Bioresource Technology 98 (2007) 429–435

IdentiWcation and quantiWcation of glycoside Xavonoidsin the energy crop Albizia julibrissin

Ching S. Lau a, Danielle J. Carrier b,¤, Robert R. Beitle a, David I. Bransby c,Luke R. Howard d, Jackson O. Lay Jr. e, Rohana Liyanage e, Edgar C. Clausen a

a Department of Chemical Engineering, University of Arkansas, 3202 Bell Engineering Center, Fayetteville, AR 72701, United Statesb Department of Biological and Agricultural Engineering, University of Arkansas, 203 Engineering Hall, Fayetteville, AR 72701, United States

c Department of Agronomy and Soils, 202 Funchess Hall, Auburn University, AL 36849, United Statesd Department of Food Science, University of Arkansas, 2650 N. Young Avenue, Fayetteville, AR 72704, United States

e Department of Chemistry and Biochemistry, University of Arkansas, 115 Chemistry Building, Fayetteville, AR 72701, United States

Received 28 July 2005; received in revised form 30 November 2005; accepted 1 December 2005Available online 14 February 2006

Abstract

Oxygen radical absorbance capacity (ORAC) values showed that methanolic extracts of Albizia julibrissin foliage displayed antioxi-dant activity. High performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques were utilized in the identiWca-tion of the compounds. The analysis conWrmed the presence of three compounds in A. julibrissin foliage methanolic extract: an unknownquercetin derivative with mass of 610 Da, hyperoside (quercetin-3-O-galactoside), and quercitrin (quercetin-3-O-rhamnoside). Fast per-formance liquid chromatography (FPLC) was employed to fractionate the crude A. julibrissin foliage methanolic extract into its individ-ual Xavonoid components. The Xavonoids were quantiWed in terms of mass and their respective contribution to the overall ORAC value.Quercetin glycosides accounted for 2.0% of total foliage.© 2005 Elsevier Ltd. All rights reserved.

Keywords: Albizia julibrissin; HPLC; LC–MS; Hyperoside; Quercitrin; Oxygen radical absorbance capacity

1. Introduction

Flavonoids, primarily categorized into Xavonols, Xava-nols, Xavones, Xavanones and anthocyanidins, are widelydistributed in nature, and are present in most fruits, vegeta-bles and plants. Flavonoids may play a preventive role in the

Abbreviations: AAPH, 2,2�-Azobis (2-amidino-propane) dihydrochloride;AUC, area under the curve; ESI, electrospray ionization; FL, Xuorescein(3�,6�-dihydroxyspiro[isobenzofuran-1[3H],9�[9H]-xanthen]-3-one); FPLC,fast performance liquid chromatography; HPLC, high performance liquidchromatography; LC, liquid chromatography; MS, mass spectrometry;MS–MS, tandem mass spectrometry; ORAC, oxygen radical absorbancecapacity; PE, phycoerythrin; TLC, thin layer chromatography; Trolox, 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid.

* Corresponding author. Tel.: +1 479 575 4993; fax: +1 479 575 2846.E-mail address: carrier@uark.edu (D.J. Carrier).

0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2005.12.011

development of cancer and heart disease due to their antiox-idant and other activities (Aviram and Fuhrman, 2002).Potential sources of antioxidant compounds have beenfound in several types of plant materials such as fruits, vege-tables, leaves, oilseeds, barks, roots, spices and herbs. Theauthors have been examining the possibility of extractingantioxidants from some common crops in the United States,especially those being studied as possible feedstock forenergy production, such as Albizia julibrissin foliage, sericealespedeza (Lespedeza cuneata), kudzu (Pueraria lobata),Arundo donax L., velvet bean (Mucuna pruriens), switchgrass(Panicum virgatum L.), and castor (Ricinus communis L.).Using the oxygen radical absorbance capacity (ORAC)assay described by Prior et al. (1998), the antioxidant poten-tial of crude methanol extracts stemming from nine diVerentenergy crops was reported (Lau et al., 2004). Of the energy

430 C.S. Lau et al. / Bioresource Technology 98 (2007) 429–435

crops studied, the antioxidants of A. julibrissin extractsshowed the strongest peroxyl radical scavenging activity.These results prompted further study of this plant.

A. julibrissin, also known in the United States as mimosa,he huan pi (pinyin), nemu (Japan), powder-puV tree or silktree, is native to Asia from Iran to Japan (Cheatham et al.,1996). It re-sprouts quickly if cut or top-killed, and theumbrella shaped A. julibrissin tree can reach a height of 6 m.The bark and Xowers of the A. julibrissin tree are used inChina as medicine. Bark extract is applied to bruises, ulcers,abscesses, boils, hemorrhoids and fractures, and has dis-played cytotoxic activity (Higuchi et al., 1992; Ikeda et al.,1997). A. julibrissin is one of several energy crops beingtested in the Auburn University energy crop research pro-gram, showing an annual forage yield of 4.5 dry tons acre¡1

(10.7 Mg ha¡1 yr¡1) from four harvests per year, and an aver-age total biomass yield of 37.3 Mg ha¡1 yr¡1 from one har-vest per year over a 4-year period (Sladden et al., 1992).

Previous work has identiWed the presence of saponinsand lignans in A. julibrissin (Ikeda et al., 1997; Kinjo et al.,1991). This paper is aimed at identifying the compound(s)that are responsible for the high total phenolic values andantioxidant activity of A. julibrissin foliage extract reportedby Lau et al. (2004). For this purpose, extract fractions wereexamined for their total phenolic content and antioxidantactivity with oxygen radical absorbance capacity (ORAC)and Folin–Ciocalteau assays, while the identiWcation ofcompounds was conducted via high performance liquidchromatography (HPLC) and mass spectrometry (MS).Fast performance liquid chromatography (FPLC) wasemployed to fractionate individual Xavonoids from thecrude extract. Each Xavonoid was later quantiWed in termsof mass and their contribution to the overall antioxidantcapacity of the A. julibrissin foliage.

2. Methods

2.1. Plant material

Samples of fresh A. julibrissin foliage that ranged from 1to 90 days of age were harvested by hand in August atAuburn, Alabama. Samples included the petiole, branchesand leaXets of the entire compound leaf. They were placedin a forced air oven at 65 °C within an hour of harvesting,dried to constant weight, ground to pass through a 1 mmscreen and stored in sealed plastic bags at room tempera-ture. A voucher specimen was deposited at the Departmentof Chemical Engineering, University of Arkansas (Fayette-ville, Arkansas). Samples of dried St. John’s Wort (Hyperi-cum perforatum L.) and hawthorn (Crataegus monogyna)berries were purchased from Ozark Natural Foods (Fayet-teville, AR). Apples were purchased from a local retailer.

2.2. Chemicals

Hyperoside (quercetin-3-O-galactoside) and rutin (querce-tin-3-O-rutinoside) were purchased from IndoWne Chemaical

Company, Inc (Somerville, NJ). Isoquercitrin (quercetin-3-O-glucoside), quercitrin (quercetin-3-O-rhamnoside), Folin–Ciocalteau reagent, chlorogenic acid, Xuorescein (3�,6�-dihydroxyspiro [isobenzofuran-1[3H], 9�-[9H]-xanthen]-3-one)(FL) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carb-oxylic acid (Trolox) were acquired from Sigma–AldrichCorporation (St. Louis, MO). Quercetin, sodium carbonate,HPLC grade methanol, acetonitrile, formic acid and hydro-chloric acid were obtained from VWR International (WestChester, PA), whereas 2,2�-azobis (2-amidino-propane) dihy-drochloride (AAPH) was purchased from Wako Chemicals(Richmond, VA). Potassium phosphate dibasic was pur-chased from J.T. Baker (Mallinckrodt Baker, Inc., Philips-burg, NJ).

2.3. Extraction

For the preparation of all extracts, 2 g of dried biomass(either A. julibrissin, St. John’s Wort, hawthorn berries orapple) were extracted with 60 ml of 60% aqueous methanolat 50 °C by blending the mixture in a household blender for10 min. To separate the supernatant from the solids, theresulting mixture of solvent and solids was centrifuged at12,096g for 30 min in an induction drive centrifuge (Beck-man Coulter, Fullerton, CA). The supernatant was thenWltered through a 0.45-�m syringe Wlter (VWR Interna-tional, West Chester, PA). The Wltered crude extracts werecollected and stored at 4 °C for subsequent fractionationand analysis.

2.4. Sample preparation for total phenolics and ORAC

A. julibrissin crude extract was fractionated with 2 mldisposable Symmetry® (Waters, Milford, MA) C18 sep-pakcartridges. The cartridges were preconditioned with 10 mlof methanol, followed by 10 ml of acidiWed water (pH 2with HCl). About 0.25 ml of Wltered A. julibrissin crudeextract was loaded onto the cartridge and eluted with 2 mlof 20% aqueous methanol. The cartridge was subsequentlyeluted with 2 ml of 60% aqueous methanol followed by100% methanol. The three fractions were dried under vac-uum using a SpeedVac Plus (Savant Instruments, Hol-brook, NY) without heat. After drying, the samples weredissolved in 2 ml of methanol. All extractions were per-formed at least twice.

2.5. Total phenolics and ORAC analysis

Total phenolics content was determined according to amodiWed Folin–Ciocalteau assay (Singleton and Rossi,1965). The absorbance was read at 726 nm on a HewlettPackard (Palo Alto, CA) 8452A Diode Array UV–visiblespectrophotometer. Data were expressed as mg of chloro-genic acid equivalents per gram of dry A. julibrissin foliage.The ORAC assay was conducted by the method of Prioret al. (2003) modiWed for use with a FLUOstar Optimamicroplate reader (BMG Labtechnologies, Durham, NC)

C.S. Lau et al. / Bioresource Technology 98 (2007) 429–435 431

using Xuorescein as Xuorescent probe. A. julibrissin extractswere diluted 1000-fold or more with phosphate buVer(75 mM, pH 7) prior to ORAC analysis. The assay was car-ried out at 37 °C in clear 48-well Falcon plates (VWR, StLouis, MO). Each well had a Wnal volume of 590�l. Ini-tially, 40 �l of diluted sample, Trolox standards (6.25, 12.5,25, 50 �M) and blank solution (75 mM, pH 7 phosphatebuVer) were added to each well using an automatic pipette.The FLUOstar Optima instrument equipped with twoautomated injectors was then programmed to add 400 �l ofXuorescein (94 nM) followed by 150 �l of AAPH (31.6 mM)to each well. Fluorescence readings (excitation 485 nm,emission 520 nm) were recorded after the addition ofAAPH and every 197 s thereafter for 112 min to reach 95%loss of Xuorescence. Final Xuorescence measurements wereexpressed relative to the initial reading. Results were calcu-lated based upon diVerences in areas under the Xuorescencedecay curve between the blank, samples and standards. Thestandard curve was obtained by plotting the four concen-trations of Trolox against the net area under the curve(AUC) of each standard. Final ORAC values were calcu-lated using the regression equation between Trolox concen-tration and AUC and are expressed as �moles of Troloxequivalents per gram dry weight.

2.6. LC–MS analysis of A. julibrissin extract

A Hewlett Packard 1100 Series (Palo Alto, CA) HPLCwith a photodiode array detector was coupled to a BrukerEsquire (Billerica, MA) mass spectrometer. The column usedwas a Symmetry® (Waters, Milford, MA) C18 column(250 mm£4.6mm). A 25-�l sample was injected via the autosampler. The mobile phase gradient employed in mass spec-trometry analysis was conducted by a modiWcation to themethod of Cho et al. (2004). Solvent A contained 5% formicacid in water, and solvent B consisted of HPLC grade meth-anol. The gradient program was initiated with 98:2 solventA:solvent B, and linearly decreased to 40:60 solvent A:sol-vent B over 60 min. The Xow rate was set to 0.7 ml min¡1. TheUV response was monitored at 360 nm. The MS was oper-ated in the positive ionization mode from the electrosprayionization (ESI) source. The temperature of the drying gas(N2) was 300 °C and Xowed at 10 ml min¡1. The nebulizingpressure (N2) was maintained at 2.1£105 Pa (30 psi). The LCsystem was directly connected to the mass spectrometerwithout stream splitting. For tandem mass spectrometryexperiments (MS–MS), the mass spectrometer was operatedin a manner similar to the LC–MS experiments, except that atarget mass (parent ion) was mass selected and separatedfrom all other ions. This mass selected parent ion was thenfragmented in collisions with helium gas in the trap. Then,the fragment ions were analyzed in the normal mode.

2.7. QuantiWcation

The quantiWcation of the Xavonoids in A. julibrissin, St.John’s Wort, hawthorn berries or apple was carried out on

a Waters HPLC Instrument, equipped with a 2996 photodi-ode array detector, 2795 separations module controlled withMass Lynx software. A 50�l sample of crude extract wasinjected through a Symmetry® C18 (50 mm£2.1 mm) col-umn (Waters, Milford, MA). Mobile phases were solvent A(0.1% formic acid in water) and solvent B (0.1% formic acidin acetonitrile). The initial condition began at 90:10 solventA:solvent B and was maintained for 5 min. The gradient waslinearly increased to 80:20 solvent A:solvent B over 30 minand was held for 1 min. The gradient was increased again to20:80 solvent A:solvent B over 2 min and was held foranother 5 min before it was decreased to 90:10 solventA:solvent B in 2 min. This process was followed by a re-equilibration of the column at 90:10 solvent A:solvent B for10 min. The Xow rate was 0.4 ml min¡1, and the column wasat room temperature. The diode array detector acquiredspectra between the wavelengths of 210 and 600 nm. Quer-cetin, hyperoside, isoquercitrin, quercitrin and rutin were alldissolved in HPLC grade methanol and kept at 4 °C. Allextraction and analyses were performed in triplicate. Aver-ages and standard deviations were calculated with Excel.

2.8. Fractionation of crude A. julibrissin

The fractionation of crude A. julibrissin was carried oututilizing AKTA (Amersham Biosciences Corp., Piscataway,NJ) fast performance liquid chromatography (FPLC) sys-tem. The 20 ml column was packed with C18 silica in 0.1%formic acid. Samples were pre-conditioned with 0.1% formicacid in 10% aqueous acetonitrile solution for 80 min before a15 ml sample of crude extract was injected. To wash outunbound compounds, the column was percolated with 10 ml0.1% formic acid in 10% aqueous acetonitrile. Mobile phasesused for the gradient consisted of solvent A (0.1% formicacid in water) and solvent B (0.1% formic acid in acetoni-trile). The gradient began at 90:10 solvent A: solvent B andlinearly changed to 40:60 solvent A:solvent B over 150 min.The gradient was increased to 0:100 solvent A:solvent B in1 min, and the Wnal gradient was held for 50 min. This wasfollowed by a re-equilibration of column with 90:10 solventA:solvent B for 30 min. The Xow rate was set at 1 ml min¡1.This equipment was equipped with a Wxed wavelengthdetector (280 nm). Consequently, compounds were moni-tored at this wavelength. Experiments were duplicated. Stan-dard deviations were calculated with Excel.

3. Results

Crude extracts of A. julibrissin foliage were separated onC18 sep-pak cartridges with increasing concentrations ofmethanol. The total phenolic values of the fractions arepresented in Table 1 and were between 16 and 30 mg ofchlorogenic acid equivalents per gram of dry A. julibrissinfoliage. Also seen in Table 1, higher ORAC values wereacquired from the cartridge eluted with 20% and 60% meth-anol than with pure methanol.

432 C.S. Lau et al. / Bioresource Technology 98 (2007) 429–435

As presented in Fig. 1A, the total ion trace of A. julibris-sin foliage crude extract obtained with 60% methanolshowed three major peaks. The ESI mass spectra of thecompounds were recorded in the positive-ion mode. Fig. 1Bshows the Wrst peak with a retention time of 42.5 min with apeak intensity of 1.2£ 105 and signiWcant ions at m/z 303and 633. The second peak (Fig. 1C) displayed a maximumintensity of 4.0£105 and was detected at 49.0 min with sig-niWcant ions at m/z 303 and 487. The third major peak(Fig. 1D) with signiWcant ions at m/z 303 and 471 wasdetected at 53.5 min, and displayed a peak intensity of3.0£105. Each of the major peaks in Fig. 1A displayed anion with a mass of 303. Based on their photodiode arrayspectra and their observed masses, the common 303 masswas assigned to protonated quercetin. The presence of pro-tonated quercetin was conWrmed based on a comparisonbetween the MS–MS product-ion spectrum obtained dur-ing the LC separation (Fig. 2a) and the reference spectrum

Table 1Total phenolics and ORAC values for the sep-pak fractionation of crudeAlbizia julibrissin extracts

a Micrograms of chlorogenic acid equivalents per gram of dry A. juli-brissin foliage. Duplicate experiment.

b Micromoles of Trolox equivalents per gram of dry A. julibrissin foli-age. Data were not duplicated.

Extraction solvent Total phenolicsa ORAC valueb

20% Methanol in water 16,000 § 3000 14060% Methanol in water 27,000 § 0 290100% Methanol 30,000 § 6000 90

from reagent grade quercetin (Fig. 2b). The aglycone of del-phinidin also has a protonated molecule at m/z 303. How-ever, the presence of delphinidin was ruled out because ofthe lack of absorbance at 510 nm (results not shown) and ofthe absence of the reddish-purple color, both characteristicof anthocyanins.

Flavones, such as quercetin, often have glycoside moie-ties attached to them. Fragmentation and loss of the glyco-side moieties to yield the parent quercetin aglycone istypical in positive ion ESI analyses (Lin et al., 2000). Thespectra presented in Fig. 1 show both the protonated mole-cule of the glycosides and the ion corresponding to the

Fig. 2a. MS/MS spectrum of m/z [M+H]+ 303 from a 60% methanol Albi-zia julibrissin foliage extract.

110.9

136.9152.9

164.9

201.0213.0

228.9

247.0

257.0

274.0

285.0

302.9

0.2

0.5

0.7

1.0

1.2

1.5

Intensity × 105

100 150 200 250 300 m/z

Fig. 1. HPLC–MS analyses of Albizia julibrissin foliage crude extract, fractionated with 60% methanol in a sep-pak column: (A) total ion trace, (B) massspectrum of peak 1, (C) mass spectrum of peak 2, (D) mass spectrum of peak 3.

303.2

325.2

471.1

642.9

0.0

0.5

1.0

1.5

2.0

2.5

3.0

100 200 300 400 500 600 700 800 900

D

Peak 3

0 10 20 30 40 50 0

20

40

60

80

A

1

2

3 303.3

331.3 405.3

465.1

487.2

591.2

633.0

779.0 0.0

0.2

0.5

0.7

1.0

1.2

Intensity x 105

Intensity x 105 Intensity x 105

100 200 300 400 500 600 700 800 900

B

Peak 1

303.3

325.2

487.1

628.9

0

1

2

3

4

100 200 300 400 500 600 700 800 900

C

Peak 2

m/z

m/z

[mAU]

m/z

Time

C.S. Lau et al. / Bioresource Technology 98 (2007) 429–435 433

protonated quercetin aglycone. All three peaks displayed acommon mass of 303 but diVerent protonated moleculemass for their conjugated glycosides. The diVerences inmasses indicate the presence of sugars or other moietiesattached to the quercetin aglycone. The diVerence in massesbetween the two main peaks in the full scan LC–MS spec-trum of peak 2 (Fig. 1C) corresponded to a sugar moietymass of 184 Da. The diVerence in mass of the quercetinmonosaccharide (molecular weight of 464) and what wasobtained (observed mass of 487) was accounted for by themass diVerence of 23, which was attributed to the chargefrom a sodium cation. The assumption was veriWed by thenegative ion detection when the deprotonated moleculesresulted in m/z of 463 instead of m/z 485, indicating that thepresence of the sodium ion occurred only in positive ionmode. To identify the sugar moiety of peak 2 (Fig. 1C),hyperoside (quercetin-3-O-galactoside) and isoquercitrin(quercetin-3-O-glucoside), two of the most common quer-cetin derivatives with a molecular mass of 464, were used incomparison studies. The masses of the quercetin glycosidesof Peak 1 (Fig. 1B) and Peak 3 (Fig. 1D) were 610 and 448,respectively when deducting the mass of sodium ion. Thus,rutin (quercetin-3-O-rutinoside) and quercitrin (quercetin-3-O-rhamnoside) were also analyzed. By co-chromatogra-phy with reference compounds, Peak 2 (Fig. 1C) and Peak 3(Fig. 1D) were identiWed as hyperoside and quercitrin,respectively.

Peak 1 (Fig. 1B) was also investigated. The parent ion ofPeak 1 had a mass of 633 Da and showed a prominent frag-

Fig. 2b. MS/MS spectrum of m/z [M+H]+ 303 of protonated referencecompound quercetin.

110.9136.9 152.9

164.9

201.0213.0

228.9

247.0

257.0

274.0

285.0

302.9

0

1

2

3

4

5Intensity × 105

100 150 200 250 300 m/z

ment ion at m/z 303, corresponding to a loss of 330 Da.Based on the presumption that the parent ion contained thesodium ion, the mass loss from a protonated moleculewould be 308 Da. A loss of 308 Da from a protonated mole-cule would correspond to cleavage of a rhamnose–hexosesugar. The lack of an intermediate rhamnose or hexose losscould be explained by the fact that it did not fragment intoits individual sugar moieties. Co-chromatography experi-ments were performed with rutin and Peak 1 (Fig. 1B),where rutin did not co-elute, establishing that rutinosidewas not the carbohydrate moiety. In this co-chromato-graphy experiment, Peak 1 (Fig. 1B) eluted before rutin.Thus, Peak 1 (Fig. 1B) was tentatively identiWed as querce-tin-rhamnosylgalactoside.

In order to relate the hyperoside and quercitrin contentof A. julibrissin to that of other plants, other hyperoside orquercitrin containing plants were extracted with 60% aque-ous methanol at the same blending condition as the A. juli-brissin foliage. St. John’s Wort (Bilia et al., 2001) and apples(Van der Sluis et al., 2001) were reported to contain hypero-side and quercitrin, whereas hawthorn (Kirakosyan et al.,2003) was reported to contain solely hyperoside. Thehyperoside and quercitrin concentrations in these plantswere compared to those of A. julibrissin foliage as shown inTable 2.

The fraction of each of the quercetin-derivatives in A.julibrissin was evaluated. The tentatively identiWed querce-tin-rhamnosylgalactoside peak (Fig. 1B), hyperoside andquercitrin were separated from the A. julibrissin crudeextract by FPLC. For 1 g of dry A. julibrissin extract, 4.7 mgof peak 1 (Fig. 1B), 7.4 mg of hyperoside and 7.4 mg ofquercitrin were obtained. These compounds correspondedto about 2% by mass of the dry A. julibrissin foliage. Table3 presents the ORAC activity of the tentatively identiWedquercetin-rhamnosylgalctoside peak (Fig. 1B), hyperosideand quercitrin.

Table 2Hyperoside and quercitrin content of A. julibrissin and other selectedmaterials

Detection limit 0.1 g kg¡1. Numbers shown show the averages of threeextraction experiments.

Crops Hyperoside(g kg¡1 dry material)

Quercitrin(g kg¡1 dry material)

Albizia julibrissin 6.1 § 2.5 9.0 § 1.8St. John’s wort 8.7 § 0.0 0.9 § 0.2Hawthorn 0.3 § 0.0 0.0 § 0.0Apple 0.1 § 0.0 0.1 § 0.1

Table 3Properties of major compounds in A. julibrissin

a Trolox equivalents. Experiments were duplicated.

Properties First peak Hyperoside Quercitrin

Percent dry weight 0.47 § 0.07 0.74 § 0.11 0.74 § 0.07ORAC activity (�mol TEa g¡1) 31.2 § 5.8 30.9 § 16.1 57.5 § 3.2Percent total ORAC 15.9 § 3.4 23.8 § 8.8 46.8 § 6.8

434 C.S. Lau et al. / Bioresource Technology 98 (2007) 429–435

4. Discussion

The total phenolic values of the fractions presented inTable 1 were between 16 and 30 mg of chlorogenic acidequivalents per gram of dry A. julibrissin foliage. Valuesreported on a chlorogenic acid basis are about 1.8 timesgreater than those reported as gallic acid equivalents. How-ever, the values presented in Table 1 as chlorogenic acidequivalents were within the range of those reported by Wuet al. (2004). Prior et al. (2001) reported that solvent com-position inXuenced selectivity, where 20% methanol/water(v/v) eluted sugars and phenolic acids, while 60% methanol/water (v/v) and 100% methanol eluted Xavonols/anthocya-nins and procyanidins, respectively. In this work, highertotal phenolics were obtained with 60% methanol and puremethanol than with 20% methanol.

Hyperoside, quercitrin and peak 1 (Fig. 1B) contain theaglycone quercetin. Interestingly, quercetin is a Xavonol,which consists of a group of naturally occurring com-pounds that are usually present in the plant as glycosidesand are colorless or light yellow (Kühnau, 1976). Quercetinis not solely found in A. julibrissin biomass, being reportedin grapes (Careri et al., 2003), apples (Van der Sluis et al.,2001), oranges (Sorrenti et al., 2004) and buckwheat (Fab-jan et al., 2003) as well as some medicinal botanicals such ashawthorn (Kirakosyan et al., 2003) and St. John’s Wort(Bilia et al., 2001). Quercetin has been associated with car-diovascular protection (Aviram and Fuhrman, 2002).Accumulating evidence is showing that oxidation of lowdensity lipids (LDL) and monocyte-endothelial cell adhe-sion are hallmark events in cardiovascular diseases (Aviramand Fuhrman, 2002). Quercetin has been shown, amongothers, to inhibit oxidation of LDL (Meyer et al., 1998), toinhibit monocyte cell adhesion to human aortic endothelialcells stimulated by interlukin-1� (Koga and Meydani, 2001)and to decrease the electrophoretic mobility of human oxi-dized LDL by 35% (Sorrenti et al., 2004). Hyperoside wasdocumented to have anti-inXammatory eVects (Krenn et al.,2004), inhibit in vitro LDL oxidation (Quettier-Deleu et al.,2003) and display antimicrobial properties (Dall’Agnolet al., 2003).

The presence of hyperoside and quercitrin has previ-ously been reported. In an unspeciWed A. julibrissin mate-rial, Kaneta et al. (1980) reported the presence ofhyperoside and quercitrin by thin layer chromatography(TLC), but not by MS. The presence of quercitrin wasreported by Li et al. (2000) and Kang et al. (2000) in Xowersof A. julibrissin, but not in foliage. Li et al. (2000) isolatedquercitrin by extraction with ether and ethyl acetate fol-lowed by a chromatographic separation. Kang et al. (2000),on the other hand, used methanol as solvent for the extrac-tion, Sephadex LH-20 multiple-column chromatographyfor the fractionation, and nuclear magnetic resonance forcompound identiWcation.

Among the four plants tested under our experimentalconditions, A. julibrissin had the highest content of querci-trin, while its hyperoside content was lower than that of St.

John’s Wort, which was in agreement with Bilia et al.(2001). The hyperoside content of hawthorn was very closeto what was reported by Kirakosyan et al. (2003), about0.3 g kg¡1 dry mass. According to Van der Sluis et al. (2001),the hyperoside and quercitrin contents of apples werereported as 0.027 and 0.029 g kg¡1 dry mass, respectively.

As shown in Table 3, quercitrin had the highest ORACactivity, followed by hyperoside, and the tentatively identi-Wed quercetin-rhamnosylgalctoside peak. This observationthat quercitrin has a higher antioxidant activity thanhyperoside is in agreement with Williamson et al. (1999).The methanol extraction of the three compounds consistedof about 2% of the mass of A. julibrissin on a dry basis. Thiswork consists of the Wrst report on hyperoside and querci-trin quantiWcation in A. julibrissin foliage extract and theirlink to ORAC activity.

In summary, the three main antioxidant constituents inA. julibrissin methanolic extracts were hyperoside, quercitrinand the tentatively identiWed quercetin-rhamnosylgalctosidepeak 1 (Fig. 1B). The combination of these three compoundsaccounted for 2% mass of the A. julibrissin foliage. From anantioxidant perspective, 119.6�Mol trolox equivalents pergram of dry foliage was obtained. This study indicates thatA. julibrissin foliage methanolic extracts have high anti-oxidant activity and are a rich source of Xavonoids, whichcould possibly be extracted prior to energy production unitoperations. Hyperoside was reported to inhibit in vitro LDLoxidation (Quettier-Deleu et al., 2003) and could possibly bebeneWcial in the management of endothelial dysfunction dis-eases. Thus, A. julibrissin extracts could possibly be includedin human or animal functional foods during the nextdecade. Flavonoid extraction from A. julibrissin biomasscould possibly occur prior to its use as an energy crop(Bransby et al., 2001), conferring added value.

Acknowledgements

This project was supported by the SoutheasternRegional Biomass Energy Program (SERBEP) and admin-istered by the Southern States Energy Board (SSEB) for theUnited States Department of Energy and by the ArkansasExperimental Station.

References

Aviram, M., Fuhrman, B., 2002. Wine Xavonoids protect against LDL oxi-dation and atherosclerosis. Ann. NY Acad. Sci. 957, 146–161.

Bilia, A., Bergonzi, M., Mazzi, G., Vincieri, F., 2001. Analysis of plant com-plex matrices by use of nuclear magnetic resonance spectroscopy: St.John’s wort extract. J. Agric. Food Chem. 49, 2115–2124.

Bransby, D., Morrison, T., Sladden, S., 2001. Advantages of mimosa (Albi-zia julibrissin) over traditional short rotation woody crops. In: 1stWorld Conference on Biomass for Energy and Industry, Sevilla, Spain,5–9 June, 2000. James and James, London, pp. 1933–1934.

Careri, M., Corradini, C., Elviri, L., Nicoletti, I., Zagnoni, I., 2003. DirectHPLC analysis of quercetin and trans-resveratrol in red wine, grape,and winemaking by-products. J. Agric. Food Chem. 51, 226–5231.

Cheatham, S., Johnston, M., Marshall, L., 1996. Albizia. In: The UsefulWild Plants of Texas, the Southeastern and Southwestern United

C.S. Lau et al. / Bioresource Technology 98 (2007) 429–435 435

States, the Southern plains, and Northern Mexico. Useful Wild PlantsInc., Texas, pp. 188–190.

Cho, M., Howard, L., Prior, R., Clark, J., 2004. Flavonoid glycosides andantioxidant capacity of various blackberry, blueberry, and red grapegenotypes determined by high-performance liquid chromatography/mass spectrometry. J. Sci. Food Agric. 84, 1771–1782.

Dall’Agnol, R., Ferrraz, A., Bernardi, A., Albring, D., Nor, C., Sarmento,K., Lamb, L., Hass, M., von Poser, G., Schapoval, E., 2003. Anti-microbial activity of some Hypercicum species. Phytomedicine 10,511–516.

Fabjan, N., Rode, J., Kosir, I., Wang, Z., Zhang, Z., Kreft, I., 2003. Tartarybuckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutinand quercitrin. J. Agric. Food Chem. 51, 6452–6455.

Higuchi, H., Kinjo, J., Nohara, T., 1992. An arrhythmic-inducing glyco-side from Albizia julibrissin Durazz. IV. Chem. Pharm. Bull. 40, 829–831.

Ikeda, T., Fujiwara, S., Araki, K., Kinjo, J., Nohara, T., Miyoshi, T., 1997.Cytotoxic glycosides from Albizia julibrissin. J. Nat. Prod. 60, 102–107.

Kaneta, M., Hikichi, H., Endo, S., Sugiyama, N., 1980. IdentiWcation ofXavones in sixteen leguminous species. Agric. Biol. Chem. 44, 1407–1408.

Kang, T., Jeong, S., Kim, N., Higuchi, R., Kim, Y., 2000. Sedative activityof two Xavonol glycosides isolated from the Xowers of Albizia julibris-sin Durazz. J. Ethnopharmacol. 71, 321–323.

Kinjo, J., Fukui, K., Higuchi, H., Nohara, J., 1991. The Wrst isolation of lig-nan tri- and tetra-glycosides. Chem. Pharm. Bull. 39, 1623–1625.

Kirakosyan, A., Seymour, E., Kaufman, P., Warber, S., Bolling, S., Chang,S., 2003. Antioxidant capacity and polyphenolic extracts from leaves ofCrataegus laevigata and Crataegus monogyna (hawthorn) subjected todrought and cold stress. J. Agric. Food Chem. 51, 3973–3976.

Koga, T., Meydani, M., 2001. EVect of plasma metabolites of (+)-catechinand quercetin on monocyte adhesion to human aortic endothelial cells.Am. J. Clin. Nutr. 73, 941–948.

Krenn, L., Beyer, G., Pertz, H., Karall, E., Kremser, M., Galambosi, B.,Melzig, M., 2004. In vitro antispasmodic and anti-inXammatory eVectsof Drosera rotundifolia. Arzneimittel Forschung 54, 402–405.

Kühnau, J., 1976. The Xavonoids. A class of semi-essential food compo-nents: their role in human nutrition. World Rev. Nutr. Diet 24, 117–191.

Lau, C., Carrier, D.J., Howard, L., Lay, J., Archambault, J., Clausen, E.,2004. Extraction of antioxidant compounds from energy crops. Appl.Biochem. Biotechnol. 114, 569–584.

Li, Z., Song, G., Hao, C., Fan, G., 2000. Studies on chemical constituentsfrom the Xower of Albizzia julibrissin Durazz. China J. Chin. Mater.Med. 25, 103–104.

Lin, L., He, X., Lindenmaier, M., Yang, J., Cleary, M., Qiu, S., Cordell, G.,2000. LC-ESI-MS study of the Xavonoid glycoside malonates of redclover (Trifolium pratense). J. Agric. Food Chem. 48, 354–365.

Meyer, A., Heinonen, M., Frankel, E., 1998. Antioxidant interactions ofcatechin, cyanidin, caVeic acid, quercetin and ellagic acid on humanLDL oxidation. Food Chem. 61, 71–75.

Prior, R., Cao, G., Martin, A., SoWc, E., McEwen, J., O’Brien, C., Lischner,N., Ehlenfeldt, M., Kalt, W., Krewer, G., Mainland, C., 1998. Antioxi-dant capacity as inXuenced by total phenolic and anthocyanin content,maturity, and variety of Vaccinium species. J. Agric. Food Chem. 46,2686–2693.

Prior, R., Lazarus, S., Cao, G., Muccitelli, H., Hammerstone, J., 2001. Iden-tiWcation of procyanidins and anthocyanidins in blueberries and cran-berries (Vaccinium spp.) using high performance liquidchromatography/mass spectrometry. J. Agric. Food Chem. 49, 1270–1276.

Prior, R., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, L., Hamp-sch-Woodill, M., Huang, D., Ou, B., Jacob, R., 2003. Assays for hydro-philic and lipophilic antioxidant capacity (oxygen radical absorbancecapacity (ORACFL)) of plasma and other biological and food samples.J. Agric. Food Chem. 51, 3273–3279.

Quettier-Deleu, C., Voiselle, G., Fruchart, J., Duriez, P., Teissier, E., Baill-eur, F., Vasseur, J., Trotin, F., 2003. Hawthorn extracts inhibit LDLoxidation. Pharmazie 58, 577–581.

Singleton, V., Rossi, J., 1965. Colorimetry of total phenolics with phospho-molybdic–phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158.

Sladden, S., Bransby, D., Aiken, G., Rose, P., 1992. Mimosa could be a newforage legume. Highlights Agric. Res. 39, 4.

Sorrenti, V., Di Giacomo, C., Russo, A., Acquaviva, R., Barcellona, M.,Vanella, A., 2004. Inhibition of LDL oxidation by red orange (Citrussinensis) extract and its active components. J. Food Sci. 69, C480–C484.

Van der Sluis, A., Dekker, M., Jager, A., Jongen, W., 2001. Activity andconcentration of polyphenolic antioxidants in apple: eVect of cultivar,harvest year, and storage conditions. J. Agric. Food Chem. 49, 3606–3613.

Williamson, G., Plumb, G., Garcia-Conesa, M., 1999. Glycosylation, ester-iWcation and polymerization of Xavonoids and hydrocinnamates:eVects on antioxidant properties. In: Gross, G.G., Hemingway, R.W.,Yoshida, T., Branham, S.J. (Eds.), Plant Polyphenols 2: Chemistry,Biology, Pharmacology, Ecology. Kluwer Academic/Plenum Publish-ers, New York, pp. 483–494.

Wu, X., Beecher, G., Holden, J., Haytowitz, D., Gebhardt, S., Prior, R.,2004. Lipophilic and hydrophilic antioxidant capacities of commonfoods in the United States. J. Agric. Food Chem. 52, 4026–4037.