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 N2(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

• MS2 for identification of glycone and aglycone flavonoids

• MS2 and MS3 fragments/relative intensities used to assign position of

conjugation/glycosylation

Extraction HPLC-PDA LC-ESI-MSn

Hydoxycinnamates

Compound [M-H]- MS2

3-Caffeoylquinic acid

353 191, 179 (42.8)

5-Caffeoylquinic acid

353 191, 179 (6.3)

3-Feruloylquinic acid

367 191, 173 (6.3)

4-Feruloylquinic acid

367 191 (51), 173

HO2C

OH

OH

OH

O

O

OH

OH

O2C

OH

OH

OH

OH

OHO2C

O

O2C

OH

OH

OH

OH

m/z: 353

m/z: 173

m/z: 191

betaelimination

acyl cleavage

3-CQA

Mono –O, -C, Glycosyl Flavones

Apigenin-7-O-Glu 431 311 (3.8), 269 (100)

Luteolin-7-O-Glu 447 327 (2.1), 285 (100)

Api-8-C-Glu 431 341 (6.3); AG;ly+71, 311 (100); AGly+41

Api-6-C-Glu 431 341 (28.9) ;AGly+71, 311 (100);AGly+41

OOH

O

OH

OH

O

OH

OH

HO

O

HO

OOH

HO O

OH

HO

HO

OH

O

OH

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 acid 45 (0.13) 9 (0.03)

3-O-feryloyl-quinic acid 1 (0.003) 1 (0.003)

5-O-caffeoyl-quinic acid 762 (2.15) 309 (0.87)

para-coumaric acid 161 (0.38) 26 (0.16)

4-O-feruloyl-quinic acid 13 (0.01) -

Lut-6-C-Pent-8-C-Hex 17 (0.03) -

Lut-O-Hex-C-Pent 49 (0.08) -

2”-O-Deoxyhex-C-Hex-Lut - 38 (0.06)

Lut-6-C-Glu 81 (0.18) 309 (0.69)

2”-O-Deoxyhex-C-Hex-Lut 114 (0.19) 384 (0.65)

Chrys/Dios-O-Hex-C-Pent 20 (0.03) 33 (0.06)

Apig-6-C-Glu - 42 (0.10)

2”-O-Deoxyhex-C-Hex-Apig 35 (0.06) 40 (0.07)

2”-O-Pent-C-Pent-Lut 75 (0.14) -

Apig-O-Hex-C-DeoxyHex 39 (0.07) 120 (0.21)

2”-O-Deoxyhex-C-Pent-Lut 164 (0.29) -

2”-O-Deoxyhex-C-Deoxyhex-Lut 123 (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)