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Flavonoid analysis of buckwheat sprouts
Food Chemistry 170 (2015) 97–101
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Analytical Methods
Flavonoid analysis of buckwheat sprouts Tae-Gyu Nam a,c, Sun Mi Lee b, Ji-Hae Park c, Dae-Ok Kim a, Nam-in Baek c, Seok Hyun Eom b,⇑ a
Department of Food Science and Technology, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea Department of Horticultural Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea c Graduate School of Biotechnology and Research Institute of Life Sciences & Resources, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 3 September 2013 Received in revised form 31 May 2014 Accepted 2 August 2014 Available online 23 August 2014 Keywords: Common buckwheat Quercetin-3-O-robinobioside Tartary buckwheat Vegetable sprout
a b s t r a c t It is known that common buckwheat sprouts contain several flavonoids, including orientin, isoorientin, vitexin, isovitexin, rutin, and quercetrin, whereas tartary buckwheat sprouts contain only rutin. In this study, we evaluated flavonoids present in buckwheat sprouts and identified a previously unreported flavonoid. Simultaneous detection by HPLC was used to separate rutin and a compound that was not separated in previous studies. We used a novel HPLC elution gradient method to successfully separate rutin and the previously unidentified compound, for which we performed structural analysis. The identification of six flavonoids by HPLC was confirmed using HPLC–ESI–MS/MS analysis. The newly identified compound, [M + H]+ = 611.17, was identified by NMR as the rutin epimer quercetin-3-O-robinobioside. Unlike common buckwheat sprout, tartary buckwheat sprout contained rutin as a main flavonoid, whereas other flavonoids appeared only in trace amounts or were not detected. Quercetin-3-O-robinobioside was not detected in tartary buckwheat sprout. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Buckwheat (Fagopyrum spp.), a member of the Polygonaceae family, is consumed all around the world. While it is technically a fruit seed, it is classified as a cereal grain because its utilisation is similar to that of other cereal grains (Kim, Kim, & Park, 2004). Common buckwheat (Fagopyrum esculentum Möench) and tartary buckwheat (Fagopyrum tataricum Gaertner) are the main species of buckwheat consumed by humans. Buckwheat has been established as a nutritional food because of its abundant levels of amino acids and protein (Bonafaccia, Marocchini, & Kreft, 2003). Moreover, common buckwheat is a major dietary source of rutin and O-glycosyl flavonols including both quercetin and rutinose moieties (Kitabayashi, Ujihara, Hirose, & Minami, 1995). The nutritional values of the edible parts of sprouts of bean sprouts, cereal crops, and vegetables have gained interest in recent years. Sprouts are recognised as outstanding dietary vegetables in Asia, Europe, and the United States, and are an important source of protein, mineral, dietary fiber, and vitamins in human diets. Especially, the contents of polyphenols, which are secondary plant metabolites, in particular, increase or are newly synthesised during sprouting, similar to peanut and broccoli (Guo, Yuan, & Wang, 2011; Wang et al., 2005). Polyphenols have attracted a great deal
⇑ Corresponding author. Tel.: +82 31 201 3860; fax: +82 31 204 8116. E-mail address: [email protected] (S.H. Eom). http://dx.doi.org/10.1016/j.foodchem.2014.08.067 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
of attention due to their various benefits for human health. For example, the total phenolic content and antioxidant capacity of mungbean sprouts is greater than that of seeds (Kim, Jeong, Gorinstein, & Chon, 2012). Likewise, the flavone and flavonol glycoside contents of buckwheat increase during sprouting (Alvarez-Jubete, Wijngaard, Arendt, & Gallagher, 2010). Buckwheat sprouts are one of the most popular forms of sprouts and are consumed as both a salad vegetable and fresh vegetable served with noodles in northeast Asia (Kim et al., 2004). Phytochemical analyses of buckwheat sprouts have been performed intensively over the last decade, and the nutritional components of seeds have been evaluated for significantly longer. Compared with buckwheat seeds, the sprouts contain relatively large amounts of rutin, which is known as a beneficial compound for health (Kim et al., 2004). Similarly, buckwheat sprouts have a significantly greater abundance of other flavonoids including orientin, isoorientin, vitexin, isovitexin, rutin, and quercetrin compared with buckwheat seeds (Kim et al., 2004, 2008; Lim, Park, Kim, Jeong, & Kim, 2012; Liu, Chen, Yang, & Chiang, 2008; Watanabe, 2007). However, the analytical high performance liquid chromatography (HPLC) techniques employed in previous studies were unable to separate and quantify individual flavonoids to a satisfactory degree. In addition, the HPLC chromatograms of numerous studies are ambiguous as to whether they represent single or multiple peaks. Reversed-phase HPLC is widely employed for quantitative and qualitative analysis of common buckwheat sprouts, and numerous
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studies have used this approach for identification of flavonoids. Several studies have reported that common buckwheat sprouts contain C-glycosyl flavones (orientin, isoorientin, vitexin and isovitexin) and rutin (Kim et al., 2008; Lim et al., 2012; Liu et al., 2008; Watanabe, 2007). In this study, we used a gradient elution, based on water-acetonitrile, to achieve optimum separation for the identification of flavonoid derivatives in common buckwheat sprouts. In order to identify a newly detected compound, we used NMR, HPLC, and Q-TOF MS to isolate and identify the quercetin derivative, O-glycosyl flavonol, in addition to the other flavonoids and their derivatives. 2. Materials and methods 2.1. Seeds Common (Cultivar, Suwon 2 Ho) and tartary (Cultivar, Dae-Guan 3-3 Ho) buckwheat seeds were purchased from a seed company (Budnara) in Gwangju, Republic of Korea in 2012. 2.2. Chemicals Orientin (P95%), isoorientin (P95%), vitexin (P95%), isovitexin (P98%), and rutin (P94%) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). All solvents used were of analytical or HPLC grade. 2.3. Sprout growth condition Buckwheat seeds were placed in a 4 °C room before initiation of experiments. After soaking in distilled water for 4 h, water saturated-seeds were placed in a dark growth chamber for 48 h to facilitate germination. Germinated seeds were planted in a light supplemented growth chamber (35 lmol m2 s1 light intensity by fluorescent light lamps) at 25 °C for 5 days. During sprout growth, water was supplied by spraying as needed. Finally, the sprouts were harvested and dried in a 30 °C dry oven equipped with ventilation for 3 days.
equipped with an auto-sampler (SIL-20A, Shimadzu), photodiode array detector (SPD-20A, Shimadzu), binary pump (LC-20AD, Shimadzu), and vacuum degasser. Chromatographic separation was performed using a ProntoSIL 120-5-C18-ace-EPS column (4.6 250 mm, 5.0 lm; Bischoff, Leonberg, Germany). For HPLC analysis, we used two different linear solvent gradients with a binary mobile phase consisting 0.1% HCOOH in water (solvent A) and 0.1% HCOOH in acetonitrile (solvent B). Elution gradient I consisted of 92% A and 8% B at 0 min, 85% A and 15% B at 4 min, 55% A and 45% B at 90 min, 5% A and 95% B at 100 min, 92% A and 8% B at 102 min, and 92% A and 8% at 105 min. Elution gradient II consisted of 92% A and 8% B at 0 min, 85% A and 15% B at 4 min, 84% A and 16% B at 8 min, 84% A and 16% B at 20 min, 83% A and 17% B at 45 min, 82% A and 18% at 75 min, 81% A and 19% at 80 min, 80% A and 20% B at 95 min, 79% A and 21% B at 102 min, 30% A and 70% B at 107 min, 92% A and 8% B at 110 min and 92% A and 8% B at 115 min. The flow rate was at 0.8 mL/min with a 20 lL injection volume. The column oven temperature was set to 25 °C, and the absorbance was monitored at 350 nm (Li et al., 2007). The extracts were analysed at least three times by HPLC. 2.6. HPLC-DAD–ESI/Q-TOF MS analysis of flavonoids in common buckwheat sprouts The molecular weights of major flavonoids in buckwheat sprouts were determined using an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight MS (Q-TOF LC/MS) with an Agilent 1200 Series Rapid Resolution LC system. The MS was operated with electrospray ionisation source in either negative or positive ion mode. A ProntoSIL 120-5-C18-ace-EPS column (4.6 250 mm, 5.0 lm; Bischoff, Leonberg, Germany) was used to separate individual flavonoids using gradient elution II at an oven temperature of 40 °C. Flavonoids were monitored at 350 nm. The MS source parameters were as follows: 350 °C drying gas (N2) temperature, 12 L/min drying gas flow, 45 psi nebulizer pressure, 200 V fragmentation voltage, and 4000 V capillary voltage. MS and MS/MS ranges were set from m/z 50 to 1700 and m/z 50 to 1100, respectively. Collision energy was set at 10 eV for positive ion mode and 40 eV for negative ion mode. 2.7. Prep-LC and TLC analysis for flavonoid separation
2.4. Extraction and fractionation of flavonoids from buckwheat sprouts Fifteen grams of dried buckwheat sprouts were processed with a commercial grinder and dispersed in 150 mL of 70% aqueous ethanol. After extraction (48 h) at room temperature, the solution was filtered through Whatman #2 filter paper (Whatman International Limited, Kent, UK). The solid filter cake was extracted again in 70% aqueous ethanol and filtered. The extraction procedure was repeated once more. Next, solvent was evaporated using a rotary evaporator at 40 °C, and 840 mg of crude extract was suspended in 40 mL of distilled water and combined with 80 mL of EtOAc/nBuOH (3:1). After shaking to mix solvents, the mixture was allowed to separate and split again between water and EtOAc/n-BuOH. A total of 250 mg of EtOAc/n-BuOH fractionated extract was gathered after solvent evaporation. The extract (250 mg) was suspended in 20 mL of water and combined with 40 mL of EtOAc. After repeating the separation described above, the aqueous fraction was collected and dried to obtain a 70 mg flavonoids-rich extract, which was kept at 4 °C until analysed. 2.5. Isolation of flavonoids in buckwheat sprouts by reversed-phase HPLC Flavonoids of buckwheat sprouts were analysed using a reversed-phase HPLC system (Shimadzu model, Kyoto, Japan)
Flavonoids from the crude extract of common buckwheat sprouts were separated and confirmed using a preparative highperformance liquid chromatography system (Prep-LC) consisting of a Waters 2555 Prep-LC controller, a 2998 Photodiode array detector, and an Empower workstation (Waters, Milford, MA, USA) with a YMC ODS column (20 250 mm, 5 lm). The binary solvent system consisted of (A) water and (B) acetonitrile, with a linear gradient of 5% (B) to 50% (A) for 10 min, isocratic for 10 min, from 50% (B) to 100% (A) for 10 min, isocratic for 10 min, and finally from 100% (B) to 5% (A) for 5 min. The flow rate was 30 mL/min and 70 mg/mL of flavonoid-rich extract was injected with 1 mL injection volume. The column oven temperature was set to 25 °C. In addition, we performed thin-layer chromatography (TLC) analysis to confirm the purity of isolated fractions. TLC analysis was carried out using Kiesel gel 60 F254 and RP-18 F254S resin (Merck, Dramstadt, Germany). TLC spots were detected using a Spectroline Model ENF-240 C/F UV lamp (Spectronics Corporation, Westbury, NY, USA) and a 10% H2SO4 solution. 2.8. NMR spectroscopy The structure of a previously unidentified flavonoid was analysed by NMR. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were recorded on a Varian Unity Inova AS-400 FT-NMR
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spectrometer (Palo Alto, CA, USA). Negative fast atom bombardment mass spectrometry (FAB-MS) was conducted using a JEOL JMSAX-700 instrument (Tokyo, Japan) described by Kim et al. (1996). 3. Results and discussion 3.1. Optimisation of gradient elution Two gradient elution systems were utilised to separate flavonoids in the buckwheat sprout extracts. Chromatograms of flavonoid derivatives are presented in Fig. 1. The pattern of flavonoids of common buckwheat sprouts eluted using gradient I were similar to previous studies (Kim et al., 2008; Lim et al., 2012; Liu et al., 2008; Watanabe, 2007), in that we did not separate successfully the individual flavonoids in peaks 5 and 6. However, peaks 5 and 6 were separated successfully using elution gradient II, where the acetonitrile concentration was increased more slowly, achieving to 21% over a period of 102 min, compared with gradient I. While common buckwheat sprouts exhibited six distinct flavonoid peaks, in tartary buckwheat sprout rutin (peak 6) was the only major component of the eluted by HPLC using gradient II (Fig. 1). Other anticipated flavonoids were present in only trace amounts or not at all. The rutin content of tartary buckwheat sprouts was approximately 4–5-fold higher than in common buckwheat sprout (Kim et al., 2008). 3.2. Q-TOF LC/MS analysis of flavonoids It is known that common buckwheat sprouts contain several flavonoids, including orientin, isoorientin, vitexin, isovitexin, rutin, and quercetrin (Kim et al., 2008; Lim et al., 2012; Liu et al., 2008; Watanabe, 2007). Among the flavonoids, quercetrin was detected late on HPLC chromatogram, whereas other flavonoids were detected within two hours of retention time. With the exception of quercetrin, chromatographic peaks of five other flavonoids and an unidentified compound shown in Fig. 1 were determined by MS/MS analysis. Q-TOF LC/MS analysis based on full scan data and MS/MS fragment patterns are shown in Table 1. Peak 1 produced an m/z 447.1004 of [M H] on MS, with MS/ MS fragments of m/z 357.0666 of [M H 90] and 327.0565 of [M H 120]. The predominant fragment patterns with product ion losses of 90 and 120 U were consistent with a C-glycosyl flavone. Peak 1 was tentatively identified as orientin (luteolin-8C-glucoside) based on the UV spectra, retention time and MS/MS ion products. In negative mode, peak 2 exhibited an m/z of 447.1015 (loss of a proton) and its fragment ions were m/z 357.0666 (loss of 90 U) and 327.0564 (loss of 120 U). Peaks 1 (orientin) and 2 possessed similar molecular masses and MS/MS spectrums, indicating that peak 2 was an isomer of orientin. Thus, peak 2 was tentatively identified as isoorientin (luteolin-6-C-glucoside). The components of peaks 1 and 2 were subsequently confirmed by Q-TOF LC/MS in comparison with the ion spectra from authentic flavone standards. Peak 3 exhibited an m/z of 431.1068 and its fragment ions were m/z 341.0711 (loss of 90 U), 311.0615 (loss of 120 U), and 283.0662 (loss of 148 U). Peak 4 also had an m/z of 431.1081 with fragment ions of m/z 341.0726, 311.0621 and 283.0666, indicating similar masses compared to peak 3. Peaks 3 and 4 were further analysed by comparison with the retention time, UV spectra, and MS/MS fragment patterns of authentic standards, which confirmed vitexin (apigenin-8-C-glucoside) and isovitexin (apigenin-6-C-glucoside), respectively. The masses of peaks 5 and 6 were m/z 609.1572 and 609.1591 in negative ion mode, and each produced a single fragment of m/z 301.0398 and 301.0397 in MS/MS analysis, respectively. For identification and
Fig. 1. Reversed-phase HPLC chromatograms of flavonoids in buckwheat sprouts using elution gradient I (A) and gradient II (B). Elution gradient II was used to separate compounds 5 and 6. Elution gradient II was applied to separate flavonoids in tartary buckwheat (C).
comparison of sugar moieties in peak 5 and 6, MS/MS analysis in positive ion mode was performed at a lower collision energy. Peak 5 presented with an m/z of 611.1736, which produced MS/MS fragment ions with m/z values of 465.0995 (loss of a methyl pentose group) and 303.0480 (loss of a hexose group). The mass and fragment ions of peak 6 were highly similar to those of peak 5, which had already been identified as rutin, based on comparison with an authentic standard. Because the mass and MS/MS patterns of peaks 5 and 6 were both similar rutin, peak 5 was considered to be an isomer of rutin. The results of orientin, isoorientin, vitexin, isovitexin, and rutin analysed in this experiment correspond with earlier results reported by several studies (Lim et al., 2012; Verardo et al., 2010; Watanabe, 2007). In our LC–MS/MS analysis, a compound
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Table 1 HPLC–ESI–MS and MS2 for identification of flavonoids in common buckwheat sprouts. Compound No. 1 2 3 4 5 6 5 6
Selected ion –
[M H] [M H]– [M H]– [M H]– [M H]– [M H]– [M + H]+ [M + H]+
Molecular ion (m/z)
MS/MS (m/z)
Tentative identification
Molecular formula
447.1004 447.1015 431.1068 431.1081 609.1572 609.1591 611.1736 611.1610
357.0666, 357.0666, 341.0711, 341.0726, 301.0398 301.0397 465.0995, 465.1023,
Orientin Isoorientin Vitexin Isovitexin Quercetin-3-O-robinobioside Rutin Quercetin-3-O-robinobioside Rutin
C21H20O11 C21H20O11 C21H20O10 C21H20O10 C27H30O16 C27H30O16 C27H30O16 C27H30O16
showed similar characteristic of rutin and had not been reported previously in buckwheat. Thus, further studies for identifying the compound were necessary. 3.3. Identification of the chemical structure in peak 5 To determine the chemical structure of peak 5 (compound 5), the flavonoid-rich fraction of the common buckwheat sprout extract was further fractionated by preparative HPLC. The eluted fractions were monitored by TLC. Finally, compound 5 [6 mg, Ve/Vt 0.76–0.89, RT 24 min, TLC (ODS F254S) Rf 0.50, MeOHH2O = 3:2] was separated. The chemical structure of compound 5 was determined based on several analyses, including 1H-NMR, 13C-NMR, DEPT, gradient correlation spectroscopy, gradient heteronuclear single-quantum coherence, gradient heteronuclear multiple-bond connectivity and fast atom bombardment mass spectrometry. Compound 5 was a yellow powder (MeOH), negative FAB-MS m/z 609 [M H]; 1 H-NMR (400 MHz, methanol-d4, dH) 7.86 (1H, d, J = 2.0 Hz, H-20 ), 7.60 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 6.86 (1H, d, J = 8.4 Hz, H-50 ), 6.38 (1H, br s, H-8), 6.20 (1H, br s, H-6), 5.05 (1H, d, J = 7.6 Hz, H-100 ), 4.51 (1H, br s, H-1000 ), 3.82 (1H, m, H-200 ), 3.80 (1H, m, H400 ), 3.74 (1H, m, H-600 b), 3.65 (1H, m, H-500 ), 3.58 (1H, m, H-2000 ), 3.55 (1H, m, H-300 ), 3.52 (1H, m, H-5000 ), 3.48 (1H, m, H-3000 ), 3.40 (1H, m, H-600 a), 3.28 (1H, m, H-4000 ), 1.18 (3H, d, J = 6.4 Hz, H-6000 ; 13 C-NMR (100 MHz, methanol-d4, dC) 179.4 (C-4), 166.4 (C-7), 163.0 (C-5), 159.3 (C-9), 158.6 (C-2), 149.8 (C-40 ), 145.9 (C-30 ), 135.6 (C-3), 123.5 (C-60 ), 123.1 (C-10 ), 117.7 (C-20 ), 116.1 (C-50 ), 105.5 (C-10), 105.8 (C-100 ), 102.4 (C-1000 ), 100.1 (C-6), 94.9 (C-8), 75.3 (C-500 ), 75.2 (C-300 ), 73.6 (C-200 ), 73.9 (C-4000 ), 72.2 (C-3000 ), 72.1 (C-2000 ), 69.7 (C-400 ), 69.7 (C-5000 ), 67.3 (C-600 ), 17.9 (C-6000 ). On the basis of spectroscopic data and chemical evidence, compound 5 was identified as quercetin-3-O-robinobioside (quercetin 3-O-(6O-a-L-rhamnopyranosyl)-b-D-galactopyranoside) (Fig. 2). These findings were confirmed by comparison with the physical and spectroscopic data reported in a literature, including the structural analysis of quercetin-3-O-robinobioside isolated from black
Fig. 2. Chemical structures of quercetin-3-O-robinobioside and rutin.
327.0565 327.0564 311.0615, 283.0662 311.0621, 283.0666
303.0480 303.0504
chokeberries (Slimestad, Torskangerpoll, Nateland, Johannessen, & Gisked, 2005). The compound is structurally similar with rutin. Rutin consists of quercetin with glucose-rhamose, while quercetin-3-O-robinobioside consists of quercetin with galactoserhamose. Although these two compounds are structurally similar, rutin has popularly known as a compound benefiting health, while the health benefits and biological activities of quercetin-3-O-robinobioside have not reported. The lack of investigation of quercetin-3-O-robinobioside with respect to health may result from difficulty in separating relative small amount in biomaterials. 4. Conclusions In this study, we analysed common buckwheat sprout, and initially identified rutin and four C-glycosyl flavones (orientin, isoorientin, vitexin, and isovitexin). Further analysis of the HPLC peak area of rutin allowed us to identify the previously undetected quercetin-3-O-robinobioside. The newly identified compound had the same molecular weight as rutin and was barely separated from rutin in the HPLC peaks reported in previous studies. Chromatographic resolution of eluted peaks varied according to the ratio of water and acetonitrile in the elution gradient. Using an optimised gradient condition, quercetin-3-O-robinobioside that was not identified previously was successfully eluted slightly ahead of rutin. Quercetin-3-O-robinobioside was not detected in tartary buckwheat sprouts. Our result may provide useful information for understanding flavonoid metabolism and synthesis during buckwheat sprout growth. Acknowledgements This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HI10C2162). References Alvarez-Jubete, L., Wijngaard, H., Arendt, E. K., & Gallagher, E. (2010). Polyphenol composition and in vitro antioxidant activity of amaranth, quinoa buckwheat and wheat as affected by sprouting and baking. Food Chemistry, 119, 770–778. Bonafaccia, G., Marocchini, M., & Kreft, I. (2003). Composition and technological properties of the flour and bran from common and tartary buckwheat. Food Chemistry, 80, 9–15. Guo, R., Yuan, G., & Wang, Q. (2011). Effect of sucrose and manitol on the accumulation of health-promoting compounds and the activity of metabolic enzymes in broccoli sprouts. Scientia Horticulturae, 128, 159–165. Kim, D.-K., Jeong, S. C., Gorinstein, S., & Chon, S.-U. (2012). Total polyphenols, antioxidant and antiproliferative activities of different extracts in mungbean seeds and sprouts. Plant Foods for Human Nutrition, 67, 71–75. Kim, S. I., Park, J. H., Ryu, J.-H., Park, J. D., Lee, Y. H., Park, J.-H., et al. (1996). Ginsenoside Rg5, a genuine dammarane glycoside from Korean red ginseng. Archives of Pharmacal Research, 19, 551–553. Kim, S.-J., Zaidul, I. S. M., Suzuki, T., Mukasa, Y., Hashimoto, N., Takigawa, S., et al. (2008). Comparison of phenolic compositions between common and tartary buckwheat (Fagopyrum) sprouts. Food Chemistry, 110, 814–820. Kim, S.-L., Kim, S.-K., & Park, C.-H. (2004). Introduction and nutritional evaluation of buckwheat sprouts as a new vegetable. Food Research International, 37, 319–327.
T.-G. Nam et al. / Food Chemistry 170 (2015) 97–101 Kitabayashi, H., Ujihara, A., Hirose, T., & Minami, M. (1995). ). On the genotypic differences for rutin content in tartary buckwheat. Fagopyrum tartaricum Gaertn. Breeding Science, 45, 189–194. Li, J., Jiang, B., Liu, X., Zhang, J., Chen, X., & Bi, K. (2007). Simultaneous determination of five bioactive flavonoids in Hypericum japonicum Thunb by high-performance liquid chromatography. Asian Journal of Traditional Medicines, 2, 75–81. Lim, J.-H., Park, K.-J., Kim, B.-K., Jeong, J.-W., & Kim, H.-J. (2012). Effect of salinity stress on phenolic compounds and carotenoids in buckwheat (Fagopyrum esculentum M.) sprout. Food Chemistry, 135, 1065–1070. Liu, C.-L., Chen, Y.-S., Yang, J.-H., & Chiang, B.-H. (2008). Antioxidant activity of tartary (Fagopyrum tataricum (L.) Gaertn.) and common (Fagopyrum esculentum Moench) buckwheat sprouts. Journal of Agricultural and Food Chemistry, 56, 173–178. Slimestad, R., Torskangerpoll, K., Nateland, H. S., Johannessen, T., & Gisked, N. H. (2005). Flavonoids from black chokeberries, Aronia melanocarpa. Journal of Food Composition and Analysis, 18, 61–68.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Flavonoid analysis of buckwheat sprouts Tae-Gyu Nam a,c, Sun Mi Lee b, Ji-Hae Park c, Dae-Ok Kim a, Nam-in Baek c, Seok Hyun Eom b,⇑ a
Department of Food Science and Technology, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea Department of Horticultural Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea c Graduate School of Biotechnology and Research Institute of Life Sciences & Resources, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 3 September 2013 Received in revised form 31 May 2014 Accepted 2 August 2014 Available online 23 August 2014 Keywords: Common buckwheat Quercetin-3-O-robinobioside Tartary buckwheat Vegetable sprout
a b s t r a c t It is known that common buckwheat sprouts contain several flavonoids, including orientin, isoorientin, vitexin, isovitexin, rutin, and quercetrin, whereas tartary buckwheat sprouts contain only rutin. In this study, we evaluated flavonoids present in buckwheat sprouts and identified a previously unreported flavonoid. Simultaneous detection by HPLC was used to separate rutin and a compound that was not separated in previous studies. We used a novel HPLC elution gradient method to successfully separate rutin and the previously unidentified compound, for which we performed structural analysis. The identification of six flavonoids by HPLC was confirmed using HPLC–ESI–MS/MS analysis. The newly identified compound, [M + H]+ = 611.17, was identified by NMR as the rutin epimer quercetin-3-O-robinobioside. Unlike common buckwheat sprout, tartary buckwheat sprout contained rutin as a main flavonoid, whereas other flavonoids appeared only in trace amounts or were not detected. Quercetin-3-O-robinobioside was not detected in tartary buckwheat sprout. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Buckwheat (Fagopyrum spp.), a member of the Polygonaceae family, is consumed all around the world. While it is technically a fruit seed, it is classified as a cereal grain because its utilisation is similar to that of other cereal grains (Kim, Kim, & Park, 2004). Common buckwheat (Fagopyrum esculentum Möench) and tartary buckwheat (Fagopyrum tataricum Gaertner) are the main species of buckwheat consumed by humans. Buckwheat has been established as a nutritional food because of its abundant levels of amino acids and protein (Bonafaccia, Marocchini, & Kreft, 2003). Moreover, common buckwheat is a major dietary source of rutin and O-glycosyl flavonols including both quercetin and rutinose moieties (Kitabayashi, Ujihara, Hirose, & Minami, 1995). The nutritional values of the edible parts of sprouts of bean sprouts, cereal crops, and vegetables have gained interest in recent years. Sprouts are recognised as outstanding dietary vegetables in Asia, Europe, and the United States, and are an important source of protein, mineral, dietary fiber, and vitamins in human diets. Especially, the contents of polyphenols, which are secondary plant metabolites, in particular, increase or are newly synthesised during sprouting, similar to peanut and broccoli (Guo, Yuan, & Wang, 2011; Wang et al., 2005). Polyphenols have attracted a great deal
⇑ Corresponding author. Tel.: +82 31 201 3860; fax: +82 31 204 8116. E-mail address: [email protected] (S.H. Eom). http://dx.doi.org/10.1016/j.foodchem.2014.08.067 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
of attention due to their various benefits for human health. For example, the total phenolic content and antioxidant capacity of mungbean sprouts is greater than that of seeds (Kim, Jeong, Gorinstein, & Chon, 2012). Likewise, the flavone and flavonol glycoside contents of buckwheat increase during sprouting (Alvarez-Jubete, Wijngaard, Arendt, & Gallagher, 2010). Buckwheat sprouts are one of the most popular forms of sprouts and are consumed as both a salad vegetable and fresh vegetable served with noodles in northeast Asia (Kim et al., 2004). Phytochemical analyses of buckwheat sprouts have been performed intensively over the last decade, and the nutritional components of seeds have been evaluated for significantly longer. Compared with buckwheat seeds, the sprouts contain relatively large amounts of rutin, which is known as a beneficial compound for health (Kim et al., 2004). Similarly, buckwheat sprouts have a significantly greater abundance of other flavonoids including orientin, isoorientin, vitexin, isovitexin, rutin, and quercetrin compared with buckwheat seeds (Kim et al., 2004, 2008; Lim, Park, Kim, Jeong, & Kim, 2012; Liu, Chen, Yang, & Chiang, 2008; Watanabe, 2007). However, the analytical high performance liquid chromatography (HPLC) techniques employed in previous studies were unable to separate and quantify individual flavonoids to a satisfactory degree. In addition, the HPLC chromatograms of numerous studies are ambiguous as to whether they represent single or multiple peaks. Reversed-phase HPLC is widely employed for quantitative and qualitative analysis of common buckwheat sprouts, and numerous
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studies have used this approach for identification of flavonoids. Several studies have reported that common buckwheat sprouts contain C-glycosyl flavones (orientin, isoorientin, vitexin and isovitexin) and rutin (Kim et al., 2008; Lim et al., 2012; Liu et al., 2008; Watanabe, 2007). In this study, we used a gradient elution, based on water-acetonitrile, to achieve optimum separation for the identification of flavonoid derivatives in common buckwheat sprouts. In order to identify a newly detected compound, we used NMR, HPLC, and Q-TOF MS to isolate and identify the quercetin derivative, O-glycosyl flavonol, in addition to the other flavonoids and their derivatives. 2. Materials and methods 2.1. Seeds Common (Cultivar, Suwon 2 Ho) and tartary (Cultivar, Dae-Guan 3-3 Ho) buckwheat seeds were purchased from a seed company (Budnara) in Gwangju, Republic of Korea in 2012. 2.2. Chemicals Orientin (P95%), isoorientin (P95%), vitexin (P95%), isovitexin (P98%), and rutin (P94%) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). All solvents used were of analytical or HPLC grade. 2.3. Sprout growth condition Buckwheat seeds were placed in a 4 °C room before initiation of experiments. After soaking in distilled water for 4 h, water saturated-seeds were placed in a dark growth chamber for 48 h to facilitate germination. Germinated seeds were planted in a light supplemented growth chamber (35 lmol m2 s1 light intensity by fluorescent light lamps) at 25 °C for 5 days. During sprout growth, water was supplied by spraying as needed. Finally, the sprouts were harvested and dried in a 30 °C dry oven equipped with ventilation for 3 days.
equipped with an auto-sampler (SIL-20A, Shimadzu), photodiode array detector (SPD-20A, Shimadzu), binary pump (LC-20AD, Shimadzu), and vacuum degasser. Chromatographic separation was performed using a ProntoSIL 120-5-C18-ace-EPS column (4.6 250 mm, 5.0 lm; Bischoff, Leonberg, Germany). For HPLC analysis, we used two different linear solvent gradients with a binary mobile phase consisting 0.1% HCOOH in water (solvent A) and 0.1% HCOOH in acetonitrile (solvent B). Elution gradient I consisted of 92% A and 8% B at 0 min, 85% A and 15% B at 4 min, 55% A and 45% B at 90 min, 5% A and 95% B at 100 min, 92% A and 8% B at 102 min, and 92% A and 8% at 105 min. Elution gradient II consisted of 92% A and 8% B at 0 min, 85% A and 15% B at 4 min, 84% A and 16% B at 8 min, 84% A and 16% B at 20 min, 83% A and 17% B at 45 min, 82% A and 18% at 75 min, 81% A and 19% at 80 min, 80% A and 20% B at 95 min, 79% A and 21% B at 102 min, 30% A and 70% B at 107 min, 92% A and 8% B at 110 min and 92% A and 8% B at 115 min. The flow rate was at 0.8 mL/min with a 20 lL injection volume. The column oven temperature was set to 25 °C, and the absorbance was monitored at 350 nm (Li et al., 2007). The extracts were analysed at least three times by HPLC. 2.6. HPLC-DAD–ESI/Q-TOF MS analysis of flavonoids in common buckwheat sprouts The molecular weights of major flavonoids in buckwheat sprouts were determined using an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight MS (Q-TOF LC/MS) with an Agilent 1200 Series Rapid Resolution LC system. The MS was operated with electrospray ionisation source in either negative or positive ion mode. A ProntoSIL 120-5-C18-ace-EPS column (4.6 250 mm, 5.0 lm; Bischoff, Leonberg, Germany) was used to separate individual flavonoids using gradient elution II at an oven temperature of 40 °C. Flavonoids were monitored at 350 nm. The MS source parameters were as follows: 350 °C drying gas (N2) temperature, 12 L/min drying gas flow, 45 psi nebulizer pressure, 200 V fragmentation voltage, and 4000 V capillary voltage. MS and MS/MS ranges were set from m/z 50 to 1700 and m/z 50 to 1100, respectively. Collision energy was set at 10 eV for positive ion mode and 40 eV for negative ion mode. 2.7. Prep-LC and TLC analysis for flavonoid separation
2.4. Extraction and fractionation of flavonoids from buckwheat sprouts Fifteen grams of dried buckwheat sprouts were processed with a commercial grinder and dispersed in 150 mL of 70% aqueous ethanol. After extraction (48 h) at room temperature, the solution was filtered through Whatman #2 filter paper (Whatman International Limited, Kent, UK). The solid filter cake was extracted again in 70% aqueous ethanol and filtered. The extraction procedure was repeated once more. Next, solvent was evaporated using a rotary evaporator at 40 °C, and 840 mg of crude extract was suspended in 40 mL of distilled water and combined with 80 mL of EtOAc/nBuOH (3:1). After shaking to mix solvents, the mixture was allowed to separate and split again between water and EtOAc/n-BuOH. A total of 250 mg of EtOAc/n-BuOH fractionated extract was gathered after solvent evaporation. The extract (250 mg) was suspended in 20 mL of water and combined with 40 mL of EtOAc. After repeating the separation described above, the aqueous fraction was collected and dried to obtain a 70 mg flavonoids-rich extract, which was kept at 4 °C until analysed. 2.5. Isolation of flavonoids in buckwheat sprouts by reversed-phase HPLC Flavonoids of buckwheat sprouts were analysed using a reversed-phase HPLC system (Shimadzu model, Kyoto, Japan)
Flavonoids from the crude extract of common buckwheat sprouts were separated and confirmed using a preparative highperformance liquid chromatography system (Prep-LC) consisting of a Waters 2555 Prep-LC controller, a 2998 Photodiode array detector, and an Empower workstation (Waters, Milford, MA, USA) with a YMC ODS column (20 250 mm, 5 lm). The binary solvent system consisted of (A) water and (B) acetonitrile, with a linear gradient of 5% (B) to 50% (A) for 10 min, isocratic for 10 min, from 50% (B) to 100% (A) for 10 min, isocratic for 10 min, and finally from 100% (B) to 5% (A) for 5 min. The flow rate was 30 mL/min and 70 mg/mL of flavonoid-rich extract was injected with 1 mL injection volume. The column oven temperature was set to 25 °C. In addition, we performed thin-layer chromatography (TLC) analysis to confirm the purity of isolated fractions. TLC analysis was carried out using Kiesel gel 60 F254 and RP-18 F254S resin (Merck, Dramstadt, Germany). TLC spots were detected using a Spectroline Model ENF-240 C/F UV lamp (Spectronics Corporation, Westbury, NY, USA) and a 10% H2SO4 solution. 2.8. NMR spectroscopy The structure of a previously unidentified flavonoid was analysed by NMR. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were recorded on a Varian Unity Inova AS-400 FT-NMR
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spectrometer (Palo Alto, CA, USA). Negative fast atom bombardment mass spectrometry (FAB-MS) was conducted using a JEOL JMSAX-700 instrument (Tokyo, Japan) described by Kim et al. (1996). 3. Results and discussion 3.1. Optimisation of gradient elution Two gradient elution systems were utilised to separate flavonoids in the buckwheat sprout extracts. Chromatograms of flavonoid derivatives are presented in Fig. 1. The pattern of flavonoids of common buckwheat sprouts eluted using gradient I were similar to previous studies (Kim et al., 2008; Lim et al., 2012; Liu et al., 2008; Watanabe, 2007), in that we did not separate successfully the individual flavonoids in peaks 5 and 6. However, peaks 5 and 6 were separated successfully using elution gradient II, where the acetonitrile concentration was increased more slowly, achieving to 21% over a period of 102 min, compared with gradient I. While common buckwheat sprouts exhibited six distinct flavonoid peaks, in tartary buckwheat sprout rutin (peak 6) was the only major component of the eluted by HPLC using gradient II (Fig. 1). Other anticipated flavonoids were present in only trace amounts or not at all. The rutin content of tartary buckwheat sprouts was approximately 4–5-fold higher than in common buckwheat sprout (Kim et al., 2008). 3.2. Q-TOF LC/MS analysis of flavonoids It is known that common buckwheat sprouts contain several flavonoids, including orientin, isoorientin, vitexin, isovitexin, rutin, and quercetrin (Kim et al., 2008; Lim et al., 2012; Liu et al., 2008; Watanabe, 2007). Among the flavonoids, quercetrin was detected late on HPLC chromatogram, whereas other flavonoids were detected within two hours of retention time. With the exception of quercetrin, chromatographic peaks of five other flavonoids and an unidentified compound shown in Fig. 1 were determined by MS/MS analysis. Q-TOF LC/MS analysis based on full scan data and MS/MS fragment patterns are shown in Table 1. Peak 1 produced an m/z 447.1004 of [M H] on MS, with MS/ MS fragments of m/z 357.0666 of [M H 90] and 327.0565 of [M H 120]. The predominant fragment patterns with product ion losses of 90 and 120 U were consistent with a C-glycosyl flavone. Peak 1 was tentatively identified as orientin (luteolin-8C-glucoside) based on the UV spectra, retention time and MS/MS ion products. In negative mode, peak 2 exhibited an m/z of 447.1015 (loss of a proton) and its fragment ions were m/z 357.0666 (loss of 90 U) and 327.0564 (loss of 120 U). Peaks 1 (orientin) and 2 possessed similar molecular masses and MS/MS spectrums, indicating that peak 2 was an isomer of orientin. Thus, peak 2 was tentatively identified as isoorientin (luteolin-6-C-glucoside). The components of peaks 1 and 2 were subsequently confirmed by Q-TOF LC/MS in comparison with the ion spectra from authentic flavone standards. Peak 3 exhibited an m/z of 431.1068 and its fragment ions were m/z 341.0711 (loss of 90 U), 311.0615 (loss of 120 U), and 283.0662 (loss of 148 U). Peak 4 also had an m/z of 431.1081 with fragment ions of m/z 341.0726, 311.0621 and 283.0666, indicating similar masses compared to peak 3. Peaks 3 and 4 were further analysed by comparison with the retention time, UV spectra, and MS/MS fragment patterns of authentic standards, which confirmed vitexin (apigenin-8-C-glucoside) and isovitexin (apigenin-6-C-glucoside), respectively. The masses of peaks 5 and 6 were m/z 609.1572 and 609.1591 in negative ion mode, and each produced a single fragment of m/z 301.0398 and 301.0397 in MS/MS analysis, respectively. For identification and
Fig. 1. Reversed-phase HPLC chromatograms of flavonoids in buckwheat sprouts using elution gradient I (A) and gradient II (B). Elution gradient II was used to separate compounds 5 and 6. Elution gradient II was applied to separate flavonoids in tartary buckwheat (C).
comparison of sugar moieties in peak 5 and 6, MS/MS analysis in positive ion mode was performed at a lower collision energy. Peak 5 presented with an m/z of 611.1736, which produced MS/MS fragment ions with m/z values of 465.0995 (loss of a methyl pentose group) and 303.0480 (loss of a hexose group). The mass and fragment ions of peak 6 were highly similar to those of peak 5, which had already been identified as rutin, based on comparison with an authentic standard. Because the mass and MS/MS patterns of peaks 5 and 6 were both similar rutin, peak 5 was considered to be an isomer of rutin. The results of orientin, isoorientin, vitexin, isovitexin, and rutin analysed in this experiment correspond with earlier results reported by several studies (Lim et al., 2012; Verardo et al., 2010; Watanabe, 2007). In our LC–MS/MS analysis, a compound
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Table 1 HPLC–ESI–MS and MS2 for identification of flavonoids in common buckwheat sprouts. Compound No. 1 2 3 4 5 6 5 6
Selected ion –
[M H] [M H]– [M H]– [M H]– [M H]– [M H]– [M + H]+ [M + H]+
Molecular ion (m/z)
MS/MS (m/z)
Tentative identification
Molecular formula
447.1004 447.1015 431.1068 431.1081 609.1572 609.1591 611.1736 611.1610
357.0666, 357.0666, 341.0711, 341.0726, 301.0398 301.0397 465.0995, 465.1023,
Orientin Isoorientin Vitexin Isovitexin Quercetin-3-O-robinobioside Rutin Quercetin-3-O-robinobioside Rutin
C21H20O11 C21H20O11 C21H20O10 C21H20O10 C27H30O16 C27H30O16 C27H30O16 C27H30O16
showed similar characteristic of rutin and had not been reported previously in buckwheat. Thus, further studies for identifying the compound were necessary. 3.3. Identification of the chemical structure in peak 5 To determine the chemical structure of peak 5 (compound 5), the flavonoid-rich fraction of the common buckwheat sprout extract was further fractionated by preparative HPLC. The eluted fractions were monitored by TLC. Finally, compound 5 [6 mg, Ve/Vt 0.76–0.89, RT 24 min, TLC (ODS F254S) Rf 0.50, MeOHH2O = 3:2] was separated. The chemical structure of compound 5 was determined based on several analyses, including 1H-NMR, 13C-NMR, DEPT, gradient correlation spectroscopy, gradient heteronuclear single-quantum coherence, gradient heteronuclear multiple-bond connectivity and fast atom bombardment mass spectrometry. Compound 5 was a yellow powder (MeOH), negative FAB-MS m/z 609 [M H]; 1 H-NMR (400 MHz, methanol-d4, dH) 7.86 (1H, d, J = 2.0 Hz, H-20 ), 7.60 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 6.86 (1H, d, J = 8.4 Hz, H-50 ), 6.38 (1H, br s, H-8), 6.20 (1H, br s, H-6), 5.05 (1H, d, J = 7.6 Hz, H-100 ), 4.51 (1H, br s, H-1000 ), 3.82 (1H, m, H-200 ), 3.80 (1H, m, H400 ), 3.74 (1H, m, H-600 b), 3.65 (1H, m, H-500 ), 3.58 (1H, m, H-2000 ), 3.55 (1H, m, H-300 ), 3.52 (1H, m, H-5000 ), 3.48 (1H, m, H-3000 ), 3.40 (1H, m, H-600 a), 3.28 (1H, m, H-4000 ), 1.18 (3H, d, J = 6.4 Hz, H-6000 ; 13 C-NMR (100 MHz, methanol-d4, dC) 179.4 (C-4), 166.4 (C-7), 163.0 (C-5), 159.3 (C-9), 158.6 (C-2), 149.8 (C-40 ), 145.9 (C-30 ), 135.6 (C-3), 123.5 (C-60 ), 123.1 (C-10 ), 117.7 (C-20 ), 116.1 (C-50 ), 105.5 (C-10), 105.8 (C-100 ), 102.4 (C-1000 ), 100.1 (C-6), 94.9 (C-8), 75.3 (C-500 ), 75.2 (C-300 ), 73.6 (C-200 ), 73.9 (C-4000 ), 72.2 (C-3000 ), 72.1 (C-2000 ), 69.7 (C-400 ), 69.7 (C-5000 ), 67.3 (C-600 ), 17.9 (C-6000 ). On the basis of spectroscopic data and chemical evidence, compound 5 was identified as quercetin-3-O-robinobioside (quercetin 3-O-(6O-a-L-rhamnopyranosyl)-b-D-galactopyranoside) (Fig. 2). These findings were confirmed by comparison with the physical and spectroscopic data reported in a literature, including the structural analysis of quercetin-3-O-robinobioside isolated from black
Fig. 2. Chemical structures of quercetin-3-O-robinobioside and rutin.
327.0565 327.0564 311.0615, 283.0662 311.0621, 283.0666
303.0480 303.0504
chokeberries (Slimestad, Torskangerpoll, Nateland, Johannessen, & Gisked, 2005). The compound is structurally similar with rutin. Rutin consists of quercetin with glucose-rhamose, while quercetin-3-O-robinobioside consists of quercetin with galactoserhamose. Although these two compounds are structurally similar, rutin has popularly known as a compound benefiting health, while the health benefits and biological activities of quercetin-3-O-robinobioside have not reported. The lack of investigation of quercetin-3-O-robinobioside with respect to health may result from difficulty in separating relative small amount in biomaterials. 4. Conclusions In this study, we analysed common buckwheat sprout, and initially identified rutin and four C-glycosyl flavones (orientin, isoorientin, vitexin, and isovitexin). Further analysis of the HPLC peak area of rutin allowed us to identify the previously undetected quercetin-3-O-robinobioside. The newly identified compound had the same molecular weight as rutin and was barely separated from rutin in the HPLC peaks reported in previous studies. Chromatographic resolution of eluted peaks varied according to the ratio of water and acetonitrile in the elution gradient. Using an optimised gradient condition, quercetin-3-O-robinobioside that was not identified previously was successfully eluted slightly ahead of rutin. Quercetin-3-O-robinobioside was not detected in tartary buckwheat sprouts. Our result may provide useful information for understanding flavonoid metabolism and synthesis during buckwheat sprout growth. Acknowledgements This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HI10C2162). References Alvarez-Jubete, L., Wijngaard, H., Arendt, E. K., & Gallagher, E. (2010). Polyphenol composition and in vitro antioxidant activity of amaranth, quinoa buckwheat and wheat as affected by sprouting and baking. Food Chemistry, 119, 770–778. Bonafaccia, G., Marocchini, M., & Kreft, I. (2003). Composition and technological properties of the flour and bran from common and tartary buckwheat. Food Chemistry, 80, 9–15. Guo, R., Yuan, G., & Wang, Q. (2011). Effect of sucrose and manitol on the accumulation of health-promoting compounds and the activity of metabolic enzymes in broccoli sprouts. Scientia Horticulturae, 128, 159–165. Kim, D.-K., Jeong, S. C., Gorinstein, S., & Chon, S.-U. (2012). Total polyphenols, antioxidant and antiproliferative activities of different extracts in mungbean seeds and sprouts. Plant Foods for Human Nutrition, 67, 71–75. Kim, S. I., Park, J. H., Ryu, J.-H., Park, J. D., Lee, Y. H., Park, J.-H., et al. (1996). Ginsenoside Rg5, a genuine dammarane glycoside from Korean red ginseng. Archives of Pharmacal Research, 19, 551–553. Kim, S.-J., Zaidul, I. S. M., Suzuki, T., Mukasa, Y., Hashimoto, N., Takigawa, S., et al. (2008). Comparison of phenolic compositions between common and tartary buckwheat (Fagopyrum) sprouts. Food Chemistry, 110, 814–820. Kim, S.-L., Kim, S.-K., & Park, C.-H. (2004). Introduction and nutritional evaluation of buckwheat sprouts as a new vegetable. Food Research International, 37, 319–327.
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