Identification and quantification of polyphenols in hull, bran and endosperm of common buckwheat (Fagopyrum esculentum) seeds

Journal of Functional Foods 38 (2017) 363–369

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Identification and quantification of polyphenols in hull, bran and endosperm of common buckwheat (Fagopyrum esculentum) seeds Weina Zhang a, Yuanyuan Zhu a, Qingqing Liu a, Jinsong Bao b,⇑, Qin Liu a,⇑ a College of Food Science and Engineering, Collaborative Innovation Center for Modern Grain Circulation and Safety, Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics, Nanjing 210023, China b Institute of Nuclear Agricultural Science, College of Agriculture and Biotechnology, Zhejiang University, Huajiachi Campus, Hangzhou 310029, China

a r t i c l e

i n f o

Article history: Received 5 April 2017 Received in revised form 9 September 2017 Accepted 14 September 2017

Keywords: Common buckwheat Seeds fraction Polypehnol Distribution

a b s t r a c t The distribution of polyphenol profiles in different fractions of buckwheat seeds is less understood. In this study, polyphenols in the hull, bran and endosperm of eight common buckwheat cultivars were studied. The results showed that the hull had highest total phenolic content, followed by bran. Total proanthocyanidin contents in the hull were similar to those in the bran. Over 40 phenolics were identified by HPLC/MS. Prominent phenolics such as 1-O-Caffeoyl-6-O-rhamnopyranosyl-glycopyranoside, epicatechin-gallate, orientin/isorientin, vitexin/isovitexin, hyperin and rutin in different seed fractions were identified and quantified. The polyphenol profile in the hull was quite different from that of bran and endosperm. Vitexin/isovitexin, hyperin and rutin were major phenolics in hulls with content ranges of 101.65–188.78 mg/100 g, 53.55–274.10 mg/100 g and 62.43–173.57 mg/100 g, respectively. Epicatechin-gallate was the predominant phenol in bran and endosperm with content ranging from 150.44 to 354.67 mg/100 g and from 19.65 to 73.92 mg/100 g, respectively. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Common buckwheat (Fagopyrum esculentum Moench) is an ancient pseudo cereal natively cultivated in northwest Yunnan province in China (Ohnishi, 1998). It is now still a cover crop wildly cultivated in plateau regions including northwest and southwest China owing to its drought resistance and short growing season. Historically, common buckwheat flour has been used to make noodle, dumpling wrap, pan cake among others as a substitute for wheat flour. Today, it is recognized as a functional food because of its nutritional values of high-quality protein, dietary fiber, essential fatty acids, vitamins and minerals as well as being rich in bioactive phytochemicals (Giménezbastida & Zielin´ski, 2015). The health attributes of buckwheat include the reduction of total cholesterol, triglycerides and low-density lipoprotein levels and the increase of high-density lipoprotein level (Hosaka et al., 2014), which result in the decreased risk of chronic disease such as diabetes (Stringer, Taylor, Appah, Blewett, & Zahradka, 2013), cancer and cardiovascular disease (Tomotake et al., 2006).Zeng et al. (2015) recently reported that people living in southwest region of China had the lowest cancer mortality in China which

⇑ Corresponding authors. E-mail addresses: [email protected] (J. Bao), [email protected] (Q. Liu). http://dx.doi.org/10.1016/j.jff.2017.09.024 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.

may result from the healthy infrastructure of food intake, in which common buckwheat is one of the main consumed functional foods. Plant phenolics and their antioxidant activities have been linked to potential health improvements (Sedej et al., 2012). Unlike other cereals, phenolics occur in grain mainly as bound and conjugated forms with ferulic acid the most abundant constituent (Adom & Liu, 2002). Phenolics in buckwheat are present mainly in a free form with complex polyphenol composition (Inglett, Chen, Berhow, & Lee, 2011). Since Watanabe (Watanabe, 1998) first isolated and identified four catechins and rutin as antioxidants in common buckwheat groats, there have been more than 30 phenolics identified including caffeic acid derivates, quercetin, hyperoside, epicatechin, vitexin/isovitexin, oreintin/isoorientin as well as rutin, among others (Verardo et al., 2011). Most studies on phenolics in common buckwheat have focused on total phenolic content (TPC), rutin content and antioxidant activities (Choy, Morrison, Hughes, Marriott, & Small, 2013). Hung and Morita (2008) reported that the outer layer had the highest TPC and antioxidant activities in different milled fractions of common buckwheat flour. Sedej et al. (2012) found that buckwheat hull showed superior antioxidant activities and high rutin content compared with buckwheat groat and whole grain. Buckwheat cultivars show significant difference in phenolics content and antioxidant activity (Kiprovski et al., 2015; Kishore, Ranjan, Pandey, & Gupta,

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2010). However, few studies have examined the distribution of polyphenols in common buckwheat seeds. In this study, polyphenol profile and distribution in common buckwheat seeds of eight cultivars were investigated. The major phenolic compounds occurring in hull, bran and endosperm flours were identified and quantified. The objective of this study is to evaluate the bioactive composition in different fractions of common buckwheat seeds that could lead to increased utilization for functional food processing. 2. Materials and methods 2.1. Materials The seed materials of eight common buckwheat (Fagopyrum esculentum Moench) cultivars were used in this study. Seeds of 9976 and 9978 were provided by the College of Agronomy at Northwest A&F University, Xi’an, China. Sichuan seed was supplied by Sichuan Academy of Agricultural Sciences (Chengdu, China). Xingnong No. 1 and Yuqiao No. 4 seeds were harvested in Yanchi County in Ningxia province. Honghua, Baihua and Yunnan were provided by the Zhaotong Institute of Agricultural Science, Yunnan. All seeds were harvested in 2014. Caffeic acid, epicatechin-gallate, isorientin, rutin, vanillin and catechin were purchased from Aladdin Industrial Corporation (Shanghai, China). Vitexin and hyperin were purchased from Sigma-Aldrich Co. LLC. Analytical grade acetone, acetic acid, hydrochloric acid, petroleum ether, sulfuric acid and sodium carbonate were purchased from Nanjing Chemical Reagent Co. Ltd (Nanjing, China). Folin-Ciocalteau was purchased from Shanghai Biochemical Reagent Factory (Shanghai China). Deionized water was prepared by Mill-Q Academic water purification system (Millipore, MA, USA). 2.2. Sample preparation Common buckwheat seeds were first dehulled by gentle grinding (3–5 s) using a cylinder grinder FW-100 (Huaxing, Tianjin, China). Hull and groat fractions were separated by low velocity air flow using a fan. The groats were further ground using a centrifugal mill (Retch ZM200, Haan, Germany) while the bran and endosperm flours were separated by passing through an 80 mesh sieve. The collected hull and bran fractions were ground to 60 mesh. All ground samples including hull, bran and endosperm flours were defatted 2 h by using petroleum ether as solvent in Soxhlet extraction equipment, and then stored at
20 °C. The hull flour accounted for about 19.50–21.45% of buckwheat seed weight while bran and endosperm flours accounted for about 12.35– 15.82% and 63.8–66.5% of seed weight, respectively, based on the milling and separation method. 2.3. Extraction of phenolic compounds Defatted sample powder (1.0 g) was extracted by 10 ml of acetone/water (v/v = 70/30) for 180 min under 50 °C controlled by water bath. The supernatant was collected and dried under 40 °C by vacuum evaporator, and the residue was reconstituted with 2 mL of methanol/water (v/v = 50/50) and stored under
20 °C until use. 2.4. Determination of total phenolic content Total phenolic content (TPC) was determined by colorimetric method using Folin-Ciocalteau reagent. Briefly, 3.5 mL of distilled water, then 0.40 mL of Folin-Ciocalteau reagent were added to

0.3 mL of extract and mixed in a vortex mixer. After 5 min, 3 ml Na2CO3 (60 g/L) was added. The mixture was incubated at 25 °C for 1.5 h. The absorbance of mixture was measured at 760 nm using an ultraviolet spectrophotometer (Hitachi U-3900, Japan). TPC was calculated using rutin as a standard, and the results expressed as mg (rutin equivalent)/100 g of sample (dry weight).

2.5. Determination of total proanthocyanidin content The total proanthocyanidin content (TPAC) was determined by using the vanillin assay with minor modification (Butler, Price, & Brotherton, 1981). Solvent of phenolics extract was removed by using a vacuum evaporator under 40 °C, and the residue was reconstituted with anhydrous methanol. 0.2 mL of reconstituted solution was mixed with 3 mL of sulfuric acid/acetic acid mixture solution (V/V = 46/54) and 3 mL of 1% vanillin acetic acid (w/v) solution. The absorbance of the mixture was determined at 510 nm. Catechin was used as a standard with the results expressed as mg (catechin equivalent)/100 g of sample (dry weight).

2.6. HPLC/MS analysis HPLC/MS analysis was performed on an Agilent 1100 HPLC/MS (Agilent, SL) system equipped with a diode array detector (DAD) (Agilent) and an auto sampler. For HPLC analysis, a reversedphase C18 analytical column (4.6  250 mm, 5 lm, Agilent, Eclipse XDB) was used for separation. The mobile phase consisted of A (1.0% acetic acid in water) and B (1.0% acetic acid in methanol). A linear gradient elution was programmed as follows: 0–30 min, 10–35% B; 30–55 min, 35–50% B; 55–60 min, 50–60% B; 60– 65 min, 60% B; 65–70 min, 60–10% B; followed by 5 min equilibration of 10% B. 10 lL of samples was injected by an auto sampler at flow rate of 0.6 mL/min. The column temperature was set at 35 °C. A T-split was used to reduce the flow before sample injection into the mass spectrometer. The mass spectrum was acquired by an electrospray ionization mass spectrometry (ESI-MS). Full mass spectra were recorded at the range of m/z 100–1000 in negative mode. The capillary voltage was set at 3.0 kV, capillary temperature was set at 350 °C with drying gas flow of 9.0 L/min and nebulizing gas pressure of 25.0 psi. The MS/MS and MS (n) spectra were acquired using smart fragment mode.

2.7. Identification and quantification of phenolic compounds Phenolic compounds in the extracts of common buckwheat seeds were identified by combining their HPLC-UV spectra and corresponding m/z values, as well as fragments measured in MS/MS. Epicatechin-gallate, isorientin, rutin, vitexin and hyperin in samples were quantified by using corresponding authorized standards. The quantification of 1-O-Caffeoyl-6-O-rhamnopyranosyl-glycopyr anoside was conducted by using caffeic acid as a standard and expressed as mg (caffeic acid equivalent)/100 g (dry weight). All experiments were conducted in triplicate.

2.8. Statistical analysis The results were reported as mean ± standard deviation (SD) for triplicate analysis. A one-way analysis of variance (ANOVA) test using SAS was performed. Least significant difference (LSD) at P < 0.05 was tested to assess the significant differences between samples.

W. Zhang et al. / Journal of Functional Foods 38 (2017) 363–369

3. Results and discussion

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correlations were found between TPC and TPAC (p < 0.05) for hull, bran and endosperm fractions, respectively.

3.1. Total phenolic content in common buckwheat seed fractions TPC of hull, bran and endosperm flours of 8 common buckwheat cultivars are shown in Fig. 1a. The TPC of hull, bran and endosperm flours ranged from 2255.2 to 7407.2 mg/100 g, 1940.5 to 2642.3 mg/100 g and 393.7 to 719.4 lmol/100 g, respectively. Hull had the highest TPC followed by bran except for Honghua variety. Among hulls, Yuqiao No. 4 had highest TPC, followed by Sichuan. Bran of Sichuan buckwheat had the highest TPC among all bran samples. Li, Yuan, Yang, Tao, and Ming (2013) compared the free and bound TPC of different cultivars of common buckwheat showing that free TPC in hull fractions were higher than that of bran, which is consistent with our results. As expected, endosperm flour had the lowest TPC in comparison to other fractions.

3.2. Total proanthocyanidin content in common buckwheat seed fractions It has been reported that common buckwheat seeds are rich in proanthocoyanins (Ölschläger, Regos, Zeller, & Treutter, 2008). TPAC in hull, bran and endosperm flours ranged from 1.52 to 4.23 mg/100 g, 1.49 to 1.93 mg/100 g and 0.57 to 0.80 mg/100 g, respectively (Fig. 1b). Similar to TPC results, hull flour of Yuqiao No. 4 had highest TPAC among all hull samples. For 9978, 9976, Xingnong No. 1 and Yuqiao No. 4, TPAC in hull was higher than that of in bran. The hull of Yunnan had similar TPAC with its bran. For the other cultivars, bran had higher TPAC than hulls. TPAC of bran flours were 2–4 times greater than those of endosperm flours. High

3.3. Identification of phenolic compounds in common buckwheat seed fractions HPLC coupled with MS combining with tandem mass is a powerful method in phenolic analysis, in which component identification is based on the UV absorbance profile, mass value and fragments produced by collision induced dissociation of the peaks (Cavaliere, Foglia, Pastorini, Samperi, & Laganà, 2005; Cuyckens & Claeys, 2004). Characterization of phenolics in common buckwheat flour has been reported using LC-ESI-TOF spectrometry (Verardo et al., 2010, 2011). Limited studies however, have been done on the comparison of phenolic profile among the hull, bran and endosperm of common buckwheat seeds. Fig. 2 and Fig. S1 illustrate HPLC chromatography of phenolic extracts of buckwheat hull, bran and endosperm flours recorded at 320 and 280 nm respectively. The retention time, corresponding m/z value and fragments of individual peaks observed are shown in Table 1. The phenolic profile of the hull flour differed from that of bran and endosperm as illustrated from HPLC recorded at 320 nm (Fig. 2). Peaks 17, 27, 28, 34, 36, 37 and 38 were dominant phenolics in buckwheat hulls, while peaks 17, 22 and 38 were major phenolics identified in bran and endosperm flours. Peak 17 with the m/ z value of 487.0 and fragments of 435.0, 179.0 and 135 in MS2 could be assigned as 1-O-Caffeoyl-6-O-rhamnopyranosyl-glycopyr anoside based on reported literature (Verardo et al., 2010). The fragment 435.0 corresponds to a loss of CO2 from parent ion 487.0, and the fragment 179.0 corresponds to the negative ion of caffeic acid resulting from the loss of rhamnopyranosyl-

Fig. 1. TPC (a) and TPAC (b) of hull, bran and endosperm flours of different cultivars of common buckwheat seeds.

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Fig. 2. HPLC chromatograms recorded at 320 nm of phenolics extracts of common buckwheat hull, bran and endosperm flours.

glycopyranose moiety (
308) from parent ion of 487.0. The fragment of 135.0 corresponds to a mass loss of CO2 (
44) from fragment 179.0. Peak 22 and 24 had the same m/z value of 441.0 and the fragment of 289.0. Those 2 peaks could be tentatively assigned as epicatechin-gallate. The fragment of 289 corresponds to negative ion of epicatechin which results from the loss of a galloyl group (
152). The fragments of 169 and 125 correspond to the negative ion of gallic acid and the daughter mass of gallic acid formed by loss of CO2 (
44). Peaks 27 and 28 eluted at 36.67 min and 37.50 min respectively had an m/z value of 447.0. The fragments of 357.0 and 327.0 in MS2 correspond to the elimination of 90 and 120 units respectively from the parent ion, which imply that the corresponding compounds had a C-linked hexose residue at the C-6-and/or C-8-positions of the flavonoid aglycone (Cuyckens & Claeys, 2004). These 2 peaks can be assigned as orientin or isorientin. Based on the same theory, peaks 34 and 36 eluted at 40.29 and 43.18 min with parent ion of 431.0 and daughter ions of 311.0 and 341.0 in MS2 suggest the presence of vitexin or isovitexin, with C-linked hexose residue at the C-6/or C-8 positions of the flavonoid nucleus (Verardo et al., 2010). Peaks 37 and 38 eluted at 44.55 and 45.26 min with m/z of 463.0 and 609.0 had the same fragment of 301.0 in MS2, which could be assigned as negative ion of quercetin based on its daughter ion of 179.0 and 151.0. The loss of 162 and 308 units from parent ions correspond to the fragments of glucose and rutinose residue, respectively.

Thus, peaks 37 and 38 can be assigned as hyperin and rutin, respectively. More peaks were observed in HPLC spectrum recorded at 280 nm (Fig. S1). It has been reported that unlike tartary buckwheat (Fagopyrum tataricum) where rutin is the predominant phenolic accounting for more than 85% of TPC (Kim et al., 2008), common buckwheat has a more diverse polyphenol composition including flavonone, proanthocyanidins, flavanols, and their Oglycoside and C-glycosides as well as many catechin and epicatechin derivates. In this study, most peaks observed at 280 nm were proanthocyanidins, and catechin or epicatechin derivates. Fortyfour compounds were tentatively identified based on information achieved from HPLC and mass spectra. Kim et al. (2008) identified 7 major phenolic compounds in common buckwheat seed sprouts, including chlorogenic acid, 4 C-glycosyl flavones (orientin, isoorientin, vitexin, and isovitexin), rutin and quercetin. However, no chlorogenic acid was detected in this study.

3.4. The distribution of major phenolics in common buckwheat seeds Since the orientin/isorientin, vitexin/isovitexin, hyperin and rutin are major phenolics in the hulls, while the 1-O-Caffeoyl-6O-rhamnopyranosyl-glycopyranoside and epicatechin gallate are the major phenolics in the bran and endosperm (Fig. 2), their

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W. Zhang et al. / Journal of Functional Foods 38 (2017) 363–369 Table 1 Retention time, mass spectral data and corresponding identified phenolics components in common buckwheat.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17* 18 19 20 21 22* 23 24* 25 26 27* 28* 29 30 31 32 33 34* 35 36* 37* 38* 39 40 41 42 43 44

tR (min)

m/z

MS2

4.734 5.575 6.272 7.397 10.305 12.822 13.698 14.241 16.345 17.142 17.571 18.583 19.374 20.638 22.765 23.939 25.346 26.536 27.182 27.806 30.369 31.029 31.733 32.665 33.021 35.872 36.668 37.503 37.987 38.126 38.828 39.021 39.606 40.293 41.784 43.186 44.546 45.263 46.672 47.108 50.105 51.273 52.135 54.958

341.0 341.0 331.0 169.0 315.1 577.0 535.0 719.0 163.1 561.0 451.0 289.0 577.0 577.0 729.0 561.0 487.0 881.0 289.0 469.1 833.0 441.0 727.0 441.0 441.0 577.0 447.0 447.0 741.0 455.0 727.0 727.0 741.0 431.0 561.0 431.0 463.0 609.0 469.0 301.0 447.0 447.0 593.0 331.0

178.9 178.9 168.9 – 152.9 424.9 266.9 451.0 – 289.0 288.9 – 424.9 424.9 577.0 289.0 443.0 – – 318.9 543.0 288.9 454.9 288.9 288.9 424.9 326.9 326.9 468.9 288.9 454.9 454.9 468.9 310.9 289.0 310.9 300.9 300.9 318.9 178.8 283.9 300.8 284.9 168.9

160.9 143.0 160.9 143.0 125.0 164.9 178.9 407.0 450.9 288.9 248.8 398.9 515.8 266.9 271.0 434.9 329.0 245.0 407.0 407.0 559.0 271.0 178.9

450.9 450.9 407.0 434.9 135.0

270.9 707.1 168.9 572.9 168.9 168.9 407.0 356.9 356.9 605.0 182.9 572.9 572.9 605.0 282.9 271.0 282.9 178.9 178.9 270.9 150.8 254.9

425.0 136.9 330.8 406.9 125.0 330.8 450.9 392.9 392.8 252.9 406.9 406.9 252.9 340.9 434.9 340.9

288.9 288.9 424.9 288.9 329.0 178.8

125.0 288.9 125.0 288.9

271.0 310.9 600.9 288.9 600.9 288.9 271.0 310.9 329.0

425.0 136.9 254.8 326.8 384.8

198.9 125.0

MS3

Compounds

– – – – – – – – – – 288.9 – – – – – – – – – 543.0 – 454.9 – – – 326.9 326.9 468.9 – 454.9 454.9 468.9 310.9 – 310.9 300.9 300.8 – – – 300.8 – –

Caffeic acid hexose Caffeic acid hexose Dihydroxy-trimethoxysilane isoflavan Gallic acid 2-Hydroxy-3-O-glucopyranoside-benzoic acid Epicatechin-(4–8)-epicatechin nd Catechin glucoside-6-O-3-O-rutinoside p-Coumaric acid (Epi) Avram catechin-(Epi) catechin Catechin glucoside Catechin Epicatechin-(4–8)-epicatechin Epicatechin-(4–8)-epicatechin Epicatechin-(4–8)-epigallocatechin-3-O-gallate acid (Epi) Avram catechin-(Epi) catechin 1-O-Caffeoyl-6-O-rhamnopyranosyl-glycopyranoside Epicatechin-gallate Catechin Epicatechin-O-3,4-dimethyl gallic acid Epiafzelechin-epicatechin-epicatechin Epicatechin-gallate Epiafzelechin-epicatechin-O-methyl gallic acid Epicatechin-gallate Epicatechin-gallate Epicatechin-(4–8)-epicatechin Orientin/isorientin Orientin/isorientin Epiafzelechin-epicatechin-O-dimethyl gallic acid (
)-Epicatechin-3-(300 -O-methyl)-gallic acid Epiafzelechin-epicatechin-O-methyl gallic acid Epiafzelechin-epicatechin-O-methyl gallic acid Epiafzelechin-epicatechin-O-dimethyl gallic acid Vitexin/isovitexin (Epi) Avram catechin-(Epi) catechin Vitexin/isovitexin Hyperin Rutin Epicatechin-O-3,4-dimethyl gallic acid Quercetin Orientin/isorientin quercitrin Kaempferol-3-rutinoside Dihydroxy-trimethoxysilane isoflavan

244.9 204.9 178.9

270.8 416.9 524.9 431.0 288.9 270.9 314.8

298.9 283.8 132.9 298.9 283.8 132.9 318.9 425.0 270.9 137.0 288.9 288.9 318.9 282.9

270.9 270.9 425.0 135.0

314.8 314.8 270.9 137.0 122.9

282.9 135.0 122.9 178.8 150.8 120.9 178.8 150.9

178.8 150.9 270.8

The peaks marked by ‘‘*” correspond to the species which were quantified in the following tables.

distribution in different fractions of seeds were quantified and shown in Tables 2–4. The content of vitexin/isovitexin, hyperin and rutin in the hull differed significantly among the eight buckwheat cultivars. Vitexin and iso-vitexin ranged from 101.65 to 188.78 mg/100 g with an average content of 146.26 mg/100 g. Hull of 9978 had the highest total vitexin/isovitexin content followed by 9976, while the lowest

total content of vitexin/isovitexin was found in the hull of Honghua. Hyperin ranged from 53.55 to 274.10 mg/100 g (average content 118.06 mg/100 g). The hull of Yuqiao No. 4 had the highest hyperin content, which was more than 5 times higher than that of Honghua with the lowest content. Rutin content in hull samples ranged from 62.43 to 173.57 mg/100 g (average content 109.63 mg/100 g). The content ranges of epicatechin-gallate,

Table 2 Content of major polyphenols in hulls of common buckwheat seeds (mg/100 g). Cultivars

9978 9976 Sichuan Xingnong No. 1 Yuqiao No. 4 Baihua Honghua Yunnan A

Polyphenol compoundsA 1-O-Caffeoyl-6-Orhamnopyranosylglycopyranoside

Epicatechin-gallate

Orientin/isorientin

Vitexin/isovitexin

Hyperin

Rutin

9.10 ± 1.21a 5.30 ± 1.02abc 2.43 ± 0.56bc 2.16 ± 0.79ab 0.60 ± 0.20c 0.28 ± 0.06c 2.43 ± 0.56bc 0.39 ± 0.10c

91.33 ± 2.17a 47.85 ± 1.87b 47.03 ± 3.64b 25.37 ± 1.07e 35.09 ± 2.60cd 31.79 ± 4.19d 38.30 ± 3.93c 25.86 ± 3.14e

16.80 ± 4.09c 21.01 ± 3.51b 9.32 ± 4.91d 26.34 ± 5.20a 26.07 ± 4.39a 13.21 ± 4.62c 8.41 ± 2.66d 15.77 ± 3.26c

188.78 ± 8.12a 179.74 ± 4.24b 106.80 ± 3.42f 154.46 ± 4.11d 119.36 ± 3.83e 155.51 ± 5.52cd 101.65 ± 4.91f 163.84 ± 4.19c

126.30 ± 5.95c 126.65 ± 6.27c 61.01 ± 4.21e 165.68 ± 8.29b 274.10 ± 11.82a 73.78 ± 2.84d 53.55 ± 3.06e 63.38 ± 6.52de

152.66 ± 3.66b 114.57 ± 1.33c 105.97 ± 3.17d 118.51 ± 5.43c 173.57 ± 4.71a 71.20 ± 7.12e 78.19 ± 4.78e 62.43 ± 4.37f

Values in each column with the different letters are significantly different (p < 0.05).

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Table 3 Content of major polyphenols in bran of common buckwheat seeds (mg/100 g). Cultivars

9978 9976 Sichuan Xingnong No. 1 Yuqiao No. 4 Baihua Honghua Yunnan A

Polyphenol compoundsA 1-O-Caffeoyl-6-Orhamnopyranosylglycopyranoside

Epicatechin-gallate

Orientin/isorientin

Vitexin/isovitexin

Hyperin

rutin

46.91 ± 4.67a 25.72 ± 3.64e 38.24 ± 3.24bc 36.79 ± 2.56bc 29.36 ± 2.89e 34.37 ± 3.67bcd 46.41 ± 4.87a 30.45 ± 4.32cde

263.48 ± 12.40bc 150.44 ± 9.94e 354.67 ± 15.24a 246.92 ± 9.43cd 241.00 ± 7.71d 257.94 ± 10.04bcd 266.20 ± 8.18b 248.87 ± 13.77bcd

6.75 ± 1.98a 5.21 ± 1.42a 4.99 ± 0.67a 6.69 ± 1.49a 4.62 ± 1.06a 6.09 ± 2.83a 5.11 ± 2.05a 6.83 ± 2.41a

19.25 ± 2.33b 15.83 ± 3.38b 30.45 ± 2.88ab 37.97 ± 3.71ab 31.52 ± 4.28a 44.77 ± 5.92ab 35.69 ± 3.18ab 54.40 ± 3.35ab

17.46 ± 2.67ef 12.69 ± 1.45f 18.49 ± 3.65de 26.52 ± 3.90bc 36.51 ± 2.36a 22.71 ± 1.09cd 17.61 ± 1.42ef 28.62 ± 3.73ab

53.22±2.92e 45.22 ± 1.46e 143.88 ± 4.26b 81.68 ± 6.62d 111.23 ± 4.69c 186.54 ± 5.40a 141.75 ± 4.75b 137.57 ± 6.06b

Values in each column with the different letters are significantly different (p < 0.05).

Table 4 Content of major polyphenols in endosperm of common buckwheat seeds (mg/100 g). Cultivars

9978 9976 Sichuan Xingnong No. 1 Yuqiao No. 4 Baihua Honghua Yunnan A

Polyphenol compoundsA 1-O-Caffeoyl-6-Orhamnopyranosylglycopyranoside

Epicatechin-gallate

Orientin/isorientin

Vitexin/isovitexin

Hyperin

rutin

7.22 ± 2.46ab 9.46 ± 2.67a 5.26 ± 1.42bcd 6.25 ± 1.65bc 2.84 ± 0.43de 6.26 ± 1.60bc 3.71 ± 1.24cde 2.14 ± 0.87e

48.87 ± 4.32b 73.92 ± 3.19a 48.36 ± 1.89b 43.50 ± 2.40c 23.08 ± 1.57e 42.35 ± 3.52c 29.75 ± 1.07d 19.65 ± 1.49e

0.99 ± 0.26a 1.05 ± 0.27a 0.40 ± 0.10bc 0.66 ± 0.29b 0.45 ± 0.07bc 0.46 ± 0.05bc 0.45 ± 0.04bc 0.30 ± 0.05c

2.41 ± 0.67b 4.00 ± 0.73ab 2.32 ± 0.55b 2.92 ± 0.31a 1.61 ± 0.32b 3.01 ± 1.04ab 2.26 ± 0.44b 1.82 ± 0.56b

1.70 ± 0.21bc 4.54 ± 1.21a 2.56 ± 0.21b 3.97 ± 0.36a 4.50 ± 0.82a 1.75 ± 0.31bc 1.17 ± 0.12c 1.33 ± 0.52c

14.45 ± 2.20c 27.40 ± 1.99b 47.57 ± 3.09a 17.70 ± 2.27c 15.27 ± 3.40c 29.60 ± 1.58b 28.56 ± 3.53b 17.71 ± 1.17c

Values in each column with the different letters are significantly different (p < 0.05).

orientin/isorientin and 1-O-Caffeoyl-6-O-rhamnopyranosyl-glyco pyranoside in hulls were 25.37–91.33 mg/100 g, 8.41– 26.34 mg/100 g and 0.28–9.10 mg/100 g, with average contents of 42.93, 17.11 and 2.84 mg/100 g, respectively. Epicatechin-gallate was the major phenolic in the bran, ranging from 150.44 to 354.67 mg/100 g with an average value of 253.69 mg/100 g. As shown in Table 3, bran of Sichuan cultivar had the highest content, and 9976 had the lowest content of epicatechin-gallate. Rutin ranked as the second highest phenolic with its content ranging from 45.22 to 186.68 mg/100 g and with an average value of 112.63 mg/100 g, which was similar to its hull content. Rutin was highest in Baihua and lowest in 9976 bran fractions. The average content of 1-O-Caffeoyl-6-O-rhamnopyranosylglycopyranoside (36.03 mg/100 g) in bran flours was higher than that of vitexin/isovitexin (33.73 mg/100 g) and hyperin (22.57 mg/100 g). Orientin/isoorientin ranked the lowest in brans ranging from 4.62 to 9.83 mg/100 g. Epicatechin-gallate and rutin were the two major phenolics in buckwheat endosperm (Table 4) ranging from 19.65 to 73.92 mg/100 g and from 14.45 to 45.75 mg/100 g with average values of 41.18 mg/100 g and 24.78 mg/100 g, respectively. The content range of 1-O-Caffeoyl-6-O-rhamnopyranosyl-glycopyrano side was 2.14–9.46 mg/100 g and the average value was 5.39 mg/100 g. The content of vitexin/isovitexin and hyperin were in similar ranges with average contents of 2.54 mg/100 g and 2.69 mg/100 g, respectively. The total content of orientin/isorientin was much lower than other phenolics with a range of 0.30– 1.05 mg/100 g (average 0.59 mg/100 g). The results indicate that bran and endosperm have a similar polyphenol profile, in which epicatechin-gallate and rutin are predominant phenolics and orientin/isorientin being minor components. Average contents of epicatechin-gallate and rutin in bran were about 6 and 4.5 times as much as those in endosperm flour

respectively. Meanwhile, vitexin/isovitexin, heprin and rutin were predominant phenolics in hulls, which show a completely different polyphenol profile compared to bran and endosperm flours. The average contents of vitexin/isovitexin and hyperin in hulls were 4.2 and 5.2 times as much as those in brans, while epicatechingallate average value in hull was less than one fifth of that in bran. 1-O-Caffeoyl-6-O-rhamnopyranosyl-glycopyranoside was a minor component in the hull, the content of which even less than that of endosperm except for the seed of 9978. Rutin had a similar content range in hull as in bran. It is interesting to note that for cultivars 9976, 9978, Xingnong No. 1, and Yuqiao No. 4, the rutin content in the hull was significantly higher than that in bran, but the reverse was found for the cultivars Honghua, Baihua and Yunnan. Buckwheat belongs to the polygonacea family, within which more than 10 varieties have been found. Common buckwheat and tartary buckwheat are two wildly cultivated species. The polyphenol composition in tartary buckwheat has been extensively studied, with rutin identified as the major phenolic. Polyphenol composition in common buckwheat is more complicated with limited studies reported. Lee et al. (2016) compared the contribution of flavonoids to the antioxidant activities of common buckwheat and tartary buckwheat from Korea. The results indicated rutin to be the major phenolic in both hull and groat fractions, while the corresponding TPC in the hull were much lower than our results. Similarly, Sedej et al. (2012) reported that rutin content in the hull, groat and whole grain of common buckwheat seeds from Ukraine was the highest among all phenolics. Quettier-Deleu et al. (2000) found proanthocyanidin dimer B2 to be the predominant phenolic in the hull of the common buckwheat seed from France. Kiprovski et al. (2015) quantified phenolics in 12 common buckwheat varieties from Europe, and they found all varieties could be divided into two groups, one with high propelargonidins content and the

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other with high proanthocyanidin content. Wide diversity in the rutin content was found with two cultivars having extremely high content, which is similar to our results. The concentrations of orientin and isoorientin in the report of Kiprovski et al. (2015) had a similar range as the endosperm flour in our study. The individual proanthocyanidin was not quantified in this study, but we did find the hull of buckwheat Yuqiao No. 4 to contain extremely high TPC and TPAC contents. It is also well established that plant phenolic content depends on a number of factors such as variety, location and environmental conditions (Guo et al., 2011), which could explain the large variation reported in different studies. Even though genotype and environment interaction had significant effect on grain phytochemicals contents (Dwivedi et al., 2016), some research showed that environment had greater contribution than genotype (Kim, Kim, Kim, & Chung, 2012). In this study, the cultivars from Yunnan showed different phenolic distributions compared to others. Weather this is resulted from the effect of genotype and environment interaction is unknown. Further studies are required to establish how the genetic and environmental conditions affect the distribution and accumulation of polyphenols in different fractions of common buckwheat cultivars. 4. Conclusions The polyphenol profiles as well as TPC and TPAC of common buckwheat hull, bran and endosperm were analyzed. We found that the hull differed in polyphenol profile from bran and endosperm. The contents of six major phenolics varied considerably in same fractions among the eight buckwheat cultivars. The wide diversity and high content of polyphenols found in seed fractions, especially in the hull suggest that they could be utilized as ingredients in the development of functional foods. Acknowledgements We are grateful to the financial supports from the National Key R & D Program (2016YFD0400104) of China, the Fundamental Research Funds for the Central Universities at Zhejiang University (2016XZZX001-09), Hangzhou, China and the Key Natural Science Foundation (11KJA550001) funded by Jiangsu Provincial Department of Education, China. Conflict of interest The authors declare that there is no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jff.2017.09.024. References Adom, K. K., & Liu, R. H. (2002). Antioxidant activity of grains. Journal of Agricultural and Food Chemistry, 50, 6182–6187. Butler, L. G., Price, M. L., & Brotherton, J. E. (1981). Vanillin assay for proanthocyanidins (condensed tannins): Modification of the solvent for estimation of the degree of polymerization. Journal of Agricultural and Food Chemistry, 30, 1087–1089. Cavaliere, C., Foglia, P., Pastorini, E., Samperi, R., & Laganà, A. (2005). Identification and mass spectrometric characterization of glycosylated flavonoids in Triticum durum plants by high-performance liquid chromatography with tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 19, 3143–3158.

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