C. S. LauD. J. Carrier
L. R. Howard J. O. Lay Jr. E. C. Clausen
University of ArkansasFayetteville, AR 72701
Extraction of Antioxidants from Energy Crops
Biomass for Energy Production
• Agricultural and
forestry wastes
• Municipal solid waste
• Energy crops
Energy Crops
• High biomass yields (~5 tons/acre/ yr)
• Able to grow on idle land and poor soils with minimal soil amendment
• Serve as income source for limited resource farmer
• Southeast is good region for energy crop development
Velvet bean
Examples
• Hybrid poplar• Arundo• Mimosa• Kudzu• Switchgrass• Sericea• Velvet bean• Castor
Sericea
Arundo Kudzu
If we use energy crops . . .
• Cost as much as $50/dry ton
• Their use might benefit from the development of alternative products
Antioxidant Extraction
• Potentially high value products
• Can be easily extracted prior to biomass conversion
• This concept is different from the biorefinery concept which concentrates on hemicellulose and lignin conversion
Purpose
• Extract a variety of potential energy crops and assay for antioxidant potential
• Identify and quantify specific antioxidant compounds in selected energy crops
Biomass Extraction
• Two grams of 0.3 mm biomass and
60 ml of 60 % aqueous methanol
• Blended at 40ºC for 1 minute in a common
household blender• After filtration . . .
ORAC Assay
• Oxygen Radical Absorbance Capacity (ORAC) assay
• Measures the degree to which a sample inhibits the action of an oxidizing agent and how long it takes to do so
ORAC Values of Crude Methanol/Water Extracts
Energy crop ORAC Value *
Mimosa foliage 470
Sericea 330
Velvet bean foliage 300
Mimosa seed 16
Kudzu 190
Arunzo 170
Switchgrass 110
Spinach 16
Castor foliage 150
* Micromoles of Trolox equivalents per gram of fresh weight
Albizia julibrissin
Common name: mimosa, silk tree
Bark extracts were applied to bruises, ulcers, abscesses, boils, hemorrhoids and fractures
Dried stem bark has been used in China to prepare tonics to ease the mind and calm the nerves
Albizia julibrissin
Advantages as energy crop:– High forage yield (4.7
tons/acre yr)
– No need for nitrogen fertilizer (legume)
– Extremely tolerant to droughtAreas with Abundant Mimosa Growth
Mimosa Sample
• Provided by Dave Bransby, Auburn University
• Dried and ground to
0.3 mm • Stored at 4°C
Overview of Methods
Extraction
High Performance Liquid Chromatography (HPLC)
analysis
Oxygen Radical Absorbance Capability
(ORAC) test
Sep-pak fractionation
Mass Spectrometer (MS) analysis
Fast Performance Liquid Chromatography
(FPLC) fractionation
Identification Method Quantification Method
• Based on polarity of components
• The higher the polarity (less MeOH), the easier the elucidation
• Elucidation with:
20% MeOH (Fraction A)
60% MeOH (Fraction B)
100% MeOH (Fraction C)
Fraction A Fraction CFraction B
Sep-pak Fractionation
Sep-pak Fractionation
Fractionation of Crude Mimosa Extract
Fraction A
FractionB
Fraction C
Total value
ORAC Value* 140 290 90 520
* Micromoles of Trolox equivalents per gram of fresh weight
HPLC Analyses of Fraction B
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0 10 20 30 40 50 60 70
Time (min)
Ab
so
rban
ce 510 nm
360 nm
320 nm
280 nm
1
2
3
Flavonoids
• Found in fruits, vegetables, and certain beverages
• Anti-viral, anti-allergic, anti-platelet, anti-inflammatory, anti-tumor and anti-oxidant activities
• Subgroups of flavonoids include anthocyanidins, flavonols, flavones, flavanones
Basic Flavan Nucleus
C6 - C3 - C6 system
Mass Spectrometer Results
Peak 1
m/z [M + H]+ 303, 633
Peak 2
m/z [M + H]+ 303, 487
Peak 3
m/z [M + H]+ 303, 471
303.3
331.3405.3
465.1
487.2
591.2
633.0
779.00.00
0.25
0.50
0.75
1.00
1.25
5x10
Intensity
100 200 300 400 500 600 700 800 900
Peak 1
303.3
325.2
487.1
628.9
0
1
2
3
4
5x10
Intensity
100 200 300 400 500 600 700 800 900
Peak 2
303.2
325.2
471.1
642.9
0.0
0.5
1.0
1.5
2.0
2.5
3.0
5x10
Intensity
100 200 300 400 500 600 700 800 900
Peak 3
Glycoside Aglycone Glycone
Flavonol Flavonol Aglycone Sugar
(e.g. glucose, galactose)
Mass Spectrometer Results
= + + Ions
Na+
633 303 308 22
Identified first
MS/MS Compound Identification
110.9136.9 152.9
164.9
201.0213.0
228.9
247.0
257.0
274.0
285.0
302.9
All, 48.7-49.2min (#412-#418)
0
1
2
3
4
5x10
Intens.
100 150 200 250
300
m/z
Common ion in mimosa foliage extract
Quercetin
110.9
136.9152.9
164.9
201.0213.0
228.9
247.0
257.0
274.0
285.0
302.9All, 48.6-49.2min (#411-#416)
0.25
0.50
0.75
1.00
1.25
1.50
4x10
Intens.
100 150 200 250 300 m/z
The presence of quercetin was suspected based on: The compound mass of 302 (mass = positive ion – 1) was
present in all three major peaks in mimosa HPLC analysis showed that the compounds were flavonols
Molecular Mass: 302
Quick Facts Flavonol that may help prevent
heart disease
Blocks sorbitol accumulation,
which seems to bring about nerve
conditions in diabetics.
An anti-histamine, anti-
inflammatory, an anti-oxidant, and
promotes proper circulation.
Can be found in apples, beans,
black tea, leafy green vegetables and
onions
Quercetin (C15H10O7)
√
√
×
×
464
464
448
611
611 464 448
Spiking of mimosa crude extract (A) with(B) rutin, (C) hyperoside, (D) quercitrin, and (E) isoquercitrin
Mimosa
+ Rutin
+ Hyperoside
+ Quercitrin
+ Isoquercitrin
Quercetin Glycosides
Hyperoside• Quercetin 3-galactoside• Anti-inflammatory,
diuretic properties
Quercitrin• Quercetin 3-rhamnoside• Intestinal repair after
chronic mucosal injury
FPLC Fractionation
Flavonoid Content
Properties 1st Peak Hyperoside Quercitrin
Weight Percent 0.48 0.90 0.92
Percent of total ORAC
19 27 39
Hyperoside and Quercitrin Content of Mimosa and Other Materials
Hyperoside (g/100g dry material)
Quercitrin (g/100g dry material)
Mimosa 0.83 0.90
St. John’s wort 0.86 0.09
Hawthorne 0.01 ~0
Apple ~0 ~0
Conclusions
Hyperoside and quercitrin are the two major quercetin glycosides found in mimosa
Quercetin compounds account for 2.3% of the total dry weight and 85% of the total antioxidant content of
mimosa foliage
FUTURE WORK
• Investigate engineering aspects of mimosa extraction (solvent, temperature, solids concentration, interfacing with hydrolysis and fermentation, etc.)
• Identify and quantify antioxidants in other energy crops
• Investigate medical applications of the extracted antioxidants
Acknowledgments
• Southeastern Regional Biomass
Energy Program (SERBEP)
• Dave Bransby, Auburn University