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Variation of Proanthocyanidins in Lotus Species
Subathira Sivakumaran &William Rumball &
Geoff A. Lane &Karl Fraser &Lai Y. Foo &
Min Yu &Lucy P. Meagher
Received: 10 May 2005 / Revised: 18 November 2005 /
Accepted: 15 March 2006 / Published online: 2 August 2006
#Springer Science + Business Media, Inc. 2006
Abstract The proanthocyanidin (PA) chemistry of 12 Lotus species of previously unknown
PA content was examined in comparison with agricultural cultivars of L. pedunculatus,L.
corniculatus, and L. tenuis and a “creeping”selection of L. corniculatus. Herbage harvested
in winter 2000 and again in spring had extractable PA concentrations, estimations of which
varied between 0.2 and 10.9% of dry matter. The four novel Lotus spp. with the highest
concentrations were selected for further evaluation together with the agricultural accessions.
PA concentrations in herbage were estimated for individual plants harvested in spring 2001
and bulk samples harvested in summer 2002–2003. PA oligomer and polymer fractions
were separated by Sephadex LH-20 chromatography from aqueous acetone PA extracts of
herbage. The chemical characteristics of the fractions were examined by acid catalyzed
degradation with benzyl mercaptan,
13
C nuclear magnetic resonance spectroscopy, elec-
trospray ionization (ESI), and matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS). A wide variation was found in the chemical composition,
mean degree of polymerization (mDP), and polydispersity of PAs from Lotus spp. Fractions
from L. americanus,L. corniculatus “creeping selection,”and L. pedunculatus consisted
predominantly of prodelphinidin (PD) units, whereas PA from L. angustissimus and L.
corniculatus consisted predominantly of procyanidin (PC) units. An approximately equal
composition in terms of PC and PD units was found in L. parviflorus and L. suaveolens.In
L. angustissimus, epicatechin is dominant in both extender and terminal units. In all Lotus
PA fractions, the 2,3-cis isomers (epicatechin or epigallocatechin) predominated. Only trace
amounts of PA were extracted from L. tenuis. The mDP of the PA fractions ranged from 8 to
97, with high mDP found only for L. pedunculatus and L. americanus. In the ESI-MS and
MALDI-TOF-MS of the L. angustissimus PA fraction, ions for homo-PC oligomers were
J Chem Ecol (2006) 32: 1797–1816
DOI 10.1007/s10886-006-9110-3
S. Sivakumaran (*):L. P. Meagher
Food and Health Group, AgResearch Ltd., Grasslands Research Centre,
Private Bag 11008, Palmerston North, New Zealand
e-mail: suba.sivakumaran@agresearch.co.nz
W. Rumball :G. A. Lane :K. Fraser :M. Yu
Applied Biotechnology Group, AgResearch Ltd., Grasslands Research Centre,
Private Bag 11008, Palmerston North, New Zealand
L. Y. Foo
Industrial Research Ltd., P.O. Box 31-310, Lower Hutt, New Zealand
dominant, whereas ions for hetero-oligomers predominated in the other Lotus spp. Ions
indicative of A-type linkages were observed in the MS of L. americanus. The results are
discussed in terms of possible relationships between the concentration and composition of
the PAs of Lotus spp. and ecological factors.
Keywords Proanthocyanidins .Thiolysis .Procyanidins .Prodelphinidins .HPLC .
13
C NMR .ESI-MS .MALDI-TOF-MS .Lotus
Introduction
Proanthocyanidins (PA) are a class of polymeric, polyphenolic, plant secondary metabolites
that are widely distributed in the plant kingdom. These polymers encompass a wide range
of structural variants. They consist of chains of flavan-3-ol (Fig. 1) units linked together
through C4–C8 and/or C4–C6 linkages (B-type) and may be doubly linked with an addi-
tional C2–O–C7 linkage (A-type). In addition to these variations in interflavanoid linkages,
PAs can vary in the hydroxylation pattern of the A- and B-rings, stereochemistry at the three
chiral centers (C2, C3, and C4) of the C ring, and the degree of polymerization (DP). The
most common PAs in forage legumes are procyanidins (PC) with a 30,40-dihydroxy sub-
stitution of B-ring (catechin and epicatechin) and prodelphinidins (PD) with 30,40,50-
trihydroxy substitution (gallocatechin and epigallocatechin).
Proanthocyanidins are not known to play any role in physiological processes of plants.
However, they are known to precipitate proteins (Hagerman and Butler, 1981) and have been
studied for likely ecological roles. They have been considered as plant defenses against
insect (Zucker, 1983; Ayres et al., 1997;Heiletal.,2002) and mammalian herbivores (reviewed
in Iason, 2005), but their role in the dynamics of nutrient cycling (Zucker, 1983; Kraus et al.,
Fig. 1 Thiolysis of proanthocyanidin terminal units released as flavan-3-ols and extension units as flavan-
3-ol thioether adducts
1798 J Chem Ecol (2006) 32: 1797–1816
2003) and in the photoprotection of plant tissues (Close and McArthur, 2002) has also come
into consideration.
In agricultural systems, high concentrations of PA [>6% of dry matter (DM)] in forage
and browse plants reduce voluntary feed intake, digestibility, and animal performance of
ruminants (Min et al., 2003), consistent with a defensive role. However, at lower concen-
trations, the protein-binding effects of PAs protect dietary protein against excessive de-
gradation in the rumen and can have beneficial effects for ruminant herbivores (Min et al.,
2003). Low to medium concentrations of PA (2–4% of DM) increase protein utilization,
which contributes to increases in lactation, wool growth, and live weight gain (Waghorn
et al., 1994; Wang et al., 1996). Other beneficial effects for agriculture include the control
of bloat (Chiquette et al., 1988) and improved tolerance against internal parasites (Niezen
et al., 1998).
Forage cultivars of Lotus spp. are of interest in agriculture, as they combine improved
protein supply for animal production through symbiotic nitrogen fixation with the protein-
protective action of PAs in the rumen. They exhibit the dichotomy described above of both
beneficial and detrimental effects on feed value and animal performance (Waghorn et al.,
1998; Aerts et al., 1999). The PA from L. corniculatus is associated with better animal
performance than that from L. pedunculatus (Waghorn et al., 1999), and this cannot be
accounted for by the lower PA content of L. corniculatus (above half that of L. pedun-
culatus; Terrill et al., 1992). The PA in L. corniculatus increased the observed absorption of
amino acids from the small intestine of sheep (Waghorn et al., 1987), whereas the PA in L.
pedunculatus (Waghorn et al., 1994) provided ruminal protein protection, but did not
improve absorption.
The primary factor in the difference in activity of the two PAs may be differences in their
structure (Min et al., 2003). Structural studies of L. pedunculatus and L. corniculatus PAs
(Foo et al., 1996,1997; Hedqvist et al., 2000; Meagher et al., 2004) have shown that L.
pedunculatus PAs have a much higher prodelphinidin (PD) content. The structure of PAs as
well as their concentration in the plant are important factors determining their activity and
role in both agricultural systems (Min et al., 2003) and the natural environment (Zucker,
1983; Clausen et al., 1990; Ayres et al., 1997; Heil et al., 2002).
The genus Lotus contains many species, several of which have been used as a forage for
ruminants (Papadopoulos and Kelman, 1999), and the initial motivation for this study was
to identify additional Lotus species of potential value as forages, which might provide PAs
at appropriate concentrations and with desirable properties. Lotus spp. are found worldwide,
except for cold arctic regions and low land tropical areas of Asia, South and Central
America (Kirkbride, 1999a). The largest collection of Lotus genetic resources is in New
Zealand (Greene, 1999). A number of groupings of Lotus spp. have been constructed based
on plant morphology (Arambarri, 2000), and within L. corniculatus, genotypes have been
grouped based on specific characteristics or habitats (Steiner and Garcia de los Santos,
2001). However, relationships between phylogeny and geographical distribution and the
phytochemistry of Lotus PAs have not been investigated.
In this study, we compare the chemistry of PAs of forage cultivars of L. corniculatus,L.
pedunculatus, and L. tenuis with those of 12 additional Lotus spp. not currently used in
agriculture. Estimates of the concentration of PAs in all 15 species are reported. In addition,
the chemical characteristics of PA fractions from L. pedunculatus and L. corniculatus
(including new data on a “creeping”selection of L. corniculatus) are compared with new
findings on the composition of PAs of four additional Lotus species found to have a high
PA content in the initial screening: L. americanus,L. angustissimus,L. parviflorus, and L.
suaveolens.
J Chem Ecol (2006) 32: 1797–1816 1799
We applied a number of complimentary and independent approaches to characterize the
structure of the Lotus PA polymer mixtures. Whereas proanthocyanidin oligomers with DP as
high as 5 have been isolated and characterized as single entities (Hemingway et al., 1982;Foo
and Karchesy, 1991), the majority of the PA polymer in plants comprises mixtures of
oligomers (DP 5–10) and higher polymeric material (DP > 10) that must be characterized
collectively. PA polymer fractions prepared by Sephadex LH-20 chromatography have been
characterized by acid catalyzed degradation in the presence of benzyl mercaptan coupled with
high-performance liquid chromatography–photodiode array (HPLC-PDA) detection to
provide information on the nature of the terminal and extension units and the mean degree
of polymerization (mDP; Guyot et al., 2001;Guetal.,2002; Taylor et al., 2003; Sivakumaran
et al., 2004). Mass spectrometric methods [liquid chromatography–electrospray ionization–
mass spectrometry (LC-ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF-MS)] have been used to provide further characterization of
the PAs in terms of their polydispersity and interflavanoid linkages (A- or B-types), without
further isolation or chromatographic separation (Le Roux et al., 1998; Foo et al., 2000a).
These estimates of composition are supported by independent nuclear magnetic resonance
(NMR) estimates (Porter et al., 1982).
The results are discussed in terms of possible relationships between the concentration
and composition of the PAs of Lotus spp. and ecological factors.
Methods and Materials
Chemical
Analytical grade acetone and dichloromethane, HPLC grade methanol, acetonitrile, and
ascorbic acid were obtained from BDH Ltd., Auckland, New Zealand. Catechin,
epicatechin, gallocatechin, and epigallocatechin were obtained from Sigma, St Louis,
MO, USA. Benzyl mercaptan was obtained from Merck, Darmstadt, Germany. 2,5-
Dihydroxybenzoic acid was obtained from L. Light & Co. Ltd., Colnbrook, UK. Sephadex
LH-20 was obtained from Pharmacia, Sweden.
Plant Material
Herbage samples of 15 Lotus species were grown in pots at Grasslands Research Centre,
Palmerston North, New Zealand; L. corniculatus (birdsfoot trefoil, cv. “Grasslands
Goldie”), L. pedunculatus (big trefoil, cv. “Grasslands Maku”), L. tenuis (narrow-leaf
trefoil, cv. “Esmeralda”), L. americanus (American birdsfoot trefoil), L. angustissimus
(slender birdsfoot trefoil), L. arenarius,L. crassifolius (big deervetch), L. creticus,L.
decumbens,L. edulis,L. japonicus (L. corniculatus L. var. japonicus), L. ornithopodioides,
L. parviflorus (small-flower birdsfoot trefoil), L. schoelleri (L. corniculatus L. var.
schoelleri), and L. suaveolens (hairy birdsfoot trefoil) were harvested in July 2000, and
regrowth was sampled again in September 2000, freeze-dried, milled, and PA content
estimated by the vanillin–HCl assay. The four species with the highest PA content were
selected for further investigation, together with the established forage species L.
pedunculatus and L. corniculatus. Herbage samples of 25 individual plants of each of the
six Lotus species grown in pots were harvested in October 2001, freeze-dried, and milled.
The extractable PA content of the individual plants was estimated by the BuOH–HCl assay.
1800 J Chem Ecol (2006) 32: 1797–1816
Herbage samples of L. corniculatus,L. pedunculatus,L. americanus,L. angustissimus,L.
parviflorus, and L. suaveolens grown in a sand-frame were harvested during December
2001 to January 2002 and were stored at −20°C for extraction. These Lotus spp. were
grown again in the sand frame and sampled during December 2002 to January 2003 along
with two additional Lotus spp. in a breeding program: L. corniculatus (birdsfoot trefoil,
“Grasslands creeping selection”) and L. tenuis (narrow-leaf trefoil, cv. “Esmeralda”).
Herbage samples harvested in summer 2002–2003 were freeze-dried, milled, and analyzed
for PA content by the BuOH–HCl assay.
Colorimetric PA Assay
Plant samples harvested in 2000 were analyzed by the vanillin–HCl assay (Scalbert, 1992).
Individual plants harvested in 2001 and bulk plant samples harvested in 2002–2003 were
analyzed by the BuOH–HCl assay (Terrill et al., 1992). Freeze-dried plant samples (leaves
and stem) were ground in a Wiley mill (1-mm screen). For the 2000 and 2001 samples, “free”
PA concentrations were estimated, and for the 2002–2003 samples, a three-step extraction
procedure was performed to determine free, protein-bound, and fiber-bound PA. Measure-
ments were performed in duplicate and the mean value reported as a percentage (%) of DM. A
purified PA fraction from L. pedunculatus was used as a standard for calibration.
Proanthocyanidin Preparative Extraction
Frozen herbage of Lotus spp. (600 g/l) were extracted with acetone/water (7:3; v/v)
containing ascorbic acid (1 g/l) in a blender (Hallde VCM62 Varning, AB Hallde Maskiner,
Kista, Sweden) for 30 min and strained through two layers of cheesecloth to remove plant
material. The extract was concentrated in vacuo (40°C) to remove acetone and the aqueous
solution defatted with dichloromethane (4 × 1l). The aqueous layer was concentrated in
vacuo and subsequently freeze-dried to yield an aqueous acetone PA extract.
Purification of Proanthocyanidin Fractions
Each freeze-dried aqueous acetone PA extract (6 g) was dissolved in aqueous methanol
(1:1, v/v, 50 ml) and centrifuged at 4500 × gfor 10 min. The PA solution was applied to a
Sephadex LH-20 column (Pharmacia, SR 25/45) preconditioned with aqueous methanol
(1:1, v/v) and connected to a Pharmacia GradiFrac system. After loading, the column was
washed with aqueous methanol (1:1, v/v; fraction 1 100 ml and fraction 2 500 ml) at a flow
rate of 5 ml/min. The PA fractions were eluted with acetone/water (7:3, v/v; fraction 1
100 ml and fraction 2 500 ml) and analyzed by HPLC–PDA at 280 nm. PA fractions were
characterized by the observation of a broad unresolved hump at 280 nm in the HPLC trace,
combined, and concentrated in vacuo (40°C). The aqueous residues were freeze-dried to
yield PA fractions.
The initial aqueous methanol (1:1, 100 ml) eluate of L.americanus was characterized as
containing PAs by the presence of a broad unresolved hump in the HPLC-PDA at 280 nm.
The aqueous methanol (1:1) fractions were rechromatographed on a Sephadex LH-20
(Pharmacia, SR 25/45) column. An initial wash with water (100 ml) was followed by
elution with aqueous methanol (1:1, 500 ml), which yielded a high molecular weight
proanthocyanidin (HMWPA) fraction, and subsequent elution with acetone/water (7:3, v/v,
500 ml) yielded a medium molecular weight proanthocyanidin (MMWPA) fraction.
J Chem Ecol (2006) 32: 1797–1816 1801
Thiolysis
A method based on that described by Guyot et al. (1998) was utilized. A PA solution (4 mg/ml
in methanol) was prepared for each PA fraction. A subsample (50 μl) was placed into a vial
to which 3.3% (v/v) hydrochloric acid in methanol (50 μl) and 5% (v/v) benzyl mercaptan
in methanol (100 μl) was added. Each solution was heated to 40°C for 30 min in a heating
block and cooled to room temperature. An internal standard, dihydroquercetin in water
(100 μl, 2.5 × 10
−2
mg/ml solution), was added and the sample analyzed immediately by
reversed phase HPLC. Concentrations of terminal flavan-3-ol units and extender flavan-3-ol
thiol adducts were estimated by peak area integration at 280 nm. Responses relative to
dihydroquercetin were determined from standards. Response factors to PC and PD flavan-3-
ols (0.26 and 0.07, respectively) were the same as the corresponding benzylthioethers,
isolated from Dorycnium rectum PA fractions according to Sivakumaran et al. (2004)as
reported by Gu et al. (2002). Thiolysis 10-μl subsamples were analyzed by the method of
Meagher et al. (2004).
Mass Spectrometry
Electrospray ionization mass spectrometry data were acquired on a Shimadzu LC-MS
QP8000αin scan mode (m/z250–1400) and detection in the negative ion mode using the
conditions described by Meagher et al. (2004).
Matrix-assisted laser desorption/ionization time-of-flight mass spectra were acquired on a
Micromass M@LDI LR time of flight mass spectrometer, equipped with delayed extraction
and a N
2
laser, set at 337 nm. For positive reflectron mode spectra, an accelerating voltage
of 15 kV and a reflectron voltage of 2 kV were used. The PA fractions were reconstituted in
acetone/water (8:2, v/v; 0.5 mg/ml) and mixed with a matrix solution of 2,5-dihydroxy-
benzoic acid in acetone/water (8:2, v/v; 10 mg/ml) at a volumetric ratio of 1:1. The PA-
matrix solutions were deionized on a cation exchange cartridge (Strata SCX, 100 mg, 1 ml)
preconditioned with HCl (1 ml, 0.1 M), Milli-Q water (5 ml), and finally acetone/water
(8:2, v/v; 2 ml). The deionized PA–matrix solutions were spiked with a NaCl solution (0.1 M,
0.5 μl) to promote the formation of single ion adducts ([M + Na]
+
), and the mixture (1 μl)
was applied to a stainless-steel target plate and crystallized at room temperature prior to analysis.
13
C NMR. NMR spectra were recorded in methanol (CD
3
OD) at 90 MHz using a Bruker
400 MHz instrument.
Results and Discussion
Estimation of Proanthocyanidin Concentration
Herbage samples of 15 Lotus species in an initial study were harvested in winter 2000, and
regrowth was again sampled in spring 2000. The extractable PA content was estimated by
the vanillin–HCl assay (Table 1) and ranged from 0.2 to 7.4% of DM in winter and from
0.9 to 10.9% of DM in spring. The four species with the highest PA content were selected
for further investigation, together with the established forage species L. corniculatus,L.
pedunculatus, and L. tenuis.
Extractable PA concentrations were estimated for individual plants in 2001, and extract-
able, protein-bound, and fiber-bound PA concentrations were estimated in bulk material
1802 J Chem Ecol (2006) 32: 1797–1816
harvested during 2002 and 2003 using the BuOH–HCl assay. The estimates of PA concen-
tration by the two methods were in reasonable agreement. Extractable PA concentrations
ranged from 0.2 to 10.9% of DM (Table 1). There were variations in PA estimates between
sampling dates, but on both occasions in 2000, PA concentrations were consistently lower
for L. creticus,L. ornithopodioides,L. arenarius,L. crassifolius,L. japonicus,L. de-
cumbens,L. edulis,L. schoelleri, and L. tenuis than for the other species. The remaining
“high PA”Lotus spp. were retained for further examination. The PAs of these species were
predominantly extractable with bound PAs comprising between 5 and 13% of DM of the
total PAs in 2002–2003. At each sampling, the PA concentration was highest for L.
americanus (9.8% DM total PA in 2002–2003), and at three of four samplings, the PA
concentration was lowest for L. corniculatus (3.0% DM total PA in 2002–2003). PA
concentrations in L. pedunculatus,L. angustissimus,L. corniculatus “creeping”selection,
and L. parviflorus were intermediate (4.2–7.2% of DM total PA in 2002–2003). The PA
content of the “creeping”selection of L. corniculatus was higher than in the standard
cultivar, consistent with previous screening and selection (Rumball, unpublished observa-
tions). Only a trace amount of PA was determined in L. tenuis in agreement with reported
values (Kelman and Tanner, 1990; Strittmatter et al., 1992; Terrill et al., 1992).
Of the Lotus species evaluated, the highest PA concentration (>6% of DM) was found for
L. americanus, a bushy branched annual about 30 cm high, present in dry prairies and
rangeland, used as forage (Table 1). Moderate PA concentrations (<6% of DM) were found
in L. angustissimus,L. parviflorus, and L. suaveolens. These three species grow in habitats
such as dry grassland that are accessible to grazing (Kirkbride, 1999a,b). The latter two also
carry “hairy”foliage, which is generally regarded as a deterrent to predation. However, both
low and high PA species are associated with Mediterranean grasslands, and there is no clear
association between annual or perennial types and PA accumulation. The four species with
the lowest PA concentrations (Table 1)areL. tenuis,L. decumbens,L. japonicus,andL. edulis.
They are, respectively, (1) vigorous annual, (2) small and weak prostrate annual, (3) prostrate
and moderately vigorous perennial, and (4) moderately vigorous short-lived species (ILDIS).
It is difficult to observe any clear ecological pattern to the occurrence of PAs in the Lotus
spp. evaluated in this study. Although the ecological role of plant PAs has been extensively
examined, a robust theoretical basis for their ecological role is not yet available. The
interactions and trade-offs between plant growth and defense are complex (Stamp, 2003),
and investigations of a single secondary metabolite class such as PAs provide a limited
view of defensive chemistry. Thus, (Berger et al. 2003) found a complementary pattern of
distribution of PAs and proteinase inhibitors in Vicia species. The Lotus spp. we have found
to have low PA content may have alternative defensive chemistry or, in the case of the more
vigorous species, have greater tolerance of herbivory. Considerations of plant defense
would suggest that high-PA-containing plants might be found in environments with low
resource availability (Coley et al., 1985). Considerations of nutrient cycling suggest that
acidic and infertile soils might be favored (Kraus et al., 2003), and considerations of
photoprotection suggest that they might be found at low latitudes and high altitudes (Close
and McArthur, 2002). To directly address these environmental factors would require an
extensive investigation of Lotus spp. in their natural environment that was beyond the scope
of the present study.
Fractionation of Proanthocyanidins
Frozen herbage of each Lotus spp. was extracted with aqueous acetone. A single extraction
provided sufficient material for fractionation. The composition of PA from a single
J Chem Ecol (2006) 32: 1797–1816 1803
Table 1 Estimated proanthocyanidin concentration (% of Dm) in the herbage of the Lotus spp
Lotus spp. 2000 2001 2002–2003 Distribution Season
e
Habitats
f
E
a
E
b
E
c
(SD) E
d
P
d
F
d
T
d
L. americanus 7.4 10.9 7.1 (0.7) 9.4 0.4 0.1 9.8 Canada
g
, USA, Mexico Annual Forage
L. pedunculatus 4.9 5.5 2.9 (0.4) 6.4 0.7 0.1 7.2 Europe
h
, Turkey, Africa, USSR,
Argentina,
Australia
Perennial Forage; Mediterranean
grasslands
L. parviflorus 3.1 5.6 2.9 (0.4) 5.3 0.5 0.1 5.9 Africa
i
, Australia, Europe, Middle
East, Azores
Annual
L. angustissimus 3.7 6.4 2.9 (0.9) 4.7 0.5 0.2 5.3 China
i
, Europe, Middle East,
Siberia, Azores
Perennial Mediterranean grasslands
L. corniculatus sel. 4.0 0.5 0.1 4.6
L. suaveolens 4.7 5.1 4.0 (0.7) 3.8 0.3 0.1 4.2 Western Mediterranean basin
h
,
UK, Africa
Annual Mediterranean grasslands
L. corniculatus 2.2 5.5 1.3 (0.6) 2.8 0.1 0.1 3.0 Widespread in Europe
h
, China Perennial
shrub
Forage; Mediterranean
grasslands; Afromontane
grassland
L. creticus 1.9 ND Africa
i
, Australia, Europe, Middle
East, Canada, Azores
Perennial Mediterranean grasslands;
Mediterranean/Sahara
regional transition
zone; grassland
L. ornithopodioides 1.3 1.7 Africa
i
, Europe, USSR, USA,
Asia
Annual Mediterranean woodland;
Mediterranean
grasslands
L. arenarius 0.7 1.7 Senegal
i
, Egypt, Morocco,
Spain
Annual Sahara regional transition
zone: desert
1804 J Chem Ecol (2006) 32: 1797–1816
L. crassifolius 0.6 ND Western US
j
, United Arab
Emirates
i
Perennial
L. japonicus 0.4 1.3 Japan
h
, Korea, China, Tibet Perennial
L. decumbens 0.3 1.4 Africa, Australia, Europe Perennial Mediterranean grasslands;
Mediterranean/Sahara
regional transition
zone; grassland
L. edulis 0.3 0.7 Africa
i
, Europe, Middle East Annual Mediterranean woodland;
Mediterranean
grasslands
L. schoelleri 0.3 2.9 Ethopia
i,k
, Kenya, Sudan Perennial
(diploid)
Afromontane grassland
L. tenuis 0.2 1.4 0.6 0.1 0.1 0.8 Africa
h
, Argentina, China, Middle
East, New Zealand
Perennial Forage
E = extractable PA; P = protein-bound PA; F = fiber-bound PA; T = total PA; SD = standard deviation; ND = not determined, insufficient herbage for sampling.
a
Plants, herbage harvested winter 2000.
b
Plants, herbage regrowth harvested spring 2000.
c
Individual plants (25), herbage harvested spring 2001.
d
Bulk plants, herbage harvested summer 2002–2003.
e
Nonclimbing herb.
f
International Legume Database and Information Service (ILDIS) at http://www.ildis.org/.
g
Steiner, 1999.
h
Kirkbride, 1999a.
i
Kirkbride, 1999b.
j
USDA Natural Resource Conservation Service Plants profile.
k
Agriculture and Agri-Food Canada Taxonomy at http://pgrc3.agr.ca.
J Chem Ecol (2006) 32: 1797–1816 1805
extraction had previously been found in the cases of L. pedunculatus and L. corniculatus
(Meagher et al., 2004) and was similar to that reported following exhaustive extraction of
plant material (Foo et al., 1997). The extracts were defatted and fractionated on a Sephadex
LH-20 column. Low molecular weight proanthocyanidin (LMWPA) polymer fractions were
recovered by elution with aqueous acetone and characterized by the presence of a broad
unresolved hump at 280 nm by HPLC-PDA. The first aqueous acetone fraction containing
flavanol monomers and dimer PAs was kept separate from the second polymeric PA
fraction. The chemical nature of these polymer fractions was determined by
13
C NMR, the
thiolysis degradation reaction, ESI-MS, and MALDI-TOF-MS.
In the case of L. americanus, a portion of the PA was not retained on the Sephadex LH-
20 column and was separated into MMWPA and HMWPA fractions that were characterized
separately (below). Similar observations have been reported for L. pedunculatus (Meagher
et al., 2004) and the PA-containing plant D. rectum (Sivakumaran et al., 2004). The PA
extracted from L. tenuis was not characterized as it was obtained in trace amounts.
Characterization of Proanthocyanidin Chemical Composition
The chemical composition of fractions was characterized by thiolysis and NMR. Fractions
were acid hydrolyzed in the presence of benzyl mercaptan, which yielded the extension
units as flavan-3-ol-4-benzylthioether adducts and terminal units as flavan-3-ols (Fig. 1).
The compositional data and mDP were determined for each Lotus spp. PA fraction
(Table 2). Catechin was the dominant terminal unit in the PA fractions from most Lotus
species, with the exceptions of L. parviflorus where epicatechin was dominant and L.
suaveolens where equal proportions of catechin and epicatechin were found. In contrast,
there was more variation in the extension units: epigallocatechin was the dominant
extension unit of L. americanus,L. corniculatus “creeping”, and L. pedunculatus, whereas
epicatechin was the dominant extension unit of L. corniculatus and L. angustissimus (53
and 66% mol/mol, respectively). Epicatechin and epigallocatechin were found in equal
proportions for L. parviflorus and L. suaveolens extension units. In contrast, L.
angustissimus is the one species dominated by epicatechin in both the extender and
terminal units.
Thiolysis gave a direct insight into the relative stereochemistry at C2 and C3 in the PA
units. The cis/trans ratio of PA fractions was determined for the terminal and extension
units for each species (Table 2). The terminal units were predominantly of trans
configuration, whereas the extensions units were predominantly cis.
For each of the Lotus spp., a low molecular weight PA fraction (LMWPA) was isolated.
The mDP determined by thiolysis for these fractions (Table 2) varied from 8.1 to 16.
Variation in mDP of these fractions may be affected by the presence of flavan-3-ol
monomers that were detected by ESI-MS (not shown in Table 3) in the LMWPA fractions
of L. angustissimus,L. corniculatus,L. parviflorus, and L. suaveolens. For L. americanus,
the mDP of the HMWPA and MMWPA fractions was estimated to be 97 and 40,
respectively. These fractions were of higher mDP than the comparable fractions previously
reported for L. pedunculatus (Meagher et al., 2004). The composition of the terminal units
of the polymer fractions (LMWPA, MMWPA, and HMWPA) of L. pedunculatus (Meagher
et al., 2004) and L. americanus was not significantly different between fractions of differing
mDP. The epigallocatechin content of the extension units increased with the increasing
mDP (Table 2) for L. pedunculatus, but such a trend was not as apparent for L. americanus.
The
13
C NMR spectrum of PA polymers from the Lotus spp. gave broad peaks,
indicating the polymeric nature of the PA. Estimates of the cis/trans ratio and PC/PD ratio
1806 J Chem Ecol (2006) 32: 1797–1816
from the NMR data based on the calculation described by Porter et al. (1982) were
generally in good agreement with the estimates from thiolysis (data not shown).
The molecular weight estimation from the carbon signal intensity gave approximate values
of 7–10 mDP for the Lotus spp. LMWPA fractions and 15–18 mDP for MMWPA fraction of
L. americanus, somewhat lower than the estimates from thiolysis. Because of the limited
dynamic range of the NMR method, calculation of a credible mDP value from the NMR
data was not feasible for the HMWPA fraction, but the NMR observations were consistent
with a large mean molecular weight as found by thiolysis. Similar higher molecular weight
PAs with mDP ranging from 33.8 to 189 have been reported for apple (Guyot et al., 2001),
grape skin (Monagas et al., 2003), and D. rectum (Sivakumaran et al., 2004).
Determination of Polydispersity of Proanthocyanidins.
The polydispersity of the PA fractions was analyzed by ESI-MS in negative ion mode and
MALDI-TOF-MS in positive mode. Although the MS ion intensities are not a quantitative
measure of oligomer species, the patterns reflect polymer heterogeneity and polydispersity.
The observed singly charged [M −H]
−
and doubly charged [M −2H]
2−
ions, corresponding
to the molecular ion masses of PA oligomers ranging from dimer (DP2) through to
heptamer (DP7) observed in negative ESI-MS, are shown in Table 3. Multiple-charged
species are observed for DP > 4, as with longer chain lengths, the charge can be better
Table 2 Comparison of chemical composition of proanthocyanidin fractions in the Lotus spp. as determined
by thiolytic degradation
Lotus spp. Terminal (%) Extender (%)
cis/trans PD/PC mDPGC EGC C EC GC EGC C EC
L. pedunculatus
LMWPA 26 14 46 14 16 68 4 14 76:24 80:20 12
MMWPA
a
23 12 51 14 6 46 2 15 88:12 80:20 18
HMWPA
a
25 11 51 13 13 72 3 13 83:17 84:16 44
L. corniculatus
LMWPA 2 2 75 21 5 39 3 53 84:16 40:60 8.7
L. corniculatus “creeping”
LMWPA 17 4 61 18 7 62 4 27 85:15 66:34 14
L. suaveolens
LMWPA 4 4 46 47 12 41 4 43 80:20 48:52 8:1
L. parviflorus
LMWPA 1 4 32 63 10 40 4 45 85:15 46:54 8.1
L. americanus
LMWPA 18 2 78 3 9 59 5 27 80:20 65:35 16
MMWPA 20 0 75 2 6 66 3 25 90:10 70:30 40
HMWPA 22 0 75 3 5 75 2 19 92:08 80:20 97
L. angustissimus
LMWPA 1 1 4 94 16 17 1 66 85:15 30:70 8.7
Percentage of extender and terminal units, PC and PD units given as mol/100 mol.
C = catechin, EC = epicatechin, EGC = epigallocatechin, GC = gallocatechin, mDP = mean degree of
polymerization, PC = procyanidin, PD = prodelphinidin.
a
From Meagher et al. (2004).
J Chem Ecol (2006) 32: 1797–1816 1807
Table 3 Observed m/zvalues of proanthocyanidin ions in negative ESI-MS analysis of lotus spp. polymer fractions
DP Ions L. pedunculatus L. corniculatus L. corniculatus
“creeping”
L. suaveolens L. parviflorus L. americanus
a
L. angustissimus
2[M−H]
−
593, 609 577, 593, 577, 593, 609 577, 593 577, 593 577, 591, 593, 607 577
3[M−H]
−
881, 897, 913 865, 881, 897 865, 881, 897 865, 881, 897 865, 881, 897 865, 881, 895, 897 865, 881, 897
4[M−H]
2−
584, 592, 600, 608 576, 584, 592 584, 592, 600, 608 584, 592 576, 584, 592 592, 608 576, 584, 592
5[M−H]
2−
720, 728, 736,
744, 752, 760
720, 728, 736, 744 720, 728, 744,
752, 760
720, 728, 736, 744 720, 728, 736, 744 720, 727, 728,
735, 736, 744,
752, 760
720, 728, 736
6[M−H]
2−
880, 896, 904, 912 872, 880, 888 864, 872, 888,
896, 904, 912
864, 880, 888, 896 864, 872, 880,
888, 896
864, 880, 888,
896, 903, 904,
911
864, 872
7[M−H]
2−
1032, 1040, 1048,
1056
1008, 1016, 1024,
1032, 1040, 1049
1008, 1016, 1024,
1032, 1040,
1008, 1016, 1024,
1033
1008, 1016, 1024,
1032, 1040, 1048
1016, 1032, 1048,
1047, 1048,
1008
1048, 1056 1056, 1063, 1064
Numbers in bold represent the most intense ion observed.
a
Trimer [M −H]
j
at m/z 895, tetramer [M −2H]
2−
or dimer [M −H]
−
at m/z607, pentamer [M −2H]
2−
at m/z735, and hexamer [M −2H]
2−
at m/z903 were observed,
supporting the presence of A-type linkages in the ESI mass spectrum (see Table 4for explanation of the calculated molecular ion masses).
1808 J Chem Ecol (2006) 32: 1797–1816
distributed, minimizing repulsive forces in the polymer chain (Foo et al., 2000b). Positive-
ion MALDI-TOF-MS of the PA fractions from the seven Lotus spp. were acquired, and
trimer (DP3) to heptamer (DP7) homo- and heteropolymers of LMWPA fractions were
detected as singly charged (M + Na)
+
adducts (Fig. 2). Monomers (DP1) and dimers (DP2)
were not detected by MALDI-TOF because of noise and matrix interference. By
comparison with the estimated mDP for these LMWPA fractions (Table 2), the ion
intensities are biased toward the lower oligomers, as the MALDI-TOF in reflectron mode
gives lower sensitivity for the larger ions because of greater breakdown as the result of
longer flight paths and the postacceleration process (Yang and Chien, 2000). The calculated
molecular ion masses for ESI and MALDI-TOF ranging from trimer (DP3) to hexamer
(DP6) are shown in Table 4. The MALDI-TOF-MS were enhanced by desalting by cation
exchange and subsequent addition of NaCl to increase the [M + Na]
+
ion signal and reduce
the [M + K]
+
ions naturally present (Ohnoshi-Kameyama et al., 1997; Krueger et al., 2003).
With [M + Na]
+
adduct ions dominant in the spectrum, the number of hydroxyl functions
present in a PA polymer unit can be identified (Table 4).
The most intense ions observed by MALDI-TOF-MS for the trimer (DP3) to heptamer
(DP7) series of Lotus spp. LMWPA fractions (Fig. 2) are generally in agreement with the
most intense ions observed by ESI-MS as singly charged trimer (DP3) species or doubly
charged tetramer (DP4) to pentamer (DP5) species (Table 3). Heteropolymer ions
(containing both PC and PD units) dominate, except in the case of L. angustissimus (PC-
only units dominant). Similar heteropolymer molecular forms and ionization patterns in a
variety of plants have been reported in the literature (Behrens et al., 2003; Krueger et al.,
2003; Taylor et al., 2003). The data for L. corniculatus is consistent with those from a study
of several L. corniculatus cultivars reported by Hedqvist et al. (2000). Ions from the
polymeric HMWPA and MMWPA fractions with mDP > 17 of L. americanus and L.
pedunculatus were not detected by ESI-MS or MALDI-TOF-MS.
Proanthocyanidin oligomer ions observed in the ESI mass spectrum of the L. americanus
LMWPA fraction suggest the presence of A-type interflavan linkages (Tables 3and 4)in
that doubly charged ions were observed that were one m/zunit less than that reported by
Foo et al. (1996,1997) for B-type linkages for Lotus PAs. Correspondingly, in the MALDI-
TOF-MS from L. americanus PA, a series of (singly charged) ions was observed that was
two m/zunits lower than the species with B-type interflavan linkages (Fig. 2f, insert),
indicative of A-type linkages (Table 4). Partial thiolysis results (not shown) suggested the
presence of A-type linkages in the terminal units. The A-type interflavan linkage is known
in PAs from cranberry, which are terminated by A-type linkages (46%; Foo et al., 2000a;
Gu et al., 2002), and cinnamon (Anderson et al., 2004), but has not been reported for forage
legume PAs.
Proanthocyanidin Structure, Geography, and Ecology
These results demonstrate that PAs from Lotus spp. differ widely in both concentration and
structure. The extractable PA fractions are structurally heterogeneous differing in terms of
constituent flavan-3-ol units, mDP, and dispersion of oligomers. Whereas the relationship
between PA concentrations and ecology and geography is weak (above), the PC/PD ratio in
the PA fractions (Table 2) shows a relationship with the geographical distribution of species
(Table 1). Lotus spp. dominated by PC-type units L. angustissimus (70%), L. corniculatus
(60%), and L. parviflorus (54%) have broad distribution and are adapted to a variety of
habitats and have moderate PA concentrations (<6% of DM). Lotus varieties adapted to
warm environments tend to be more widely distributed than those adapted to colder envi-
J Chem Ecol (2006) 32: 1797–1816 1809
Table 4 Calculated masses of poly flavan-3-ol ions
Oligomers PC PD No. of linkages ESI-MS MALDI-TOF-MS
A-type B-type [M −H]
1−
[M −H]
2−
[M + Na]
+
Trimer 3 0 0 2 865 889
3 0 1 1 863 887
2 1 0 2 881 905
2 1 1 1 879 903
1 2 0 2 897 921
1 2 1 1 895 919
0 3 0 2 913 937
0 3 1 1 911 935
Tetramer 4 0 0 3 576 1177
4 0 1 2 575 1175
3 1 0 3 584 1193
3 1 1 2 583 1191
2 2 0 3 592 1209
2 2 1 2 591 1207
1 3 0 3 600 1225
1 3 1 2 599 1223
0 4 0 3 608 1241
0 4 1 2 607 1239
Pentamer 5 0 0 4 720 1466
5 0 1 3 719 1464
4 1 0 4 728 1482
4 1 1 3 727 1480
3 2 0 4 736 1498
3 2 1 3 735 1496
2 3 0 4 744 1514
2 3 1 3 743 1512
1 4 0 4 752 1530
1 4 1 3 751 1528
0 5 0 4 760 1546
0 5 1 3 759 1544
Hexamer 6 0 0 5 864 1754
6 0 1 4 863 1752
5 1 0 5 872 1770
5 1 1 4 871 1768
4 2 0 5 880 1786
4 2 1 4 879 1784
3 3 0 5 888 1802
3 3 1 4 887 1800
2 4 0 5 896 1818
2 4 1 4 895 1816
1 5 0 5 904 1834
1 5 1 4 903 1832
0 6 0 5 912 1850
0 6 1 4 911 1848
Mass calculations were based on the equation 2 + 288a+ 304b+ 23, where 2 is the molecular weight of two
additional hydrogen atoms of terminal flavan 3-ol units, ais the number of PC units, bis the number of the
PD units, and 23 is the atomic weight of sodium. Formation of the one A-type linkage results in the loss of
two hydrogen atoms.
1810 J Chem Ecol (2006) 32: 1797–1816
Fig. 2 MALDI-TOF-MS (positive reflectron mode) of a series of polymeric proanthocyanidin fractions from
(a) L. pedunculatus, (b) L. corniculatus, (c) L. corniculatus “creeping”, (d) L. suaveolens, (e). L. parviflorus,
(f) L. americanus, and (g) L. angustissimus. The most intense ions are shown. The inset is an enlarged
spectrum of the tetramer series showing different chemical constitutions. See Table 4for explanation of the
calculated molecular ion masses
J Chem Ecol (2006) 32: 1797–1816 1811
ronments (Steiner, 1999). L. pedunculatus and L. americanus PAs differ from other Lotus
spp. examined here not only in having the highest PA concentrations, but also in polymer
dispersion, containing higher mDP (>30) HMWPA polymer fractions, and in polymer
composition, being dominated by PD-type extender units. An intermediate composition was
observed for the “creeping”selection of L. corniculatus with a PD content of extender
groups in the LMWPA fraction much higher than that for the standard L. corniculatus
Fig. 2 (continued)
1812 J Chem Ecol (2006) 32: 1797–1816
cultivar and comparable to that for L. americanus. However, no HMWPA fraction was
separated in this case. The “creeping”selection is derived from material collected in
Morocco, subsequently crossed and introgressed with standard agricultural cultivars of L.
corniculatus (Rumball, unpublished observations). The observed structural differences in
PAs suggest that there may be considerable variation between L. corniculatus populations
in the wild. The sole “New World”species studied, L. americanus, contains PAs with
doubly linked (A-type) units, and this is unique among reported PAs of not only these seven
Fig. 2 (continued)
J Chem Ecol (2006) 32: 1797–1816 1813
Lotus spp. but also legumes in general (Koupai-Abyazani et al., 1993; Foo et al., 1982,
1996,1997,2000b; Sivakumaran et al., 2004).
Functional relationships between the differences observed and between the structures
and estimated concentrations of PAs in these Lotus spp. and the ecological niche the plants
occupy remain to be elucidated. This would require study of the variations in chemical com-
position of Lotus PAs in terms of growth period, season, and other environmental factors, as
well as a detailed examination of their interactions with the environment. However, these
findings draw attention to the need to consider the complexities of PA structure as well as
the concentration of PAs in considering the ecological role of Lotus PAs and their effects on
herbivores both in nature and in agricultural systems.
Acknowledgments We thank the New Zealand Foundation for Research Science and Technology Sustainable
Development portfolio for funding. We acknowledge Reg Keogh and Willy Martin for their assistance with
analyses of PA content of Lotus species.
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