Soluble Phenolic Compounds in Fresh and Ensiled Orchard Grass(Dactylis glomerata L.), a Common Species in Permanent Pastureswith Potential as a Biomass FeedstockBarbara Hauck, Joe A. Gallagher, S. Michael Morris, David Leemans, and Ana L. Winters*

Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Gogerddan, Aberystwyth, Ceredigion SY23 3EE,United Kingdom

*S Supporting Information

ABSTRACT: High-value coproducts can greatly improve the feasibility of utilizing plant feedstocks for biorefining and biofuelproduction. Plant polyphenolics have potential application in the pharmaceutical and cosmetic industries. Orchard grass varietieshave been noted for accumulation of polyphenolic compounds, and the current study determined the soluble phenol profile andcontent in the orchard grass variety ‘Abertop’. Hydroxycinnamates and flavonoids were monitored during the transition fromvegetative to flowering stage at maximum crop yield. Caffeic acid derivatives, related to bioactives in the Asian medicinal herbSalvia miltiorrhiza, and novel hydroxycinnamate−flavone conjugates were also identified in extracts. Harvest yields ofhydroxycinnamates and flavonoids ranged from 2.6 to 4.0 kg/ha and from 2.1 to 5.1 kg/ha, respectively. Abundant compoundsshowed high levels of antioxidant activity comparable with that of trolox. Minimal changes in soluble phenol content andcomposition were observed after ensiling with the exception of increases in caffeic acid, a caffeic acid derivative, and a caffeic acidbreakdown product, dihydroxystyrene.

KEYWORDS: Dactylis glomerata, orchard grass, phenolic acid esters, flavonoid glycosides, caffeic acid derivatives

■ INTRODUCTION

Orchard grass (Dactylis glomerata) is a relatively coarse, tussock-forming grass that is native to Europe, temperate Asia, and NorthAfrica.1 It has been introduced as a forage grass into mosttemperate countries in the world and is one of the mostimportant sown species in Australia and New Zealand.2 Plantsare either diploid (2n = 14) or, more commonly, autotetraploid(2n = 28), and subspecies are morphologically distinguishableaccording to area of origin. For example, plants of the Eurasian(or Northern) group are typically large with broad leaves andwinter-dormant, whereas plants from the Mediterranean regionare relatively small, narrow-leaved, and summer-dormant.Although this species has a lower nutritional value comparedto other forage species such as perennial ryegrass, it is moretolerant to heat and drought, resulting in higher productionduring summer dry periods.Phenolic compounds are generally abundant in orchard grass

compared with other temperate grass species. The presence ofphenolic compounds in this species was first reported in 1926 byLandsborough-Thompson,3 who described the isolation of aflavone glycoside and a free flavone from plant extracts. Morerecently, Parveen et al.4 identified a number of hydroxcinnamateesters in a commercial variety of orchard grass (D. glomerata).There is a strong tendency for chemotaxonomically relatedplants to produce similar types of flavonoids,5 and a number ofstudies have reported characteristic variation in the flavonoidprofile of different populations of orchard grass.6,7

Although temperate grasses have a fundamental role inruminant nutrition, recent studies have focused on their potentialas feedstocks for biorefining.8−10 Increased recovery ofcomponents with a marketable value can improve the feasibility

of grasses as biorefining feedstocks. Valuable components can berecovered from the liquid fractions of ensiled grass biomassincluding lactic acid and amino acids,11 whereas the residualpresscake has potential as a combustible feedstock.12 Alter-natively, residual biomass may be used to produce bioethanol9 orbiogas.13 Polyphenols have been identified as potential high valuecoproducts in a biorefinery, and processes have been developedfor their recovery from olive mill wastewater.14 These classes ofcompounds are of interest to the pharmaceutical, cosmetics, andnutraceutical sectors, and there is an increasing body of evidencedemonstrating the health benefits of plant phenols.15 Orchardgrass is reported to be a traditional remedy for tumors, kidney,and bladder ailments.16

Here we report on the identification and quantitative analysisof flavonoids/phenolic compounds in a commercial variety oforchard grass, ‘Abertop’. This variety has been developed toprovide a high-yielding conservation cut and good ground cover.We monitor the phenol profile during the transition fromvegetative stage to flowering stage, which corresponds withmaximum yield. We also present data on changes in solublephenol profile following a short period of ensiling.

■ MATERIALS AND METHODSChemicals. HPLC grade methanol was obtained from Fisher

Scientific (Loughborough, UK), and commercial standards for phenolicacids and flavone aglycones were from Sigma-Aldrich (Gillingham, UK)and PhytoLab (Vestenbergsgreuth, Germany).

Received: September 11, 2013Revised: December 16, 2013Accepted: December 17, 2013Published: December 17, 2013

Article

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Plant Material. D. glomerata cv. Abertop was established in 1.2 m2

plots (replicated × 4) at Aberystwyth University, Gogerddan,Aberystwyth (52°19′22″ N, 3°56′54″ W). Plots were harvested with aHaldrop plot harvester at regular intervals during May 2010. Plant yieldswere recorded during harvesting. Following harvesting, plant materialwas transported to the laboratory within a period of 1 h and stored at−20 °C for subsequent analysis.Ensiling. Plant material was harvested from plots on May 8, 2010.

Leaf material was chopped into 1 in. lengths and packed into three silagetubes (approximately 200 mL size) and capped with silo air traps. Tubeswere incubated at 25 °C and opened after 2 weeks. Freshly sampledmaterial was extracted for phenols, and remaining material was frozen at−20 °C for further chemical analysis. Samples were analyzed for drymatter content, pH, volatile fatty acids, and water-soluble carbohydrates.Extraction and Analysis of Water-Soluble Carbohydrates and

Organic Acids. Fresh and ensiled material was extracted by immersing2.5 g in 10 mL of water. Samples were homogenized and centrifuged at3000g for 15 min. The supernatant was recovered and the pelletdiscarded. Fifty microliters of extract was added to 950 μL of a buffercomprising 5 μM H2SO4, containing 5 μM crotonic acid as an internalstandard. These samples were analyzed with a Jasco HPLC system(Jasco Ltd., Great Dunmow, Essex, UK), and the column used was a 150mm × 7.8 mm Rezex ROA-Organic Acid (Phenomenex, Macclesfield,UK) with a mobile phase of 5 μM H2SO4 at 0.6 mL/min. Peaks weredetected with a refractive index detector, identified using JascoEZChrom Elite HPLC software (Jasco Ltd., Essex, UK), and quantitatedby comparison with standard samples of fructan, sucrose, glucose,fructose, lactic acid, and acetic acid.Phenol Extraction. Plant material was chopped and immersed in

70% methanol at 80 °C for 5 min to inactivate polyphenol oxidases(typically 1 g FW of sample was extracted for monitoring variation over agrowing season, and a bulk sample of approximately 6 g was extracted fordetailed profiling of phenolic compounds). The supernatant wasdecanted, and the pellet was extracted with methanol/water (8:2) andcentrifuged for 20 min at 3000g. The fractions were then combined togive a total soluble phenol extract. Methanol was removed from theextracts by rotary evaporation, and extracts were then partially purifiedon a 500 mg Sep-Pak C18 3 cc Vac RC cartridge (Waters Ltd., Elstree,UK). Cartridges were prepared with 4 mL of methanol (100%) followedby 4 mL of 5% acetic acid. Samples were then loaded, washed with 2.5mL of water, and subsequently eluted with 4 mL of methanol (100%),dried down at 50 °C under nitrogen, and redissolved in 70% MeOH.HPLC. Flavonoid and phenolic end products were analyzed by

reverse-phase HPLC with online photodiode array detection with andwithout electrospray ionization−ion trap mass spectrometry.HPLC analysis was carried out on a Waters system with a 996

photodiode array detector (PDA) and a 8 mm × 100 mm i.d., 4 μm, C18Nova-Pak radial compression column (Waters), equilibrated with 100%solvent A (5% acetic acid) at a flow rate of 2 mL/min.Compounds were eluted by linear gradient to 100% solvent B (100%

methanol) over 50 min and monitored from 240 to 400 nm.Compounds were quantitated by comparison to calibration curves forappropriate standards.HPLC−ion trap mass spetrometry (HPLC-MSn) analysis was

performed on a Thermo Finnigan LC-MS system (Thermo ElectronCorp., Waltham, MA, USA) comprising a Finnigan Surveyor PDA Plusdetector and a Finnigan LTQ linear ion trap with ESI source, and thecolumn used was a 3.9 mm × 100 mm i.d., 4 μm, C18 Nova-Pak(Waters). The autosampler tray temperature wasmaintained at 5 °C andthe column temperature at 30 °C. Sample injection volume was typically10 μL, the detection wavelength was set to 240−400 nm, and the flowrate was 1 mL/min, with 100 μL/min going to the mass spectrometer.The mobile phase consisted of water/formic acid (A; 100:0.1, v/v) andMeOH/formic acid (B; 100:0.1, v/v). The columnwas equilibrated with95% solvent A, and the percentage of B increased linearly to 60% over 65min.Ionization parameters were optimized by infusion of chlorogenic acid

standard at a constant rate into the LC flow. Mass spectra were acquiredin negative ionization mode with the following interface and MSparameters: nitrogen sheath gas, 30 arbitrary units; nitrogen auxiliary

gas, 15 units; spray voltage, 4 kV; capillary temperature, 320 °C; capillaryvoltage, −1 V; and tube lens offset, −68 V. MS/MS fragmentation wascarried out at a normalized collision energy of 35% and isolation widthm/z 2.0.

Purification of Selected Compounds and Acid Hydrolysis.Abundant phenolic compounds were purified by reverse-phasechromatography with the Waters system described above. Fractionswere concentrated by rotary evaporation, and a portion was retained foracid and alkali hydrolysis. Acid hydrolysis was performed by combining avolume of purified compound with an equal volume of 2 M HCl. Thesolution was heated to 90 °C for 1 h, and the pHwas then adjusted to 4−5 with NaOH. Alkali hydrolysis was performed by combining a volumeof purified compound with an equal volume of 2MNaOH. The solutionwas incubated for 16 h under N2, and the pH was then adjusted to 4−5with HCl. Purified compounds and neutralized hydrolysates werepartially purified on a 500 mg Sep-Pak C18 3 cc Vac RC cartridge(Waters Ltd.) as described above, dried down at 50 °C under nitrogen,and redissolved in 70% MeOH.

Antioxidant Assays. Antioxidant activity was expressed as troloxequivalents (TE) by comparison of tested compounds to troloxcalibration curves in the range of 5−50 μmol/L. The diphenylpicrylhy-drazyl (DPPH) radical scavanging assay was carried out according to themethod of Brand-Williams et al.17 A 140 μM solution of DPPH wasprepared in 50:50 methanol/water. This was combined with an equalvolume of sample in a 1.5 mL cuvette, with 50:50 methanol/water as acontrol. Mixtures were left to stand for 30 min at room temperature,after which time absorbance at 517 nm was determined. A range ofsample concentrations were tested to identify the range giving partialreduction of the DPPH in the reaction mixture.

A second radical-scavanging assay based on the positive radical ion,ABTS•+, was carried out according to the protocol of Re et al.18 Theradical form was prepared by adding potassium persulfate to a 7 mMstock solution of ABTS to give a final concentration of 2.45 mM andallowing the mixture to stand for 16 h at room temperature in the dark.At the time of assay, the ABTS•+ solution was diluted with water to givean absorbance of 0.700 (±0.2) at 743 nm. Sample volumes of 25 μLwere added to 1475 μL of reagent in a 1.5 mL cuvette, and absorbancewas measured after 6 min. A range of sample concentrations were testedto identify the range giving partial reduction of the ABTS•+ in thereaction mixture.

The ferric reducing capacity of samples was measured by the FRAPassay, which detects reduction of the ferric ion (Fe3+) to the ferrous ion(Fe2+).19 The assay reagent was prepared by combining 10 mM TPTZ,20 mM ferric chloride, and 300 mM sodium acetate buffer (pH 3.6) inthe ratio of 1:1:10. Sample volumes of 25 μL were added to 1475 μL ofreagent in a 1.5 mL cuvette, and absorbance was measured at 593 nmafter 10 min.

■ RESULTS AND DISCUSSION

Phenol Profile. Figure 1 shows a typical profile of solublephenols extracted from ‘Abertop’. Analysis by LC ESI-MS withPDA detection revealed approximately 50 compounds that couldbe easily distinguished (Table 1). Several compounds werereadily identified by comparison of retention times andfragmentation patterns with known standards. Others weretentatively identified by the similarity of UV absorbancecharacteristics and fragmentation patterns with known standardsand compounds reported in the literature.5,20,21 Representativestructures are shown in Figure 2.Compounds identified by LC-MS include hydroxycinnamoyl

esters, flavone glycosides, salvianolic acids, and hydroxycinna-moyl flavone conjugates. Relatively abundant hydroxycinnamateesters, which have been previously reported by Parveen et al.4 inorchard grass, were identified in extracts including 5-caffeoylquinate (chlorogenic acid), 2-O-caffeoylisocitrate, and 2-O-caffeoylisocitrate 6-methyl ester (compounds 5, 9, and 18a asshown in Table 1).

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Hydroxycinnamate Esters.A number of hydroxycinnamateesters that have not previously been reported in orchard grasswere detected in ‘Abertop’ extracts. Compound 7withMr 268 Daat tR 11 min showed characteristics consistent with a caffeoyl/feruloyl ester with UVmaxima at 303sh and 326 nm. MS2 ofm/z267 in negative mode produced a base peak at m/z 161 [caffeicacid − H2O − H+]− and a secondary peak at m/z 105 [glycericacid−H+]−. Further fragmentation of them/z 105 ion produceda base peak at m/z 75 [glyceric acid − HCOH − H+]− andcorresponded with the fragmentation pattern observed forglyceric acid. This compound is identified as caffeoylglycerate. 2-Caffeoylglycerate has previously been identified in extracts of themedicinal plant, Mercurialis perennis L.22

A second hydroxycinnamoyl ester (MR 352 Da), compound22a, was detected at tR 24.8 min, showing maximal UVabsorbance at 315 nm. This compound produced an MS2 basepeak atm/z 163 [coumaric acid−H+]− and secondary ions atm/z 169 [methylisocitric acid − 2 × H2O − H+]−, 205[methylisocitric acid − H+]−, and 119 [coumaric acid − CO2−H+]−. Further fragmentation of the 205 ion in MS3 produced abase peak at m/z 173 [methylisocitric acid − CH3OH − H+]−

and secondary ions at m/z 111 [methylisocitric acid − CH3OH− CO2 − H2O − H+]− and 155 [methylisocitric acid− CH3OH− H2O − H+]−. Caffeoylisocitrate methyl ester has beenpreviously identified in orchard grass,4 and this compoundedyielded an ion at m/z 205 in MS2 with an equivalentfragmentation pattern in MS3 to the 205 ion reported here. Onthis basis this compound is identified as coumaroylisocitratemethyl ester.Compound 8 withMr 430 Da showed a fragmentation pattern

related to that of compound 7 and was detected at tR 11.4 min.This compound also showed a UV profile consistent with acaffeoyl/feruloyl ester with UV maxima at 300sh and 324 nm. Itproduced an MS2 base peak in negative mode at m/z 267corresponding to the neutral loss of 162 Da and secondary peaksat m/z 179 and 411 [M − H2O − H+]−. The 179 ion wasconfirmed as [caffeic acid −H+]− in MS3. Further fragmentationof the 267 ion inMS3 yielded a base peak atm/z 249 [267−H2O

− H+]− and secondary peaks at m/z 113, 175, 85, 87, and 205,and the 249 ion in MS4 yielded a base peak at 113 and secondarypeaks at 205, 157, 87, and 85. This indicates that the 157 ionyielded inMS4 is released from the 175 ion yielded inMS3. Thesefragments are consistent with neutral losses of CO2 (−44 Da),dehydrophenoxy radical (−92 Da), dehydrophenoxy radical andCO2 (−136 Da), and dehydrophenoxy radical, CO2, and C2H2/C2H4 (−162/−164 Da). These fragments may be accounted forby whole or partial loss of a coumaroyl moiety, and the ion atm/z87 is consistent with a dehydro-glycerate moiety. On this basis itis proposed that compound 8 is caffeoylglycerate conjugatedwith coumaric acid formed via an oxidative radical couplingreaction.

Caffeic Acid Derivatives. Antioxidant, antithrombotic,antitumor, and antiviral properties of the popular Asianmedicinal herb, danshen (Salvia miltiorrhiza), have beenattributed to constituent caffeic acid derivatives known assalvianolic and lithospermic acids.23 A number of compoundswith structural properties consistent with these caffeic acidderivatives were detected in orchard grass extracts. Theseincluded compounds 15, 19a, 19b, 22b, 23c, 23d, 25a, and25b withMr 360, 492, 538, 556, and 736 Da. ESI MSn spectra ofnegative ions showed characteristic fragmentation patterns oflithospermic and salvianolic acids with products derived fromneutral losses of 198, 180, 162, 110, and 44 Da corresponding tolosses of 3,4-dihydroxyphenyl-lactic acid (danshensu), caffeicacid, caffeic acid dehydrate, catechol, and CO2 moieties,respectively. They also yielded characteristic fragment ions ofsalvianolic acids at m/z 537, 493, 383, 339, 313, 295, 197, and179.24

Compound 25b with Mr 360 Da shows retention time, UVabsorption profile (maxima at 276 and 331 nm), andfragmentation pattern consistent with those of rosmarinic acid(MS2 fragments at m/z 161, 179, and 197). The remainingmembers of the group have been identified on the basis of theirMS and UV spectra where detected.Compound 23d with Mr 492 Da at tr 26.0 min shows

characteristic features of caffeic acid derivatives. This compoundshowed a base peak in negative mode atm/z 339 and a secondarypeak at m/z 447 corresponding to neutral losses of 152 and 44Da. The fragmentation pattern suggests a molecular structureconsistent with lithospermic acid less CHOOH. The loss of 152Da is equivalent to the loss of 198 Da observed duringfragmentation of lithospermic acid in negative mode resultingfrom a cleavage of the ester bond between the carboxyl oxygenand second oxygen atom, yielding a negative ion at m/z 339 asobserved here. Other fragments in common with those yieldedby lithospermic acid include negative ions at m/z 295 and 185.Other fragments observed include ions at 447 and 337, whichmay correspond with ions observed at m/z 493 and 383 inlithospermic MS spectra (447 + CHOOH and 337 + CHOOH,respectively).Compounds 19b and 25a with Mr 556 and 736 Da at tr 21.6

and 27.3 min, respectively, yielded fragment ions in negative MSat m/z 537 and 717, respectively, corresponding withlithospermic acid and lithospermic acid B. Similar compoundswith equivalent masses have been reported in sage tea.25 A 736Da isomer gave rise to a base peak at m/z 537 (198 Da loss),which in MS3 yielded fragment ions typical of lithospermic acidas reported by Zeng et al.24

Caffeic acid derivatives, including rosmarinic, lithospermic,and salvianolic acids, have potent radical-scavenging activityrelated to the number of catechol moieties contained in the

Figure 1.HPLC chromatogram showing separation of soluble phenoliccompounds in extracts of orchard grass detected by photodiode array at340 nm. Peak numbering corresponds with Table 1.

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structure and are reported to have a range of protective andantimicrobial properties.26,27

Hydroxycinnamate Esters and Flavone GlycosideConjugates. UV combined with MS analysis revealed a novelgroup of compounds in D. glomerata extracts with distinctivefragmentation patterns in negative mode MS. These compoundsshow neutral losses consistent with cleavage of ester-linked

organic acids and, where detected, UV absorption maxima at269−270 and 349 nm. UV profiles were typical of the profileproduced by orientin (luteolin-8-C-glucoside) standard. Com-pound 24 with Mr 800 Da eluted at tR 26.6 min and in negativemode produced amass spectrum with a base peak atm/z 581 andfurther peaks at m/z 607 and 625. This corresponds to neutrallosses of 218, 192, and 174 Da, respectively. Interestingly, full MS

Table 1. Phenolic Compounds in Orchard Grass Leaf Extracts

peak/ID tr

[M − H]−

(m/z) main fragments(m/z) λmax(nm) tentative compound identification

1 4.9 353 191, 179 (45), 135 (8), 173 (3) 299sh, 323 3-caffeoylquinate2 5.7 297 135, 179 (29), 279 (5), 229 (3) 301sh, 326 2-O-caffeoylthreonate3 6.5 297 135, 179 (29), 279 (3), 229 (2), 117 (1) 299sh, 326 2-O-caffeoylthreonate4a 8.9 179 135, 179 (76), 151 (1) 300sh,322 caffeic acid4b 367 193, 134 (5), 173 (3), 191 (2) 300sh, 322 feruloyl quinate5 9.7 353 191, 179 (5), 173 (1), 135 (0.5), 161 (1) 300sh, 321 5-caffeoylquinate6 10.0 353 173, 179 (54), 191 (33), 135 (5) 301sh, 323 4-caffeoylquinate7 11.0 267 161, 105 (64), 179 (33), 221 (6), 135(5) 303sh, 326 caffeoylglycerate8 11.4 429 267, 411 (3), 179 (3) 300sh, 324 caffeoylglycerate p-coumaric conjugate9 12.1 353 173, 191 (75), 111 (13), 155 (6), 179 (2) 304sh, 328 2-O-caffeoylisocitrate10 12.8 163 119, 163 (8) 312 p-coumaric acid11 15.7 337 173, 111 (13), 155 (5), 191 (3) 314 2-O-coumaroylisocitrate12 16.4 609 489, 519 (26), 490 (12), 399 (9), 591 (8), 369 (7) 270, 342 luteolin-6-C-hexoside-8-C-hexoside13a 18.0 593 473, 503 (32), 353 (27), 474 (22), 383 (14), 575 (11) ND apigenin-6-C-hexoside-8-C-hexoside13b 367 173, 111 (13), 155 (7), 179 (7), 135 (3) ND 2-O-caffeoylisocitrate methyl ester14 18.4 609 327, 357 (65), 411 (25), 328 (11), 591 (10), 358 (8), 447 (7) ND iso-orientin-O-hexoside15 18.8 537 339, 493 (5), 357 (1), 252, 285,

318sh, 344lithospermic acid isomer

16 19.8 579 489, 459 (20), 519 (17), 561 (16), 399 (12), 369 (8), 471 (6) 270, 349 luteolin-6-C-pentoside-8-C-hexoside17 20.5 447 327, 357 (46), 369 (6), 393 (4) ND orientin18a 20.8 367 179, 205 (56), 169 (8), 135 (7), 173 (5), 187 (1), 111 (1) 302sh, 330 2-O-caffeoylisocitrate 6-methyl ester18b 579 429, 459 (20), 357 (19), 309 (14), 561 (6), 447 (4) ND luteolin-C-hexosyl pentoside19a 21.6 537 493, 295 (61), 357 (6), 383 (6), 313 (6), 337 (4) ND lithospermic acid isomer19b 555 537, 511 (62), 269 (8), 493 (7), 313 (4) ND salvianolic acid K isomer20a 21.8 447 327, 357 (49), 429 (15), 387 (3) ND iso-orientin20b 563 443, 473 (60), 353 (20), 383 (15), 545 (14), 503 (7) ND apigenin-6-C-hexoside-8-C-pentoside21a 23.8 563 413, 293 (20), 443 (3) apigenin-C-hexosyl pentoside21b 593 473, 503 (83), 383 (33), 413 (21), 474 (20), 504 (17), 575 (15), 533 (7) chryseriol-6-C-hexoside-8-C-pentoside22a 24.8 351 163, 169 (78), 205 (38), 119 (5) 315 2-O-coumaroylisocitrate methyl ester22b 555 511, 537 (64), 357 (61), 269 (47), 285 (38), 313 (33), 449 (27), 241 (22),

339 (18), 197 (10)ND caffeic acid derivative

22c 431 311, 341 (28), 312 (10), 413 (7) ND isovitexin22d 563 443, 473 (50), 444 (18), 353 (16), 545 (11), 474 (10), 383 (8), 455 (4) ND apigenin-6-C-hexoside-8-C-pentoside23a 26.0 743 607, 581 (10), 487 (7), 625 (1), 553 (1) ND 2-O-caffeoylthreonate orientin conjugate23b 26.6 713 607, 581 (20), 607 (17), 487 (15), 581 (3), 517 (3), 553 (2), 625 (2) ND 2-O-caffeoylglycerate orientin conjugate23c 735 493, 537 (66), 717 (50), 313 (45), 691 (20), 475 (18), 555 (15), 431 (11),

519 (11), 295 (10), 339 (8)ND caffeic acid derivative

23d 491 339, 447 (33) ND caffeic acid derivative24 799 581, 607 (2), 461 (1), 625 (1), 755 (1), 491 (1) 269, 349 2-O-caffeoylisocitrate orientin conjugate25a 27.3 735 537, 717 (80), 493 (26), 339 (22), 475 (20), 449 (15), 519 (13), 465 (12),

313 (12), 673 (10), 357 (9), 295 (8)ND salvianolic acid isomer

25b 27.3 359 161, 179 (29), 197 (24), 223 (15), 133 (3), 135 (2) ND rosmarinic acid26 28.8 813 581, 607 (10), 461 (2), 625 (2), 487 (1), 491 (1) 270, 349 2-O-caffeoylisocitrate methyl ester orientin

conjugate27 30.0 491 329, 330 (5), 371 (3), 314 (1) 260, 343 tricin-O-hexoside28 30.8 813 595, 639 (19), 581 (8), 607 (1), 769 (1), 625 (1), 461 (1) 270, 345 2-O-caffeoylisocitrate methyl ester orientin

conjugate29 32.8 827 595, 639 (3) 271, 335 2-O-caffeoylisocitrate methyl ester luteolin-

methyl-ether conjugate30 34.1 577 533, 491 (1) 260, 345 tricin-O-hexosyl -malonate31 34.4 687 491, 669 (49), 643 (33), 329 (30), 492 (13), 525 (12), 326 tricin conjugate32 41.4 329 314, 329 (50), 299 (1) 267, 349 tricin33 42.5 525 329, 477 (2), 195 (2), 314 (2), 507 (2), 165 (1) 270, 331 tricin conjugate

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in positive mode revealed major ions at m/z 801 and 627 at tR26.6, whereas only one ion was observed in negative mode,suggesting a neutral fragment of 174 Da is readily lost in positivemode. Losses of 192 and 174 Da correspond to cleavage of anisocitrate or quinate moiety on either side of the ether linkage inan ester bond. The neutral loss of 218 Da could be accounted forby cleavage at the far side of the carbonyl group (174 + 44 Da).Further fragmentation of the 581 ion in MS3 yielded ions at m/z461 and 491, which correspond to neutral losses of 120 and 90Da, typical of C-linked flavonoid hexosides.28 Additionalfragments at m/z 471 and 351 correspond to neutral losses of110 and 230Da. These may be accounted for by loss of a catecholmoiety (110 Da) and a catechol moiety combined with a C-linked hexoside fragment (110 and 120 Da). Spectra in MS4 ofthe ions at m/z 491 and 461 showed base peaks at m/z 381 and351, respectively, corresponding to the loss of a catechol moiety.Compound 24 was purified by reverse phase chromatographyand incubated with 1 M HCl with heat or with 1 M NaOH atroom temperature to hydrolyze ester and O-glycosidic linkages.Major products of acid and alkali hydrolysis experimentsincluded compounds with Mr 626, 582, and 472 Da. Thesecompounds yielded identical fragment ions in MS2 correspond-ing to fragment ions described above and may be accounted forby hydrolysis of ester linkages, acid-catalyzed decarboxylation,and release of pyrocatechol by decomposition. Minor productsincluded compounds with Mr 192, 354, and 447 Da. Thesecompounds showed fragmentation patterns identical to those ofisocitric acid, 2-O-caffeoylisocitrate, and orientin.On the basis of this evidence, this molecule is composed of 2-

O-caffeoylisocitrate and orientin residues. The fragmentation

patterns observed in MSn experiments including the release ofisocitrate and catechol moieties and CO2 indicate that thiscompound is formed by a condensation reaction with bondsformed between C2 and C3 on the alkyl side chain of the caffeoylmoiety and the luteolin B-ring. Similar condensation productsinclude naturally occurring oxyneolignans, americanin A29 andeusiderin,30 and the coumarinolignoid, daphneticin.31 Otherrelated compounds include a hydroxcinnamate−mandelonitrileconjugate, which has been reported in Miscanthus × giganteusstem extracts.32 These compounds are postulated to be formedby oxidative coupling mechanisms, and conjugates identified inorchard grass extracts may be formed by similar pathways.Activity of plant oxidative enzymes during the extraction processcould potentially catalyze formation of these products. Toeliminate this possibility, we repeated the extraction procedureincluding 50 mM ascorbic acid in the extraction solvent. Anidentical profile of phenolic compounds was obtained by thismethod (data not presented), indicating that these conjugateswere not formed during the extraction procedure.

Figure 2. Structures of phenolic compounds in orchard grass.

Figure 3. Caffeic acid ester−orientin conjugates detected in orchardgrass extracts including caffeoylthreonate orientin conjugate (23a),caffeoylglycerate orientin conjugate (23b), and 2-O-caffeoylisocitratemethyl ester orientin conjugate (26).

Table 2. Variation in Composition and Yield of OrchardGrassduring the Transition from Vegatative to Flowering Stage

May 8 May 13 May 18 May 23 May 29

crude protein (g/kg DM) 166.6 161.3 129.1 126.9 106.9WSC (g/kg DM) 152.7 114.3 165.4 150.9 130.6NDF (g/kg DM) 430.9 480.0 465.1 469.1 522.4ADF (g/kg DM) 206.0 236.2 230.8 234.7 264.0DMD (g/kg DM) 759.6 722.4 744.8 720.2 692.1yield (t/ha) 2.58 3.05 3.68 3.87 6.36

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Other compounds assigned to this group on the basis offragmentation patterns include compounds 23a, 23b, 26, 28, and29 withMr 744, 714, 814, 814, and 828 Da. Common fragmentsdetected in negative mode ESI MSn spectra include ions at m/z625, 607, 581, 491, 487, and 461 Da. Compounds with Mr 714,744, and 814 Da have been tentatively identified as conjugates oforientin with caffeoylthreonate, caffeoylglycerate, and caffeoyli-socitrate methyl ester, respectively (Figure 3), and a compoundwith Mr 828 Da has a fragmentation pattern consistent with aconjugate of hispidulin-C-glycoside with caffeoylisocitratemethyl ester.Variation in Phenolic Profiles during Transition from

Vegetative to Flowering Stage. Orchard grass production

was monitored during transition from vegetative to floweringstage, which corresponds with maximum yield of biomass. Table2 shows changes in composition in harvested material. Changesin composition reflect the alteration in morphology that occursduring flowering, that is, increasing proportion of stem tissuewith a corresponding decrease in leaf tissue. Neutral detergentfiber (NDF) and acid detergent fiber (ADF) show an increasingtrend, whereas the opposite pattern is observed with dry matterdigestibility and crude protein, which generally decrease withplant maturity. Biomass yield showed a steady increase duringthis period. Figure 4A shows changes in total hydroxycinnamateand flavonoid content during flowering. Levels remained highthroughout this period, with potential yields of hydroxycinna-mates and flavonoids ranging from 2.6 to 4.0 kg/ha and from 2.1to 5.1 kg/ha, respectively. Levels of the most abundant phenoliccompounds are shown in Figure 4B. These included compounds2-O-caffeoylisocitrate, 9; 2-O-caffeoylisocitrate methyl ester, 18a;lithospermic acid isomer, 19; 2-O-caffeoylisocitrate orientinconjugate, 24; and 2-O-caffeoylisocitrate methyl ester orientinconjugate, 26. Some variation in content was observed with 2-O-caffeoylisocitrate concentrations decreasing over time. Analysisof the antioxidant activity of these compounds by a range ofassays, including DPPH, ABTS, and FRAP, showed levels ofactivity comparable with that of trolox (Table 3) and indicatethat the lithospermic isomer is the most potent antioxidant of thecompounds tested.

Ensiling. Orchard grass was ensiled in laboratory silos for 14days to investigate the effect of conservation on the phenolprofile of the plant material. The composition of the silage isshown in Table 4. Limited fermentation occurred during thisperiod with reduced production of fermentation acids, minimaldepletion of WSC content, and little change in pH.Analysis of phenolic compounds in ensiled orchard grass

showed that many of these compounds persisted during theensiling period (Figure 5).Whereas all compounds were not fullyresolved in these extracts, differences in compound abundancewere verified by total ion counts. There was evidence ofhydrolysis of caffeoyl esters with accumulation of free caffeic acid,4a. There was also evidence of further catabolism of caffeic acidwith accumulation of dihydroxystyrene (X, Figure 5). 2-O-Caffeoylisocitrate, 9, showed a decrease, while an increase in thelithospermic acid isomer, 15, was also evident. It is noteworthy

Figure 4. Variation in phenolic composition of orchard grass during thetransition from vegetative to flowering stage: (A) changes in totalflavonoids and hydroxycinnamates; (B) changes in abundant phenoliccompounds. CI, 2-O-caffeoylisocitrate, 9; Lith, lithospermic, 15; CMI,2-O-caffeoylisocitrate 6-methyl ester, 18a; CI-Or, 2-O-caffeoylisocitrateorientin conjugate, 26; CMI-Or, 2-O-caffeoylisocitrate methyl esterorientin conjugate, 28.

Table 3. Antioxidant Capacity of Abundant Phenolic Compounds in Orchard Grass

TE (μM trolox/μM phenol)

compound DPPH ABTS FRAP

2-O-caffeoylisocitrate, 9 0.531 ± 0.033 0.911 ± 0.054 1.500 ± 0.009lithospermic acid, 15 1.652 ± 0.011 3.565 ± 0.049 4.133 ± 0.0032-O-caffeoylisocitrate 6-methyl ester, 18a 0.388 ± 0.007 0.725 ± 0.024 1.325 ± 0.0292-O-caffeoylisocitrate orientin conjugate, 26 1.187 ± 0.008 0.581 ± 0.099 0.884 ± 0.0412-O-caffeoylisocitrate methyl ester orientin conjugate, 28 1.535 ± 0.016 0.831 ± 0.064 2.283 ± 0.054

Table 4. Composition of Fresh and Ensiled ‘Abertop’ PlantMaterial

fresh ensiled

DM (g/kg fw) 22.00 19.13pH 6.40 5.95WSC (g/kg dm) 167.41 147.59lactic acid (g/kg dm) 23.86acetic acid (g/kg dm) 14.31

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that dramatic increases in levels of the compound salvianolic acidB, which is a bioactive component in the medicinal herb danshen(Salvia miltiorrhiza), were observed during postharvest drying ofthe herbal material.33 This observation may be significant as thiscomponent is potentially of therapeutic value.This study suggests that restricted silage fermentation may be

advantageous from a biorefining perspective. Limited degrada-tion of metabolites offers increased scope for recovery of high-value components, and a high WSC content provides afermentable substrate in extracted juice for the production ofethanol or lactic acid.In summary, there is currently much interest in developing

biorefineries based on grass feedstocks, and ensilage is thepreferred method for stabilizing harvested biomass forbiorefining and biofuel production. Processes have beendeveloped at pilot scale for the extraction of amino acids andlactic acid from silage juice.10 Recovery of other potential high-value components such as soluble phenolic compounds withbioactive properties can improve the economic feasibility of thesebiorefineries. It is noteworthy that we have demonstrated inprevious experiments that compounds identified in orchard grasscan also be extracted with ethanol, a solvent compatible with theproduction of nutraceuticals. Moreover, biorefining can enablerecovery of these compounds on a large scale as coproducts when

profit margins would prohibit an operation based solely on thesemolecules.

■ ASSOCIATED CONTENT

*S Supporting InformationProposed fragmentation route for compound 24, which isrepresentative of fragmentation patterns observed for othercompounds in this group. This material is available free of chargevia the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*(A.L.W.) Phone: +44 (0) 1970 823207. Fax: +44 (0) 1970828357. E-mail: alg@aber.ac.uk.

FundingThis work was supported by funding from the Biotechnology andBiological Sciences Research Council (BBSRC) InstituteStrategic Programme Grant (BB/J0042/1), the EuropeanRegional Development Fund through funding provided for theBEACON project by the Welsh European Funding Office, andthe Technology Strategy Board through the Succinic Esters fromRenewable Feedstocks (SERF) project.

Figure 5. LC chromatogram showing separation of soluble phenolic compounds detected by photodiode array at 340 nm: (A) before ensiling; (B) afterensiling. Peaks that showed a change in content during ensiling are labeled according to numbering in Table 1. X denotes dihydroxystyrene peak.

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NotesThe authors declare no competing financial interest.

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