Screening for platform chemicals in a novel Miscanthus sinensis mapping family Tom Wilson, Dr Ifat Parveen, Dr Ana Winters

Overview Identification of potential co-products from Miscanthus as part of the bio refinery Screening a M. sinensis mapping family for high value chemicals Simple extraction procedure for total phenol content Over 30 hydroxycinnamtes and flavonoid glycosides isolated from leaf tissues First study profiling flavonoid glycoside content of Miscanthus Potential for identified compounds to be screened for biological activity

Bio- Refining Creating valuable products from readily available feedstocks Fibres, proteins, nutraceuticals, high value organic compounds can all be obtained via extraction and separation Fermentation and thermochemical processing yields platform chemicals and bio fuels

Why Miscanthus? C4 Rhizamatous grass Greater resistance to drought and frost (compared with sugar cane) Tolerant of marginal land and flooding conditions Extremely low carbon impact High lignocellulose yield (>x2 switchgrass (P. virgatum)) Fermentable feedstock

M. Sinensis Mapping Family Bi-paternal cross Maternal = stay-green trait Paternal = high biomass yielder & high seed producer 200 Progeny sampled after 4 weeks growth (seedling stage) and after 35 weeks growth (mature vegetative stage)

Frozen in N 2(l) to minimise enzyme activity Extracted into 75% MeOH (aq) and semi purified on C18 sep-pak column All phenolics quantified relative to the Rf of either 5-CQA or Apigenin, and expressed as mg/g FW MS 2 for identification of glycone and aglycone flavonoids MS 2 and MS 3 fragments/relative intensities used to assign position of conjugation/glycosylation ExtractionHPLC-PDALC-ESI-MS n

Hydoxycinnamates Compound[M-H] - MS 2 3-Caffeoylquinic acid , 179 (42.8) 5-Caffeoylquinic acid , 179 (6.3) 3-Feruloylquinic acid , 173 (6.3) 4-Feruloylquinic acid (51), CQA

Mono –O, -C, Glycosyl Flavones Apigenin-7-O-Glu (3.8), 269 (100) Luteolin-7-O-Glu (2.1), 285 (100) Api-8-C-Glu (6.3); AG;ly+71, 311 (100); AGly+41 Api-6-C-Glu (28.9) ;AGly+71, 311 (100);AGly+41 Apigenin-7-O-Glucoside Apigenin-8-C-Glucoside

Compound Paternal Leaf (µg/g fresh weight) (mmol g -1 ) Maternal Leaf (µg/g fresh weight) (mmol g -1 ) 3-O-caffeoyl-quinic acid45 (0.13)9 (0.03) 3-O-feryloyl-quinic acid1 (0.003) 5-O-caffeoyl-quinic acid762 (2.15)309 (0.87) para-coumaric acid161 (0.38)26 (0.16) 4-O-feruloyl-quinic acid13 (0.01)- Lut-6-C-Pent-8-C-Hex17 (0.03)- Lut-O-Hex-C-Pent49 (0.08)- 2”-O-Deoxyhex-C-Hex-Lut-38 (0.06) Lut-6-C-Glu81 (0.18)309 (0.69) 2”-O-Deoxyhex-C-Hex-Lut114 (0.19)384 (0.65) Chrys/Dios-O-Hex-C-Pent20 (0.03)33 (0.06) Apig-6-C-Glu-42 (0.10) 2”-O-Deoxyhex-C-Hex-Apig35 (0.06)40 (0.07) 2”-O-Pent-C-Pent-Lut75 (0.14)- Apig-O-Hex-C-DeoxyHex39 (0.07)120 (0.21) 2”-O-Deoxyhex-C-Pent-Lut164 (0.29)- 2”-O-Deoxyhex-C-Deoxyhex-Lut123 (0.21)-

Total phenols of maternal plant (mb 255)

Total phenols of paternal plant (mb 111)

Comparison of seedling and mature stage phenol content

Change in total phenol content (seedlings vs mature stage)

Conclusions Rapid screening tool for qualitative and quantitative determination of soluble phenols in Miscanthus Over 30 different polyphenols identified from leaf tissue of progeny and parents Concentrations of phenols decreased as leaves matured; total polyphenolic concentration varied between 0.53 and 7.6 mg/g FW Potentially eleven novel flavone glycosides identified Genotypes with high phenolic content can be selected for use as a source of platform chemicals Composition at seedling and mature stage, are not closely correlated

Acknowledgements Dr Ifat Parveen, Dr Ana Winters, Dr Barbara Hauck, Dr Paul Robson, Ruth Roberts and Jakob Luyten (IBERS, Aberystwyth University) Professor Mike Threadgill (School of Pharmacy and Pharmacology, Bath University) Funding from BEACON (ERDF) and the Biotechnology and Biological Sciences Research Council (BBSRC)