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Characterisation of Proanthocyanidins from seeds of Perennial Ryegrass (Lolium perenne L.) and Tall Fescue (Festuca arundinacea) by Liquid Chromatography/Mass Spectrometry. Karl Fraser, Vern Collette, and Kerry R. Hancock J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02563 • Publication Date (Web): 17 Aug 2016 Downloaded from http://pubs.acs.org on August 19, 2016

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Journal of Agricultural and Food Chemistry

Characterisation of Proanthocyanidins from seeds of Perennial Ryegrass (Lolium perenne L.) and Tall Fescue (Festuca arundinacea) by Liquid Chromatography/Mass Spectrometry.

Karl Fraser, Vern Collette and Kerry R. Hancock.

Forage Improvement, AgResearch Grasslands, Private Bag 11008, Palmerston North, New Zealand.

Corresponding Author: Karl Fraser Tel: +64-6-351-8222 Fax: +64-6-351-8042 E-Mail: [email protected]

1 ACS Paragon Plus Environment


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1

ABSTRACT:

2

Perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) are

3

forage species of the grass family (Poaceae) that are key components of temperate

4

pasture-based agricultural systems. Proanthocyanidins (PAs) are oligomeric

5

flavonoids which when provided as part of a farm animal’s diet, have been reported

6

to improve animal production and health. Up to now, forage grasses have been

7

deemed not to produce PAs. We report here for the first time the detection of

8

polymerised PAs in aqueous methanolic extracts of seed tissue of both perennial

9

ryegrass and tall fescue, using LC-MS/MS. We have determined the structure of the

10

PAs to be trans-flavan-3-ol based, consisting predominantly of afzelechin and

11

catechin, and linked primarily by B-type bonds. Investigations into the leaf tissue of

12

both species failed to detect any PAs. This discovery opens the possibility of using

13

genetic engineering tools to achieve tannin accumulation in leaf tissue of perennial

14

ryegrass and tall fescue.

15

16

KEYWORDS: Proanthocyanidin, Lolium perenne, Festuca arundinacea, forage

17

grasses, perennial ryegrass, tall fescue, procyanidins, propelargonidins, afzelechin,

18

oligomers, condensed tannins, electrospray mass spectrometry, LC-MS/MS.


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INTRODUCTION:

20

Proanthocyanidins (PAs), also known as condensed tannins, are polyphenolic plant

21

secondary metabolites which accumulate in a wide range of plant tissues

22

Moderate concentrations of PAs can play an important role in forage–based animal

23

production systems where forages with foliar PAs have been shown to protect

24

excess dietary protein from rumen degradation, leading to an increase of protein by-

25

pass to the ruminants’ gut with subsequent increase in milk and meat production

26

PAs also reduce the risk of excessive foam and gas accumulation in the rumen

27

(bloat) 5, which is a serious problem associated with low tannin pastures and which,

28

if untreated, can result in animal losses. Moreover, by protecting forage protein from

29

degradation in the rumen, PAs help to reduce both ammonium excretion in urine and

30

methane production, resulting in significant reductions in emissions of the potent

31

greenhouse gases methane and nitrous oxide from pastures 6, 7.

32

Proanthocyanidins can be divided into three types based on the hydroxylation

33

patterns of their monomer units and the linkages between the multiple monomer

34

units that form the oligomers/polymers (Figure 1). These monomer units are the

35

(epi)afzelechins, (epi)catechins, and (epi)gallocatechins, giving rise to the more

36

common description of the monomer units based on the number of hydroxylations as

37

propelargonidin (PP), procyanidin (PC), and prodelphinidin (PD) respectively (Figure

38

1A). The mixtures of oligomers and polymers composed of flavan-3-ol units are

39

linked primarily via C4→C8 or C4→C6 bonds, and these linkages are designated B-

40

type. The flavan-3-ol units can also be doubly linked by an additional ether bond

41

between C2→O7 (known as A-type) (Figure 1B). In general, the most common form

42

of PAs found in nature are the procyanidins and prodelphinidins, while the

43

propelargonidins are much less common 8, 9. The number of monomer units can vary

1

2-4

.

.



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and this is described as the degree of polymerisation (DP). Thus, given the different

45

monomer units, potentially mixed stereochemistry and linkages, and varying DP,

46

PAs can occur as highly complex mixtures of chemical structures, which in turn could

47

produce differing levels of biological properties 10.

48

PAs have been observed to be widely distributed in forage and grain legumes,

49

shrubs, cereals, and can also be found in the cell walls and vacuoles of bark, stem,

50

leaf, flower and seeds of many dicotyledonous plants

51

PAs are depleted in the vacuoles of mature leaves entering senescence, suggesting

52

that plants may recycle these compounds

53

has been observed in a number of forage species and has been recently

54

summarized 4.

55

A number of monocot species, such as sorghum (Sorghum bicolor), barley

56

(Hordeum vulgare L.), rice (Oryza sativa), wheat (Triticum aestivum L.) and some

57

red finger millets (Eleusine coracana) have been shown to contain forms of

58

oligomeric or polymeric PAs solely found in the seed

59

from elite cultivars of other monocots such as maize, although wild ecotypes

60

producing other secondary compounds including flavones and flavonoids such as

61

phlobaphenes and anthocyanin are known 13, 16.

62

Perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) are

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forage species of the monocot grass family (Poaceae) and are key components of

64

temperate pasture systems, providing a cost-effective source of high quality foliage

65

feed for grazing ruminants, with improved animal performance and productivity.

66

However, information regarding the presence or absence of PAs within species of

67

Poaceae is very limited. Previous published studies of the Poaceae family have used

12

11

. It has been suggested that

. The location of PAs in specific tissues

13-15

. PAs are typically lacking


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chemical staining techniques which are not specific for PAs. Ellis (1990) reported

69

varieties of tropical grasses, primarily from southern Africa contained tannin–like

70

substances (TLS) deposits in epidermal cells, within 1104 species from 290 genera,

71

suggesting that these grasses contained polyphenols and possibly PAs

72

generally been considered to be absent from all temperate and high latitude grasses

73

18, 19

74

measurable levels of tannin in both leaf and flower tissue

75

concentration in perennial ryegrass using staining approaches determined that levels

76

in leaf tissue were indistinguishable from zero

77

amounts of PAs in leaf tissue of perennial ryegrass and summer grass (Digiteria

78

sanguinalis) has previously been observed

79

conflicting information and warrant further investigation with more sensitive

80

techniques like LC-MS/MS.

17

. PAs have

, with the exception of Holcus lanatus which was found to contain low but

22

20

. Early studies into CT

21

. However, the presence of low

. These previous reports contain

81



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MATERIALS AND METHODS:

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Chemicals and Reagents:

84

Analytical-grade methanol and acetic acid were obtained from BDH (Poole, UK). (+)-

85

Catechin hydrate, (–)-epicatechin, (–)-epigallocatechin, (–)-gallocatechin and formic

86

acid were purchased from Sigma-Aldrich (St Louis, MO). (–)-Epiafzelechin was

87

obtained from TransMIT GmbH (Gießen, Germany). Ultrapure water was obtained

88

from a Milli-Q® system (Millipore, Bedford, MA). OPTIMA LC-MS grade acetonitrile

89

was purchased from ThermoFisher (Auckland, New Zealand).

90 91

Sample Preparation:

92

Seed of fourteen perennial ryegrass and fifteen tall fescue accessions were obtained

93

from the Margot Forde Germplasm Centre (see Supplementary Table S1 for

94

accession numbers). Plants were grown and leaf material harvested and extracted

95

and analysed along with the parent seed. To extract flavonoids for LC-MS/MS

96

analysis, seed (50 mg) or leaf tissue (500 mg fresh weight) were frozen in liquid N2,

97

ground to a fine powder with a mortar and pestle and extracted with either 2 mL or 5

98

mL respectively of aqueous acetic acid (0.1% v/v): methanol (80:20 v/v) for 30 min at

99

4oC. Debris was pelleted in a microcentrifuge at 13,000 rpm for 10 min. The

100

supernatant was removed and placed at -20oC for 30 min. The extract was

101

evaporated to dryness under a stream of nitrogen and reconstituted in 500 µL of

102

100% water to yield the crude proanthocyanidin extract.

103 104

LC-MS/MS Analysis Conditions:

105

The analyses were conducted using a Thermo Finnigan Surveyor HPLC coupled to a

106

linear ion trap mass spectrometer (Thermo LTQ, both San Jose, CA, USA) with the 6 ACS Paragon Plus Environment

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23

107

same column and detection parameters as described previously

. Briefly, a 5 µL

108

aliquot of sample was injected onto a 150 x 2.1mm Luna C18(2) column

109

(Phenomenex, Torrance, CA, USA) held at a constant 25°C. The flow rate was 200

110

µL min-1 and gradient elution was performed with: solvent A = 0.1 % (v/v) formic acid

111

in water; solvent B = 0.1% (v/v) formic acid in acetonitrile. The mass spectrometer

112

was set for electrospray ionisation in positive mode, with a spray voltage of 4.5 kV,

113

capillary temperature 275°C. Flow rates of nitrogen sheath gas, auxiliary gas, and

114

sweep gas were set (in arbitrary units/min) to 20, 10, and 5, respectively. The MS

115

was programmed to scan from 150-2000 m/z (MS1 scan), then sequentially perform

116

product ion scans for selected masses on the different combinations of PP, PC and

117

PD monomer and dimer masses in the first MS method, and the combinations of

118

trimer masses in the second method (Table 1), with isolation windows for each

119

selected m/z value of 2.0 mu and a collision energy of 35%. Each sample was

120

measured twice by LC-MS/MS, using each of the MS data collection methods listed

121

in Table 1. The separation of the monomer/dimer and trimer MS/MS scan events into

122

two methods was required to ensure adequate MS scans were collected across the

123

chromatographic peaks.

124 125

126

127

RESULTS AND DISCUSSION:

128

Our previous studies using this MS/MS approach on a linear ion-trap

129

demonstrated that results from the positive ESI analysis were up to twice as

130

sensitive as the negative ESI for the PAs we monitored. Furthermore, similar to

131

observations of Li et al., (2007)

23

24

, we noted that proanthocyandin product ion 7

ACS Paragon Plus Environment


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spectra collected in positive mode contained a greater number of diagnostic ions for

133

compound identification than negative mode, thus all data reported here was

134

collected in positive ESI.

135 136

PA composition of seed extracts:

137

Initial LC-MS investigations into the averaged LC-MS1 spectra of both perennial

138

ryegrass and tall fescue seed extracts revealed the presence of [M+H]+ ions

139

consistent with the masses of PP and PC monomers, and both homogenous and

140

mixed B-type oligomers up to a chain length of DP6 (Figure 2). The composition of

141

the monomer and major dimer and trimer oligomers were further investigated and

142

confirmed by examination of the MS/MS fragmentation patterns (see section below).

143

Upon closer examination of the oligomer ion groupings observed in the MS1

144

spectrum, it was possible to detect the presence of ions corresponding to oligomers

145

with a single A-type bond in both perennial ryegrass (Figure 3) and tall fescue seed

146

extracts (data not shown). The A-type oligomers (bridged) can be distinguished in

147

the ESI-MS1 spectrum as the ion masses are 2 Daltons (Da) lower than the un-

148

bridged counterpart. Of note, no ions were detected for oligomers containing more

149

than one A-type bond (e.g. 4 or 6 Da lower than the un-bridged counterpart),

150

potentially due to them being formed in very low abundance. The low levels of A-type

151

oligomer PAs tentatively assigned in the MS1 spectra of the perennial ryegrass and

152

tall fescue seed extracts were not unexpected, as low levels of A-type PAs have

153

previously been observed in many plants in which B-type PAs are observed

154

26

155

reversed phase chromatography method used here does not create sharp well-

156

defined peaks of these higher molecular weight oligomers. Thus the practical limit

8, 14, 25,

. While it is possible that oligomers of DP >6 also occur in the seed extracts, the


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ion detection comes more from the issue of chromatographic broadening than

158

ionisation and MS scan range. Techniques such as hydrophilic interaction

159

chromatography with ESI detection or MALDI-TOF MS have been used to detect

160

oligomers of larger DP

27, 28

.

161 162

To investigate the PA composition further, a targeted MS2 method was developed,

163

based on modifications to a procedure previously used in our laboratory

164

the predicted parent masses of the monomers, dimers and trimers were isolated with

165

the ion-trap, fragmented, and the MS2 spectral data collected and filtered post-run for

166

known PA fragmentation ions. This approach collects fragmentation data useful for

167

structural identification/classification, but is also a more selective and sensitive

168

method for detecting the presence and abundance of these compounds compared to

169

LC-MS1 spectral data alone. Thus the samples were run with this approach, and all

170

further results and discussion refer to data from the targeted MS2 scans.

23

, where

171 172

PA composition of leaf extracts:

173

No PA monomers or oligomers were detected in either the MS1 or targeted MS2

174

chromatograms from the leaf tissue extracts of either the perennial ryegrass or tall

175

fescue accessions used in this study (data not shown). This confirms the earlier

176

findings of Terrill et al. (1992), and conflicts with the previous study of Jackson et al.

177

(1996). The perennial ryegrass accessions used in the previous studies were not

178

described, so while our study screened 14 major accessions, it is possible the

179

Jackson study used a different cultivar from those used in this study.

180 181

PA monomer identification and composition in seed extracts:



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Monomer composition was assigned based on comparison of retention times with

183

authentic purchased standards and matching observed MS2 spectra. As there is no

184

commercially available standard for afzelechin, an epiafzelechin standard was

185

purchased and an epimerisation reaction performed by heating an aqueous solution

186

of 100 µg mL-1 to 120°C for 30 minutes

187

cis and trans PP monomers. This resulted in a 50:50 mixture of afzelechin and

188

epiafzelechin, from which the afzelechin peak matched both the retention time and

189

MS2 spectrum of the detected PP monomer peak in the extracts of both perennial

190

ryegrass (Figure 4) and tall fescue (data not shown).

191

The monomer composition of both the perennial ryegrass and tall fescue seed

192

extracts consisted exclusively of trans-flavan-3-ols, predominantly afzelechin and

193

catechin, with traces of gallocatechin detected in tall fescue only (Figure 5,

194

components 1-3 in Table 2, Table S2). Thus, the seed extracts were completely

195

devoid of any cis-flavan-3-ols (epiafzelechin, epicatechin, epigallocatechin),

196

suggesting the biochemical pathway for producing cis-flavan-3-ols is not active (or

197

does not exist) in the seeds of either species. The monomer percentage

198

compositional patterns were similar across the 14 perennial ryegrass accessions

199

monitored, with afzelechin ranging from 16.1%–41.6%, and correspondingly, the

200

catechin ranging from 58.4%–83.9%. Consistent monomer composition was also

201

observed across 14 of the 15 tall fescue accessions monitored, with afzelechin

202

ranging from 34.0%–57.9%, catechin ranging from 36.6%–61.4%, and gallocatechin

203

ranging from 0.2%–9.0%. One tall fescue cultivar, Argelia Soft, contained a

204

monomer composition dominated almost exclusively by afzelechin (99.5%). The

205

monomer composition suggested that while most accessions within each species

206

were similar, there were both species differences and outliers within species,

29

to generate a solution containing both the


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207

suggesting some biochemical variation for trans-flavan-3-ol composition within the

208

species is possible.

209 210

PA dimer and trimer identification in seed extracts:

211

The PA dimers and trimers observed in both perennial ryegrass and tall fescue seed

212

were composed of homogenous and heterogeneous mixtures of afzelechin and

213

catechin, and linked predominantly by the conventional B-type bonds, although low

214

levels of the less common A-type bonded oligomers were also observed.

215

Interestingly these low abundant A-type oligomers were only detectable in the mixed

216

(afzelechin:catechin) oligomers, and only oligomers containing a single A-type bond

217

were detected. These oligomers have previously not been reported within any

218

monocot species. No catechin:gallocatechin or pure gallocatechin dimers or trimers

219

were detected in any extract.

220

Characterisation of the dimer and oligomer composition based on precursor mass

221

and MS2 fragmentation spectra collected can be performed using a tandem mass

222

spectrometer such as an ion trap in this instance. For example, the 819, 835 and 851

223

m/z ions in Figure 2 correspond to the [M+H]+ ion for B-type trimers of 3 PP units, 2

224

PP units + 1 PC unit, and 1 PP unit + 2 PC units, respectively. While it is theoretically

225

possible that the 851 m/z ion be constructed from 2 PP units + 1 PD unit, the PD

226

monomer unit was not observed in the perennial ryegrass extract, and the m/z ion

227

can only be detected at very low levels in the tall fescue extract and as such it is

228

unlikely that any PD units were components of the oligomers detected. This was

229

further confirmed by examination of the MS2 fragmentation patterns as shown in

230

Table 2.



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ESI-MS/MS fragmentation has been used many times to interpret the structure of

232

dimer and oligomer PAs, so there are a well-defined set of fragmentation rules and

233

observations and many publications utilising these to identify PAs in foods and plants

234

14, 24-26, 30, 31

235

Diels-Alder (RDA) and heterocyclic ring fission (HRF) are listed in the MS fragments

236

detected in Table 2. PA dimers (and oligomers) consist of an extension unit and a

237

terminal unit and key fragments for determining the extension and terminal units are

238

those resulting from quinone-methide (QM) cleavage of the interflavan bond

239

Due to the wealth of understanding on PA fragmentation patterns already published,

240

we only briefly describe the key diagnostic MS2 fragment ions, and combine this

241

information with chromatographic retention times to provide tentative assignments

242

for the dimers and major trimers detected in both perennial ryegrass and tall fescue

243

seed.

. Fragments from the well described PA fragmentations such as retro

14, 24

.

244 245

Seven B-type dimers were tentatively identified (Table 2, Supplementary Figure S1),

246

two pure afzelechin dimers, three mixed afzelechin:catechin dimers and two pure

247

catechin dimers. In each these dimer species, the most abundant dimer detected

248

possessed the C4→8 interflavanoid linkages, however low levels of C4→6 linked

249

dimers were also detected for each. For both the pure afzelechin and catechin

250

dimers the C4→8 were the most intense species. In each of these we have shown

251

that the monomer species only existed as trans-flavan-3-ols. It is very unlikely any

252

cis-flavan-3-ol units existed only in dimers or oligomers, and thus we annotate the

253

species as containing only the trans- PA units. Peak 4 had the correct parent mass

254

([M+H]+ = 547 m/z) for an afzelechin dimer and exhibited QM MS2 fragments of 273

255

m/z and 275 m/z and classical RDA and HRF fragmentation and consequently is


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tentatively assigned as afzelechin-(4→8)-afzelechin. Peak 5 also had the same

257

parent mass and has been tentatively assigned as afzelechin-(4→6)-afzelechin as

258

the MS2 spectrum yielded very weak QM fragments and a base peak of 411 m/z

259

from HRF loss of 126 Da (trihydroxybenzene) from ring A of the extension unit. This

260

change in fragmentation to a more stable fragment after HRF has previously been

261

documented and is due to the increased stability of the π-π conjugated system

262

which requires higher collision energy to fragment further

263

literature data on the chromatographic behaviour of procyanidins

264

PAs consisting of epicatechin units or possessing C4→6 interflavan linkage(s)

265

usually elute later than the corresponding catechin derivatives and C4→8 linked

266

PAs. Thus, the increased retention time for peak 5 compared to the other afzelechin

267

dimer (peak 4) was further evidence of it containing a C4→6 linkage.

268

The same arguments existed for the two catechin dimers detected (peaks 9 and 10),

269

with peak 9 tentatively assigned as catechin-(4→8)-catechin based on the parent

270

mass of 579 m/z ([M+H]+), QM MS2 fragment ions of 289 m/z and 291 m/z and the

271

classical RDA and HRF fragmentation. As observed for peak 5, peak 10 likely

272

contained the C4→6 interflavan linkage, based on very weak QM fragments, an

273

intense HRF fragment of 453 m/z (loss of 126 Da) and a later elution time, thus was

274

tentatively assigned as catechin-(4→6)-catechin.

275

When the dimer/oligomer contains monomer units of different mass, it was possible

276

to apply the rules of QM formation to sequence the dimer/oligomer. Thus peak 6 was

277

tentatively identified as catechin-(4→8)-afzelechin, based on the presence in the

278

MS2 spectrum of the 275 m/z and 289 m/z fragments corresponding to the terminal

279

QM fragment (afzelechin) and the extension QM fragment (catechin), respectively.

280

The inversely assembled dimer was observed as the major mixed dimer (peak 7),

24

. A meta-analysis of the 31

revealed that



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afzelechin-(4→8)-catechin, tentatively annotated using the MS2 fragments 273 m/z

282

and 291 m/z, corresponding to the terminal QM fragment (catechin) and the

283

extension QM fragment (afzelechin), respectively. Peak 8 is an example of a mixed

284

dimer with the less common C4→6 interflavan linkage and is annotated as

285

afzelechin-(4→6)-catechin based on the weak intensity of QM fragments 273 m/z

286

and 291 m/z, an intense HRF fragment of 437 m/z (loss of 126 Da) and, as

287

described above, a later elution time.

288

Multiple pure afzelechin, mixed afzelechin:catechin and pure catechin trimers were

289

detected. The overall pattern of abundances for these trimers was highly consistent

290

across both the tall fescue and perennial ryegrass populations, so we have provided

291

tentative assignments to the six most abundant trimers (Supplementary Figure S2

292

and Table 2, peaks 11 – 16) for which good quality MS2 spectra were collected.

293

Annotations of the sequence of monomer units in the trimers are shown in Table 2,

294

with the extension unit listed first and the terminal unit listed last. This sequence was

295

determined by the fragment ions produced by QM fissions, with fragments observed

296

for both the extender and terminal units, along with the corresponding dimer

297

fragment ion resulting from loss of each extender or terminal unit fission (listed in

298

brackets in Table 2). The fragmentation interpretation and sequence deduction is

299

exemplified with peak 12, which was tentatively assigned as a mixed trimer,

300

catechin→afzelechin→afzelechin from the parent mass 835.3 m/z and the MS2

301

fragmentation pattern. The observation of the QM fission ions 289 m/z and 275 m/z

302

(and the complementary dimer fragments 547 m/z and 561 m/z, respectively)

303

indicated that a catechin unit is the extension unit and an afzelechin unit the terminal

304

unit. The central unit of the trimer was deduced to be another afzelechin unit from

305

the parent mass and the two dimer fragments produced by the QM fission. By the


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306

same process, peak 13 was deduced to be a trimer of the same parent mass

307

containing a different sequence order. This peak was also tentatively assigned as a

308

mixed trimer, afzelechin→afzelechin→catechin based on its parent ion mass of

309

835.3 m/z and the MS2 fragmentation pattern. The observation of the QM fission ions

310

273 m/z and 291 m/z (and the complementary dimer fragments 563 m/z and 545

311

m/z, respectively) indicated that an afzelechin unit must be the extension unit and a

312

catechin unit must be the terminal unit. The central unit was again deduced to be

313

another afzelechin unit from the parent mass and the two dimer fragments produced

314

by the QM fission.

315

Thus the mixed trimer composition revealed multiple assemblies of the monomer

316

units to create a complex mixture. This pattern is most likely continued for oligomers

317

containing >3 monomer units, suggesting there will be a rich complexity in the

318

perennial ryegrass and tall fescue seed PAs at higher molecular weights.

319 320

PA dimer and trimer composition in seed extracts:

321

As reported for the monomer composition above, there were similar percentage

322

compositional patterns (Figure 2, Table S3) across the 14 perennial ryegrass

323

accessions monitored, with pure afzelechin dimer and trimer percentages ranging

324

from 19.3%–54.5% and 16.0%–53.0%, respectively. Mixed dimers and trimers

325

ranged

326

afzelechins:1 catechin trimer), and 10.2%–32.4% (1 afzelechin:2 catechin trimer),

327

while the pure catechin dimer and trimer percentages ranged from 7.2%–29.2% and

328

1.1%–10.5%, respectively. Tall fescue dimer and trimer composition were again

329

consistent across 14 of the 15 tall fescue accessions monitored, with pure afzelechin

330

dimer and trimer percentages ranging from 76.3%–88.0% and 71.9%–89.4%,

from

36.2%–51.4%

(afzelechin:catechin

dimer),

28.3%–42.6%

(2



Mixed

dimers

and

trimers

ranged

from

Page 16 of 35

331

respectively.

11.4%–22.2%

332

(afzelechin:catechin dimer), 9.7%–24.6% (2 afzelechin:1 catcechin trimer), and

333

0.9%–4.1% (1 afzelechin:2 catechin trimer), while the pure catechin dimer and trimer

334

percentages ranged from 0.2%–2.8% and 0.0%–0.3% respectively. As observed

335

with monomer composition, the tall fescue cultivar Argelia Soft, contained dimer and

336

trimer composition dominated almost exclusively by afzelechin (99.7% and 99.8%

337

respectively).

338

These data highlight considerable contrast between the perennial ryegrass and tall

339

fescue dimer and trimer composition, with the tall fescue containing (percentage

340

wise) considerably more afzelechin than perennial ryegrass (Figure 5). The relative

341

compositional pattern of the dimers and trimers within species is generally similar,

342

however there were inter-species differences (Table S3) in the oligomer patterns. It

343

is worth noting that the dimer and trimer compositions differ from the monomer

344

ratios. For perennial ryegrass, 71% of the monomer composition observed is

345

catechin and yet only 16.4% of the dimers is pure catechin (i.e. containing two

346

catechin units), suggesting that afzelechin is more readily polymerised (29%

347

monomer and 39% pure dimer). Likewise for tall fescue, the catechin monomer

348

constitutes 42% of the monomer composition, but only 1.3% and 0.1% of the pure

349

dimer and trimer composition, respectively. Again, afzelechin dominates the dimer

350

and trimer compositions reinforcing the concept that afzelechin polymerisation is

351

preferred or up-regulated.

352 353

Comparison of results with previous studies

354

These results have reconciled the different evidence reported concerning the

355

presence or absence of PAs in two forage grasses. We have clearly shown that both


Page 17 of 35


356

perennial ryegrass and tall fescue contain a relatively rare polymerised PA within the

357

seed tissues. In contrast to Jackson et al. (1996), no PA monomers or oligomers of

358

any kind were detected within the leaf tissue of cultivars investigated using this LC-

359

MS/MS analysis technique (data not shown).

360

Within the perennial ryegrass population sampled, the principal PAs detected were

361

catechin-based, which contributed about 60% of the PAs in seed, with lesser

362

amounts of afzelechin-based PAs. Whereas, the principal PAs detected in tall fescue

363

were afzelechin-based, which contributed about 75% of the PAs in the seed tissue,

364

with lesser amounts of catechin-based PAs. This significantly differs from that

365

reported for other monocot PAs, which are all devoid of any PP type monomers.

366

Sorghum and rice PAs are principally PC based, while barley and finger millet PAs

367

consist of a mixture of oligomeric PDs and PCs 32-34.

368 369

Propelargonidin PAs have only been reported for dicot species (Porter 1994). The

370

highest reported proportion of (epi)afzelechin units in PAs is found in red kidney

371

beans (14.6%

372

prunes, raspberries, strawberries and pinto beans and only 5.4–7.8% of the PAs in

373

strawberry contained at least one (epi)afzelechin subunit. All these species contain

374

significantly higher percentages of both epicatechin and catechin units. Furthermore,

375

perennial ryegrass and tall fescue PAs contained low levels of the less common A-

376

type bonds. All 39 species investigated by Gu et al. (2003) contained B-type

377

linkages, while only 12% also contained A-type linkages. A-type bonds were

378

detected in the dicots plum, peanut, avocado, cranberries, and in the spices curry

379

and cinnamon. Until now, A-type bonds have not been reported in PAs from another

380

monocot species, with barley and sorghum PAs being solely linked via B-bonds 14.

14

). Lesser amounts of (epi)afzelechin units are found in almonds,



Page 18 of 35

381

Other methods used for the detection of PAs in grasses have included vanillin, p-

382

dimethylaminocinnamaldehyde (DMACA), and acid–butanol, however, there can be

383

problems associated with these assays due to their non-specificity (they can also

384

measure flavonols, dihydrochalcones, anthocyanins, as well as PAs). LC-MS/MS on

385

the other hand, is an accurate and definitive approach allowing the unequivocal

386

detection and identification of proanthocyanidin oligomers by initial chromatographic

387

separation, and then utilising mass spectrometry to measure the degree of

388

polymerization and individual oligomer composition.

389 390

The efficacy with which afzelechin based PAs bind to and precipitate proteins is the

391

first step in considering the practical implications to animal science for the discovery

392

of PAs in ryegrass and tall fescue. If this is shown to be at least equivalent to the

393

protein binding properties of the more common catechin and gallocatechin derived

394

PAs, then it warrants the development of genetic tools to modify the expression of

395

key regulatory genes, controlling PA accumulation in the seeds, towards achieving

396

the accumulation of PAs in leaf tissue. The PA pathway is regulated in plants by a

397

suite of transcription factors (TFs) and novel phenotypes can be achieved by altering

398

the expression of TFs 34-35.

399

The results clearly show that by using a LC-MS/MS approach it is possible to detect

400

and identify PAs in Lolium perenne and Festuca arundinacea seeds, and that these

401

PAs were composed of afzelechin and catechin monomers linked primarily by B-type

402

bonds, although low levels of A-type bonded PAs were also detected. This study has

403

revealed that the percentage composition of these PAs is very homogenous within

404

species, with only one outlier detected in the tall fescue accessions screened

405

producing almost exclusively afzelechin based PAs.


Page 19 of 35


406 407 408

SUPPORTING INFORMATION:

409

Details of the Lolium perenne and Festuca arundinacea accessions studied

410

(Supplementary Table S1) and percent compositions of the PA monomers, dimers

411

and trimers (Supplementary Table S2) along with extracted ion chromatograms of

412

the dimers (Supplementary Figure S1) and trimers (Supplementary Figure S2) are

413

provided.

414 415

ACKNOWLEDGEMENTS:

416

The authors would like to thank Tom Featonby and Leo Liu for their valuable

417

assistance in sample preparation and LC-MS analysis.



Page 20 of 35

418

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reactivity of condensed tannin with ribulose- 1,5-bis-phosphate carboxylase

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(Rubisco) protein. J. Sci. Food Agr. 1996, 72, 483-492.

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mass spectrometry and characterization of varieties by quantitative phenolic

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Monagas, M.; Quintanilla-López, J. E.; Gómez-Cordovés, C.; Bartolomé, B.;

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Lebrón-Aguilar, R., MALDI-TOF MS analysis of plant proanthocyanidins. J. Pharm.

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Biomed. Anal. 2010, 51, 358-372.

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hydrophilic interaction×reversed phase liquid chromatographic analysis of green tea

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phenolics. J. Sep. Sci. 2010, 33, 853-863.

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29.

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polyphenols in tea drinks. J. Sci. Food Agr. 2003, 83, 1617-1621.

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30.

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R.,

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responsible

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Mesembryanthemum edule L. Food Chem. 2011, 127, 1732-1738.

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31.

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propelargonidins and procyanidins in buckwheat grain and quantification of rutin and

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flavanols from homostylous hybrids originating from F. esculentum × F.

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homotropicum. Phytochem. 2008, 69, 1389-1397.

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32.

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procedure for the isolation of dimeric and trimeric proanthocyanidins from barley. J.

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Agric. Food Chem. 1996, 44, 1731-1735.

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33.

Min, B.; Gu, L.; McClung, A. M.; Bergman, C. J.; Chen, M.-H., Free and bound

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total

phenolic

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proanthocyanidins and anthocyanins in whole grain rice (Oryza sativa L.) of different

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bran colours. Food Chem. 2012, 133, 715-722.

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34.

514

Cereal Sci. 2006, 44, 236-251.

Kalili, K. M.; de Villiers, A., Off-line comprehensive two-dimensional

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Falleh, H.; Oueslati, S.; Guyot, S.; Dali, A. B.; Magné, C.; Abdelly, C.; Ksouri, LC/ESI-MS/MS for

the

characterisation strong

of

procyanidins

antioxidant

activity

of

and the

propelargonidins edible

halophyte

Ölschläger, C.; Regos, I.; Zeller, F. J.; Treutter, D., Identification of galloylated

McMurrough, I.; Madigan, D.; Smyth, M. R., Semipreparative chromatographic

concentrations,

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capacities,

and

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Dykes, L.; Rooney, L. W., Sorghum and millet phenols and antioxidants. J.



Page 24 of 35

515

516

Note: This research was supported through funding from Grasslanz Technologies

517

Ltd., NZ and AgResearch Core Funding.

518


Page 25 of 35


Figure Captions

Figure 1: Flavan-3-ol structure/variations for monomer units (A) and an example of a procyanidin trimer containing a single A-type bond and a single B-type bond (B).

Figure 2: Examples of averaged MS1 spectra from the LC-MS chromatograms of perennial ryegrass ‘Grasslands Nui’ (A) or tall fescue ‘Grasslands Advance’ (B) seed extracts, from 18.5-35 minutes. Labels refer to ions representing oligomer groups with differing degrees of polymerisation (DP), monomers catechin (Cat) and afzelechin (Azf) and major sample dependant ions, ergovaline (Ergov) and thesinine rhamnoside (Thes-rham) for perennial ryegrass and perloline for tall fescue.

Figure 3: Zoomed averaged MS1 spectra from the LC-MS chromatogram of perennial ryegrass ‘Grasslands Nui’ from 18.5-35.0 minutes, demonstrating the presence of trimer (A) and tetramer (B) oligomers containing either all B-type bonds or a single A-type bond. PP = propelargondin unit, PC = procyanidin unit.

Figure 4: Extracted ion chromatograms of the 275.3-> 139 m/z transition in the MS2 spectra of epiafzelechin (28.9 min) standard before the epimerisation heating reaction (A), epiafzelechin standard after epimerisation showing the formation of an afzelechin peak (24.8 min) in addition to the residual epiafzelechin (B), and a perennial ryegrass ‘Grasslands Nui’ seed extract (C). The corresponding MS2 spectra for the afzelechin peak at 24.8 min are shown for the standard (D) and perennial ryegrass extract (E).



Page 26 of 35

Figure 5: Figure showing percent composition of monomers, dimers and trimers in both perennial ryegrass and tall fescue, calculated from cumulative peak areas for each monomer and oligomer type from MS2 chromatograms. Afz = afzelechin unit, Cat = catechin unit, GC = gallocatechin unit.


Page 27 of 35


Table 1: Selected Precursor Ion Masses For Product Ion Scans Of Monomers, Dimers And Trimers In Positive ESI Mode ([M+H]+): Precursor ion

MS2 scan range (m/z)

Target compounds

mass (m/z) Analysis 1 MS2 Scan Settings Monomers 275.3

80-700

PP monomers

291.3

80-700

PC monomers

307.3

80-700

PD monomers

547.3

150-2000

PP:PP dimers

563.3

155-2000

PP:PC dimers

579.3

155-2000

PP:PD or PC:PC dimers

595.3

160-2000

PC:PD dimers

611.3

165-2000

PD:PD dimers

Dimers

Analysis 2 MS2 Scan Settings Trimers 819.3

225-2000

PP3 trimers

835.3

225-2000

PP2:PC trimers

851.3

230-2000

PP:PC2 or PP2:PD trimers

867.3

235-2000

PP:PC:PD or PC3 timers

883.3

240-2000

PP:PD2 or PC2:PD trimers

899.3

245-2000

PC:PD2 trimers

915.3

250-2000

PD3 trimers



Page 28 of 35

Table 2: Proanthocyanidin Monomers, Dimers And Trimers In Perennial Ryegrass And Tall Fescue Seed. Rt = Chromatographic Retention Time, QM ext = Quinone Methide Fragment From The Extension Unit, QM term = Quinone Methide Fragment From The Terminal Unit. Peak # Name

Rt (min)

[M+H]+ Key MS2 ions for QM peak

assignment ext

QM term

and area 1

Afzelechin

25.8

275.3

139, 149

2

Catechin

20.0

291.3

123, 139, 151, 165

3

Gallocatechin 13.8

307.3

139, 151,289

4

Afz-(4-8)-Afz

547.3

273, 275, 393, 411, 273

27.2

275

421 5

Afz-(4-6)-Afz

33.3

547.3

273, 275, 393, 403, 273*

275*

411, 421 6

Cat-(4-8)-Afz

22.4

563.3

275, 287, 289, 393, 275

289

409, 411, 427, 437 7

Afz-(4-8)-Cat

23.0

563.3

273, 291, 393, 409, 273

291

411, 427, 437 8

Afz-(4-6)-Cat

30.0

563.3

273, 291, 393, 409, 273*

291*

411, 419, 427, 437 9

Cat-(4-8)-Cat

18.7

579.3

289, 291, 409, 427, 289

291

435, 453 10

Cat-(4-6)-Cat

25.3

579.3

289, 291, 409, 427, 289*

291*

435, 453


Page 29 of 35


11

Afz-Afz-Afz

31.3

819.3

273, 275, 391, 409, 273

275

421, 527, 545, 547, (547)

(545)

683 12

13

Cat-Afz-Afz

Afz-Afz-Cat

26.6

28.3

835.3

835.3

275, 289, 391, 409, 289

275

421, 547, 561, 699

(561)

(547)

273, 291, 391, 409, 273

291

421, 527, 545, 563, (563)

(545)

699 14

Cat-Cat-Afz

23.4

851.3

289, 291, 391, 409, 289

291

421, 543, 561, 563, (563)

(561)

699 15

16

Cat-Cat-Afz

Cat-Cat-Cat

24.8

20.4

851.3

867.3

273, 291, 407, 427, 273

291

561, 561, 579, 699

(561)

(579)

289, 291, 407, 425, 289

291

559, 577, 579, 715

(577)

(579)

Note - Extender unit listed first, Terminal unit listed last; * = weak fragment; Base peak in MS2 spectrum displayed in bold.



Page 30 of 35

Figure 1:


Page 31 of 35


Figure 2:



Page 32 of 35

Figure 3:


Page 33 of 35


Figure 4:



Page 34 of 35

Figure 5:

100.0 Ryegrass

90.0

Tall fescue

80.0

Percent composition

70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 Afz

Cat

Monomers

GC

Afz:Afz

Afz:Cat

Cat:Cat

Afz3

Dimers

Afz2:Cat Afz:Cat2

Cat3

Trimers


Page 35 of 35


Table of Contents Graphic


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