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Analysis of Flavonoids and Hydroxycinnamic Acid Derivatives in Rapeseeds (Brassica napus L. var. napus) by HPLC-PDA−ESI(−)-MSn/ HRMS Yanlin Shao,†,§ Jinjin Jiang,†,§ Liping Ran,† Chunliang Lu,‡ Cunxu Wei,† and Youping Wang*,† †

Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology and ‡Test Center, Yangzhou University, Yangzhou, 225009 Jiangsu, China ABSTRACT: A comprehensive description of flavonoids and hydroxycinnamic acid derivatives in Brassica napus L. var. napus seeds is important to improve rapeseed quality. HPLC-PDA−ESI(−)-MSn/HRMS has been broadly applied to study phenolic compounds in plants. In the present study, crude phenolic compounds extracted from rapeseed were subjected to column chromatography, alkaline hydrolysis, and HPLC-PDA−ESI(−)-MSn/HRMS analysis. A total of 91 flavonoids and hydroxycinnamic acid derivatives were detected, including 39 kaempferol derivatives, 11 isorhamnetin derivatives, 5 quercetin derivatives, 6 flavanols and their oligomers, and 30 hydroxycinnamic acid derivatives. A total of 78 of these compounds were tentatively identified; of these, 55 were reported for the first time in B. napus L. var. napus and 24 were detected for the first time in the genus Brassica. This research enriches our knowledge of the phenolic composition of rapeseed and provides a reliable guide for the selection of rapeseed with valuable breeding potential. KEYWORDS: Brassica napus L. var. napus, flavonoids, hydroxycinnamic acid, HPLC-PDA−ESI(−)-MSn/HRMS

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INTRODUCTION Brassica napus L. is one of the most economically important oil crops. Cultivating rapeseed with high quality is the main objective of most breeders.1 Over the past decade, edible plants, including crops with enriched secondary metabolites, have gained increased research attention because of their economic and nutritional value. Phenolic compounds, including flavonoids and hydroxycinnamic acid derivatives, are a group of phytochemicals with antioxidant activity.2 Besides its oil and protein contents, rapeseed also contains flavonoids and hydroxycinnamic acid derivatives. Recent studies have confirmed that some polyphenols prevent cardiovascular disease and cancer, especially cancers of the gastrointestinal tract.3−5 Selective breeding of yellow-seeded B. napus is an optimized method of improving rapeseed quality because this variant has a thinner seed coat and higher oil and protein contents than black-seeded B. napus.6 Flavonoids are correlated with the yellow-seeded phenotype.7 To date, over 200 flavonoids have been identified in Brassica species.8 Hydroxycinnamic acid derivatives deposited in B. napus provide the bitter taste of rapeseed; conjugation of these derivatives with proteins during the maturation of rapeseed reduces rapeseed nutrition.9 Thus, comprehensive interpretation of flavonoids and hydroxycinnamic acid derivatives in rapeseed is necessary to breed B. napus with improved quality and determine the benefits of B. napus to human health. The chemical structures of flavonoids and hydroxycinnamic acid derivatives have been well elaborated. Flavonoids in Brassica plants mainly include kaempferol, isorhamnetin, and quercetin and their corresponding glycosylated and acylated derivatives.8,10−17 The sugars conjugated to flavonols in Brassica are most commonly found as O-glycosides; they are also commonly acylated by different hydroxycinnamic acids.2,18 Sinapic, ferulic, and p-coumaric acids, the main hydroxycin© 2014 American Chemical Society

namic acids in Brassica crops, are often conjugated with sugar or other hydroxycinnamic acids.19,20 While the discovery of phenolic components in Brassica has undergone rapid expansion in recent years, a comprehensive analysis of rapeseed phenolics, particularly flavonoids and hydroxycinnamic acid derivatives, has yet to be reported. Only six flavonoids have been identified by Olsson et al.,21 who used UV-B radiation to analyze B. napus. Fourteen flavonoids have recently been discovered in the developing seeds of B. napus.22 Eighteen phenolic components have also been determined in a comparative study of yellow- and black-seeded B. napus, 14 of which were proven to be flavonoids.23 Farag et al.24 provided a highly comprehensive investigation of polyphenols in different organs of B. napus, including 30 flavonoids and hydroxycinnamic acid derivatives. Multiple phenolic compounds exhibit similar retention times in traditional high-performance liquid chromatography (HPLC) because of the poor separation efficiency of phenolic extracts. These compounds are difficult to identify, which explains the lack of chemical information on phenolics in B. napus. HPLC is a highly selective and sensitive analytical technique that allows structure elucidation when combined with MSn and high-resolution mass spectrometry (HRMS).25 In this work, we aimed to identify the phenolic compounds in the seeds of B. napus L. var. napus via HPLC-PDA−ESI(−)-MSn/ HRMS using extracts separated by Sephadex LH-20. Several phenolic compounds, including flavonoids and hydroxycinnamic acid derivatives, were successfully identified in rapeseed. Received: Revised: Accepted: Published: 2935

October 25, 2013 February 26, 2014 March 12, 2014 March 12, 2014 dx.doi.org/10.1021/jf404826u | J. Agric. Food Chem. 2014, 62, 2935−2945

Journal of Agricultural and Food Chemistry

Article

concentrated using a vacuum rotary evaporator at 35 °C, and the slurry was washed with ddH2O to a final volume of 3 mL, which was transferred into a 10 mL glass tube and evaporated using nitrogen flow. Finally, the slurry was washed with 1 mL to 2 mL 80% methanol, filtered with Teflon membrane, and analyzed with HPLC−PDA− ESI(−)/MSn (LCQ) and HPLC−DAD−ESI(−)/HRMS (Q-TOF). Alkaline Hydrolysis of Crude Extracts. Alkaline hydrolysis of the crude extracts was carried out according to the method described by Lin et al.8 with minor modifications. The crude extract (0.4 mL) was evaporated by nitrogen flow, and the residue was mixed with 0.3 mL of 2 N NaOH under a N2 atmosphere for 18 h. Then, 0.1 mL of HCl (37%) was added to the reaction mixture to adjust the pH to 1.0, and 1.6 mL of methanol was added. After filtering through a Teflon membrane, the extract was analyzed by HPLC-PDA−ESI(−)-MSn (IonTrap) and HPLC-DAD−ESI(−)-HRMS (Q-TOF). HPLC-PDA−ESI(−)-MSn (IonTrap) Analysis. The crude extract, alkaline-hydrolyzed extract, and eight fractions of the crude extract were analyzed using an LCQ Deca XP MAX (ThermoFinnigan, San Jose, CA) instrument to determine the UV absorption characteristics and MSn fragmentation patterns of the flavonoids and other compounds. The instrument was operated using Xcalibur version 1.4 software. An LCQ Deca XP MAX system with an ion-trap mass spectrometer including an LC pump, a Finnigan Surveyor autosampler, and a Thermo Finnigan Surveyor PDA detector was used. Separation was performed on an RP-C18 column (150 mm × 4.6 mm, 5 μm, Agilent Technologies, Waldbronn, Germany) at 30 °C. The flow rate was set to 0.8 mL min−1, and the injection volume was set to 10 μL. The mobile phase consisted of a combination of solvent A (0.1% formic acid in water, v/v) and solvent B (0.1% formic acid in acetonitrile, v/v). The linear gradient was as follows: 4%−20% B (v/v) for 40 min, 35% B for 60 min, 100% B for 61 min, hold at 100% B for 76 min, 4% B for 77 min, and hold at 4% B to 86 min. UV−vis spectra were recorded from 200 to 600 nm. The mass analyzer was a ThermoFinnigan LCQ IonTrap instrument coupled with an ESI source. The instrument was set to negative ion mode with an m/z range of 90−2000. The following conditions were adopted: capillary temperature of 300 °C, sheath gas of 70 arbitrary units, capillary voltage of 20 V, and tube lens of 70 V. Under 50% normalized collision energy, the most intense ion was selected to obtain the MS2, MS3, and MS4 data. The collision gas used was nitrogen. HPLC-DAD−ESI(−)-HRMS (Q-TOF) Analysis. The extracts were subjected to HPLC-DAD−ESI(−)-HRMS (Q-TOF) analysis to determine their exact molecular mass, identify the elemental composition of the compounds, and verify the result of MSn (IonTrap) analysis. An Ultimate 3000 series instrument (Dionex Sunnyvale, CA) coupled with an Ultimate 3000 pump including an autosampler, a column compartment, and a DAD detector was used for liquid chromatography. Separation was performed using the same gradient described above; here, an Ultimate XB-C18 column (3.5 μm, 2.1 mm × 150 mm, Welch) and a Phenomenex precolumn (2 mm × 4 mm, Torrance, CA) were used at 30 °C. The flow rate was set to 0.2 mL min−1, and the injection volume was 10 μL. The DAD was set to 520, 330, and 280 nm to record peaks, and UV−vis spectra were recorded from 190 to 800 nm. A Bruker Daltonik (Bremen, Germany) maXis Q-TOFHRMS instrument with an ESI source was used for mass analysis. The instrument was operated by the microTOF control (Bruker Daltonik) software package. Negative ion mode and an m/z range of 100−2000 were selected during operation. The nebulizer gas (N2) pressure was 1.5 bar, the drying gas flow was 6 L min−1, the drying gas temperature was 180 °C, and the capillary voltage was 2500 V.

Some of these compounds were detected for the first time in the genus Brassica and species B. napus.

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MATERIALS AND METHODS

Chemicals. NaOH and HCl were purchased from Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, China). Methanol, formic acid, acetone, and acetonitrile were of HPLC grade (Sigma, St. Louis, MO). Water was purified using a MilliQ system (Millipore, Bedford, MA). Procyanidin B2, (−)-epicatechin, and quercetin-3-O-β-D-glucoside were purchased from Sigma. Isorhamnetin-3-O-glucoside was purchased from ChromaDex (Irvine, CA). Sephadex LH-20 was purchased from GE Healthcare BioSciences AB (Uppsala, Sweden). Plant Materials and Sample Collection. According to previous research, some flavonoids and hydroxycinnamic acid derivatives normally combine with other seed coat components, which makes them difficult to identify. The flavonoid and hydroxycinnamic acid derivative contents of rapeseed accumulate in the early periods of seed development and reach their peak about 5 weeks after flowering (WAF).23 In the present study, B. napus L. var. napus (cv.Yang 6) grown in a greenhouse at approximately 25 °C with a 16 h light/8 h dark cycle was used as the plant material. Two weeks after seedling development, rapeseed lines were planted into 15 cm diameter pots filled with the same soil (20% vermiculite and 80% orchid soil). The plants were vernalized for 2 months at 4 °C. Each flower on the primary raceme was tagged on the day of pollination. Developing seeds were harvested at 5 WAF for phenolic analysis. Five pods from individual plants of one rapeseed line were collected, pooled, and immediately frozen in liquid nitrogen for further analysis. Preparation of Crude Methanol Extract. The powder (40 g) of dissected rapeseeds was extracted with 200 mL of 80% methanol in an ultrasonicator bath at 4 °C for 1 h. The supernatant was collected after centrifugation (10 000 rpm, 10 min, 4 °C) and stored at −80 °C. The pellet was again extracted overnight with 100 mL of the same extraction buffer at 4 °C under agitation (200 rpm). The supernatant from the second extraction was collected and pooled with the supernatant collected from the previous day. The extracts were subsequently concentrated using a vacuum rotary evaporator at 35 °C, and the slurry was washed with ddH2O to a final volume of 4 mL. Finally, the obtained crude extract was diluted with 5 volumes of methanol, filtered through a 0.45 μm Teflon membrane (Interchim, Montluc-on, France), and analyzed by HPLC-PDA−ESI(−)-MSn (IonTrap) and HPLC-DAD−ESI(−)-HRMS (Q-TOF). Preparation of Sephadex LH-20 Column Chromatography Fractions. Column chromatography of the crude extract described above was carried out according to the method described by Yang et al.26 with several modifications. The crude extract (2 mL) was loaded onto a 22 mm i.d. × 470 mm length glass column packed with 25 g of Sephadex LH-20 medium resin (dry particle size 18−118 μm); the resin was swelled in ddH2O overnight and equilibrated with ddH2O before use. Finally, the column was eluted with the solvents listed in Table 1. The elution speed was controlled to 1 mL min−1, and 150 mL of each collection was produced. The column was regenerated by washing with 10 column volumes of ddH2O. Each fraction was again

Table 1. Eluotropic Series for Sephadex LH-20 Column Chromatography fraction

H2O concn (%)

MeOH concn (%)

acetone concn (%)

F1 F2 F3 F4 F5 F6 F7 F8

100 70 50 50 50 50 50 40

0 30 50 40 30 20 10 0

0 0 0 10 20 30 40 60

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RESULTS AND DISCUSSION Confirmation of Flavonol Glycosides. The structures of the compounds identified in seeds of B. napus L. var. napus are shown in Figure 1. The HPLC−UV (330 nm) profiles of flavonoids and hydroxycinnamic acid derivatives obtained from the crude extracts and chromatography fractions are presented in Figure 2. Fifteen flavonol glycosides were successfully 2936

dx.doi.org/10.1021/jf404826u | J. Agric. Food Chem. 2014, 62, 2935−2945


Article

Figure 1. Structures of compounds identified in seeds of B. napus L. var. napus.

identified according to their retention times, UV−vis wavelength maxima (λmax), MS2, MS3, and MS4 ion characteristics, alkaline hydrolysis patterns, and HRMS profiles. The corresponding chemical information of the identified phenolic compounds is listed in Table 2. On the basis of previous conclusions,8,14,16 the identified flavonol glycosides were analyzed according to their UV wavelength maxima (λmax) and MSn fragments. For example, the UV spectra of the 3-O-glycosides and 3-O-,7-O-diglycosides in both quercetin and isorhamnetin showed λ max at approximately 255−270 nm (band II) and 345−355 nm (band I), respectively. The 7-O-glycosides in quercetin and isorhamnetin showed λmax at 255−270 nm (band II) and 370 nm (band I), respectively. By contrast, the λmax values of 3-Oglycosides, 3-O-,7-O-diglycosides, and 7-O-glycosides on kaempferol were detected at around 255−270 nm (band II), 345−350 nm (band II), and 365 nm (band I), respectively. During the formation of flavonol derivatives in Cruciferae, glycosylation initially occurs at 3-OH and then at 7-OH. During MSn analysis with negative ionization, the major MS2 product ions resulting from the loss of 7-O-glycosides were first detected, followed by the main MS3 product ions formed by the loss of 3-O-glycosides.16,27 According to the size of the MSn

fragments, losses of glucosyl (162 Da) and sophorosyl (2-β-Dglucopyranosyl-D-glucopyranosyl)/sophorotriosyl (2-β-D-glucopyranosyl-20-β-D-glucopyranosyl-D-glucopyranosyl) (120 Da/ 180 Da) were also detected. Several compounds could be confirmed on the basis of the chemical formulas obtained from HRMS analysis and alkaline hydrolysis characteristics. For instance, kaempferol-3-O-sophoroside-7-O-glucoside (peak 5), which has been previously reported,21 exhibited UV spectra with λmax at 255 and 350 nm. The major MS2 fragment at m/z 609 obtained from the loss of a glucosyl (162 Da) was first detected, followed by two major MS3 fragments, including an ion fragment at m/z 429 from the partial loss of a sophorosyl (180 Da) and a parent ion at m/z 285 from the complete loss of a sophorosyl. The presence of peak 5 after alkaline hydrolysis indicates the presence of glucosyl rather than caffeoyl in this compound, which is in agreement with the HRMS analysis. In summary, 15 glucosylated flavonols were successfully identified (Table 2), including 6 isomers of kaempferol diglucoside and 2 isomers of quercetin triglucoside. We found that the number of glycosides varied among the different flavonol derivatives. The 15 glucosylated flavonols consisted of 9 glycosylated kaempferol compounds, 3 glycosylated quercetin compounds, and 3 glycosylated isorhamnetin compounds. Five 2937



Article

Figure 2. LC chromatograms (330 nm) for the crude extracts and column chromatography fractions (F1−F8) of phenolic compounds in rapeseed. Peaks: (1) qn-3-O-sophoroside-7-O-glucoside; (4) km-O-diglucoside; (5) km-3-O-sophoroside-7-O-glucoside; (6) sinapoylhexose; (7) km-3-Ocaffeoylsophoroside-7-O-glucoside; (9) procyanidin B2; (10) qn-3-O-sinapoylsophoroside-7-O-glucoside; (11) cis-sinapic acid; (12) sinapoylhexose; (16) km-3-O-sinapoylsophorotrioside-7-O-glucoside; (17) (−)-epicatechin; (18) km-3-O-sinapoylsophoroside-7-O-glucoside; (20) putative hydroxycinnamic acid derivative; (21) km-3-O-glucoside-7-O-glucoside; (22) km-3-O-feruloylsophoroside-7-O-glucoside; (23) sinapoylhexose; (25) km-3-O-p-coumaroylsophoroside-7-O-glucoside; (26) is-3-O-glucoside-7-O-glucoside; (28) sinapoylgentiobiose; (29) sinapoylhexose; (30) [DP 3]; (31) km-3-O-sophoroside; (32) qn-3-O-sophoroside; (33) km-3-O-diglucoside; (35) [DP 4]; (37) km-3-O-caffeoyldiglucoside-7-Oglucoside; (38) km-3-O-sinapoylsophotrioside-7-O-glucoside; (39) is-3-O-diglucoside; (40) km-3-O-disinapoylgalloyldiglucoside; (41) km-Odihydroxyferuloylsinapoyldiglucoside; (42) km-7-O-sophoroside; (43) qn-3-O-sinapoylsophoroside-7-O-glucoside; (45) km-3-O-sinapoyldiglucoside-7-O-glucoside; (47) is-3-O-sinapoyldiglucoside-7-O-glucoside; (48) is-O-glucoside-sulfate; (50) [DP 2]; (51) sinapoylmalic acid; (53) km-3-Osinapoylsophoroside; (57) km-3-O-sinapoyldiglucoside-7-O-glucoside; (58) km-3-O-sinapoylsophoroside-7-O-glucoside; (60) is-3-O-sinapoylglucoside-sulfate-7-O-glucoside; (61) km-3-O-glucoside; (62) km-3-O-sophoroside-7-O-sinapoylglucoside; (64) km-3-O-disinapoylhydroxyferuloyldiglucoside; (65) is-3-O-glucoside; (66) km-3-O-sinapoylsophoroside-7-O-sinapoylglucoside; (67) km-3-O-disinapoylhydroxyferuloyldiglucoside; (68) km-3-O-sinapoyltriglucoside-7-O-sinapoylglucoside; (69) disinapoylgentiobiose; (71) km-3-O-sinapoyldiglucoside-7-O-sinapoylglucoside; (72) is-3O-sinapoylglucoside-7-O-glucoside; (73) km-3-O-sinapoylglucoside-7-O-glucoside; (76) putative hydroxycinnamic acid derivative; (77) km-3-Otrisinapoylhydroxyferuloylsophoroside; (78) 1,2-disinapoylglucoside; (82) km-3-O-disinapoylhydroxyferuloylglucoside-7-sinapoylglucoside; (83) 1,2,2′-trisinapoylgentiobiose; (84) putative hydroxycinnamic acid derivative; (85) km-3-O-sinapoylsophoroside; (86) km-3-O-sinapoylsophoroside7-O-sinapoylglucoside; (87) putative hydroxycinnamic acid derivative; (89) putative hydroxycinnamic acid derivative. 2938


tR (min)

16.57

17.40 17.63 17.89 18.50

21.00 21.96

22.21 22.50 22.82 23.26 23.40

23.92 23.93

24.08 24.17

24.46 24.84

24.91

25.28 25.50 25.80

25.99 26.64 26.71

26.81 27.02 27.26 28.21 28.50 29.31 30.02 30.60 30.85 30.90

peak

1

2 3 4 5

6 7

8 9 10 11 12

13 14

15 16

17 18

2939

19

20 21 22

23 24 25

26 27 28 29 30 31 32 33 34 35

639.1561 465.0715 547.1666 385.1135 865.1994 609.1460 625.1410 609.1452 753.2260 1153.2634

385.1144 547.1662 917.2351

729.2621 609.1479 947.2457

1045.2459

289.0714 977.2581

719.1148 1139.3100

547.1670 977.2584

431.1923 577.1361 993.2525 223.0616 385.1141

385.1137 933.2307

787.1938 933.2529 609.1469 771.1997

787.1945

measd m/z [M − H]−

C28H31O17 C13H21O18 C23H31O15 C17H21O10 C45H37O18 C27H29O16 C27H29O17 C27H29O16 C34H41O19 C60H49O24

C17H21O10 C23H31O15 C42H45O23

C33H45O18 C27H29O16 C43H47O24

C47H49O27

C15H13O6 C44H49O25

C28H31O20S C50H59O30

C23H31O15 C44H49O25

C20H31O10 C30H25O12 C44H49O26 C11H11O5 C17H21O10

C17H21O10 C42H45O24

C33H39O22 C39H49O26 C27H29O16 C33H39O21

C33H39O22

calcd formula [M − H]−

0.8 4.0 0.5 1.3 −1.0 0.2 0.0 1.4 −1.5 −1.3

477 (100), 315 (21) 421 (44), 379 (100), 283 (11), 223 (26) 385 (34), 223 (100) 325 (69), 295 (100), 265 (81), 223 (24) 577 (62), 451 (71), 407 (100), 289 (78) 447 (100), 429 (31) 463 (100), 445 (44), 300 (81) 44 (53), 285 (100) 529 (100), 487 (42) 865 (43), 575 (100), 413 (35), 289 (42)

325 (71), 295 (100), 265 (63),223 (21) 223 (100), 205 (27) 755 (100)

−0.1 1.2 0.7

−1.3 −2.9 0.6

977 (49), 815 (100), 609 (39), 447 (20), 285 (9) 533 (17), 341 (100), 179 (29) 489 (35), 447 (100), 285 (22) 785 (100), 609 (19)

245 (100), 205 (38), 179 (22) 815 (100), 609 (10)

0.7

1.2 −1.3

639 (100), 477 (50), 315 (23) 977 (100), 771 (21), 609 (12), 285 (15)

−1.9 −0.3

(69), 281 (83), 223 (100) (100), 407 (44), 289 (18) (78), 625 (100) (100), 164 (74) (69), 223 (100)

411 (36), 223 (100) 815 (100), 785 (24), 771 (57)

359 425 831 193 247

247 (100), 223 (11), 205 (6) 771 (100)

(100) (100) (100), 285 (30) (100)

0.9 −1.6

−0.1 −1.7 0.5 −1.6 −0.3

0.9 0.0

625 771 447 609

625 (100)

−0.9 0.0 −1.1 −1.3 −1.0

major and important MS2 ions (m/z) (%)

error (ppm)

315 nd 208 223 nt 285 300 257 427 nd

(77) (7)

(51) (79)

(100) (100) (100), 241 (41) (22), 223 (100)

(100), 179 (42) (100)

(40),314 (100)

nd 284 (100), 243 609 (100), 623 591 (62) 223 (100) 208 (100), 164 609 (100), 591

203 (100), 187 (39) 609 (100), 591 (33), 285 (7) nd

nd nd

445 (23), 301 (85), 300 (100) 463 (100), 300 (42) 429 (51), 284 (100) nd 429 (33), 285 (48), 284 (100) nt 609 (100), 447 (11), 429 (29), 285 (5) nt nt nd nt 208 (32), 179 (21), 164 (100) 208 (100), 179 (40) 609 (100)


nd nd nd nt nd nd nd nd nt nd

nt nt nd

nd nd 429 (100), 284 (27)

nt 429 (100), 284 (71), 285 (23) nd

nd nd

nd nd

nd nd nd nd nd

nd nd

ndc nt nd nt

nt

b


nd nd 240, 240, 240, nd 265, 265, nd nd

345 335

325 325 280

240, 325 nd nd

nd nd 270, 330

nd

220, 280 270, 335

nd 270, 335

nd nd

nd 202, 280 255, 330 nd nd

240, 330 268, 330

nd 265, 345 nd 255, 350

255, 355

UV λmax (nm)

putative hydroxycinnamic acid derivative km-3-O-glucoside-7-O-glucosided km-3-O-feruloylsophoroside-7-Oglucosided sinapoylhexosed sinapoylgentiobiosed km-3-O-p-coumaroylsophoroside-7-Oglucosided is-3-O-glucoside-7-O-glucosided putative hydroxycinnamic acid derivative sinapoylgentiobiosed sinapoylhexosed [DP 3] km-3-O-sophorosided qn-3-O-sophorosided km-3-O-diglucosided disinapoylgentiobiosed [DP 4]

putative kaempferol derivative

sinapoylgentiobiosed km-3-O-sinapoyldiglucoside-7-Oglucosided is-O-diglucoside-sulfatee km-3-O-sinapoylsophorotrioside-7-Oglucosided (−)-epicatechin km-3-O-sinapoylsophoroside-7-O-glucoside

sinapoylhexosed km-3-O-caffeoylsophoroside-7-Oglucosided putative hydroxycinnamic acid derivative procyanidin B2 ([DP 2]) qn-3-O-sinapoylsophoroside-7-O-glucoside cis-sinapic acid sinapoylhexose

qn-3-O-diglucoside-7-O-glucosided km-3-O-triglucoside-7-O-glucosided km-O-diglucoside km-3-O-sophoroside-7-O-glucoside

qn-3-O-sophoroside-7-O-glucoside

tentative identificationa

Table 2. HPLC-PDA−ESI(−)-MSn/HRMS Data and Putative Identification of Flavonoids and Hydroxycinnamic Acid Derivatives in Seeds of B. napus L. var. napus

Journal of Agricultural and Food Chemistry Article


tR (min)

31.34 31.73 32.02

32.40 33.28 33.74

34.10

34.44

34.61 35.20

35.23 35.70

36.65 37.28 37.42 37.58

37.60 37.86 37.92 38.15 38.31 39.23

40.26

40.40 41.05

42.74 43.40 43.84 44.28

44.25 45.27

45.90

peak

36 37 38

39 40 41

42

43

44 45

46 47

48 49 50 51

52 53 54 55 56 57

2940

58

59 60

61 62 63 64

65 66

67

1213.3039

477.1047 1183.3156

447.0934 977.2574 947.2471 1213.3035

947.2469 925.1709

977.2585

223.0616 815.2046 679.1519 917.2144 865.2005 977.2586

557.0624 977.2578 577.1359 339.0720

609.1467 1007.2683

681.1662 977.2586

993.2521

609.1469

639.1570 1173.2715 1199.2885

719.1154 933.2311 1139.3083


Table 2. continued

C59H57O28

C22H21O12 C55H59O29

C21H19O11 C44H49O25 C43H47O24 C59H57O28

C43H47O24 C39H41O24S

C44H49O25

C11H11O5 C38H39O20 C30H31O18 C45H41O21 C45H37O18 C44H49O25

C22H21O15S C44H49O25 C30H25O12 C15H15O9

C27H29O16 C45H51O26

C30H33O18 C44H49O25

C44H49O26

C27H29O16

C28H31O17 C56H53O28 C58H55O28

C28H31O20S C42H45O24 C50H59O30

calcd formula [M − H]− 639 (100), 403 (27), 315 (12) 771 (100), 609 (10) 977 (100), 815 (53) 477 (45), 315 (100), 313 (46) 815 (100) 993 (11), 771 (27), 609 (49), 427 (100), 285 (12) 447 (9), 429 (100), 285 (91) 831 (100)

−2.6 −0.5 1.2 −0.5 1.2 0.5

−0.3

609 (100), 815 (36) 785 (100) 763 (100) 357 609 644 815

315 (43), 314 (100) 977 (15), 815 (100), 609 (21), 285 (7)

−1.7 −0.7 0.5 −0.3 −0.6 −0.8 0.6 −1.8 −0.7 0.2

208 (100), 179 (32), 164 (51) 623 (66), 609 (100), 591 (34) 631 (82), 479 (89), 223 (100) 639 (19), 577 (67), 407 (100), 223 (79) 695 (37), 587 (76), 407 (84), 289 (100) 815 (100), 653 (30)

−1.6 −0.7 −0.4 0.2 −2.3 −1.8

1080 (11), 609 (100), 285 (21)

(23), 285 (100). 284 (80) (100) (35), 609 (100), 285 (38) (21), 609 (100), 285 (29)

477 (100), 395 (7), 315 (18) 815 (100) 451 (35), 425 (100), 407 (41), 289 (19) 223 (100)

490 (7), 429 (21), 355 (10), 284 (100) 845 (100)

−1.0 −0.9 1.4 −0.9 −1.3 0.3

476 (100), 315 (29) 815 (100), 653 (28), 609 (4)

1.5 −1.8

−1.3


error (ppm)

285 (100), 243 (55) 609 (100), 429 (42), 285 (23) nd

609 (47), 591 (100) 601 (100), 557 (57), 315 (29) nt 429 (71), 285 (100) nd nd

369 (44), 339 (100). 327 (65), 309 (72) 669 (67), 625 (100), 463 (32), 301 (29) 315 (100) 653 (100), 447 (14), 285 (4) nt 683 (100), 639 (21), 477 (43), 315 (14) 315 (100) 653 (100) 407 (100) 208 (100), 179 (41), 164 (55) nt 429 (100), 284 (20) nt nt nt 653 (100), 447 (59), 285 (7) 429 (100), 285 (65)

300 (100) 609 (100) nd

315 (100), 211 (69) 609 (100) nd


nd

nd nd

nd nd nd nd

nd nd

nd

nd nd nd nd nd nd

nd 353 (68), 285 (100) nt nd

nd nd

nt nd

nd

nd

nt nd nd

nd 447 (100), 285 (46) nd


nd

265, 355 270, 330

265, 345 265, 340 nd 270, 330

nd 275, 330

265, 330

nd nd nd nd nd nd

265, 320 nd nd 240,330

nd 265, 330

nd 265, 330

265, 330

nd

256, 350 275, 330 nd

nd nd 270, 320

UV λmax (nm)

km-3-O-sinapoylsophoroside-7-Oglucosided km-3-O-feruloyldiglucoside-7-O-glucosidee is-3-O-sinapoylglucoside-sulfate-7-Oglucosidee km-3-O-glucoside km-3-O-sophoroside-7-O-sinapoylglucoside km-3-O-diglucoside-7-O-feruloylglucosidee km-3-Odisinapoylhydroxyferuloyldiglucosidee is-3-O-glucoside km-3-O-sinapoylsophoroside-7-Osinapoylglucosidee km-3-Odisinapoylhydroxyferuloyldiglucosidee

trans-sinapic acid km-3-O-sinapoylsophorosided putative hydroxycinnamic acid derivative putative hydroxycinnamic acid derivative [DP 3] km-3-O-sinapoyldiglucoside-7-O-glucosidee

is-O-glucoside-sulfate km-3-O-sinapoyldiglucoside-7-O-glucosidee [DP 2] sinapoylmalic acid

km-O-sophorosided is-3-O-sinapoyldiglucoside-7-O-glucoside

qn-3-O-sinapoylsophoroside-7-Oglucosided is-3-O-glucoside-7-O-acetylglucosidee km-3-O-sinapoyldiglucoside-7-O-glucosidee

is-O-diglucoside-sulfatee km-3-O-caffeoyldiglucoside-7-O-glucosidee km-3-O-sinapoylsophotrioside-7-Oglucosided is-3-O-diglucosided km-3-O-disinapoylgalloyldiglucosidee km-Odihydroxyferuloylsinapoyldiglucosidee km-7-O-sophorosided




2941

53.78

54.49

55.15

55.62 56.92 57.87

86

87

88

89 90 91

757.2359 959.2828 739.2108

591.1728

841.2780

1183.3155

815.2053

787.2461

959.2833

1419.3643

1419.3629

1237.2697

1205.3333

591.1725

1419.3633

949.2999 757.2353

815.2042 753.2259

845.2161

1183.3162

845.2157

753.2253

1345.3681


529 (100) 683 (100), 477 (13), 459 (10) 815 (100), 623 (37) 683 (100), 477 (21), 357 (7) 653 (100), 447 (11), 284 (4) 529 (100) 753 (39), 725 (100) 561 (100), 337 (49), 223 (19) 1213 (2), 1195 (2), 815 (100), 609 (16) 367 (100), 223 (91) 1130 (7), 941 (8), 835 (6), 613 (100), 427 (11), 223 (12) 1013 (23), 911 (34), 705 (100), 613 (67), 223 (19), 1051 (45), 889 (29), 815 (100), 653 (24), 591 (19) 1213 (25), 1051 (100), 815 (39), 609 (16), 591 (23) 735 (100), 529 (8), 511 (11) 739 (15), 591 (100), 367 (56), 223 (31) 623 (77), 609 (100), 591 (41) 815 (80), 609 (100) 635 (100), 617 (23), 371 (39) 531 (33), 367 (100), 205 (31) 595 (100), 223 (47) 735 (100) 515 (100), 353 (41), 223 (10)

−0.7 −1.3 −1.2 −1.8 −0.2 −1.6 −1.6 −0.5 −0.8 −0.9 −1.1

−0.7 −0.8 −1.6 −0.6 −1.0 −1.5 −0.5 −0.1 −1.5

C28H31O14

C40H69O41

C38H49O21

C28H31O14

C37H41O17 C45H51O23 C33H39O19

C55H59O29

C38H39O20

C38H43O18

C45H51O23

C70H68O32

C70H68O32

C60H53O29

C70H68O32

C44H53O23 C37H41O17

C38H39O20 C34H41O19

C39H41O21

C55H59O29

C39H41O21

C34H41O19

−1.5

−0.6

−1.5

977 (100), 815 (23)

−0.4

C61H69O34


error (ppm)

calcd formula [M − H]−

575 (40), 473 (82), 455 (100), 429 (62) 223 (45), 205 (100), 190 (41) 401 (100) 331 (71), 247 (100) 379 (87), 353 (100), 191(33)

429 (70), 308 (29), 285 (100) 429 (33), 285 (100)

528 (87), 511 (100), 385 (21), 223 (27) 223 (100)

609 (38), 591 (89), 285 (100), 751 (100), 591 (79)

nd

353 (100), 329 (57), 315 (63) 353 (100), 285 (29) 289 (61), 223 (21), 205 (100) 419 (100), 223 (11) 337 (100), 223 (22), 216 (40) 609 (100), 591 (16), 284 (14) 352 (40), 223 (37), 205 (100), 179 (4) nd

785 (17), 771 (100), 753 (21) 289 (64), 223 (76), 205 (100), 477 (38), 353 (100), 315 (30) 609 (100)


nd nd nd

nd

nd

nd

nd

nt

nd

nd

nd

nd

nd

nd

429 (100), 284 (43)

nd nd

nd nt

nt

nd

nd

nt

nd


nd 235, 330 235, 330

235, 330

nd

nd

235, 290, 325 265, 330

230, 330

nd

nd

nd

nd

245, 320

270, 330

nd 235, 330

265, 330 nd

270, 330

270, 330

nd

nd

275, 330

UV λmax (nm)

putative hydroxycinnamic acid derivative trisinapoylgentiobiosed sinapoylhydroxyferuloylgentiosided

1,6-disinapoylglucoside

km-3-O-sinapoylsophoroside-7-Osinapoylglucosidee putative hydroxycinnamic acid derivative

km-3-O-sinapoylsophorosided

putative hydroxycinnamic acid derivative

km-3-Otrisinapoylhydroxyferuloyldiglucosidee km-3-Odisinapoylhydroxyferuloylglucoside-7sinapoylglucosidee 1,2,2′-trisinapoylgentiobiose



km-3-Otrisinapoylhydroxyferuloylsophorosidee 1,2-disinapoylglucoside

putative hydroxycinnamic acid derivative putative hydroxycinnamic acid derivative

km-3-O-sinapoylglucoside-7-O-glucosidee disinapoylgentiobiosed

km-3-O-sinapoyldiglucoside-7-Osinapoylglucosidee is-3-O-sinapoylglucoside-7-O-glucosidee

is-3-O-sinapoylglucoside-7-O-glucosidee

km-3-O-sinapoyltriglucoside-7-Osinapoylglucosidee disinapoylgentiobiosed


a Abbreviations: km, kaempferol; qn, quercetin; is, isorhamnetin; DP, degree of polymerization of the epicatechin unit. bnt = no test was made. cnd = not detected. dChemicals identified for the first time in B. napus. eChemicals identified for the first in the genus Brassica.

53.32

85

50.63

79

53.11

50.60

78

84

49.75

77

51.67

49.54 49.64

75 76

83

48.29 49.45

73 74

51.50

48.01

72

82

47.6

71

50.94

46.82

70

81

46.71

69

50.66

46.00

68

80

tR (min)

peak

Table 2. continued




Article

Figure 3. MSn analysis of kaempferol-3-O-trisinapoylhydroxyferuloylsophoroside (peak 77), (km = kaempferol). (A) [M − H]−, (B) MS2 [M − H]−, (C) MS3 [(M − H) → (M − H − 2Sina − hydroxyfer)]−, (D) MS4 [(M − H) → (M − H − 2Sina − hydroxyfer) → (M − H − 3Sina − Hydroxyfer)]−.

of these compounds have previously been reported in B. napus and genus Brassica; these compounds include quercetin-3-Osophoroside-7-O-glucoside,11,21,28 kaempferol-O-diglucoside, 24 kaempferol-3-O-sophoroside-7-O-glucoside,11,21,28 kaempferol3-O-glucoside,11,16,20,29 and isorhamnetin-3-O-glucoside.11,22 Ten of the flavonols, including quercetin-3-O-diglucoside-7-Oglucoside,13,16,17,27 kaempferol-3-O-triglucoside-7-O-glucoside,12,13,16,17,27 kaempferol-3-O-glucoside-7-O-glucoside,8,11,16 isorhamnetin-3-O-glucoside-7-O-glucoside,8,11,28 kaempferol-3O-sophoroside,8,10,11,28 quercetin-3-O-sophoroside,8,10 kaempferol-3-O-diglucoside,13,14,16,29 isorhamnetin-3-O-diglucoside,16 kaempferol-7-O-sophoroside,8 and kaempferol-O-sophoroside,8 have previously been reported in the genus Brassica. For example, km-3-O-sophoroside (peak 31) presented MS 2 fragments at m/z 447 and 429 from the loss of a glucosyl (162 Da) and the partial loss of a sophorosyl (180 Da). Qn-3O-sophoroside (peak 32) presented MS2 fragments at m/z 463 and 445 from the loss of a glucosyl (162 Da) and the partial loss of a sophorosyl (180 Da). The parent ions of peaks 31 and 32 were respectively observed at m/z 285 and 300 from the complete loss of a glucosyl. Thus, peaks 31 and 32 may be concluded to contain sophorose. Consistent with the alkaline hydrolysis characteristics and HRMS data, we found that peaks 31 and 32 feature λmax at 265 and 345 nm, respectively, which indicate that peaks 31 and 32 are kaempferol-3-O-sophoroside and quercetin-3-O-sophoroside, respectively. Confirmation of Acylated Flavonol Glycosides. In Brassica, acyl groups on flavonol glycosides were mainly observed in p-coumaroyl, caffeoyl, feruloyl, hydroxyferuloyl, and sinapoyl. Acylation greatly affects the UV absorption of flavonol glycosides. For example, the λmax of flavonol glycosides (band I) normally shifts to 310−312 nm when they are combined with p-coumaroyl. However, the λmax of band I may also shift to 320−340 nm when the flavonol glycosides are

combined with caffeoyl, feruloyl, hydroxyferuloyl, and sinapoyl.14,16,25,28 Acylated flavonol glycosides, such as flavonol-3-O-acylglycoside-7-O-acylglycoside, produce a major MS2 ion peak from the loss of 7-acyl and glucosyl as well as a parent peak of flavonol from the loss of 3-acyl and glucosyl. p-coumaroyl, galloyl, caffeoyl, feruloyl, hydroxyferuloyl, and sinapoyl could be identified from losses of 146, 152, 162, 176, 192, and 206 Da, respectively. As such, deduction of unknown compounds according to the glycosyl products of flavonols after alkaline hydrolysis of acylated flavonol glycosides, accompanied by their UV absorption characteristics, MSn fragments, and HRMS analysis results, is possible. For instance, Olsson et al.21 characterized kaempferol-3-O-sinapoylsophoroside-7-O-glucoside (peak 18) with λmax at 265 and 330 nm, which are considered typical UV absorption peaks of acylated flavonol glycosides. This compound presents a major MS2 fragment at m/z 815 from the loss of 162 Da and three MS3 fragments at m/z 609, 591, and 285 from the loss of 206 Da (sinapoyl), 224 Da (sinapic acid), and all acyl and glycosyl groups, respectively. The MS3 fragment at m/z 609 can be further fragmented into an ion peak at m/z 429, which indicates the presence of sophorosyl. In this study, we identified 35 acylated flavonol glycosides, including 29 kaempferol derivatives, 3 quercetin derivatives, and 4 isorhamnetin derivatives (Table 2). These flavonols were also clarified in terms of different numbers of glucosyls. Of these 35 compounds, 31 were found for the first time in rapeseed and 21 were discovered for the first time in the genus Brassica. Seven derivatives of kaempferol sinapoyltriglucosides were distributed as peaks 14, 18, 45, 49, 57, 58, and 62. Peaks 14, 45, 49, and 57 were identified as isomers of kaempferol-3-Osinapoyldiglucoside-7-O-glucoside, and peaks 18 and 58 were 2942



Article

side,8,11 kaempferol-3-O-sinapoylsophoroside-7-O-glucoside,12 and kaempferol-3-O-sinapoylsophoroside.8 Confirmation of Sulfated Flavonol Glycosides. In the present study, we identified four sulfated isorhamnetin compounds. Peak 48 was previously reported by Auger et al.,22 whereas peaks 15, 36, and 60 have never been reported in the genus Brassica. Peak 36 was identified as an isorhamnetinO-diglucoside-sulfate, which presents an MS2 fragment at m/z 639 from the loss of a SO32− (80 Da) and an MS3 parent fragment at m/z 315. Peaks 15 and 60 were confirmed as isorhamnetin-O-diglucoside-sulfate and isorhamnetin-3-O-sinapoylglucoside-sulfate-7-O-glucoside, respectively. Among the four isorhamnetins found, three were reported for the first time in the genus Brassica; only isorhamnetin-O-glucoside-sulfate has been previously reported.22 Confirmation of Flavanol and its Oligomers. Flavanols show λmax at 202 and 280 nm. The molecular mass of flavanol polymers can be determined as follows: 290 + 288(n − 1), where n stands for the number of polymers. A previous report found that fragments of 290 or 288 Da are always lost during the fragmentation of these polymers.32 Using (−)-epicatechin and procyanidin B2 as standards, we identified six (−)-epicatechins and their oligomers [DP 2], [DP 3], and [DP 4] [DP = degree of polymerization of the (−)-epicatechin unit] on the basis of their UV absorption characteristics, MSn fragments, and HRMS analysis results. Identification of these compounds was performed in accordance with previous research.22,23 We observed that flavanols and their oligomers are generally found in F4 fractions obtained by column chromatography and that isomers of flavanol oligomers in F4 are obviously more abundant than those in crude extracts. These results indicate the possibility of polymer degradation during the experiment and that flavanol polymers are abundant in seeds of B. napus. Confirmation of Hydroxycinnamic Acid Derivatives. Hydroxycinnamic acid derivatives in Brassica principally include p-coumalic acid, caffeic acid, ferulic acid, hydroxyferulic acid, and sinapic acid, which could be combined with glucose, gentiobiose, quinic acid, malic acid, and malonic acid to form hydroxycinnamic acid derivatives.19,23,33 The λmax of these derivatives ranged from 230 to 250 nm and from 320 to 330 nm.14 Hydroxycinnamoyl or hydroxycinnamic acid may be lost during MSn analysis, and the presence of gentiobiose can be deduced from the loss of 306 Da. Peak 78 was defined as 1,2disinapoylglucoside, which has been previously reported.30 1,2Disinapoylglucoside shows an MS2 ion fragment at m/z 367 from the loss of 224 Da and MS3 fragments at m/z 352, 223, and 179 from losses of 15 Da (a methyl on sinapoyl), 144 Da (a glucosyl without H2O), and 188 Da (a sinapoyl without H2O), respectively. Eighteen phenypropanoid derivatives were determined according to their retention times, UV absorption characteristics, MS2 and MS3 fragments, and HRMS molecular ions [M − H]−. Most of these compounds were derivatives of sinapic acid in conjunction with monosaccharides or disaccharides. While all of the hydroxycinnamic acid derivatives obtained have been previously reported in the genus Brassica, 11 of them were discovered for the first time in B. napus (Table 2). Four isomers of sinapoylhexose (peaks 6, 12, 23, and 29) were further identified; of these, peaks 12, 23, and 29 were reported for the first time in B. napus. Three isomers of sinapoylgentiobiose (peaks 24, 28, and 34) and three isomers of disinapoylgentio-

identified as isomers of kaempferol-3-O-sinapoylsophoroside-7O-glucoside on the basis of ion peaks (loss of 180 Da) in the MS3 spectra. Peak 62 was detected with an ion peak at m/z 609 from losses of (206 Da) and glucosyl (162 Da). We thus hypothesize that sinapoyl is ligated to 7-glucosyl. Peaks 18, 58, and 62 have been previously reported in rapeseed.21,30 Peak 14 was reported for the first time in B. napus, and peaks 45, 49, and 57 were discovered for the first time in the genus Brassica. Ion fragments of peaks 3, 7, and 37 obtained from lowresolution MS in negative mode were found at m/z 933. However, the λmax (band 1) of peak 7 observed at 330 nm was obviously smaller than that of peak 3; this result indicates that peak 7 is an acylated flavonol glycoside. Whereas peak 3 was observed even after alkaline hydrolysis, peak 7 disappeared. HRMS analysis demonstrated that peak 7 (C42H45O24) possesses three more carbon atoms than peak 3 (C39H49O26). An MS3 ion fragment from the loss 180 Da was observed after further analysis of peak 7, which indicates that the peak is kaempferol-3-O-caffeoylsophoroside-7-O-glucoside. We also confirmed that peak 37 is kaempferol-3-O-caffeoyldiglucoside7-O-glucoside. Peaks 66, 71, and 86 showed the same molecular mass, and peaks 66 and 71 were further ascertained as acylated flavonol glycosides according to their UV absorption characteristics. The major MS2 fragment of peaks 66 and 71 was found at m/z 815 (from the loss of 368 Da), while the major MS3 fragment of peak 86 was observed at m/z 609 (from the loss of 206 Da). The parent ions of these compounds were about 285 Da, arising from the complete loss of acyl and glucosyl. The m/z 609 ions of peaks 66 and 86 could be fragmented into other ions at m/z 429 from the loss of 180 Da. Thus, we can identify peaks 66, 71, and 86 as kaempferol-3-O-sinapoylsophoroside-7O-sinapoylglucoside, kaempferol-3-O-sinapoyldiglucoside-7-Osinapoylglucoside, and kaempferol-3-O-sinapoylsophoroside-7O-sinapoylglucoside, respectively. Figure 3 depicts the MSn spectra of peak 77, which was identified as kaempferol-3-O-trisinapoylhydroxyferuloylsophoroside. The spectra of peak 77 show λmax at 270 and 330 nm, which implies that peak 77 is an acylated flavonol glycoside. Further MSn analysis proved the presence of sinapoyl and hydroxyferuloyl in this compound. Two isomers of kaempferol-3-O-trisinapoylhydroxyferuloylsophoroside at peaks 81 and 82 were successfully identified. Peaks 64 and 67 were similarly identified as isomers of kaempferol-3-Odisinapoylhydroxyferuloyldiglucoside. Peaks 40 and 44 were identified as kaempferol-3-O-disinapoylgalloyldiglucoside and 3-O-glucoside-7-O-acetylglucoside, respectively. Multiple acylated flavonol glycosides have never been reported in rapeseed, and 21 of these compounds were discovered for the first time in the genus Brassica (Table 2). Several compounds found in our work have been previously reported in the genus Brassica; these compounds include quercetin-3-O-sinapoylsophoroside-7-Oglucoside,21 kaempferol-3-O-sinapoylsophoroside-7-O-glucoside,21,31 isorhamnetin-3-O-sinapoyldiglucoside-7-O-glucoside,22 kaempferol-3-O-sophoroside-7-O-sinapoylglucoside,30 kaempferol-3-O-caffeoylsophoroside-7-O-glucoside,8,11 kaempferol-3-O-sinapoyldiglucoside-7-O-glucoside,11,16,17,29 kaempferol-3-O-sinapoylsophorotrioside-7-O-glucoside,8 kaempferol3-O-feruloylsophoroside-7-O-glucoside,8,28 kaempferol-3-O-pcoumaroylsophoroside-7-O-glucoside,8,28 kaempferol-3-O-sinapoylsophotrioside-7-O-glucoside,8,10,12 quercetin-3-O-sinapoylsophoroside-7-O-glucoside,11 kaempferol-3-O-sinapoylsophoro2943



Article

biose (peaks 34, 69, and 74) were reported for the first time in B. napus. A trisinapoylgentiobiose (peak 90) and a sinapoylferuloylgentiobiose (peak 91) were also discovered in this study. Among the hydroxycinnamic acid derivatives observed, seven have been previously reported in both B. napus and genus Brassica; these compounds include cis-sinapic acid,33 sinapoylhexose,19,24 sinapoylmalic acid,30 trans-sinapic acid,33 1,2disinapoylglucoside,30 1,2,2′-trisinapoylgentiobiose,14,16,30 and 1,6-disinapoylglucoside.8,12,30 Another 11 of these acid derivatives, including sinapoylhexose,12 sinapoylgentiobiose,8 sinapoylhexose, 8 sinapoylgentiobiose, 8,12 sinapoylgentiobiose,8,12 sinapoylhexose,17 disinapoylgentiobiose,8,12,14 disinapoylgentiobiose,8,12 disinapoylgentiobiose,12 trisinapoylgentiobiose,12 and sinapoylhydroxyferuloylgentioside,8 have also been previously reported in the genus Brassica Unknown Flavonoids and Hydroxycinnamic Acid Derivatives. Although multiple phenolic compounds were successfully identified in this research, many chemical compounds remain unknown. These compounds may include specific hydroxycinnamic acid derivatives and flavonoids and always present in conjunction with several unknown chemical groups. For instance, peak 19 was ascertained as a kaempferol combined with three hexoses, a sinapine, and an unknown 68 Da group. Twelve unknown hydroxycinnamic acid derivatives (peaks 8, 20, 27, 54, 55, 75, 79, 80, 84, 87, and 89) produced MSn fragments of sinapoyl, feruloyl, and gentiobiose. Peaks 20, 75, 76, and 84 exhibited an unknown compound (196 Da) during MSn analysis. Further confirmation of these unknown compounds must be carried out by purification of single components and subsequent application of infrared spectroscopy and/or nuclear magnetic resonance spectroscopy. The UV absorption characteristics and MSn fragments of the target compounds were principally investigated to identify new components by HPLC analysis. HPLC analysis of the crude extracts of flavonoids and hydroxycinnamic acid derivatives in rapeseed is limited by the fact that many of the compounds are simultaneously washed away during the determination, thereby leading to UV spectrum overlaps and identification difficulties.23 As well, minute amounts of certain compounds may be neglected because of their weak signals in the UV spectra. Sephadex LH-20 column chromatography was performed to separate the crude methanol extract of rapeseed into different fractions that could be further analyzed by HPLC-PDA− ESI(−)-MSn (LCQ) and HPLC-DAD−ESI(−)-HRMS (QTOF), thereby allowing comprehensive and detailed characterization of the crude extract. Although some of the compounds may be dehydrated during subsequent concentration, no adverse effects on their identification were noted. Alkaline hydrolysis transformed acylated flavonol glycosides into glycosylated chemicals of the parent ion, thereby promoting the identification of acylated compounds. Compounds released after alkaline hydrolysis also directly demonstrate the type of phenypropanoid available.14,16,34 The development of HPLC-PDA−ESI(−)-MSn/HRMS greatly facilitates the identification of unknown compounds according to their molecular mass; this analytical technique further allows calculation and deduction of the element composition of relevant chemical compounds. Several flavonol derivatives were identified in this research, 70% of which were confirmed as kaempferol derivatives, 20% of which were isorhamnetin derivatives, and 10% of which were quercetin derivatives. In a previous study, the kaempferol content was found to be higher than those of quercetin and isorhamnetin.23

Epicatechin, a compound related to the flavonoid biosynthetic pathway, varies obviously in content in differently colored rapeseed.23 A comprehensive dissection of flavonoid biosynthesis patterns indicates that kaempferol formation is related to epicatechin accumulation.35,36 In the present study, we found that sinapic acid, the main derivative of phenypropanoids in rapeseed, greatly increases after alkaline hydrolysis. This result suggests that sinapic acid is the main antinutritional factor of rapeseed. In conclusion, we detected 91 flavonoids and hydroxycinnamic acid derivatives in B. napus L. var. napus seeds through HPLC-PDA−ESI(−)-MSn/HRMS (IonTrap and Q-TOF) analysis. Seventy-eight of these compounds were identified tentatively; 55 of the compounds were reported for the first time in B. napus, and 24 were reported for the first time in the genus Brassica. Comprehensive interpretation of flavonoids and hydroxycinnamic acid derivatives in rapeseed is fundamental to studies on the antinutrition properties of rapeseed. The results of this study improve the scientific knowledge of the phenolic composition of rapeseed and provide a reliable guide for selecting rapeseed for breeding with better seed quality. We also successfully prove that HPLC-PDA−ESI(−)-MSn/HRMS is a highly efficient and elaborate technique for plant phenolic analysis and chemical identification.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 514 87997303. E-mail: [email protected]. Author Contributions §

Y.S. and J.J. contributed equally to this work.

Funding

This study was supported by NSFC projects (Grants 31330057 and 31171581), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and an SRFDP project (Grant 20123250110009). Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED HPLC, high-performance liquid chromatography; HRMS, highresolution mass spectrometry; PDA, photodiodide array; ESI, electrospray ion source

■

REFERENCES

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Analysis of Flavonoids and Hydroxycinnamic Acid ... - ACS Publications

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