Phytochemistry 73 (2012) 95–105 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Analysis of commercial proanthocyanidins. Part 1: The chemical composition of quebracho (Schinopsis lorentzii and Schinopsis balansae) heartwood extract Pieter B. Venter, Mirek Sisa, Marthinus J. van der Merwe, Susan L. Bonnet, Jan H. van der Westhuizen ⇑ Department of Chemistry, University of the Free State, Nelson Mandela Avenue, Bloemfontein 9301, South Africa a r t i c l e i n f o Article history: Received 4 April 2011 Received in revised form 23 June 2011 Available online 5 November 2011 Keywords: Schinopsis lorentzii and Schinopsis balansae Anacardiaceae Quebracho Electrospray mass spectrometry Proanthocyanidins Natural polymer a b s t r a c t Quebracho (Schinopsis lorentzii and Schinopsis balansae) extract is an important source of natural polymers for leather tanning and adhesive manufacturing. We combined established phyto- and synthetic chemistry perspectives with electrospray mass spectrometry experiments to prove that quebracho proanthocyanidin polymers consist of an homologous series of flavan-3-ol based oligomers. The starter unit is always catechin which is angularly bonded to fisetinidol extender units. By comparison of the MS2 fragmentation spectra of the oligomer with product ion scans of authentic catechin and robinetinidol samples, we proved that quebracho extract contains no robinetinidol, as is often reported. Quebracho proanthocyanidins have acid resistant interflavanyl bonds, due to the absence of 5-OH groups in fisetinidol, and the aDP cannot be determined via conventional thiolysis and phloroglucinolysis. We used the MS data to estimate a conservative (minimum value) aDP of 3.1. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction The wild quebracho forests in the Gran Chaco region of Argentina, Bolivia, and Paraguay have been harvested for more than 100 years as an important source of vegetable tannins and timber. The timber is durable and extremely hard and the name quebracho is derived from the Spanish word quiebrahacha which means ‘‘axebreaker’’. To obtain a warm water soluble quebracho extract, the heartwood is stripped of its bark, chipped, and extracted with boiling water. A cold water soluble extract (sulfited extract) is obtained upon treatment of the warm water soluble extract with bisulfite or direct extraction of wood chips with a boiling aqueous bisulfite solution. Higher extraction rates are obtained with boiling aqueous bisulfite solution than with boiling water alone. Quebracho extract is obtained from Schinopsis balansae (red ‘‘chaqueno’’ quebracho, pure tannin content 20–21%) from the Eastern Chaco region and Schinopsis lorentzii (red ‘‘santiagueno’’ quebracho, pure tannin content 15–18%) from the Western Chaco region. These two species were previously referred to as Quebracho colorado chaqueño and Quebracho colorado santiagueño (Schinopsis quebracho-colorado) and belongs to the family Anacardiaceae. A third tree species, Aspidosperma quebracho-blanco of the family Apocynaceae, is commonly referred to as white quebracho. ⇑ Corresponding author. Tel.: +27 51 4012782; fax: +27 51 4448463. E-mail address: vdwestjh@ufs.ac.za (J.H. van der Westhuizen). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.10.006 Quebracho extract consists of about 95% proanthocyanidins (PAs) and 5% water soluble sugars on a dry basis. The term proanthocyanidin (PA) refers to the characteristic development of a red color upon heating PAs with dilute acid (Roux, 1992). PAs are also referred to as condensed tannins to distinguish them from hydrolysable tannins which do not produce a red color when heated with aqueous acid. Hydrolysable tannin oligomers are esters of gallic acid and D-glucose. Important industrial sources of PAs are mimosa bark extract (Acacia mearnsii) and quebracho heartwood extract, and of hydrolysable tannins, tara pods, chestnut bark, and oak gall extracts. Progress in defining quebracho PA composition has been slow, mainly due to the complexity of the extracts and the difficulty of isolating pure PAs with silica gel based chromatography materials. Uncertainties include different hydroxylation patterns of the constituent flavan-3-ol aromatic rings, different configurations at the C-2, C-3 and C-4 stereogenic centers, the possibility of a second ether interflavanyl bond (A-type PAs), the average chain length (degree of polymerization), and the presence of angular oligomers. Progress is further hampered by the absence of 5-OH groups in the constituent monomers, which imparts stability to the interflavanyl bond against acid hydrolysis (Roux and Paulus, 1962; Roux et al., 1975). This renders the classical method to analyse PAs via acid hydrolysis of the interflavanyl bond and subsequent trapping of intermediates with toluene-a-thiol or phloroglucinol (thiolysis and phloroglucinolysis) (Thompson et al., 1972; Foo and Porter, 1978; Kennedy and Taylor, 2003; Rigaud et al., 1991) and analysis 96 P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 Fig. 1. Flavan-3-ol and flavan-3,4-diol monomers from the heartwood of S. lorentzii (putative building blocks of quebracho PAs). Fig. 3. Trimer isolated from S. balansae [ent-fisetinidol-(4b ? 8)-catechin-(6 ? 4b)ent-fisetinidol]. Fig. 2. Quebracho dimers from S. balansae. of such trapped intermediates with HPLC (Shen et al., 1986; Koupai-Abyazani et al., 1993; Rigaud et al., 1991; Kennedy and Taylor, 2003), unreliable. Vivas et al. (2004), for example, failed to isolate any known flavan-3-ol toluene-a-thiol adducts upon thioacidolysis of quebracho tannins. Most of the properties and industrial applications of vegetable tannins are attributed to the ability of the constituent PAs or hydrolysable tannins to form complexes with proteins via hydrogen bonds (Haslam, 1974, 1988, 1997). This includes astringency in tea and red wine (interactions between tannins and protein based taste receptors in the mouth) (Bate-Smith, 1954; Hofmann et al., 2006), anti-feeding properties (the indigestibility of tannin–protein complexes) (Hagerman et al., 1992), and growth inhibition of many micro-organisms (irreversible deactivation of enzymes) (Akin, 1982). Complexation of vegetable tannins with hide proteins transform biodegradable raw hide into leather which resists bacterial degradation, has a nice touch and is abrasion, heat, and water resistant (Haslam, 2005). Quebracho is extensively used to produce vegetable tanned leather. It is also used to manufacture adhesives via cross linking of the nucleophilic aromatic A-rings of the constituent PAs with formaldehyde (Pizzi, 1978). It is a source of oenological tannins, used to enhance the ‘‘mouth feel’’ properties of young or poor quality red wines. The absence of the 5-OH group and corresponding stability of the PA oligomer to interflavanyl bond fission (Roux and Paulus, 1962; Roux et al., 1975) is probably an important factor in the industrial application of quebracho and mimosa PAs as it imparts longevity to leather and adhesives manufactured from it. A better understanding of the molecular composition of vegetable tannins will assist industrial applications. The relative affinity for collagen, rate of penetration into hides and skins during commercial tannage, mobility within leather, and desorption from finished leather under moist conditions are determined by oligomer composition (Covington, 2009). The availability of nucleophilic centers for cross linking with formaldehyde on the periphery of oligomers determines curing time and pot life of thermosetting PA based adhesives. Fig. 4. Tetramer synthesized by Viviers and co-workers. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are soft ionization techniques that can fractionate a mixture of oligomers, such as quebracho PA extract, into fractions of different degrees of polymerization (DP) and estimate the average degree of polymerization (aDP). Soybean seed coat extract (Takahata et al., 2001) and hop PAs (Taylor et al., 2003) with a DP of 30 and 22, respectively, have been characterised by MALDI-TOF MS, and litchi PAs with a DP of 22 (Le Roux et al., 1998) with ESI. Mouls and co-workers (2011) compared aDP values obtained from thiolysis of PAs with the aDP values obtained from ESI-MS. They confirmed that poorer ionization of high DP PAs led to the underestimation of the aDP with MS, but concluded that ESI is appropriate to analyse low molecular weight PA samples (aDP below 20). Pasch et al. (2001) investigated commercial sulfited quebracho tannin extract using MALDI-TOF mass spectrometry and observed oligomers to a maximum of decamers (2798 Da) (c.f. octamers for mimosa PAs). This is in line with the aDP of 6.74 (c.f. 4.9 for mimosa PAs) found by Thompson and Pizzi (1995) and Fechtal and Riedl (1993) with NMR methods. The individual PA oligomers consisting of clusters of ions 16 Da apart, was attributed to combinations and permutations of fisetinidol (274 Da) and robinetinidol (290 Da) constituent units. They concluded that quebracho PAs consist mostly of profisetinidins. The same authors claim that quebracho PAs were, in contrast with angular mimosa PAs, linear and that this linear structure explains the relative ease with which quebracho PAs undergo acid catalysed hydrolysis compared to smaller, less viscous oligomers. 97 P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 Table 1 ESI (negative mode and positive mode) ions for hot water soluble quebracho extract. Oligomer m/z Value (negative mode) m/z Value (positive mode) Catechin Fisetinidol Dimer Trimer Tetramer Pentamer Hexamer Heptamer 561 833 1105 1377a (1649)b (1921)c 563 835 1107 1379d 1651e (1923)f 1 1 1 1 1 1 1 2 3 4 5 6 (Ions in brackets were not detected directly but indirectly as water adducts). a The 13C isotope peak at m/z 1378 was automatically annotated in Fig. 5a. The slightly less intensive 12C peak at m/z 1377 is also visible. Water adducts (+18 Da) of these two peaks are visible at m/z 1395 and 1396. b The m/z 1649 value was indirectly detected as a water adduct of the 13C isotope peak at m/z 1668. Close inspection of a magnified spectrum reveals the presence of a 12C water adduct at m/z 1667. c The expected heptamer was not detected in negative mode at m/z 1921 in Fig. 5a. d The m/z 1379 peak is also detected as the 13C isotope peak at m/z 1380, and as their water adducts (+18 Da) at m/z 1397 and 1398, respectively. e The m/z 1651 peak is also detected in Fig. 5c as single and double water adducts at m/z 1669 and 1687, respectively. Magnification of the spectrum also reveals the corresponding 13C isotope peaks at 1652, 1670, and 1688, respectively. f The heptamer is mainly detected as a double water adduct (+36 Da) at m/z 1959 (and the corresponding 13C isotope peak at 1960). NOPQ NP_Pol_100216_23 11 (0.126) Cm (11:14-1:4) 561.1 TOF MS ES374 100 833.1 834.1 % 562.1 552.1 1105.2 835.1 601.1 287.0 238.9 423.0449.1 495.1 602.1 688.1 688.6 603.1 689.1 849.1 831.1 343.0 0 200 1106.2 1107.2 850.1 985.1 869.1 1009.2 1123.2 1395.2 1378.2 1396.3 1668.3 m/z 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 Fig. 5a. Negative mode ESI spectrum of hot water soluble quebracho extracts (m/z 200 to 2000 m/z range). 2. Phytochemistry Roux and Evelyn (1960) found only catechin 1 and ent-fisetinidol-4b-ol [( )-leucofisetinidin] 2 (Fig. 1) as monomeric constituents in the heartwood of S. lorentzii. This suggests that 1 and 2 are the precursors of quebracho PAs. The flavan-3,4-diol 2 is present in high concentrations at the sapwood/heartwood interface and declines rapidly from the heartwood edge and is absent from the center heartwood of mature (120–140 year old) trees. An increase in average molecular weight from 910 in the outer heartwood to 1784 Da in the central heartwood PAs (determined with ebulliometry) suggests that PA oligomer formation continues away from the sapwood after heartwood formation. Viviers and co-workers (1983) isolated the two diastereoisomers ent-fisetinidol-(4b ? 8)-catechin 3 and ent-fisetinidol-(4a ? 8)-catechin 4 (m/z 562) from S. balansae. Smaller quantities of ent-fisetinidol-(4b ? 6)-catechin and ent-fisetinidol-(4a ? 6)-catechin diastereoisomers 5 and 6 were also isolated (Fig. 2). The ratio of 3:4:5:6 approximated 2.5:1:0.6:0.2. The same team also isolated the angular trimer entfisetinidol-(4b ? 8)-catechin-(6 ? 4b)-ent-fisetinidol 7 (4,6;4,8bis-ent-fisetinidol-catechin) (m/z 834) (Fig. 3) and three diastereoisomers from S. balansae. However, no tetramers were reported. 98 P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 NOPQ NP_Pol_100216_11 31 (0.339) Cn (Cen,4, 70.00, Ar); Sm (SG, 1x5.00); Sb (1,40.00 ); Cm (23:31-2:4) 563.1 TOF MS ES+ 2.00e4 100 683.2 835.2 % 564.2 684.2 955.2 836.2 857.2 725.2 411.1 393.1 1107.3 956.2 437.1 545.1 565.2 858.2 1108.3 681.2 287.1 485.1 301.1 1129.3 726.2 585.1 873.2 817.2 801.2 273.1 271.1 231.1 874.2 315.1 1130.3 997.2 998.3 1227.3 1228.3 1229.3 1131.3 1033.3 1692.4 1693.4 1694.4 1669.4 1414.4 1499.4 1270.3 365.1 0 200 1686.4 1397.4 1148.3 1959.5 1965.5 m/z 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 Fig. 5b. Positive mode ESI spectrum of hot water soluble quebracho extracts (m/z 200 to 2000 m/z range). NOPQ NP_Pol_100216_11 31 (0.339) Cn (Cen,4, 70.00, Ar); Sm (SG, 1x5.00); Sb (1,40.00 ); Cm (23:31-2:4) TOF MS ES+ 1.86e3 1686.4 100 1687.5 1397.4 1691.4 1692.4 1414.4 1379.3 1419.3 1959.5 1669.4 1958.5 % 1420.3 1693.4 1964.5 1499.4 1500.4 1421.3 1668.4 1965.5 1651.4 1517.4 1694.4 1518.4 1943.5 1941.5 1300.4 1433.3 1435.3 1445.4 1361.3 1307.3 1329.4 0 1300 1519.4 1979.5 1771.4 1577.4 1481.4 1451.3 1790.5 1709.4 1559.4 1839.5 1711.4 1862.5 1634.4 1924.5 1923.5 1982.5 1864.5 m/z 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 Fig. 5c. Positive mode ESI spectrum (expansion of 5b) of hot water soluble quebracho extracts (m/z 1300 to 2000 m/z range). 3. Synthesis Viviers and co-workers (1983) investigated the biomimetic synthesis of quebracho PAs via acid catalysed condensation of catechin 1 and ent-fisetinidol-4b-ol 2. The products closely resemble those isolated by the same authors. Condensation of 1 eq. of catechin 1 with ent-fisetinidol-4b-ol 2 (1 eq.) gives mainly ent-fisetinidol-(4b ? 8)-catechin 3 and small quantities of the epimeric ent-fisetinidol-(4a ? 8)-catechin 4 (Fig. 2). The presence of a second equivalent of 2 led to formation of the trimer, ent-fisetinidol-(4b ? 8)-catechin-(6 ? 4b)-entfisetinidol 7 (Fig. 3). A further equivalent of 2 leads to the 99 P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 Fig. 6. Structures of rDA fragments of m/z 563, 835 and 1107 dimers, trimers and tetramers. (HOMO) exhibits it’s maximum amplitude (Elliot et al., 1982). The first condensation product (dimer) will thus predominantly be ent-fisetinidol-(4b ? 8)-catechin 3. The C-6 position of the phloroglucinol A-ring of catechin (two enolic OH groups, one enolic ether group) is more nucleophic than the resorcinol A-ring (one enolic OH and one enolic ether group) of the competing ent-fisetinidol unit. The second condensation product (trimer) will thus predominantly be ent-fisetinidol-(4b ? 8)-catechin-(6 ? 4b)-ent-fisetinidol 7. In constructing the tetramers from the trimers (Young et al., 1985), it must be emphasized that both reactive positions of the phloroglucinol A-ring of the catechin moiety are occupied in the trimer. Thus, the resorcinol A-ring of the upper ent-fisetinidol moiety is the most reactive remaining nucleophilic position. The trimer will thus react via the sterically less hindered C-6 position with a third ent-fisetinidol-4b-ol molecule to yield the tetramer Table 2 Diagnostic rDA fragments associated with their corresponding oligomer precursors. a Oligomer ESI+ mass Rel. comp.a RDA mass Rel. comp.a Dimer Trimer Tetramer Pentamer Hexamer Heptamer 563 835 1107 1379 1651 1923 125 79 33 8 3 61 411 683 955 1227 1499 1771 36 110 49 13 4 1.5 Relative composition is based on peak height. formation of the tetramer, ent-fisetinidol-(4b ? 6)-ent-fisetinidol(4b ? 8)-catechin-(6 ? 4b)-ent-fisetinidol 8 (Fig. 4). The most reactive nucleophilic position on catechin 1 is C-8 since at this position the highest occupied molecular orbital -MS2 (561.20): 0.312 to 3.359 min from Sample 1 (TuneSampleID) of MT20110310130141.wiff (Heated Nebulizer) Max. 4366.1 cps. 289.3 100% 95% 90% 85% 80% 75% 70% 65% Rel. Int. (%) 60% 55% 50% 45% 40% 35% 561.3 30% 25% 161.3 409.5 20% 15% 271.3 125.1 10% 137.3 5% 205.0 151.1 113.2 50 100 391.3 245.4 109.0 253.1 286.9 295.0 313.4 189.3 150 200 257.0 250 300 m/z, amu 451.3 328.5 350 400 450 Fig. 7a. Product ion scan of the m/z 561 dimer ion (APCI in the negative mode). 500 550 600 100 P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 -MS2 (833.30): 2.057 to 21.201 min from Sample 1 (TuneSampleID) of MT20110310142843.wiff (Heated Nebulizer) Max. 122.8 cps. 289.5 100% 95% 90% 561.2 833.5 85% 80% 75% 70% 65% 529.3 Rel. Int. (%) 60% 409.4 55% 50% 45% 40% 161.0 391.8 600.4 35% 270.8 30% 680.8 25% 20% 109.1 15% 203.6 154.9 192.2 1 198..8 377.5 267.5 294.8 8 310.9 329.8 359. 92 451.4 41 13.9 510.8 433 3.3 475.5 593.1 1 635.9 663.3 719.1 824.2 2 10% 100 150 200 250 300 350 400 450 500 m/z, amu 550 600 650 700 750 800 Fig. 7b. Product ion scan of the m/z 833 trimer (APCI in the negative mode). ent-fisetinidol-(4b ? 6)-ent-fisetinidol-(4b ? 8)-catechin-(6 ? 4b) -ent-fisetinidol 8 (Fig. 4). Owing to the increased thermodynamic stability of 3,4-trans compared to 3,4-cis isomers (Forest et al., 2004), the isolated and synthesised oligomers possess predominantly, but not exclusively, 3,4-trans configured constituent units. Owing to the fact that mass spectrometry cannot distinguish between diastereoisomers or regioisomers, implies that we will not refer in our further discussion to configuration or position of the interflavanyl link and replace, e.g., the terms ent-fisetinidol-(4b ? 6)-catechin 5 and ent-fisetinidol-(4a ? 6)-catechin 6 with fisetinidol-catechin. Phytochemistry thus suggests that: 1. Quebracho heartwood contains only catechin and ent-fisetinidol-4-ol and no robinetinidol-4-ol. 2. Dimers and trimers consist of a catechin starter unit and one or two fisetinidol extender units. The trimer is angular with one fisetinidol in the ‘‘upper’’ C-8 position and the other in the ‘‘terminal’’ C-6-position. No linear fisetinidol–fisetinidol dimers, fisetinidol–fisetinidol–fisetinidol trimers, or robinetinidol containing dimers and trimers were reported in the literature. Synthetic organic chemistry suggests that: 1. The fisetinidol–catechin–fisetinidol trimer will be the sole intermediate in the construction of all higher oligomers and all higher oligomers will have this moiety attached to one or more additional fisetinidol extender units. 2. The formation of tetramers and higher oligomers are inhibited by the lower reactivity of the 5-deoxy fisetinidol A-ring. We thus expect that dimers and trimers will be the major compo- nents in quebracho (and mimosa) PAs and higher oligomers will be relatively less common. This is not the case with 5-oxy PAs where reactive catechin-4-ol or gallocatechin-4-ol are the extender units and large oligomers (DP of 20 and more) are common (Takahata et al., 2001; Taylor et al., 2003; Le Roux et al., 1998). We thus postulate that quebracho PA oligomers consist of a homologous series of flavan-3-ol based oligomers. The starter unit is always catechin which is angularly bonded to fisetinidol extender units. Herein, we report our results on the composition of a hot water soluble (unsulfited) quebracho extract from S. lorentzii with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) mass spectrometry and use these results to test our hypothesis. When comparing our ESI and APCI data with published MALDI data, it should be taken into account that MALDI-ionization is observed via sodium [M+23]+ or potassium [M+39]+ adducts. The two MALDI m/z values are 16 Da apart and can be misinterpreted in PA mass spectra as evidence for the presence of oligomers with additional OH-groups (Reed et al., 2005). 4. Experimental Spray dried, hot water soluble quebracho extract from S. lorentzii was supplied by Mimosa Extract Company (Pty) Ltd., 24 van Eck Place, Pietermaritzburg, 3201, South Africa. HPLC grade (P99.9% purity) methanol and water were purchased from Merck. The mass spectrometer was a Sciex API 2000 MS/MS system, equipped with an ESI or APCI source and operated in the negative ion mode. The operating conditions in the ESI 101 P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 Scheme 1. Fragmentation of m/z 561 quebracho dimer (M H) . source were as follows: ionspray voltage, 4500 V; declustering potential, 40 V; probe temperature, 450 °C. Nitrogen was used as the nebulizer gas (20 units) curtain gas (20 units), and the collision gas (5 units). The operating conditions in the APCI source were as follows: nebulizer current, 2.0 lA; probe temperature, 450 °C; declustering potential 20 V. Nitrogen was also used as -MS2 (289.00): 1.150 to 5.635 min from Sample 1 (TuneSample ID) of MT20110310140838.wiff (Heated Nebulizer) Max. 2640.7 cps. 109.0 100% 95% 90% 85% 80% 75% 70% 123.2 65% Rel. Int. (%) 60% 55% 50% 45% 40% 137.5 35% 151.4 97.3 30% 121.6 203.2 25% 95.1 20% 161.4 81.3 149.2 15% 57.2 10% 110.8 92.8 5% 41.1 44.9 59.1 40 60 69.3 80.2 80 135.3 139.3 85.296.2 100 107.8 160.6 145.2 117.0 133.4 142.9 120 140 154.4 163.2 205.0 175.0 187.2 174.1 177.1 171.3 160 m/z, amu 185.7 180 289.4 221.1 201.7 200 Fig. 8a. Product ion scan of the m/z 289 fragment in Fig. 7a. 217.4 220 245.4 227.5 230.2 247.4 261.4 270.9 240 260 287.9 280 300 102 P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 -MS2 (289.30): 2.695 to 6.316 min from Sample 1 (TuneSampleID) of MT20110310162022.wiff (Turbo Spray) Max. 2.8e4 cps. 108.8 100% 95% 90% 85% 80% 75% 70% 124.8 65% 289.4 60% 123.2 Rel. Int. (%) 55% 50% 45% 40% 35% 137.6 151.4 203.3 30% 97.3 245.5 205.1 25% 121.4 94.9 20% 15% 161.5 149.3 81.4 83.3 10% 57.6 135.3 93.0 5% 38.9 41.5 40 67.3 69.3 43.2 55.2 60 80.1 80 105.4 110.9 100 120 139.1 179.5 187.2 165.1 175.0 221.1 159.1 167.2 133.3 144.6 140 157.1 185.5 201.7 227.6 217.6 181.3 160 180 200 220 230.2 247.6 240 257.1 260 288.1 271.2 280 300 m/z, amu Fig. 8b. Product ion scan of an authentic catechin sample. the nebulizer gas (60 units), curtain gas (40 units), and the collision gas (5 units). The collision energy used for both the APCI and ESI source was 30 eV. The chromatograph consisted of an Agilent 1200 series auto-sampler, pump and column department. The injection solvent consisted of water and methanol (1:1, v/v) with a flow speed of 50 lL/min. Additional mass spectrometric information was obtained by direct infusion of a solution of quebracho extract into a Waters API Q-TOF Ultima mass spectrometer, using a carrier solution of acetonitrile:water:formic acid (80:20:0.1, v/v/v) delivered by a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system at a flowrate of 0.3 lL/min. The operating conditions for the ESI source in the negative ion mode were as follows: capillary voltage, 3.5 kV; cone voltage, 35 kV; source temperature, 100 °C; desolvation temperature, 350 °C; desolvation gas, 350 L/h; cone gas, 50 L/h. 5. Results and discussion 5.1. Q1 scan of hot water soluble quebracho extract The negative mode ESI mass spectrum of hot water soluble quebracho extract (Fig. 5a) has salient m/z values at 561.1 and 833.1 Da, and less intense ions at m/z 1105, 1378, and 1668. These ions correspond with fisetinidol–catechin dimers, fisetinidol– catechin–fisetinidol trimers, and higher oligomers corresponding with one catechin starter unit and three to five fisetinidol extender units. The pentamer, with a small peak at m/z 1377 and 13C isotope peak at 1378, is additionally observed as a more intense water adduct (+18 Da) at m/z 1395. The pentamer is further confirmed by doubly charged ions at m/z 688.1, 688.6, and 689.1 corresponding with singly charged m/z values of 1376, 1377 and 1378 (13C isotope peak), respectively. The 1376 value indicates neutral hydrogen radical transfer between ionic species. The oligomers identified are in accordance with our predictions based on isolated monomers, dimers, and trimers, and in vitro reactions of catechin with ent-fisetinidol-4b-ol. We cannot say whether extension to the higher oligomers takes place via the ‘‘upper’’ or ‘‘lower’’ fisetinidol unit. The structural conclusions from Fig. 5a are summarised in Table 1. The spectrum is relatively simple. No monomers (m/z 273 or 289) and little fragmentation are observed. The positive mode ESI mass spectrum of hot water soluble quebracho extract (Fig. 5b) is similar to the negative mode spectrum (Fig. 5a), but with more evidence of fragmentation. Prominent fragments at m/z 411, 683 and 955 correspond with a retro Diels–Alder (rDA) fragmentation of the m/z 563 (dimer), 835 (trimer), and 1107 (tetramer) ions, respectively (Fig. 6). Interestingly, only the 103 P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 -MS2 (289.20): 2.606 to 3.270 min from Sample 1 (TuneSampleID) of MT20110310161127.wiff (Turbo Spray) Max. 3.6e5 cps. 139.3 100% 95% 90% 85% 80% 75% 70% 65% Rel. Int. (%) 60% 55% 50% 110.9 45% 40% 35% 30% 25% 289.4 20% 125.0 15% 149.3 108.8 10% 123.2 5% 121.4 94.8 41.2 44.5 40 59.1 65.5 77.1 81.1 92.8 60 80 165.2 137.5 148.2 163.5 97.2 105.5 100 179.5 188.8 120 140 160 m/z, amu 180 205.1 214.9 200 220 228.9 243.5 252.9 255.8 271.0 240 260 288.1 280 300 Fig. 8c. Product ion scan of an authentic robinetinidol sample. catechin moiety undergoes RDA fragmentation and no similar fragmentation of the fisetinidol moieties is evident. The hexamer that appear as a minor peak at m/z 1651 is observed as more intense single and double water adducts at m/z 1669 and 1687, respectively, and the heptamer that should appear at m/z 1923 as a double water adduct (+36 Da) at m/z 1959. These water adducts are not observed in the smaller oligomers, indicating that molecular size plays a role in their stability. It is well known that PAs are very hydrophilic and water adducts are not unexpected. Similarly, PAs are antioxidants, indicating the presence of labile hydrogen radicals (Wright et al., 2001) and thus possible neutral hydrogen transfer between ionic species. Fragments corresponding to extra oxygen (+16 or +32 Da), are however not observed. PAs that contain more than six flavan-3-ol building blocks (hexamers and upwards) have 90 and more carbon atoms and explains the more intense 13C isotope peaks (1 Da bigger) than 12C peaks. Table 2 collates the oligomers up to the heptamer level and their corresponding rDA fragments. The rDA fragments have m/z values that correspond exactly with the calculated value without any evidence of water adducts. Rather important signals at m/z 725 and 997 correspond to the loss of a resorcinol or catechol moiety (110.0 Da) from either the A- or B-ring of fisetinidol extender units. 5.2. Product ion scans of the dimers (m/z 561) and trimers (m/z 833) in hot water soluble quebracho extract A product ion scan (APCI in the negative mode) of both the m/z 561.2 (dimer) (Fig. 7a) and m/z 833.3 (trimer) (Fig. 7b) yields the m/z 289.4 product ion as base peak as would be expected from fission of a fisetinidol–catechin interflavanyl bond. The complementary m/z 273 ion, associated with fisetinidol, is not observed (although the loss of a neutral 273 Da is observed). The m/z 409 and 391 ions are the result of rDA fragmentation (Scheme 1). 5.3. Product ion scans of the m/z 289 fragment (MS2), pure robinetinidol, and catechin Comparison of the product ion scan of the m/z 289 fragment (Fig. 8a) with that of pure catechin 1 (Fig. 8b) and robinetinidol 9 (Fig. 8c) confirms the aforementioned conclusion that the m/z 289 fragment is catechin and not robinetinidol as was previously reported by Pasch et al. (2001) and Vivas et al. (2004). 5.4. Relative composition of quebracho PAs Reliable quantification with mass spectrometry requires internal standards that are not available for complex PA mixtures. PAs in quebracho, however, form a homologous series of oligomers that differs only in the number of ent-fisetinidol extender units per molecule. We thus assume that the amount of each oligomer present is related to the intensity of the corresponding peak and that 104 P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 Table 3 Composition of quebracho extract calculated from ESI (Fig. 5a). Oligomer M [M H] b 1 (int.) c Dimer 562 561 (124) Trimer 834 833 (118) Tetramer 1106 1105 (32) Pentamer 1378 1377 (0) Hexamer 1650 1649 (0) Number average molecular mass Degree of polymerization Weight average molecular mass 13 C[M H] 1a 562 (44) 834 (66) 1106 (22) 1378 (5 + 5) 1650 (0) (Int.) [M H+H2O] 1 580 (0) 852 (0) 1124 (0) 1396 (4) 1668 (6) (int.) [M 2H] 2 (int.) 280 (0) 416 (0) 552 (31) 688 (15) 824 (0) Mn = 854.96 DP = 3.14 Mw = 938.76 13 C[M 2H] 280.5 416.5 552.5 688.5 824.5 (0) (0) (22) (10) (0) 2a (Int.) Total int. Weighted compos. 168 184 107 41 6 33 37 21 8 1 a 13 b c C isotope peaks. Observed peak. Measured intensity. measurement of peak intensities will give a rough estimate of the relative composition of quebracho PAs. At worst we believe that mass discrimination will underestimate the relative amount of higher oligomers present. 13C isotope ions become an important factor with oligomers and these were taken into account in our ESI quantification. The absence of significant fragments smaller than m/z 561 (dimer) in Fig. 5a (negative mode ESI) allows us to assume that quebracho extract contains almost no flavan-3-ol monomers. This is in agreement with the conclusion by Roux and Evelyn (1960) that catechin and ent-fisetinidol-4-ol is virtually absent in the central heartwood of old quebracho trees. A calculation of the composition of quebracho extract based on the intensities of peaks in the ESI (negative mode, Fig. 5a) gave a number average degree of polymerization (aDP) of 3.1 (Table 3). This is more conservative than the values of 4.5, 6.25, and 6.74 determined for sulfited quebracho extract with gel permeation chromatography (Covington et al., 2005), MALDI-TOF (Pasch et al., 2001), and 13C NMR (Thompson and Pizzi, 1995), respectively. These values agree with Mouls et al. (2011) observation that a PA extract with an aDP of 6.7 determined by thiolysis gave an aDP value of 4.9 with ESI (about 1.8 lower). Larger polymers no doubt exist, but probably in small quantities. A conservative aDP of 3.1 was calculated from the intensity of MS fragments. Taking Mouls’s results, that the aDP of small oligomers is underestimated by about 1.8 with ESI relative to the thiolysis-HPLC method, the aDP of quebracho should be about 4.9. The relatively poor solubility of hot water soluble quabraco extract, as compared to mimosa extract (soluble in cold water and does not require sulfitation for complete extraction) is attributed to the absence of robinetinidol extender units. Robinetinidol has one more aromatic OH than fisetinidol which increases water solubility via hydrogen bonding. Acknowledgments Thanks are due to Prof. H. Pasch for recording the ESI spectra (Figs. 5a–c) of hot water soluble quebracho extract. Mimosa Extract Company (Pty) Ltd. and the Technology and Human Resources for Industry Programme (THRIP) for financial support. References 6. Conclusion Phytochemistry and established synthetic organic chemistry perspectives were combined with a mass spectrometry investigation (ESI, APCI, and product ion scans as fingerprints) to probe the chemical composition of the PAs in commercial hot water soluble (unsulfited) quebracho extract. Comparison of the fragmentation spectrum of the m/z 289 fragment in the product ion scans of dimers and trimers, with the fragmentation spectra of authentic samples of catechin and robinetinidol, assigns this fragment unequivocally to catechin. The starter unit in our sample was thus always catechin and the extender unit always ent-fisitinedol. The quebracho extract sample does not contain detectable quantities of robinetinidol, either in the monomeric form or as extender unit in prorobinetinidin type oligomers. Quebracho PAs thus consist of a homologous series of one molecule of catechin (starter unit) linked to one, two, three, etc. ent-fisetinidol extender units. This conclusion is further supported by the isolation of exclusively catechin and ent-fisetinidol-4b-ol monomers from quebracho heartwood, the structure of the dimers and trimers determined by NMR from the same heartwood, and biomimetic synthesis of dimers, trimers, and a tetramer from catechin and ent-fisetinidol4b-ol. The relatively simple nature of quebracho extract and the absence of large oligomers (the biggest oligomer observed was a heptamer) allowed us to estimate the composition of quebracho extract with ESI. Quebracho PAs consist roughly of 33% dimers, 37% trimers, 21% tetramers, 8% pentamers, and 1% heptamers. Akin, D., 1982. Forage cell wall degradation and q-coumaric, ferulic, and sinapic acids. Agron. J. 74, 424–428. Bate-Smith, E., 1954. Astringency in foods. Food Proc. Packag. 23, 124–127. Covington, A.D., 2009. Tannning Chemistry: The Science of Leather. The Royal Society of Chemistry, Cambridge, pp. 304–309. Covington, A.D., Lilley, T.H., Song, L., Evans, C.S., 2005. Collagen and polyphenols: new relationships and new outcomes. Part 1. Flavonoid reactions for new tanning processes. J. Am. Leather Chem. Ass. 100, 325. Elliot, R.J., Sackwild, V., Richards, J.J., 1982. Quantitative frontier orbital theory: part I. Electrophilic aromatic substitution. Mol. Struct. 86, 301–314. Fechtal, M., Riedl, B., 1993. Use of eucalyptus and Acacia mollissima bark extractformaldehyde adhesives in particleboard manufacture. Holzforschung 47, 349. Foo, L.Y., Porter, L.J., 1978. Prodelphinidin polymers: definition of structural units. J. Chem. Soc. Perkin Trans. I, 1186–1190. Forest, K., Wan, P., Preston, C.M., 2004. Catechin and hydroxybenzhydrols as models for the environmental photochemistry of tannins and lignins. Photochem. Photobiol. 3, 463–472. Hagerman, A.E., Robbins, C.T., Weerasuriya, Y., Wilson, T.E., Mcarthur, C.J., 1992. Tannin chemistry in relation to digestion. Range Manage. 45, 57–62. Haslam, E., 1974. Polyphenol–protein interactions. Biochem. J. 139, 285–288. Haslam, E., 1988. Twenty-second procter memorial lecture: vegetable tannins – renaissance and reappraisal. J. Soc. Leather Technol. Chem. 72, 45. Haslam, E., 1997. Vegetable tannage: where do the tannins go? J. Soc. Leather Technol. Chem. 82, 45. Haslam, E., 2005. Polyphenols, Collagen and Leather In: Practical Polyphenolics. From Structure to Molecular Recognition and Physiological Action. Cambridge University Press, Cambridge, p. 378. Hofmann, T., Glabasnia, A., Schwarz, B., Wisman, K.N., Gangwer, K.A., Hagerman, A.E., 2006. Protein binding and astringent taste of a polymeric procyanidin, 1,2,3,4,6-penta-O-galloyl-b-D-glucopyranose, castalagin, and grandinin. J. Agric. Food Chem. 54, 9503–9509. Kennedy, J.A., Taylor, A.W., 2003. Analysis of proanthocyanidins by high performance gel permeation chromatography. J. Chromatogr. A 995, 99–107. Koupai-Abyazani, M.R., Muir, A.D., Bohm, B.A., Towers, G.H.N., Gruber, M.Y., 1993. The proanthocyanidin polymers in some species of Onobrychis. Phytochemistry 34, 113–117. P.B. Venter et al. / Phytochemistry 73 (2012) 95–105 Le Roux, E., Doco, T., Sarni-Manchado, P., Lozano, Y., Cheynier, V., 1998. A-type proanthocyanidins form pericarp of Litchi chinensis. Phytochemistry 48, 1251– 1258. Mouls, L., Mazauric, J.P., Sommerer, N., Fulcrand, H., Mazerolles, G., 2011. Comprehensive study of condensed tannins by ESI mass spectrometry: average degree of polymerisation and polymer distribution determination from mass spectra. Anal. Bioanal. Chem. 400, 613–623. Pasch, H., Pizzi, A., Rode, K., 2001. MALDI-TOF mass spectrometry of polyflavonoid tannins. Polymer 42, 7531–7539. Pizzi, A., 1978. The chemistry and development of tannin-based adhesives for exterior plywood. J. Polym. Sci. 22, 2397–2399. Reed, J.D., Krueger, C.G., Vestling, M.M., 2005. MALDI-TOF mass spectrometry of oligomeric food polyphenols. Phytochemistry 66, 2248–2263. Rigaud, J., Perez-Ilzarbe, J., Ricardo da Silva, J.M., Cheynier, V., 1991. Micro method for the identification of proanthocyanidin using thiolysis monitored by highperformance liquid chromatography. J. Chromatogr. A 540, 401–405. Roux, D.G., 1992. Introduction. In: Hemingway, R.W., Laks, P.E. (Eds.), Plant Polyphenols. Plenum Press, New York, pp. 7–39. Roux, D.G., Evelyn, S.R., 1960. The distribution and deposition of tannins in the heartwoods of Acacia mollissima and Schinopsis spp. Biochem. J. 76, 17–23. Roux, D.G., Paulus, E., 1962. Polymeric leuco-fisetinidin tannins from the heartwood of Acacia mearnsii. Biochem. J. 82, 320–324. Roux, D.G., Ferreira, D., Hundt, H.K.L., Malan, E., 1975. Structure, stereochemistry, and reactivity of natural condensed tannins as basis for their extended industrial application. Appl. Polym. Symp. 28, 335–353. Shen, Z., Haslam, E., Falshaw, C.P., Begley, M.J., 1986. Procyanidins and polyphenols of Larix gmelini bark. Phytochemistry 25, 2629–2635. Takahata, Y., Ohnishi-Kameyama, M., Furuta, S., Takahashi, M., Suda, I., 2001. Highly polymerized procyanidins in brown soybean seed coat with a high radicalscavenging activity. J. Agric. Food Chem. 49, 5843–5847. 105 Taylor, A.W., Barofsky, E., Kennedy, J.A., Deinzer, M.L., 2003. Hop (Humulus lupulus L.) proanthocyanidins characterized by mass spectrometry, acid catalysis, and gel permeation chromatography. J. Agric. Food Chem. 51, 4101–4110. Thompson, D., Pizzi, A., 1995. Simple 13C NMR methods for quantitative determinations of polyflavonoid tannin characteristics. J. Appl. Polym. Sci. 55, 107. Thompson, R.S., Jacques, D., Haslam, E., Tanner, R.N.J., 1972. Plant proanthocyanidins. Part I. Introduction; the isolation, structure, and distribution in nature of plant procyanidins. J. Chem. Soc. Perkin Trans. 1, 1387–1399. Vivas, N., Nonier, M.F., Vivas de Gaulejac, N., Absalon, C., Bertrand, A., Mirabel, M., 2004. Differentiation of proanthocyanidin tannins from seeds, skins and stems of grapes (Vitis vinifera) and heartwood of quebracho (Schinopsis balansae) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thioacidolysis/liquid chromatography/electrospray ionization mass spectrometry. Anal. Chim. Acta 513, 256–274. Viviers, P.M., Kolodziej, H., Young, D.A., Ferreira, D., Roux, D.G., 1983. Synthesis of condensed tannins. Part 11. Intramolecular enantiomerism of the constituent units of tannins from the Anacardiaceae:Stoicheometric control in direct synthesis: derivation of 1H nuclear magnetic resonance parameters applicable to higher oligomers. J. Chem. Soc. Perkin Trans. 1, 2555–2562. Wright, J.S., Johnson, E.R., DiLabio, G.A., 2001. Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants. J. Am. Chem. Soc. 123, 1173– 1183. Young, D.A., Ferreira, D., Roux, D.G., 1985. Synthesis of condensed tannins. Part 15. Structure of natural ‘angular’ profisetinidin tetraflavanoids: asymmetric induction during oligomeric synthesis. J. Chem. Soc. Perkin Trans. 1, 2529– 2535.