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 poly-
mers for leather tanning and adhesive manufacturing. We combined established phyto- and synthetic
chemistry perspectives with electrospray mass spectrometry experiments to prove that quebracho pro-
anthocyanidin polymers consist of an homologous series of avan-3-ol based oligomers. The starter unit
is always catechin which is angularly bonded to setinidol extender units. By comparison of the MS
2
frag-
mentation spectra of the oligomer with product ion scans of authentic catechin and robinetinidol sam-
ples, we proved that quebracho extract contains no robinetinidol, as is often reported. Quebracho
proanthocyanidins have acid resistant interavanyl bonds, due to the absence of 5-OH groups in setin-
idol, 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.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
The wild quebracho forests in the Gran Chaco region of Argen-
tina, 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 axe-
breaker. To obtain a warm water soluble quebracho extract, the
heartwood is stripped of its bark, chipped, and extracted with boil-
ing water. A cold water soluble extract (sulted extract) is obtained
upon treatment of the warm water soluble extract with bisulte or
direct extraction of wood chips with a boiling aqueous bisulte
solution. Higher extraction rates are obtained with boiling aqueous
bisulte solution than with boiling water alone.
Quebracho extract is obtained from Schinopsis balansae (red
chaqueno quebracho, pure tannin content 2021%) from the
Eastern Chaco region and Schinopsis lorentzii (red santiagueno
quebracho, pure tannin content 1518%) from the Western Chaco
region. These two species were previously referred to as Quebracho
colorado chaqueo and Quebracho colorado santiagueo (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.
Quebracho extract consists of about 95% proanthocyanidins
(PAs) and 5% water soluble sugars on a dry basis. The term pro-
anthocyanidin (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, chest-
nut bark, and oak gall extracts.
Progress in dening quebracho PA composition has been slow,
mainly due to the complexity of the extracts and the difculty of
isolating pure PAs with silica gel based chromatography materials.
Uncertainties include different hydroxylation patterns of the con-
stituent avan-3-ol aromatic rings, different congurations at the
C-2, C-3 and C-4 stereogenic centers, the possibility of a second
ether interavanyl 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 interav-
anyl 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 interavanyl 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
0031-9422/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.10.006

Corresponding author. Tel.: +27 51 4012782; fax: +27 51 4448463.

E-mail address: vdwestjh@ufs.ac.za (J.H. van der Westhuizen).
Phytochemistry 73 (2012) 95105
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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 avan-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 hydro-
gen 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 tan-
ninprotein complexes) (Hagerman et al., 1992), and growth inhi-
bition 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 proper-
ties of young or poor quality red wines. The absence of the 5-OH
group and corresponding stability of the PA oligomer to interav-
anyl bond ssion (Roux and Paulus, 1962; Roux et al., 1975) is
probably an important factor in the industrial application of que-
bracho 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 afnity for collagen, rate of penetration
into hides and skins during commercial tannage, mobility within
leather, and desorption from nished leather under moist condi-
tions are determined by oligomer composition (Covington, 2009).
The availability of nucleophilic centers for cross linking with form-
aldehyde on the periphery of oligomers determines curing time
and pot life of thermosetting PA based adhesives.
Electrospray ionization (ESI) and matrix-assisted laser desorp-
tion 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 esti-
mate 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 conrmed 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 sulted 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 mimo-
sa 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 setinidol (274 Da) and robinetinidol (290 Da)
constituent units. They concluded that quebracho PAs consist
mostly of prosetinidins. 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.
Fig. 1. Flavan-3-ol and avan-3,4-diol monomers from the heartwood of S. lorentzii
(putative building blocks of quebracho PAs).
Fig. 2. Quebracho dimers from S. balansae.
Fig. 3. Trimer isolated from S. balansae [ent-setinidol-(4b ?8)-catechin-(6 ?4b)-
ent-setinidol].
Fig. 4. Tetramer synthesized by Viviers and co-workers.
96 P.B. Venter et al. / Phytochemistry 73 (2012) 95105
2. Phytochemistry
Roux and Evelyn (1960) found only catechin 1 and ent-se-
tinidol-4b-ol [()-leucosetinidin] 2 (Fig. 1) as monomeric con-
stituents in the heartwood of S. lorentzii. This suggests that 1
and 2 are the precursors of quebracho PAs. The avan-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 (120140 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 diastereoiso-
mers ent-setinidol-(4b ?8)-catechin 3 and ent-setinidol-(4a
?8)-catechin 4 (m/z 562) from S. balansae. Smaller quantities of
ent-setinidol-(4b ?6)-catechin and ent-setinidol-(4a ?6)-cat-
echin 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 ent-
setinidol-(4b ?8)-catechin-(6 ?4b)-ent-setinidol 7 (4,6;4,8-
bis-ent-setinidol-catechin) (m/z 834) (Fig. 3) and three diastereo-
isomers from S. balansae. However, no tetramers were reported.
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 561 563 1 1
Trimer 833 835 1 2
Tetramer 1105 1107 1 3
Pentamer 1377
a
1379
d
1 4
Hexamer (1649)
b
1651
e
1 5
Heptamer (1921)
c
(1923)
f
1 6
(Ions in brackets were not detected directly but indirectly as water adducts).
a
The
13
C isotope peak at m/z 1378 was automatically annotated in Fig. 5a. The slightly less intensive
12
C 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
13
C isotope peak at m/z 1668. Close inspection of a magnied
spectrum reveals the presence of a
12
C 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
13
C 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. Magnication of
the spectrum also reveals the corresponding
13
C 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
13
C isotope peak at 1960).
NOPQ
m/z
1700 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1800 1900
%
0
100
NP_Pol_100216_23 11 (0.126) Cm (11:14-1:4) TOF MS ES-
374 561.1
552.1
287.0
238.9
449.1 423.0
343.0
495.1
833.1
562.1
601.1
688.1
602.1
603.1
688.6
689.1
831.1
834.1
1105.2
835.1
849.1
850.1
985.1
869.1
1009.2
1106.2
1107.2
1395.2 1123.2
1378.2 1668.3 1396.3
Fig. 5a. Negative mode ESI spectrum of hot water soluble quebracho extracts (m/z 200 to 2000 m/z range).
P.B. Venter et al. / Phytochemistry 73 (2012) 95105 97
3. Synthesis
Viviers and co-workers (1983) investigated the biomimetic syn-
thesis of quebracho PAs via acid catalysed condensation of catechin
1 and ent-setinidol-4b-ol 2. The products closely resemble those
isolated by the same authors.
Condensation of 1 eq. of catechin 1 with ent-setinidol-4b-ol 2
(1 eq.) gives mainly ent-setinidol-(4b ?8)-catechin 3 and small
quantities of the epimeric ent-setinidol-(4a ?8)-catechin 4
(Fig. 2). The presence of a second equivalent of 2 led to formation
of the trimer, ent-setinidol-(4b ?8)-catechin-(6 ?4b)-ent-
setinidol 7 (Fig. 3). A further equivalent of 2 leads to the
NOPQ
m/z
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100
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+
2.00e4 563.1
411.1
393.1
301.1 287.1
273.1
271.1
231.1
315.1
365.1
437.1
545.1
485.1
683.2
564.2
565.2
681.2
585.1
835.2
684.2
725.2
726.2
817.2
801.2
955.2
836.2
857.2
858.2
873.2
874.2
1107.3 956.2
997.2
998.3
1033.3
1108.3
1129.3
1227.3
1130.3
1131.3
1148.3
1686.4
1397.4
1228.3
1229.3
1270.3
1414.4
1669.4
1499.4
1692.4
1959.5
1693.4
1694.4
1965.5
Fig. 5b. Positive mode ESI spectrum of hot water soluble quebracho extracts (m/z 200 to 2000 m/z range).
NOPQ
m/z
1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950
%
0
100
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
1397.4
1379.3
1300.4
1361.3
1307.3
1329.4
1414.4
1419.3
1669.4
1420.3
1499.4
1421.3
1433.3
1435.3
1445.4
1481.4
1451.3
1500.4
1668.4
1651.4
1517.4
1518.4
1559.4
1519.4
1577.4
1634.4
1687.5
1691.4
1692.4
1959.5
1958.5
1693.4
1694.4
1943.5
1941.5
1790.5 1709.4
1771.4
1711.4
1924.5
1923.5 1862.5
1839.5
1864.5
1964.5
1965.5
1979.5
1982.5
Fig. 5c. Positive mode ESI spectrum (expansion of 5b) of hot water soluble quebracho extracts (m/z 1300 to 2000 m/z range).
98 P.B. Venter et al. / Phytochemistry 73 (2012) 95105
formation of the tetramer, ent-setinidol-(4b ?6)-ent-setinidol-
(4b ?8)-catechin-(6 ?4b)-ent-setinidol 8 (Fig. 4).
The most reactive nucleophilic position on catechin 1 is C-8
since at this position the highest occupied molecular orbital
(HOMO) exhibits its maximum amplitude (Elliot et al., 1982). The
rst condensation product (dimer) will thus predominantly be
ent-setinidol-(4b ?8)-catechin 3. The C-6 position of the phloro-
glucinol 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-setinidol unit.
The second condensation product (trimer) will thus predominantly
be ent-setinidol-(4b ?8)-catechin-(6 ?4b)-ent-setinidol 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-setinidol moi-
ety is the most reactive remaining nucleophilic position. The trimer
will thus react via the sterically less hindered C-6 position with a
third ent-setinidol-4b-ol molecule to yield the tetramer
Fig. 6. Structures of rDA fragments of m/z 563, 835 and 1107 dimers, trimers and tetramers.
Table 2
Diagnostic rDA fragments associated with their corresponding oligomer precursors.
Oligomer ESI+ mass Rel. comp.
a
RDA mass Rel. comp.
a
Dimer 563 125 411 36
Trimer 835 79 683 110
Tetramer 1107 33 955 49
Pentamer 1379 8 1227 13
Hexamer 1651 3 1499 4
Heptamer 1923 61 1771 1.5
a
Relative composition is based on peak height.
-MS2 (561.20): 0.312 to 3.359 min from Sample 1 (TuneSam . s p c 1 . 6 6 3 4 . x a M ) r e z i l u b e N d e t a e H ( f f i w . 1 4 1 0 3 1 0 1 3 0 1 1 0 2 T M f o ) D I e l p
50 100 150 200 250 300 350 400 450 500 550 600
m/z, amu
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
R
e
l
.

I
n
t
.

(
%
)
289.3
561.3
161.3
409.5
271.3
391.3 125.1
245.4
109.0
205.0 257.0
137.3
253.1
451.3 151.1
313.4 328.5
295.0 189.3 9 . 6 8 2 2 . 3 1 1
Fig. 7a. Product ion scan of the m/z 561 dimer ion (APCI in the negative mode).
P.B. Venter et al. / Phytochemistry 73 (2012) 95105 99
ent-setinidol-(4b ?6)-ent-setinidol-(4b ?8)-catechin-(6 ?4b)
-ent-setinidol 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 congured 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 conguration or position of the interavanyl link
and replace, e.g., the terms ent-setinidol-(4b ?6)-catechin 5
and ent-setinidol-(4a ?6)-catechin 6 with setinidol-catechin.
Phytochemistry thus suggests that:
1. Quebracho heartwood contains only catechin and ent-setin-
idol-4-ol and no robinetinidol-4-ol.
2. Dimers and trimers consist of a catechin starter unit and one or
two setinidol extender units. The trimer is angular with one
setinidol in the upper C-8 position and the other in the
terminal C-6-position. No linear setinidolsetinidol dimers,
setinidolsetinidolsetinidol trimers, or robinetinidol con-
taining dimers and trimers were reported in the literature.
Synthetic organic chemistry suggests that:
1. The setinidolcatechinsetinidol 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 setinidol extender units.
2. The formation of tetramers and higher oligomers are inhibited
by the lower reactivity of the 5-deoxy setinidol 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 avan-3-ol based oligomers. The starter unit
is always catechin which is angularly bonded to setinidol exten-
der units. Herein, we report our results on the composition of a
hot water soluble (unsulted) quebracho extract from S. lorentzii
with electrospray ionization (ESI) and atmospheric pressure
chemical ionization (APCI) mass spectrometry and use these re-
sults 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 pres-
ence 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 pur-
chased 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
. s p c 8 . 2 2 1 . x a M ) r e z i l u b e N d e t a e H ( f f i w . 3 4 8 2 4 1 0 1 3 0 1 1 0 2 T M f o ) D I e l p m a S e n u T ( 1 e l p m a S m o r f n i m 1 0 2 . 1 2 o t 7 5 0 . 2 : ) 0 3 . 3 3 8 ( 2 S M -
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
m/z, amu
10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
R
e
l
.

I
n
t
.

(
%
)
289.5
833.5 561.2
529.3
409.4
161.0 600.4 391.8
270.8
680.8
451.4 510.8 5 . 7 7 3 6 . 3 0 2 1 . 9 0 1
310.9 329.8 359. 9999 2 222222294.88 267.5 41113.9 4 33 33 33.3 4475.5 198..8 192.21 593.111 154.9 635.9 663.3 719.1 824.2222
Fig. 7b. Product ion scan of the m/z 833 trimer (APCI in the negative mode).
100 P.B. Venter et al. / Phytochemistry 73 (2012) 95105
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
Scheme 1. Fragmentation of m/z 561 quebracho dimer (MH)

.
-MS2 (289.00): 1.150 to 5.635 min from Sample 1 (TuneSample ID) Max. 2640.7 cps. ated Nebulizer) e H ( f f i w . 8 3 8 0 4 1 0 1 3 0 1 1 0 2 T M f o
40 60 80 100 120 140 160 180 200 220 240 260 280 300
m/z, amu
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
R
e
l
.

I
n
t
.

(
%
)
109.0
123.2
137.5
151.4
97.3
121.6
203.2
95.1
161.4
81.3
149.2
205.0
289.4
57.2
175.0
139.3
160.6
245.4 135.3 187.2
221.1
110.8
92.8
177.1
145.2 174.1
201.7
163.2 227.5
230.2 247.4 107.8 69.3
142.9 80.2
59.1 41.1 217.4 133.4 185.7
96.2 117.0
44.9
270.9 85.2
9 . 7 8 2 4 . 1 6 2 3 . 1 7 1
154.4
Fig. 8a. Product ion scan of the m/z 289 fragment in Fig. 7a.
P.B. Venter et al. / Phytochemistry 73 (2012) 95105 101
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 ow 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 owrate 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 que-
bracho 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 setinidolcatechin dimers, setinidol
catechinsetinidol trimers, and higher oligomers corresponding
with one catechin starter unit and three to ve setinidol extender
units. The pentamer, with a small peak at m/z 1377 and
13
C isotope
peak at 1378, is additionally observed as a more intense water
adduct (+18 Da) at m/z 1395. The pentamer is further conrmed
by doubly charged ions at m/z 688.1, 688.6, and 689.1 correspond-
ing with singly charged m/z values of 1376, 1377 and 1378 (
13
C
isotope peak), respectively. The 1376 value indicates neutral
hydrogen radical transfer between ionic species. The oligomers
identied are in accordance with our predictions based on isolated
monomers, dimers, and trimers, and in vitro reactions of catechin
with ent-setinidol-4b-ol. We cannot say whether extension to
the higher oligomers takes place via the upper or lower setin-
idol 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 que-
bracho extract (Fig. 5b) is similar to the negative mode spectrum
(Fig. 5a), but with more evidence of fragmentation. Prominent frag-
ments at m/z 411, 683 and 955 correspond with a retro DielsAlder
(rDA) fragmentation of the m/z 563 (dimer), 835 (trimer), and 1107
(tetramer) ions, respectively (Fig. 6). Interestingly, only the
-MS2 (289.30): 2.695 to 6.316 min from Sample 1 (TuneSampleID) of MT20110310162022.wiff (Turbo Spray) Max. 2.8e4 cps.
40 60 80 100 120 140 160 180 200 220 240 260 280 300
m/z, amu
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
R
e
l
.

I
n
t
.

(
%
)
108.8
124.8
289.4
123.2
137.6 151.4
203.3
97.3 245.5
205.1
121.4
94.9 161.5
179.5
149.3
81.4
187.2 83.3
175.0
165.1
221.1 139.1
159.1
57.6 135.3
167.2
227.6
93.0
185.5 201.7 217.6 105.4 247.6 110.9 133.3
230.2 144.6 288.1 271.2 69.3 157.1 41.5 80.1 67.3
55.2 181.3 43.2 38.9 257.1
Fig. 8b. Product ion scan of an authentic catechin sample.
102 P.B. Venter et al. / Phytochemistry 73 (2012) 95105
catechin moiety undergoes RDA fragmentation and no similar frag-
mentation of the setinidol 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, respec-
tively, and the heptamer that should appear at m/z 1923 as a dou-
ble 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 radi-
cals (Wright et al., 2001) and thus possible neutral hydrogen trans-
fer between ionic species. Fragments corresponding to extra
oxygen (+16 or +32 Da), are however not observed. PAs that
contain more than six avan-3-ol building blocks (hexamers and
upwards) have 90 and more carbon atoms and explains the more
intense
13
C isotope peaks (1 Da bigger) than
12
C 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 moi-
ety (110.0 Da) from either the A- or B-ring of setinidol 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
ssion of a setinidolcatechin interavanyl bond. The comple-
mentary m/z 273 ion, associated with setinidol, 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 (MS
2
), 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) conrms 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 quantication with mass spectrometry requires inter-
nal standards that are not available for complex PA mixtures. PAs
in quebracho, however, forma homologous series of oligomers that
differs only in the number of ent-setinidol extender units per
molecule. We thus assume that the amount of each oligomer pres-
ent is related to the intensity of the corresponding peak and that
-MS2 (289.20): 2.606 to 3.270 min from Sample 1 (TuneSampleID) of MT20110310161127.wiff (Turbo Spray) Max. 3.6e5 cps.
40 60 80 100 120 140 160 180 200 220 240 260 280 300
m/z, amu
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
R
e
l
.

I
n
t
.

(
%
)
139.3
110.9
289.4
125.0
149.3
108.8
123.2
165.2
121.4
137.5 163.5 94.8
148.2
179.5
205.1
92.8 271.0 252.9 288.1 97.2 81.1 105.5 243.5 214.9 188.8 65.5 228.9 77.1 41.2 44.5 255.8 59.1
Fig. 8c. Product ion scan of an authentic robinetinidol sample.
P.B. Venter et al. / Phytochemistry 73 (2012) 95105 103
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.
13
C isotope ions become an important
factor with oligomers and these were taken into account in our
ESI quantication.
The absence of signicant fragments smaller than m/z 561
(dimer) in Fig. 5a (negative mode ESI) allows us to assume that
quebracho extract contains almost no avan-3-ol monomers. This
is in agreement with the conclusion by Roux and Evelyn (1960)
that catechin and ent-setinidol-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 sulted quebracho extract with gel permeation
chromatography (Covington et al., 2005), MALDI-TOF (Pasch
et al., 2001), and
13
C NMR (Thompson and Pizzi, 1995), respec-
tively. 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).
6. Conclusion
Phytochemistry and established synthetic organic chemistry
perspectives were combined with a mass spectrometry investiga-
tion (ESI, APCI, and product ion scans as ngerprints) to probe
the chemical composition of the PAs in commercial hot water sol-
uble (unsulted) quebracho extract. Comparison of the fragmenta-
tion 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-sitinedol. 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-setinidol extender units. This
conclusion is further supported by the isolation of exclusively cat-
echin and ent-setinidol-4b-ol monomers from quebracho heart-
wood, 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-setinidol-
4b-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.
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 Moulss results, that the aDP of small oligo-
mers 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 ex-
tract, as compared to mimosa extract (soluble in cold water and
does not require sultation for complete extraction) is attributed
to the absence of robinetinidol extender units. Robinetinidol has
one more aromatic OH than setinidol which increases water sol-
ubility via hydrogen bonding.
Acknowledgments
Thanks are due to Prof. H. Pasch for recording the ESI spectra
(Figs. 5ac) of hot water soluble quebracho extract.
Mimosa Extract Company (Pty) Ltd. and the Technology and
Human Resources for Industry Programme (THRIP) for nancial
support.
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Composition of quebracho extract calculated from ESI (Fig. 5a).
Oligomer M [MH]
1
(int.)
13
C[MH]
1a
(Int.) [MH+H
2
O]
1
(int.) [M2H]
2
(int.)
13
C[M2H]
2a
(Int.) Total int. Weighted compos.
Dimer 562 561
b
(124)
c
562 (44) 580 (0) 280 (0) 280.5 (0) 168 33
Trimer 834 833 (118) 834 (66) 852 (0) 416 (0) 416.5 (0) 184 37
Tetramer 1106 1105 (32) 1106 (22) 1124 (0) 552 (31) 552.5 (22) 107 21
Pentamer 1378 1377 (0) 1378 (5 + 5) 1396 (4) 688 (15) 688.5 (10) 41 8
Hexamer 1650 1649 (0) 1650 (0) 1668 (6) 824 (0) 824.5 (0) 6 1
Number average molecular mass Mn = 854.96
Degree of polymerization DP = 3.14
Weight average molecular mass Mw = 938.76
a 13
C isotope peaks.
b
Observed peak.
c
Measured intensity.
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