Soluble Phenolic Compounds in Fresh and Ensiled Orchard Grass

Dec 17, 2013 - Salvia miltiorrhiza, and novel hydroxycinnamate−flavone conjugates were also identified in extracts. Harvest yields of hydroxycinnama...
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Soluble Phenolic Compounds in Fresh and Ensiled Orchard Grass (Dactylis glomerata L.), a Common Species in Permanent Pastures with Potential as a Biomass Feedstock Barbara 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 biofuel production. Plant polyphenolics have potential application in the pharmaceutical and cosmetic industries. Orchard grass varieties have been noted for accumulation of polyphenolic compounds, and the current study determined the soluble phenol profile and content in the orchard grass variety ‘Abertop’. Hydroxycinnamates and flavonoids were monitored during the transition from vegetative to flowering stage at maximum crop yield. Caffeic acid derivatives, related to bioactives in the Asian medicinal herb Salvia miltiorrhiza, and novel hydroxycinnamate−flavone conjugates were also identified in extracts. Harvest yields of hydroxycinnamates and flavonoids ranged from 2.6 to 4.0 kg/ha and from 2.1 to 5.1 kg/ha, respectively. Abundant compounds showed high levels of antioxidant activity comparable with that of trolox. Minimal changes in soluble phenol content and composition were observed after ensiling with the exception of increases in caffeic acid, a caffeic acid derivative, and a caffeic acid breakdown product, dihydroxystyrene. KEYWORDS: Dactylis glomerata, orchard grass, phenolic acid esters, flavonoid glycosides, caffeic acid derivatives

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INTRODUCTION Orchard grass (Dactylis glomerata) is a relatively coarse, tussockforming grass that is native to Europe, temperate Asia, and North Africa.1 It has been introduced as a forage grass into most temperate countries in the world and is one of the most important sown species in Australia and New Zealand.2 Plants are either diploid (2n = 14) or, more commonly, autotetraploid (2n = 28), and subspecies are morphologically distinguishable according to area of origin. For example, plants of the Eurasian (or Northern) group are typically large with broad leaves and winter-dormant, whereas plants from the Mediterranean region are relatively small, narrow-leaved, and summer-dormant. Although this species has a lower nutritional value compared to other forage species such as perennial ryegrass, it is more tolerant to heat and drought, resulting in higher production during summer dry periods. Phenolic compounds are generally abundant in orchard grass compared with other temperate grass species. The presence of phenolic compounds in this species was first reported in 1926 by Landsborough-Thompson,3 who described the isolation of a flavone glycoside and a free flavone from plant extracts. More recently, Parveen et al.4 identified a number of hydroxcinnamate esters in a commercial variety of orchard grass (D. glomerata). There is a strong tendency for chemotaxonomically related plants to produce similar types of flavonoids,5 and a number of studies have reported characteristic variation in the flavonoid profile of different populations of orchard grass.6,7 Although temperate grasses have a fundamental role in ruminant nutrition, recent studies have focused on their potential as feedstocks for biorefining.8−10 Increased recovery of components with a marketable value can improve the feasibility © 2013 American Chemical Society

of grasses as biorefining feedstocks. Valuable components can be recovered from the liquid fractions of ensiled grass biomass including lactic acid and amino acids,11 whereas the residual presscake has potential as a combustible feedstock.12 Alternatively, residual biomass may be used to produce bioethanol9 or biogas.13 Polyphenols have been identified as potential high value coproducts in a biorefinery, and processes have been developed for their recovery from olive mill wastewater.14 These classes of compounds are of interest to the pharmaceutical, cosmetics, and nutraceutical sectors, and there is an increasing body of evidence demonstrating the health benefits of plant phenols.15 Orchard grass is reported to be a traditional remedy for tumors, kidney, and bladder ailments.16 Here we report on the identification and quantitative analysis of flavonoids/phenolic compounds in a commercial variety of orchard grass, ‘Abertop’. This variety has been developed to provide a high-yielding conservation cut and good ground cover. We monitor the phenol profile during the transition from vegetative stage to flowering stage, which corresponds with maximum yield. We also present data on changes in soluble phenol profile following a short period of ensiling.

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MATERIALS AND METHODS

Chemicals. HPLC grade methanol was obtained from Fisher Scientific (Loughborough, UK), and commercial standards for phenolic acids and flavone aglycones were from Sigma-Aldrich (Gillingham, UK) and PhytoLab (Vestenbergsgreuth, Germany). Received: Revised: Accepted: Published: 468

September 11, 2013 December 16, 2013 December 17, 2013 December 17, 2013 dx.doi.org/10.1021/jf4040749 | J. Agric. Food Chem. 2014, 62, 468−475

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gas, 15 units; spray voltage, 4 kV; capillary temperature, 320 °C; capillary voltage, −1 V; and tube lens offset, −68 V. MS/MS fragmentation was carried out at a normalized collision energy of 35% and isolation width m/z 2.0. Purification of Selected Compounds and Acid Hydrolysis. Abundant phenolic compounds were purified by reverse-phase chromatography with the Waters system described above. Fractions were concentrated by rotary evaporation, and a portion was retained for acid and alkali hydrolysis. Acid hydrolysis was performed by combining a volume of purified compound with an equal volume of 2 M HCl. The solution was heated to 90 °C for 1 h, and the pH was then adjusted to 4− 5 with NaOH. Alkali hydrolysis was performed by combining a volume of purified compound with an equal volume of 2 M NaOH. The solution was incubated for 16 h under N2, and the pH was then adjusted to 4−5 with HCl. Purified compounds and neutralized hydrolysates were partially 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 trolox equivalents (TE) by comparison of tested compounds to trolox calibration curves in the range of 5−50 μmol/L. The diphenylpicrylhydrazyl (DPPH) radical scavanging assay was carried out according to the method of Brand-Williams et al.17 A 140 μM solution of DPPH was prepared in 50:50 methanol/water. This was combined with an equal volume of sample in a 1.5 mL cuvette, with 50:50 methanol/water as a control. Mixtures were left to stand for 30 min at room temperature, after which time absorbance at 517 nm was determined. A range of sample concentrations were tested to identify the range giving partial reduction 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 The radical form was prepared by adding potassium persulfate to a 7 mM stock solution of ABTS to give a final concentration of 2.45 mM and allowing 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 give an absorbance of 0.700 (±0.2) at 743 nm. Sample volumes of 25 μL were added to 1475 μL of reagent in a 1.5 mL cuvette, and absorbance was measured after 6 min. A range of sample concentrations were tested to identify the range giving partial reduction of the ABTS•+ in the reaction mixture. The ferric reducing capacity of samples was measured by the FRAP assay, 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) in the ratio of 1:1:10. Sample volumes of 25 μL were added to 1475 μL of reagent in a 1.5 mL cuvette, and absorbance was measured at 593 nm after 10 min.

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 a Haldrop plot harvester at regular intervals during May 2010. Plant yields were recorded during harvesting. Following harvesting, plant material was 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 silage tubes (approximately 200 mL size) and capped with silo air traps. Tubes were incubated at 25 °C and opened after 2 weeks. Freshly sampled material was extracted for phenols, and remaining material was frozen at −20 °C for further chemical analysis. Samples were analyzed for dry matter 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 immersing 2.5 g in 10 mL of water. Samples were homogenized and centrifuged at 3000g for 15 min. The supernatant was recovered and the pellet discarded. Fifty microliters of extract was added to 950 μL of a buffer comprising 5 μM H2SO4, containing 5 μM crotonic acid as an internal standard. These samples were analyzed with a Jasco HPLC system (Jasco Ltd., Great Dunmow, Essex, UK), and the column used was a 150 mm × 7.8 mm Rezex ROA-Organic Acid (Phenomenex, Macclesfield, UK) with a mobile phase of 5 μM H2SO4 at 0.6 mL/min. Peaks were detected with a refractive index detector, identified using Jasco EZChrom Elite HPLC software (Jasco Ltd., Essex, UK), and quantitated by 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 a growing season, and a bulk sample of approximately 6 g was extracted for detailed profiling of phenolic compounds). The supernatant was decanted, and the pellet was extracted with methanol/water (8:2) and centrifuged for 20 min at 3000g. The fractions were then combined to give a total soluble phenol extract. Methanol was removed from the extracts by rotary evaporation, and extracts were then partially purified on a 500 mg Sep-Pak C18 3 cc Vac RC cartridge (Waters Ltd., Elstree, UK). Cartridges were prepared with 4 mL of methanol (100%) followed by 4 mL of 5% acetic acid. Samples were then loaded, washed with 2.5 mL 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 and without 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, C18 Nova-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 for appropriate standards. HPLC−ion trap mass spetrometry (HPLC-MSn) analysis was performed on a Thermo Finnigan LC-MS system (Thermo Electron Corp., Waltham, MA, USA) comprising a Finnigan Surveyor PDA Plus detector and a Finnigan LTQ linear ion trap with ESI source, and the column used was a 3.9 mm × 100 mm i.d., 4 μm, C18 Nova-Pak (Waters). The autosampler tray temperature was maintained at 5 °C and the column temperature at 30 °C. Sample injection volume was typically 10 μL, the detection wavelength was set to 240−400 nm, and the flow rate 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) and MeOH/formic acid (B; 100:0.1, v/v). The column was equilibrated with 95% solvent A, and the percentage of B increased linearly to 60% over 65 min. Ionization parameters were optimized by infusion of chlorogenic acid standard at a constant rate into the LC flow. Mass spectra were acquired in negative ionization mode with the following interface and MS parameters: nitrogen sheath gas, 30 arbitrary units; nitrogen auxiliary

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RESULTS AND DISCUSSION Phenol Profile. Figure 1 shows a typical profile of soluble phenols extracted from ‘Abertop’. Analysis by LC ESI-MS with PDA detection revealed approximately 50 compounds that could be easily distinguished (Table 1). Several compounds were readily identified by comparison of retention times and fragmentation patterns with known standards. Others were tentatively identified by the similarity of UV absorbance characteristics and fragmentation patterns with known standards and compounds reported in the literature.5,20,21 Representative structures are shown in Figure 2. Compounds identified by LC-MS include hydroxycinnamoyl esters, flavone glycosides, salvianolic acids, and hydroxycinnamoyl flavone conjugates. Relatively abundant hydroxycinnamate esters, which have been previously reported by Parveen et al.4 in orchard grass, were identified in extracts including 5-caffeoyl quinate (chlorogenic acid), 2-O-caffeoylisocitrate, and 2-Ocaffeoylisocitrate 6-methyl ester (compounds 5, 9, and 18a as shown in Table 1). 469

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− 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 secondary peaks at 205, 157, 87, and 85. This indicates that the 157 ion yielded in MS4 is released from the 175 ion yielded in MS3. These fragments are consistent with neutral losses of CO2 (−44 Da), dehydrophenoxy radical (−92 Da), dehydrophenoxy radical and CO2 (−136 Da), and dehydrophenoxy radical, CO2, and C2H2/ C2H4 (−162/−164 Da). These fragments may be accounted for by whole or partial loss of a coumaroyl moiety, and the ion at m/z 87 is consistent with a dehydro-glycerate moiety. On this basis it is proposed that compound 8 is caffeoylglycerate conjugated with coumaric acid formed via an oxidative radical coupling reaction. Caffeic Acid Derivatives. Antioxidant, antithrombotic, antitumor, and antiviral properties of the popular Asian medicinal herb, danshen (Salvia miltiorrhiza), have been attributed to constituent caffeic acid derivatives known as salvianolic and lithospermic acids.23 A number of compounds with structural properties consistent with these caffeic acid derivatives were detected in orchard grass extracts. These included compounds 15, 19a, 19b, 22b, 23c, 23d, 25a, and 25b with Mr 360, 492, 538, 556, and 736 Da. ESI MSn spectra of negative ions showed characteristic fragmentation patterns of lithospermic and salvianolic acids with products derived from neutral losses of 198, 180, 162, 110, and 44 Da corresponding to losses of 3,4-dihydroxyphenyl-lactic acid (danshensu), caffeic acid, caffeic acid dehydrate, catechol, and CO2 moieties, respectively. They also yielded characteristic fragment ions of salvianolic acids at m/z 537, 493, 383, 339, 313, 295, 197, and 179.24 Compound 25b with Mr 360 Da shows retention time, UV absorption profile (maxima at 276 and 331 nm), and fragmentation pattern consistent with those of rosmarinic acid (MS2 fragments at m/z 161, 179, and 197). The remaining members of the group have been identified on the basis of their MS and UV spectra where detected. Compound 23d with Mr 492 Da at tr 26.0 min shows characteristic features of caffeic acid derivatives. This compound showed a base peak in negative mode at m/z 339 and a secondary peak at m/z 447 corresponding to neutral losses of 152 and 44 Da. The fragmentation pattern suggests a molecular structure consistent with lithospermic acid less CHOOH. The loss of 152 Da is equivalent to the loss of 198 Da observed during fragmentation of lithospermic acid in negative mode resulting from a cleavage of the ester bond between the carboxyl oxygen and second oxygen atom, yielding a negative ion at m/z 339 as observed here. Other fragments in common with those yielded by lithospermic acid include negative ions at m/z 295 and 185. Other fragments observed include ions at 447 and 337, which may correspond with ions observed at m/z 493 and 383 in lithospermic 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 MS at m/z 537 and 717, respectively, corresponding with lithospermic acid and lithospermic acid B. Similar compounds with equivalent masses have been reported in sage tea.25 A 736 Da isomer gave rise to a base peak at m/z 537 (198 Da loss), which in MS3 yielded fragment ions typical of lithospermic acid as reported by Zeng et al.24 Caffeic acid derivatives, including rosmarinic, lithospermic, and salvianolic acids, have potent radical-scavenging activity related to the number of catechol moieties contained in the

Figure 1. HPLC chromatogram showing separation of soluble phenolic compounds in extracts of orchard grass detected by photodiode array at 340 nm. Peak numbering corresponds with Table 1.

Hydroxycinnamate Esters. A number of hydroxycinnamate esters that have not previously been reported in orchard grass were detected in ‘Abertop’ extracts. Compound 7 with Mr 268 Da at tR 11 min showed characteristics consistent with a caffeoyl/ feruloyl ester with UV maxima at 303sh and 326 nm. MS2 of m/z 267 in negative mode produced a base peak at m/z 161 [caffeic acid − H2O − H+]− and a secondary peak at m/z 105 [glyceric acid − H+]−. Further fragmentation of the m/z 105 ion produced a base peak at m/z 75 [glyceric acid − HCOH − H+]− and corresponded with the fragmentation pattern observed for glyceric acid. This compound is identified as caffeoylglycerate. 2Caffeoylglycerate has previously been identified in extracts of the medicinal plant, Mercurialis perennis L.22 A second hydroxycinnamoyl ester (MR 352 Da), compound 22a, was detected at tR 24.8 min, showing maximal UV absorbance at 315 nm. This compound produced an MS2 base peak at m/z 163 [coumaric acid − H+]− and secondary ions at m/ 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 a base 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 been previously identified in orchard grass,4 and this compounded yielded an ion at m/z 205 in MS2 with an equivalent fragmentation pattern in MS3 to the 205 ion reported here. On this basis this compound is identified as coumaroylisocitrate methyl ester. Compound 8 with Mr 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 a caffeoyl/feruloyl ester with UV maxima at 300sh and 324 nm. It produced an MS2 base peak in negative mode at m/z 267 corresponding to the neutral loss of 162 Da and secondary peaks at m/z 179 and 411 [M − H2O − H+]−. The 179 ion was confirmed as [caffeic acid − H+]− in MS3. Further fragmentation of the 267 ion in MS3 yielded a base peak at m/z 249 [267 − H2O 470

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Table 1. Phenolic Compounds in Orchard Grass Leaf Extracts peak/ ID 1 2 3 4a 4b 5 6 7 8 9 10 11 12 13a 13b 14 15

tr 4.9 5.7 6.5 8.9 9.7 10.0 11.0 11.4 12.1 12.8 15.7 16.4 18.0 18.4 18.8

λmax(nm)

main fragments(m/z)

353 297 297 179 367 353 353 267 429 353 163 337 609 593 367 609 537

191, 179 (45), 135 (8), 173 (3) 135, 179 (29), 279 (5), 229 (3) 135, 179 (29), 279 (3), 229 (2), 117 (1) 135, 179 (76), 151 (1) 193, 134 (5), 173 (3), 191 (2) 191, 179 (5), 173 (1), 135 (0.5), 161 (1) 173, 179 (54), 191 (33), 135 (5) 161, 105 (64), 179 (33), 221 (6), 135(5) 267, 411 (3), 179 (3) 173, 191 (75), 111 (13), 155 (6), 179 (2) 119, 163 (8) 173, 111 (13), 155 (5), 191 (3) 489, 519 (26), 490 (12), 399 (9), 591 (8), 369 (7) 473, 503 (32), 353 (27), 474 (22), 383 (14), 575 (11) 173, 111 (13), 155 (7), 179 (7), 135 (3) 327, 357 (65), 411 (25), 328 (11), 591 (10), 358 (8), 447 (7) 339, 493 (5), 357 (1),

579 447 367 579 537 555 447 563 563 593 351 555

299sh, 323 301sh, 326 299sh, 326 300sh,322 300sh, 322 300sh, 321 301sh, 323 303sh, 326 300sh, 324 304sh, 328 312 314 270, 342 ND ND ND 252, 285, 318sh, 344 270, 349 ND 302sh, 330 ND ND ND ND ND

23d 24 25a

27.3

491 799 735

25b 26

27.3 28.8

359 813

489, 459 (20), 519 (17), 561 (16), 399 (12), 369 (8), 471 (6) 327, 357 (46), 369 (6), 393 (4) 179, 205 (56), 169 (8), 135 (7), 173 (5), 187 (1), 111 (1) 429, 459 (20), 357 (19), 309 (14), 561 (6), 447 (4) 493, 295 (61), 357 (6), 383 (6), 313 (6), 337 (4) 537, 511 (62), 269 (8), 493 (7), 313 (4) 327, 357 (49), 429 (15), 387 (3) 443, 473 (60), 353 (20), 383 (15), 545 (14), 503 (7) 413, 293 (20), 443 (3) 473, 503 (83), 383 (33), 413 (21), 474 (20), 504 (17), 575 (15), 533 (7) 163, 169 (78), 205 (38), 119 (5) 511, 537 (64), 357 (61), 269 (47), 285 (38), 313 (33), 449 (27), 241 (22), 339 (18), 197 (10) 311, 341 (28), 312 (10), 413 (7) 443, 473 (50), 444 (18), 353 (16), 545 (11), 474 (10), 383 (8), 455 (4) 607, 581 (10), 487 (7), 625 (1), 553 (1) 607, 581 (20), 607 (17), 487 (15), 581 (3), 517 (3), 553 (2), 625 (2) 493, 537 (66), 717 (50), 313 (45), 691 (20), 475 (18), 555 (15), 431 (11), 519 (11), 295 (10), 339 (8) 339, 447 (33) 581, 607 (2), 461 (1), 625 (1), 755 (1), 491 (1) 537, 717 (80), 493 (26), 339 (22), 475 (20), 449 (15), 519 (13), 465 (12), 313 (12), 673 (10), 357 (9), 295 (8) 161, 179 (29), 197 (24), 223 (15), 133 (3), 135 (2) 581, 607 (10), 461 (2), 625 (2), 487 (1), 491 (1)

27 28

30.0 30.8

491 813

329, 330 (5), 371 (3), 314 (1) 595, 639 (19), 581 (8), 607 (1), 769 (1), 625 (1), 461 (1)

260, 343 270, 345

29

32.8

827

595, 639 (3)

271, 335

30 31 32 33

34.1 34.4 41.4 42.5

577 687 329 525

533, 491 (1) 491, 669 (49), 643 (33), 329 (30), 492 (13), 525 (12), 314, 329 (50), 299 (1) 329, 477 (2), 195 (2), 314 (2), 507 (2), 165 (1)

260, 345 326 267, 349 270, 331

16 17 18a 18b 19a 19b 20a 20b 21a 21b 22a 22b 22c 22d 23a 23b 23c

19.8 20.5 20.8

[M − H]− (m/z)

21.6 21.8 23.8 24.8

26.0 26.6

431 563 743 713 735

structure and are reported to have a range of protective and antimicrobial properties.26,27 Hydroxycinnamate Esters and Flavone Glycoside Conjugates. UV combined with MS analysis revealed a novel group of compounds in D. glomerata extracts with distinctive fragmentation patterns in negative mode MS. These compounds show neutral losses consistent with cleavage of ester-linked

tentative compound identification 3-caffeoylquinate 2-O-caffeoylthreonate 2-O-caffeoylthreonate caffeic acid feruloyl quinate 5-caffeoylquinate 4-caffeoylquinate caffeoylglycerate caffeoylglycerate p-coumaric conjugate 2-O-caffeoylisocitrate p-coumaric acid 2-O-coumaroylisocitrate luteolin-6-C-hexoside-8-C-hexoside apigenin-6-C-hexoside-8-C-hexoside 2-O-caffeoylisocitrate methyl ester iso-orientin-O-hexoside lithospermic acid isomer

315 ND

luteolin-6-C-pentoside-8-C-hexoside orientin 2-O-caffeoylisocitrate 6-methyl ester luteolin-C-hexosyl pentoside lithospermic acid isomer salvianolic acid K isomer iso-orientin apigenin-6-C-hexoside-8-C-pentoside apigenin-C-hexosyl pentoside chryseriol-6-C-hexoside-8-C-pentoside 2-O-coumaroylisocitrate methyl ester caffeic acid derivative

ND ND ND ND ND

isovitexin apigenin-6-C-hexoside-8-C-pentoside 2-O-caffeoylthreonate orientin conjugate 2-O-caffeoylglycerate orientin conjugate caffeic acid derivative

ND 269, 349 ND

caffeic acid derivative 2-O-caffeoylisocitrate orientin conjugate salvianolic acid isomer

ND 270, 349

rosmarinic acid 2-O-caffeoylisocitrate methyl ester orientin conjugate tricin-O-hexoside 2-O-caffeoylisocitrate methyl ester orientin conjugate 2-O-caffeoylisocitrate methyl ester luteolinmethyl-ether conjugate tricin-O-hexosyl -malonate tricin conjugate tricin tricin conjugate

organic acids and, where detected, UV absorption maxima at 269−270 and 349 nm. UV profiles were typical of the profile produced by orientin (luteolin-8-C-glucoside) standard. Compound 24 with Mr 800 Da eluted at tR 26.6 min and in negative mode produced a mass spectrum with a base peak at m/z 581 and further peaks at m/z 607 and 625. This corresponds to neutral losses of 218, 192, and 174 Da, respectively. Interestingly, full MS 471

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

Figure 2. Structures of phenolic compounds in orchard grass.

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

in positive mode revealed major ions at m/z 801 and 627 at tR 26.6, whereas only one ion was observed in negative mode, suggesting a neutral fragment of 174 Da is readily lost in positive mode. Losses of 192 and 174 Da correspond to cleavage of an isocitrate or quinate moiety on either side of the ether linkage in an ester bond. The neutral loss of 218 Da could be accounted for by cleavage at the far side of the carbonyl group (174 + 44 Da). Further fragmentation of the 581 ion in MS3 yielded ions at m/z 461 and 491, which correspond to neutral losses of 120 and 90 Da, typical of C-linked flavonoid hexosides.28 Additional fragments at m/z 471 and 351 correspond to neutral losses of 110 and 230 Da. These may be accounted for by loss of a catechol moiety (110 Da) and a catechol moiety combined with a Clinked hexoside fragment (110 and 120 Da). Spectra in MS4 of the ions at m/z 491 and 461 showed base peaks at m/z 381 and 351, respectively, corresponding to the loss of a catechol moiety. Compound 24 was purified by reverse phase chromatography and incubated with 1 M HCl with heat or with 1 M NaOH at room temperature to hydrolyze ester and O-glycosidic linkages. Major products of acid and alkali hydrolysis experiments included compounds with Mr 626, 582, and 472 Da. These compounds yielded identical fragment ions in MS2 corresponding to fragment ions described above and may be accounted for by hydrolysis of ester linkages, acid-catalyzed decarboxylation, and release of pyrocatechol by decomposition. Minor products included compounds with Mr 192, 354, and 447 Da. These compounds showed fragmentation patterns identical to those of isocitric acid, 2-O-caffeoylisocitrate, and orientin. On the basis of this evidence, this molecule is composed of 2O-caffeoylisocitrate and orientin residues. The fragmentation

crude protein (g/kg DM) WSC (g/kg DM) NDF (g/kg DM) ADF (g/kg DM) DMD (g/kg DM) yield (t/ha)

May 8

May 13

May 18

May 23

May 29

166.6 152.7 430.9 206.0 759.6 2.58

161.3 114.3 480.0 236.2 722.4 3.05

129.1 165.4 465.1 230.8 744.8 3.68

126.9 150.9 469.1 234.7 720.2 3.87

106.9 130.6 522.4 264.0 692.1 6.36

patterns observed in MSn experiments including the release of isocitrate and catechol moieties and CO2 indicate that this compound is formed by a condensation reaction with bonds formed between C2 and C3 on the alkyl side chain of the caffeoyl moiety and the luteolin B-ring. Similar condensation products include naturally occurring oxyneolignans, americanin A29 and eusiderin,30 and the coumarinolignoid, daphneticin.31 Other related compounds include a hydroxcinnamate−mandelonitrile conjugate, which has been reported in Miscanthus × giganteus stem extracts.32 These compounds are postulated to be formed by oxidative coupling mechanisms, and conjugates identified in orchard grass extracts may be formed by similar pathways. Activity of plant oxidative enzymes during the extraction process could potentially catalyze formation of these products. To eliminate this possibility, we repeated the extraction procedure including 50 mM ascorbic acid in the extraction solvent. An identical profile of phenolic compounds was obtained by this method (data not presented), indicating that these conjugates were not formed during the extraction procedure. 472

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Table 4. Composition of Fresh and Ensiled ‘Abertop’ Plant Material DM (g/kg fw) pH WSC (g/kg dm) lactic acid (g/kg dm) acetic acid (g/kg dm)

fresh

ensiled

22.00 6.40 167.41

19.13 5.95 147.59 23.86 14.31

was monitored during transition from vegetative to flowering stage, which corresponds with maximum yield of biomass. Table 2 shows changes in composition in harvested material. Changes in composition reflect the alteration in morphology that occurs during flowering, that is, increasing proportion of stem tissue with a corresponding decrease in leaf tissue. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) show an increasing trend, whereas the opposite pattern is observed with dry matter digestibility and crude protein, which generally decrease with plant maturity. Biomass yield showed a steady increase during this period. Figure 4A shows changes in total hydroxycinnamate and flavonoid content during flowering. Levels remained high throughout this period, with potential yields of hydroxycinnamates and flavonoids ranging from 2.6 to 4.0 kg/ha and from 2.1 to 5.1 kg/ha, respectively. Levels of the most abundant phenolic compounds are shown in Figure 4B. These included compounds 2-O-caffeoylisocitrate, 9; 2-O-caffeoylisocitrate methyl ester, 18a; lithospermic acid isomer, 19; 2-O-caffeoylisocitrate orientin conjugate, 24; and 2-O-caffeoylisocitrate methyl ester orientin conjugate, 26. Some variation in content was observed with 2-Ocaffeoylisocitrate concentrations decreasing over time. Analysis of the antioxidant activity of these compounds by a range of assays, including DPPH, ABTS, and FRAP, showed levels of activity comparable with that of trolox (Table 3) and indicate that the lithospermic isomer is the most potent antioxidant of the compounds tested. Ensiling. Orchard grass was ensiled in laboratory silos for 14 days to investigate the effect of conservation on the phenol profile of the plant material. The composition of the silage is shown in Table 4. Limited fermentation occurred during this period with reduced production of fermentation acids, minimal depletion of WSC content, and little change in pH. Analysis of phenolic compounds in ensiled orchard grass showed that many of these compounds persisted during the ensiling period (Figure 5). Whereas all compounds were not fully resolved in these extracts, differences in compound abundance were verified by total ion counts. There was evidence of hydrolysis of caffeoyl esters with accumulation of free caffeic acid, 4a. There was also evidence of further catabolism of caffeic acid with accumulation of dihydroxystyrene (X, Figure 5). 2-OCaffeoylisocitrate, 9, showed a decrease, while an increase in the lithospermic acid isomer, 15, was also evident. It is noteworthy

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

Other compounds assigned to this group on the basis of fragmentation patterns include compounds 23a, 23b, 26, 28, and 29 with Mr 744, 714, 814, 814, and 828 Da. Common fragments detected in negative mode ESI MSn spectra include ions at m/z 625, 607, 581, 491, 487, and 461 Da. Compounds with Mr 714, 744, and 814 Da have been tentatively identified as conjugates of orientin with caffeoylthreonate, caffeoylglycerate, and caffeoylisocitrate methyl ester, respectively (Figure 3), and a compound with Mr 828 Da has a fragmentation pattern consistent with a conjugate of hispidulin-C-glycoside with caffeoylisocitrate methyl ester. Variation in Phenolic Profiles during Transition from Vegetative to Flowering Stage. Orchard grass production

Table 3. Antioxidant Capacity of Abundant Phenolic Compounds in Orchard Grass TE (μM trolox/μM phenol) compound

DPPH

ABTS

FRAP

2-O-caffeoylisocitrate, 9 lithospermic acid, 15 2-O-caffeoylisocitrate 6-methyl ester, 18a 2-O-caffeoylisocitrate orientin conjugate, 26 2-O-caffeoylisocitrate methyl ester orientin conjugate, 28

0.531 ± 0.033 1.652 ± 0.011 0.388 ± 0.007 1.187 ± 0.008 1.535 ± 0.016

0.911 ± 0.054 3.565 ± 0.049 0.725 ± 0.024 0.581 ± 0.099 0.831 ± 0.064

1.500 ± 0.009 4.133 ± 0.003 1.325 ± 0.029 0.884 ± 0.041 2.283 ± 0.054

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

profit margins would prohibit an operation based solely on these molecules.

that dramatic increases in levels of the compound salvianolic acid B, which is a bioactive component in the medicinal herb danshen (Salvia miltiorrhiza), were observed during postharvest drying of the herbal material.33 This observation may be significant as this component is potentially of therapeutic value. This study suggests that restricted silage fermentation may be advantageous from a biorefining perspective. Limited degradation of metabolites offers increased scope for recovery of highvalue components, and a high WSC content provides a fermentable substrate in extracted juice for the production of ethanol or lactic acid. In summary, there is currently much interest in developing biorefineries based on grass feedstocks, and ensilage is the preferred method for stabilizing harvested biomass for biorefining and biofuel production. Processes have been developed at pilot scale for the extraction of amino acids and lactic acid from silage juice.10 Recovery of other potential highvalue components such as soluble phenolic compounds with bioactive properties can improve the economic feasibility of these biorefineries. It is noteworthy that we have demonstrated in previous experiments that compounds identified in orchard grass can also be extracted with ethanol, a solvent compatible with the production of nutraceuticals. Moreover, biorefining can enable recovery of these compounds on a large scale as coproducts when

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ASSOCIATED CONTENT

S Supporting Information *

Proposed fragmentation route for compound 24, which is representative of fragmentation patterns observed for other compounds in this group. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(A.L.W.) Phone: +44 (0) 1970 823207. Fax: +44 (0) 1970 828357. E-mail: [email protected]. Funding

This work was supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant (BB/J0042/1), the European Regional Development Fund through funding provided for the BEACON project by the Welsh European Funding Office, and the Technology Strategy Board through the Succinic Esters from Renewable Feedstocks (SERF) project. 474

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Notes

of dog’s mercury (Mercurialis perennis L.) by LC-MS/MS and GC-MS analyses. Phytochem. Anal. 2012, 23, 60−71. (23) Chen, J.; Wang, F.; Lee, F. S-C.; Wang, X.; Xie, M. Separation and identification of water-soluble salvianolic acids from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography and ESI-MS analysis. Talanta 2006, 69, 172−179. (24) Zeng, G.; Xiao, H.; Liu, J.; Liang, X. Identification of phenolic constituents in Radix Salvia miltiorrhizae by liquid chromatography/ electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2006, 20, 499−506. (25) Zimmermann, B. F.; Walch, S. G.; Tinzoh, L. N.; Stühlinger, W.; Lachenmeier, D. W. Rapid UHPLC determination of polyphenols in aqueous infusions of Salvia of f icinale L. (sage tea). J. Chromatogr., B 2011, 879, 2459−2464. (26) Nuengchamnong, N.; Krittasilp, K.; Ingkaninan, K. Characterisation of phenolic antioxidants in aqueous extract of Orthosiphon grandif lorus tea by LC-ESI-MS/MS coupled to DPPH assay. Food Chem. 2011, 127, 1287−1293. (27) Ho, J. H. C.; Hong, C.-Y. Salvianolic acids: small compounds with multiple mechanisms for cardiovascular protection. J. Biomed. Sci. 2011, 18, 30. (28) Ferreres, F.; Silva, B. M.; Andrade, P. B.; Seabra, R. M.; Ferreira, M. A. Approach to the study of C-glycosyl flavones by ion trap HPLCPAD-ESI/MS/MS: application to seeds of quince (Cydonia oblonga). Phytochem. Anal. 2003, 14, 352−359. (29) Woo, W. S.; Kang, S. S.; Wagner, H.; Chari, V. M. The structure of Americanin, a neolignan from Phytolacca americana. Tetrahedron Lett. 1978, 25, 3239. (30) Dias, M. C.; Fernandes, J. B.; Maia, J. G. S.; Gottlieb, O. R.; Gottlieb, H. E. Eusiderins and other neolignans from an Aniba species. Phytochemistry 1985, 25, 213−217. (31) Zhuang, L.-G.; Seligmann, O.; Wagner, H. Daphneticin, a coumarinolignoid from Daphne tangutica. Phytochemistry 1983, 22, 617−619. (32) Parveen, I.; Threadgill, M. D.; Hauck, B.; Donnison, I.; Winters, A. Isolation, identification and quantification of hydroxycinnamic acid conjugates, potential platform chemicals, in leaves and stems of Miscanthus × giganteus using LC-ESI-MSn. Phytochemistry 2011, 72, 2376−2384. (33) Li, X. B.; Wang, W.; Zhou, G. J.; Li, Y.; Xie, X. M.; Zhou, T. S. Production of salvianolic acid B in roots of Salvia miltiorrhiza (Danshen) during the post-harvest drying process. Molecules 2012, 17, 2388−2407.

The authors declare no competing financial interest.

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REFERENCES

(1) Beddows, A. R. Biological flora of the British Isles. Dactylis glomerata L. J. Ecol. 1959, 47, 223−239. (2) Lolicato, S.; Rumball, W. Past and present improvement of orchard grass (Dactylis glomerata L.) in Australia and New Zealand. N. Z. J. Agric. Res. 1994, 37, 379−390. (3) Landsborough-Thompson, D. The pigments of butterflies’ wings. II. Occurrence on the pigment of Melanargia galatea in Dactylis glomerata. Biochem. J. 1926, 20, 1026−1027. (4) Parveen, I.; Winters, A.; Threadgill, M. D.; Hauck, B.; Morris, P. Extraction, structural characterisation and evaluation of orchard grass (Dactylis glomerata) as substrates for polyphenol oxidase. Phytochemistry 2008, 69, 2799−2806. (5) Cuyckens, F.; Claeys, M. Mass spectrometry in the structural analysis of flavonoids. J. Mass Spectrom. 2004, 39, 1−15. (6) Jay, M.; Plenet, D.; Ardouin, P.; Lumaret, R.; Jacquard, P. Flavonoid variation in seven tetraploid populations of Dactylis glomerata L. (Graminae). Biochem. Syst. Ecol. 1984, 12, 193−198. (7) Jay, M.; Lumaret, R. Variation in the subtropical group of Dactylis glomerata L. 2. Evidence from phenolic compound pattern. Biochem. Syst. Ecol. 1995, 23, 523−531. (8) Mandl, M. G. Status of biorefining in Europe. Biofuels, Bioprod. Biorefin. 2010, 4, 268−274. (9) Sieker, T.; Neuner, A.; Dimitrova, D.; Tippkötter, N.; Muffler, K.; Bart, H. J.; Heinzle, E.; Ulber, R. Ethanol production from grass silage by simultaneous pretreatment, saccharification and fermentation: first steps in process development. Eng. Life. Sci. 2011, 11, 436−442. (10) Ecker, J.; Schaffenberger, M.; Koschuh, W.; Mandl, M.; Böchzelt, H. G.; Schnitzer, H.; Harasek, M.; Steinmüller, H. Green biorefinery Upper Austria − pilot plan operation. Sep. Purif. Technol. 2012, 96, 237− 247. (11) Schaffenberger, M.; Ecker, J.; Koschuh, W.; Essl, R.; Mandl, M. G.; Boechzelt, H. G.; Steinmueller, H.; Schnitzer, H. Green biorefinery − production of amino acids from grass silage juice using an ion exchanger device at pilot scale. Chem. Eng. Trans. 2012, 29, 505−510. (12) McEniry, J.; Finnan, J.; King, C.; Kiely, P. The effect of ensiling on the suitability for combustion of three common grassland species at sequential harvest dates. Grass Forage Sci. 2012, 67, 559−568. (13) O’Keeffe, S. O.; Schulte, R. P. O.; Sanders, J. P. M.; Struik, P. C., II Economic assessment for first generation green biorefinery (GBR): scenarios for an Irish GBR blueprint. Biomass Bioenergy 2012, 41, 1−13. (14) Bertin, L.; Ferri, F.; Scoma, A.; Marchetti, L.; Fava, F. Recovery of high added value natural polyphenols from actual olive mill wastewater through solid phase extraction. Chem. Eng. J. 2011, 171, 1287−1293. (15) Boudet, A. Evolution and current status of research in phenolic compounds. Phytochemistry 2007, 68, 2722−2735. (16) Duke, J. A.; Wain, K. K. Medicinal Plants of the World, computer index with more than 85000 entries, 1981; 3 vol. (17) Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT−Food Sci. Technol. 1995, 28, 25−30. (18) Re, R.; Pellegrini, N.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical decolorization assay. Free Radical Biol. Med. 1999, 26, 1231−1237. (19) Benzie, I. F. F.; Strain, J. J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”. The FRAP assay. Anal. Biochem. 1996, 239, 70−76. (20) Ferreres, F.; Andrade, P.; Valentão, P.; Gil-Izquierdo, A. Further knowledge on barley (Hordeum vulgare L.) leaves O-glycosyl-C-glycosyl flavones by liquid chromatography-UV diode array detection-electrospray ionisation mass spectrometry. J. Chromatogr., A 2007, 22, 56−64. (21) Vukics, V.; Guttman, A. Structural characterization of flavonoid glycosides by multi-stage mass spectrometry. Mass Spectrom. Rev. 2010, 29, 1−16. (22) Lorenz, P.; Conrad, J.; Bertrams, J.; Berger, M.; Duckstein, S.; Meyer, U.; Stintzing, F. C. Investigations into the phenolic constituents 475

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