Phaseolus Vulgaris L.)

Available online at www.sciencedirect.com Food Chemistry Food Chemistry 107 (2008) 399–410 www.elsevier.com/locate/foodchem

The polyphenolic proﬁles of common bean (Phaseolus vulgaris L.)

Long-Ze Lin a,*, James M. Harnly a, Marcial S. Pastor-Corrales b, Devanand L. Luthria a

a Food Composition and Method Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, USDA, 103000 Baltimore Avenue, Beltsville, MD 20705-3000, USA b Vegetable Laboratory, Plant Sciences Institute, Agricultural Research Service, USDA, 103000 Baltimore Avenue, Beltsville, MD 20705-3000, USA

Received 3 January 2007; received in revised form 21 June 2007; accepted 14 August 2007

Abstract

Based on the phenolic profiles obtained by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS), 24 common bean samples, representing 17 varieties and 7 generic off-the-shelf items, belonging to ten US commercial market classes can be organized into six different groups. All of them contained the same hydroxycinnaminic acids, but the flavonoid components showed distinct differences. Black beans contained primarily the 3-O-glucosides of delphinidin, petunidin and malvidin, while pinto beans contained kaempferol and its 3-O-glycosides. Light red kidney bean contained traces of quercetin 3-O-glucoside and its malonates, but pink and dark red kidney beans contained the diglycosides of quercetin and kaempferol. Small red beans contained kaempferol 3-O-glucoside and pelargonidin 3-O-glucoside, while no flavonoids were detected in alubia, cranberry, great northern, and navy beans. This is the first report of the tentative identification of quercetin 3-O-pentosylhexoside and flavonoid glucoside malonates, and the first detailed detection of hydroxycinnamates, in common beans. Published by Elsevier Ltd.

Keywords: 24 Common beans; Phaseolus vulgaris L.: Market classes; Polyphenol proﬁle; Hydroxycinnamates; Glycosylated ﬂavonols; Anthocyanin; HPLC-DAD-ESI/MS

1. Introduction The common bean is primarily consumed as dry seeds (dry beans) but also as green pods (snap beans) and green The common bean (Phaseolus vulgaris L.) is by far the shelled seeds. There are 10 major US commercial market most important pulse crop (i.e., annual leguminous food classes of dry beans: black beans (Aparicio-Fernades crops, such as chickpea, cowpea, lentils, pea and others et al., 2006; Aparicio-Fernandez, Yousef, Loarca-Pina, that are harvested for dry seeds) in the world (Singh, Gonzales de Mejia, & Lila, 2005; Takeoka et al., 1997), 1999). Among all the major food legumes, the common pinto beans (Beninger et al., 2005; Beninger & Hosfield, bean is the world’s third most important bean after soy- 2003), light and dark red kidney beans (Beninger & Hos- beans (Glycine max (L.) Merr.) and peanut (Arachis hypo- field, 1999; Choung, Choi, An, Chu, & Cho, 2003), pink gea L.). The common bean is an important source of beans, navy beans, great northern beans (Kelly et al., protein, dietary fiber, iron, complex carbohydrates, miner- 2003; Luthria & Pastor-Corrales, 2006), alubia beans (Kelly als, and vitamins for millions of people in developing and & Copeland, 1998; Kelly, Hosfield, Varner, Uebersax, & developed nations and is one of the basic foods of the Taylor, 1999) cranberry beans (Beaver, 1999), and small indigenous populations of South America and Eastern red beans, also known as red Mexican beans (Hosfield, Var- and Southern Africa. ner, Uebersax, & Kelly, 2004). Common dry beans contain a wide range of flavonoids, including flavonols, their glycosides, anthocyanins, proanthocyanidins, and isoflavones, as well as some phenolic acids (Aparicio-Fernades & Gari- * Corresponding author. Tel.: +1 301 504 9136; fax: +1 301 504 8314. E-mail address: [email protected] (L.-Z. Lin). ca-Gasca et al., 2006; Aparicio-Fernades & Yousef et al.,

0308-8146/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.foodchem.2007.08.038 400 L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410

2005; Beninger & Hosfield, 1999; Beninger & Hosfield, 2003; acid derivatives, but the flavonoid components were signif- Choung et al., 2003; Diaz-Batalla, Widholm, Fahey, icantly different allowing the beans to be classified into six Casano-Tostado, & Paredes-Lo´pez, 2006; Macz-Pop, Gon- groups. Acid and base hydrolysis of the dry bean samples za´lez-Parama´s, Pe´rez-Alonso, & Rivas-Gonzalo, 2006; was carried out to isolate and identify the aglycones. This Romani et al., 2004; Takeoka et al., 1997)(Fig. 1). study reports the first detection of quercetin 3-O-pentosylh- A previous study from our laboratory characterized the exoside and flavonoid glucoside malonates and the first phenolic acids obtained following alkaline hydrolysis of fif- detailed detection of hydroxycinnamates in common beans. teen dry edible bean varieties from ten bean classes (Luthria & Pastor-Corrales, 2006). However, there is no systematic 2. Materials and methods study of the polyphenolic profiles of common beans. As a part of a project to determine naturally occurring flavonoids 2.1. Plant materials and other polyphenolic compounds in food plants, we examined seeds of 24 dry bean samples, representing 10 Common dry beans from 10 different US commercial US commercial market classes (17 were identified varieties, market classes were analyzed: alubia, black, cranberry, dark and 7 generic off-the-shelf items from the local grocery red kidney, great northern, light red kidney, navy, pink, store). Using high performance liquid chromatography with pinto, and small red. Eight varieties, belonging to four mar- diode array and electrospray ionization mass spectrometric ket classes, pinto (Buster and Othello), black (Jaguar and T- detection (HPLC-DAD-ESI/MS) (Lin & Harnly, 2007), 39), navy (Seahawk and Vista) and great northern (Matter- retention times and UV/Vis (ultraviolet/visible) and mass horn and Weihing) were obtained from experimental plots spectra were acquired for each of the 24 samples. In general, grown under field conditions in the ARS-USDA South all of the tested beans contained the same hydroxycinnamic Farm in Beltsville, MD. Nine other varieties belonging to

R1 R1

OH OR3 3' 3' 2' 4' 2' 4' B 1' 5' + B 5' HO O 6' HO O 6' 8 8 R2 7 2 R2 7 2 A C A C 6 3 6 3 5 4 5 4 OR OH OH O OH Anthocyanidins Flavonols + Pelargonidin (M 271): R1=R2=R3=H + Kaempferol: R=R1=R2=H Cyanidin (M 287): R1=OH, R2=R3=H + Quercetin: R=R2=H, R1=OH Delphinidin (M 301): R1=R2=OH, R3=H + Myricetin: R=H, R1=R2=OH Petunidin (M 317): R1=OMe, R2=R3=H + Malvidin (M 331): R1=OMe, R2=Me, R3=H Glycosylated flavonoids Anthocyanins The 3-O-glycosides of kaempferol, quercetin and myricetin Their 3-O-glucoside or 3,5-O,O-diglucoside

R1 1 COOH 1 COOH 1 COOH OH 2 2 2 3 H C OH H C OH HO C H 4 2 3 3 3 8 5 HO C H HO C H H C OH HO 6 R 4 4 4 7 2 HO C H H C OH H C OH O 5 5 5 H C OH H C OH H C OH Trans-Hydroxycinnamic acids 66COOH COOH 6COOH 1 2 3 p-Coumaric acid: R1=R2=H Ferulic acid: R1=OMe, R2=H 2,3,4,5-Tetrahydroxy-hexanedioic acids Sinapic acid: R1= R2=OMe (MW=210) 1=D-Galactaric acid 2=D-Glucaric acid 3=D-Altraric acid,

Fig. 1. Structures of flavonols, anthocyanins, and hexanedioic acids and hydroxycinnamic acids. Table 1 Identification of flavonoids and phenolic acids Market Class Variety F- F- F- F- F- F- F- F- F- F- F- F- A- A- A- A- A- A- A- A- Ag- Ag- Ag- Ag- Ag- P- P- P- P- P- P- P- P- P- P- P- P- P- P- P- P- 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Alubia Beluga EEEEEEEE E EEE

Black Generic E EEEE/HE H H E E E H H H EEE EEEE EHHH EEE 399–410 (2008) 107 Chemistry Food / al. et Lin L.-Z. T-39 E EEEE E EEE EEE EEEE E EEE Eclipse E EEEE E EEE EEE EEEE E EEE Jaguar E EEEE E EEE EEE EEEE E EEE Cranberry Taylor EEEEEEEEEE EEE cranberry Great Generic EEE EEEE EHHH EEE Northern Matterhorn EEE EEEE E EEE Norstar EEE EEEE E EEE Weihing EEE EEEE E EEE Kidney, Generic E E E E E/H H E E E EEEEEEEHHH EEE Light Red Cal early E E E E EEEEEEEEEE EEE Kidney, Red hawk E E E E H H E E E EEEE EHHH EEE Dark Red Navy Generic EEE EEEE EHHE/HEEE Sea hawk EEE EEEE E EEE Vista EEE EEEE E EEE Pink Bgeneric E E H H E E E EEEE EHHH EEE UI-537 E E EEE EEEE E EEE Pinto Generic E E E E/H E E E EEEE EHHH EEE Buster E E E E E E E EEEE E EEE Maverick E E E E E E E EEEE E EEE Othello E E E EEE EEEE E EEE Red Mexican Red merlot E E E E E/H E E E E E H H E E E EEEE EHHH EEE UI-239 E E E E E E E E E E E E E EEEE E EEE E – Extract. H – Hydrolyzed extract. 401 402 L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410 nine market classes, pinto (Maverick), black (Eclipse), navy NI were used. The LC system was directly coupled to the (Norstar), dark red kidney (Red Hawk), light red kidney MSD without stream splitting. (California Early), small red also known as Red Mexican The hydrolyzed bean extracts were used to detect the fla- (Red Merlot and UI-239), Alubia (Beluga), cranberry (Tay- vonoid aglycones and some hydroxycinnamic acids. PI/NI lor Hort), and pink (UI 535) beans were obtained from selective ion monitoring (SIM) detection was used to con- Greg Varner, Research Director, Michigan Dry Bean firm the existence of trace amount of some aglycones in Research Board. In addition, generic pinto, black, navy, the hydrolyzed extracts. MS2 and MS3, using an LCQ great northern, light red kidney, and pink beans (Goya ion trap MS (ThermoQuest Corp., San Jose, CA, USA), products) (see Table 1) were bought from a local food mar- were used to determine two minor diglycosylated flavonols ket in Maryland. All the beans were finely powdered and in pink bean. passed through a 20 mesh sieve prior to extraction. 3.2. Sample preparation 3. Flavonoid standards and chemicals 3.2.1. Plant extracts Myricetin (85% purity), quercetin dihydrate (98% pur- Approximately 50 g of dried beans were ground in a cof- ity), kaempferol (minimum 90% purity), rutin trihydrate, fee grinder and the powdered beans were sieved through a p-coumaric acid, sinapic acid, ferulic acid and isofeluric standard sieve (number 20 corresponding to a sieve open- acid were purchased from Sigma Chemical Co. (Saint ing size of 0.85 mm, respectively). Powdered dried bean Louis, MO, USA). HPLC grade quercetin 3-O-gluctoside (250 mg, particle size 60.85 mm) was extracted with meth- and kaempferol 3-O-gluctoside were purchased from anol–water (5.0 ml, 60:40, v/v) using sonication (FS30 Extrasynthese (Genay, Cedex, France). Cyanidin chloride, Ultrasonic sonicator, 40 KHz, 100 W, Fisher Scientific, pelargonidin chloride, petunidin chloride, delphinidin chlo- Pittsburgh, PA, USA) for 60 min at room temperature ride, malvidin chloride, pelargonidin 3-O-glucoside chlo- (from 22 to <40 °C at the end). The extract was separated ride, pelargonidin 3,5-O,O-diglucoside chloride, cyanidin from the solid by centrifuging at 420 g for 15 min (IEC 3-O-glucoside chloride and malvidin 3-O-glucoside chlo- Clinical Centrifuge, Damon/IEC Division, Needham, ride were purchased from Indofine Chemical Co. (Somer- MA, USA). The solvent was removed and filtered through ville, NJ, USA). Formic acid, hydrochloric acid (37%), a 0.45 lm 13 mm PVDF syringe filter (VWR Scientific, sodium hydroxide, HPLC grade acetonitrile, and methanol Seattle, WA, USA). Twenty-five microlitre samples were were purchased from VWR Scientific (Seattle, WA, USA). injected onto the column for LC-DAD-ESI/MS analysis. HPLC grade water was prepared from distilled water using a Milli-Q system (Millipore Lab., Bedford, MA, USA). 3.2.2. Acidic hydrolysis of the glycosylated flavonoids in bean extracts 3.1. LC-DAD and ESI-MSD conditions Bean extract (0.5 ml, each) was mixed with 0.1 ml of concentrated HCl (12 N), and heated in a covered tube at The LC-DAD-ESI/MS instrument has been described 85 °C for 2 h. After cooling to room temperature, 0.4 ml previously (Lin & Harnly, 2007). Briefly, it consisted of of methanol was added and the mixture was sonicated an Agilent 1100 HPLC (Agilent, Palo Alto, CA, USA) cou- for 10 min. The solution was re-filtered prior to HPLC pled with a diode array detector (DAD) and mass spec- injection, and 50 ll of the extract was injected into the col- trometer (MSD, SL mode). A Waters (Waters Corp., umn for the analysis of aglycones (Lin & Harnly, 2007). Milford, MA, USA) Symmetry column (C18, 5 lm, 250 4.6 mm) with a sentry guard column (C18, 5 lm, 3.2.3. Alkaline hydrolysis of the hydroxycinnamic acid 3.9 20 mm) was used at flow rate of 1.0 ml/min. The col- derivatives in bean extracts umn oven temperature was set at 25 °C. The mobile phase A 1 ml aliquot of bean extract was taken to dryness. consisted of a combination of A (0.1% formic acid in Three hundred microlitres of 4 N NaOH was added to water) and B (0.1% formic acid in acetonitrile). The gradi- the dried residue and kept at room temperature under N2 ent was varied linearly from 10–26% B (v/v) in 40 min, to atmosphere for 18 h. Then, 0.15 ml of HCl (12 N) was 65% B at 70 min, and finally to 100% B at 71 min and held added to the reaction mixture to make it pH 1. Then, at 100% B to 75 min. The DAD was set at 270, 310, 350 0.55 ml of MeOH was added, the solution filtered, and and 520 nm for real-time read-out and UV/VIS spectra, 25 ll injected into the column (Lin & Harnly, 2007). from 190 to 650 nm, were continuously collected. Mass spectra were simultaneously acquired using electrospray 4. Results and discussion ionization in the positive and negative ionization (PI and NI) modes at low and high fragmentation voltages 4.1. Identification of bean flavonoids (100 V and 250 V, respectively) for the mass range of 100–2000 amu. A drying gas flow of 13 l/min, a drying A qualitative overview for the 24 bean samples analyzed gas temperature of 350 °C, a nebulizer pressure of 50 psi, in this study is presented in Table 1. In general, the flavo- and capillary voltages of 4000 V for PI and 3500 V for noid profiles tend to follow market classes. Grouping L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410 403 according to flavonoid content will be discussed in the last delphinidin, petunidin, malvidin, cyanidin and pelargoni- section. In this section, the identification of specific flavo- din (Table 3). noids will be discussed on a market class basis. The results Specific identification of these compounds was made by can be assumed to be similar regardless of whether a gen- comparison of the retention time and UV/Vis and mass eric sample or a specific variety was used as an example. spectral data of standards for 3-O-glucosides of cyanidin, Only two dry bean market classes [the black and small pelargonidin and malvidin (separated using the same con- red (red Mexican) beans] contained anthocyanins. The ditions) with that of the sample peaks. All the peaks (with HPLC chromatograms (520 nm) for extracts of black and the exception of peak A-4) were confirmed to be 3-O-glu- small red beans and their hydrolyzed extracts are shown cosides (Table 2). Of these, 3-O-glucosides of delphinidin, in Fig. 2. The retention times (tR), UV/Vis absorption max- petunidin and malvidin had been previously isolated from ima (kmax), and PI/NI molecular and aglycone ions for black beans (Aparicio-Fernades & Garica-Gasca et al., each flavonoid peak are listed in Table 2. Based on this 2006; Aparicio-Fernades & Yousef et al., 2005; Romani data, peaks A-1 to A-3 of the black bean and peaks A5 et al., 2004; Takeoka et al., 1997). Pelargonidin 3-O-glu- and A6 of the Red Merlot bean (Fig. 2) were identified coside and cyanidin 3-O-glucoside had been previously as anthocyanins. From the molecular ions (m/z 465, 479, reported to exist in red kidney beans (Choung et al., 493, 449 and 433) and aglycone ions (m/z 303, 317, 331, 2003). Based on the data presented in Table 2, the trace 287 and 271) of the unhydrolyzed and hydrolyzed extracts, peaks A-4, A-8 and A-7 of small red beans were tenta- peaks A-1 to A-6 were tentatively identified as hexosides of tively identified as pelargonodin 3,5-O,O-diglucoside,

A DAD1 E, Sig=520,20 Ref=700,100 (D:\L-BEAN\LBEAN005.D) mAU 10 A-1 8 A-2 Black bean 6 A-3 4 2 0 5 7.5 10 12 .5 15 17 .5 20 22 .5 min

B DAD1 E, Sig=520,20 Ref=700,100 (D:\L-BEAN\LBEAN029.D) mAU

25 Hydrolyzed black bean 20 Ag-1 Ag-2 15 Ag-3 10 5 0 5 7.5 10 12 .5 15 17 .5 20 22 .5 min

C DAD1 E, Sig=520,20 Ref=700,100 (D:\L-BEAN\LBEAN009.D) mAU

4 A-6 Red merlot bean 3 A-5 2 A-8 1 A-4 A-7 0 5 7.5 10 12 .5 15 17 .5 20 22 .5 min

D DAD1 E, Sig=520,20 Ref=700,100 (D:\L-BEAN\LBEAN042.D) mAU 20 + Pelargonidin 3-O-glucoside 15 + Cyanidin 3-O-glucoside Red merlot bean + two standards 10

0 5 7.5 10 12 .5 15 17 .5 20 22 .5 min

E DAD1 E, Sig=520,20 Ref=700,100 (D:\L-BEAN\LBEAN035.D) mAU Hydrolyzed red merlot bean Ag-5 15 Ag-4

0 5 7.5 10 12 .5 15 17 .5 20 22 .5 min Fig. 2. Chromatograms (520 nm) of the anthocyanins in (A) black bean extract, (B) hydrolyzed black bean extract, (C) small Red Merlot bean extract, (D) small Red Merlot bean extract with added standards, and (E) hydrolyzed hydrolyzed black bean extract. 404 L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410

Table 2 Retention time and UV/Vis and mass spectra of the ﬂavonoids in common bean extract and hydrolyzed extractsa + Peak Flavonoid tR (min) [M+H] /[MH] (m/z) PI/NI (m/z) Aglycone and other ions kmax (nm) Flavonols F-1 Myricetin 3-O-glucoside 21.28 481/479 319/317 260, 358 F-2 Quercetin 3-O xylosylglucoside 22.02 597/595 465, 303/301 256, 356 F-3 Rutin (Quercetin 3-O-rutinoside)b 24.62 611/609 465, 303/301 258, 266sh, 356 F-4 Kaempferol 3-O xylosylglucoside 26.84 581/579 449, 287/285 266, 348 F-5 Quercetin 3-O-glucosideb 27.06 465/463 303/301 258, 266sh, 356 F-6 Quercetin 3-O-(600-O-malonyl)glucoside 31.14 551/549 303/301 258, 266sh, 356 F-7 Kaempferol 3-O-glucosideb 31.97 449/447 287/285 266, 348 F-8 Myricetinb 36.65 319/317 – 256, 274 F-9 Kaempferol 3-O-(600-O-malonyl)glucoside 37.11 535/533 287/285 266, 348 F-10 Kaempferol 3-O-(malonyl)glucoside 38.76 535/533 287/285 266, 348 F-11 Quercetinb 48.44 303/301 – ndc F-12 Kaempferolb 54.46 287/275 – 266, 366 Anthocyanins A-1 Delphinidin 3-O-glucoside 5.68 465 303 278, 524 A-2 Pelargonidin 3,5-O-diglucoside 6.18 595 271 ndc A-3 Petunidin 3-O-glucoside 8.52 479 317 280, 526 A-4 Malvidin 3-O-glucosideb 11.68 493 331 270, 516 A-5 Cyanidin 3-O-glucosideb 7.65 449 287 274, 516 A-6 Pelargonidin 3-O-glucosideb 9.81 433 271 272, 502 A-7 Cyanidin 3-O-(600-malonyl)glucoside 15.11 535 287 ndc A-8 Pelargonidin 3-O-(600-malonyl)glucoside 17.89 519 271 ndc Anthocyanidins in Hydrolyzed extracts Ag-1 Delphinidinb 11.46 – 303 274, 532 Ag-2 Petunidinb 16.93 – 317 276, 532 Ag-3 Malvidinb 20.91 – 331 270, 308, 534 Ag-4 Cyanidinb 16.76 – 287 276, 522 Ag-5 Pelargonidinb 21.15 – 271 268, 512 a This determination is able to detect the ﬂavonoid content in bean equal to 0.0008 % (w/w) and larger. The tR, mass and UV data were taken from one of the tested sample. b Directly compared with standard. c Undetectable.

Table 3 Retention time and UV/Vis and mass spectra of the hydroxycinnamaic acid and derivatives in common bean extracts and their hydrolyzed extractsa Peak tR (min) [MH] (m/z) kmax (nm) Identification Hydroxycinnamic acid derivatives P-1 7.04 355 232, 312 P-coumaric acid derivative P-2 8.43 385 240, 326 Ferulic acid derivativesb P-3 9.05 355 232, 314 P-coumaric acid derivative P-4 9.31 355 232, 314 P-coumaric acid derivative P-5 10.64 355 232, 314 P-coumaric acid derivative P-6 10.95 385 240, 328 Ferulic acid derivativesb P-7 12.32 385 240, 328 Ferulic acid derivativesb P-8 13.19 355 232, 314 P-coumaric acid derivative P-9 13.79 355 232, 314 P-coumaric acid derivative P-10 15.24 385 240, 328 Ferulic acid derivativesb P-13 25.22 193 220, 234sh, 324 Ferulic acidd P-14 60.05 ndc 226, 314 – P-15 61.86 ndc 224, 296 – P-16 62.68 ndc 224, 296 – Hydroxycinnamic acids P-11 22.30 163 224, 310 P-coumaric acidd P-12 24.82 223 238, 324 Sinapic acidd P-13 25.22 193 220, 234sh,324 Ferulic acidd a The tR, mass and UV data were taken from one of the tested navy bean and red kidney bean samples. b Contained around 10% of sinapic acid derivatives. c nd: un-detectable. d By direct comparison with standard. L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410 405 pelargonidin 3-O-(600-malonyl)glucoside and cyandin 3-O- side, kaempferol 3-O-glucoside, myricetin, quercetin, and (600-malonyl) glucoside, respectively. The later two antho- kaempferol, respectively. F1 has UV absorption maxima cyanins had molecular ions that were 248 amu larger than at 260 and 358 nm, PI/NI molecular ions of m/z 481/479, their aglycones and 86 amu larger than that of their glu- and aglycone ions of m/z 319/317, suggesting that it is a cosides; a typical pattern for flavonoid malonylglucosides myricetin 3-O-hexoside. Positive identification as myricetin (Cuyckems & Cleays, 2004; Lin et al., 2000). 3-O-glucoside was obtained by comparison of the data in Five of the market classes (black, pinto, dark red kid- Table 2 with that previously obtained for myricetin 3-O- ney, light red kidney, and small red beans) contained glucoside in cranberries (Lin & Harnly, 2007). detectable levels of flavonoids. The HPLC chromatograms Peak F-6 had the same UV spectra and aglycone ion as (350 nm) for the extracts of black, pinto, red hawk, light peak F-5 (quercetin 3-O-glucoside), but its molecular ion is red kidney beans, and Red Merlot bean and a mixture of 86 amu larger than that of F5 and or 248 amu larger than 6 flavonol standards are shown in Fig. 3. The retention that of its aglycone. Thus, F-6 was tentatively identified as 00 times (tR), UV/Vis absorption maxima (kmax), and PI/NI quercetin 3-O-(6 -malonyl) glucoside (Cuyckems & Cleays, molecular and aglycone ions for each flavonoid peak are 2004; Lin et al., 2000). Similarly, peaks F-9 and F-10 were listed in Table 2. identified as kaempferol 3-O-(600-malonyl)glucoside and A comparison of the data in Table 2 with that obtained kaempferol 3-O-(malonyl)glucoside. F-9 was identified as for standards revealed that peaks F-3, F-5, F-7, F-8, F-11 kaempferol 3-O-(600-malonyl)glucoside because linkage of were rutin (quercetin 3-O-rutinoside), quercetin 3-O-gluco- the malonyl at the 600-position of the sugar is far more

A DAD1 A, Sig=350,4 Ref=700,100 (D:\L-BEAN\LBEAN045.D) mAU 10 0 Flavonol standards F-5 F-8 F-11 F-12 75 F-7 50 F-3 25 0 10 20 30 40 50 min

B DAD1 A, Sig=350,4 Ref=700,100 (D:\L-BEAN\LBEAN005.D) mAU 15 F-1 Black bean 10 F-5 F-7 F-8 5 F-6 F-9 0 10 20 30 40 50 min

C DAD1 A, Sig=350,4 Ref=700,100 (D:\L-BEAN\LBEAN016.D) mAU 30 F-7 Pinto bean 20 F-9 F-12 10 F-10 0 10 20 30 40 50 min

D DAD1 A, Sig=350,4 Ref=700,100 (D:\L-BEAN\LBEAN002.D) mAU

30 F-2 F-5 Red Hawk bean 20 F-6 10 F-4 0 10 20 30 40 50 min

E DAD1 A, Sig=350,4 Ref=700,100 (D:\L-BEAN\LBEAN012.D) mAU 10 F-5 7.5 Red kidney bean F-6 5 F-3 2.5 F-7 F-11 0 10 20 30 40 50 min

F DAD1 A, Sig=350,4 Ref=700,100 (D:\L-BEAN\LBEAN009.D) mAU 10 0 F-7 Red merlot bean 75 F-9 50 F-4 F-12 25 F-10 0 10 20 30 40 50 min

Fig. 3. Chromatograms (350 nm) of (A) 6 flavonol standards and extracts from (B) black, (C) pinto, (D) red hawk, (E) red kidney, and (F) red merlot beans. 406 L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410 common (Lin et al., 2000; Svelikova et al., 2004). The posi- 287/285, should be kaempferol 3-O-pentosylhexoside or tion of malonyl group on the sugar moiety was not deter- kaempferol 3-O-hexosylpentoside. The order of the sugars mined for peak F-10. cannot be determined by LC-DAD-ESI/MS, since the mass Peak F-2 of Red Hawk bean, of the dark red kidney spectra did not offer any fragments to indicate the mass of market class (Fig. 3) showed PI/NI molecular ions at m/z the secondary sugar. Both sugar orders, kaempferol 3-O- 597/595, aglycone ions at m/z 303/301, and fragment ions glucosylxylose in Montcalm dark kidney beans seed coats at m/z 465/463 (i.e., M+-132, 132 for a pentosyl) from loss (Beninger & Hosfield, 2003; Beninger & Hosfield, 1999) of a secondary sugar. The mass spectra of the hydrolyzed and keampferol 3-O-xylosylglucoside in the Zolfino com- extract indicated that aglycone was quercetin. The data mon bean landraces (P. vulgaris L., cv. Zolfino) (Romani suggest that this quercetin glycoside contains one pentose et al., 2004), have been reported. MS2 experiments support more than that of peak F-5 (quercetin 3-O-glucoside). the existence of kaempferol 3-O-xylosylglucoside in the Since the UV absorption spectrum is the same as that tested dark red kidney bean extract based on observation for peak F-5, peak F-2 is most likely quercetin 3-O- of an ion with m/z 447 formed by the loss of pentosyl pentosylhexoside. (132 amu) from the diglycosylated flavonoid (Fig. 4). The minor peak, F-4 of Red Hawk beans, with PI/NI Otherwise, the existence of kaempferol 3-O-glucosylxolo- molecular ions of m/z 581/579 and aglycone ions of m/z side would have required an ion at m/z 417 formed by

A 06/06/2006 11:49:32 AM C:\Xcalibur\data\BEANS\pinbeansrm-2

300.04 100

Relative Abundance 40

20 255.40 427.11 462.63 595.36 178.63 282.54 340.89 0 200 250 300 350 400 450 500 550 600 m/z

B 271.20 100

60 255.27 179.00 Relative Abundance 40 150.80 20 228.80 244.00 203.13 257.07 300.27 0 100 120 140 160 180 200 220 240 260 280 300 m/z C 283.94 100 429.10 80

60 447.20 Relative Abundance 40 579.19 257.13 326.92 20 236.84 380.74 401.10 227.33 265.25 298.11 368.03 458.91 524.24 178.69 534.95 561.10 0 200 250 300 350 400 450 500 550 600 m/z D 255.13 100

Relative Abundance 197.53 40 215.07 255.87 20 150.87 184.00 241.07 266.93 163.07 213.13 229. 20 0 100 120 140 160 180 20 0 22 0 24 0 260 280 30 0 m/z

Fig. 4. MS2 and MS3 spectra of peaks F-2 (quercetin 3-O-pentosylhexoside) and F-4 (kaempferol 3-O-xylosylglucoside, bottom two) of red hawk bean: (A) MS2 of F-2, (B) MS3 of F-2, (C) MS2 of F-4, and (D) MS3 of F-4. L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410 407 the loss of glucosyl from the diglycoside. This ion cannot (Fig. 5). The profiles of red kidney, cal early, and cranberry be seen in Fig. 4. beans showed slightly greater amounts of P-4, and P-9 than Similar reasoning was used to identify peak F-4 for the the profiles of the other common beans. Their total pink bean as quercetin 3-O-pentosylhexoside. The peak at hydroxycinnamate content, however, is slightly lower than m/z 463 corresponds to the mass of the ion formed by the the others. Fig. 5 shows the chromatograms for the extracts loss of the pentosyl (132 amu) from the diglycosylated fla- of light red kidney and navy bean extracts, the NaOH vonoid. Based on the logic that both 3-O-pentosylhexo- hydrolyzed extracts of navy and black bean, and a mixture sides of quercetin and kaempferol in same beans might of hydroxycinnamic acid standards (ferulic acid, sinapic be formed through the same biogenetic pathway, peak acid, p-coumaric acid, and isoferulic acid). Peaks P-1 to F-2 is most likely quercetin 3-O-xylosylglucoside. P-10 are hydroxycinnamic acid derivatives and P-11 to P-13 It is worth noting that this may be the first determina- are hydroxycinnamic acids (Table 2). Peaks P-14 to P-16 tion of quercetin 3-O-xylosylglucoside and all the malonyl were not identified. esters of the 3-O-glucosylated flavonols and anthocyani- Peaks P-2, P-6, P-7, and P-10 (Fig. 5) appear to be feru- dins in these beans. lic acid derivatives. They have UV absorption maxima at 326–328 nm, similar to that of ferulic acid (324 nm), and 4.2. Identification of hydrolysable hydroxycinnamates the strongest NI ions at m/z 385 (Fig. 6). These peaks disappear upon alkaline hydrolysis of the sample (Fig. 5) and After hydrolysis, all the dry beans of the US. commer- release ferulic acid (peak P-13, MW = 194). Thus, peaks cial market classes had very similar hydroxycinnamic acid P-2, P-6, P-7 and P-10 are suggested to be ferulate deriva- profiles. Prior to hydrolysis, there were minor variations tives (MW = 386) of 2,3,4,5-tetrahydroxy-hexanedioic

A DAD1 B, Sig=310,20 Ref=700,100 (D:\L-BEAN\LBEAN012.D) mAU P-6 P-7 P-10 P-15 P-16 15 P-5 P-14 10 P-2 P-4 P-9 Light red kidney bean extract P-3 P-8 5 P-1 0 10 20 30 40 50 60 min

B DAD1 B, Sig=310,20 Ref=700,100 (D:\L-BEAN\LBEAN022.D) mAU P-7 40 P-6 P-15 30 P-2 P-10 P-16 P-5 Nave bean extract 20 P-3 P-8 10 P-1 P-13 P-14 0 10 20 30 40 50 60 min DAD1 B, Sig=310,20 Ref=700,100 (D:\L-BEAN\LBEAN033.D) C mAU P-13 80 60 P-11 Alkaline hydrolyzed nave bean extract 40 P-12 20 0 10 20 30 40 50 60 min DAD1 B, Sig=310,20 Ref=700,100 (D:\L-BEAN\LBEAN030.D) D mAU

60 P-13 Alkaline hydrolyzed black bean extract 40 P-11

20 P-12

0 10 20 30 40 50 60 min DAD1 B, Sig=310,20 Ref=700,100 (D:\L-BEAN\LBEAN032.D) E mAU P-13 20 0 P-12 P-17 (isoferulic acid) 15 0 Four hydroxycinnamic acid standards P-11 10 0

0 10 20 30 40 50 60 min

Fig. 5. Chromatograms (310 nm) of the extract of (A) light red kidney and (B) navy beans, alkaline hydrolyzed extracts of (C) navy and (D) black beans, and (E) four hydroxycinnamic acid standards (P-17, isoferulic acid). 408 L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410

*MSD4 SPC, time=12.384 of LIN\ETEST534.D API-ES, Neg, Scan, Frag:2 Max: 2752

80 385.1 60 40 191. 1 41 3.1

20 38 7.2 20 9.1 14 7.1 13 4.0 36 9.0 284. 2

200 300 m/z

Fig. 6. The NI mass spectrum (250 V) of peak P-10, a ferulic acid derivative. acids (such as galactaric acid, glucaric acid, and altraric 4.3. Grouping common bean according to their phenolic acid) with NI ions at m/z 209 (Fig. 6). The mass spectra profiles of these peaks also contained an ion of m/z 415 (about 10% the intensity ratio of the strongest ion of m/z 385) indi- An examination of the phenolic profiles of all the dry cating the presence of similar derivatives for sinapic acid bean cultivars analyzed in this study shows that all of the (MW = 224, UV: 324 nm). beans contained the same hydroxycinnamic acids in the Similarly, peaks P-1, P-3, P-4, P-5, P-8 and P-9 are sug- hydrolyzed extract and very similar hydroxycinnamic acid gested to be p-coumaric acid (peak P9, MW = 164, UV: derivatives in the extract. The hydroxycinnamic acid deriv- 310 nm) derivatives. These peaks have UV absorption atives constituted the main phenolic component of beans. maxima at 312–314 nm and their strongest NI ion at m/z By comparison, their flavonoid components were less 355. These peaks disappear a upon NaOH hydrolysis and prominent, with the exception of small red dry bean culti- give rise to peak P-9. Mass spectra (not shown) suggest that vars (Red Merlot and UI 237) (Figs. 2 and 3). However, peaks P-1, P-3, P-4, and P-7 are p-coumaric acid condensed their flavonoid components of beans showed some distinct with galactaric acid, glucaric acid, or altraric acid. differences which allow us to separate the common beans Navel orange peel [Citrus sinensis (L.) Osbeck (navel into 6 groups. In general, these groups follow the lines of group)] have been reported to contain hydrolyzable market class. hydroxycinnamtes which also produce p-coumaric acid, The black bean group (Jaguar, T-39, Eclipse and generic,) ferulic acid, and sinapic acid upon alkaline hydrolysis has chromatographic profiles (not shown) similar to that of (Fig. 7D) (Lin & Harnly, 2007). Some of the orange generic black bean shown in Figs. 2 and 3. All of the black hydroxycinnamates were identified as 20-(E)-O-feruloylga- beans contained three anthocyanins (the 3-O-glucosides of lactaric acid (MW = 386), 20-(E)-O-p-coumaroylgalactaric delphinidin, petunidin and malvidin) as their main flavonoid acid, and (E) O p-coumaroyl-derivatives of glucaric components. In addition, they each contained myricetin 3- acid (MW = 356) (Risch, Hermann, & Wray, 1988; Risch, O-glucoside and trace amounts of 3-O-glucosides of querce- Hermann, Wray, & Grotjahn, 1987). Altraric acid, another tin and kaempfeorl and their malonyl esters. isomeric acid, has also been reported to form hydroxycin- The pinto bean group (Othello, Maverick, Buster and namic acid derivatives in yacon root (Takenka et al., generic) contained kaempferol 3-O-glycosides and their 2003). Fig. 7 shows chromatograms for navy bean and free aglycone (Fig. 3). The chromatograms of other three navel orange peel extracts. Several peaks (P-1, P-2, P-3, pinto beans (not shown) were very similar, except Buster P-6, P-7 and P-10) in both plant materials had similar and Maverick bean had a much lower kaempferol aglycone retention times, UV/Vis, and mass spectral data. Spiking content (much smaller absorbance peak). the navy bean extract with the navel orange peel extract The pink bean group was composed of pink bean (UI (Fig. 7B) confirmed the correspondence of these peaks. 537 and generic) and dark red kidney bean (Red Hawk). This strongly suggests that both orange peel and navy bean These beans contained 3-O-pentosylhexosides of quercetin contain hydroxycinnamic acids derivatized with 2,3,4,5-tet- and kaempferol, and quercetin 3-glucoside and its malonyl rahydroxy-hexanedioic acids. derivatives. However, the Red Hawk bean a much higher Each of the three tetrahydroxy-hexanedioic acids can quercetin 3-O-pentosylhexoside content (the peak absor- produce some possible positional isomers with a hydroxy- bance was approximately 17 times greater) than the other cinnamic acid. This accounts for the complexity of the two beans. chromatograms in Fig. 7 between 5 and 18 min. The tech- The light red kidney bean group consisted of Cal Early niques used in this study, LC-MS and MSn, were insuffi- and a generic light red kidney bean. Both beans contained cient to determine the exact linkage. Further studies with queretin 3-O-glucoside and its malonyl derivatives. The nuclear magnetic resonance spectroscopy are necessary to chromatogram of Cal Early bean (not shown) is close to confirm their structures. that of red kidney as shown in Fig. 3. L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410 409

A DAD1 B, Sig=31 0,20 Re f=700 ,100 (D:\L-BEAN\LBEAN022 .D) mAU P-6 30 P-10 P-7 Navy bean (A) 25 P-2 20 P-5 15 P-1 P-3 P-8 10

6 8 10 12 14 16 18 min

B DAD1 B, Sig=31 0,16 Re f=700 ,100 (LIN\ETEST-7\ETEST 63 0.D) mAU

35 Navy bean + Navel orange peel (B) 30

6 8 10 12 14 16 18 min

C DAD1 B, Sig=31 0,16 Re f=700 ,100 (LIN\ETEST-7\ETEST 63 1.D) mAU 60 Navel orange peel (C) 50 P-7 40 P-6 P-10 30 P-3

20 P-1 P-2 10

0 6 8 10 12 14 16 18 min

Fig. 7. Chromatorams of extracts of (A) navy bean, (B) a mixture of navy bean and navel orange peel, and (C) navel orange peel.

The small red bean group (Red Merlot and UI 239) con- 5. Conclusion tained 3-O-glucosides of pelargonidin and cyanidin and kaempferol 3-O-glucoside and its malonyl derivative. Their All of the beans examined in this study contain similar kaempferol 3-O-glucoside content is about 5 times higher hydroxycinnaminic acid derivatives as their main phenolic than that of the pinto bean group and their total flavonoid component. Their flavonoid content is less prominent, but content is the highest among all the beans. The chromato- more varied. The flavonoid components allow the beans to gram of UI 239 (not shown) is very similar to that of Red be separated into 6 groups. These groups follow taxonomic Merlot bean. lines except for the largest group, which is composed of The navy bean group consists of Norstar, Vista, Sea- beans with no detectable flavonoid component. hawk and a generic bean; the great northern bean group was comprised of Matterhorn, Weihing and a generic bean; Acknowledgements the Alubia market class was represented by the bean variety Beluga, and the cranberry market class by the Taylor This research was supported by an Interagency Agree- Hort variety. Except for ferulic acid (present as a trace ment with the Office of Dietary Supplement at the National quantity in navy bean), all of the beans in this group had Institutes of Health, Health and Human Services chromatographic profiles (not shown) very similar to that Department. of navy beans (Fig. 5), i.e., they contained the same hydroxycinnamic acid derivatives as navy beans. However, References no flavonoids were detectable for any of the beans in this group. No significant flavonoid peaks were detectable in Aparicio-Fernades, X., Garica-Gasca, T., Yousef, G. G., Lila, M. A., their chromatographic profiles. Gonzales de Mejia, E., & Loarca-Pina, G. (2006). Chemopreventive 410 L.-Z. Lin et al. / Food Chemistry 107 (2008) 399–410

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