Identification and quantification of glycoside flavonoids in the energy crop Albizia julibrissin

Bioresource Technology 98 (2007) 429–435

IdentiWcation and quantiWcation of glycoside Xavonoids in 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 b

a

a Department of Chemical Engineering, University of Arkansas, 3202 Bell Engineering Center, Fayetteville, AR 72701, United States 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 States d 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 2005 Available online 14 February 2006

Abstract Oxygen radical absorbance capacity (ORAC) values showed that methanolic extracts of Albizia julibrissin foliage displayed antioxidant activity. High performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques were utilized in the identiWcation of the compounds. The analysis conWrmed the presence of three compounds in A. julibrissin foliage methanolic extract: an unknown quercetin derivative with mass of 610 Da, hyperoside (quercetin-3-O-galactoside), and quercitrin (quercetin-3-O-rhamnoside). Fast performance liquid chromatography (FPLC) was employed to fractionate the crude A. julibrissin foliage methanolic extract into its individual 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, Xavanols, Xavones, Xavanones and anthocyanidins, are widely distributed in nature, and are present in most fruits, vegetables 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 liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; MS–MS, tandem mass spectrometry; ORAC, oxygen radical absorbance capacity; PE, phycoerythrin; TLC, thin layer chromatography; Trolox, 6hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid. * Corresponding author. Tel.: +1 479 575 4993; fax: +1 479 575 2846. E-mail address: [email protected] (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 antioxidant and other activities (Aviram and Fuhrman, 2002). Potential sources of antioxidant compounds have been found in several types of plant materials such as fruits, vegetables, leaves, oilseeds, barks, roots, spices and herbs. The authors have been examining the possibility of extracting antioxidants from some common crops in the United States, especially those being studied as possible feedstock for energy production, such as Albizia julibrissin foliage, sericea lespedeza (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 potential of crude methanol extracts stemming from nine diVerent energy crops was reported (Lau et al., 2004). Of the energy

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crops studied, the antioxidants of A. julibrissin extracts showed 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 silk tree, is native to Asia from Iran to Japan (Cheatham et al., 1996). It re-sprouts quickly if cut or top-killed, and the umbrella shaped A. julibrissin tree can reach a height of 6 m. The bark and Xowers of the A. julibrissin tree are used in China as medicine. Bark extract is applied to bruises, ulcers, abscesses, boils, hemorrhoids and fractures, and has displayed cytotoxic activity (Higuchi et al., 1992; Ikeda et al., 1997). A. julibrissin is one of several energy crops being tested in the Auburn University energy crop research program, 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 average total biomass yield of 37.3 Mg ha¡1 yr¡1 from one harvest per year over a 4-year period (Sladden et al., 1992). Previous work has identiWed the presence of saponins and 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 and antioxidant activity of A. julibrissin foliage extract reported by Lau et al. (2004). For this purpose, extract fractions were examined for their total phenolic content and antioxidant activity with oxygen radical absorbance capacity (ORAC) and Folin–Ciocalteau assays, while the identiWcation of compounds was conducted via high performance liquid chromatography (HPLC) and mass spectrometry (MS). Fast performance liquid chromatography (FPLC) was employed to fractionate individual Xavonoids from the crude extract. Each Xavonoid was later quantiWed in terms of mass and their contribution to the overall antioxidant capacity of the A. julibrissin foliage. 2. Methods 2.1. Plant material Samples of fresh A. julibrissin foliage that ranged from 1 to 90 days of age were harvested by hand in August at Auburn, Alabama. Samples included the petiole, branches and leaXets of the entire compound leaf. They were placed in a forced air oven at 65 °C within an hour of harvesting, dried to constant weight, ground to pass through a 1 mm screen and stored in sealed plastic bags at room temperature. A voucher specimen was deposited at the Department of Chemical Engineering, University of Arkansas (Fayetteville, Arkansas). Samples of dried St. John’s Wort (Hypericum perforatum L.) and hawthorn (Crataegus monogyna) berries were purchased from Ozark Natural Foods (Fayetteville, AR). Apples were purchased from a local retailer. 2.2. Chemicals Hyperoside (quercetin-3-O-galactoside) and rutin (quercetin-3-O-rutinoside) were purchased from IndoWne Chemaical

Company, Inc (Somerville, NJ). Isoquercitrin (quercetin-3-Oglucoside), 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-carboxylic acid (Trolox) were acquired from Sigma–Aldrich Corporation (St. Louis, MO). Quercetin, sodium carbonate, HPLC grade methanol, acetonitrile, formic acid and hydrochloric acid were obtained from VWR International (West Chester, PA), whereas 2,2⬘-azobis (2-amidino-propane) dihydrochloride (AAPH) was purchased from Wako Chemicals (Richmond, VA). Potassium phosphate dibasic was purchased from J.T. Baker (Mallinckrodt Baker, Inc., Philipsburg, NJ). 2.3. Extraction For the preparation of all extracts, 2 g of dried biomass (either A. julibrissin, St. John’s Wort, hawthorn berries or apple) were extracted with 60 ml of 60% aqueous methanol at 50 °C by blending the mixture in a household blender for 10 min. To separate the supernatant from the solids, the resulting mixture of solvent and solids was centrifuged at 12,096g for 30 min in an induction drive centrifuge (Beckman Coulter, Fullerton, CA). The supernatant was then Wltered through a 0.45-m syringe Wlter (VWR International, West Chester, PA). The Wltered crude extracts were collected and stored at 4 °C for subsequent fractionation and analysis. 2.4. Sample preparation for total phenolics and ORAC A. julibrissin crude extract was fractionated with 2 ml disposable Symmetry® (Waters, Milford, MA) C18 sep-pak cartridges. The cartridges were preconditioned with 10 ml of methanol, followed by 10 ml of acidiWed water (pH 2 with HCl). About 0.25 ml of Wltered A. julibrissin crude extract was loaded onto the cartridge and eluted with 2 ml of 20% aqueous methanol. The cartridge was subsequently eluted with 2 ml of 60% aqueous methanol followed by 100% methanol. The three fractions were dried under vacuum using a SpeedVac Plus (Savant Instruments, Holbrook, NY) without heat. After drying, the samples were dissolved in 2 ml of methanol. All extractions were performed at least twice. 2.5. Total phenolics and ORAC analysis Total phenolics content was determined according to a modiWed Folin–Ciocalteau assay (Singleton and Rossi, 1965). The absorbance was read at 726 nm on a Hewlett Packard (Palo Alto, CA) 8452A Diode Array UV–visible spectrophotometer. Data were expressed as mg of chlorogenic acid equivalents per gram of dry A. julibrissin foliage. The ORAC assay was conducted by the method of Prior et al. (2003) modiWed for use with a FLUOstar Optima microplate reader (BMG Labtechnologies, Durham, NC)

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

using Xuorescein as Xuorescent probe. A. julibrissin extracts were diluted 1000-fold or more with phosphate buVer (75 mM, pH 7) prior to ORAC analysis. The assay was carried out at 37 °C in clear 48-well Falcon plates (VWR, St Louis, MO). Each well had a Wnal volume of 590 l. Initially, 40 l of diluted sample, Trolox standards (6.25, 12.5, 25, 50 M) and blank solution (75 mM, pH 7 phosphate buVer) were added to each well using an automatic pipette. The FLUOstar Optima instrument equipped with two automated injectors was then programmed to add 400 l of Xuorescein (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 of AAPH and every 197 s thereafter for 112 min to reach 95% loss of Xuorescence. Final Xuorescence measurements were expressed relative to the initial reading. Results were calculated based upon diVerences in areas under the Xuorescence decay curve between the blank, samples and standards. The standard curve was obtained by plotting the four concentrations of Trolox against the net area under the curve (AUC) of each standard. Final ORAC values were calculated using the regression equation between Trolox concentration and AUC and are expressed as moles of Trolox equivalents per gram dry weight. 2.6. LC–MS analysis of A. julibrissin extract A Hewlett Packard 1100 Series (Palo Alto, CA) HPLC with a photodiode array detector was coupled to a Bruker Esquire (Billerica, MA) mass spectrometer. The column used was a Symmetry® (Waters, Milford, MA) C18 column (250 mm £ 4.6 mm). A 25-l sample was injected via the auto sampler. The mobile phase gradient employed in mass spectrometry analysis was conducted by a modiWcation to the method of Cho et al. (2004). Solvent A contained 5% formic acid in water, and solvent B consisted of HPLC grade methanol. The gradient program was initiated with 98:2 solvent A:solvent B, and linearly decreased to 40:60 solvent A:solvent B over 60 min. The Xow rate was set to 0.7 ml min¡1. The UV response was monitored at 360 nm. The MS was operated in the positive ionization mode from the electrospray ionization (ESI) source. The temperature of the drying gas (N2) was 300 °C and Xowed at 10 ml min¡1. The nebulizing pressure (N2) was maintained at 2.1 £ 105 Pa (30 psi). The LC system was directly connected to the mass spectrometer without stream splitting. For tandem mass spectrometry experiments (MS–MS), the mass spectrometer was operated in a manner similar to the LC–MS experiments, except that a target mass (parent ion) was mass selected and separated from all other ions. This mass selected parent ion was then fragmented 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

431

a Waters HPLC Instrument, equipped with a 2996 photodiode array detector, 2795 separations module controlled with Mass Lynx software. A 50 l sample of crude extract was injected through a Symmetry® C18 (50 mm £ 2.1 mm) column (Waters, Milford, MA). Mobile phases were solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The initial condition began at 90:10 solvent A:solvent B and was maintained for 5 min. The gradient was linearly increased to 80:20 solvent A:solvent B over 30 min and was held for 1 min. The gradient was increased again to 20:80 solvent A:solvent B over 2 min and was held for another 5 min before it was decreased to 90:10 solvent A:solvent B in 2 min. This process was followed by a reequilibration of the column at 90:10 solvent A:solvent B for 10 min. The Xow rate was 0.4 ml min¡1, and the column was at room temperature. The diode array detector acquired spectra between the wavelengths of 210 and 600 nm. Quercetin, hyperoside, isoquercitrin, quercitrin and rutin were all dissolved in HPLC grade methanol and kept at 4 °C. All extraction and analyses were performed in triplicate. Averages and standard deviations were calculated with Excel. 2.8. Fractionation of crude A. julibrissin The fractionation of crude A. julibrissin was carried out utilizing AKTA (Amersham Biosciences Corp., Piscataway, NJ) fast performance liquid chromatography (FPLC) system. The 20 ml column was packed with C18 silica in 0.1% formic acid. Samples were pre-conditioned with 0.1% formic acid in 10% aqueous acetonitrile solution for 80 min before a 15 ml sample of crude extract was injected. To wash out unbound compounds, the column was percolated with 10 ml 0.1% formic acid in 10% aqueous acetonitrile. Mobile phases used for the gradient consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The gradient began at 90:10 solvent A: solvent B and linearly changed to 40:60 solvent A:solvent B over 150 min. The gradient was increased to 0:100 solvent A:solvent B in 1 min, and the Wnal gradient was held for 50 min. This was followed by a re-equilibration of column with 90:10 solvent A:solvent B for 30 min. The Xow rate was set at 1 ml min¡1. This equipment was equipped with a Wxed wavelength detector (280 nm). Consequently, compounds were monitored at this wavelength. Experiments were duplicated. Standard deviations were calculated with Excel. 3. Results Crude extracts of A. julibrissin foliage were separated on C18 sep-pak cartridges with increasing concentrations of methanol. The total phenolic values of the fractions are presented in Table 1 and were between 16 and 30 mg of chlorogenic acid equivalents per gram of dry A. julibrissin foliage. Also seen in Table 1, higher ORAC values were acquired from the cartridge eluted with 20% and 60% methanol than with pure methanol.

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

Table 1 Total phenolics and ORAC values for the sep-pak fractionation of crude Albizia julibrissin extracts Extraction solvent

Total phenolicsa

ORAC valueb

20% Methanol in water 60% Methanol in water 100% Methanol

16,000 § 3000 27,000 § 0 30,000 § 6000

140 290 90

Intensity × 105

1.5

302.9

1.2 1.0

a

Micrograms of chlorogenic acid equivalents per gram of dry A. julibrissin foliage. Duplicate experiment. b Micromoles of Trolox equivalents per gram of dry A. julibrissin foliage. Data were not duplicated.

0.7 0.5

228.9

0.2

As presented in Fig. 1A, the total ion trace of A. julibrissin foliage crude extract obtained with 60% methanol showed three major peaks. The ESI mass spectra of the compounds were recorded in the positive-ion mode. Fig. 1B shows the Wrst peak with a retention time of 42.5 min with a peak intensity of 1.2 £ 105 and signiWcant ions at m/z 303 and 633. The second peak (Fig. 1C) displayed a maximum intensity of 4.0 £ 105 and was detected at 49.0 min with signiWcant ions at m/z 303 and 487. The third major peak (Fig. 1D) with signiWcant ions at m/z 303 and 471 was detected at 53.5 min, and displayed a peak intensity of 3.0 £ 105. Each of the major peaks in Fig. 1A displayed an ion with a mass of 303. Based on their photodiode array spectra and their observed masses, the common 303 mass was assigned to protonated quercetin. The presence of protonated quercetin was conWrmed based on a comparison between the MS–MS product-ion spectrum obtained during the LC separation (Fig. 2a) and the reference spectrum

100

3

201.0 213.0

150

247.0

200

250

274.0

300

m/z

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

from reagent grade quercetin (Fig. 2b). The aglycone of delphinidin also has a protonated molecule at m/z 303. However, the presence of delphinidin was ruled out because of the lack of absorbance at 510 nm (results not shown) and of the absence of the reddish-purple color, both characteristic of anthocyanins. Flavones, such as quercetin, often have glycoside moieties attached to them. Fragmentation and loss of the glycoside moieties to yield the parent quercetin aglycone is typical in positive ion ESI analyses (Lin et al., 2000). The spectra presented in Fig. 1 show both the protonated molecule of the glycosides and the ion corresponding to the

2

A

285.0

136.9 152.9 110.9

Intensity x 10

[mAU]

257.0

164.9

1.2

5

Peak 1 633.0

B

303.3

80 1.0

60 0.7

40

0.5

1

20

0.2

0

0.0

0

10

Intensity x 10 4

C

20

30

40

100

50 Time

300

400

500

600

779.0 700

800

900

m/z

487.1

303.3

200

Intensity x 10

Peak 2

5

331.3 465.1 591.2 405.3 487.2

3.0

5

Peak 3 303.2

D

471.1

2.5 3 2.0 1.5

2

1.0 1 325.2

0.5 325.2

642.9

628.9 0.0

0 100

200

300

400

500

600

700

800

900

m/z

100

200

300

400

500

600

700

800

900

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) mass spectrum of peak 1, (C) mass spectrum of peak 2, (D) mass spectrum of peak 3.

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

433

Table 2 Hyperoside and quercitrin content of A. julibrissin and other selected materials

Intensity × 105 5 302.9

4

3

2

1

228.9

164.9

Crops

Hyperoside (g kg¡1 dry material)

Albizia julibrissin St. John’s wort Hawthorn Apple

6.1 § 2.5 8.7 § 0.0 0.3 § 0.0 0.1 § 0.0

Quercitrin (g kg¡1 dry material) 9.0 § 1.8 0.9 § 0.2 0.0 § 0.0 0.1 § 0.1

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

257.0 285.0

110.9

0

100

136.9 152.9

150

201.0 213.0

247.0

200

250

274.0

300

m/z

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

protonated quercetin aglycone. All three peaks displayed a common mass of 303 but diVerent protonated molecule mass for their conjugated glycosides. The diVerences in masses indicate the presence of sugars or other moieties attached to the quercetin aglycone. The diVerence in masses between the two main peaks in the full scan LC–MS spectrum of peak 2 (Fig. 1C) corresponded to a sugar moiety mass of 184 Da. The diVerence in mass of the quercetin monosaccharide (molecular weight of 464) and what was obtained (observed mass of 487) was accounted for by the mass diVerence of 23, which was attributed to the charge from a sodium cation. The assumption was veriWed by the negative ion detection when the deprotonated molecules resulted in m/z of 463 instead of m/z 485, indicating that the presence of the sodium ion occurred only in positive ion mode. 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 quercetin derivatives with a molecular mass of 464, were used in comparison studies. The masses of the quercetin glycosides of 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 (quercetin3-O-rhamnoside) were also analyzed. By co-chromatography 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 of Peak 1 had a mass of 633 Da and showed a prominent frag-

ment ion at m/z 303, corresponding to a loss of 330 Da. Based on the presumption that the parent ion contained the sodium ion, the mass loss from a protonated molecule would be 308 Da. A loss of 308 Da from a protonated molecule would correspond to cleavage of a rhamnose–hexose sugar. The lack of an intermediate rhamnose or hexose loss could be explained by the fact that it did not fragment into its individual sugar moieties. Co-chromatography experiments were performed with rutin and Peak 1 (Fig. 1B), where rutin did not co-elute, establishing that rutinoside was not the carbohydrate moiety. In this co-chromatography experiment, Peak 1 (Fig. 1B) eluted before rutin. Thus, Peak 1 (Fig. 1B) was tentatively identiWed as quercetin-rhamnosylgalactoside. In order to relate the hyperoside and quercitrin content of A. julibrissin to that of other plants, other hyperoside or quercitrin containing plants were extracted with 60% aqueous methanol at the same blending condition as the A. julibrissin foliage. St. John’s Wort (Bilia et al., 2001) and apples (Van der Sluis et al., 2001) were reported to contain hyperoside and quercitrin, whereas hawthorn (Kirakosyan et al., 2003) was reported to contain solely hyperoside. The hyperoside and quercitrin concentrations in these plants were compared to those of A. julibrissin foliage as shown in Table 2. The fraction of each of the quercetin-derivatives in A. julibrissin was evaluated. The tentatively identiWed quercetin-rhamnosylgalactoside peak (Fig. 1B), hyperoside and quercitrin were separated from the A. julibrissin crude extract by FPLC. For 1 g of dry A. julibrissin extract, 4.7 mg of peak 1 (Fig. 1B), 7.4 mg of hyperoside and 7.4 mg of quercitrin were obtained. These compounds corresponded to about 2% by mass of the dry A. julibrissin foliage. Table 3 presents the ORAC activity of the tentatively identiWed quercetin-rhamnosylgalctoside peak (Fig. 1B), hyperoside and quercitrin.

Table 3 Properties of major compounds in A. julibrissin Properties Percent dry weight ORAC activity (mol TEa g¡1) Percent total ORAC a

Trolox equivalents. Experiments were duplicated.

First peak 0.47 § 0.07 31.2 § 5.8 15.9 § 3.4

Hyperoside 0.74 § 0.11 30.9 § 16.1 23.8 § 8.8

Quercitrin 0.74 § 0.07 57.5 § 3.2 46.8 § 6.8

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4. Discussion The total phenolic values of the fractions presented in Table 1 were between 16 and 30 mg of chlorogenic acid equivalents per gram of dry A. julibrissin foliage. Values reported on a chlorogenic acid basis are about 1.8 times greater than those reported as gallic acid equivalents. However, the values presented in Table 1 as chlorogenic acid equivalents were within the range of those reported by Wu et al. (2004). Prior et al. (2001) reported that solvent composition inXuenced selectivity, where 20% methanol/water (v/v) eluted sugars and phenolic acids, while 60% methanol/ water (v/v) and 100% methanol eluted Xavonols/anthocyanins and procyanidins, respectively. In this work, higher total phenolics were obtained with 60% methanol and pure methanol than with 20% methanol. Hyperoside, quercitrin and peak 1 (Fig. 1B) contain the aglycone quercetin. Interestingly, quercetin is a Xavonol, which consists of a group of naturally occurring compounds that are usually present in the plant as glycosides and are colorless or light yellow (Kühnau, 1976). Quercetin is not solely found in A. julibrissin biomass, being reported in grapes (Careri et al., 2003), apples (Van der Sluis et al., 2001), oranges (Sorrenti et al., 2004) and buckwheat (Fabjan et al., 2003) as well as some medicinal botanicals such as hawthorn (Kirakosyan et al., 2003) and St. John’s Wort (Bilia et al., 2001). Quercetin has been associated with cardiovascular protection (Aviram and Fuhrman, 2002). Accumulating evidence is showing that oxidation of low density lipids (LDL) and monocyte-endothelial cell adhesion are hallmark events in cardiovascular diseases (Aviram and Fuhrman, 2002). Quercetin has been shown, among others, to inhibit oxidation of LDL (Meyer et al., 1998), to inhibit monocyte cell adhesion to human aortic endothelial cells stimulated by interlukin-1 (Koga and Meydani, 2001) and to decrease the electrophoretic mobility of human oxidized LDL by 35% (Sorrenti et al., 2004). Hyperoside was documented to have anti-inXammatory eVects (Krenn et al., 2004), inhibit in vitro LDL oxidation (Quettier-Deleu et al., 2003) and display antimicrobial properties (Dall’Agnol et al., 2003). The presence of hyperoside and quercitrin has previously been reported. In an unspeciWed A. julibrissin material, Kaneta et al. (1980) reported the presence of hyperoside and quercitrin by thin layer chromatography (TLC), but not by MS. The presence of quercitrin was reported by Li et al. (2000) and Kang et al. (2000) in Xowers of A. julibrissin, but not in foliage. Li et al. (2000) isolated quercitrin by extraction with ether and ethyl acetate followed by a chromatographic separation. Kang et al. (2000), on the other hand, used methanol as solvent for the extraction, Sephadex LH-20 multiple-column chromatography for the fractionation, and nuclear magnetic resonance for compound identiWcation. Among the four plants tested under our experimental conditions, A. julibrissin had the highest content of quercitrin, 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 close to what was reported by Kirakosyan et al. (2003), about 0.3 g kg¡1 dry mass. According to Van der Sluis et al. (2001), the hyperoside and quercitrin contents of apples were reported as 0.027 and 0.029 g kg¡1 dry mass, respectively. As shown in Table 3, quercitrin had the highest ORAC activity, followed by hyperoside, and the tentatively identiWed quercetin-rhamnosylgalctoside peak. This observation that quercitrin has a higher antioxidant activity than hyperoside is in agreement with Williamson et al. (1999). The methanol extraction of the three compounds consisted of about 2% of the mass of A. julibrissin on a dry basis. This work consists of the Wrst report on hyperoside and quercitrin quantiWcation in A. julibrissin foliage extract and their link to ORAC activity. In summary, the three main antioxidant constituents in A. julibrissin methanolic extracts were hyperoside, quercitrin and the tentatively identiWed quercetin-rhamnosylgalctoside peak 1 (Fig. 1B). The combination of these three compounds accounted for 2% mass of the A. julibrissin foliage. From an antioxidant perspective, 119.6 Mol trolox equivalents per gram of dry foliage was obtained. This study indicates that A. julibrissin foliage methanolic extracts have high antioxidant activity and are a rich source of Xavonoids, which could possibly be extracted prior to energy production unit operations. Hyperoside was reported to inhibit in vitro LDL oxidation (Quettier-Deleu et al., 2003) and could possibly be beneWcial in the management of endothelial dysfunction diseases. Thus, A. julibrissin extracts could possibly be included in human or animal functional foods during the next decade. Flavonoid extraction from A. julibrissin biomass could possibly occur prior to its use as an energy crop (Bransby et al., 2001), conferring added value. Acknowledgements This project was supported by the Southeastern Regional Biomass Energy Program (SERBEP) and administered by the Southern States Energy Board (SSEB) for the United States Department of Energy and by the Arkansas Experimental Station. References Aviram, M., Fuhrman, B., 2002. Wine Xavonoids protect against LDL oxidation and atherosclerosis. Ann. NY Acad. Sci. 957, 146–161. Bilia, A., Bergonzi, M., Mazzi, G., Vincieri, F., 2001. Analysis of plant complex 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 (Albizia julibrissin) over traditional short rotation woody crops. In: 1st World 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. Direct HPLC 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 Useful Wild Plants of Texas, the Southeastern and Southwestern United

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