Changes in phenolic content, phenylalanine ammonia-lyase (PAL) activity, and antioxidant capacity of two buckwheat sprouts in relation to germination

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Changes in phenolic content, phenylalanine ammonia-lyase (PAL) activity, and antioxidant capacity of two buckwheat sprouts in relation to germination Shun-Cheng Rena,*, Jun-Tao Sunb a

School of Food Science and Technology, Henan University of Technology, Zhengzhou 450001, PR China College of Food Science and Engineering, Xuchang University, Xuchang 461000, PR China

b

A R T I C L E I N F O

A B S T R A C T

Article history:

Total phenol, total flavonoid, rutin and quercetin contents as well as antioxidant activity in

Received 4 November 2013

ethanol extracts of common buckwheat (Fagopyrum esculentum Moench) and tartary

Received in revised form

buckwheat (Fagopyrum tararicum Gaertn) sprouts were determined, and the phenylalanine

28 January 2014

ammonia-lyase (PAL) activity in the sprouts were estimated. Total flavonoid content and

Accepted 28 January 2014

rutin in the sprouts of both buckwheat species gradually increased and reached the max-

Available online 22 February 2014

imum levels on days 7 and 9 of germination, respectively; total phenol and free phenylalanine content also had the same trend. However, quercetin in tartary buckwheat decreased

Keywords:

from day 5 and became undetectable on day 7, similar to that observed in common

Buckwheat sprouts

buckwheat sprouts beyond the germinating period. An obvious positive linear relationship

Phenols

(r2 = 0.9792, P < 0.01) was found between PAL activity and flavonoid accumulation. The phe-

PAL activity Antioxidant activity Radical-scavenging activity

1.

nolic extracts of the sprouts of both buckwheat species exhibited strong antioxidant activity. This study suggests that buckwheat sprouts are a good health-promoting food source.

Introduction

Buckwheat belongs to the plant family of Polygonaceae. Both Fagopyrum esculentum and Fagopyrum tartaricum, which are known as common and tartary buckwheat, respectively, are usually cultivated (Krkosˇkova´ & Mra´zova´, 2005). The main producers of buckwheat are China, the Russian Federation, Ukraine, and Kazakhstan (Bonafaccia, Marocchini, & Kreft, 2003; Li & Zhang, 2001), but it is also harvested in Slovenia, Poland, Hungary, and Brazil (Kreft et al., 1999). Buckwheat is an important medicinal and edible plant that is rich in protein, vitamins B1, B2, and PP, as well as iron, zinc,

* Corresponding author. Tel.: +86 371 68883238; fax: +86 371 67789817. E-mail address: [email protected] (S.-C. Ren). http://dx.doi.org/10.1016/j.jff.2014.01.031 1756-4646/ 2014 Elsevier Ltd. All rights reserved.

 2014 Elsevier Ltd. All rights reserved.

selenium, and other trace elements. Buckwheat has a high antioxidant activity and is rich in rutin, catechins, and other polyphenols, which have significant dietary value (Oomah & Mazza, 1996; Watanabe, 1998). The main flavonoid of buckwheat also has a positive effect on reducing capillary vessel brittleness, improving microcirculation, and strengthening body immunity (Tian & Ren, 2007). However, the digestibility of buckwheat protein is relatively low because of the existence of proteinase inhibitors (Ikeda, Arioka, Fujii, Kusano, & Oku, 1984). Buckwheat germination can reduce the content of proteinase inhibitors and improve the quality of protein. In addition, the nutritional value of fatty acid composition can

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also be improved during seed germination (Cheng, Chen, & Liu, 2003a; Ikeda et al., 1984). Bean sprouts, which are rich in dietary fibre, various nutrients, and bioactive components, are important food of plant origin consumed in Asian countries. Beans have now become more popular in the United States and European countries. Although the most popular bean sprouts are cultivated from mungbean and soybean, buckwheat seeds are also a good source of sprouts. At present, several studies about the flavonoid content and antioxidation activity of buckwheat grains have been reported. However, reports about the influence of buckwheat seed germination on the change in rutin and quercetin content are few. The present study aims to reveal these changes and to study the antioxidant activities of buckwheat sprouts to have a better understanding of their health-promoting food source properties.

2.

Materials and methods

2.1.

Buckwheat materials and chemicals

Jinqiao No. 1 (common buckwheat, Fagopyrum esculentum Moench) and Jinqiao No. 2 (tartary buckwheat, Fagopyrum tararicum Gaertn) were both purchased from Shanxi Academy of Agricultural Science (Taiyuan, China). Rutin (>99% purity) was purchased from Sigma Chemicals Co. (St. Louis, MO, USA); quercetin (>99% purity) was purchased from Sinopharm Chemical Reagent Co., Ltd., (Shanghai, China); and 1,1–diphenyl–2–picrylhydrazyl (DPPH) was purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.

2.2.

Sample preparation

2.2.1.

Preparation of buckwheat flour and bran

Buckwheat seeds were dried at 50 C in an electric blast drying oven and then crushed using a mortar. Buckwheat hull were sought out by forceps, and the buckwheat bran were separated using a sifter. Finally, buckwheat flour and bran were crushed to 200 meshes by using a high-speed pulverizer (FW-200, Beijing Zhongxingweiye Instrument Co., Ltd., Beijing, China) and stored at 4 C until use.

2.2.2.

Preparation of buckwheat sprouts

Fifteen grams of buckwheat seeds, including common buckwheat and tartary buckwheat, were soaked in water for 8 h at room temperature, placed into a plate for germination in the same environmental conditions (namely at 25 C, with 12 h sunlight and roughly 75% humidity), and watered every 4 h with de-ionized water (mean pH 6.0) or calcium ion water. Buckwheat sprouts were harvested after a few days of seeding, washed, and immediately dried in a freeze-dryer (FD12-2S-6P-S, Kingmech Co., Taipei, Taiwan). The dried buckwheat sprouts were powdered to 200 meshes by using a high-speed pulverizer (FW-200, Beijing Zhongxingweiye Instrument Co., Ltd., Beijing, China) and stored at 4 C until use.

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2.3.

Determination of phenolic content

2.3.1.

Preparation of extracts

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One gram each of buckwheat flour, bran, or sprouts was extracted with 95% ethanol (m/v, 1:20) at 70 C for 6 h in a water boiler at constant temperature. The ethanol solution was filtered using a vacuum pump, moved into a 100 mL container filled with 95% ethanol, and stored at 4 C until use (Liu, Chen, Yang, & Chiang, 2008; Umma, Salma, Shinya, Emiko, & Murakami, 2012).

2.3.2.

Determination of total phenol content

The total phenols were estimated via the Folin–Ciocalteu method (Singleton, Orthofer, & Lamuela-Ravento 0 s, 1999). Approximately 50 lL of the sample were added to 250 lL of undiluted Folin–Ciocalteu phenol reagent. After 1 min, 750 lL of 20% (w/v) aqueous Na2CO3 were added, and the volume was made up to 5.0 mL with H2O. The control samples contained all the reaction reagents except for the extract. After 2 h of incubation at 25 C, the absorbance was read at 760 nm on a spectrophotometer (UV-2000, Unico (Shanghai) Instrument Co., Ltd., China) and compared with a gallic acid calibration curve. Total phenols were expressed as gallic acid equivalents (mg gallic acid eq/g buckwheat sprouts powder), and the values are presented as means of triplicate analyses.

2.3.3.

Determination of total flavonoid content

The total flavonoid content was measured via the aluminum nitrate colorimetry method (Xue, Yuan, Wang, Yao, & Niu, 2006). An aliquot (1 mL) of phenolic extract was mixed with 1.4 mL of solution containing 25 g/L sodium nitrite and 50 g/L aluminum nitrate. After 6 min, 5 mL of 1 M sodium hydrate solution were added up to a total volume of 25 mL with 95% ethanol. The solution was mixed and incubated at room temperature for 10 min. The absorbance was read at 510 nm on a spectrophotometer (UV-2000, Unico (Shanghai) Instrument Co., Ltd., China), and the flavonoid content was calculated with a calibration curve of rutin and expressed as mg of rutin equivalents per g of buckwheat sprout powder.

2.3.4. Determination of rutin and quercetin via highperformance liquid chromatography (HPLC) Buckwheat seed (or sprouts) contain many kinds of phenolic compounds. Rutin and quercetin are the most typical active components in buckwheat seed (or sprouts) for their strong health care functions. In fact, rutin, comprised of quercetin and rhamnose, was usually used for an adjuvant therapy of hypertension and diabetes in clinical (Tian & Ren, 2007). Therefore, the changes in rutin and quercetin contents were monitored during buckwheat germination by the following method. Phenolic extract was filtered through a nylon filter (0.45 lm). The rutin and quercetin contents in the extracts were determined via HPLC (Hewlett Packard series 1100, Agilent Technologies, Inc., Santa Clara, CA, USA) by using the rutin and quercetin standard curve method. Chromatogram conditions: HPLC was equipped through the betabasic-C18 column (250 nm · 456 mm i.d., Thermo Finnigan Company, Waltham, MA, USA). The column was packed with a particle stationary phase with a diameter of 5 lm. The column oven was set to 50 C, ultraviolet detection was at 350 nm, and

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the injection volume was 5 lL. The mobile phase consisted of 0.2% acetic acid in water (mobile phase A) and of 0.2% acetic acid in methanol (mobile phase B), and the gradient program was 20% to –60% B at 20 min.

2.4. Extraction and assays of phenylalanine ammonialyase (PAL) Phenylalanine ammonia-lyase was extracted according to Liu, Li, and Chen (2005). All steps were processed at 4 C. Buckwheat sprouts (1 g of fresh weight) were ground into powder in liquid nitrogen by using a mortar and pestle. The powder was extracted for 20 min with 5 mL of 0.1 mol/L borax-borate buffer (pH 8.7) containing 20 mmol/L mercaptoethanol and 1 mmol/L of ethylenediaminetetraacetic acid disodium salt (Na2 EDTA). The enzyme extract was centrifuged for 30 min at 10,000 · g, and the supernatant was collected for the determination of PAL activity. PAL activity was assayed based on the method of Modern Plant Physiology Laboratory Manual (2004) by using a reaction mixture of 3.8 mL of 0.1 M borax-borate buffer (pH 8.7) containing 1 mL of 0.6 mM l-phenylalanine and 0.2 mL of enzyme solution. The samples were incubated for 1 h at 40 C. In the control sample, the enzyme extract was replaced by 0.2 mL of 0.1 M borax-borate buffer (pH 8.7). The reaction was stopped by adding 0.2 mL of 6 M trichloroacetic acid. One unit of enzymatic activity was defined as the amount of the enzyme that caused a change of 0.01 in the absorbance at 290 nm within an hour, and results were converted into per unit dry weight.

2.5.

Determination of free phenylalanine content

Phenylalanine content was determined by L-8800 high-speed amino acid analyzer (Hitachi, Tokyo, Japan). One gram each of buckwheat flour, bran, or sprouts were diluted with 10 ll of 3% trichloroacetic acid solution. The sample was left at the room temperature for 1 h, centrifuged at 10,000 g for 15 min. The collected supernatant was filtered with Millipore 0.45 lm syringe filters (Milford, MA, USA). The filtrate was loaded on amino acid analyzer (Kim, Kim, Park, 2004b). The standard amino acid solutions were obtained from Sigma Chemicals Co. (St. Louis, MO, USA).

2.6.

Evaluation of the DPPH radical-scavenging activity

The DPPH radical scavenging activity of buckwheat sprout extracts was according to Sa´nchez-Moreno, Larrauri, and SauraCalixto (1999), with a slight modification. An aliquot of 7.8 mL of 25 mg/L DPPH in ethanol was added to 0.2 mL of the extracts. The mixture was then shaken well and incubated at 30 C. A spectrophotometer was used to record the absorbance at 517 nm every 10 min after the initial mixing and recorded until the endpoint of 40 min. The absorbance of the control sample was read using the same procedure, except that ethanol was used instead of the extracts. The degree of decoloration of the solution indicates the scavenging efficiency of the extracts. DPPH radical-scavenging activity was calculated using the following equation:   Ai % discoloration ¼ 1   100% A0

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where Ai denotes the absorbance for buckwheat sprout phenolic extract and A0 denotes the absorbance for the control sample. Each sample was carried out in triplicate, and the average was used to calculate for the first-order derivative of absorbance, which was plotted against incubation time to illustrate the kinetic scavenging rate of legume extracts.

2.7. Evaluation of the Inhibitory effect on lipid oxidation by the Rancimat method The Rancimat test of the samples was performed using a 743 Rancimat analyzer based on the method of Ho, Chen, Shi, Zhang, and Rosen (1992) with a minor modification. Because commercially commercial vegetable oil often contain antioxidants, lard was freshly made as the source of oxidants. Aliquots (3 mL) of phenolic extracts with freshly lab made lard (3 g) were subjected to oxidation at 110 C (air flow 20 L/h). A control sample containing 95% ethanol instead of phenols was used. Induction periods (h) were automatically recorded.

2.8.

Statistical analysis

Three independent trials (n = 3) with triplicate sample analyses were performed. Data were presented as the mean ± standard derivation. The P values less than 0.05 were regarded as significant differences, and the P values less than 0.01 were very significant differences.

3.

Results and discussion

3.1.

Phenolic content

3.1.1.

Total phenol content (TPC)

Phenolic substances are responsible for the antioxidant activity of plant materials (Rice-Evans, Miller, & Paganga, 1996). Therefore, the amount of total phenols in the buckwheat sprouts was investigated using the Folin–Ciocalteu method. During the test, the total phenol content in both common buckwheat and tartary buckwheat sprouts increased, and the total phenol level of both common buckwheat and tartary buckwheat on the ninth day were 7.7 and 3.3 times more than that of 1-day sprouts, respectively (Table 1). During buckwheat germination, phenolic accumulation increased with increasing flavonoid accumulation. An obvious positive linear relationship (r2 = 0.9943, P < 0.01) between phenolic accumulation and flavonoid accumulation suggested that the change in phenolic accumulation is involved with the change in flavonoid accumulation.

3.1.2.

Total flavonoid content (TFC)

The flavonoid content of buckwheat sprouts constantly varied during seed germination. The total flavonoid content decreased in the sequence of 7-day sprouts, bran, and flour (Table 1). During the early stage of germination, the total flavonoid content in both common buckwheat and tartary buckwheat increased during seed germination, and the total flavonoid level of both common and tartary buckwheat on the seventh day were 7.1 and 3.2 times more than that of

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Table 1 – Total phenolic (TPC), total flavonoid (TFC), and rutin and quercetin contents in buckwheat samples (mg/g) a. Common buckwheat TPC Flour Bran Sprouts

Day 1 Day 3 Day 5 Day 7 Day 9

1.9 ± 0.16 150.2 ± 6.01 53.1 ± 2.63C 56.3 ± 2.86C 132.0 ± 6.47B 402.3 ± 12.01A 406.5 ± 12.56A

TFC 0.4 ± 0.03 30.4 ± 1.22 11.2 ± 0.65C 11.6 ± 0.53C 26.5 ± 1.36B 79.9 ± 3.15A 77.6 ± 3.31A

Tatary buckwheat Rutin 0.14 ± 0.01 4.10 ± 0.17 0.30 ± 0.03D 0.39 ± 0.02D 2.41 ± 0.13C 5.97 ± 0.26B 7.60 ± 0.38A

Quercetin ND ND ND ND ND ND ND

b

TPC

TFC

Rutin

Quercetin

4.7 ± 0.21 324.7 ± 13.45 177.3 ± 7.43C 178.6 ± 8.72C 237.5 ± 9.58B 539.0 ± 21.84A 553.4 ± 23.15A

0.9 ± 0.06 72.5 ± 3.62 36.2 ± 1.83BC 36.2 ± 1.76C 47.6 ± 1.95B 115.0 ± 4.80A 108.2 ± 5.92A

1.36 ± 0.05 36.37 ± 1.45 17.82 ± 0.78C 17.20 ± 0.88C 22.51 ± 0.91B 41.46 ± 1.65A 42.24 ± 1.71A

ND ND 15.22 ± 0.68 15.48 ± 0.64 4.53 ± 0.17 ND ND

a Each value was expressed as mean ± standard deviation (n = 3). Means within the same sprout sample germination with different letters differ significantly (P < 0.05). b ND, not detected.

1-day sprouts, respectively. Our previous studies showed that sunlight, temperature, and calcium ion water had a great effect on the flavonoid content of buckwheat sprouts. Long exposure to sunlight, reasonable temperature, and calcium ion water can promote flavonoid synthesis in buckwheat sprouts (Sun, 2008).

3.1.3.

Content of rutin and quercetin

The rutin and quercetin contents in common buckwheat flour, bran, and sprouts are shown in Table 1. The rutin level was higher, especially in bran and sprouts, but no quercetin was detected. The rutin content increased with seed germination and reached 7.60 mg/g on the ninth day, which is 25 and 54 times more than those of 1-day sprouts and flour, respectively. The rutin and quercetin contents in the tartary buckwheat flour, bran, and sprouts are shown in Table 1. Quercetin was detected from 1-day to 5-day sprouts, but not in flour, bran, and 6-day sprouts. Rutin existed in the flour, bran, and sprouts, and the rutin level was higher in bran than in flour. The rutin content significantly increased with seed germination, especially from the fifth day to the ninth day, and the maximum concentration of rutin reached 42.24 mg/g on the ninth day, which was 2.4 and 31 times more than those of buckwheat flour and 1-day sprouts, respectively. Our research results were interesting. Although no quercetin was detected in tartary buckwheat flour and bran, quercetin can be found in sprouts during the early stages of germination. However, its concentration was low, and its content decreased gradually from 15.2 mg/g (1-day sprouts) to 0 mg/g (7-day sprouts), probably because more quercetin was used for the synthesis of rutin. However, no quercetin can be detected in common buckwheat flour, bran, and sprouts (Table 1) probably because the metabolisms in two different buckwheat varieties are different. Although rutin (retention time: 14.7 min) was the main flavonoid in common buckwheat flour, bran, and sprouts, other types of flavonoid probably showed an obvious difference. In other words, the HPLC results of common buckwheat flour, bran, and 1-day and 3-day sprouts was were similar but changed obviously from the 5-day sprouts (Fig. 1(a–f)). Rutin was also the main flavonoid in tartary buckwheat flour, bran, and sprouts; however, other types of flavonoid did not show

obvious difference, and the difference was whether the samples contained querentin or not (retention time: 20.4 min) (Fig. 1(g–l)). TFC was higher than the total content of rutin and quercetin (Table 1). Two reasons were suggested for this phenomenon as in follows: First, except for rutin and quercetin, other trace flavonoids, such as orientin, isoorientin, vitexin and isovietexin are also present in buckwheat seed (or sprouts) (Dorota & Wieslaw, 1999). Second, buckwheat seed (or sprouts) might contain some non-flavonoids (protocatechualdehyde, protocatechuic acid, caffeic acid, and chlorogenic acid) with strong absorption at 510 nm, which can affect the detection (Guo, Fan, Wang, & Zhang, 2002).

3.2. Relevance of PAL activity and total flavonoid and total phenol content During buckwheat germination, flavonoid accumulation increased as the PAL activity was enhanced. An obvious positive linear relationship (r2 = 0.9792, P < 0.01) between PAL activity and flavonoid accumulation suggested that the change in PAL activity is involved with the change in flavonoid accumulation (Fig. 2). Similarly, a positive linear relationship was also observed between PAL activity and phenolic accumulation (r2 = 0.9761, P < 0.01). PAL is the key synthesis enzyme of flavonoids and phenols, which can catalyze the L-phenylalanine produced by the shikimic acid pathway to remove ammonia residues and produce trans-cinnamic acid. Trans-cinnamic acid can be turned into an intermediate product via the phenylpropanoid metabolic pathway. The intermediate products include coumaric acid, asafetida, sinapic acid, and so on, which can further turn into coumarin, chlorogenic acid, CoA ester, and finally change into secondary metabolites, including lignin, flavonoids, and other compounds (Cheng et al., 2003b; Kim, Kim, & Park, 2004).

3.3.

Free phenylalanine content (FPC)

Table 2 shows the free phenylalanine content in buckwheat flour, bran, and in 1, 3, 5, 7, and 9-day buckwheat sprouts. The free phenylalanine content in buckwheat sprouts increased with seed germination and the total free phenylalanine level of both common and tartary buckwheat on the

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Fig. 1 – Selected HPLC chromatograms of phenolic extracts (a–f for common buckwheat flour, bran, and sprouts at days 1, 3, 5, and 9 of germination; g–l for tartary buckwheat flour, bran, and sprouts at days 1, 3, 5, and 9 of germination).

ninth day was almost 15 and 12 times higher than that of 1-day sprouts, respectively. The highly active PLA and sufficient quantity of free phenylalanine could promote the biosynthesis process for phenol and flavonoid compounds in buckwheat sprouts.

3.4. Antioxidant activities of buckwheat sprout phenolic extracts The DPPH method is related to the hydrogen-donating ability of phenols and the stability of the formed phenoxyl radicals (Rice-Evans et al., 1996). After 40 min of reaction, the percentage

of DPPH radical-scavenging is shown in Fig. 3. The phenol in buckwheat flour, bran, and sprouts exhibited DPPH radicalscavenging activity, but the differences are significant. The DPPH radical-scavenging activity of tartary buckwheat flour, bran, and 1–5-day sprouts were stronger than that in common buckwheat. The scavenging activity gradually increased as germination progressed, and the strongest radical-scavenging activity was reached from 7-day to 9-day sprouts. For example, the radical-scavenging activity of tartary buckwheat sprouts increased to 99.7% on the 7th day from 35.9% on the 1st day, and common buckwheat sprouts also exhibited similar radicalscavenging activity (increased to 98.8% on the 7th day from

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303

8000

Total phenol content Total flavonoid content

7000

y=62.593x+30.315 2 R =0.9743

6000

PAL activity (units/g sprous)

5000

y=12.618x+18.107 2 R =0.9698

4000 3000 2000 1000 0 0

100

200

300

400

500

600

Total phenol content(mg/g sprouts) 0

20

40

60

80

100

120

Fig. 4 – Antioxidation of buckwheat flour, bran, and sprouts phenolic extracts in lard system.

Total flavnoid content(mg/g sprouts)

Fig. 2 – Correlations between PAL activity and total flavonoid content, total phenol content during buckwheat germination.

Table 2 – Free phenylalanine content (FPC) in buckwheat samples (mg/100 g) a.

Flour Bran Sprouts

Day 1 Day 3 Day 5 Day 7 Day 9

Common buckwheat

Tatary buckwheat

2.5 ± 0.2 3.6 ± 0.3 6.1 ± 0.4D 14.3 ± 1.4C 21.2 ± 1.9C 54.8 ± 3.1B 90.5 ± 2.9A

3.8 ± 0.2 5.8 ± 0.3 10.3 ± 0.6E 21.6 ± 2.1D 33.4 ± 2.8C 87.6 ± 3.2B 123.2 ± 3.3A

a Each value was expressed as mean ± standard deviation (n = 3). Means within the same sprout sample germination with different letters differ significantly (P < 0.05).

buckwheat sprouts. TPC and TFC showed a similar correlation with the DPPH radical-scavenging activity (r2 = 0.9022 for TPC, P < 0.01, and r2 = 0.8991 for TFC, P < 0.01). The Rancimat method was used to monitor the change in the electrical conductivity of water. In this method, lipid oxidation resulted in the formation of volatile secondary oxidation products at elevated temperature and accelerated aeration (Ho et al., 1992). The inhibitory effect of phenolic extracts on oxidation-induced lipid fragmentation by the antioxidants was evaluated in the induction period (h) and shown in Fig. 4. When the phenolic extracts of buckwheat flour, bran, and sprouts were added to a lard system, the oxidation rate of the lard slowed down. Buckwheat flour, bran, and sprouts demonstrated antioxidation activity, and the antioxidation activities of common and tartary buckwheat sprouts were stronger than that of flour. The 9-day tartary buckwheat sprouts demonstrated the strongest antioxidation activity, and the phenolic extract can effectively prolong the induction period of lard oxidation from 2.7 to 7.1 h. TPC and TFC showed similar correlation with the induction period of lard oxidation (r2 = 0.7134 for TPC, P < 0.01, and r2 = 0.6966 for TFC, P < 0.01).

4.

Fig. 3 – DPPH radical-scavenging activity of buckwheat flour, bran, and sprouts phenolic extracts. 13% at day 1). This result may be attributed to the fact that the amount of other flavonoid ingredients significantly increased during common buckwheat germination (Fig. 1(e and f)), although they had lower TFC (or TPC) levels than tartary

Conclusion

This study demonstrated that during the early stage of seed germination, TPC, TFC and FPC gradually increase and reach the highest level on days 9, 7 and 9, respectively. In common buckwheat flour, bran, and sprouts, rutin can be detected, but no quercetin was detected. Quercetin can be detected in tartary buckwheat sprouts from days 1 to 5, but none was detected in flour, bran, and 6-day sprouts. The maximum concentration of rutin was reached on day 9. An obvious positive linear relationship between PAL activity and phenolic accumulation (r2 = 0.9761, P < 0.01) and between PAL activity and flavonoid accumulation (r2 = 0.9792, P < 0.01) suggests that the change in PAL activity was probably involved with the change in phenolic (or flavonoid) accumulation. Buckwheat sprouts exhibited the strongest antioxidant activity on day 9. A significant correlation existed between DPPH free radical scavenging and TPC (r2 = 0.9022, P < 0.01), induction period of lard oxidation and TPC (r2 = 0.7134, P < 0.01), DPPH radical scavenging and TFC (r2 = 0.8991, P < 0.01), and

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induction period of lard oxidation and TFC (r2 = 0.6966, P < 0.01). Thus, findings of this study demonstrated that buckwheat sprouts are excellent sources of health-promoting food.

Acknowledgements We thank Dr. Zelong Liu for helpful advice and discussions. This research was financially supported by the Doctoral Foundation of Henan University of Technology grants 150164 (to S. R.).

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