c. s. lau d. j. carrier l. r. howard j. o. lay jr. e. c. clausen university of arkansas...

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