C. S. Lau D. J. Carrier L. R. Howard J. O. Lay Jr. E. C. Clausen University of Arkansas Fayetteville, AR 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 ArundoKudzu

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 drought Areas 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 MethodQuantification 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 AFraction C Fraction B Sep-pak Fractionation

Fractionation of Crude Mimosa Extract Fraction A Fraction B Fraction C Total value ORAC Value* * Micromoles of Trolox equivalents per gram of fresh weight

HPLC Analyses of Fraction B 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, x10 Intensity Peak 2

GlycosideAglyconeGlycone Flavonol Flavonol Aglycone Sugar (e.g. glucose, galactose) Mass Spectrometer Results =++ Ions Na Identified first

MS/MS Compound Identification All, min (#412-#418) x1 0 Inten s m/z Common ion in mimosa foliage extract Quercetin All, min (#411-#416) x10 Intens 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 (C 15 H 10 O 7 )

√ √ × × 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 Properties1 st PeakHyperosideQuercitrin Weight Percent Percent of total ORAC

Hyperoside and Quercitrin Content of Mimosa and Other Materials Hyperoside (g/100g dry material) Quercitrin (g/100g dry material) Mimosa St. John’s wort Hawthorne0.01~0 Apple ~0 ~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