Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles

Phytochemistry xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles Luka Mihajlovic a, Jelena Radosavljevic a, Lidija Burazer b, Katarina Smiljanic a, Tanja Cirkovic Velickovic a,⇑ a b

Center of Excellence in Molecular Food Sciences, University of Belgrade – Faculty of Chemistry, Belgrade, Serbia Institute of Virology, Vaccines and Sera ‘‘Torlak’’, Belgrade, Serbia

a r t i c l e

i n f o

Article history: Received 11 July 2014 Received in revised form 13 October 2014 Available online xxxx Keywords: Ambrosia Common ragweed Pollen Sub-pollen particles Flavonoids UHPLC/ESI-LTQ-Orbitrap-MS–MS Polyphenolics Polyamides Immunomodulatory role

a b s t r a c t Phenolic composition of Ambrosia artemisiifolia L. pollen and sub-pollen particles (SPP) aqueous extracts was determined, using a novel extraction procedure. Total phenolic and flavonoid content was determined, as well as the antioxidative properties of the extract. Main components of water-soluble pollen phenolics are monoglycosides and malonyl-mono- and diglycosides of isorhamnetin, quercetin and kaempferol, while spermidine derivatives were identified as the dominant polyamides. SPP are similar in composition to pollen phenolics (predominant isorhamnetin and quercetin monoglycosides), but lacking small phenolic molecules (<450 Da). Ethanol-based extraction protocol revealed one-third lower amount of total phenolics in SPP than in pollen. For the first time in any pollen species, SPP and pollen phenolic compositions were compared in detail, with an UHPLC/ESI-LTQ-Orbitrap-MS–MS approach, revealing the presence of spermidine derivatives in both SPP and pollen, not previously reported in Ambrosia species. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Ambrosia artemisiifolia L. (short or common ragweed, A. artemisiifolia L.) is the most widespread plant of the genus Ambrosia in North America. It is naturalized in Europe, where it grows mostly in the central and southern parts of the continent, spreading quickly each year, with large tracts of land falling from agricultural use. Pollen of A. artemisiifolia L. is the most important seasonal allergen in Europe, that triggers rhinitis, conjunctivitis, asthma and is an exacerbating factor in atopic dermatitis, affecting more than 36 million people each year (Wopfner et al., 2009). Several studies demonstrated episodes of grass pollen-induced allergic asthma after heavy rain falls. It was shown that these asthma attacks might be due to the release of respirable, allergen-bearing sub-pollen particles (SPP) from the pollen cytoplasm (Grote et al., 2000). Because of its size, the relevance of A. artemisiifolia L. pollen grain penetration and subsequent lung inflammation and asthma is unclear. The same mechanism of SPP release upon increased humidity and rainfalls from the pollen of A. artemisiifolia L. explains the increased allergen levels in the ⇑ Corresponding author at: Center of Excellence in Molecular Food Sciences, University of Belgrade – Faculty of Chemistry, Studentski trg 16, 11 000 Belgrade, Serbia. Tel.: +381 113336608; fax: +381 112184330. E-mail address: [email protected] (T. Cirkovic Velickovic).

absence of identifiable pollen grains and the severe asthma symptoms associated with its pollen season (Bacsi et al., 2006). While a large body of research was done on characterization of A. artemisiifolia L. pollen and sub-pollen allergenic proteins (Bacsi et al., 2006; Wopfner et al., 2009), very few contributed to the preliminary determination of the phenolics composition (Kanter et al., 2013). To the best of our knowledge, the phenolic and polyamide composition of A. artemisiifolia L. pollen and its sub-pollen particles, that are important causes of allergic diseases, has not yet been investigated in detail. The precise identification of phenolic compounds can be a complex task as they contain a variety of structures (Ferreres et al., 2014). In this context, HPLC/ESI-MS–MS has proved to be a very useful tool in the structural characterization of small molecules, because compounds in the real samples co-elute and single stage MS is unable to distinguish between substances with the same mass and different structures (Quirantes-Pine et al., 2013). Polyphenolic and polyamide compounds are known to be present in a variety of plant tissues, including the pollen, due to their diverse biological functions. Practically all research on phenolic composition of pollen performed so far, has been done on honeybee collected pollen, mostly on pollen mixtures, focusing on beneficial effects on the human health or the fingerprint authentication studies. The main constituents are flavonoid glycosides of quercetin, isorhamnetin, kaempferol, as well as different less characterized derivatives (Negri et al., 2011).

http://dx.doi.org/10.1016/j.phytochem.2014.10.022 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Mihajlovic, L., et al. Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.022

2

L. Mihajlovic et al. / Phytochemistry xxx (2014) xxx–xxx

Phenolics have the ability to modulate immune response, and may also play a role in the allergenic response to the pollen (Yoon et al., 2006). The IgE-binding of allergens may be influenced by membrane attached flavonoids (Hendrich, 2006) and a direct interaction of allergens with biologically important flavonoids, has already been shown (Ognjenovic et al., 2014; Stojadinovic et al., 2013). Moreover, polyamides possess an important intrinsic toxicity and it was shown that they can contribute to the suppression of immunologic reactions in the lung (Hoet and Nemery, 2000). Establishment of their detailed composition in the pollen and its sub-particles is an important task, with perspectives in alleviation of allergy symptoms. To achieve this, a novel procedure of water-soluble polyphenolics and polyamides extraction from the pollen and SPP was employed, as well as powerful UHPLC/ ESI-LTQ-Orbitrap-MS–MS method for their detection. 2. Results and discussion 2.1. Phenolics and flavonoid concentration in the pollen and SPP extracts of A. artemisiifolia L Separation scheme for extraction of phenolic compounds is given in Fig. 1. It was determined that the ethanol-based extract of A. artemisiifolia L. pollen contains phenolics at a concentration of 6.79 ± 0.05 mg GAE/g pollen, while its SPP value is 4.56 ± 0.06 mg GAE/g SPP, being 33% significantly lower from the whole pollen grain extract. In addition, flavonoid content in these extracts mounted up to 98% (4.45 ± 0.01 mg/g) of total phenolics in SPP and 70% for whole pollen extract (4.71 ± 0.04 mg/g). This is in accordance with structural data (Tables 1 and 2). Water-soluble phenolics are present at 4.51 ± 0.02 mg GAE/g pollen. This value of phenolics in A. artemisiifolia L. pollen is significantly lower than the value previously reported for the phenolic

content of A. artemisiifolia L. herb, which can go as high as 4.35% of total dry weight (43.5 mg/g dry herb) (Maksimovic, 2008). This can be due to the different roles phenolics might play in pollen vs. plant leaves and stalks (Falasca et al., 2010). The amount of flavonoids obtained from the aqueous pollen extract is 3.95 ± 0.24 mg/g pollen, which is 87.5% of total soluble phenolic content. It can be concluded that the flavonoids are the main components of pollen and SPP phenolic extracts, as opposed to dry plant flowering summits, where they make up less than 5% of total phenolics content (Maksimovic, 2008). In addition, values obtained for water- and ethanol extracts in A. artemisiifolia L. pollen are also lower than majority of bee pollen (flowering) species, while results on sub-pollen particle phenolics amount are novel and thus incomparable to literature data. However it is noticeable that SPP have one-third lower content of the total phenolics than the whole pollen grains, which could be important when concerned with allergy modulating properties of polyphenols. It is known that beside direct anti-allergic effect (Yoon et al., 2006), polyphenolic compounds could alleviate allergy by cross-linking with allergens and thus reducing their allergenicity (Tantoush et al., 2011). 2.2. Antioxidative properties of the pollen and SPP extract DPPH assay is commonly used to assess free radical scavenging of a compound or a mixture. Ambrosia pollen extract showed 73 ± 2% free radical scavenging, as compared to a methanol blank. This value is equivalent to the scavenging activity of 28.4 lM butylated hydroxyl toluene (BHT) standard. SPP extract exhibited lower activity, with 56 ± 2% of free radical scavenging activity (23% decrease). The differences are statistically significant (p < 0.001) (Fig. 2A). Both values can be considered as significant free radical scavenging activity and are corroborated by papers by other authors (Negri et al., 2011). ABTS+ assay was used to measure the antioxidative ability of the extracts. Ambrosia pollen extract showed activity equivalent to 5.9 + 0.2 lM quercetin, while the activity of SPP extract was significantly lower (p < 0.001) at 4.1 + 0.1 lM quercetin equivalent (31% decrease). The results are shown in Fig. 2B. Both values are lower than expected from the previously obtained values of pollen phenolics concentration. Although quercetin was used as a standard due to its structural similarity to main components of the mixture, most of the phenolics present in the extract are glycosylated or otherwise modified, which can affect the antioxidative capacity of the mixture. 2.3. Characterization of A. artemisiifolia L. pollen water-soluble phenolics on Orbitrap LTQ XL

Fig. 1. Schematic representation of aqueous phenolic extraction from A. artemisiifolia L. pollen and sub-pollen particles (SPP).

An Orbitrap analysis showed that A. artemisiifolia L. pollen phenolics have a complex and diverse composition, including phenolic derivates of polyamide spermidine, which we were unable to detect with the triple quadrupole detector (data not shown). All of the phenolic glycosides detected with high resolution and sensitive UHPLC/ESI-LTQ-MS–MS Orbitrap, including m/z higher than 570 (di- and tri-glycosides of isorhamnetin), are shown Table 1 and its base peak chromatogram is shown in Fig. 3. All detected glycosides are derivatives of three main flavonols: quercetin, O-methylquercetin (isorhamnetin), and kaempferol (Fig. 4). For each aglycone, a series of peaks (satellite sets) was observed, representing unsubstituted and malonylated hexosides and hexuronides. Fragment analysis allowed identification of [Y02HCO] ions, a characteristic ion-peak of 3-O-monoglucosides, confirming the structures proposed with preliminary results (Vukics and Guttman, 2010).

Please cite this article in press as: Mihajlovic, L., et al. Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.022

3

L. Mihajlovic et al. / Phytochemistry xxx (2014) xxx–xxx Table 1 Compounds detected in A. artemisiifolia L. pollen aqueous extract on the UHPLC/ESI-LTQ Orbitrap mass spectrometer.

a

Retention time (min)

Measured m/z

Diagnostic fragments (m/z)

0.38 0.49 0.52 1.72 1.87 1.89 1.93 1.95 1.97 2.01 2.07 2.07 2.15 2.20 2.21 2.25 2.34 2.38 2.38 2.93 2.96 3.03 4.68

179.06 191.02 162.98 447.12 625.14 609.15 639.16 625.35 609.15 595.14 463.09 593.15 549.09 490.98 505.10 477.10 533.09 519.12 563.11 598.26 582.26 787.36 353.20

161.02 126.82 147.03 285.07 463.07 463.08 477.16 477.19 445.15 463.07 301.04 447.09 505.13 301.03 461.10 357.09 489.15 477.14 519.03 478.20 462.22 477.20 309.25

131.30 111.02 119.05 151.01 301.07 301.05 315.11 315.20 315.10 301.06 151.10 315.07 463.07 179.10 315.04 314.03 428.89 403.11 403.11 436.17 436.17 374.06 295.09

115.13 85.2 93.03 133.2 151.02 151.02 300.08 151.03 300.05 255.01 149.02 241.09 300.87 151.01 151.06 285.10 285.05 315.05 315.06 358.19 342.16 315.20 262.48

Molecular formula

Compound name

C9H8O4 C7H13O6 C9H8O3 C21H20O11 C27H29O17 C27H30O16 C28H32O17 C28H32O16 C27H30O16 C26H28O16 C21H20O12 C27H30O15 C24H22O15 C22H22O13 C23H24O13 C22H22O12 C24H20O14 C25H24O13 C25H23O15 C34H37N3O7 C34H37N3O6 C36H47O19 C16H18O9

Caffeic acida Quinic acida p-Coumaric acida Kaempferol 3-O-glucosidea Quercetin diglucoside Quercetin rutinoside isomer 1a Isorhamnetin 3-O-diglucosidea Isorhamnetin rutinosidea Quercetin rutinoside isomer 2a Quercetin 3-O-glucosyl-6-O-pentoside Quercetin 3-O-glucoside Kaempferol rutinosidea Quercetin 3-O-malonyl glucosidea Quercetin 3-O-methyl hexuronide Isorhamnetin 3-O-methyl hexuronide Isorhamnetin 3-O-glucoside Kaempferol O-malonyl-acetyl glucoside Isorhamnetin 3-O-acetyl glucoside Isorhamnetin 3-O-malonyl glucoside di Coumaroyl caffeoyl spermidine tri-p-Coumaroyl spermidine Isorhamnetin rutinosyl glucoside Caffeoyl quinic acida

Compounds found solely in whole pollen grains and not in SPP.

Table 2 Compounds detected in A. artemisiifolia L. sub-pollen particle aqueous extract on the UHPLC/ESI-Orbitrap mass spectrometer. Retention time (min)

Measured m/z

Diagnostic fragments (m/z)

1.87 2.08 2.13 2.18 2.21 2.29 2.34 2.37 2.43 2.94 2.97 3.03

625.14 595.14 463.09 490.98 505.10 477.11 533.09 519.12 563.11 598.26 582.26 787.36

463.06 463.07 301.03 301.04 461.10 357.08 489.15 487.11 519.02 478.20 462.22 477.20

301.07 301.06 179.10 179.10 315.04 314.03 428.89 403.11 487.13 436.17 436.17 374.06

151.02 255.02 151.03 151.06 285.08 285.05 315.03 403.11 358.19 342.16 315.20

Molecular formula

Compound name

C27H29O17 C26H28O16 C21H20O12 C22H22O13 C23H24O13 C22H22O12 C24H20O14 C25H24O13 C25H23O15 C34H37N3O7 C34H37N3O6 C36H47O19

Quercetin di-glucoside Quercetin 3-O-glucosyl-6-O-pentoside Quercetin 3-O-glucoside Quercetin O-methyl hexuronide Isorhamnetin O-methyl hexuronide Isorhamnetin 3-O-glucoside Kaempferol O-malonyl-acetyl glucoside Isorhamnetin O-acetyl glucoside Isorhamnetin O-malonyl glucoside di Coumaroyl caffeoyl spermidine tri-p-Coumaroyl spermidine Isorhamnetin rutinosyl glucoside

Fig. 2. (A) Relative free radical scavenging activity of A. artemisiifolia L. pollen and sub-pollen particles (SPP) aqueous extracts according to DPPH assay (p < 0.001). (B) Antioxidative activity of A. artemisiifolia L. pollen and sub-pollen particles (SPP) aqueous extracts according to ABTS+ assay (p < 0.001). ABTS assay showed similar results to the DPPH assay, with SPP extract exhibiting significantly diminished activity.

In addition to the aglycone (Y0) fragment, an abundant radical aglycone [Y0H] product ion is also formed, indicating a loss of 3O-glucosyl fragment (all fragments with m/z of aglycone 285, 301, 315 accompanied by ions at masses 284, 300, 314), further proving that mostly 3-O-glycosilated fragments are present in the mixture (Hvattum and Ekeberg, 2003). Fragmentation of the flavonol derivative quercetin-3-glucoside-3-malonate is shown in Fig. 5.

The abundance ratio of the radical aglycone to the regular aglycone product ion originating from cleavage at the 3-O glycosidic bond increases with increasing OH substitution on the B-ring, whereas the opposite holds for 7-O-glycosides (March et al., 2006). This phenomenon can be observed when comparing kaempferol and quercetin derivatives (peaks at retention time (RT) 1.72 and 2.09, as well as 1.89 and 2.09). Aglycone identities

Please cite this article in press as: Mihajlovic, L., et al. Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.022

4

L. Mihajlovic et al. / Phytochemistry xxx (2014) xxx–xxx

Fig. 3. Base peak chromatogram of the A. artemisiifolia L. aqueous pollen extract analysed on UHPLC/ESI-LTQ-Orbitrap mass spectrometer.

Fig. 4. Main aglycones detected in the A. artemisiifolia L. aqueous pollen extract. (A) quercetin, (B) kaempferol, (C) isorhamnetin.

were confirmed by following 1,3B0 and 1,3A0 fragments present in the MS2 spectra. Both quercetin and isorhamnetin can produce 1,3 A0 fragment with m/z of 151 in negative mode, in a reaction that proceeds via retro Diels–Alder mechanism (RDA). These compounds were distinguished according to their molecular ions and Y02HCO ions. Compounds having a methoxy substituent show relatively weaker RDA fragmentation, observable when comparing the fragmentation spectra of quercetin and isorhamnetin derivatives (RT 2.2 and 2.21, 1.72 and 1.93) (Fabre et al., 2001). 2.4. Spermidine derivatives in the pollen extract of A. artemisiifolia L Phenolamides constitute a diverse and quantitatively major group of secondary metabolites resulting from the conjugation of a phenolic moiety with polyamides, such as spermidine (Bassard et al., 2010). Several spermidine amide derivatives have been detected in the A. artemisiifolia L. pollen extract. Among them, the most prominent were di-coumaroyl-caffeoyl and tri-coumaroyl containing compounds. Fragmentation patterns for spermidine derivatives are shown in Fig. 6. Peak at retention time 2.93, with m/z of the [MH] ion of 598.2573 showed characteristic fragmentation pattern (Sobolev

et al., 2008) with fragments at m/z 478, attributed to the loss of 120 Da fragment from p-coumaric residue ([MH]AHOAC6A H4ACH@CH), m/z 436 (loss of coumaroyl residue) and m/z 358 (loss of 120 Da fragment at N5 and breakage of CAC bond of the caffeoyl residue linked at N10). The compound was identified as di-coumaroyl caffeoyl spermidine. Observed masses can be explained by formation of homolytic cleavage/deprotonation products during fragmentation. Few papers have been published so far regarding the fragmentation of polyamide phenolics (Narvaez-Cuenca et al., 2013). To the best of our knowledge, formation of radical products of spermidine derivatives in MS2 has not been described so far. Similarly, at 2.96 min retention time, a peak with m/z of 582.262 for the [MH] ion, with corresponding fragments in MS2 at m/z 462, 436 and 358 has identical fragmentation mechanism as for the previous compound, with the only 16 Da difference for the m/z 462 and m/z 436 fragments. The difference stems from the replacement of a caffeoyl residue with a coumaroyl residue. The compound was identified as tri-p-coumaroyl spermidine. Spermidine and other polyamide compounds are widely present in all organisms, known to affect many processes, in both plants (Falasca et al., 2010), and animals (Igarashi and Kashiwagi, 2010). They have been shown to function naturally as free radical

Please cite this article in press as: Mihajlovic, L., et al. Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.022

L. Mihajlovic et al. / Phytochemistry xxx (2014) xxx–xxx

5

et al., 2006). UHPLC/ESI-MS–MS analysis was conducted on the Orbitrap instrument, using the same method as the one used for whole pollen analysis. The results show that flavonoid-O-glycosides are the principal components of SPP phenolic fraction in A. artemisiifolia L. (Table 2). Compared to the whole pollen phenolics composition, SPP exhibit similar composition but with lower total phenolic amount and lack of the small phenolic molecules, below 450 Da, that are extensively present in pollen. Main components are monoglycoside derivatives of isorhamnetin and quercetin, with characteristic fragmentation patterns (1,3A0 and 1,3B0 fragments) (Table 2). Di-glycosides of isorhamnetin found in the whole pollen extract haven’t been detected and this could be due to the mode of their formation from pollen cytoplasm. Spermidine derivatives are present, with implications regarding the immunomodulatory effects they could exert. 3. Concluding remarks

Fig. 5. Fragmentation pattern of quercetin-3-O-(3-O-malonyl) glycoside.

scavengers (Ha et al., 1998). Spermidine acyl derivatives such as its amides have already been reported in pollen (Bassard et al., 2010; Kite et al., 2013). Recently, polyamides were reviewed by Bassard et al. (2010), who concluded that di- and tri-substituted hydroxy-cinnamoyl conjugates, particularly of spermidine and putrescine, are major metabolites of pollen and suggested to have an ecological role as defense compounds against viruses, bacteria, and fungi, and could deter herbivores from eating plants. The spermidine conjugates are implicated in protection against pathogens, detoxifying phenolic compounds, and/or serving as a reserve of polyamines that are available to actively proliferating tissues, although not always essential for survival. In addition, polyamides and their derivatives play a regulatory role in several immunologic processes, including allergic reactions (Bueb et al., 1991; Hoet and Nemery, 2000), regulation of T cell function, cell migration and growth in local inflammation (Ferioli et al., 2000). 2.5. Characterization of the SPP phenolic extract So far, comparison and characterization of pollen and sub-pollen particle’s phenolic extract of any plant species has not been undertaken. We aimed to simultaneously characterize the A. artemisiifolia L. pollen and SPP phenolic extract, since it was shown that phenolic compounds posses immunomodulatory effects (González Romano et al., 1996; Hoet and Nemery, 2000; Koistinen et al., 2005), and that SPP have more profound effect on asthma development compared to whole pollen grains (Bacsi

The compositions of water soluble phenolics from A. artemisiifolia L. pollen and SPP were established by UHPLC/ESI-LTQ-MS–MS, and their total phenolic content in ethanol extract, including flavonoid fraction were determined. We have shown that principal components of the aqueous pollen and SPP extracts are polyphenols and polyamides. Main flavonoids are glycosides and malonyl glycosides of quercetin, kaempferol and isorhamnetin, while coumaroyl- and caffeoyl-spermidine derivatives were identified as polyamide components. The role of these compounds as allergy modulating factors needs corroborative investigations. Similar phenolic content was determined in A. artemisiifolia L. SPP, with isorhamnetin and quercetin mono-glycosides being predominant, but lacking small phenolics molecules (below 450 Da). Polyamide molecules are present in SPP particles as well, accentuating the need for further research on the immune effects of this ubiquitous class of compounds. Ethanolbased extraction protocol revealed one-third lower amount of total phenolics in SPP than in the whole pollen grains. In view of the importance of SPP-induced allergy and asthma due to particles penetrability into lower airways and the immunomodulatory role of pollen phenolics and polyamide compounds in allergy, further investigations will include the effects of pollen and SPP – phenolics in basophil activation assays as well as the effects on relevant immune cell lines such as dendritic cells and T cells. 4. Experimental 4.1. Chemicals Organic solvents were purchased from J.T. Baker (Mallinckrodt Baker, Phillipsburg, USA). Fetal bovine albumin, L-glutamine, penicillin, streptomycin, gentamicin and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) were purchased from Merck (Merck KGaA, Darmstadt, Germany). HPLC grade water (18 mX) was prepared using a Smart2Pure3™ purification system (ThermoFisher Scientific, MA, USA). 4.2. Pollen samples A. artemisiifolia L. pollen was obtained from the Institute of Virology, Vaccines and Sera ‘‘Torlak’’ in Belgrade, Serbia. The pollen was collected in the surrounding area of Belgrade during the summer season of 2012. 4.3. Extraction and isolation procedure of A. artemisiifolia L. pollen and SPP water-soluble phenolics The main intention of this study was to obtain and characterize aqueous phenolic extract from A. artemisiifolia L. pollen and SPP

Please cite this article in press as: Mihajlovic, L., et al. Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.022

6

L. Mihajlovic et al. / Phytochemistry xxx (2014) xxx–xxx

Fig. 6. Fragmentation patterns of spermidine derivatives detected in the A. artemisiifolia L. aqueous pollen extract.

that will mimic natural conditions of SPP creation and that is excessive pollen water-soaking and hydration during the heavy rains, resulting in its osmolysis and SPP release. Water-soluble phenolics from A. artemisiifolia L. pollen were isolated by a novel extraction procedure. Additional concentrating step in the extraction with ethyl-acetate was needed in order to facilitate and improve structural and further biological activity analyses. In a novel, aqueous extraction of pollen, the first step was to remove the biologically active lipid molecules (2 g of A. artemisiifolia L. pollen was defatted 3 times with 20 mL of hexane, after which the pollen was left to dry at a room temperature). 15 mL of distilled water was added, and the mixture was left at 4 °C with shaking for 4 h. After this step, the mixture was centrifuged for 20 min at 3000g, and 15 mL of CCl4 was added to the supernatant. The water layer was removed after 30 min and it was mixed with an equal volume of ethyl-acetate, and left at 4 °C for 10 h. The ethyl-acetate layer was removed and the solvent was evaporated. The dry residue was dissolved in 1 mL distilled water and this solution was used in further analyses (Fig. 1). All extractions were done in triplicates. 4.4. Sub-pollen particle (SPP) isolation and water-soluble phenolic extraction procedure Sub-pollen particles were isolated according to Bacsi et al. (2006), with minor modifications. A. artemisiifolia L. pollen (1 g) was osmolysed with 10 mL of distilled water for 4 h and the pollen grains were removed by centrifugation at 1600g for 15 min. This step was repeated and remaining suspension was centrifuged for

20 min at 10,000g and the pellet containing purified SPP was collected and dried at room temperature, overnight (Fig. 1). Phenolic extraction from the sub-pollen particles was performed according to our procedure already described, with some modifications. Briefly, approximately 35 mg of SPP were defatted with 1 mL of hexane, mixed for 15 min and centrifuged at 10,000g for 30 min. Most hexane (950 lL) was removed and the procedure was repeated. The defatted SPP were left to dry at room temperature, overnight. SPP polyphenolics were extracted with 0.3 mL of ethyl-acetate overnight and the mixture was centrifuged for 30 min at 10,000g. The ethyl-acetate layer was evaporated and the dry residue was dissolved in 1 mL water (Fig. 1). This solution was used in phenolic characterization on UHPLC/ESI-Orbitrap tandem mass spectrometer. Due to the limited amount of material, defatting with carbon tetrachloride extraction step was skipped, as well as the reverse phase HPLC. 4.5. Ethanol extraction of phenolics from A. artemisiifolia L. pollen and its sub pollen particles Fast ethanol extraction of phenolic compounds from defatted pollen and defatted SPP was done to assess the ratio of total phenolic content in the pollen and its SPP, since low amount of SPP aqueous phenolic extract was sufficient only for MS Orbitrap structural analyses and antioxidative properties determination. SPP and pollen phenolics were extracted with 70% ethanol solution in the ratio 1:20 w/v (pollen or SPP/ethanol), with shaking during 50 min on 50 °C. The supernatant was separated and the solid residue was re-extracted under the same conditions. Afterwards, the

Please cite this article in press as: Mihajlovic, L., et al. Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.022

L. Mihajlovic et al. / Phytochemistry xxx (2014) xxx–xxx

ethanol extracts were combined, evaporated in the vacuum and reconstituted in water at 1:20 w/v ratio, for the total phenolic and flavonoid concentration measurement. All samples were extracted in triplicates. 4.6. Preparative chromatography Water-soluble phenolics from the pollen extract were further purified by reverse phase chromatography on a C18 column. A linear gradient elution was performed by using a mobile phase A represented by water acidified with formic acid (0.1%) and a mobile phase B represented by 90% ethanol acidified with formic acid (0.1%) at a flow rate of 1 mL/min. The column used was Spherisorb ODS 2 250  4.6 mm, 5 lm (Waters, Milford, MA, USA) on Acta Purifier 10 system with UV detection (GE Healthcare, Uppsala, Sweden). Fractions containing phenolics (as determined by the absorption maxima at k = 215 and k = 260 nm) were pooled together and submitted to further analysis. Preparative chromatography was done in order to remove any protein and peptide contaminants that might be present in the sample, which might interfere with the results of ongoing immunological studies by our group. 4.7. Measurement of the total phenolic concentration The method used to determine the concentration of water- and ethanol-soluble phenolics in pollen and SPP extracts, was a modified Folin–Ciocalteau protocol (Chun et al., 2003) adjusted for measurements in 96-well micro titer plates. In brief, 100 lL of 10 times diluted Folin–Ciocalteau reagent was added to 10 lL of pollen or SPP aqueous extracts, and was left in the dark at room temperature to incubate for 5 min. After that, 140 lL of freshly made 7.5% Na2CO3 was added, and the mixture was incubated in the dark for 2 h. The absorbance of the mixture was measured at 620 nm. All measurements were conducted in triplicates. Total phenolics were expressed as milligrams of gallic acid equivalents (GAE) per gram of pollen/SPP using a standard calibration curve constructed of different concentrations of gallic acid with linearity between 0.01 and 0.5 mg/mL of gallic acid.

7

containing methanol instead of test-solutions (Kikuzaki et al., 2002). 4.10. Antioxidative test with 2,20 -azino-bis(3-ethylbenzothiazoline-6sulfonate) cation (ABTS+ assay) The procedure followed the method of Re and coworkers (Re et al., 1999), with some modifications. 7.4 mM ABTS+ solution and 2.6 mM potassium persulfate solution were mixed in equal quantities and allowed to react for 12 h at the room temperature in the dark. The solution was then diluted to 60 mL in methanol to obtain an absorbance of 0.7 units at 734 nm. Fresh ABTS+ solution was prepared for each assay. Pollen extracts (20 lL) were allowed to react with 180 lL of the ABTS+ solution for 2 h in the dark, after which the absorbance was measured at 734 nm. The percentage inhibition of the absorbance at 734 nm is calculated as a function of concentration of pollen phenolics and the standard reference data (in this case obtained with quercetin, 0.01–0.5 mg/mL) and expressed as the concentration equivalent of the standard antioxidant used in the assay. 4.11. UHPLC/ESI-LTQ-Orbitrap-MS–MS analyses Water-based phenolic pollen and SPP extract components were analyzed on an Accela UHPLC with Orbitrap linear trap quadrupole (LTQ) XL (ThermoFisher Scientific, Bremen, Germany). The separation was achieved using a 50 mm  2.3 mm 1.8 lm Hypersil Gold rapid resolution reverse-phase column (ThermoFisher Scientific, Bremen, Germany). The binary mobile phase consisted of 0.1% aqueous formic acid (A) and 98% acetonitrile (B) and was delivered at a flow rate of 0.3 mL/min. Components were eluted in linear gradient mode, 5–95% B in 9 min. Injection volume was 1 lL. Analysis was performed with the heated ESI source (HESI) set at 350 °C and capillary temperature of 275 °C. Orbitrap detector was set to resolution 30,000 with automatic gain control set to 1  106 ions. Xcalibur 2.1 and MassFrontier 6.0 software were used for instrument control, data acquisition and analysis. 4.12. Statistical analysis

4.8. Measurement of flavonoid concentration The flavonoid concentration was measured in ethanol-based phenolic extracts of SPP and whole pollen, as well as in aqueous phenolic pollen extract according to the scaled-down protocol of Penarrieta et al. (2008). The method is based on the formation of a coloured flavonoid-aluminum complex. 10 lL 10% NaNO2, 20 lL 20% AlCl3 and 70 lL 1M NaOH were mixed and 100 lL of water was added. To this reaction mixture, 30 lL of pollen/SPP was added and the mixture was incubated for 5 min at room temperature, after which the absorbance at 405 nm was measured. Quercetin was used as a standard, with a linear calibration curve ranging 0.01–0.125 mg/mL and results were expressed as milligrams of quercetin equivalents per gram of pollen/SPP. All measurements were conducted in triplicates. 4.9. Antioxidative test with 2,2-diphenyl-1-picrylhydrazyl compound (DPPH assay) The antioxidant activity of the extract was determined through the use of the DPPH assay. In brief, 10 lL of the antioxidant solution was mixed with 190 lL of 6  105 mol/L solution of DPPH and was incubated for 30 min in the dark at the room temperature. Afterwards, the absorbance at 540 nm was measured and the scavenging ability was calculated, against methanol as a blank. The percentage of inhibition was calculated against the control solution,

Where applicable, results were expressed as mean ± standard deviation. Calibration curves were constructed by linear regression analysis of absorbance of the compounds used as standards against their concentration. Statistical analyses were performed with Graph Pad Prism 6.0 with two-tail sample t-test when comparing two groups and unpaired one-way ANOVA when comparing three or more groups. Differences were considered significant at p < 0.05. Acknowledgments This research was carried out with the support from the Ministry of Education and Science of the Republic of Serbia, project No. 172024, and by the European Commission, under the Framework 7, project Reg-Pot FCUB-ERA, GA No. 256716. References Bacsi, A., Choudhury, B.K., Dharajiya, N., Sur, S., Boldogh, I., 2006. Sub-pollen particles: carriers of allergenic proteins and oxidases. J. Allergy Clin. Immunol. 118, 844–850. Bassard, J.-E., Ullmann, P., Bernier, F., Werck-Reichhart, D., 2010. Phenolamides: bridging polyamines to the phenolic metabolism. Phytochemistry 71, 1808– 1824. Bueb, J.L., Mousli, M., Landry, Y., 1991. Molecular basis for cellular effects of naturally occurring polyamines. Agents Actions 33, 84–87. Chun, O.K., Kim, D.-O., Lee, C.Y., 2003. Superoxide radical scavenging activity of the major polyphenols in fresh plums. J. Agric. Food Chem. 51, 8067–8072.

Please cite this article in press as: Mihajlovic, L., et al. Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.022

8

L. Mihajlovic et al. / Phytochemistry xxx (2014) xxx–xxx

Fabre, N., Rustan, I., de Hoffmann, E., Quetin-Leclercq, J., 2001. Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 12, 707–715. Falasca, G., Franceschetti, M., Bagni, N., Altamura, M.M., Biasi, R., 2010. Polyamine biosynthesis and control of the development of functional pollen in kiwifruit. Plant Physiol. Biochem. 48, 565–573. Ferioli, M.E., Pirona, L., Pinotti, O., 2000. Prolactin and polyamine catabolism: specific effect on polyamine oxidase activity in rat thymus. Mol. Cell. Endocrinol. 165, 51–56. Ferreres, F., Oliveira, A.P., Gil-Izquierdo, A., Valentao, P., Andrade, P.B., 2014. Piper betle leaves: profiling phenolic compounds by HPLC/DAD-ESI/MS and anticholinesterase activity. Phytochem. Anal.. http://dx.doi.org/10.1002/pca.2515. González Romano, M.L., Gallego, M.T., Berrens, L., 1996. Extraordinary stability of IgE-binding Parietaria pollen allergens in relation to chemically bound flavonoids. Mol. Immunol. 33, 1287–1293. Grote, M., Vrtala, S., Niederberger, V., Valenta, R., Reichelt, R., 2000. Expulsion of allergen-containing materials from hydrated rye grass (Lolium perenne) pollen revealed by using immunogold field emission scanning and transmission electron microscopy. J. Allergy Clin. Immunol. 105, 1140–1145. Ha, H.C., Yager, J.D., Woster, P.A., Casero Jr., R.A., 1998. Structural specificity of polyamines and polyamine analogues in the protection of DNA from strand breaks induced by reactive oxygen species. Biochem. Biophys. Res. Commun. 244, 298–303. Hendrich, A.B., 2006. Flavonoid–membrane interactions: possible consequences for biological effects of some polyphenolic compounds. Acta Pharmacol. Sin. 27, 27–40. Hoet, P.H., Nemery, B., 2000. Polyamines in the lung: polyamine uptake and polyamine-linked pathological or toxicological conditions. Am. J. Physiol. Lung Cell. Mol. Physiol. 278, L417–L433. Hvattum, E., Ekeberg, D., 2003. Study of the collision-induced radical cleavage of flavonoid glycosides using negative electrospray ionization tandem quadrupole mass spectrometry. J. Mass Spectrom. 38, 43–49. Igarashi, K., Kashiwagi, K., 2010. Modulation of cellular function by polyamines. Int. J. Biochem. Cell Biol. 42, 39–51. Kanter, U., Heller, W., Durner, J., Winkler, J.B., Engel, M., Behrendt, H., Holzinger, A., Braun, P., Hauser, M., Ferreira, F., Mayer, K., Pfeifer, M., Ernst, D., 2013. Molecular and immunological characterization of ragweed (Ambrosia artemisiifolia L.) pollen after exposure of the plants to elevated ozone over a whole growing season. PLoS ONE 8, e61518. Kikuzaki, H., Hisamoto, M., Hirose, K., Akiyama, K., Taniguchi, H., 2002. Antioxidant properties of ferulic acid and its related compounds. J. Agric. Food Chem. 50, 2161–2168. Kite, G.C., Larsson, S., Veitch, N.C., Porter, E.A., Ding, N., Simmonds, M.S., 2013. Acyl spermidines in inflorescence extracts of elder (Sambucus nigra L., Adoxaceae) and elderflower drinks. J. Agric. Food Chem. 61, 3501–3508. Koistinen, K.M., Soininen, P., Venalainen, T.A., Hayrinen, J., Laatikainen, R., Perakyla, M., Tervahauta, A.I., Karenlampi, S.O., 2005. Birch PR-10c interacts with several biologically important ligands. Phytochemistry 66, 2524–2533.

Maksimovic´, Z., 2008. In vitro antioxidant activity of ragweed (Ambrosia artemisiifolia L., Asteraceae) herb. Ind. Crops Prod. 28, 356–360. March, R.E., Lewars, E.G., Stadey, C.J., Miao, X.-S., Zhao, X., Metcalfe, C.D., 2006. A comparison of flavonoid glycosides by electrospray tandem mass spectrometry. Int. J. Mass Spectrom. 248, 61–85. Narvaez-Cuenca, C.E., Vincken, J.P., Zheng, C., Gruppen, H., 2013. Diversity of (dihydro) hydroxycinnamic acid conjugates in Colombian potato tubers. Food Chem. 139, 1087–1097. Negri, G., Teixeira, E.W., Alves, M.L., Moreti, A.C., Otsuk, I.P., Borguini, R.G., Salatino, A., 2011. Hydroxycinnamic acid amide derivatives, phenolic compounds and antioxidant activities of extracts of pollen samples from Southeast Brazil. J. Agric. Food Chem. 59, 5516–5522. Ognjenovic´, J., Stojadinovic´, M., Milcˇic´, M., Apostolovic´, D., Vesic´, J., Stambolic´, I., Atanaskovic´-Markovic´, M., Simonovic´, M., Cirkovic Velickovic, T., 2014. Interactions of epigallo-catechin 3-gallate and ovalbumin, the major allergen of egg white. Food Chem. 164, 36–43. Penarrieta, J.M., Alvarado, J.A., Akesson, B., Bergenstahl, B., 2008. Total antioxidant capacity and content of flavonoids and other phenolic compounds in canihua (Chenopodium pallidicaule): an Andean pseudocereal. Mol. Nutr. Food Res. 52, 708–717. Quirantes-Pine, R., Lozano-Sanchez, J., Herrero, M., Ibanez, E., Segura-Carretero, A., Fernandez-Gutierrez, A., 2013. HPLC–ESI–QTOF–MS as a powerful analytical tool for characterising phenolic compounds in olive-leaf extracts. Phytochem. Anal. 24, 213–223. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237. Sobolev, V.S., Sy, A.A., Gloer, J.B., 2008. Spermidine and flavonoid conjugates from peanut (Arachis hypogaea) flowers. J. Agric. Food Chem. 56, 2960–2969. Stojadinovic, M., Radosavljevic, J., Ognjenovic, J., Vesic, J., Prodic, I., Stanic-Vucinic, D., Cirkovic Velickovic, T., 2013. Binding affinity between dietary polyphenols and beta-lactoglobulin negatively correlates with the protein susceptibility to digestion and total antioxidant activity of complexes formed. Food Chem. 136, 1263–1271. Tantoush, Z., Stanic, D., Stojadinovic, M., Ognjenovic, J., Mihajlovic, L., AtanaskovicMarkovic, M., Cirkovic Velickovic, T., 2011. Digestibility and allergenicity of blactoglobulin following laccase-mediated cross-linking in the presence of sour cherry phenolics. Food Chem. 125, 84–91. Vukics, V., Guttman, A., 2010. Structural characterization of flavonoid glycosides by multi-stage mass spectrometry. Mass Spectrom. Rev. 29, 1–16. Wopfner, N., Jahn-Schmid, B., Schmidt, G., Christ, T., Hubinger, G., Briza, P., Radauer, C., Bohle, B., Vogel, L., Ebner, C., Asero, R., Ferreira, F., Schwarzenbacher, R., 2009. The alpha and beta subchain of Amb a 1, the major ragweed-pollen allergen show divergent reactivity at the IgE and T-cell level. Mol. Immunol. 46, 2090– 2097. Yoon, M.S., Lee, J.S., Choi, B.M., Jeong, Y.I., Lee, C.M., Park, J.H., Moon, Y., Sung, S.C., Lee, S.K., Chang, Y.H., Chung, H.Y., Park, Y.M., 2006. Apigenin inhibits immunostimulatory function of dendritic cells: implication of immunotherapeutic adjuvant. Mol. Pharmacol. 70, 1033–1044.

Please cite this article in press as: Mihajlovic, L., et al. Composition of polyphenol and polyamide compounds in common ragweed (Ambrosia artemisiifolia L.) pollen and sub-pollen particles. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.022