Isoflavone Content and Composition in Chickpea ( Cicer Arietinum L.) Sprouts Germinated Under Different Conditions

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Isoflavone Content and Composition in Chickpea ( Cicer arietinum L.) Sprouts Germinated under Different Conditions

ARTICLE in JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY · JANUARY 2015 Impact Factor: 3.11 · DOI: 10.1021/jf5057524 · Source: PubMed

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Isoﬂavone Content and Composition in Chickpea (Cicer arietinum L.) Sprouts Germinated under Diﬀerent Conditions † ‡ † § ∥ § Yue Gao, , Yang Yao, , Yingying Zhu, and Guixing Ren*, ‡ Qilu University of Technology, 3501 Daxue Road, Western University Science Park, Jinan, Shandong 250353, People’s Republic of China § Institute of Crop Science, Chinese Academy of Agricultural Sciences (CAAS), 80 South Xueyuan Road, Haidian, Beijing 100081, People’s Republic of China ∥ Laboratory of Biophysics and Formulation Engineering, Gembloux Agro-Bio Tech, University of Liege,̀ Passage des Deporté s2,́ B-5030 Gembloux, Belgium

ABSTRACT: The influence of different germination conditions on isoflavone contents in chickpea sprouts was investigated in this study. Chickpea sprouts were germinated under different experimental conditions, including germination in the dark (GD), in the light (GL), under ethanol stress (GE), or under salt stress (GS) in the dark. The results demonstrated that the isoflavone contents in chickpea sprouts germinated with these various conditions significantly increased (p < 0.05) compared to those in untreated chickpea seeds. The maximum amount of total isoflavones was obtained from chickpea sprouts in the GL group on day 8. The contents of formononetin and biochanin A in this group were 154 and 130 times higher, respectively, than in untreated seeds and 1.2 times higher than in sprouts in the GD group. Moreover, the isoflavone contents of chickpea sprouts in the GE and GS groups were also higher (p < 0.05) than those in the GD group. A solution of 3% ethanol and 0.03 mol/L salt seemed to be the most optimal for isoflavone production among the solutions selected for this study. Most of the isoflavone contents significantly increased (p < 0.05), especially formononetin and biochanin A, while the genistein content decreased with germination. Ononin, pseudobaptigenin, and glycitein glucoside acetylated were only detected in germinated chickpeas. This finding could expand the potential for the development of chickpea sprouts as a functional food. KEYWORDS: germination, external stress, isoflavones, chickpea sprouts

■ INTRODUCTION associated with a lower risk of type 2 diabetes among ≥ 2 Chickpeas (Cicer arietinum L.) are an ancient pulse crop and are overweight women [body mass index (BMI) 25 kg/m ]in Japan.12 It is reported that the amount of isoflavones in legumes widely consumed because of their high nutritional value. ff 13 Chickpeas are a rich source of dietary protein and are used as a varies greatly at di erent growth stages of the plant. Additionally, both the isoflavone contents and composition protein supplement in many countries, including Pakistan, 14 India, and European countries.1 Sprouting is a traditional can be altered by germination. The study by Sharma had observed that isoflavones in chickpeas increased during processing method used to convert chickpeas into consumable 15 7 forms.2 Sprouted chickpea seeds are eaten as a vegetable or germination. In the study by Wu et al., who investigated 3 sprouted chickpeas germinated with water in the dark, 25 added to salads according to Khattak et al. Germination is fl fl known to enhance the nutritional value of legumes by in- iso avones were detected and the total iso avone content creasing protein digestibility,4 essential amino acids,5 and increased more than 100 times compared to the untreated 6 fl chickpea seeds. ascorbic acid. Furthermore, the iso avone contents in fl chickpeas dramatically increase during germination.7 Current We have an interest in enriching the iso avone contents of chickpea sprouts, in view of a study by Simons et al.,16 who studies have also reported that the consumption of chickpea fl sprouts is strongly associated with a reduced risk of type 2 reported that iso avone contents and diversity could vary diabetes8 and gastrointestinal disorders.9 depending upon the experimental conditions of the induction fl process. Meanwhile, Aisyah et al.17 found that germination Iso avones, a large group of plant secondary metabolites, in fl legumes are known to be important components that exhibit increased the total iso avone content of soybeans over 2-fold strong antiestrogenic, antioxidative, and antimicrobial activ- compared to untreated beans and can be further enhanced with ities.10 Over the past few decades, isoflavones have attracted wounding and light treatments (3-fold). Moreover, Abdul fi ff et al.18 found that germination was delayed at lower salinity more attention because of their bene cial e ects and are ff believed to be responsible for the protective role of legumes levels and salt stress can a ect the formation of the metabolites against the development of type 2 diabetes. Isoflavones have a in the plant, especially in leguminous plants. similar structure to endogenous estrogen, which is reportedly beneficial for preventing and treating type 2 diabetes by Received: November 27, 2014 attenuating insulin resistance, improving insulin secretion, and Revised: January 28, 2015 increasing β-cell mass.11 An epidemiologic study has shown Accepted: January 29, 2015 that a higher intake of isoflavone-rich soy products was Published: January 29, 2015

However, only the influence of germination with water in the dark on the isoflavone contents of chickpeas has been studied. 0.20 b 0.21 d 0.16 e 0.11 f 0.21 c 0.22 a Therefore, in this study, we carried out a quantitative analysis of 0.03 g ± ± ± ± ± ± ± isoflavone contents and composition in chickpeas and chickpea sprouts by growing them in different conditions, including in light, under ethanol stress, and under salt stress. Employing this analysis, we determined the optimal germination time to nd = not detected. maximize isoflavone contents for the purpose of maximizing b 0.01 b 6.21 0.01 c 5.34 0.00 d 3.93 0.00 e 2.69 0.01 b 6.46 the nutraceutical benefits of chickpea sprout isoflavone con- 0.01 a 6.82 ± ± ± ± ± sumption. ± acetylated total < 0.05). p ■ MATERIALS AND METHODS glycitein glucoside Materials. Chickpeas, 88-1, were provided by the Institute of Food erent ( Crops, Xinjiang Academy of Agricultural Sciences in China. ff Reagent. Authentic standards of biochanin A and formononetin 0.01 c 0.17 0.00 d 0.15 0.00 e 0.05 0.01 a 0.17 0.01 b 0.18 ± ± ± ± were purchased from Sigma-Aldrich (Shanghai, China). Acetonitrile, ± cantly di

acetic acid, and methanol used were all high-performance liquid ﬁ chromatography (HPLC)-grade and purchased from Fisher Chemicals (Shanghai, China). NaCl, NaSO4, NaClO, and ethanol were all analytical-grade and purchased from the Beijing Chemical Industry Group (Beijing, China). 0.00 a 0.05 0.00 a nd 0.03 0.00 b nd nd 0.60 ± ± Germination of Chickpeas. Chickpea treatments were performed ± in sterilized sprouting cartridges, which accommodated 300 g of dry chickpeas. The chickpeas were subjected to four diﬀerent treatments: germination in the dark (GD), germination in the light (GL), germination under ethanol stress (GE), or germination under salt 0.03 b nd 0.13 0.05 c nd 0.08 0.04 d 0.01 0.03 b nd 0.14 0.03 e 0.01 0.05 a nd 0.14 stress (GS) in the dark. The treatment methods and experimental 0.01 e 0.06 ± ± ± ± ± ± ± stages are presented in Table 1. In all of the treatments, chickpeas were a

a Table 1. Summary of Diﬀerent Chickpea Treatments

stage 0.04 b 1.95 0.03 c 1.61 0.03 c 1.32 0.05 a 1.99 0.03 d 0.61 0.04 a 2.10 0.01 d 0.18 ± ± ± ± ± ± treatment soaking germination ethanol soaking ± untreated −−− GD √√(dark) − GL √√(light) − GE √√(dark) √ (1, 3, and 5%) GS √ (salt soaking) √ (dark) − a 0.04 b 1.28 0.04 c 1.16 0.04 d 1.11 0.02 e 1.41 0.01 f 0.56 0.03 a 1.42 GD, germination in the dark; GL, germination in the light. GE, 0.00 f 0.10 ± ± ± ± ± ± germination under ethanol stress; GS, germination under salt stress; −, ± treatment mentioned was not performed; and √,treatment malonylated formononetin biochanin A genistein pseudobaptigenin mentioned was performed. biochanin A glucoside sequentially subjected to soaking (20 h) and germination stages (12 days). The chickpeas were ﬁrst surface-sterilized by immersing them in a 1% (w/v) NaClO solution (5 L/kg of chickpeas) for 1 h at room 0.03 a 0.75 0.04 b 0.63 0.03 b 0.50 0.02 d 0.33 0.04 c 0.60 0.02 e 0.19 temperature and then rinsed 4 times with distilled pall water (3 L/kg 0.00 e 0.06 ± ± ± ± ± ± ± glucoside of chickpeas). Then, the sterilized chickpeas were soaked for 20 h at biochanin 25 °C in sterilized distilled water in the absence of light, except for the salinity stress chickpeas (GS), which were soaked in a saline solution ﬀ (1:1 NaCl/NaSO4)ofdi erent concentrations (0.01, 0.02, 0.03, and 0.04 mol/L) for 20 h. Subsequently, the soaked chickpeas were put in 0.02 a 1.38 0.01 b 1.28 0.02 c 1.23 0.02 d 0.68 0.03 e 1.39 0.01 e 0.43 ° 0.01 f 0.08 ± ± ± ± ± ± sterilized plastic cartridges and germinated at 25 C and 95% relative ± humidity (RH) in a sprouting machine that was sterilized according to standard deviation of triplicate samples. Values in the same column that do not share the same letter are signi 0.12

the instructions of the manufacturer in advance. Then, for the ± experiments with light (GL), a light incubator was placed on top of the avone Contents during the Germination of Chickpeas in the Dark (mg/g)

machine and used to simulate natural sunlight. The light was applied fl 0.03 a 0.35 0.02 b 0.29 0.03 c 0.24 0.01 e 0.19 0.02 d 0.31 during the all of the germination stages. In the experiments with 0.01 f 0.12 ± ± ± ± ± ethanol treatments (GE), chickpea sprouts were soaked in solutions of ± b 1, 3, and 5% (v/v) ethanol for 12 h at room temperature for elicitation. ononin daidzein All of the experiments were performed in triplicate. Chickpea sprouts )nd in each group were collected on days 2, 4, 6, 8, 10, and 12 and 1 − ° − immediately stored at 20 C. 10 Extraction of Isoflavones. Extractions were carried out according × to the method previously described, with slight modifications.19 All samples were dried at 50 °C and milled with a laboratory mill using a Data are expressed as the mean 10 days 0.50 8 days 0.48 6 days 0.37 4 days 0.24 12 days 0.45 2 days 0.15 untreated ( 60 mesh screen sieve. A total of 1 g of the ground samples was Table 2. Changes in Iso a

2702 DOI: 10.1021/jf5057524 J. Agric. Food Chem. 2015, 63, 2701−2707 Journal of Agricultural and Food Chemistry Article

Figure 1. UHPLC−UV profile of 70% aqueous EtOH extracts of germinated chickpeas in the light, with HPLC chromatograms shown at 280 nm. Peaks: 1, glycitein glucoside acetylated; 2, ononin; 3, daidzein; 4, biochanin A glucoside; 5, biochanin A glucoside malonylated; 6, genistein; 7, pseudobaptigenin; 8, formononetin; and 9, biochanin A. extracted twice and put into 10 mL of 70% ethanol solutions for 3 h ■ RESULTS AND DISCUSSION ° at 70 C. After extraction, the mixture was centrifuged at 3000g for fl 10 min to separate the supernatant. The supernatants were combined Iso avone Contents in Germinated Chickpea Seeds and stored at 4 °C for later analysis. under Water in the Dark. The variation of isoflavone Analysis of Isoflavones. The identification of isoflavones was contents in chickpea seeds and sprouts during germination are performed on the basis of ultraviolet (UV) and mass spectrometry shown in Table 2. The contents of formononetin and biochanin (MS) spectra according to Simons et al.16 and Aisyah et al.17 The A were 0.10 ± 0.01 and 0.18 ± 0.02 mg/10 g, respectively, in samples were analyzed by ultrahigh-pressure liquid chromatography the untreated chickpea seeds. During germination, the contents − (UHPLC) MS. The UHPLC system (San Jose, CA) was equipped of formononetin and biochanin A increased; the maximum with two Shimadzu pumps, a Shimadzu autosampler, and a photodiode amount was obtained on day 10 (1.42 ± 0.06 and 2.10 ± array (PDA) detector. Each sample (1 μL) was injected onto a Waters × 0.08 mg/g, respectively). These results suggest that formono- Acquity UPLC BEH shield RP18 column (2.1 150 mm, 1.7 mm fl particle size) with a Waters Acquity UPLC shield RP18 Vanguard pre- netin and biochanin A are principal iso avones in both chickpea column (2.1 × 5 mm, 1.7 mm particle size, Waters, Milford, MA). seeds and sprouts and are consistent with results reported by Water acidified with 0.1% (v/v) acetic acid, eluent A, and acetonitrile Ma et al.20 Except for genistein, all of the other isoflavones that (ACN) acidified with 0.1% (v/v) acetic acid, eluent B, were used as we tested increased with treatment and reached their peak solvents at a flow rate of 300 μL/min. The temperature of the column content on day 10 (p < 0.05). However, genistein showed an ° oven was controlled to be 35 C, and the PDA detector was set to opposite tendency and was undetectable after 4 days. Because measure a range of 200−400 nm. The elution profile was as follows: − − genistein is formed by the hydrolysis of glycosides through 0 5 min, isocratic on 15% (v/v) B; 5 15 min, linear gradient from β 15 to 30% (v/v) B; 15−25 min, linear gradient from 30 to 60% (v/v) -glucosidase, the decrease of genistein observed in this study B; 25−45 min, linear gradient from 60 to 80% (v/v) B; 45−50 min, may have resulted from the inhibition of the activity of β 21 linear gradient from 80 to 95% (v/v) B; 50−55 min, linear gradient -glycosidase during germination. Moreover, the total from 95 to 15% (v/v) B; and 55−58 min, isocratic on 15% (v/v) B. A isoflavone content showed a similar pattern to formononetin mass spectrometric analysis was performed on a Thermo Scientific and biochanin A from seeds (0.59 ± 0.06 mg/10 g) to 10 day LTQ-XL (San Jose, CA) equipped with an electrospray ionization sprouts (6.82 ± 0.33 mg/g). This increase in isoflavone (ESI)−MS probe coupled with reversed-phase (RP)-UHPLC. Nitro- contents may be induced by the metabolic pathways of naring- gen was used as a sheath and auxiliary gas. Data were obtained in the fl − n eninchalcone and isoliquiritigenin, the precursors of iso avo- m/z range of 150 1500. Data-dependent MS analysis was performed noids commonly found in legumes.22 However, a slight with a normalized collision energy of 35%. To this end, the system was decrease in the isoflavone contents was observed between tuned with biochanin A in both the positive ionization (PI) and 23 negative ionization (NI) modes. For both modes, the ion transfer tube days 10 and 12. Terrence et al. reported that the primary (ITT) temperature was 350 °C and the source voltage was 4.8 kV. soybean leaf tissues underwent a programmed shift from Quantification was performed on the basis of the absorption at 280 nm isoflavonoid to flavonoid metabolism 3 days after germination. using Xcalibur (version 2.1.0, Thermo Scientific). The decrease observed in this study may be due to a conversion For many compounds, no commercial standards were available. The of isoflavonoids to other isoflavones. In addition, the chickpea fl concentrations of individual iso avonoids were evaluated using the sprouts contained ononin, pseudobaptigenin, and glycitein − 2 calibration curve of biochanin A (mg/g; 10 0.0001 mg/mL; R = glucoside acetylated, which were undetected in the chickpea 0.9999). fl Statistical Analysis. All values were expressed as the mean ± seeds. Our results showed that the iso avone composition standard deviation (X̅± SD). Data were analyzed by one-way analy- changed during germination. It has been reported that sprouts sis of variance (ANOVA), followed by post-hoc Dunnett’s t test, grown in the dark have a lower overall nutritional value than using the SAS software. Differences of p < 0.05 were considered sprouts grown under light, because of the lack of photosyn- significant. thesis in dark conditions.14 Thus, the following section

2703 DOI: 10.1021/jf5057524 J. Agric. Food Chem. 2015, 63, 2701−2707 Journal of Agricultural and Food Chemistry Article investigates the changes of isoflavones in chickpea sprouts germinated in the light. 0.26 b 0.21 c Isoflavone Contents in Germinated Chickpea Seeds 0.03 g 0.11 f 0.14 e 0.21 d 0.21 a ± ± ± ± ± ± ± under Water in the Light. We used HPLC−MS to quantify the individual isoflavones of these chickpea sprouts (Figure 1). The isoflavone contents of the chickpea sprouts germinated in the light are listed in Table 3. Exposing the chickpea seedlings nd = not detected. to light boosted the accumulation of all of the isoflavones in our b 0.01 a0.01 c 8.05 7.89 experiments, except genistein, which was undetectable after 0.00 f0.00 e0.01 d 2.40 0.01 b 4.86 6.38 8.14 ± ± 2 days. The chickpea sprouts germinated on day 8 and had ± ± ± ± acetylated total ± < 0.05). the highest formononetin and biochanin A contents (1.54 p 0.04 and 2.34 ± 0.04 mg/g, respectively), which were 154 and glycitein glucoside 130 times higher, respectively, than their contents in untreated erent ( chickpea seeds and 1.2 times higher than their contents in the ff 0.02 a0.01 c 0.14 0.12 GD group. The same trend was also observed in other 0.00 f0.00 e0.01 d 0.04 0.01 b 0.07 0.11 0.13 ± ± isoflavones compared to the GD group, except genistein. These ± ± ± ± 24 cantly di results were consistent with a study by Phommalth et al., who fi reported that soybean seeds germinated with light treatments could produce over 1.53 times more isoflavones than when grown under dark conditions. Moreover, the total isoflavone ± 0.00 b0.00 a nd 0.02 nd 0.60

content in chickpea sprouts dramatically increased, from 2.40 ± ± 0.11 to 4.86 ± 0.14 mg/g during days 2 and 4, and the maximum amount (8.14 ± 0.21 mg/g) of total isoflavones was obtained on day 8. Isoflavones were synthesized in the malonate and phenylpropanoid pathways using three malonyl- 0.05 a nd 0.21 0.04 a nd 0.18 0.01 e 0.06 CoAs and one coumaroyl-CoA, which are produced during 0.02 c0.02 b 0.01 0.04 b nd0.04 a nd nd 0.10 0.15 0.20 ± ± ± ± ± ± ± photosynthesis.14 Light has been thought to increase the a amount of malonyl-CoA and coumaroyl-CoA by promoting photosynthesis, thus, in turn, enhance the precursor for isoflavone production commonly found in legumes.21 Light may 0.05 b 2.27 0.03 b 2.21 0.01 d 0.18 influence the phenylalanine ammonia-lyase (PAL), which is 0.02 a0.03 d 0.56 0.03 c 1.58 0.04 a 1.78 2.34 ± ± ± reported to be an initial enzyme for synthesizing the isoflavone ± ± ± ± and result in the biosynthesis of isoflavone compounds25 apart from the efficiency of photosynthesis. Isoflavone Content in Germinated Chickpea Seeds in the Ethanol Treatment. The changes of isoflavone contents during germination using ethanol are presented in Table 4. The 0.05 a 1.50 0.03 b 1.49 0.00 f 0.10 isoflavone contents of chickpea sprouts significantly increased 0.01 e0.02 d0.03 c0.04 a 0.46 1.05 1.36 1.54 ± ± (p < 0.05) compared to the untreated chickpea seeds. The ± ± ± ± ± maximum amount of formononetin and biochanin A (1.53 ± malonylated formononetin biochanin A genistein pseudobaptigenin

0.05 and 2.21 ± 0.04 mg/g, respectively) in the chickpea biochanin A glucoside sprouts was observed in 3% ethanol-treated chickpea sprouts on day 10. Moreover, the highest total isoflavone content (7.01 ± 0.18 mg/g) was also obtained in the 3% ethanol- 0.04 b 0.99 0.00 f 0.06 treated group. However, in comparison to the GD group, only a 0.02 e0.04 d0.05 c0.04 a 0.20 0.04 a 0.46 0.73 0.99 0.97 ± ± fi fl ± ± ± ± ± glucoside slight increase (not signi cant) in iso avone contents and slight biochanin changes in the composition of isoflavones were observed. Moreover, there was no significant difference among 1% ethanol, 5% ethanol, and GD groups. 0.02 b 1.43 0.01 f 0.08 Stimulation of the defense response of plants has a large 0.01 c0.01 e 0.35 0.01 d 0.81 0.01 a 1.26 0.03 a 1.45 1.45 ± ± potential to alter the metabolites of the plant, especially when ± ± ± ± ±

26 standard deviation of triplicate samples. Values in the same column that do not share the same letter are signi germinated seeds are abruptly exposed to external stresses. In 0.12 this study, ethanol was used as an external stress to stimulate ± avone Contents during the Germination of Chickpeas in the Light (mg/g)

the defense response of the chickpeas, thus increasing the fl 0.03 c 0.50 isoflavone contents. Moreover, ethanol has been reported to 0.03 f0.02 e 0.23 0.03 d 0.28 0.02 b 0.35 0.01 a 0.60 0.61 ± ± ± ± ± ± b induce alcohol dehydrogenase (ADH) activity and could ononin daidzein stimulate alcohol fermentation via the action of ADH under aerobic conditions,27 which, in turn, could induce the con- )nd 1 version of other compounds to other isoflavones. − 10

Isoﬂavone Contents in Germinated Chickpea Seeds × after Salinity Stress Treatment. The isoﬂavone contents of the chickpea sprouts germinated in saline solutions at the con- Data are expressed as the mean 12 days 0.85 2 days4 days6 days8 days10 0.35 days 0.44 0.64 0.89 0.90 untreated ( centrations of 0.01, 0.02, 0.03, and 0.04 mol/L are summarized Table 3. Changes in Iso a

2704 DOI: 10.1021/jf5057524 J. Agric. Food Chem. 2015, 63, 2701−2707 a Chemistry Food and Agricultural of Journal Table 4. Changes in Isoﬂavone Contents during the Germination of Chickpeas Treated with 3% Ethanol (mg/g)

a Table 5. Changes in Isoﬂavone Contents during the Germination of Chickpeas Treated with 0.03 mol/L Saline Solution (mg/g)

biochanin biochanin A glucoside glycitein glucoside ononin daidzein glucoside malonylated formononetin biochanin A genistein pseudobaptigenin acetylated total untreated (×10−1)ndb 0.12 ± 0.01 e 0.08 ± 0.00 f 0.06 ± 0.00 f 0.10 ± 0.01 e 0.18 ± 0.01 e 0.06 ± 0.00 nd nd 0.60 ± 0.03 g 2 days 0.29 ± 0.01 f 0.10 ± 0.01 e 0.28 ± 0.01 e 0.13 ± 0.01 e 0.75 ± 0.02 d 0.90 ± 0.02 e nd 0.03 ± 0.00 e 0.03 ± 0.00 e 2.51 ± 0.08 f 4 days 0.41 ± 0.01 e 0.21 ± 0.01 d 0.77 ± 0.03 e 0.27 ± 0.01 d 0.87 ± 0.02 d 1.06 ± 0.02 d nd 0.05 ± 0.00 d 0.06 ± 0.00 d 3.70 ± 0.10 e 6 days 0.66 ± 0.01 d 0.24 ± 0.01 c 0.88 ± 0.01 d 0.57 ± 0.01 c 1.10 ± 0.03 c 1.26 ± 0.04 c nd 0.10 ± 0.01 c 0.10 ± 0.00 c 4.91 ± 0.12 d 8 days 0.91 ± 0.02 b 0.43 ± 0.01 b 1.25 ± 0.03 b 0.61 ± 0.02 b 1.18 ± 0.01 b 1.37 ± 0.05 b nd 0.17 ± 0.00 b 0.17 ± 0.01 b 6.09 ± 0.15 c 10 days 0.98 ± 0.01 a 0.50 ± 0.01 a 1.33 ± 0.02 a 0.65 ± 0.02 a 1.29 ± 0.03 a 1.77 ± 0.02 a nd 0.21 ± 0.01 a 0.20 ± 0.01 a 6.93 ± 0.13 a 12 days 0.86 ± 0.02 c 0.45 ± 0.01 b 1.12 ± 0.03 c 0.57 ± 0.02 c 1.28 ± 0.04 a 1.75 ± 0.03 a nd 0.17 ± 0.00 b 0.19 ± 0.00 a 6.39 ± 0.15 b a Data are expressed as the mean ± standard deviation of triplicate samples. Values in the same column that do not share the same letter are significantly different (p < 0.05). b nd = not detected. .Arc odChem. Food Agric. J. O:10.1021/jf5057524 DOI: 05 3 2701 63, 2015, Article − 2707 Journal of Agricultural and Food Chemistry Article here. In comparison to chickpea sprouts that germinated in (5) Kuo, Y. H.; Rozan, P.; Lambein, F.; Frias, J.; Vidal-Valverde, C. 0.01, 0.02, and 0.04 mol/L saline solutions, the isoflavone Effects of different germination conditions on the contents of free contents in 0.03 mol/L saline solution were higher, as shown in protein and non-protein amino acids of commercial legumes. Food Chem. 2004, 86, 537−545. Table 5. The formononetin content of samples germinated in ff 0.03 mol/L saline solutions increased to 0.75 ± 0.02 mg/g, and (6) Finney, P. L. E ect of germination on cereal and legume nutrient the biochanin A content increased to 0.90 ± 0.02 mg/g on day 2, changes and food or feed value: A comprehensive review. In Mobilization of Reserves in Germination; Nozzolillo, C., Lea, P. J., which was significantly higher (p < 0.05) than in untreated Loewus, F. A., Eds.; Springer: New York, 1983; Recent Advances in chickpea seeds. Genistein was undetected in all of the stages of Phytochemistry, Vol. 17, Chapter 12, pp 229−305. germination, and ononin, pseudobaptigenin, and glycitein (7) Wu, Z.; Song, L.; Feng, S.; Liu, Y.; He, G.; Yioe, Y.; Huang, D. glucoside acetylated were only detected in chickpea sprouts. Germination dramatically increases isoflavonoid content and diversity Moreover, the 0.03 mol/L salt stress significantly enhanced in chickpea (Cicer arietinum L.) seeds. J. Agric. Food Chem. 2012, 60, the total isoflavone content, and it reached a peak value (6.93 ± 8606−8615. 0.02 mg/g) on day 10. (8) Mao, X.; Zhang, L.; Xia, Q.; Sun, Z.; Zhao, X.; Cai, H.; Tang, Y. Salinity can induce osmotic and ionic imbalances inside plant Vanadium-enriched chickpea sprout ameliorated hyperglycemia and cells, thus affecting plant growth and metabolism.28 However, impaired memory in streptozotocin-induced diabetes rats. BioMetals our results showed that a low-concentration salt stress could 2008, 21, 563−570. increase the total isoflavone content in chickpea sprouts. (9) Kolonel, L. N.; Hankin, J. H.; Whittemore, A. S.; Wu, A. H.; Similar results on soybeans have been reported by Abdul Gallagher, R. P.; Wilkens, L. R.; Paffenbarger, R. S. Vegetables, fruits, 18 legumes and prostate cancer: A multiethnic case-control study. Cancer et al., who stated that a low concentration of salt stress − exposure on a plant could promote photosynthesis, thus Epidemiol., Biomarkers Prev. 2000, 9, 795 804. (10)Zhao,S.;Zhang,L.;Gao,P.;Shao,Z.Isolationand enhancing the accumulation of metabolites in the plant. characterisation of the isoflavones from sprouted chickpea seeds. In conclusion, in this study, we demonstrated that different − ff fl Food Chem. 2009, 114, 869 873. stimuli had positive e ects on the iso avone content and (11) Choi, S. B.; Jang, J. S.; Park, S. Estrogen and exercise may composition of chickpea sprouts, and the maximum amount of enhance beta-cell function and mass via insulin receptor substrate 2 total isoflavones was obtained on day 10, except for in the GL induction in ovariectomized diabetic rats. Endocrinology 2005, 146, group, in which the maximum amount was obtained on day 8. 4786−4794. On the basis of our results, we suggest that the application of (12) Nanri, A.; Mizoue, T.; Takahashi, Y.; Kirii, K.; Inoue, M.; Noda, light is the most effective method for producing functional M.; Tsugane, S. Soy product and isoflavone intakes are associated with foods with high isoflavone contents, which might help to a lower risk of type 2 diabetes in overweight Japanese women. J. Nutr. improve the nutritional quality and health benefits of chickpea 2010, 109−115. sprouts. (13) Lee, S. J.; Ahn, J. K.; Kim, S. H.; Kim, J. T.; Han, S. J.; Jung, M. Y.; Chung, I. M. Variation in isoflavone of soybean cultivars with − ■ AUTHOR INFORMATION location and storage duration. J. Agric. Food Chem. 2003, 51, 3382 3389. Corresponding Author (14) Kim, E. H.; Kim, S. H.; Chung, J. I.; Chi, H. Y.; Kim, J. A.; *E-mail: [email protected]. Chung, I. M. Analysis of phenolic compounds and isoflavones in Author Contributions soybean seeds (Glycine max (L.) Merill) and sprouts grown under † − Yue Gao and Yang Yao contributed equally to this work. different conditions. Eur. Food Res. Technol. 2006, 222, 201 208. (15) Sharma, R. D. Isoflavone content of Bengalgram (Cicer Funding arietinum) at various stages of germination. J. Plant Foods 1981, 3, This work was supported by the Hong Kong, Macao, and 259−264. Taiwan Science and Technology Cooperation Program of (16) Simons, R.; Vincken, J. 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2706 DOI: 10.1021/jf5057524 J. Agric. Food Chem. 2015, 63, 2701−2707 Journal of Agricultural and Food Chemistry Article

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2707 DOI: 10.1021/jf5057524 J. Agric. Food Chem. 2015, 63, 2701−2707