa,b

Wiedemannioside Da

10

11

12 13

14 15 16 17

18

b

a

3.4 6.9 9.4 10.1 10.3 10.5 11.3 11.5

Asperulosidea Echinacoside Arenariosidea Acteosidea Tubuloside Aa Poliumosidea Isoacteosidea Crenatosidea

2 3 4 5 6 7 8 9

Isolated and elucidated by NMR. Novel compound.

12.5

2.2

Asperulosidic acida

1

a

RT (min)

Identity

No

C38H50O20

C31H38O16 C37H48O20 C31H38O16 C37H48O19

C36H46O20 C30H38O15

C36H48O19

C35H46O18

C18H22O11 C35H46O20 C34H44O19 C29H36O15 C37H48O21 C35H46O19 C29H36O15 C29H34O15

C18H24O12

Formula

330 330 330 330 330 328 330

290sh, 330

290sh, 330 290sh, 330 290sh, 328 317

290sh, 332 290sh, 330

290sh, 330

317

237 290sh, 290sh, 290sh, 290sh, 290sh, 290sh, 290sh,

235

UVmax (nm)

−3.7

2.2 1.5 2.2 2.8

1.1 1.8

3

2.5

0.8 0.9 1.1 1 0.2 1.2 2 2.5

2

Error (ppm)

15.5

20.9 10.8 27 16.2

20.7 26.8

29.6

31.2

11.2 18 20.9 12 17.4 19.6 13.9 20.8

10.7

mσ

825.2853

665.2073 811.2654 665.2072 795.2695

797.2501 637.2126

783.2694

753.2593

413.1083 785.2502 755.2395 623.1975 827.2613 769.2551 623.1969 621.1809

431.1186

Observed [M-H]431.1186 (100); 251.0558 (66); 165.0555 (61); 225.0765 (19); 147.0441 (18) 147.0449 (100); 413.1083 (29); 191.0349 (20); 251.0556 (7); 233.0463 (3) 161.0240 (100); 623.2190 (20); 133.0293 (13); 477.1606 (6); 179.0350 (6) 161.0242 (100); 593.2084 (21); 133.0296 (9); 755.2396 (5); 135.0449 (5) 161.0241 (100); 623.1975 (21); 461.1658 (20); 133.0294 (6); 135.0450 (4) 161.0241 (100); 623.2189 (21); 133.0294 (13); 477.1610 (5); 135.0450 (4) 161.0244 (100); 607.2237 (18); 133.0297 (10); 461.1661 (6); 769.2550 (6) 161.0241 (100); 623.1970 (41); 461.1657 (37); 179.0348 (6); 133.0294 (4) 161.0240 (100); 179.0345 (82); 621.1810 (18); 151.0399 (14); 135.0450 (13); 459.1502 (6) 145.0294 (100); 607.2234 (26); 117.0347 (12); 461.1661 (10); 153.0557 (10) 175.0397 (100); 160.0171 (33); 607.2233 (24); 193.0504 (14); 153.0555 (14) 161.0239 (100); 593.2079 (22); 133.0292 (12); 447.1502 (5); 135.0448 (4) 175.0399 (100); 461.1659 (35); 160.0178 (20); 193.0506 (18); 135.0450 (9) 161.0239 (100); 461.1656 (22); 133.0292 (8); 665.2073 (7); 503.1765 (4) 161.0244 (100); 607.2238 (18); 133.0297 (12); 461.1663 (6); 135.0453 (4) 161.0240 (100); 461.1657 (26); 665.2072 (11); 503.1759 (6); 133.0292 (6) 145.0299 (100); 607.2251 (26); 461.1670 (12); 117.0354 (10); 163.0407 (6) 175.0403 (100); 160.0176 (41); 607.2246 (29); 461.1667 (13); 193.0511 (10)

Major fragments (%)

2108 ± 35

1716 ± 26 2115 ± 35 not determined 1831 ± 31

2352 ± 33 not determined

1910 ± 27

1785 ± 28

not determined not determined 2186 ± 35 1766 ± 25 2099 ± 27 2084 ± 36 1250 ± 20 1757 ± 26

not determined

Molar absorption coefficient (ε) at 330 nm (m2·mol−1)

Table 1 UHPLC-QTOF-MS/MS data of metabolites identified in the studied Polish broomrape species, and molar absorption coefficients (ε) of isolated phenylethanoid glycosides determined at 330 nm.

D. Jedrejek, et al.

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Fig. 2. Chemical structures of 18 phytochemicals identified in six studied broomrape species, including two iridoid glycosides (1 and 2) and 16 phenylethanoid glycosides (3–18).

Nevertheless, the occurrence of phenylethanoid glycosides has also been reported in many other non-parasitic plants, especially from the order Lamiales, including the families Oleaceae, Plantaginaceae, and Scrophulariaceae (Jiménez and Riguera, 1994). The physiological role of specialized metabolites in broomrapes, such as PhGs, is not fully understood (the lack of literature data in this topic). Due to the demonstrated antibacterial and antimicrobial activity of phenylethanoid glycosides, it has been proposed that their role in the plants may be associated with the resistance to, or protection from, pathogenic microorganisms attacks (Jiménez and Riguera, 1994). There are only a few publications dealing with the quantification of phenylethanoid glycosides in holoparasitic plants of Orobanchaceae, and they basically concern Herba Cistanches (Cistanche species – C. deserticola Y.C.Ma, C. tubulosa (Schenk) Wight, and C. sinensis Beck) (Lu et al., 2013; Yang et al., 2013; Yan et al., 2017). In addition, remarkable and difficult to explain differences have been revealed among the contents of PhGs in different species and batches of Cistanches Herba. For example, either acteoside or echinacoside has been described as the main components of C. deserticola, and their content in different samples varied in the range 0.8–31.4 mg/g or 2.3–27.1 mg/g, respectively, (Lu et al., 2013; Yang et al., 2013); while echinacoside has been found as the main constituent of C. tubulosa, and its level in different samples varied greatly in the range 1.4–378.5 mg/g (Lu et al., 2013; Yan et al., 2017); in turn, the dominant compound of C. sinensis was poliumoside, and its concentration in different batches varied in the range 3.4–36.2 mg/g (Lu et al., 2013). The above quantitative differences have been linked to multiple factors, including environmental conditions and genetic variations (Lu et al., 2013). For quantitative analysis of PhGs, the main phytochemical constituents of broomrapes, in Orobanche spp. and Phelipanche spp. extracts, a developed UHPLC-PDA method (UV 330 nm detection) and external standard method (acteoside as group standard) were employed. Results of quantification, calculated per dry plant matter (DW), are presented in Table 3. Acteoside (5) was found as the major constituent of all studied Orobanche spp., and its concentrations were very high, ranging from 183.4 to 284.2 mg/g of dry weight. The amounts of

other determined PhGs (isoacteoside, crenatoside, leucosceptoside A, and 2′-acetylacteoside) were considerably (several times) lower in comparison to acteoside in four Orobanche species (Table 3). The main constituent of P. arenaria was arenarioside (4, about 80 mg/g DW), followed by acteoside (about 70 mg/g DW), while the levels of other measured PhGs (echinacoside, isoacteoside, pheliposide, leucosceptoside A, and 2′-acetylacteoside) were several times lower compared to both compounds 4 and 5. Poliumoside (7) and its acylated derivative (2′-acetylpoliumoside, 15) were found as dominant components of both P. ramosa samples, where they represented on average 55.8% (208.1 mg/g DW) and 23.8% (88.6 mg/g DW), respectively, of total PhGs. While, the concentrations of other 8 determined phenylethanoid glycosides (acteoside, isoacteoside, ramoside A, 3-methylpoliumoside, leucosceptoside A, 2′-acetylacteoside, 2′-acetylramoside A, and wiedemannioside D) were much lower (Table 3). The examined broomrape species also differed in the overall content of PhGs, the highest value was described in P. ramosa (about 370 mg/g DW), followed by O. coerulescens (about 315 mg/g DW), while the lowest in P. arenaria (about 180 mg/g DW) and O. picridis (about 205 mg/g DW). To visualize chemical similarities and/or differences between studied broomrape species, and relationships between determined compounds and examined plant samples, the principal component analysis (PCA), and cluster analysis (CA) were performed. The PCA enabled the reduction of all quantitative data to four principal components (PC1PC4) which explained 99% of the entire variation (Fig. 4A). The inspection of Fig. 4B1-2 reveals that the four examined Orobanche species showed a great similarity (created one cluster in projection PC1 vs PC2), while both Phelipanche spp. created their own separate groups that were formed by relevant variables (compounds present only in a given species). In this way, our results, showing the phytochemical distinctiveness of the Orobanche and Phelipanche plants, are consistent with previous morphological and molecular studies (Schneeweiss, 2007; Joel, 2009), which resulted in taxonomic separation of both genera; and confirm the usefulness of chemotaxonomic studies (combination of phytochemical and chemometric analyses) as complementary tool in plant classification. In addition, other very 5

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cluster analysis. The CA dendrograms for plant samples and PhGs are presented in the Supplementary Materials (Fig. 5S). Finally, two broomrape species collected from two different hosts, O. lutea parasitizing on Medicago falcata and M. sativa, and P. ramosa parasitizing on tomato and tobacco, showed a far-reaching similarity regardless of the host plant, both in terms of the qualitative and quantitative phytochemical profiles (Fig. 4B and C and 5S, Table 3). The antiradical scavenging capacity of 13 isolated PhG compounds (4–12, 14–15, 17–18) was expressed as Trolox Equivalents (TE) and IC50. The results are shown in Table 4. Additionally, curves (inhibition % vs sample concentration) obtained for Trolox and tested phenylethanoid glycosides are presented in the Supplementary Materials (Fig. 6S). All tested phenolic compounds exhibited dose-dependent activity to scavenge DPPH free radicals in vitro. Nevertheless, differences in the demonstrated antioxidant potential have been observed between investigated PhGs. The estimated TE values varied from 31% to 87% of Trolox activity (1.00). With regard to the IC50 values, the obtained results were between 1.2 and 3.1-fold higher (46–122 μg DW/ mL) compared to the positive control (Trolox, 40 μg DW/mL). The highest anti-oxidative action was shown by acteoside (5), followed by isoacteoside (8) and 2′-O-acetylacteoside (14), however, with the exception of 4 compounds (10, 11, 17, and 18) marked in green in Table 5, differences in activity between the tested compounds were not very large (compounds marked in red/pink in Table 4; 4–9, 12, 14–15). Whereas, four abovementioned PhGs (10, 11, 17, and 18), having substituted caffeoyl moiety by feruloyl or p-coumaroyl moiety, were about 3-fold less active in comparison to the positive control (Trolox). In general, the results of our test are in accordance and confirm the findings of the previous antioxidant in vitro experiments on phenylethanoids. In a study by Heilmann et al. (2000), the authors measured the effects of a number of PhGs, differing in the molecular structure, on the production of reactive oxygen species in a luminol-enhanced chemiluminescence assay with FMLP stimulated human polymorphonuclear leukocytes. As a result, the antioxidant activity of phenylethanoid glycosides has been mainly related to the structure of the acyl moieties (phenolic acid and phenylethanoid unit), including the presence and/or modification of catechol moiety. For example, replacement of the caffeoyl by a feruloyl moiety has resulted in a slight but significant decrease in the radical scavenger activity. In turn, the position of the acyl moiety (acteoside activity vs. isoacteoside activity), as well as modification of the sugar chain has been found to be of minor importance (Heilmann et al., 2000). Similarly, all tested by us polyphenolic compounds acylated with caffeic acid and hydroxytyrosol (both containing catechol moiety) demonstrated strong and comparable antioxidant activity, whereas compounds having replaced caffeoyl moiety by either feruloyl (3-O-methylpoliumoside and wiedemannioside D) or p-coumaroyl (ramoside A and 2′-acetylramoside A) moiety possessed significantly decreased anti-oxidative potential.

Table 2 1 H (500 MHz) and13C-NMR (125 MHz) data for compound 10 (ramoside A) and 17 (2′-acetylramoside A) (δ in ppm, J in Hz, both measured in MeOH-d4, at 30 °C). Position

10 δH

Hydroxytyrosol 1 – 2 6.57 dd (8.0, 2.0) 3 6.69 d (8.0) 4 – 5 – 6 6.70 d (2.0) 7 2.82 dt (14.2, 7.3) 2.79 dt (14.2, 6.7) 8 3.99 ddd (9.9, 8.4, 6.7) 3.73 ddd (9.9, 8.4, 7.3) 8hydroxytyrosol-O-β-Glucoside 1′ 4.37 d (7.9) 2′ 3.39 dd (9.2, 8.0) 3′ 3.81 t (9.0) 4′ 5.00 t (9.2) 5′ 3.69 ddd (10.1, 5.5, 2.1) 6′ 3.75 dd (11.3, 2.1) 3.48 dd (11.3, 5.4) 3′glc-O-α-Rhamnoside 1″ 5.19 d (1.8) 2″ 3.92 dd (3.3, 1.8) 3″ 3.58 dd (9.5, 3.3) 4″ 3.29 t (9.5) 5″ 3.56 dq (9.5, 6.2) 6″ 1.08 d (6.2) 6′glc-O-α-Rhamnoside 1‴ 4.63 d (1.7) 2‴ 3.84 dd (3.5, 1.7) 3‴ 3.67 dd (9.5, 3.5) 4‴ 3.34 t (9.5) 5‴ 3.60 dq (9.5, 6.2) 6‴ 1.20 d (6.2) 4′glc-O-p-Coumaric acid 1‴′ – 2‴′, 6‴′ 7.47 d (8.7) 3‴′, 5‴′ 6.81 d (8.7) 4‴′ – 7‴′ 7.67 d (15.9) 8‴′ 6.34 d (15.9) 9‴′ – 2′glc-O-Acetyl COCH3 – – COCH3

17 δC

δH

δC

131.4 121.3 116.4 144.7 146.1 117.1 36.7

– 6.53 dd (8.0, 2.1) 6.68 d (8.0) – – 6.64 d (2.1) 2.72 dt (14.1, 6.1) 2.69 dt (14.1, 7.2) 4.03 dt (9.7, 6.1) 3.64 dt (9.7, 7.2)

131.7 121.3 116.3 144.6 146.1 117.2 36.4

104.4 76.2 81.6 70.4 74.7 67.6

4.53 d (8.0) 4.87 dd (9.5, 8.0) 4.01 t (9.3) 5.09 t (9.5) 3.73 ddd (10.0, 5.5, 2.1) 3.77 dd (11.2, 2.1) 3.49 dd (11.2, 5.4)

101.8 75.1 80.5 70.5 74.8 67.3

103.0 72.4 72.1 73.8 70.4 18.4

4.80 d (1.8) 3.63 dd (3.4, 1.8) 3.51 dd (9.5, 3.4) 3.26 t (9.5) 3.51 dq (9.5, 6.2) 1.06 d (6.2)

103.3 72.6 71.9 73.6 70.8 18.4

102.3 72.0 72.3 74.0 69.9 18.0

4.63 d (1.7) 3.85 dd (3.5, 1.7) 3.69 dd (9.5, 3.5) 3.35 t (9.5) 3.61 dq (9.5, 6.2) 1.21 d (6.3)

102.3 72.0 72.3 74.0 69.9 18.0

127.1 131.3 116.9 161.4 147.7 114.8 168.0

– 7.47 d 6.81 d – 7.67 d 6.34 d –

127.1 131.4 116.9 161.5 147.8 114.7 167.8

– –

– 1.98, s

72.4

(8.7) (8.7) (15.9) (15.9)

71.9

171.4 20.9

interesting chemotaxonomic conclusions can be drawn based on our PCA and CA analyses (using phytochemical quantitative data), for example, the O. caryophyllacea species, due to the presence of the relatively high amount of compound 14 (2′-O-acetylacteoside), was the most detached from the monolithic Orobanche spp. cluster (Fig. 4C1-2). Moreover, the distinctiveness of O. caryophyllacea should be even greater when considering the exclusive presence of iridoid glycosides in this species, which were not quantified in the current study (Fig. 1). This is the first report on the occurrence of iridoids in Orobanche plant, and as the presence of asperuloside and asperulosidic acid has been confirmed so far only in O. caryophyllacea (while not present in O. coerulescens, O. lutea, and O. picridis), these compounds can serve as chemotaxonomic markers of this species. In addition, our quantitative and chemometric analyses (CA and PCA) showed that P. arenaria species appears to be more phytochemically related rather to Orobanche plants than to another examined Phelipanche species – P. ramosa, which is mainly associated with the increased content of compounds 5 (acteoside) and 13 (leucosceptoside A) and significantly lower total PhGs content (Fig. 4C1-2 and 5S; Table 3). Results complementary to the PCA were obtained after applying

3. Conclusions The current phytochemical study is the first report on the occurrence of phenylethanoid glycosides (PhGs) and iridoids in six broomrape species growing in Poland, including Orobanche picridis, O. caryophyllacea, O. coerulescens, O. lutea, Phelipanche arenaria, and P. ramosa. Among the 18 described major compounds in the set of investigated broomrapes, we found in Phelipanche ramosa two novel phenylethanoids acylated with p-coumaric acid, named ramoside A and 2′-acetylramoside A. Due to limited literature data on the quantity of PhGs in holoparasites of Orobanchaceae (research focused exclusively on Herba Cistanches), we performed quantification of phenylethanoids in the whole plants (flower shoots with underground haustoria) of six Polish broomrape species. It was found that all tested Orobanche and Phelipanche plants are rich in PhGs (180–380 mg/g DW), and their amount in the studied plant material is even greater than that reported for Herba Cistanches. The results of the antiradical DPPH test of 13 6

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Fig. 3. Key COSY, HMBC and NOESY correlations for novel compound (17–2′-O-Acetylramoside A) isolated from Phelipanche ramosa.

isolated polyphenolic compounds demonstrated high and comparable antioxidant potential of PhGs containing two catechol moieties in their structure (caffeic acid and hydroxytyrosol), regardless of the modification of either the acylation site or the sugar chain. While the modification of catechol moiety of phenolic acid has caused a significant decrease in the activity. The present study provides insights for the potential applications of broomrapes as a component of a nutraceutical and medical preparation, similarly to the Herba Cistanches.

Acetonitrile (isocratic grade and LC-MS grade), methanol (isocratic grade), formic acid (98–100% purity), and deuterated solvents – DMSO‑d6 and MeOH-d4 (both 99.96% D) were purchased from Merck (Darmstadt, Germany). MS-grade formic acid, D and L enantiomers of monosaccharides (glucose, xylose, rhamnose), L-cysteine methyl ester hydrochloride, o-tolyl isothiocyanate, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Trolox and acteoside standard (≥99%, HPLC) were was purchased from Sigma-Aldrich (Steinheim, Germany). Ultrapure water was prepared in-house using a Milli-Q water purification system (Millipore, Milford, MA, USA). All other reagents of analytical grade were provided by commercial suppliers.

column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA, USA). The MS ion source was operated in the negative and positive electrospray ionization mode, with the following settings: mass scan range 50–2000 m/z; capillary voltage 3.0 kV and 4.5 kV, respectively; nebulizer and drying gas (N2) 0.7 bar and 6 L/min, respectively; dry gas temperature 200 °C. The MS/MS spectra were acquired using variable collision energy in the range from 2.5 to 80 eV. The acquired data were calibrated internally with sodium formate introduced to the ion source via a 20 μL loop at the beginning of each analysis. The UV absorbance detection in the 200–600 nm wavelength range (5 nm bandwidth, 10 Hz frequency) was combined with MS analyses. Data were collected and processed using DataAnalysis 4.3 software (Bruker). The final step of purification of compounds was performed on HPLC system (Dionex) equipped with a photodiode array detector PDA-100 and fraction collector FC 204 (Gilson, Middleton, WI, USA). Separations were carried out on a semipreparative reversed-phase column Kromasil C18 (10 × 250 mm, 5 μm, AkzoNobel, Bohus, Sweden). Quantitative UHPLC analyses were performed using a Waters ACQUITY chromatographic system equipped with PDA detector and Triple Quadrupole (TQD) Mass Detector (Waters). Separations were carried out on a BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters). Plant material and pure isolated compounds were dried using Gamma 2–16 LSC freeze dryer (Christ, Osterode am Harz, Germany). Automated extractions were made using Dionex ASE 200 extractor (Dionex).

4.2. Equipment

4.3. Plant material

The 1D and 2D NMR spectra (1H, 13C DEPTQ, 1H–13C HSQC, 1H–13C H2BC, 1H–13C HMBC, 1H–13C HSQC-TOCSY, 2D 1H–13C, F2-coupled perfect-CLIP HSQC, 1H–1H COSY DQF, 1H–1H TOCSY, 1H–1H TROESY, 1D–TOCSY, 1D-TROESY) were measured at a Bruker Avance III HD Ascend-500 spectrometer (Bruker BioSpin, Rheinstetten, Germany), equipped with 5 mm broad-band inverse (BBI) probe. Data processing was performed with the Topspin software (version 3.5pl2, Bruker BioSpin). High resolution MS spectra, exact masses of compounds, MS/ MS fragmentation patterns, and molecular formulas were received using a Thermo Scientific Ultimate 3000 RS chromatographic system coupled with a Bruker Impact II HD (Bruker, Billerica, MA, USA) quadrupole time-of-flight (Q-TOF) mass spectrometer and a BEH C18

The six broomrape species, including Orobanche caryophyllacea Sm., O. coerulescens Stephan ex Willd., O. lutea Baumg. (two different hosts), O. picridis F.W. Schultz, Phelipanche arenaria Pomel, and P. ramosa (L.) Pomel (two different hosts), together with their host plants, were identified and harvested from a natural source (the belt of the Polish Uplands, Lubelskie, Śląskie, and Świętokrzyskie voivodships, as well as Podlaskie voivodship) in 2014 by prof. Renata Piwowarczyk (Department of Botany, Institute of Biology, Jan Kochanowski University, Kielce, Poland). Information about the collected plant material (host plant, and place and time of harvest) is presented in Table 5. Demonstrative photographs of all collected broomrape species are shown in Fig. 5. Three independent plant samples, consisting of

4. Experimental 4.1. Reagents

7

Phytochemistry 170 (2020) 112189 ND ND 13.98 ± 2.85a ND 195.86 ± 18.37 0.21 ± 0.02a ND 8.09 ± 1.23 30.14 ± 3.63 ND 0.32 ± 0.02a 4.63 ± 0.23a 91.23 ± 9.67 ND 5.59 ± 1.01 13.67 ± 2.55 365.12 ± 28.19de

flowering aerial shoots with underground haustoria, were collected from the place of occurrence of each broomrape species. Voucher specimens have been deposited at the herbarium of the Jan Kochanowski University in Kielce (KTC). Collected plant material was freeze-dried, finely ground with an electric grinder and sieved through a 0.5 mm sieve, and stored at 4 °C before extraction.

NDa ND 13.90 ± 1.16a ND 220.40 ± 19.49 0.30 ± 0.02a ND 7.18 ± 0.84 28.50 ± 2.79 ND 0.58 ± 0.04a 5.24 ± 0.39a 84.65 ± 7.28 ND 5.67 ± 0.55 14.14 ± 1.39 380.56 ± 33.36e

4.4. Extraction and isolation

ND ND 185.64 ± 4.24b ND ND 6.02 ± 0.42c 14.42 ± 1.93b ND ND ND 0.70 ± 0.11a ND ND ND ND ND 206.77 ± 5.09 ab

4.28 ± 0.16 80.66 ± 7.15 72.79 ± 10.40a 4.68 ± 0.85 ND 2.24 ± 0.42 ab ND ND ND 11.64 ± 1.98 1.41 ± 0.01b 4.10 ± 0.71a ND < LLOQ ND ND 181.80 ± 16.94a

For isolation of compounds the three broomrape species: O. caryophyllacea (O.C.), P. arenaria (P.A.) and P. ramosa (P.R.), differing in phytochemical composition (according to preliminary UHPLC-PDA-MS analyses), were chosen. Powdered plant material (O.C. – 2 g; P.A. and P.R. – 3 g) was extracted with accelerated solvent extractor Dionex ASE using 80% MeOH (v/v), at 40 °C and 1500 psi (solvent pressure), and three repeated static extraction cycles. The obtained extracts were evaporated and freeze-dried. The yields of extraction were 55% (O.C.), 36.7% (P.A.), and 43.3% (P.R.). The crude methanol extracts were purified by reverse-phase (RP) chromatography, in a stepwise manner. First, the polar constituents (mainly sugars) were removed from the extracts, and they were initially divided into three fractions by solidphase extraction (SPE). The extracts were applied to a preconditioned RP-C18 column (30 × 80 mm, Cosmosil 40C18-PREP, 40 μm; Nacalai Tesque, INC, Kyoto, Japan), followed by elution with 0.2% MeOH (v/v, containing 0.1% FA,v/v), 20% MeOH (v/v, fraction 1), 40% MeOH (v/ v, fraction 2), and then 80% MeOH (v/v, fraction 3). The fractionation process was monitored by UHPLC-PDA-MS analyses. Individual compounds were obtained from the O.C., P.A. and P.R. fractions 1–3 by means of semi-preparative HPLC. The conditions of chromatographic separation were individually optimized for each fraction. Separations were carried out in isocratic or gradient mode, using aqueous 31–44% MeOH (containing 0.1% formic acid). The mobile phase flow rate was 2.5 mL/min, and the column temperature was maintained between 21 and 35 °C. The PDA detector was operated at 210 and 330 nm wavelengths. As a result of chromatographic separation of the O.C. fractions the following compounds were isolated: Fr. 1 yielded compound 1 (2.5 mg) and 2 (11 mg), Fr. 3 yielded compound 5 (240 mg), 8 (4.5 mg), 9 (15.5 mg), 13 (1.1 mg), and 14 (23 mg). Separation of the P.A. fraction 2 produced compound 4 (36 mg), 5 (24.7 mg), 6 (11.5 mg), 12 (186.4 mg) and 14 (8.9 mg). Chromatographic separation of the P.R. fractions enabled to obtain the following compounds: from Fr. 2 was obtained compound 5 (7.3 mg) and 7 (9.2 mg), and Fr. 3 yielded compound 7 (9.5 mg), 10 (3.2 mg), 11 (12.8 mg), 15 (9.3 mg), 17 (3.4 mg) and 18 (7 mg).

ND – not detected. * Different letter within a row indicates significant differences (p < 0.05). ** < LLOQ (below lower limit of quantification).

ND ND 284.16 ± 32.92c ND ND 15.95 ± 1.56f 14.03 ± 2.54b ND ND ND 2.04 ± 0.34bc ND ND ND ND ND 316.18 ± 35.94cde ND ND 265.16 ± 51.01c ND ND 9.17 ± 1.91d 7.37 ± 1.39a ND ND ND 2.28 ± 0.33c ND ND ND ND ND 283.97 ± 54.33bcd ND ND 269.95 ± 24.15c ND ND 12.71 ± 1.02e 7.75 ± 0.71a ND ND ND 2.13 ± 0.24c ND ND ND ND ND 292.54 ± 25.68bcd 1 Echinacoside (3) 2 Arenarioside (4) 3 Acteoside (5) 4 Tubuloside A (6) 5 Poliumoside (7) 6 Isoacteoside (8) 7 Crenatoside (9) 8 Ramoside A (10) 9 3-O-Methylpoliumoside (11) 10 Pheliposide (12) 11 Leucosceptoside A (13) 12 2′-O-Acetylacteoside (14) 13 2′-O-Acetylpoliumoside (15) 14 2′-O-Acetylisoacteoside (16) 15 2′-O-Acetylramoside A (17) 16 Wiedemannioside D (18) Total PhGs

ND ND 183.35 ± 16.11b* ND ND 4.45 ± 0.59bc 14.27 ± 1.55b ND ND ND 1.81 ± 0.34bc 35.36 ± 5.82b ND < LLOQ** ND ND 239.24 ± 18.29abc

O. lutea II O. lutea I O. caryophyllacea

Content [mg/g DW] (Mean ± SD, n = 3) Compound (number) No

Table 3 Comparison of phenylethanoid glycosides content in the six studied broomrape species.

O. coerulescens

O. picridis

P. arenaria

P. ramosa I

P. ramosa II

D. Jedrejek, et al.

4.5. Determination the absolute configuration of monosaccharides The absolute configuration of monosaccharides was determined after acid hydrolysis of each isolated compound (1–2, 4–15, 17–18) in 1 mL of 4M HCl solution at 100 °C. The sugar-containing aqueous layer, obtained after extraction with ethyl acetate, was neutralized with Amberlite IRA-400 (OH − form). Released monosaccharides were derivatized and determined according to the modified method of Tanaka et al. (2007) (Pérez et al., 2014). On the basis of the retention time of authentic standards of glucose (D-Glc 10.40 min, L-Glc 10.20 min), rhamnose (D-Rha 9.96 min, L-Rha 11.56 min) and xylose (D-Xyl 10.72 min, L-Xyl 10.78 min), the three monosaccharide isomers: Dglucose, D-xylose and L-rhamnose, were identified among isolated compounds. 4.6. Characteristic data of undescribed compounds 4.6.1. Compound 10 2-(3,4-dihydroxyphenyl)ethyl O-α-L-rhamnopyranosyl-(1 → 3)-[α-Lrhamnopyranosyl-(1 → 6)]-4-O-trans-p-coumaroyl-β-D-glucopyranoside (ramoside A, 10) white amorphous powder; UV (PDA, MeCN/H2O) λmax (nm) 8

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Fig. 4. Results of PCA analysis of examined broomrape species and determined phenylethanoid glycosides (3–15, 17–18): the 4 derived PCs with explained variance % (A); representation of broomrape samples (B1) and PhGs (B2) as functions of the PC1 vs PC2, with color-coded relationships between compounds and plant samples; representation of broomrape samples (C1) and PhGs (C2) as functions of the PC1 vs PC3, with color-coded relationships between compounds and plant samples. (O. caryophyllacea – O. caryo; O. coerulescens – O. coer; O. lutea – O. lut; O. picridis – O. pic; P. arenaria – P. aren; P. ramosa – P. ram). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

317; HR-Q-TOF-MS (neg.) m/z 753.2593 [M-H]- (calc. for C35H45O18); HRQ-TOF-MS (pos.) m/z 772.3013 (M+NH4)+ (calc. for C35H50NO18), 755.2756 (M+H)+ (calc. for C35H45O18); HR-MS/MS of m/z 755.2756 give diagnostic fragment ions at m/z 609.2171 [M+H-146(deoxyhexosyl)]+, 463.1587 [M+H-146(deoxyhexosyl)-146(deoxyhexosyl)]+, 455.1539 [M +H-146(deoxyhexosyl)-154(hydroxytyrosol)]+, 309.0957 [M+H-146 (deoxyhexosyl)-146(deoxyhexosyl)-154(hydroxytyrosol)]+, 147.0434 [pCoA+H–H2O]+. For 1H and 13C-NMR data see Table 2.

C18 classic cartridges (360 mg, Waters). The compounds of interest were eluted with 80% MeOH. The samples, after evaporation, were dissolved in 1 mL of 50% MeOH. Before analysis samples were centrifuged (16,000×g) and appropriately diluted with 50% MeOH. 4.7.2. UHPLC-PDA-MS analyses UHPLC analyses were performed using Waters ACQUITY chromatographic system with PDA detector and TQD-MS detector and BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters). The chromatographic conditions were as follows: linear gradient 10 → 25% of mobile phase B (0.1% formic acid in acetonitrile) in mobile phase A (0.1% formic acid in H2O) over 12 min; flow rate – 0.4 mL/min; oven temperature – 25 °C; injection volume – 2 μL. The UV spectra were recorded in the 190–490 nm wavelength range (3.6 nm resolution). The MS analyses were performed in negative ion mode with electrospray ionization (ESI). The other settings of MS detector were as follows: scan range – 100–1200 m/z; capillary voltage – 2.8 kV; cone voltage – 50 V; source temperature – 140 °C, desolvation temperature – 350 °C, desolvation gas flow – 800 L/h; cone gas flow – 100 L/h. For quantitative analysis, the phenylethanoid glycosides in plant samples, and the used reference compound – acteoside (≥99%, HPLC, Sigma-Aldrich), were detected at UV 330 nm. The external calibration curve was prepared in six concentrations within the range of 0.8–200 μg/mL. The regression coefficient (R2) for the calibration curve was calculated to be 0.9999, and the lower limit of quantification (LLOQ) to be 0.78 μg/mL. Three independent chromatographic runs were performed for each sample/ standard working solution. To calculate the quantitative content of individual compounds, using an external calibration curve, the areas of the chromatographic peaks for the isolated compounds (4–15, 17–18)

4.6.2. Compound 17 2-(3,4-dihydroxyphenyl)ethyl O-α-L-rhamnopyranosyl-(1 → 3)-[α-Lrhamnopyranosyl-(1 → 6)]-2-O-acetyl-4-O-trans-p-coumaroyl-β-D-glucopyranoside (2′-O-acetylramoside A 17) white amorphous powder; UV (PDA, MeCN/H2O) λmax (nm) 317; HR-Q-TOF-MS (neg.) m/z 795.2695 [M-H](calc. for C37H47O19); HR-Q-TOF-MS (pos.) m/z 814.3121 (M+NH4)+ (calc. for C37H52NO19), 797.2832 (M+H)+ (calc. for C37H47O19); HR-MS/ MS of m/z 797.2832 give diagnostic fragment ions at m/z 651.2276 [M+H146(deoxyhexosyl)]+, 497.1636 [M+H-146(deoxyhexosyl)-154(hydroxytyrosol)]+, 351.1061 [M+H-146(deoxyhexosyl)-154(hydroxytyrosol)146(deoxyhexosyl)]+, 147.0434 [p-CoA+H–H2O]+. For 1H and 13C-NMR data see Table 2. 4.7. UHPLC-PDA-MS and quantitative analyses 4.7.1. Extraction and sample preparation For quantitative analyses, the 100 mg of powdered plant material was extracted with Dionex ASE 200 extractor using the same conditions as for the isolation of compounds (section 4.4). Obtained extracts were concentrated and cleaned to remove the sugars by SPE using Sep-Pak 9

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Fig. 5. Demonstrative photographs of collected and studied 6 species of broomrapes: Orobanche caryophyllacea (A); O. coerulescens (B); O. lutea (C); O. picridis (D); Phelipanche arenaria (E); P. ramosa (F) (photographs were taken by R. Piwowarczyk).

were corrected based on the calculated molar absorption coefficients (ε) at a wavelength of 330 nm (Table 1). For non-isolated PhG compounds 3 and 16, and compound 13 (isolated in very low amount) a raw chromatographic data (without correction) was used. Molar absorption coefficient values for PhGs were calculated based on absorbance measurements (at 330 nm) of compounds dissolved in 15% MeCN (at least three different concentrations within the range of 0.25–8 mM) using Thermo Scientific Evolution 260 Bio spectrophotometer. Quantitative results were calculated per plant dry weight (DW).

4.8. Determination of free radical scavenging activity of phenylethanoid glycosides The free radical scavenging activity of the isolated phenylethanoid glycosides against DPPH radical was determined using the method of Brand-Williams et al. (1995), with slight modifications. First, DPPH solution with methanol (100 μM), and solutions of freeze-dried compounds with 50% methanol (four different concentrations in the range of 10–70 μg/mL (compounds 4–9, 12, 14, 15) or 20–140 μg/mL 10

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Table 4 Antioxidant activity of isolated phenylethanoid glycosides estimated by DPPH method (Mean ± SD, n ≥ 4).

Table 5 Information about the collected plant material (host plant, and place and time of harvest). Broomrape Species

Host Plant

Place and time of harvest

Orobanche caryophyllacea Sm. Orobanche coerulescens Stephan ex Willd. Orobanche lutea Baumg. Orobanche lutea Baumg. Orobanche picridis F.W. Schultz Phelipanche arenaria (Borkh.) Pomel Phelipanche ramosa (L.) Pomel Phelipanche ramosa (L.) Pomel

Galium boreale L. Artemisia campestris L. Medicago falcata L. Medicago sativa L. Picris hieracioides Sibth. & Sm. Artemisia campestris L. Nicotiana tabacum L. Solanum lycopersicum L.

Chomentówek (50.3349° N, 20.4000° E), May 2014 Dobrowoda (50.2401° N, 20.4617° E), June 2014 Zakawie (50.1852° N, 19.2016° E), May 2014 Chomentówek (50.3349° N, 20.4000° E), May 2014 Pęczelice (50.2655° N, 20.4716° E), June 2014 Zwierzyniec (50.3652° N, 22.5801° E), June 2014 Brzeziny (50.3021° N, 22.5957° E), September 2014 Szewce (50.3553° N, 22.3038° E), September 2014

(compound 10, 11, 17, 18)), and Trolox solutions with 50% methanol (five different concentrations ranging from 5 to 60 μg/mL) were prepared. Subsequently, a 30 μL of compound solution or Trolox solution or 50% methanol (control) was added to 170 μL of DPPH solution in a well of a 96-well plate. The reaction mixture was mixed gently, incubated in the dark for 30 min, and then the absorbance was recorded at 517 nm using a microplate reader (Infinite 200 Pro, Tecan, Austria). The percentage of absorbance inhibition was calculated from the equation:

Inhibition (%) = 100 × [(Acontrol

Author contribution D.J. and R.P. designed the study. R.P. identified and collected plant material. D.J. and S.P. performed the extraction, compound isolation, DPPH test, and participated in qualitative data analysis. Ł.P. carried out NMR analyses and elucidated the structures of compounds. D.J. performed quantitative UPLC-PDA-MS analyses and multivariate analyses. D.J., Ł.P, and S.P. wrote the manuscript. R.P. and A.S. commented on the manuscript. All authors approved the final version of the manuscript.

Asample)/Acontrol]

Declaration of competing interest

where Acontrol and Asample are the absorbance values of the control and test samples at t = 30 min, respectively. Linear curves were obtained for Trolox and each tested compound using the relationship of standard/sample concentration (μg/mL) to absorbance inhibition (%). To obtain Trolox Equivalents (TE) for the thirteen tested compounds slopes of their curves were divided by the slope of the Trolox curve. The IC50 values of isolated phenylethanoid glycosides on DPPH (defined as the concentration of sample necessary to cause 50% inhibition) were determined from the same linear curves (the percentage of scavenging activity plotted against the sample concentration), and Trolox was used as a positive control.

The authors declare no conflict of interest. Acknowledgments The authors would like to thank dr Mariusz Kowalczyk for performing UHPLC-HR-MS/MS analyses, and dr Marcin Przybys for help in conducting the antioxidant DPPH test. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112189.

4.9. Statistical analysis The statistical analysis which included one-way ANOVA (followed by Tukey's multiple comparisons test), principal component analysis (PCA) and cluster analysis (CA) was performed using Statistica 10 (StatSoft), and Quick Analysis tool using Microsoft Excel. Multivariate analysis (CA and PCA analysis) was conducted using quantitative data for PhG compounds after standardization. Significance was considered at p < 0.05. Data are expressed as means ± SD.

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