ACTAACTA UNIVERSITATISUNIVERSITATIS CIBINIENSISCIBINIENSIS

10.2478/aucft-2020-0010 SeriesSeries E: E: Food Food technology technology

PHYTOCHEMICAL AND BIOACTIVE PROPERTIES OF PHELYPAEA TOURNEFORTII – EFFECT OF PARASITIC LIFESTYLE AND ENVIRONMENTAL FACTORS

– Research paper –

Renata PIWOWARCZYK*, Ireneusz OCHMIAN**, Sabina LACHOWICZ***, Ireneusz KAPUSTA****, Zofia SOTEK*****

*Department of Microbiology and Parasitology, Institute of Biology, Jan Kochanowski University, Uniwersytecka 7 Street, 25-406 Kielce, Poland; [email protected] **Department of Horticulture, West Pomeranian University of Technology Szczecin, Słowackiego 17 Street, 71-434 Szczecin, Poland; [email protected] ***Department of Fermentation and Cereals Technology, Wrocław University of Environmental and Life Sciences, Chełmońskiego 37 Street, 51-630 Wrocław, Poland; [email protected] ****Department of Food Technology and Human Nutrition, University of Rzeszów, Zelwerowicza 4 Street, 35-601 Rzeszów, Poland; [email protected] *****Institute of Marine and Environmental Sciences, University of Szczecin, Adama Mickiewicza 16 Street, 70-383 Szczecin, Poland; [email protected]

Abstract: Some holoparasitic species can become cultivated due to their unique chemical composition. A lot of bioactive contents are characteristic for them. Holoparasites of the family are known to be an important and rich source of polyphenols, especially metabolites of the phenylethanoid glycosides (PhGs) group. However, only a minority of the species in this family have been phytochemically tested. They are reported to have multiple biological and therapeutic effects and have been used for centuries in traditional Chinese medicine. This is the first study to present phytochemical profiling for a representative of genus Phelypaea. The chemical composition and biological activity in particular organs of the parasite, P. tournefortii, were determined. The interaction with its host, Tanacetum polycephalum (), from different places and altitudes was also studied. We presented the determination of polyphenolic compounds with the UPLC-PDA-MS/MS method, antioxidative effects and inhibitory activities, polyphenols, and nitrates content, ABTS•+, DPPH, FRAP, as well as colour parameters. The polyphenols profile of the parasite and host were different in quality and quantity. Identification of polyphenolic compounds revealed 41 compounds, 15 in the parasite (12 phenylethanoids and 3 anthocyanins), and 26 in the host (mainly flavonoids and phenolic acids). The amount and biological activity of polyphenolic compounds present in Phelypaea was very diverse and depended on the host and the parasite's organs, as well as on population altitude. The results show that P. tournefortii is a potential source of functional and pro-health components. They also direct researchers' attention to the parasite's organs, host, and environmental influence.

Key words: anthocyanins, holoparasite, host, Orobanchaceae, parasitic plant, altitude factor

INTRODUCTION and 2,100 species (Nickrent 2020). They occur The extraordinary characteristics of holoparasitic worldwide, but show the greatest diversity in the species are the reason for the increasing popularity Mediterranean basin, and western and central Asia. of some of these plants as 'super foods' (functional The Caucasus, the Near East, and central Asia are foods), characterised by a high amount of bioactive the probable centres of origin of certain contents. Except for a few non-parasitic autotrophs, holoparasitic genera. However, these areas have not the family Orobanchaceae exclusively includes root been sufficiently explored thus far (Piwowarczyk et hemiparasites and holoparasites with 102 genera al., 2019). The Republic of Armenia is part of the Caucasus, one of the most important biodiversity Received: 20.05.2020 hotspots in the world (Mittermeier et al., 2005). In Accepted in revised form:15.06.2020 Armenia occurs about 40 representatives of 1 Corresponding author. E-Mail address: [email protected]

Acta Universitatis Cibiniensis Series E: FOOD TECHNOLOGY 113 Vol. XXIV (2020), no. 1 Orobanchaceae holoparasites. These include, occurring primarily in the Caucasus, and less L., Phelipanche Pomel, frequently in the Middle East, Asia Minor, Crimea, Hoffmanns & Link, and Phelypaea L. (syn. and Balkans. They occur as parasites on the roots of Diphelypaea Nicolson) genera (Piwowarczyk et al., Asteraceae species like Tanacetum, Centaurea, 2019). Holoparasites of the Orobanchaceae family Psephellus, and rarely, Cousinia. Species belonging are non-photosynthetic, achlorophyllous, root to this genus have an unusual appearance compared parasites. They are fully host-dependent for their to other plants; they are parasitic and have nutritional needs which they obtain through an adaptations for the parasitic lifestyle. These intrusive organ called a haustorium. The preferred perennial herbs are achlorophyllous and possess one hosts vary from species to species and can be of the most intense red flowers among all plants classified into three categories: monophagous, worldwide. Phelypaea tournefortii Desf. primarily oligophagous (dominant), and polyphagous occurs in the Caucasus (Armenia, Azerbaijan, (Piwowarczyk et al., 2019). Holoparasites from the Georgia, and Iran) and in Turkey. It occurs as a Orobanchaceae family possess a significant and rich parasite on the roots of Tanacetum. It generally source of metabolites belonging to the class, grows in the central and southern part of Armenia, phenylethanoid glycosides (PhGs). Only a small in the steppe or on rocky slopes, (1200)1800 - part of holoparasitic species from Orobanchaceae in 2100(2500) m above sea level. Its flowering season the world have been studied for their phytochemical is from May to June, and it usually occurs in properties. Despite this, they were found to have populations of a few dozens to a few hundred shoots many biological and therapeutic effects (Piwowarczyk et al., 2019). The host species, (Scharenberg and Zidorn, 2018). PhG species have Tanacetum polycephalum subsp. argyrophyllum (K. pharmacodynamic properties. They also act as a Koch) Podlech [syn. T. argyrophyllum (C. Koch) neuroprotective, anti-inflammatory, antioxidant, Tzvel.], belonging to the Asteraceae family, is seen cytotoxic, antimicrobial, antiviral, in Transcaucasia and Turkey, and is an important immunomodulating, and enzyme inhibitory factors folk medicinal plant with wide biological activities (Xue and Yang, 2016). To date, over 40 different (Mahdavi et al., 2015). PhGs, predominantly including acteoside and To date, the Phelypaea genus has not been studied echinacoside, have been reported in Cistanche spp. for chemical composition. This is the first attempt (Wang et al., 2015; Piwowarczyk et al., 2020). to characterise P. tournefortii for phytochemical Presumably, 70 natural products have been composition and biological activity. The aim of our described in the Orobanche (among 200 species, ca. research was to assess the nutritional value, 30 species have undergone phytochemical analysis) phytochemical composition, and biological activity and Phelipanche genera of the Orobanchaceae of P. tournefortii. Chemical composition of family and 19 natural products have been individual parts of the parasite (flowers and stem sequestered from the host (Scharenberg and Zidorn, with a tuber, separately) and host species were 2018). The Polish team studied six species compared - T. polycephalum, in various localities, belonging to the genera Orobanche and at different altitudes. Additionally, it was unclear Phelipanche and reported that they are rich in PhGs whether the compounds identified were synthesised and their number is greater than in Herba by the holoparasite or obtained from their host Cistanches. This herb has a high and comparable plants. Therefore, we examined the effect of the host antioxidant potential. It is widely used in Chinese species on the chemical composition and bioactivity medicine as a part of nutraceutical and medicinal of P. tournefortii. We have also identified preparations (Jedrejek et al., 2020). Owing to their phytochemical components associated with the unique properties, certain holoparasitic species like antioxidant properties of the parasite and host and Cistanche are being considered for cultivation in the interactions between them. Our research on the specific regions of China (Xu et al., 2009). chemical composition and beneficial health Orobanche crenata is a possible novel functional properties of this species can be used to create an food. In southern Italy, it is used to prepare several innovative product. This species can be treated as a traditional meals (Renna et al., 2018). The genus functional food as well as a pharmacological and Phelypaea includes 3 holoparasitic species therapeutic agent.

Piwowarczyk et al., Phytochemical and bioactive properties of Phelypaea tournefortii 114 – effect of parasitic lifestyle and environmental factors MATERIALS AND METHODS MPW-251 (MPW MED. INSTRUMENTS, Warsaw, Poland) at 19,000 × g for 10 min. Before Species studied and plant material analysis, the supernatant was purified using a The study was conducted between May and June hydrophilic PTFE membrane (Millex Samplicity 2017, in four localities of central (Kotayk province) Filter, Merck) of 0.20 µm pore size. All extractions and southern (Vayots Dzor province) Armenia were performed in triplicate (Oszmiański et al., (Western Asia), at different altitudes: 2018). 1. Kotayk prov., Garni N, rocky slopes, 40°07’50”N, 44°42’50”E, 1480 m. Analysis of antioxidant effect and inhibitory 2. Kotayk prov., NW Goght, rocky slopes, activity 40°08’18”N, 44°45’45”E, 1620 m 2,2’-azo-bis-3-ethylbenzothiazoline-6-sulfonic acid 3. Kotayk prov., N of Geghard, rocky slopes, (ABTS•+) was used to observe the enzyme kinetics 40°09’08”N, 44°47’26”E, 1800 m. of assays (Arnao et al., 2001). Additionally, DPPH 4. Vayots Dzor prov., N of Sers, slope, (1,1-diphenyl-2-picrylhydrazyl) and the ferric 39°34’22”N, 45°29’03”E, 2050 m. reducing antioxidant property (FRAP) were also The holoparasite Phelypaea tourneforti and its host determined (Yen and Chen, 1995; Benzie and species Tanacetum polycephalum subsp. Strain, 1996). The antioxidant capacity was argyrophyllum were examined (Figure 1). Samples expressed as millimoles of Trolox per 100 g (flowers and stems with a tubers, separately) of the distilled water. ABTS•+ and FRAP assay P. tournefortii parasite were rinsed from dirt and measurements were carried out on a UV-2401 PC soil before analysis. Samples (stem with leaves and spectrophotometer. The effect of the activity of the flowers) of the host species, T. polycephalum subsp. extracts on the enzymes, α-glucosidase lipase, and argyrophyllum, infected by the parasite, were α-amylase, was determined (Podsedek et al., 2014; collected from the aforementioned localities. Nickavar and Yousefian, 2011). The result is Following harvesting under natural conditions, the expressed as IC50. IC50 means the amount of materials were dried. Plant specimens were inhibitor (expressed as mg of powder samples of deposited in the herbariums of the Universities: plants per mL of the reaction mixture, under assay Yerevan State University (ERCB) and Jan conditions) required to inhibit 50% of enzyme Kochanowski University in Kielce (KTC). activity. The IC50 value of the plants tested was obtained from a plot of the concentration in 1 mL of Sample preparation reaction mixture against the % inhibition. For the Extraction - antioxidant effect and inhibitory measurement of L-ascorbic acid and nitrates, the activity RQflex 10 (Merck) requantometer was used Extraction of a compound mixture of parts (flowers, (Mijowska et al., 2016, Ochmian et al., 2019). All and stem with a tuber, separately) from the parasite samples were assayed in triplicate. and host was performed using 70% ethanol, at 20°C, for 60 min, with constant stirring. Afterwards they Analysis of polyphenolic compounds using the were centrifuged at 4000 rpm for 10 minutes. The UPLC-PDA-ESI-MS/MS method supernatant was filtered under reduced pressure Polyphenolic compounds were analysed using a through a cellulose membrane with 1.2 μm pore UPLC-PDA-ESI-MS/MS Waters ACQUITY diameter and the supernatant was concentrated at system (Waters, Milford, MA, USA). It consists of 40°C in a vacuum evaporator, to remove acetone. a binary pump manager, sample manager, column Subsequently, the aqueous phase was diluted with manager, PDA detector, and tandem quadrupole water. The fruit extract was serially diluted using mass spectrometer (TQD) with electrospray redistilled water to obtain a gradient of ionisation (ESI). The separation was carried out on concentrations for use in analytical and biological a BEH C18 column (100 mm × 2.1 mm internal activity assays. diameter, 1.7 µm, Waters) at 50°C. For anthocyanin investigation, the following solvent system was Extraction - polyphenols used: mobile phase A (2% formic acid in water v/v) The flowers and stem with tuber from the parasite and mobile phase B (2% formic acid in 40% ACN and host herb were extracted using 30% aqueous in water v/v). For other polyphenolic compounds, a methanol. The separation was performed twice by lower concentration of formic acid was used (0.1% incubating in a sonicator (Sonic 6D, Polsonic, v/v). The program was set as follows: 0 min 5% B; Warsaw, Poland) for 20 min, followed by shaking 0 to 8 min, linear to 100% B; 8 to 9.5 min, washing, from time to time (a few times or rarely). and back to initial condition. The sample injection Subsequently, the suspension was centrifuged in an volume was 5 µl (partial loop with needle overfill)

Acta Universitatis Cibiniensis Series E: FOOD TECHNOLOGY 115 Vol. XXIV (2020), no. 1 and the flow rate was 0.35 mL/min. The following a photodiode array (CP Pigment Analyzer PA1101 parameters were used for TQD: capillary voltage produced by Control in Applied Physiology GbR., 3.5 kV; con voltage, 30 V in positive and negative Germany), which was used to capture a range of mode; the source temperature was 250°C, and the wavelengths from 450-1100 nm. The instrument desolvation temperature was 350°C; con gas flow, consists of 9 LEDs (660–780 nm) and a PDA 100 L/h; and desolvation gas flow 800 L/h. Argon, (Photodiode Array Detector), which measures at a flow rate of 0.3 mL/min, was used as the photons within the range of 450–1100 nm, remitted collision gas. The polyphenolic compound from the plant's epidermis. The pigment contents detection and identification were based on the are displayed on the screen as a normalised specific PDA spectra, mass to charge ratio, and difference vegetation index (NDVI) and normalised fragment ions obtained following collision-induced anthocyanin index (NAI). Indices have been dissociation (CID). Additional settings are enlisted optimised for the determination of pigments, and in Table 1. Prior to injection, samples were filtered calculated according to the equations: through a 0.45 µm pore size membrane filter NDVI=(I780−I660)/(I780+I660) and (Merck Millipore) and injected directly on to the NAI=(I780−I550)/(I780+I570), with a disposition chromatographic column. Quantification was of both parameters normalised between -1 (lack of achieved by injecting solutions of known green or redness) and +1 (green or red) (Ochmian et concentrations, ranging from 0.05 to 5 mg/mL (R al., 2013). The dried plant material (at 25°C) was 2 ≤ 0.9998), of phenolic compounds as standards. ground in a laboratory mill in triplicate. About 3 g The measurements were performed in triplicate and of ground plants were poured into the glass cuvette, expressed as mg/L. Waters MassLynx software and 35 measurements were made in triplicate. The v.4.1 was used for data acquisition and processing. samples were mixed before each measurement. The polyphenolic compounds were evaluated in the positive and negative ion modes. The sample Statistical analysis measurement was done in triplicate and expressed The Statistica 13.1 program was used to perform all as mg/100 g dry weight (DM). analyses (StatSoft Poland, Cracow, Poland). If no homogeneity of variance or normality of Analysis of colour and pigment parameters distribution was found before, non-parametric The evaluated colour indicators are L* (L* = 100 methods were used (Kruskal-Wallis test). Statistical means white; L* = 0 means black), a* (+a* means significance of the differences between means was red; -a* means green), b* (+b* means yellow; -b* determined by testing the homogeneity of variance means blue). Measurements were performed with a and normality of distribution, followed by ANOVA 3 mm hole diameter; the colour was measured in with Tukey's post hoc test. The results were glass cells, using a 10° observer and a D65 analysed and classified into groups in a hierarchical illuminant. CIE L*a*b* and CMC (Colour order by means of Ward's method. This was to Measurement Committee of the Society of Dyes and characterise the relationship between phenol Colourists) were measured with a content, antioxidant and inhibitory activity. spectrophotometer (Konica Minolta CM-700d) Multidimensional analysis was carried out using the (Chełpiński et al. 2019). The non-destructive analysis of the main components (PCA). The data measurement of quality parameters was performed were scaled during pre-processing automatically. using a hand-held spectrophotometer equipped with

Figure 1. Habitat of the holoparasite Phelypaea tournefortii and its host Tanacetum polycephalum. Phot. R. Piwowarczyk

Piwowarczyk et al., Phytochemical and bioactive properties of Phelypaea tournefortii 116 – effect of parasitic lifestyle and environmental factors RESULTS AND DISCUSSION respectively. Fragmentation peaks 14-16, and 20, with m/z=563, generated an ion at m/z=285, which Determination and content of polyphenolic was tentatively noted as Kaempferol–pentoside. compounds Four common kaempferol derivatives like 3-O- The 41 compounds (Table 1) were identified by the rutinoside (peak 10–m/z=593), 3-O-galactoside determination of bioactive compounds using (peak 26–m/z=447), glucopyranoside (peak 38– UPLC-PDA-ESI-MS/MS (typically the CID m/z=461), and glucoside acetate (peak 39– method). The profile of the bioactive compound of m/z=491), were also found in the host. The the holoparasite, P. tournefortii, and the host, T. identified compounds belonging to the flavonol polycephalum, were different, qualitatively and group have been reported extensively previously quantitatively. The bioactive compound profile of (Han et al., 2007; Karioti et al., 2007). However, the host included 26 phenols out of which 4 they have not yet been identified in the host (peak belonged to phenylethanoids, 9 to phenolic acids, 12 37), identified as apigenin – glucopyranoside to flavonols, and 1 to flavones. Conversely, the (m/z=445), which belongs to flavones. This bioactive compound profile of the holoparasite compound has been previously reported in a included 15 compounds, out of which 12 belonged Chinese herb by Han et al. (2007). to phenylethanoids, and 3 to anthocyanins. The The polyphenolic content in the stem of the parasite three anthocyanins in P. tournefortii were caffeoyl was similar (in the Sers locality), or greater (from and p-coumaroyl derivatives. This group was 2.1 to 3.8), as compared to the host. In the flowers detected using the positive mode as flavylium of the parasite, these differences were even greater cations, in an acidified milieu. - from 2.5 to 6.6. Habitat had a very large impact on The structures of few compounds identified in the the examined feature. Polyphenols were most parasite (flowers and stems) namely, pelargonidin abundant in the flowers and stem of the parasite, and (peak 4–m/z=593), delphinidin (peak 5–m/z=611), in the host herb collected from the highest location cyanidin (peak 8–m/z=595), were identified. Similar (2050 m above sea level – Sers). results were noted in grapes (Kapusta et al., 2017), The bioactive profile of P. tournefortii, regardless while in P. tournefortii, this was the first time the of the site, primarily included phenylethanoids anthocyanins compound was detected. The host, T. (PhGs). Twelve of the compounds identified polycephalum, was studied for hydroxycinamic (constituting about 92.1% of the polyphenolic acids. The determination of feruloylquinic acid compounds) belonged to PhGs, and the remaining (peak 17–m/z=367), caffeic acid 4-glucoside (peak compounds were anthocyanins. 7–m/z=341), caffeoylquinic acid in item 3-O and 5- The polyphenol concentration in P. tournefortii O (peaks 2 and 6–m/z=353), di-caffeoylquinic (peak varied depending on the plant part. On average, 31, 32, 34, and 37–m/z=515), and p- flowers contained 1.8 times more total polyphenols coumaroylquinic acid (peak 3–m/z=377) was compared to stems. A similar relationship was performed with available standards and data based observed in C. violacea (Debouba, et al., 2012). on the MS and MS/MS profile. These compounds However, the opposite was observed in C. armena, were previously described either in grapes or in regardless of whether the host was Salsola Fallopia (Lachowicz and Oszmiański, 2019). dendroides or Alhagi maurorum (Piwowarczyk, et The next group identified in the host was flavonol. al., 2020). On average, the concentration of the This group primarily comprised of kaempferol, polyphenols pectolinaroside and eukovoside were quercetin, and isorhamnetin derivatives with a 85 and 36 times more in flowers than in stems, characteristic fragment ion at m/z=285, 301, 315, respectively. Eukovoside was previously identified respectively. Two peaks, 19 and 41, produced the in the semiparasitic species [M-H]- ion at m/z=463 and 345, which indicates the (Orobanchaceae), and in Verbascum loss of 44 Da and 162 Da. These compounds were (Scrophulariaceae) and Aloysia citrodora identified as quercetin 3-O-glucoside, and (Verbenaceae) (Tóth, et al., 2014; Pereira et al., quercetin-acetate. Peaks 28 and 40, with a 2017). However, its presence in the holoparasitic pseudomolecular ion at m/z=623 and 315, species from Orobanchaceae family has not been respectively, and a fragment ion at m/z=315, established till date. The flowers of P. tournefortii indicated the loss of the rutinoside moiety (148 Da). contained eukovoside at a concentration greater Two peaks, 19 and 41, produced the [M-H]- ion at than that in A. citrodora (Pereira et al., 2017). m/z=463 and 345, and these compounds were noted Eukovoside contributes to the formation of ferulic as quercetin 3-O-glucoside and quercetin-acetate acid, which possesses a wide range of health (which indicates the loss of 44 Da and 162 Da), benefits.

Acta Universitatis Cibiniensis Series E: FOOD TECHNOLOGY 117 Vol. XXIV (2020), no. 1 Table 1. Identification of phenyloethanoids and their glycosides in flowers and stem with tuber of parasite Phelypaea tournefortii and infected host – Tanacetum polycephalum No Rt [min] UV-Vis [nm] MS [m/z] MS/MS [m/z] Tenative compounds P. tournefortii T. polycephalum Phenylethanoids 1 2.38 288sh,329 487 179/161/135 Cistanoside F X 9 3.50 288 sh,324 639 621/477/179 B-hydroxyverbascoside X 11 3.63 253,347 637 461/193/175 Eukovoside X 621/477/459/ 12 3.68 286sh,328 639 B-hydroxtverbascoside X 179/161/135 13 3.74 290sh,329 785 623/461/161 Echinacoside X 18 4.17 267,332 621 475/313 Pectolinaroside X 21 4.42 253,341 651 505/475/193 Martynoside X 22 4.46 288sh,324 533 371 Glycetein malonylglucoside X 23 4.66 286sh,329 623 461/161 Acteoside (Verbascoside) X 24 4.85 281,333 461 299 Chrysoeriol 7-O-glucoside X 25 4.92 284sh,329 621 459/487/179 Crenatoside X 27 4.96 288sh,327 477 315/179 Calceolarioside B X 29 5.07 288,327 623 461/175 Isoverbascoside X 30 5.09 288sh,327 639 621/477/179 B-hydroxyverbascoside X 33 5.26 288sh,329 639 621/459/179 Campneoside II X Tubuloside B 35 5.40 278sh,329 655 623/461/161 X (2'-acetyloverbascoside) Anthocyanins Delphinidin 3-O-(6''- 4 2.61 277,524 611 303 X coumaryl)-glucoside Cyanidin 3-O-(6''-coumaryl)- 5 2.99 281,517 595 287 X glucoside 8 3.37 281,502 579 271 Pelargonidin 3-O-rutinoside X Phenolic acids 2 2.46 288sh,324 353 191 3-O-Caffeoylquinic acid X 3 2.59 309 337 163 3-O-p-Coumaryloquinic acid X 6 3.07 288sh,327 353 191 5-O-Caffeoylquinic acid X 7 3.20 288sh,324 341 179/161/173 Caffeic acid 4-glucoside X 17 4.15 288sh,326 367 191 Feruloylquinic acid X 31 5.17 288sh,327 515 353 3,4-Di-caffeoylquinic acid X 32 5.24 288sh,327 515 353 1,4-Di-caffeoylquinic acid X 34 5.29 288sh,327 515 353 3,5-Di-caffeoylquinic acid X 37 5.62 288sh,327 515 353 4,5-Di-caffeoylquinic acid X Flavonols 10 3.58 271,331 593 285 Kaempferol 3-O-rutinoside X 14 3.95 270,329 563 285 Kaempferol –pentoside X 15 4.07 271,333 563 285 Kaempferol –pentoside X 16 4.12 271,332 563 285 Kaempferol –pentoside X 19 4.25 254,338 463 301 Quercetin 3-O-glucoside X 20 4.29 272,329 563 285 Kaempferol –pentoside X 26 4.92 267,345 447 285 Kaempferol 3-O-galactoside X 28 4.99 257,364 623 315 Isorhamnetin 3-O-rutinoside X Kaempferol – 38 5.74 269,338 461 285 X glucopyranoside Kaempferol –glucoside- 39 5.77 267,338 491 285 X acetate 40 6.36 255,366 315 Isorhamnetin X 41 7.54 255,352 345 301 Quercetin–acetate X Flavones 36 5.60 266,336 445 269 Apigenin – glucopyranoside X

Piwowarczyk et al., Phytochemical and bioactive properties of Phelypaea tournefortii 118 – effect of parasitic lifestyle and environmental factors Among the compounds identified, those present in apoptosis and have a wide array of biological high concentrations regardless of the plant part were activities (Deng et al., 2004; Liu et al., 2018). These acteoside (575.31–740.21 mg/100 g in flowers and compounds were detected in the flower and stem 455.02–750.95 mg/100 g in the stem) and extracts of P. tournefortii in low concentration crenatoside (193.54–261.72 mg/100 g in flowers (14.96–23.61 and 20.22–32.67 mg/100 g in flowers, and 73.85–210.11 mg/100 g in the stem). and 4.86–11.38 and 11.34–24.66 in stems, Eukovoside was found in high concentration in respectively). The echinacoside content in parasite flowers (199.07–308.89 mg/100 g). However, its flowers was similar to that in the C. armena flowers, content was much lower in the stem (4.86 to 11.38 which dwell as parasites on Salsola dendroides. It mg/100 g). Pectolinaroside was also present in the was found in lower concentration in the flowers of flowers in large quantities. The stem contained the parasitic plant, Alhagi maurorum (Piwowarczyk cistanoside F, calceolarioside B, and tubuloside B at et al., 2020). The polyphenol profile of P. a concentration similar to or greater than that in tournefortii (parasite) was different from that of T. flowers. In Acteoside plants, it is widespread given polycephalum (host), indicating the presence of an its diverse biological activity. According to Taskova independent biosynthetic pathway in the et al. (2005) it has already been detected in over 200 holoparasite (Table 2), similar to C. armena plant species belonging to 23 families. Crenatoside (Piwowarczyk et al., 2020) However, in both T. was first isolated from Orobanche crenata, from the polycephalum and P. tournefortii, phenylethanoid part of the plant above ground. It is a natural glycosides (PhGs) were among the compounds bioactive compound found in Pogostemon cable identified. They are considered to be a characteristic (Chen, et al., 2016). It has long been used in of parasites, and they usually do not occur in the traditional Chinese medicine. It is used to treat host (Scharenberg and Zidorn, 2018). In T. colds, nausea, diarrhea, headaches, and fevers. polycephalum, PhGs were detected in small Compounds like echinacoside and tubuloside B, quantities. However, these compounds were along with acteoside, inhibit chemically-induced different from those in P. tournefortii.

Table 2. Content of phenolic compounds in the parasite, Phelypaea tournefortii parasite – Phelypaea tournefortii flower stem with tuber Tenative 11 3 5 7 2 4 6 8 Delphinidin 3-O-(6''-p-coumaryl)- 58.36e2 27.29c 45.19d 65.76f 12.61a 10.07a 14.60b 12.58ab glucoside Cyanidin 3-O-(6''-p-coumaryl)- 48.50e 20.74c 34.93d 43.63e 10.84ab 8.84a 10.91ab 12.12b glucoside Pelargonidin 3-O-rutinoside 91.14e 47.41c 84.15d 104.49f 24.06b 20.96a 23.78b 24.67b 198.00 95.44 164.27 213.88 47.51 39.87 49.29 49.37 ANTHOCYANIDIN E C D F B A B B Cistanoside F 16.84b 17.40b 24.22c 17.85b 22.31c 10.76a 24.81c 30.26d Eukovoside 229.79d 199.07c 298.98e 308.89f 11.38b 4.86a 10.56b 5.61a B-hydroxtverbascoside 17.94f 15.83e 18.24f 24.29g 5.91c 0.91a 3.48b 8.19d Echinacoside 17.50e 14.96d 17.88e 24.32f 8.08c 1.36a 4.02b 8.71c Pectolinaroside 160.05e 152.19d 234.69f 295.85g 7.43c 4.49b 4.18b 0.76a Martynoside 17.11d 31.12e 31.53e 40.27f 2.77a 2.62a 5.55c 3.43b Acteoside – Verbascoside 740.21d 575.31b 733.93d 727.51d 606.08b 455.02a 686.57c 750.95d Crenatoside 219.16d 193.54c 213.87d 261.72e 210.11d 73.85a 173.97b 185.19c Calceolarioside B 16.86b 16.08b 24.79d 19.13c 19.85c 11.97a 42.11f 35.53e B-hydroxyverbascoside 9.61e 7.33d 9.40e 14.53f 2.75b 0.82a 4.74c 4.60c Campneoside II 3.43b 5.70d 10.42f 7.92e 2.58a 2.43a 4.49c 2.77a 2'-acetyloverbascoside (Tubuloside B) 21.19c 20.22bc 25.04d 33.65e 21.45c 11.34a 18.56b 24.66d 1469.69 1248.75 1642.99 1775.92 920.7 580.43 983.04 1060.66 PHENYLOETHANOIDS E D F G B A B C 1668 1344 1807 1990 968 620 1032 1110 TOTAL E D F G B A B C 1Garni – flower 1, stem with tuber 2; Goght – flower 3, stem with tuber 4; Geghard – flower 5, stem with tuber 6; Sers – flower 7, stem with tuber 8; 2 means followed by the same letter in lines do not differ significantly at P=0.05 according to Tukey multiple ranges

Acta Universitatis Cibiniensis Series E: FOOD TECHNOLOGY 119 Vol. XXIV (2020), no. 1 PhGs were also found in few other non-parasitic populations in Iran (Malekpoor et al., 2015). In T. plants. They are a characteristic constituent of the polycephalum the essential oil concentration was families belonging to the order, and they between 63 mg GA/100 g and 153 mg GA/100 g. perform a protective, antimicrobial, anti-stress, or Among the bioactive compounds found in the host, stress-resistant function in them. For example, in phenolic acids were dominant. On average, the Plantago coronopus (Plantaginaceae), verbascoside phenolic acid content was 65.2%, flavonol content (acteoside) was present as a concentration of 4621 was 17.6%, flavone content was 14.1%, and mg/100 g, and isoverbascoside (isoacteoside) was phenylethanoid content was 5.4%. present as a concentration of 147 mg/100 g, while Of all the identified compounds in T. polycephalum, in Orobanche crenata they were present at a 5-O-caffeoylquinic acid (5-CQA) was found in the concentration of 3389 mg/100 g and 200 mg/100 g, highest concentration (73.79–188.94 mg/100 g). It respectively (Gatto et al., 2011). plays an important role in the stress response of the In the flowers and stem of the parasite, 3 pigments plant (Dawidowicz and Typek 2010), and therefore classified as anthocyanin in the glycosylated form the presence of the parasite might influence its high were found, at a concentration ranging from 95.44 content. Moreover, it was found in most plants mg/100 g (stand 3) to 213.88 mg/100 g (stand 4). harvested from high altitudes in Sers. Many other They were responsible for the very intense red compounds could also be involved in the stress colour of the flowers (Table 5). The flowers were response, resulting from the harsh climatic rich in anthocyanins, and such large amounts have conditions at a high altitude. Amigoni et al. (2017), not been reported previously. Anthocyanins were performed in vivo studies on the model organism also present in smaller quantities in the stems Caenorhabditis elegans, and showed that the green (39.87–49.37 mg/100 g). Cyanidine 3-O-glucoside coffee extract containing 5-CQA, and pure 5-CQA, was the main anthocyanin found in the majority of improve the body's resistance to oxidative stress, plants. However, in P. tournefortii, pelargonidin 3- and contribute to prolonging life and delaying O-rutinoside was the dominant form, particularly ageing. T. polycephalum, which has a significant present in the flowers collected from the highest concentration of 5-CQA, can be used to develop elevation in the Sers locality. novel nutraceutical and pharmaceutical products. Anthocyanins were present in much larger quantities in P. tournefortii flowers compared to Antioxidant effect and inhibitory activity wild edible flowers, among which Fuchsia hybrida Functional food ingredients must possess a high had the highest content (47.52 mg/100 g) content of substances with health benefits, namely (Benvenuti et al., 2016). The anthocyanin content in vitamins, polyphenols, or other compounds with these flowers was comparable and sometimes antioxidant properties, or compounds which inhibit higher, than those in fruits considered as rich adverse processes. The antioxidant and antidiabetic sources of anthocyanins (Ramos et al., 2014). To features of the flowers and stem of the parasite P. date, the holoparasitic Orobanchaceae family is tournefortii, and host T. polycephalum were habitat- known for plants rich in PhGs. However, reports on dependent (Table 4). Regardless of the habitat, P. the availability of anthocyanins in this group of tournefortii flowers were characterised by a higher plants are limited. Luteolin 7-O-glucoside has been content of antioxidants, as determined by reported in Orobanche minor, and cyanidin 3-O- ABTS•+and DPPH, compared to the stem of the rutinoside has been reported in indica host T. polycephalum. The most distinct differences (Iwashina, 2010). Anthocyanin pigments have been were found when the DPPH reagent was applied. traditionally used for food colouring. They possess The flowers showed six times higher content of various health benefits including antioxidant and antioxidants compared to the shoots of P. antimicrobial activities. They have also been used tournefortii. For measuring the antioxidant as nutraceuticals (Khoo et al., 2017). In Armenia, P. capacity, DPPH reagent is commonly used (Nenadis tournefortii flowers have been used extensively to and Tsimidou, 2002). The flowers of P. tournefortii, dye foods (e.g., eggs) and wool. on each habitat, had a very high content of phenolic In T. polycephalum, the total polyphenol level was compounds (Table 2). The host of T. polycephalum very different between populations. Its content was was several times stronger in the reduction of metal more than double in one site, 12–Sers (561 mg/100 ions, which was determined by FRAP. PCA g), compared to another site, 10–Goght (274 analysis showed the positive effects of polyphenols, mg/100 g) (Table 3). Very large differences were their antioxidant property, and inhibitory activity also found in the amount of individual compounds (Figure 2). Polyphenols from the flowers and stems obtained from the host herb at different sites. The of the parasite and host had varied health benefits. total polyphenol content also differed among the

Piwowarczyk et al., Phytochemical and bioactive properties of Phelypaea tournefortii 120 – effect of parasitic lifestyle and environmental factors Table 3. Content of phenolic compounds in the host, Tanacetum polycephalum host – Tanacetum polycephalum Tenative 91 10 11 12 3-O-Caffeoylquinic acid 4.59d2 1.08a 2.79c 1.77b 3-O-p-Coumaryloquinic acid 0.23a 0.68b 0.66b 2.01c 5-O-Caffeoylquinic acid 165.53b 73.79a 188.94c 169.45b Caffeic acid 4-glucoside 1.83b 1.21a 3.49d 2.48c Feruloylquinic acid 0.07a 5.32c 0.19a 3.74b 3,4-Di-caffeoylquinic acid 2.56b 4.17c 1.62a 2.66b 1,4-Di-caffeoylquinic acid 29.88b 61.17c 4.61a n.d. 3,5-Di-caffeoylquinic acid 64.00c 26.28b 8.56a 92.18d 4,5-Di-caffeoylquinic acid 18.75b 4.59a 73.21d 33.79c 287.44 178.29 284.07 308.08 PHENOLIC ACIDS B A B B B-hydroxyverbascoside 1.39b 1.34b 1.96c 0.90a Chrysoeriol 7-O-glucoside 4.09b 7.25c 0.37a 54.43d Isoverbascoside 2.09b 0.13a 29.31c nd Glycetein malonylglucoside 1.38c 0.65ab 0.71b 0.61a 8.95 9.37 32.35 55.94 PHENYLETHANOIDS A A B C Kaempferol 3-O-rutinoside 0.51c 0.08a 0.86d 0.20b Kaempferol pentoside 1.39c 1.01b 3.51d 0.13a Kaempferol pentoside 0.43a 0.42a 1.62c 0.84b Kaempferol pentoside 0.80a 2.61d 1.04b 1.53c Quercetin 3-O-glucoside 1.60c 3.97d 0.28a 0.83b Kaempferol pentoside 0.40a 1.18b 4.03c 5.50d Kaempferol 3-O-galactoside 4.57b 8.55c 0.51a 25.88d Isorhamnetin 3-O-rutinoside 9.00b 6.39a 34.59c nd Kaempferol glucopyranoside 0.95a 2.15a 19.83b 22.35b Kaempferol glucoside-acetate 0.48a 10.37b 10.22b 10.48b Isorhamnetin 0.82a 2.05b 6.87d 2.75c Quercetin acetate 5.40a 18.10c 19.43d 8.18b 27.73 57.53 103.50 79.28 FLAVONOLS A B D C Apigenin glucopyranoside 10.65a 29.95b 111.20c 119.11d 333 274 530 561 TOTAL B A C C 1Garni 9; Goght 10; Geghard 11; Sers 12, 2 designations according to Table 2

Table 4. Antioxidant effect and inhibitory activity of the indicator parts of the parasite, Phelypaea tournefortii, and host, Tanacetum polycephalum, based on locality α-amylase α-glucosidase lipase L-ascorbic nitrates V ABTS•+ DPPH FRAP IC50 IC50 IC50 acid (NO3) (μmol T/g) (μmol T/g) (μmol T/g) (mg/ml) (mg/ml) (mg/ml) (mg/100 g) (mg/1000 g) parasite – Phelypaea tournefortii flower 11 137.3e2 709.3f 97.4d 11.26c 17.82b 45.33c 68.9b 12.7a 3 144.5ef 574.2d 75.4b 9.34b 13.44a 26.73a 77.8bc 19.0b 5 95.6c 645.6e 103.5d 6.78a 27.88d 37.58b 46.3a 13.5a 7 153.7f 765.0g 114.7e 5.78a 11.59a 42.71c 81.6c 15.6ab mean 132.78C 673.53C 97.75B 8.29C 17.68C 38.09C 68.65B 15.20A

Acta Universitatis Cibiniensis Series E: FOOD TECHNOLOGY 121 Vol. XXIV (2020), no. 1 DPPH FRAP α-amylase α-glucosidase lipase nitrates V ABTS•+ L-ascorbic acid (μmol (μmol IC50 IC50 IC50 (NO3) (μmol T/g) (mg/100 g) T/g) T/g) (mg/ml) (mg/ml) (mg/ml) (mg/1000 g) stem with tuber 2 78.5b 103.7ab 55.8a 24.51f 28.96d 73.56f 211.5c 28.5b 4 96.1c 89.5a 62.5ab 17.12e 22.52c 42.80c 228.9c 35.4c 6 104.2d 145.9c 72.1b 13.53d 33.91e 52.30d 166.5b 38.1d 8 64.9a 128.7bc 83.2c 17.93e 25.92c 63.04e 139.4a 23.3a mean 85.93B 116.95A 68.40A 18.27B 27.83B 57.93B 186.58C 31.33B host – Tanacetum polycephalum 9 37.9B 194.5A 234.3B 53.85B 42.32B 97.89B 27.0AB 205.2C 10 32.4A 203.0A 216.1A 58.50A 45.28B 88.35C 35.8BC 219.5C 11 48.9C 271.2C 289.4C 51.07B 38.90C 79.08D 24.5A 184.0B 12 53.4D 245.4B 248.9B 46.61C 51.41A 112.04A 42.3C 164.7A mean 43.15A 228.53B 247.18C 52.51A 44.48A 94.34A 32.40A 193.35C 1,2 designation according to Table 2

1,0 1,0 α-amylase

ABTS L* polifenolsNDVI α-amylase lipase 0,5 L* 0,5

NAI b*

NAI 0,0 0,0 a* NDVI

26,27% polifenols 28,67% a*b*

ABTS DPPH α-glucosida -0,5 -0,5 lipase FRAP α-glucosida FRAP DPPH

-1,0 -1,0

-1,0 -0,5 0,0 0,5 1,0 -1,0 -0,5 0,0 0,5 1,0 55,78% 60,11%

parasite flower parasite stem with tuber

1,0

FRAP

DPPH 0,5

α-amy lase ABTS L* NAI 0,0

36,43% NDVI

polif enols -0,5

α-glucosida a* b* lipase -1,0

-1,0 -0,5 0,0 0,5 1,0

58,67%

host herb Figure 2. Correlation between the content of polyphenolic compounds and antioxidant activity using PCA test of flower and stem with tuber of the Phelypaea tournefortii parasite and host Tanacetum polycephalum. Location of charge vectors against two main components (1 and 2 factors)

Piwowarczyk et al., Phytochemical and bioactive properties of Phelypaea tournefortii 122 – effect of parasitic lifestyle and environmental factors The flowers of the parasite were also characterised defined in the DPPH test was 23.08 μmol TE/g FW by very high inhibitory activity owing to the and FRAP was 11.68 μmol TE/g (Peng et al., 2016). presence of α-amylase, α-glucosidase, and lipase. The antioxidant property of the dried flowers of P. The inhibitory activity of the flowers was higher tournefortii was much higher compared to these than that of the stem of the tested parasite and much studies. On average, DPPH was 673.53 μmol T/g higher compared to the host. The parasitic flowers and FRAP was 97.75 μmol T/g. Stem DPPH was collected from the highest altitude of the Sers site about six times lower (116.95 μmol T/g) compared possessed the maximum health benefit. The impact to the studied flowers of P. tournefortii. For of location (altitude above sea level) was confirmed ABTS•+ and FRAP, the differences were not by Ward analysis. Flowers and stems collected from significant (Table 4). In contrast, other studies the Sers site constituted a separate group. The other found higher free radical scavenging activity in the stands had a similar impact on the chemical stem, compared to flowers and roots of C. compound of the flowers and stems with tuber phelypaea, as determined by the DPPH and (Figure 3). ABTS•+ radicals (Trampetti et al., 2019). Extracts from Cistanche phelypaea described by The daily vitamin C requirement of an adult is about Trampetti et al. (2019), had a lower inhibitory 60–75 mg. In the studied parasite, the L-ascorbic activity to carbohydrate hydrolysing enzymes than acid content of the stem (186.58 mg/100 g) was P. tournefortii investigated, except for the root comparable to that of the fruit (sea buckthorn), extract, which showed good inhibitory activity to which is considered to be rich in this compound glucosidase. Other studies have shown that C. (about 200 mg/100 g). Conversely, the vitamin C tubulosa significantly suppressed elevated blood content of the flowers of the parasite was at a level glucose levels and improved insulin resistance and comparable with other fruits commonly considered dyslipidemia in mice. Studies have shown that the to be rich sources of vitamin C, like strawberry (65 antioxidant activity of C. tubulosa was ABTS•+ mg/100 g), lemon (74.3 mg/100 g), mandarin (37.7 1.39 mM TEAC/g and FRAP 1.61 mM FEAC/g mg/100 g), or blackberry (21 mg/100 g) (Teleszko respectively (Xiong et al., 2013). In other studies, et al., 2015). the maximum free radical scavenging activity as

Garni flower (1)

Garni stem (2)

Goght stem (4)

Goght flower (3)

Sers flower (7)

Geghard flower (5)

Geghard stem (6)

Sers stem (8)

0 500 1000 1500 2000 2500 Linkage distance

Figure 3. Dendrogram of cluster analysis of polyphenols, antioxidant effect, and inhibitory activity of Phelypaea tournefortii (flower versus stem with tuber), depending on the altitude of the locality. The vertical line indicates the cut- off used to form the 5 groups

Acta Universitatis Cibiniensis Series E: FOOD TECHNOLOGY 123 Vol. XXIV (2020), no. 1 In addition to large health benefits, plants that are to (12) site possessed the darkest flowers (L* value of be used as functional foods must also possess low 32.2) and stem (L* value of 48.1). Also, the flowers levels of harmful substances. Regardless of the and the stem of the parasite, P. tournefortii, location, the flowers and stem of the parasite collected from the host in this site were contained very little nitrate (on average, 15.2 mg characterised by more intense chromatic indices and 31.33 mg, respectively). However, many (flowers a* 51.4, b* 22.3 and stem a* 42.4, b* harmful compounds were found in the host herb T. 37.9). The flowers had a high NAI index, which was polycephalum (193.35 mg). According to applicable also confirmed by the high content of regulations, these fruits are considered safe for the anthocyanidins (Table 2). consumer. According to the EU legislation, a limit The indicator is highly correlated with the on the nitrate content is set only for green leafy anthocyanin content of plants (Seifert et al., 2015). vegetables. According to the EC regulation, fresh It was characterised by a low NDVI index. lettuce may contain up to 5000 mg/1000 g and the Conversely, the host from Sers had low colour- nitrate content in foods for infants and young determining indices. Both, the parasite and the host, children must be significantly lower and should not had low NAI and high NDVI. The NDVI, which exceed 200 mg/1000 g. indicates the vegetative potential of plants, was significant in host plants. In parasites, on the other Colour and pigment parameters hand, it was very low - this is understandable for a Fast and relatively accurate methods are being holoparasite plant. Both indicators are used to assess sought for assessing plant quality, under field plant quality: NAI and NDVI (Rutkowski et al., conditions. However, this demands the 2008; Seifert et al., 2015). identification of appropriate measuring methods For saturated (so-called 'pure') colours, the colour and indicators, which would allow for evaluating differences in the samples may be greater compared the parameters of individual species, and sometimes to colours with lower saturation. The human eye even cultivars (Zanella et al., 2013). Various finds it harder to notice differences in chromaticity, methods like spectrophotometry, time-resolved and easier to notice differences in shade. Flowers reflectance spectroscopy, hyper-spectral back- and stems of the parasite collected from the locality scattering imaging, or chlorophyll fluorescence can which showed the most intense colour (Sers), were be used for these purposes. These techniques are characterised by the highest values of colour based on the light exposure of a given object: the deviations: saturation (dC), and hue (dH). light is either absorbed, radiated, reflected or The host plant influences the colour of the indicator scattered by the sample. Recently, non-destructive elements of the parasite, as indicated by the results measuring methods have been sought. Optical of the colour analysis. The colour is also methods such as visible and near-infrared undoubtedly influenced by the location in which the spectroscopy are used (Zude et al., 2011). During host plant grows. The colour of the flowers, fruit, production, colour changes are used to measure stems, and leaves of the plant is mainly caused by food quality (Yusufe et al., 2017). The aerial parts anthocyanins. They protect the plant against biotic of the host, T. polycephalum, which was infected by and abiotic stresses. At high altitudes, they protect P. tournefortii, was characterised by varied CIE the plants against strong UV radiation and lower L*a*b* and CMC colour parameters (Table 5). The temperatures (Sivankalyani et al., 2016). At high studied parameters depended on the place (altitude) altitudes, anthocyanin was found to accumulate from which the plants were collected, and more rapidly in the later phases of the fruit ripening differences were observed in the stems and flowers process (Spinardi et al., 2019). In our study, the Sers of P. tournefortii. The L* parameter is associated site was located at the highest altitude (2050 m), with the physiological attributes of the visual several hundred meters above sea level compared to response. Each colour is characterised by other sites. Many of the parameters studied in the brightness, hue, and saturation, which cannot be experiment indicate that the parasite and host which systematised on a plane, and therefore it is presented grew there were more intensively coloured, and in a coordinate system (on the x and y axes). possessed higher polyphenol content (Table 2). The host herb harvested from Sers (12) and Geghard The flowers of P. tournefortii have an intense red (11) was much darker (52.4 and 57.8, respectively) colour, while that of T. polycephalum are yellow. compared to plants harvested from other sites. This The colour intensity of the parasite's indicator was a significant difference compared to plants elements is influenced by the host plant and the harvested from Garni and Goght sites 1 and 2, where environmental condition - especially altitude. This the L* value was 66.9 and 64.9, respectively. This is indicated by the colour analysis of the species relationship was also observed in the indicator parts tested. However, this requires further investigation. of the parasite. The parasite collected from the Sers

Piwowarczyk et al., Phytochemical and bioactive properties of Phelypaea tournefortii 124 – effect of parasitic lifestyle and environmental factors Table 5. Colour and pigment parameters of the holoparasite, Phelypaea tournefortii, and its host, Tanacetum polycephalum Color parameters CIE Pigment parameters place1 L* a* b* dC dH NAI NDVI parasite – Phelypaea tournefortii flower 11 36.2bc2 48.4a 18.7a 3.20a 2.37a 0.85bc -0.64b 3 37.3c 47.5a 18.7a 2.95a 2.72ab 0.79ab -0.62ab 5 35.6b 47.7a 19.0a 3.17a 3.12bc 0.71a -0.71c 7 32.2a 51.4b 22.3b 6.25b 3.91c 0.92c -0.58a mean 35.3A 48.7C 19.7A 3.89A 3.03A 0.82C -0.64B stem with tuber 2 54.6c 38.0b 35.4b 8.85b 5.61b 0.56b -0.69a 4 53.6c 36.8a 32.6a 5.95a 5.13a 0.54b -0.78b 6 51.2b 37.5ab 32.8a 6.26a 5.02a 0.57b -0.66a 8 48.1a 42.4c 37.9c 11.08c 5.01a 0.45a -0.88c mean 51.9B 38.7B 34.7C 8.03B 5.19B 0.53B -0.75A host – Tanacetum polycephalum 9 66.9c -40.0b 28.3b 10.31b 12.77bc -0.46c 0.79b 10 64.9c -44.8c 31.2c 13.28c 13.24c -0.50c 0.75a 11 57.8b -50.4d 24.3a 6.42a 11.19a -0.33b 0.83b 12 52.4a -37.6a 35.6d 8.06a 11.95ab -0.25a 0.89c mean 60.5C -43.2A 29.8B 9.52C 12.29C -0.38A 0.82C 1,2 designation according to Table 2

CONCLUSIONS DPPH, and FRAP assays), and higher inhibitory activity (as determined by α-amylase, α- Our research has shown significant differences in glucosidase, and lipase activities), compared to the the amount of phytochemicals, colour, and health stem. The stem was characterised by a high content benefits shown by Phelypaea tournefortii, with of L-ascorbic acid. On average, the pectolinaroside respect to particular parts of the parasite. This was and eukovoside content were 85 and 36 times more influenced by the location of the host and especially respectively, in flowers compared to stems. The the altitude of the habitat above sea level. The parasite flowers collected from the highest altitude quality of the profile as well as the amount of were more intensively coloured, possessed higher polyphenols of the parasite differed from the host polyphenol content, and the greatest health benefit. profile. A total of 41 polyphenolic compounds The ability of the host, T. polycephalum, to reduce (primarily flavonols and phenolic acids) were metal ions was several times higher compared to the identified, of which 15 (12 phenylethanoid, and 3 parasite, as determined by FRAP. Our study shows anthocyanins) were found in the parasite, and 26 in that P. tournefortii harvested from potential their host. The parasite contained significant cultivations (and not from natural endangered sites) amounts of valuable PhGs like acteoside, can be a promising source of functional and pectolinaroside, crenatoside, and anthocyanins bioactive ingredients and pigments. Due to the (particularly, pelargonidin 3-O-rutinoside). growing popularity of crops and herbs based on Anthocyanins were found in unprecedented large different parasitic plants, our research also draws quantity in flowers. Large amounts of eukovoside attention to different bioactive components, the were also identified for the first time in the activity of individual parts of the parasite, key holoparasite. The flowers of the parasite contained parameters associated with their parasitic lifestyles, 1.8 times more total polyphenols, displayed greater especially the host, and the impact of environmental antioxidant effect (as determined by ABTS•+, conditions.

Acta Universitatis Cibiniensis Series E: FOOD TECHNOLOGY 125 Vol. XXIV (2020), no. 1 ACKNOWLEDGEMENTS The field research in this study was financed by the National Geographic grant "Fascinating parasitic plants – Orobanchaceae in the Caucasus (Western Asia) – phytogeography, , host range, and evolution”, no. GEFNE 192-16 (2017), and the Jan Kochanowski University grant, no. SMGR.20.208-615 (2020) for R. Piwowarczyk. This research was supported by the West Pomeranian University of Technology of grants no. 518-07-014-3171-03/18. Two anonymous reviewers are thanked for their helpful comments on the manuscript.

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Piwowarczyk et al., Phytochemical and bioactive properties of Phelypaea tournefortii 128 – effect of parasitic lifestyle and environmental factors