Research article Received: 4 September 2013
Revised: 29 January 2014
Accepted: 3 February 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3344
Profiles analysis of proanthocyanidins in the argun nut (Medemia argun—an ancient Egyptian palm) by LC–ESI–MS/MS Arafa I. Hamed,a,b* Abdulla S. Al-Ayed,c Jaroslaw Moldoch,b Sonia Piacente,d Wieslaw Oleszekb and Anna Stochmalb Medemia argun is an ancient palm rich in proanthocyanidins (PACs). These polyphenolic compounds are widely distributed in plants and are an integral part of the human diet. A sensitive high-performance liquid chromatography and electrospray ionization mass spectrometry (HPLC–ESI–MS) method in the negative ion mode for sequencing these ubiquitous and highly beneficial antioxidants is described in order to profile different PACs in M. argun nuts. The analytical protocol based on tandem mass spectrometry was used to sequence dimers, trimers, tetramers and pentamers with different A-type, B-type and A/B-type linkages. Diagnostic ions resulting from heterocyclic ring fission and retro-Diels–Alder reaction of flavan-3-ol provided information on the hydroxylation pattern and the type of interflavan bond. The sequences were discovered through ions derived from quinone methide cleavage of the interflavan bond. The identification of PACs linkages through LC–MSn eliminates a number of tedious separation steps. The method was successfully applied to give a view of PAC profile in M. argun nuts. M. argun nuts contained 636.88 mg/g PACs (as equivalent of (þ)-catechin). The data obtained in our research show that M. argun is a rich source of hydrolyzable PACs. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Medemia argun nut; proanthocyanidins; liquid chromatography; electrospray ionization; mass spectrometry
Introduction
306
Medemia argun (Syns. = Hyphaene argun = Medemia abiadensis = Areca abiadensis: Coryphoideae: Borasseae: Hyphaeninae) is a mysterious and little known species of fan palm from the Nubian Desert Oases of Southern Egypt and Northern Sudan. M. argun fruits have been found in the tombs from the 5th Dynasty (ca. 2500 BC) to Roman times (6–7th century AD) including the celebrated tomb of Tutankhamun.[1–5] It seems that M. argun fruits were highly valued in ancient Egypt, although the reason for that is still unknown. An Arabic name for M. argun is ‘argun’.[6] It also has a hieroglyphic name, Mama-n-Khanen (or Mama-n-Xanin). [7–9] Different authors give various claims on the edibility of the fruit. Some of them state that they are bitter and inedible (Von Martius, 1823–1850),[10] while others suggest that the thin fruit flesh is sweet and might have been acceptable to the ancient tastes, especially to those living in the desert where the fruits were scarce.[11,12] According to Loutfy Boulos,[12] the fruits were buried under sand for a period, during which the endosperm (nut) developed a sweet taste similar to coconut. Proanthocyanidins (PACs) are oligomeric and polymeric end products of the flavonoid biosynthetic pathway (syn. condensed tannins) which constitute one of the ubiquitous groups of all plant phenols. The size of PAC molecules can be described by their degree of polymerization.[13] PACs containing afzelechin, catechin or gallocatechin as subunits are named PAC. They are present in fruits, barks, leaves and seeds of many plants, where they provide protection against predation. At the same time, they give flavor and astringency to beverages such as wine, fruit juices and teas, and are increasingly recognized as having beneficial effects on human health. Nuts of Areca catechu, a palm tree
J. Mass Spectrom. 2014, 49, 306–315
indigenous to the South- and Southeast Asia, had been evaluated for its phenolic constituents and their biological activity.[14–17] Euterpe oleraceae (Acai), a large palm tree indigenous to the Amazon River, had been evaluated for phenolic constituents and their biological activity.[18–20] Due to the structural complexity of PAC derivatives and high difficulties in their separation, studies on these compounds are limited in comparison with other polyphenols.[21,22] Usually, catechins give unresolved high-performance liquid chromatography (HPLC) peaks, due to the similarity in their structures and also to the large number of phenolic groups that can give the same interactions with chromatographic stationary phase.[23,24] Reversed phase (RP)-HPLC is the most popular method used in the analysis of these compounds.[24,25] Nowadays, little detailed phytochemical and pharmaceutical studies have been reported for M. argun.[26] Our previous study exhibited that the PAC fraction from M. argun nuts can be useful
* Correspondence to: Arafa I. Hamed, Department of Botany, Faculty of Science, Aswan University, Aswan 81528, Egypt. E-mail: [email protected] a Department of Botany, Faculty of Science, Aswan University, Aswan 81528, Egypt b Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State of Research Institute, ul. Czartoryskich 8, 24-100 Pulawy, Poland c Department of Chemistry, College of Science & Arts at Al-Rass, Qassim University, P.O. 53, Kingdom of Saudi Arabia d Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, I-84084 Fisciano, Salerno, Italy
Copyright © 2014 John Wiley & Sons, Ltd.
Analysis of PACs in M. argun nut by LC–ESI–MS/MS as a protecting factor against oxidative/nitrative stress associated with different diseases (cancer, cardiovascular and neurodegenerative diseases) and PACs of M. argun nuts may be promising antioxidants.[27] Due to the complexity of PACs structures, we have proposed to rationalize the presence of these compounds on the basis of electrospray ionization mass spectrometry (ESI–MS) and ESI–MS/MS profiles. Direct flow injection/electrospray ionization/ion trap tandem mass spectrometry was used to investigate polyphenolic compounds in the methanolic extract of M. argun. In a second stage, analytical HPLC–ESIMS and HPLC–ESI–MS/MS were developed. In the present work, we focused on the analysis of the phenolic constituents of this palm which resulted to be a rich novel source of PACs with strong free radical scavenging activity.
Materials and methods Plant material Fresh fruits of M. argun were collected from the Dungul Oasis (Aswan, Egypt) in October, 2010 and dried in dark at room temperature. The plant material was identified by Prof Arafa I. Hamed according to Täckholm (1974), and the voucher specimens were deposited in Botany Department Herbarium, Aswan Faculty of Science (Egypt). Extraction and sample preparation Dried powdered nuts of M. argun (450 g) were first extracted with hexane and then with 80% MeOH. The aqueous methanolic extract was concentrated under reduced pressure to give 50 g of crude extract (ca: 11% crude extract). Ten grams of extract was loaded onto a C18 column (4 × 50 cm, 40–63 μm LiChroprep, Millipore Corp, Bedford) and eluted with 30, 50 and 100% of methanol. Fractions 30 and 50% were combined together to give 7 g of MA fraction. Of this fraction, 0.5 g was infused with water (100 ml) for 10 min. An aliquot of the infusion was filtered through a Millex filter (0.45 nm) and analyzed by HPLC–MS. Proanthocyandin content The content of active PACs in M. argun fraction was determined using standard methods given Broadhurst and Jones.[28] In order to determine the amount of PACs, 15 mg of PAC fraction was dissolved in 0.25 ml of 60% (v v 1) MeOH and then 1.50 ml of vanillin reagent (4% v v 1 in methanol) and 0.75-ml concentrated (37%) HCl were added. The reaction was carried out in glass cuvettes at temperature 22 °C and analyzed by the spectrophotometer Hewlett-Packard 6453A, at a 500-nm wavelength. The (þ)-catechin was used as a standard for the standard curve preparation. It was shown that the dried PAC fraction from M. argun nuts contained 636.88 mg/g PACs (as equivalent of (þ)-catechin). HPLC and ESI–MS of fraction MA
J. Mass Spectrom. 2014, 49, 306–315
Results and discussion The UV spectra of the MA fraction obtained from M. argun nuts showed absorption maxima bands at 205–215 nm and 270–285 nm, suggesting the presence of polymeric PACs. Due to the weak acidic nature of PACs, the ESI–MS profile of this fraction was obtained operating in negative ion mode since the proton dissociation can be produced much more easily than operating in positive mode and gives simpler mass spectra due to the absence of intense ion species adducts.[29,30] In order to investigate the presence of different compounds with the same molecular weight and then to realize a qualitative analysis on the PAC constituents occurring in the MA fraction from MeOH extract of M. argun nuts, MS experiments were performed using LC–MS system equipped with an ESI source and an Ion Trap analyzer. A full MS scan, in the form of a total ion current chromatogram (TIC), was acquired, and reconstructed ion chromatograms (RICs) were derived for each of the expected m/z values based on the molecular weights of the possible constituents (Fig. 1A–E). ESI–MS and ESI–MS/MS fingerprint of MA fraction The negative ion ESI–MS profile of the MA fraction showed deprotonated ions from monomers, dimers, trimers, tetramers, pentamers and hexamers (Fig. 2A) of PACs. Observed ions indicated the presence of PACs made up of afzelechin, catechin and gallocatechin. The negative ion mass spectra of the MA fraction revealed two ions at m/z 289 with different retention times, due to the presence of diastereoisomers (catechin and epicatechin). The MS2 spectrum of the ion at m/z 289 (Figs. 1B, 3A and Scheme 1) showed the major product ions at m/z 271, 247, 245, 205, 179, 165, 151, 137, 125 and 109. The ion at m/z 271 was derived from the loss of water (18 Da) and those at m/z 247 and 245 from the losses of 42 Da (HC ≡ C OH) and 44 Da (CH2 = CH OH), respectively (Table 1). The ion at m/z 165 [M–125-H] was attributed to the elimination of ring A by heterocyclic ring fission (HRF) fission, confirmed by the ion at m/z 125 [M–165–H] . The ion at m/z 137 resulted from a retro-Diels–Alder (RDA) cleavage of ring C which was confirmed by the presence of
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
307
The components of the infusion were separated using Thermo Surveyor HPLC system and a C18 RP column (5 μm, 2.1 mm × 150 mm; Symmetry, Waters, MA, USA). The gradient of mobile phases A (water containing 0.03% formic acid) and B (MeCN containing 0.03% formic acid) was as follows: 0 min (10% B); 20 min (30% B); 40 min (45%, B); 45 min (100% B); 50 min (100% B). The flow rate was 0.4 ml/min, and column temperature was 50 °C.
The flow generated by the chromatographic system was introduced directly into the ESI source. MS analysis was performed using an LCQ Advantage Max ion trap instrument (Thermo, San Jose, CA, USA) controlled by Xcalibur 1.3 SR1 software. The ion source operated in the negative ion mode with the following parameters: capillary voltage 4 V, spray voltage 4.1 kV, tube lens offset 0 V, capillary temperature 240 °C and sheath gas (N2) flow rate 50 (arbitrary units). Ions were scanned in the m/z range from 150 to 2000 with three microscans and maximum injection time of 150 ms (meaning the time of filling the trap). Two scan events were set to run sequentially. The first event was a full scan to acquire ions in the selected range. The second scan event was MS/MS fragmentation experiment performed on the most intense ion from the acquired set. Normalized collision energy of 35% was used to generate fragment ions. To obtain general ESI–MS fingerprint of the infusion, it was diluted ten times with 40% MeOH and introduced directly into the ion source of the mass spectrometer at a flow rate of 5 μl/min using syringe pump. The source and the scanning parameters were essentially as described above, except that in this case data-dependent acquisition was not performed.
A. I. Hamed et al.
Figure 1. (A) LC–ESI–MS–TIC (negative ion mode) (base peak in the range 150–2000 m/z) and RICs (reconstructed ion chromatograms) of monomers (B), dimers (C), trimers (D), tetramers (E).
308
the ion peak at m/z 151. The ion at m/z 179 [M–110–H] was due to the loss of dihydroxybenzene structure, which was confirmed by the presence of the ion at m/z 109 [M–179–H] . The ion at m/z 167 [M–122–H] may be formed by BFF fission. The ESI–MSn spectra of afzelechin (m/z 273) and gallocatechin (m/z 305) derivatives were similar to those of catechin and epicatechin.[31] In Figs. 1C, 3B–E and Table 2, the spectra of argun nut PAC dimers are shown in the range m/z 543–609 for both A-type and B-type compounds. PAC dimers with A-type linkage were identified by their [M–H] ions being 2 mass units lower than those of the B-type PACs.[31,32] MS/MS spectra demonstrated that the relative abundance of major product ions (e.g. 271, 285, 287, 289, 305, 407, 409, 417, 421, 425, 433, 439, 441, 449) varied, indicating that they represented different isomeric forms, arising from the different linkage of monomeric flavan-3-ol units. The analysis of mass spectra also evidenced the presence of precursor ions in the range m/z 813–913 corresponding to A-type, B-type and A/B-type trimers (Table 3, Figs. 1D and 3F–H). MS/MS spectra demonstrated, also in this case, that the relative abundance of major product ions (e.g. 271, 285, 287, 289, 305, 407, 439, 449, 711 and 713) varied due to different isomeric forms and different
wileyonlinelibrary.com/journal/jms
linkage of monomeric flavan-3-ol units. PAC trimers with two A-type linkages were identified by MS on the basis of their [M–H] ions being 4 Da less than those of the B-type PACs. PAC trimers with one A/B-type linkage were identified by MS since their [M–H] ions were 2 Da less than those of the B-type PACs and 2 Da more than those of the A-type PACs.[31] According to scheme 1, some rules can be applied to aid in sequencing of PAC dimers. The main fragmentation mechanisms can be rationalized by RDA, quinine methide (QM), and the HRF mechanisms.[31] Moreover, the monomeric building blocks afzelechin, catechin and gallocatechin can be detected at m/z 273, m/z 289 and m/z 305, respectively. Since mass spectrometry cannot distinguish between stereoisomers like (+)/( )-afzelechin/ epiafzelechin, (+)/( )-catechin/epicatechin or (+)/( )-gallocatechin/ epigallocatechin, the names (E)afzelechin, (E)catechin and (E) gallochatechin for all isomers are used. Two anthocyanins were detected in M. argun nuts which were previously detected from strawberries and blackberries.[33] A peak with a retention time of 26.30 min showed a molecular ion at m/z 433 and a fragment ion at m/z 271. The MS data indicated that it was a glucoside or galactoside of pelargonidin. The other
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 306–315
Analysis of PACs in M. argun nut by LC–ESI–MS/MS
(A)
(B)
(C)
(D)
(E)
Figure 2. Typical deprotonated ESI–IT–MS fingerprint of methanolic extract of the MA fraction of Medemia argun nuts obtained in negative ion mode: (A) mass scan in the range of m/z 150–2000, (B) mass scan in the range of m/z 500–660, (C) mass scan in the range of m/z 800–920, (D) mass scan in the range of m/z 1080–1200 and (E) mass scan in the range of m/z 1360–1480.
peak had a molecular ion at m/z 449 and a fragment ion at m/z 287 and was supposed as cyanidin-3-O-hexoside.[34] LC–ESI–MS analysis of PAC dimers
J. Mass Spectrom. 2014, 49, 306–315
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
309
The PAC dimers with A-type (m/z 543, m/z 559, m/z 575, m/z 591 and m/z 607) and B-type (m/z 545, m/z 561, m/z 577, m/z 593 and m/z 609) linkages were readily identified by their [M–H] ions (Table 2). To describe the structure of dimers, we define the
two flavan-3-ol units in the dimers as the top unit and the base unit, and we used the general fission rules illustrated in scheme 1. The product ion spectrum of the deprotonated dimer at m/z 543 produced the base peak at m/z 407 [M–136–H], formed by RDAF fragmentation and loss of the 1,3E0 (C8H8O2) fragment, which indicated that the E ring had a single hydroxyl group at the 4 position (Fig. 2B). The RDA reaction could take place on the base unit of this dimer. The ion at m/z 417 derived from the ion at m/z 543 (Scheme 1), due to the HRF fragmentation and
A. I. Hamed et al.
Scheme 1. The main fragmentation pathways of monomers shown in negative ion mode (RDA = retro-Diels–Alder fission, QM = quinine methide cleavage, and HRF = heterocyclic ring fission, BFF = benzene furan fission).
Table 1. Neutral losses of small molecules from proanthocyanidin [M–18] [M–26] [M–28] [M–36] [M–42] [M–46] [M–54] [M–74] [M–102] [M–112]
[M–H2O] [M–HC ≡ CH] [M–CO] [M–2 x H2O] [M–HC ≡ C–OH] [M–CO–H2O] [M–HC ≡ CHCO] [M–2CO–H2O] [M–H2O–2(HC ≡ C–OH)] [M–CO–2(HC ≡ COH)]
310
the loss of the A ring ([M–126–H]). This fragment indicated that the A ring had a 1,3,5-trihydroxybenzene structure. HRF yielded the product ion at m/z 269, due to the loss of 148 Da from the product ion at m/z 417. Identification of this dimer was confirmed by the fragment ion [M–271–H] at m/z 272 and the ion at m/z 269 [M–274–H]. This ion originated from the quasi-molecular at m/z 543 after quinone methide (QM) cleavage of the interflavan bond. Hence, both the top and base units of this dimer could be identified as (E)afzelechin. The occurrence of HRF fission on the top unit and formation of ions at m/z 272, 271 and 269 through QM cleavage indicated the monomers were linked together through C–C and ether linkages. Therefore, this compound was identified as a doubly linked PAC dimer. Thus, this
wileyonlinelibrary.com/journal/jms
dimer has an A-type structure, and the connection sequence of it has been deduced to be (E)afzelechin–A–(E)afzelechin (1). The [M–H] ion at m/z 545, 2 mass units higher than the corresponding ion of compound 1, potentially indicated a B-type structure. This product ion produced the base peak at m/z 419 [M–126–H], which again derived from the HRF fragmentation and the loss of A ring. HRF also yielded the ion at m/z 271 [M–419–148–H], due to the fission of 2,3 double bond and the loss of the B ring (C9H8O2), indicating a single hydroxyl group located at the 4 position of the B ring (Fig. 3C and Scheme 1). RDA produced the ion at m/z 271 [M–136–H] due to the loss of C8H8O2, followed by elimination of 138 mass units. The ion at m/z 391 [M–170–H] resulted from RDA of the heterocyclic rings and loss of water. A minor ion at m/z 438 derived from the ion at m/z 545 due to neutral loss of water (18 Da) and phenol (94 Da) (Scheme 1). The RDA reaction can take place on either the top or the base unit of the dimer. However, elimination of ring B of the base unit gives rise to an ion with a smaller π–π conjugated system than RDA of the top unit. The ion at m/z 391 [M–18–136–H] derived from the ion at m/z 545 by neutral loss of water molecule (18 Da) and RDA fission, confirming the occurrence of a single hydroxyl group located at the 4 position of the B ring. RDA reaction yielded the ion at m/z 273 [M–136– 136–H] due to the loss of two molecules of C8H8O2 from both units of the dimer. Also, this ion product derived from QM cleavage. Identification of this dimer was confirmed by the fragment ion at m/z 273 [M–271–H]. This ion was formed from the product ion spectrum of the deprotonated molecule at m/z 545 after QM cleavage of the interflavan bond. Hence, the top and base units
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 306–315
Analysis of PACs in M. argun nut by LC–ESI–MS/MS Table 2. Negative fragment ions (m/z) of proanthocyanidin dimers from argun nut Compound (tR) 1 (10.11) 2 (21.11) 3 (12.47) 4 (12.06) 5 (11.96) 6 (16.93) 7 (13.21) 8 (14.81) 9 (15.90) 10 (13.60) 11 (7.35) 12 (15.26) 13 (15.26) 14 (6.58) 15 (7.40) 16 (16.28) 17 (8.67)
Subunit sequence
[M–H]
HRF
RDA–t
RDA–b
QM–t
QM–b
(E)Afz–A–(E)Afz (E)Afz–(E)Afz (E)Afz–A–(E)Cat (E)Cat–A–(E)Afz (E)Afz–(E)Cat (E)Cat–(E)Afz (E)Afz–A–(E)Gal (E)Gal–A–(E)Afz (E)Cat–A–(E)Cat (E)Afz–(E)Gal (E)Cat–(E)Cat (E)Gal–A–(E)Cat (E)Cat–A–(E)Gal (E)Cat–(E)Gal (E)Gal–(E)Cat (E)Gal–A–(E)Gal (E)Gal–(E)Gal
543 545 559 559 561 561 575 575 575 577 577 591 591 593 593 607 609
417 419 433 417 435 419 449 417 433 451 435 433 449 451 435 449 451
– 409 – – 425 409 – – – 441 425 – – 441 425 – 441
407 409 407 423 407 425 407 439 423 409 425 439 423 407 441 439 441
269 271 269 285 273 287 269 301 285 271 287 301 285 289 303 301 303
273 273 289 273 289 273 305 273 289 305 289 289 305 305 289 305 305
Afz, Cat and Gal are abbreviation for afzlechin, catechin and gallocatechin; the symbol (E) indicates that there are two possibilities: ‘afzelechin or epiafzelechin; catechin or epicatechin; gallocatechin or epigallocatechin’ A = A-type; B = B-type; A/B = A/B- type, tR = retention time.
J. Mass Spectrom. 2014, 49, 306–315
The ion at m/z 451 derived from the ion at m/z 609 after a 158-Da neutral loss [M–140–H2O–H] (Scheme 1) due to elimination of C7H8O3 (140 Da), suggesting that ring B contained three hydroxyl groups. Elimination of 162 Da from the ion at m/z 451 indicated that the top unit of the dimer was (E)gallocatechin. The presence of the ions at m/z 303 [M–306–H] and 289 [M–302–H2O–H] suggested this dimer as (E)gallocatechin–(E) gallocatechin (17) (Fig. 3E). LC–ESI–MS analysis of PAC trimers Mass spectra also exhibited precursor ions corresponding to PAC trimers in the range 813–913 amu for B-type and A/B-type (Table 3 and Figs. 1D and 2C). To describe the structure of trimers, we define the three flavan-3ol units as the top unit, the middle unit and the base unit. Twenty-six trimers could be identified by selecting their [M–H] pseudomolecular ions (Scheme 1, Table 3 and Figs. 3F–H). For the sake of simplicity, we have selected only one A-type and A/B-type for the discussion. The pseudomolecular ion [M–H] at m/z 847 produced the base peak at m/z 711 [M–152–H] due to the RDA fragmentation and loss of the 1,3B group (Scheme 1). The RDA rearrangement was found to prevail especially on the C-ring of catechin and epicatechin derivatives, in which two possible fragment ions are formed by loss of 136 Da of a B-ring, indicating that B-ring contained a single hydroxyl group.[35,36] RDA yielded the product ion at m/z 575 [M–136–136–H] due to the loss of C7H4O3, probably by elimination of 3,4 double bond and A-ring. The product ion at m/z 722 derived by elimination of the A-ring (126 Da) from the top unit of the trimer by HRF fragmentation of the ion peak at m/z 847, indicating that the A ring of the top unit had a 1,3,5-trihydroxybenzene structure (Fig. 3F). HRF yielded the product ion at m/z 575 [M–126–147–H] due to the loss of C9H8O2, probably by elimination of the 3,4 double bond and B-ring from the ion peak at m/z 722. Formation of the product ion at m/z 575 also indicated that there was one hydroxyl group located at C4 of the B ring. Hence, the top unit was afzelechin which was confirmed by the presence of the ion peak at m/z 575 [M–273–H] (QM).
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
311
of this dimer were identified as (E)afzelechin. The mass spectra resulting from MS/MS experiment of the ion at m/z 545 in the negative ion mode showed product ions at m/z 283 [M–H–262] (HRF + RDA), 273 (QM) and 271 (QM), typical of B-type PAC dimer. Therefore, this compound was identified as a singly linked PAC dimer corresponding to (E)afzelechin–(E)afzelechin (2) (Fig. 4). The [M–H] ion at m/z 559 and the ion at m/z 433 formed by the neutral loss of 126 mass units as a result of HRF. As before, the loss of 126 Da indicates 1,3,5-trihydroxybenzene structure for the A ring. As shown in Table 2, CID spectrum of 3 at m/z 559 gave fragment ions at m/z 407 [M–152–H] and 389 [M–170–H] from RDAF of the heterocyclic rings. This cleavage points out that the E ring contains two hydroxyl groups located at 3 and 4 positions. The mass spectra resulting from MS/MS experiment of the ion at m/z 559 in the negative ion mode showed product ions at m/z 423 [M–H–136] (RDAC) after QM1 and 271 [M–152–H] (QM2), typical for A-type PAC dimer. Fragment ions at m/z 287 [M–272–H], 271 [M–287–H] and 269 [M–271–H2O–H] indicated afzelechin as the top unit and catechin as the base unit. Therefore, the structure of the dimer is (E) afzelechin–A–(E)catechin (3) (Fig. 4). Another peak with the same [M–H] ion at m/z 559 but with different retention time and fragmentation profile has also been recorded (Table 2). In this case, the structure has been deduced as (E)catechin–A–(E)afzelechin (4). Figure 3D shows a base ion peak at m/z 481 derived from the peak at m/z 607 after elimination of ring A (126 amu) by HRF reaction. Another ion peak at m/z 463 derived from the subsequent loss of a water molecule from the F ring. BFF fission generated an ion peak at m/z 471 [M–137–H] (Scheme 1). Hence, the base unit of this dimer was identified as gallocatechin. Another minor peak at m/z 405 [M–168–32–H] was due to RDA reaction of ring F after elimination of 32 Da from the deprotonated ion peak at m/z 607. Loss of 168 Da indicated that ring F had three hydroxyl groups located at 3, 4 and 5. QM cleavage gave ion peaks at m/z 301 [M–306–H] and 287 [M–302–H2O–H], indicating this dimer as a doubly linked PAC dimer made up of two gallocatechin units. Therefore, this compound has an A-type structure and has been deduced as (E)gallocatechin–A–(E)gallocatechin (16).
A. I. Hamed et al. Table 3. Negative fragment ions (m/z) of proanthocyanidin trimmers and tetramers from argun nut Compound (tR)
Subunit sequence
[M–H]
18 19 (18.33) 20 (11.27)
(E)Afz–(E)Afz–(E)Afz (E)Afz–(E)Afz–(E)Afz (E)Afz–(E)Afz–(E)Afz
813 815 817
21 22 (10.70)
(E)Afz–(E)Afz(E)Cat (E)Cat–(E)Afz–(E)Afz
829 831
23 (12.28) 24 25 (11.69)
(E)Afz–(E)Afz–(E)Cat (E)Afz–(E)–Cat–(E)Cat (E)Afz–(E)Cat–(E)Cat
833 845 847
26 (10.50) 27 (10.67) 28 (12.21) 29 (9.87)
(E)Afz–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat
849 861 863 865
30 (13.92) 31 (8.64) 32 (15.07 33 (16.00) 34(16.00) 35 (16.00) 36 37 (8.48) 38 (9.29) 39(8.17) 40 (9.66) 41 42 (12.10) 43 (12.22) 44 (7.73) 45 (7.90) 46 (7.99) 47 (15.72) 48 (7.55) 49 (8.09) 50 (12.70)
(E)Cat–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Gal–(E)Cat (E)Gal–(E)Gal–(E)Cat (E)Gal–(E)Gal–(E)Cat (E)Gal–(E)Gal–(E)Gal (E)Gal–(E)Gal–(E)Gal (E)Gal–(E)Gal–(E)Gal (E)Afz–(E)Afz–(E)Afz–(E)Cat (E)Afz–(E)Afz–(E)Afz–(E)Cat (E)Afz–(E)Afz–(E)Cat–(E)Cat (E)Afz–(E)Afz–(E)Cat–(E)Cat (E)Afz–(E)Cat–(E)Cat–(E)Cat (E)Afz–(E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat–(E)Cat
865 877 879 881 881 881 893 895 897 909 911 913 1103 1105 1119 1121 1135 1137 1149 1151 1153
51 (7.86) 52 (7.86) 53 (7.70) 54 (7.60) 55 (7.73)
(E)Cat–(E)Cat–(E)Cat–(E)Gal (E)Cat–(E)Cat–(E)Cat–(E)Gal (E)Cat–(E)Cat–(E)Gal–(E)Gal (E)Cat–(E)Cat–(E)Gal–(E)Gal (E)Cat–(E)Cat–(E)Gal–(E)Gal
1165 1167 1181 1183 1199
Major fragments (m/z) N.D. 797, 783, 753, 735, 679, 543, 529, 343, 271 799, 775, 755, 706, 665, 637, 578, 555, 545, 529, 419, 407, 365, 271 N.D. 813, 769, 695, 679, 633, 551, 532, 523, 460, 419, 411, 405, 335, 711, 704, 561, 477, 449, 435, 417, 407, 289 N.D. 829, 785, 722, 711, 693, 645, 575, 559, 547, 439, 362, 351, 295, 289, 285, 265 849, 723, 653, 589, 561, 433, 415, 289, 273 553, 435, 425, 407, 735, 712, 695, 575, 451, 407, 289 847, 821, 755, 739, 712, 647, 627, 617, 577, 449, 404, 381, 327, 287 847, 803, 713, 561, 543, 449, 423, 289, 269 467, 451, 425, 407, 381, 309, 289, 245 861, 773, 727, 714, 665, 617, 576, 529, 443, 409, 285 853, 801, 771, 743, 609, 513, 451, 305 853, 801, 771, 743, 592, 513, 429, 305 853, 801, 771, 743, 577, 513, 451, 305 N.D. 877, 863, 815, 756, 607, 599, 471, 413, 303 879, 817, 745, 647, 607, 536, 445, 381, 285 787, 607, 475, 435, 407 869, 851, 743, 703, 607, 559, 481, 323, 303 N.D. 1042, 997, 947, 833, 559, 545, 425, 407, 289, 273 846, 577, 434, 425, 407, 289 1105, 1055, 833, 561, 543, 289 1008, 865, 729, 577, 407, 289 985, 849, 713, 559, 407, 289 1105, 1042, 1013, 863, 711, 575, 433, 425, 289 865, 701, 577, 451, 425, 407, 289, 245 1099, 1065, 1043, 983, 947, 905, 865, 863, 711, 701, 693, 651, 605, 575, 574, 549, 425, 407, 395 N.D. 999, 879, 865, 713, 577, 452, 425, 407, 289 1074, 996, 879, 741, 729, 577, 425, 407 879, 742, 719, 577, 451, 425, 289 1137, 910, 727, 608, 577, 451, 425, 289
Type A g A/B B A A/B B A A/B B A A/B B B A A/B B B B A A/B B A A/B B A/B B A/B B A/B B A/B A/B B A/B A/B A/B A/B A/B
Afz, Cat and Gal are abbreviation for afzlechin, catechin and gallocatechin; the symbol (E) indicates that there are two possibilities: ‘afzelechin or epiafzelechin; catechin or epicatechin; gallocatechin or epigallocatechin’ A = A-type; B = B-type; A/B = A/B- type, tR = retention time.
312
On the other hand, the presence of the ion peak at m/z 575 indicated that the middle and base units had A-type linkage. Another product ion was observed at m/z 439 [M–575–136–H] due to the loss of C7H4O3 and at m/z 285 ([M–288–H], resulting from the cleavage of the ion peak at m/z 439 through the QM mechanism. It seems that the A-type interflavan bonds do not undergo observable QM cleavage in the presence of a B-type interflavan bond in the same molecule. The fragmentation profile of the ion peak at m/z 575 indicated that the middle and base units were (E)catechin. Thus, the structure of this trimer has been deduced as (E)afzlechin–(E)catechin–A–(E)catechin (25) (Fig. 4).
wileyonlinelibrary.com/journal/jms
The pseudomolecular ion [M–H] at m/z 849 produced the base peak at m/z 723 [M–126–H] which was due to the RDA fragmentation and loss of the A-ring (trihydroxybenzene) (Scheme 1). Loss of the 2, 3, 4 E group of 162 Da by HRF produced the ion peak at m/z 561, indicating that the B ring of the top unit had a 3,4-dihydroxybenzene structure, corresponding to a catechin derivative (Fig. 3G). RDA of the ion peak at m/z 561 yielded the product ion at m/z 433 [M–561–126– 162–126–H] due to the loss of a trihydroxybenzene, probably by elimination of D-ring from the middle unit. Loss of 160 Da by HRF of the ion peak at m/z 433 produced the ion peak at
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 306–315
Analysis of PACs in M. argun nut by LC–ESI–MS/MS
2
Figure 3. Product ion spectra of argun nut components ([M–H] ): MS of monomer at m/z 289 (A), dimers at m/z 543 (B), 545 (C), 607 (D) and at m/z 609 (E), trimmers at m/z 847 (F), 849 (G) and at m/z 881 (H), and tetramer at m/z 1153 (I).
J. Mass Spectrom. 2014, 49, 306–315
LC–ESI–MS analysis of PAC tetramers The analysis of mass spectra also evidenced the presence of precursor ions corresponding to tetramers in the m/z range 1089–1199 (Table 3 and Figs. 1E and 2D). The obtained m/z values evidenced the presence of PACs belonging to B-type and A/B-type and the absence of A-type series. The pseudomolecular ion [M–H] at m/z 1153 [288 × 3 + 289–H] and UV spectra are similar with those of (E)catechin. As shown in Fig. 3I and Table 3, the fragmentation spectrum of compound 50 at m/z 1153 gave fragment ions at m/z 865 [M–288–H] due to the quinine methide reaction (QM1) of the top unit, m/z 701 [M–288–164–H] due to HRF reaction of the interflavone unite ([M–288–C9H8O3–H]), m/z 575 ([M–288–C9H8O3–126–H] due to the loss of a trihydroxybenzene, probably by elimination of D-ring from the middle unit. Also, product ion at m/z 575 [M–289 × 2H] due to the quinine methide reaction (QM1, QM4) of the top and lower units. The fragment ion at m/z 449 [M–575–126–H] due to loss of another trihydroxybenzene, probably by elimination of D-ring or G-ring from one of the two middle units. The presence of product ion at
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
313
m/z 273 indicating that the middle unit was a catechin derivative and the base unit was an afzelechin derivative. Another product ion was observed at m/z 561 [M–288–H] due to the loss of a catechin unit, confirming the type of the top monomer. Two base peaks at m/z 289 and m/z 273 due to the quinine methide reaction (QM) of the ion peak at m/z 561 confirmed that the middle unit was a catechin derivative and the base unit was afzelechin. Thus, for this trimer the structure (E)catechin–(E)catechin–(E)afzlechin (26) (Fig. 4) is proposed. Figure 3H shows that the unique chromatographic peak at m/ z 881 produced three peaks at m/z 609 ([M–272–H]), m/z 592 ([M–289–H]) and m/z 577 ([M–304–H]), due to the QM1 mechanism (Table 2). Moreover, only one peak raised from QM2 at m/z 305, indicating that the chromatogram contained three trimers. Two of them had similar monomers, but their linkages were alternate ([M–272–304–H]) and ([M–304–272–H]), and the last one had different monomers ([M–289–287–H]). Their structures were deduced as (E)afzlechin–(E)gallocatechin– (E)gallocatechin (34), (E)gallocatechin–(E)afzlechin–(E)catechin (35) and (E)catechin–(E)catechin–(E)gallocatechin (36), respectively (Fig. 4).
A. I. Hamed et al.
Figure 4. Structure of the monomers, dimers and trimers proanthocyanidins in Medemia argun nuts.
m/z 425 [M–575–C6H6O3–H2O–H] is due to the BFF mechanism from the lower unit of the two intermediate flavone units (Scheme 1). Its structure was (E)catechin–(E)catechin–(E) catechin (50). The analysis of mass spectra also evidenced the presence of precursor ions corresponding to pentamers in the m/z range 1377–1454 (Fig. 2E). The obtained m/z values evidenced the presence of PACs belonging to B-type and A/B-type and the absence of A-type series. However, no single pentameric and hexameric signals were observed, but a group of signals with a mass difference of 16 amu.
quinonemethide (QM) cleavage of the interflavan bond. The rationalization of oligomers can be of interest for plant physiologists and can be crucial for nutritionists who are interested in studying their bioavailability and possible human health effects. The discovery of PACs with well-known antioxidant properties can explain the good conservation of argun nuts during the time and may bring us closer to answering about potential use of this fruits in the ancient time. The data obtained in our research show that M. argun is a novel rich source of hydrolyzable PACs made up of afzelechin, catechin and gallocatechin units.
Conflict of Interest Conclusion
314
The analytical procedure described in this work provides a valuable profile of PACs occurring in M. argun nuts and represents a rapid, simple and sensitive method to rationalize PACs in plants and derived preparations. In particular, this strategy based on HPLC coupled to electrospray ionization mass spectrometry (HPLC–ESI–MS) in the negative ion mode allows to achieve deep structural information on the nature of the flavane monomers which make up proantocyanidins, their sequence and the type of flavane bond. In particular, diagnostic ions resulting from HRF and RDA reaction of flavan-3-ol provide information on the hydroxylation pattern and the type of interflavan bond. The flavane sequences can be ascertained through ions derived from
wileyonlinelibrary.com/journal/jms
The present work was supported by the European Project Proficiency (FP7-REGPOT-2009-1) no. 245751. Acknowledgements We are thankful to the European Project Proficiency (FP7-REGPOT2009-1) no. 245751.
References [1] C.S. Kunth. Recherches sur les plantes trouvées dans les tombeaux égyptiens par M. Passalacqua. Annales des Sciences Naturelles. Botanique (Paris), 1826, 8, 418. [2] H. Wendland. Beitrage zu den Borassinen. Botanische Zeitung 1881, 39, 89.
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 306–315
Analysis of PACs in M. argun nut by LC–ESI–MS/MS [3] V. Täckholm, M. Drar. Flora of Egypt 2. Bull. Fac. Sci. (Egypt University) 1950, 28, 296. [4] C. Newton. Le palmier Argoum, Medemia argun (Mart.), Württemb. ex Wendl. P., Encyclopedie Religieuse de l’Univers Vegetal. Croyances phytoreligieuses de l’Egypte ancienne (ERUV) II. OrMonsp 2001, XI, 141. [5] S. Pain. Fruits of the tomb. New Sci. 2006, 190, 554. [6] V. Täckholm. Students’ Flora of Egypt, 2nd ed. Cairo University: Egypt, 1974, 888. [7] V. Loret. La Flore pharaonique d’apres les documents hieroglyphiques et les speciments decouverts dans le tombes, 2nd ed. J. B. Bailliere and sons: Paris, 1887. [8] A. R. David, E. V. Tapp. The Mummy’s Tale, The Scientific and Medical Investigation of Natsif-Amun, Priest in the Temple of Karnak. Michael O’Mara Books Ltd., (V. ), London, 1972, 176. [9] H. Ibrahim, W. J. Baker. Medemia argun – past, present and future. Palms 2009, 53, 9. [10] C. F. P. Von Martius. Historia Naturalis Palmarum. Leipz. (Germany) 1823-1850, 3 Bände, 790. [11] M. Gibbons, T. W. Spanner. Medemia argun lives. Principl. 1996, 40, 65. [12] L. Boulos, The discovery of Medemia argun palm in the Nubian Desert of Egypt. Bot. Notebook 1968, 121, 117. [13] J. Porter. In methods in plant Biochemistry: 1- Plant phenolics, Tannins, P. M. Dey, J. B. Harborne (Eds). Academic Press Inc.: San Diego, 1989. [14] G. Nonaka, F. L. Hasu, I. Nishioka. Structures of dimeric, trimeric, and tetrameric procyanidins from Areca catechu L. J. Chem. Soc. Chem. Commun. 1981, 15, 781. [15] C. K. Wang, W. H. Lee. Separation, characteristics, and biological activities of phenolics in areca fruit. J. Agric. Food Chem. 1996, 44, 2014. [16] A. Nunez-de la Mora, R. T. Chatterton, E. T. Mateo, F. Jesmin, G. R. Bentley. Effect of chewing betel nut on measurements of salivary progestrone and estradiol. Americ. J. Physic. Anthrop. 2007, 132, 311. [17] Q. Wu, Y. Yang, J. E. Simon. Qualitative and Quantitative HPLC/MS Determination of Proanthocyanidins in Areca Nut (Areca catechu). Chem. Biodiver. 2007, 4, 2817. [18] R. B. Rodrigues, R. Lichtenthaler, B. F. Zimmermann. Total oxidant scavenging capacity of Euterpe oleracea Mart. (Acai) seeds and identificationof their polyphenolic compounds. J. Agric. Food Chem. 2006, 54, 4126. [19] L. A. Paacheco-Palencia, S. Mertens-Talcott, S. T. Talcott. Chemical composition, antioxidant properties, and thermal stability of a phytochemical enriched oil from Acai (Euterpe oleracea Mart.). J. Agric. Food Chem. 2008, 56, 4631. [20] G. D. Noratto, G. Angel-Morales, S. T. Talcott, S. U. Mertens-Talcott. Polyphenolics from Acai (Euterpe oleracea Mart.) and red muscadine grape (Vitis rotundifolia) protect human umbilical vascular endothelial cells (HUVEC) from glucose- and lipopolysacharide (LPS)- induced inflammation and target microRNA-126. J. Agric. Food Chem. 2011, 59, 7999. [21] C. M. Rodrigues, D. Rinaldo, L. C. Dos Santos, P. Montoro, S. Piacente, C. Pizza, C. A. Hiruma-Lima, A. R. M. Souza Brito, W. Vilega. Metabolic fingerprinting using direct flow injection electrospray ionization tandem mass spectrometry for the characterization of proanthocyanidins
[22]
[23] [24] [25] [26] [27]
[28] [29]
[30] [31] [32]
[33]
[34]
[35]
[36]
from the barks of Hancornia speciosa. Rapid Commun. Mass Spectr. 2007, 21, 1907. M. Maldini, P. Montora, A. I. Hamed, U. A. Mahalel, W. Oleszek, S. Piacente. Strong antioxidant phenolics from Acacia nilotica: profiling by ESI-MS and quali-quantitative determination by LC-ESI-MS. J. Pharm. Biomed. Anal. 2011, 56, 228. V. Cheynier, T. Doco, H. Fulcrand, S. Guyot, E. Le Roux, J. M. Souquet, J. Rigaud, M. Moutounet. ESI-MS analysis of polyphenolic oligomers and polymers. Anal. 1997, 25, 32. L. Poblocka-Olech, M. Krauze-Baranowska. SPE-HPTLC of procyanidins from the barks of different species and clones of Salix. J. Pharm. Biomedical Anal. 2008, 48, 965. J. Valls, S. Milan, M. P. Marti, E. Borras, L. Areola. Advanced separation methods of food anthocyanins, isoflavones and flavanols. J. Chromatogr. A 2009, 1216, 7143. A. I. Hamed, M. Leonardi, A. Stochmal, W. Oleszek, L. Pistelli. Flavone and flavonol glycosides from Astragalus eremophilus and Astragalus vogelii. Nat. Prod. Commun. 2012, 7, 633. A. Morel, A. I. Hamed, W. Oleszek, A. Stochmal, R. Glowacki, B. Olas. Protective action of proanthocyanidin fraction from Medemia argun against Oxidative/nitrative damages of blood platelet and plasma components. Platelet 2014, 25(1), 75. R. B. Broadhurst, W. T. Jones. Analysis of condensed tannins using acidified vanillin. J. Sci. Food Agric. 1978, 29, 788. A. Mari, D. Eletto, C. Pizza, P. Montoro, S. Piacente. Integrated mass spectrometry approach to profile proanthocyanidins occurring in food supplements: Analysis of Potentilla erecta L. rhizomes. Food Chem. 2013, 141(4), 4171. Y. Hayasaka, E. Waters, V. Cheynier, M. J. Herderich, S. Vidal. Characterization of proanthocyanidins in grape seeds using electrospray mass spectrometry. Rapid Commun. Mass Spectr. 2003, 17, 9. H. J. Li, M. L. Deinzer. Tandem mass spectrometry for sequencing proanthocyanidins. Anal. Chem. 2007, 79, 1739. L. Gu, A. M. Kelm, F. J. Hammerstone, G. Beecher, J. Holden, D. Hytowitz, L. R. Prior. Screening of foods containing proanthocyanidins and their structural characterization using LC-MS/MS and thiolytic degradation. J. Agric. Food Chem. 2003, 51, 7513. X. Wu, G. Beecher, J. M. Holden, D. B. Haytowitz, S. E. Gebhardt, R. L. Prior. Concentration of anthocyanins in common foods in the united states and estimation of normal consumption. J. Agric. Food Chem. 2006, 54, 4069. W. Mullen, C. A. Edwards, M. Serafini, A. Crozier. Bioavailability of pelargonidin-3-O-glucoside and its metabolites in humans following the ingestion of strawberries with and without cream. J. Agric. Food Chem. 2008, 56, 713. S. de Pascual-Teresa, J. C. Rivas-Gonzalo. Application of LC-MS for the identification of polyphenols. In Methods in Polyphenol Analysis, C. Santos-Buelga, G. Williamson (Eds). Royal Soci. Chem.: Cambridge, U.K., 2003, 48. S. Guyot, J. Vercauteren, V. Cheynier. Structure determination of colourless and yellow dimers resulting from (+)-catechin coupling catalyzed by grape polyphenoloxidase. Phytochem. 1996, 42, 5.
315
J. Mass Spectrom. 2014, 49, 306–315
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
Revised: 29 January 2014
Accepted: 3 February 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3344
Profiles analysis of proanthocyanidins in the argun nut (Medemia argun—an ancient Egyptian palm) by LC–ESI–MS/MS Arafa I. Hamed,a,b* Abdulla S. Al-Ayed,c Jaroslaw Moldoch,b Sonia Piacente,d Wieslaw Oleszekb and Anna Stochmalb Medemia argun is an ancient palm rich in proanthocyanidins (PACs). These polyphenolic compounds are widely distributed in plants and are an integral part of the human diet. A sensitive high-performance liquid chromatography and electrospray ionization mass spectrometry (HPLC–ESI–MS) method in the negative ion mode for sequencing these ubiquitous and highly beneficial antioxidants is described in order to profile different PACs in M. argun nuts. The analytical protocol based on tandem mass spectrometry was used to sequence dimers, trimers, tetramers and pentamers with different A-type, B-type and A/B-type linkages. Diagnostic ions resulting from heterocyclic ring fission and retro-Diels–Alder reaction of flavan-3-ol provided information on the hydroxylation pattern and the type of interflavan bond. The sequences were discovered through ions derived from quinone methide cleavage of the interflavan bond. The identification of PACs linkages through LC–MSn eliminates a number of tedious separation steps. The method was successfully applied to give a view of PAC profile in M. argun nuts. M. argun nuts contained 636.88 mg/g PACs (as equivalent of (þ)-catechin). The data obtained in our research show that M. argun is a rich source of hydrolyzable PACs. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Medemia argun nut; proanthocyanidins; liquid chromatography; electrospray ionization; mass spectrometry
Introduction
306
Medemia argun (Syns. = Hyphaene argun = Medemia abiadensis = Areca abiadensis: Coryphoideae: Borasseae: Hyphaeninae) is a mysterious and little known species of fan palm from the Nubian Desert Oases of Southern Egypt and Northern Sudan. M. argun fruits have been found in the tombs from the 5th Dynasty (ca. 2500 BC) to Roman times (6–7th century AD) including the celebrated tomb of Tutankhamun.[1–5] It seems that M. argun fruits were highly valued in ancient Egypt, although the reason for that is still unknown. An Arabic name for M. argun is ‘argun’.[6] It also has a hieroglyphic name, Mama-n-Khanen (or Mama-n-Xanin). [7–9] Different authors give various claims on the edibility of the fruit. Some of them state that they are bitter and inedible (Von Martius, 1823–1850),[10] while others suggest that the thin fruit flesh is sweet and might have been acceptable to the ancient tastes, especially to those living in the desert where the fruits were scarce.[11,12] According to Loutfy Boulos,[12] the fruits were buried under sand for a period, during which the endosperm (nut) developed a sweet taste similar to coconut. Proanthocyanidins (PACs) are oligomeric and polymeric end products of the flavonoid biosynthetic pathway (syn. condensed tannins) which constitute one of the ubiquitous groups of all plant phenols. The size of PAC molecules can be described by their degree of polymerization.[13] PACs containing afzelechin, catechin or gallocatechin as subunits are named PAC. They are present in fruits, barks, leaves and seeds of many plants, where they provide protection against predation. At the same time, they give flavor and astringency to beverages such as wine, fruit juices and teas, and are increasingly recognized as having beneficial effects on human health. Nuts of Areca catechu, a palm tree
J. Mass Spectrom. 2014, 49, 306–315
indigenous to the South- and Southeast Asia, had been evaluated for its phenolic constituents and their biological activity.[14–17] Euterpe oleraceae (Acai), a large palm tree indigenous to the Amazon River, had been evaluated for phenolic constituents and their biological activity.[18–20] Due to the structural complexity of PAC derivatives and high difficulties in their separation, studies on these compounds are limited in comparison with other polyphenols.[21,22] Usually, catechins give unresolved high-performance liquid chromatography (HPLC) peaks, due to the similarity in their structures and also to the large number of phenolic groups that can give the same interactions with chromatographic stationary phase.[23,24] Reversed phase (RP)-HPLC is the most popular method used in the analysis of these compounds.[24,25] Nowadays, little detailed phytochemical and pharmaceutical studies have been reported for M. argun.[26] Our previous study exhibited that the PAC fraction from M. argun nuts can be useful
* Correspondence to: Arafa I. Hamed, Department of Botany, Faculty of Science, Aswan University, Aswan 81528, Egypt. E-mail: [email protected] a Department of Botany, Faculty of Science, Aswan University, Aswan 81528, Egypt b Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State of Research Institute, ul. Czartoryskich 8, 24-100 Pulawy, Poland c Department of Chemistry, College of Science & Arts at Al-Rass, Qassim University, P.O. 53, Kingdom of Saudi Arabia d Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, I-84084 Fisciano, Salerno, Italy
Copyright © 2014 John Wiley & Sons, Ltd.
Analysis of PACs in M. argun nut by LC–ESI–MS/MS as a protecting factor against oxidative/nitrative stress associated with different diseases (cancer, cardiovascular and neurodegenerative diseases) and PACs of M. argun nuts may be promising antioxidants.[27] Due to the complexity of PACs structures, we have proposed to rationalize the presence of these compounds on the basis of electrospray ionization mass spectrometry (ESI–MS) and ESI–MS/MS profiles. Direct flow injection/electrospray ionization/ion trap tandem mass spectrometry was used to investigate polyphenolic compounds in the methanolic extract of M. argun. In a second stage, analytical HPLC–ESIMS and HPLC–ESI–MS/MS were developed. In the present work, we focused on the analysis of the phenolic constituents of this palm which resulted to be a rich novel source of PACs with strong free radical scavenging activity.
Materials and methods Plant material Fresh fruits of M. argun were collected from the Dungul Oasis (Aswan, Egypt) in October, 2010 and dried in dark at room temperature. The plant material was identified by Prof Arafa I. Hamed according to Täckholm (1974), and the voucher specimens were deposited in Botany Department Herbarium, Aswan Faculty of Science (Egypt). Extraction and sample preparation Dried powdered nuts of M. argun (450 g) were first extracted with hexane and then with 80% MeOH. The aqueous methanolic extract was concentrated under reduced pressure to give 50 g of crude extract (ca: 11% crude extract). Ten grams of extract was loaded onto a C18 column (4 × 50 cm, 40–63 μm LiChroprep, Millipore Corp, Bedford) and eluted with 30, 50 and 100% of methanol. Fractions 30 and 50% were combined together to give 7 g of MA fraction. Of this fraction, 0.5 g was infused with water (100 ml) for 10 min. An aliquot of the infusion was filtered through a Millex filter (0.45 nm) and analyzed by HPLC–MS. Proanthocyandin content The content of active PACs in M. argun fraction was determined using standard methods given Broadhurst and Jones.[28] In order to determine the amount of PACs, 15 mg of PAC fraction was dissolved in 0.25 ml of 60% (v v 1) MeOH and then 1.50 ml of vanillin reagent (4% v v 1 in methanol) and 0.75-ml concentrated (37%) HCl were added. The reaction was carried out in glass cuvettes at temperature 22 °C and analyzed by the spectrophotometer Hewlett-Packard 6453A, at a 500-nm wavelength. The (þ)-catechin was used as a standard for the standard curve preparation. It was shown that the dried PAC fraction from M. argun nuts contained 636.88 mg/g PACs (as equivalent of (þ)-catechin). HPLC and ESI–MS of fraction MA
J. Mass Spectrom. 2014, 49, 306–315
Results and discussion The UV spectra of the MA fraction obtained from M. argun nuts showed absorption maxima bands at 205–215 nm and 270–285 nm, suggesting the presence of polymeric PACs. Due to the weak acidic nature of PACs, the ESI–MS profile of this fraction was obtained operating in negative ion mode since the proton dissociation can be produced much more easily than operating in positive mode and gives simpler mass spectra due to the absence of intense ion species adducts.[29,30] In order to investigate the presence of different compounds with the same molecular weight and then to realize a qualitative analysis on the PAC constituents occurring in the MA fraction from MeOH extract of M. argun nuts, MS experiments were performed using LC–MS system equipped with an ESI source and an Ion Trap analyzer. A full MS scan, in the form of a total ion current chromatogram (TIC), was acquired, and reconstructed ion chromatograms (RICs) were derived for each of the expected m/z values based on the molecular weights of the possible constituents (Fig. 1A–E). ESI–MS and ESI–MS/MS fingerprint of MA fraction The negative ion ESI–MS profile of the MA fraction showed deprotonated ions from monomers, dimers, trimers, tetramers, pentamers and hexamers (Fig. 2A) of PACs. Observed ions indicated the presence of PACs made up of afzelechin, catechin and gallocatechin. The negative ion mass spectra of the MA fraction revealed two ions at m/z 289 with different retention times, due to the presence of diastereoisomers (catechin and epicatechin). The MS2 spectrum of the ion at m/z 289 (Figs. 1B, 3A and Scheme 1) showed the major product ions at m/z 271, 247, 245, 205, 179, 165, 151, 137, 125 and 109. The ion at m/z 271 was derived from the loss of water (18 Da) and those at m/z 247 and 245 from the losses of 42 Da (HC ≡ C OH) and 44 Da (CH2 = CH OH), respectively (Table 1). The ion at m/z 165 [M–125-H] was attributed to the elimination of ring A by heterocyclic ring fission (HRF) fission, confirmed by the ion at m/z 125 [M–165–H] . The ion at m/z 137 resulted from a retro-Diels–Alder (RDA) cleavage of ring C which was confirmed by the presence of
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
307
The components of the infusion were separated using Thermo Surveyor HPLC system and a C18 RP column (5 μm, 2.1 mm × 150 mm; Symmetry, Waters, MA, USA). The gradient of mobile phases A (water containing 0.03% formic acid) and B (MeCN containing 0.03% formic acid) was as follows: 0 min (10% B); 20 min (30% B); 40 min (45%, B); 45 min (100% B); 50 min (100% B). The flow rate was 0.4 ml/min, and column temperature was 50 °C.
The flow generated by the chromatographic system was introduced directly into the ESI source. MS analysis was performed using an LCQ Advantage Max ion trap instrument (Thermo, San Jose, CA, USA) controlled by Xcalibur 1.3 SR1 software. The ion source operated in the negative ion mode with the following parameters: capillary voltage 4 V, spray voltage 4.1 kV, tube lens offset 0 V, capillary temperature 240 °C and sheath gas (N2) flow rate 50 (arbitrary units). Ions were scanned in the m/z range from 150 to 2000 with three microscans and maximum injection time of 150 ms (meaning the time of filling the trap). Two scan events were set to run sequentially. The first event was a full scan to acquire ions in the selected range. The second scan event was MS/MS fragmentation experiment performed on the most intense ion from the acquired set. Normalized collision energy of 35% was used to generate fragment ions. To obtain general ESI–MS fingerprint of the infusion, it was diluted ten times with 40% MeOH and introduced directly into the ion source of the mass spectrometer at a flow rate of 5 μl/min using syringe pump. The source and the scanning parameters were essentially as described above, except that in this case data-dependent acquisition was not performed.
A. I. Hamed et al.
Figure 1. (A) LC–ESI–MS–TIC (negative ion mode) (base peak in the range 150–2000 m/z) and RICs (reconstructed ion chromatograms) of monomers (B), dimers (C), trimers (D), tetramers (E).
308
the ion peak at m/z 151. The ion at m/z 179 [M–110–H] was due to the loss of dihydroxybenzene structure, which was confirmed by the presence of the ion at m/z 109 [M–179–H] . The ion at m/z 167 [M–122–H] may be formed by BFF fission. The ESI–MSn spectra of afzelechin (m/z 273) and gallocatechin (m/z 305) derivatives were similar to those of catechin and epicatechin.[31] In Figs. 1C, 3B–E and Table 2, the spectra of argun nut PAC dimers are shown in the range m/z 543–609 for both A-type and B-type compounds. PAC dimers with A-type linkage were identified by their [M–H] ions being 2 mass units lower than those of the B-type PACs.[31,32] MS/MS spectra demonstrated that the relative abundance of major product ions (e.g. 271, 285, 287, 289, 305, 407, 409, 417, 421, 425, 433, 439, 441, 449) varied, indicating that they represented different isomeric forms, arising from the different linkage of monomeric flavan-3-ol units. The analysis of mass spectra also evidenced the presence of precursor ions in the range m/z 813–913 corresponding to A-type, B-type and A/B-type trimers (Table 3, Figs. 1D and 3F–H). MS/MS spectra demonstrated, also in this case, that the relative abundance of major product ions (e.g. 271, 285, 287, 289, 305, 407, 439, 449, 711 and 713) varied due to different isomeric forms and different
wileyonlinelibrary.com/journal/jms
linkage of monomeric flavan-3-ol units. PAC trimers with two A-type linkages were identified by MS on the basis of their [M–H] ions being 4 Da less than those of the B-type PACs. PAC trimers with one A/B-type linkage were identified by MS since their [M–H] ions were 2 Da less than those of the B-type PACs and 2 Da more than those of the A-type PACs.[31] According to scheme 1, some rules can be applied to aid in sequencing of PAC dimers. The main fragmentation mechanisms can be rationalized by RDA, quinine methide (QM), and the HRF mechanisms.[31] Moreover, the monomeric building blocks afzelechin, catechin and gallocatechin can be detected at m/z 273, m/z 289 and m/z 305, respectively. Since mass spectrometry cannot distinguish between stereoisomers like (+)/( )-afzelechin/ epiafzelechin, (+)/( )-catechin/epicatechin or (+)/( )-gallocatechin/ epigallocatechin, the names (E)afzelechin, (E)catechin and (E) gallochatechin for all isomers are used. Two anthocyanins were detected in M. argun nuts which were previously detected from strawberries and blackberries.[33] A peak with a retention time of 26.30 min showed a molecular ion at m/z 433 and a fragment ion at m/z 271. The MS data indicated that it was a glucoside or galactoside of pelargonidin. The other
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 306–315
Analysis of PACs in M. argun nut by LC–ESI–MS/MS
(A)
(B)
(C)
(D)
(E)
Figure 2. Typical deprotonated ESI–IT–MS fingerprint of methanolic extract of the MA fraction of Medemia argun nuts obtained in negative ion mode: (A) mass scan in the range of m/z 150–2000, (B) mass scan in the range of m/z 500–660, (C) mass scan in the range of m/z 800–920, (D) mass scan in the range of m/z 1080–1200 and (E) mass scan in the range of m/z 1360–1480.
peak had a molecular ion at m/z 449 and a fragment ion at m/z 287 and was supposed as cyanidin-3-O-hexoside.[34] LC–ESI–MS analysis of PAC dimers
J. Mass Spectrom. 2014, 49, 306–315
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
309
The PAC dimers with A-type (m/z 543, m/z 559, m/z 575, m/z 591 and m/z 607) and B-type (m/z 545, m/z 561, m/z 577, m/z 593 and m/z 609) linkages were readily identified by their [M–H] ions (Table 2). To describe the structure of dimers, we define the
two flavan-3-ol units in the dimers as the top unit and the base unit, and we used the general fission rules illustrated in scheme 1. The product ion spectrum of the deprotonated dimer at m/z 543 produced the base peak at m/z 407 [M–136–H], formed by RDAF fragmentation and loss of the 1,3E0 (C8H8O2) fragment, which indicated that the E ring had a single hydroxyl group at the 4 position (Fig. 2B). The RDA reaction could take place on the base unit of this dimer. The ion at m/z 417 derived from the ion at m/z 543 (Scheme 1), due to the HRF fragmentation and
A. I. Hamed et al.
Scheme 1. The main fragmentation pathways of monomers shown in negative ion mode (RDA = retro-Diels–Alder fission, QM = quinine methide cleavage, and HRF = heterocyclic ring fission, BFF = benzene furan fission).
Table 1. Neutral losses of small molecules from proanthocyanidin [M–18] [M–26] [M–28] [M–36] [M–42] [M–46] [M–54] [M–74] [M–102] [M–112]
[M–H2O] [M–HC ≡ CH] [M–CO] [M–2 x H2O] [M–HC ≡ C–OH] [M–CO–H2O] [M–HC ≡ CHCO] [M–2CO–H2O] [M–H2O–2(HC ≡ C–OH)] [M–CO–2(HC ≡ COH)]
310
the loss of the A ring ([M–126–H]). This fragment indicated that the A ring had a 1,3,5-trihydroxybenzene structure. HRF yielded the product ion at m/z 269, due to the loss of 148 Da from the product ion at m/z 417. Identification of this dimer was confirmed by the fragment ion [M–271–H] at m/z 272 and the ion at m/z 269 [M–274–H]. This ion originated from the quasi-molecular at m/z 543 after quinone methide (QM) cleavage of the interflavan bond. Hence, both the top and base units of this dimer could be identified as (E)afzelechin. The occurrence of HRF fission on the top unit and formation of ions at m/z 272, 271 and 269 through QM cleavage indicated the monomers were linked together through C–C and ether linkages. Therefore, this compound was identified as a doubly linked PAC dimer. Thus, this
wileyonlinelibrary.com/journal/jms
dimer has an A-type structure, and the connection sequence of it has been deduced to be (E)afzelechin–A–(E)afzelechin (1). The [M–H] ion at m/z 545, 2 mass units higher than the corresponding ion of compound 1, potentially indicated a B-type structure. This product ion produced the base peak at m/z 419 [M–126–H], which again derived from the HRF fragmentation and the loss of A ring. HRF also yielded the ion at m/z 271 [M–419–148–H], due to the fission of 2,3 double bond and the loss of the B ring (C9H8O2), indicating a single hydroxyl group located at the 4 position of the B ring (Fig. 3C and Scheme 1). RDA produced the ion at m/z 271 [M–136–H] due to the loss of C8H8O2, followed by elimination of 138 mass units. The ion at m/z 391 [M–170–H] resulted from RDA of the heterocyclic rings and loss of water. A minor ion at m/z 438 derived from the ion at m/z 545 due to neutral loss of water (18 Da) and phenol (94 Da) (Scheme 1). The RDA reaction can take place on either the top or the base unit of the dimer. However, elimination of ring B of the base unit gives rise to an ion with a smaller π–π conjugated system than RDA of the top unit. The ion at m/z 391 [M–18–136–H] derived from the ion at m/z 545 by neutral loss of water molecule (18 Da) and RDA fission, confirming the occurrence of a single hydroxyl group located at the 4 position of the B ring. RDA reaction yielded the ion at m/z 273 [M–136– 136–H] due to the loss of two molecules of C8H8O2 from both units of the dimer. Also, this ion product derived from QM cleavage. Identification of this dimer was confirmed by the fragment ion at m/z 273 [M–271–H]. This ion was formed from the product ion spectrum of the deprotonated molecule at m/z 545 after QM cleavage of the interflavan bond. Hence, the top and base units
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 306–315
Analysis of PACs in M. argun nut by LC–ESI–MS/MS Table 2. Negative fragment ions (m/z) of proanthocyanidin dimers from argun nut Compound (tR) 1 (10.11) 2 (21.11) 3 (12.47) 4 (12.06) 5 (11.96) 6 (16.93) 7 (13.21) 8 (14.81) 9 (15.90) 10 (13.60) 11 (7.35) 12 (15.26) 13 (15.26) 14 (6.58) 15 (7.40) 16 (16.28) 17 (8.67)
Subunit sequence
[M–H]
HRF
RDA–t
RDA–b
QM–t
QM–b
(E)Afz–A–(E)Afz (E)Afz–(E)Afz (E)Afz–A–(E)Cat (E)Cat–A–(E)Afz (E)Afz–(E)Cat (E)Cat–(E)Afz (E)Afz–A–(E)Gal (E)Gal–A–(E)Afz (E)Cat–A–(E)Cat (E)Afz–(E)Gal (E)Cat–(E)Cat (E)Gal–A–(E)Cat (E)Cat–A–(E)Gal (E)Cat–(E)Gal (E)Gal–(E)Cat (E)Gal–A–(E)Gal (E)Gal–(E)Gal
543 545 559 559 561 561 575 575 575 577 577 591 591 593 593 607 609
417 419 433 417 435 419 449 417 433 451 435 433 449 451 435 449 451
– 409 – – 425 409 – – – 441 425 – – 441 425 – 441
407 409 407 423 407 425 407 439 423 409 425 439 423 407 441 439 441
269 271 269 285 273 287 269 301 285 271 287 301 285 289 303 301 303
273 273 289 273 289 273 305 273 289 305 289 289 305 305 289 305 305
Afz, Cat and Gal are abbreviation for afzlechin, catechin and gallocatechin; the symbol (E) indicates that there are two possibilities: ‘afzelechin or epiafzelechin; catechin or epicatechin; gallocatechin or epigallocatechin’ A = A-type; B = B-type; A/B = A/B- type, tR = retention time.
J. Mass Spectrom. 2014, 49, 306–315
The ion at m/z 451 derived from the ion at m/z 609 after a 158-Da neutral loss [M–140–H2O–H] (Scheme 1) due to elimination of C7H8O3 (140 Da), suggesting that ring B contained three hydroxyl groups. Elimination of 162 Da from the ion at m/z 451 indicated that the top unit of the dimer was (E)gallocatechin. The presence of the ions at m/z 303 [M–306–H] and 289 [M–302–H2O–H] suggested this dimer as (E)gallocatechin–(E) gallocatechin (17) (Fig. 3E). LC–ESI–MS analysis of PAC trimers Mass spectra also exhibited precursor ions corresponding to PAC trimers in the range 813–913 amu for B-type and A/B-type (Table 3 and Figs. 1D and 2C). To describe the structure of trimers, we define the three flavan-3ol units as the top unit, the middle unit and the base unit. Twenty-six trimers could be identified by selecting their [M–H] pseudomolecular ions (Scheme 1, Table 3 and Figs. 3F–H). For the sake of simplicity, we have selected only one A-type and A/B-type for the discussion. The pseudomolecular ion [M–H] at m/z 847 produced the base peak at m/z 711 [M–152–H] due to the RDA fragmentation and loss of the 1,3B group (Scheme 1). The RDA rearrangement was found to prevail especially on the C-ring of catechin and epicatechin derivatives, in which two possible fragment ions are formed by loss of 136 Da of a B-ring, indicating that B-ring contained a single hydroxyl group.[35,36] RDA yielded the product ion at m/z 575 [M–136–136–H] due to the loss of C7H4O3, probably by elimination of 3,4 double bond and A-ring. The product ion at m/z 722 derived by elimination of the A-ring (126 Da) from the top unit of the trimer by HRF fragmentation of the ion peak at m/z 847, indicating that the A ring of the top unit had a 1,3,5-trihydroxybenzene structure (Fig. 3F). HRF yielded the product ion at m/z 575 [M–126–147–H] due to the loss of C9H8O2, probably by elimination of the 3,4 double bond and B-ring from the ion peak at m/z 722. Formation of the product ion at m/z 575 also indicated that there was one hydroxyl group located at C4 of the B ring. Hence, the top unit was afzelechin which was confirmed by the presence of the ion peak at m/z 575 [M–273–H] (QM).
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
311
of this dimer were identified as (E)afzelechin. The mass spectra resulting from MS/MS experiment of the ion at m/z 545 in the negative ion mode showed product ions at m/z 283 [M–H–262] (HRF + RDA), 273 (QM) and 271 (QM), typical of B-type PAC dimer. Therefore, this compound was identified as a singly linked PAC dimer corresponding to (E)afzelechin–(E)afzelechin (2) (Fig. 4). The [M–H] ion at m/z 559 and the ion at m/z 433 formed by the neutral loss of 126 mass units as a result of HRF. As before, the loss of 126 Da indicates 1,3,5-trihydroxybenzene structure for the A ring. As shown in Table 2, CID spectrum of 3 at m/z 559 gave fragment ions at m/z 407 [M–152–H] and 389 [M–170–H] from RDAF of the heterocyclic rings. This cleavage points out that the E ring contains two hydroxyl groups located at 3 and 4 positions. The mass spectra resulting from MS/MS experiment of the ion at m/z 559 in the negative ion mode showed product ions at m/z 423 [M–H–136] (RDAC) after QM1 and 271 [M–152–H] (QM2), typical for A-type PAC dimer. Fragment ions at m/z 287 [M–272–H], 271 [M–287–H] and 269 [M–271–H2O–H] indicated afzelechin as the top unit and catechin as the base unit. Therefore, the structure of the dimer is (E) afzelechin–A–(E)catechin (3) (Fig. 4). Another peak with the same [M–H] ion at m/z 559 but with different retention time and fragmentation profile has also been recorded (Table 2). In this case, the structure has been deduced as (E)catechin–A–(E)afzelechin (4). Figure 3D shows a base ion peak at m/z 481 derived from the peak at m/z 607 after elimination of ring A (126 amu) by HRF reaction. Another ion peak at m/z 463 derived from the subsequent loss of a water molecule from the F ring. BFF fission generated an ion peak at m/z 471 [M–137–H] (Scheme 1). Hence, the base unit of this dimer was identified as gallocatechin. Another minor peak at m/z 405 [M–168–32–H] was due to RDA reaction of ring F after elimination of 32 Da from the deprotonated ion peak at m/z 607. Loss of 168 Da indicated that ring F had three hydroxyl groups located at 3, 4 and 5. QM cleavage gave ion peaks at m/z 301 [M–306–H] and 287 [M–302–H2O–H], indicating this dimer as a doubly linked PAC dimer made up of two gallocatechin units. Therefore, this compound has an A-type structure and has been deduced as (E)gallocatechin–A–(E)gallocatechin (16).
A. I. Hamed et al. Table 3. Negative fragment ions (m/z) of proanthocyanidin trimmers and tetramers from argun nut Compound (tR)
Subunit sequence
[M–H]
18 19 (18.33) 20 (11.27)
(E)Afz–(E)Afz–(E)Afz (E)Afz–(E)Afz–(E)Afz (E)Afz–(E)Afz–(E)Afz
813 815 817
21 22 (10.70)
(E)Afz–(E)Afz(E)Cat (E)Cat–(E)Afz–(E)Afz
829 831
23 (12.28) 24 25 (11.69)
(E)Afz–(E)Afz–(E)Cat (E)Afz–(E)–Cat–(E)Cat (E)Afz–(E)Cat–(E)Cat
833 845 847
26 (10.50) 27 (10.67) 28 (12.21) 29 (9.87)
(E)Afz–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat
849 861 863 865
30 (13.92) 31 (8.64) 32 (15.07 33 (16.00) 34(16.00) 35 (16.00) 36 37 (8.48) 38 (9.29) 39(8.17) 40 (9.66) 41 42 (12.10) 43 (12.22) 44 (7.73) 45 (7.90) 46 (7.99) 47 (15.72) 48 (7.55) 49 (8.09) 50 (12.70)
(E)Cat–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Cat–(E)Cat (E)Gal–(E)Gal–(E)Cat (E)Gal–(E)Gal–(E)Cat (E)Gal–(E)Gal–(E)Cat (E)Gal–(E)Gal–(E)Gal (E)Gal–(E)Gal–(E)Gal (E)Gal–(E)Gal–(E)Gal (E)Afz–(E)Afz–(E)Afz–(E)Cat (E)Afz–(E)Afz–(E)Afz–(E)Cat (E)Afz–(E)Afz–(E)Cat–(E)Cat (E)Afz–(E)Afz–(E)Cat–(E)Cat (E)Afz–(E)Cat–(E)Cat–(E)Cat (E)Afz–(E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat–(E)Cat (E)Cat–(E)Cat–(E)Cat–(E)Cat
865 877 879 881 881 881 893 895 897 909 911 913 1103 1105 1119 1121 1135 1137 1149 1151 1153
51 (7.86) 52 (7.86) 53 (7.70) 54 (7.60) 55 (7.73)
(E)Cat–(E)Cat–(E)Cat–(E)Gal (E)Cat–(E)Cat–(E)Cat–(E)Gal (E)Cat–(E)Cat–(E)Gal–(E)Gal (E)Cat–(E)Cat–(E)Gal–(E)Gal (E)Cat–(E)Cat–(E)Gal–(E)Gal
1165 1167 1181 1183 1199
Major fragments (m/z) N.D. 797, 783, 753, 735, 679, 543, 529, 343, 271 799, 775, 755, 706, 665, 637, 578, 555, 545, 529, 419, 407, 365, 271 N.D. 813, 769, 695, 679, 633, 551, 532, 523, 460, 419, 411, 405, 335, 711, 704, 561, 477, 449, 435, 417, 407, 289 N.D. 829, 785, 722, 711, 693, 645, 575, 559, 547, 439, 362, 351, 295, 289, 285, 265 849, 723, 653, 589, 561, 433, 415, 289, 273 553, 435, 425, 407, 735, 712, 695, 575, 451, 407, 289 847, 821, 755, 739, 712, 647, 627, 617, 577, 449, 404, 381, 327, 287 847, 803, 713, 561, 543, 449, 423, 289, 269 467, 451, 425, 407, 381, 309, 289, 245 861, 773, 727, 714, 665, 617, 576, 529, 443, 409, 285 853, 801, 771, 743, 609, 513, 451, 305 853, 801, 771, 743, 592, 513, 429, 305 853, 801, 771, 743, 577, 513, 451, 305 N.D. 877, 863, 815, 756, 607, 599, 471, 413, 303 879, 817, 745, 647, 607, 536, 445, 381, 285 787, 607, 475, 435, 407 869, 851, 743, 703, 607, 559, 481, 323, 303 N.D. 1042, 997, 947, 833, 559, 545, 425, 407, 289, 273 846, 577, 434, 425, 407, 289 1105, 1055, 833, 561, 543, 289 1008, 865, 729, 577, 407, 289 985, 849, 713, 559, 407, 289 1105, 1042, 1013, 863, 711, 575, 433, 425, 289 865, 701, 577, 451, 425, 407, 289, 245 1099, 1065, 1043, 983, 947, 905, 865, 863, 711, 701, 693, 651, 605, 575, 574, 549, 425, 407, 395 N.D. 999, 879, 865, 713, 577, 452, 425, 407, 289 1074, 996, 879, 741, 729, 577, 425, 407 879, 742, 719, 577, 451, 425, 289 1137, 910, 727, 608, 577, 451, 425, 289
Type A g A/B B A A/B B A A/B B A A/B B B A A/B B B B A A/B B A A/B B A/B B A/B B A/B B A/B A/B B A/B A/B A/B A/B A/B
Afz, Cat and Gal are abbreviation for afzlechin, catechin and gallocatechin; the symbol (E) indicates that there are two possibilities: ‘afzelechin or epiafzelechin; catechin or epicatechin; gallocatechin or epigallocatechin’ A = A-type; B = B-type; A/B = A/B- type, tR = retention time.
312
On the other hand, the presence of the ion peak at m/z 575 indicated that the middle and base units had A-type linkage. Another product ion was observed at m/z 439 [M–575–136–H] due to the loss of C7H4O3 and at m/z 285 ([M–288–H], resulting from the cleavage of the ion peak at m/z 439 through the QM mechanism. It seems that the A-type interflavan bonds do not undergo observable QM cleavage in the presence of a B-type interflavan bond in the same molecule. The fragmentation profile of the ion peak at m/z 575 indicated that the middle and base units were (E)catechin. Thus, the structure of this trimer has been deduced as (E)afzlechin–(E)catechin–A–(E)catechin (25) (Fig. 4).
wileyonlinelibrary.com/journal/jms
The pseudomolecular ion [M–H] at m/z 849 produced the base peak at m/z 723 [M–126–H] which was due to the RDA fragmentation and loss of the A-ring (trihydroxybenzene) (Scheme 1). Loss of the 2, 3, 4 E group of 162 Da by HRF produced the ion peak at m/z 561, indicating that the B ring of the top unit had a 3,4-dihydroxybenzene structure, corresponding to a catechin derivative (Fig. 3G). RDA of the ion peak at m/z 561 yielded the product ion at m/z 433 [M–561–126– 162–126–H] due to the loss of a trihydroxybenzene, probably by elimination of D-ring from the middle unit. Loss of 160 Da by HRF of the ion peak at m/z 433 produced the ion peak at
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 306–315
Analysis of PACs in M. argun nut by LC–ESI–MS/MS
2
Figure 3. Product ion spectra of argun nut components ([M–H] ): MS of monomer at m/z 289 (A), dimers at m/z 543 (B), 545 (C), 607 (D) and at m/z 609 (E), trimmers at m/z 847 (F), 849 (G) and at m/z 881 (H), and tetramer at m/z 1153 (I).
J. Mass Spectrom. 2014, 49, 306–315
LC–ESI–MS analysis of PAC tetramers The analysis of mass spectra also evidenced the presence of precursor ions corresponding to tetramers in the m/z range 1089–1199 (Table 3 and Figs. 1E and 2D). The obtained m/z values evidenced the presence of PACs belonging to B-type and A/B-type and the absence of A-type series. The pseudomolecular ion [M–H] at m/z 1153 [288 × 3 + 289–H] and UV spectra are similar with those of (E)catechin. As shown in Fig. 3I and Table 3, the fragmentation spectrum of compound 50 at m/z 1153 gave fragment ions at m/z 865 [M–288–H] due to the quinine methide reaction (QM1) of the top unit, m/z 701 [M–288–164–H] due to HRF reaction of the interflavone unite ([M–288–C9H8O3–H]), m/z 575 ([M–288–C9H8O3–126–H] due to the loss of a trihydroxybenzene, probably by elimination of D-ring from the middle unit. Also, product ion at m/z 575 [M–289 × 2H] due to the quinine methide reaction (QM1, QM4) of the top and lower units. The fragment ion at m/z 449 [M–575–126–H] due to loss of another trihydroxybenzene, probably by elimination of D-ring or G-ring from one of the two middle units. The presence of product ion at
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
313
m/z 273 indicating that the middle unit was a catechin derivative and the base unit was an afzelechin derivative. Another product ion was observed at m/z 561 [M–288–H] due to the loss of a catechin unit, confirming the type of the top monomer. Two base peaks at m/z 289 and m/z 273 due to the quinine methide reaction (QM) of the ion peak at m/z 561 confirmed that the middle unit was a catechin derivative and the base unit was afzelechin. Thus, for this trimer the structure (E)catechin–(E)catechin–(E)afzlechin (26) (Fig. 4) is proposed. Figure 3H shows that the unique chromatographic peak at m/ z 881 produced three peaks at m/z 609 ([M–272–H]), m/z 592 ([M–289–H]) and m/z 577 ([M–304–H]), due to the QM1 mechanism (Table 2). Moreover, only one peak raised from QM2 at m/z 305, indicating that the chromatogram contained three trimers. Two of them had similar monomers, but their linkages were alternate ([M–272–304–H]) and ([M–304–272–H]), and the last one had different monomers ([M–289–287–H]). Their structures were deduced as (E)afzlechin–(E)gallocatechin– (E)gallocatechin (34), (E)gallocatechin–(E)afzlechin–(E)catechin (35) and (E)catechin–(E)catechin–(E)gallocatechin (36), respectively (Fig. 4).
A. I. Hamed et al.
Figure 4. Structure of the monomers, dimers and trimers proanthocyanidins in Medemia argun nuts.
m/z 425 [M–575–C6H6O3–H2O–H] is due to the BFF mechanism from the lower unit of the two intermediate flavone units (Scheme 1). Its structure was (E)catechin–(E)catechin–(E) catechin (50). The analysis of mass spectra also evidenced the presence of precursor ions corresponding to pentamers in the m/z range 1377–1454 (Fig. 2E). The obtained m/z values evidenced the presence of PACs belonging to B-type and A/B-type and the absence of A-type series. However, no single pentameric and hexameric signals were observed, but a group of signals with a mass difference of 16 amu.
quinonemethide (QM) cleavage of the interflavan bond. The rationalization of oligomers can be of interest for plant physiologists and can be crucial for nutritionists who are interested in studying their bioavailability and possible human health effects. The discovery of PACs with well-known antioxidant properties can explain the good conservation of argun nuts during the time and may bring us closer to answering about potential use of this fruits in the ancient time. The data obtained in our research show that M. argun is a novel rich source of hydrolyzable PACs made up of afzelechin, catechin and gallocatechin units.
Conflict of Interest Conclusion
314
The analytical procedure described in this work provides a valuable profile of PACs occurring in M. argun nuts and represents a rapid, simple and sensitive method to rationalize PACs in plants and derived preparations. In particular, this strategy based on HPLC coupled to electrospray ionization mass spectrometry (HPLC–ESI–MS) in the negative ion mode allows to achieve deep structural information on the nature of the flavane monomers which make up proantocyanidins, their sequence and the type of flavane bond. In particular, diagnostic ions resulting from HRF and RDA reaction of flavan-3-ol provide information on the hydroxylation pattern and the type of interflavan bond. The flavane sequences can be ascertained through ions derived from
wileyonlinelibrary.com/journal/jms
The present work was supported by the European Project Proficiency (FP7-REGPOT-2009-1) no. 245751. Acknowledgements We are thankful to the European Project Proficiency (FP7-REGPOT2009-1) no. 245751.
References [1] C.S. Kunth. Recherches sur les plantes trouvées dans les tombeaux égyptiens par M. Passalacqua. Annales des Sciences Naturelles. Botanique (Paris), 1826, 8, 418. [2] H. Wendland. Beitrage zu den Borassinen. Botanische Zeitung 1881, 39, 89.
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 306–315
Analysis of PACs in M. argun nut by LC–ESI–MS/MS [3] V. Täckholm, M. Drar. Flora of Egypt 2. Bull. Fac. Sci. (Egypt University) 1950, 28, 296. [4] C. Newton. Le palmier Argoum, Medemia argun (Mart.), Württemb. ex Wendl. P., Encyclopedie Religieuse de l’Univers Vegetal. Croyances phytoreligieuses de l’Egypte ancienne (ERUV) II. OrMonsp 2001, XI, 141. [5] S. Pain. Fruits of the tomb. New Sci. 2006, 190, 554. [6] V. Täckholm. Students’ Flora of Egypt, 2nd ed. Cairo University: Egypt, 1974, 888. [7] V. Loret. La Flore pharaonique d’apres les documents hieroglyphiques et les speciments decouverts dans le tombes, 2nd ed. J. B. Bailliere and sons: Paris, 1887. [8] A. R. David, E. V. Tapp. The Mummy’s Tale, The Scientific and Medical Investigation of Natsif-Amun, Priest in the Temple of Karnak. Michael O’Mara Books Ltd., (V. ), London, 1972, 176. [9] H. Ibrahim, W. J. Baker. Medemia argun – past, present and future. Palms 2009, 53, 9. [10] C. F. P. Von Martius. Historia Naturalis Palmarum. Leipz. (Germany) 1823-1850, 3 Bände, 790. [11] M. Gibbons, T. W. Spanner. Medemia argun lives. Principl. 1996, 40, 65. [12] L. Boulos, The discovery of Medemia argun palm in the Nubian Desert of Egypt. Bot. Notebook 1968, 121, 117. [13] J. Porter. In methods in plant Biochemistry: 1- Plant phenolics, Tannins, P. M. Dey, J. B. Harborne (Eds). Academic Press Inc.: San Diego, 1989. [14] G. Nonaka, F. L. Hasu, I. Nishioka. Structures of dimeric, trimeric, and tetrameric procyanidins from Areca catechu L. J. Chem. Soc. Chem. Commun. 1981, 15, 781. [15] C. K. Wang, W. H. Lee. Separation, characteristics, and biological activities of phenolics in areca fruit. J. Agric. Food Chem. 1996, 44, 2014. [16] A. Nunez-de la Mora, R. T. Chatterton, E. T. Mateo, F. Jesmin, G. R. Bentley. Effect of chewing betel nut on measurements of salivary progestrone and estradiol. Americ. J. Physic. Anthrop. 2007, 132, 311. [17] Q. Wu, Y. Yang, J. E. Simon. Qualitative and Quantitative HPLC/MS Determination of Proanthocyanidins in Areca Nut (Areca catechu). Chem. Biodiver. 2007, 4, 2817. [18] R. B. Rodrigues, R. Lichtenthaler, B. F. Zimmermann. Total oxidant scavenging capacity of Euterpe oleracea Mart. (Acai) seeds and identificationof their polyphenolic compounds. J. Agric. Food Chem. 2006, 54, 4126. [19] L. A. Paacheco-Palencia, S. Mertens-Talcott, S. T. Talcott. Chemical composition, antioxidant properties, and thermal stability of a phytochemical enriched oil from Acai (Euterpe oleracea Mart.). J. Agric. Food Chem. 2008, 56, 4631. [20] G. D. Noratto, G. Angel-Morales, S. T. Talcott, S. U. Mertens-Talcott. Polyphenolics from Acai (Euterpe oleracea Mart.) and red muscadine grape (Vitis rotundifolia) protect human umbilical vascular endothelial cells (HUVEC) from glucose- and lipopolysacharide (LPS)- induced inflammation and target microRNA-126. J. Agric. Food Chem. 2011, 59, 7999. [21] C. M. Rodrigues, D. Rinaldo, L. C. Dos Santos, P. Montoro, S. Piacente, C. Pizza, C. A. Hiruma-Lima, A. R. M. Souza Brito, W. Vilega. Metabolic fingerprinting using direct flow injection electrospray ionization tandem mass spectrometry for the characterization of proanthocyanidins
[22]
[23] [24] [25] [26] [27]
[28] [29]
[30] [31] [32]
[33]
[34]
[35]
[36]
from the barks of Hancornia speciosa. Rapid Commun. Mass Spectr. 2007, 21, 1907. M. Maldini, P. Montora, A. I. Hamed, U. A. Mahalel, W. Oleszek, S. Piacente. Strong antioxidant phenolics from Acacia nilotica: profiling by ESI-MS and quali-quantitative determination by LC-ESI-MS. J. Pharm. Biomed. Anal. 2011, 56, 228. V. Cheynier, T. Doco, H. Fulcrand, S. Guyot, E. Le Roux, J. M. Souquet, J. Rigaud, M. Moutounet. ESI-MS analysis of polyphenolic oligomers and polymers. Anal. 1997, 25, 32. L. Poblocka-Olech, M. Krauze-Baranowska. SPE-HPTLC of procyanidins from the barks of different species and clones of Salix. J. Pharm. Biomedical Anal. 2008, 48, 965. J. Valls, S. Milan, M. P. Marti, E. Borras, L. Areola. Advanced separation methods of food anthocyanins, isoflavones and flavanols. J. Chromatogr. A 2009, 1216, 7143. A. I. Hamed, M. Leonardi, A. Stochmal, W. Oleszek, L. Pistelli. Flavone and flavonol glycosides from Astragalus eremophilus and Astragalus vogelii. Nat. Prod. Commun. 2012, 7, 633. A. Morel, A. I. Hamed, W. Oleszek, A. Stochmal, R. Glowacki, B. Olas. Protective action of proanthocyanidin fraction from Medemia argun against Oxidative/nitrative damages of blood platelet and plasma components. Platelet 2014, 25(1), 75. R. B. Broadhurst, W. T. Jones. Analysis of condensed tannins using acidified vanillin. J. Sci. Food Agric. 1978, 29, 788. A. Mari, D. Eletto, C. Pizza, P. Montoro, S. Piacente. Integrated mass spectrometry approach to profile proanthocyanidins occurring in food supplements: Analysis of Potentilla erecta L. rhizomes. Food Chem. 2013, 141(4), 4171. Y. Hayasaka, E. Waters, V. Cheynier, M. J. Herderich, S. Vidal. Characterization of proanthocyanidins in grape seeds using electrospray mass spectrometry. Rapid Commun. Mass Spectr. 2003, 17, 9. H. J. Li, M. L. Deinzer. Tandem mass spectrometry for sequencing proanthocyanidins. Anal. Chem. 2007, 79, 1739. L. Gu, A. M. Kelm, F. J. Hammerstone, G. Beecher, J. Holden, D. Hytowitz, L. R. Prior. Screening of foods containing proanthocyanidins and their structural characterization using LC-MS/MS and thiolytic degradation. J. Agric. Food Chem. 2003, 51, 7513. X. Wu, G. Beecher, J. M. Holden, D. B. Haytowitz, S. E. Gebhardt, R. L. Prior. Concentration of anthocyanins in common foods in the united states and estimation of normal consumption. J. Agric. Food Chem. 2006, 54, 4069. W. Mullen, C. A. Edwards, M. Serafini, A. Crozier. Bioavailability of pelargonidin-3-O-glucoside and its metabolites in humans following the ingestion of strawberries with and without cream. J. Agric. Food Chem. 2008, 56, 713. S. de Pascual-Teresa, J. C. Rivas-Gonzalo. Application of LC-MS for the identification of polyphenols. In Methods in Polyphenol Analysis, C. Santos-Buelga, G. Williamson (Eds). Royal Soci. Chem.: Cambridge, U.K., 2003, 48. S. Guyot, J. Vercauteren, V. Cheynier. Structure determination of colourless and yellow dimers resulting from (+)-catechin coupling catalyzed by grape polyphenoloxidase. Phytochem. 1996, 42, 5.
315
J. Mass Spectrom. 2014, 49, 306–315
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
