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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)
Extraction HPLC-PDA LC-ESI-MSn
• 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
Hydoxycinnamates
OH
3-CQA HO Compound [M-H]- MS2
HO 2C OH
3-Caffeoylquinic 353 191, 179 (42.8)
O
acid
HO O
acyl 5-Caffeoylquinic 353 191, 179 (6.3)
beta
elimination OH cleavage acid
m/z: 353
OH O 2C OH
3-Feruloylquinic 367 191, 173 (6.3)
O 2C
acid
4-Feruloylquinic 367 191 (51), 173
HO HO OH acid
OH
OH
m/z: 191
O 2C OH
HO
O
m/z: 173
Mono –O, -C, Glycosyl Flavones
Apigenin-7-O-Glucoside
Apigenin-7-O-Glu 431 311 (3.8), 269 (100)
Luteolin-7-O-Glu 447 327 (2.1), 285 (100)
Apigenin-8-C-Glucoside
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
Compound Paternal Leaf (µg/g fresh weight) Maternal Leaf (µg/g fresh weight)
(mmol g-1) (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)
0.40
0.35
0.30
0.25
mg / g FW
0.20
Oct-11
Mar-11
0.15
0.10
0.05
0.00
5.3 8.2 9.1 9.6 11.5 13.5 13.9 14.5 16.6 17.0 17.6 18.2 19.5 19.9 20.3 20.7 21.9 22.3 23.7 24.5 24.7 25.8 26.2 26.3 27.9 28.5 29.4
Retention time (min)
Total phenols of paternal plant (mb 111)
0.80
0.70
0.60
0.50
mg /g FW
0.40
Oct-11
Mar-11
0.30
0.20
0.10
0.00
5.1 8.0 8.1 9.5 10.711.513.914.515.315.716.116.617.117.618.318.919.319.620.020.420.721.322.122.423.824.925.928.228.732.2
Retention tim (min)
Comparison of seedling and mature stage phenol content
9.0
March
8.0
October
7.0
Total phenols (mg/g FW)
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Plant
178
315
285
207
273
232
260
267
Paternal
Change in total phenol content
262
294
284
(seedlings vs mature stage)
342
210
337
314
223
203
353
195
240
236
345
239
197
263
182
274
339
212
330
Maternal
310
290
-1.00
-2.00
-3.00
-4.00
-5.00
-6.00
-7.00
-8.00
-9.00
1.00
0.00
Total Phenols (mg/g FW)
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)
