Comparison of the Phenolic Profiles of Soaked and Germinated Peanut Cultivars via UPLC-QTOF-MS

Diverse peanut varieties are widely cultivated in China. However, few studies have investigated the effects of germination on the phenolic profiles and antioxidant activities of specific Chinese peanut cultivars. Therefore, this study was conducted to evaluate the effects of germination on total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, and phenolic profiles of seven peanut cultivars in China. The TPC, TFC, and antioxidant activities were determined by spectrophotometry, while phenolic profiles were analyzed by using ultra-high performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS). The results found that germination significantly increased TPC, TFC, and antioxidant activity. Antioxidant activity was found to be closely related to TPC in germinated peanut extracts, which indicates that phenolics are the main contributors of antioxidants in germinated peanuts. In addition, germination induced significant changes in polyphenolic profiles. In the analyzed samples, 36 phenolic compounds were identified in which most were flavonoids. Overall, these findings highlight that germinated peanuts can be a good natural source of natural antioxidants for human consumption and functional food development.


Introduction
Germination of edible seeds typically involves de novo synthesis of bioactive compounds in which many are considered health-enhancing ingredients [1]. Many edible seeds, such as lentils (Lens culinaris L.), kidney bean (Phaseolus vulgaris L.), mung bean (Phaseolus aureus L.), faba bean (Vicia faba L.), lupin (Lupinus angustifolius L.), and soybean (Glycine max), have been germinated to obtain sprouts with improved nutritional and medicinal values [2][3][4][5]. It has been demonstrated that germination can significantly accumulate diverse bioactive compounds such as polyphenols, gamma-aminobutyric acid, and vitamins, and enhance antioxidant capability in germinated seeds [1]. As a result, germinated seeds have received greater attention due to their better health benefits when compared to seeds [6].
The peanut (Arachis hypogaea L.) or groundnut is a multi-purpose oil seed widely cultivated in China with many health benefits. Peanut seeds and germinated peanuts are nutritious and have high medicinal and economic values [7]. It has been reported that germinated peanut contains higher levels of flavonoids and other phenolic compounds than peanut seeds [8]. Especially, compared to peanut seeds, germinated peanuts exhibit enhanced antioxidant activity [8]. These phenolic compounds have been associated with a wide range of health benefits such as the prevention of coronary heart diseases, different types of cancer, diabetes, and obesity [9]. Given the health benefits and commercial value, it is useful to comprehensively analyze the phenolic profiles and bioactivities of germinated peanuts.
The evaluations of phenolic profiles and antioxidant activity of local peanut seeds were performed in Canada [10], Japan [11], Argentina [12], and many other countries. Variability of bioactive components and their antioxidant activities in peanut skin extracts have been found due to different growing conditions and cultivars [10][11][12]. Different peanut cultivars have also been widely cultivated in China and the planting area is increasing annually. However, little research has been done on the effects of germination on the phenolic profiles and antioxidant activities of peanut cultivars in China.
Therefore, the purpose of this study was to evaluate the effects of germination on phenolic profiles and antioxidant activities of seven peanut cultivars grown in China. We hope that germination can increase phenolic content and produce some new antioxidant compounds in germinated peanuts. The results of this research can provide valuable information for germinated peanuts as nutraceuticals or functional foods.

Preparation of Soaked and Germinated Peanuts
Seven peanut cultivars were purchased from online shops of the Taobao Mall. Peanuts were soaked and germinated, according to a previous study [13] with minor modification. After rinsing three times with deionized water, the peanuts were soaked in deionized water (1:10, w/v) for 10 h at room temperature (22 ± 1 • C). The soaked peanuts were drained, rinsed three times with deionized water, and then germinated in a domestic semi-automatic germination machine (Model CB-A323B, Connie, Guangdong, China) for 10 days in the dark. During the germination process, the temperature was automatically controlled at about 25 • C, and the peanuts were automatically watered every 10 min with fresh deionized water. After germination, germinated peanuts (Day 10) and corresponding soaked peanuts (Day 0) were manually peeled, lyophilized, ground into a fine powder, and stored at 4 • C for further analysis. Germination was performed in triplicate.

Extraction Procedure
According to the previous study and considering its green chemistry property, 70% of ethanol was selected as the extraction solvent [14]. Each sample (0.5 g) was extracted with 10 mL 70% ethanol in a shaker (130 rpm) for 24 h at 50 • C. The extract was then centrifuged at 3000× g for 15 min at room temperature (22 ± 1 • C), the supernatant was collected and stored at −20 • C, and it was then analyzed within one week.

Determination of Total Phenolic and Flavonoid Contents
The total phenolic content (TPC) was determined by the Folin-Ciocalteu method, as previously described [15], and the results were expressed as milligrams of gallic acid equivalent (mg GAE)/100 g DW of samples. Total flavonoid content (TFC) was determined by the AlCl 3 -based colorimetric method, as previously described [8], and the results were expressed as mg catechin equivalent (CE mg)/100 g dry weight (DW) of samples.

Determination of Free Radical Scavenging Activity by 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Method
The DPPH assay was carried out as previously described [16] with minor modification. The DPPH solution was prepared by dissolving DPPH in 80% methanol to obtain an absorbance of 0.70 ± 0.05 at 515 nm. The reaction was conducted by mixing 3.9 mL of DPPH solution with 100 µL of the sample and then reacted at room temperature in dark for 2 h. After that, a UV-Vis spectrophotometer (UV1800PC, Jinghua Technology Instrument Co., Ltd. Shanghai, China) was used to record the absorbance of the solution at 515 nm. A Trolox external calibration curve was established (y = 0.5085x − 0.009, R 2 = 0.9988), and the results were expressed as mmol Trolox/g DW.

Ferric Ion Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was carried out as previously described [16] with some modifications. Fresh FRAP reagent was prepared by adding 300 mM sodium acetate buffer, 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) solution, and 20 mM FeCl 3 solution in the ratio of 10:1:1, and then warmed at 37 • C. The test was conducted by adding 100 µL of sample solution to 3 mL of FRAP reagent, and then reacted for 4 min. After that, a UV-Vis spectrophotometer was used to record the absorbance of the solution at 593 nm. A FeSO 4 solution external calibration curve was established (y = 0.45x + 0.00993, R 2 = 0.9984), and the results were expressed as mmol Fe (II)/g DW.

UPLC-QTOF-MS Analysis
A Primer ultra-high performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS) (Waters, Milford, MA, USA) equipped with an electrospray ionization source was used to analysis the phenolic profile of soaked and germinated peanut extracts. Samples were separated on a BEH C18 column (2.1 mm × 100 mm, 1.7 µm) (Waters, Milford, MA, USA) with the following settings: column temperature, 45 • C, flow rate, 0.4 mL/min, mobile phase A, ultrapure water containing 0.1% (v/v) formic acid, mobile phase B, acetonitrile containing 0.1% (v/v) formic acid, and injection volume, 1 µL. The gradient conditions were: 0 min, 5% B, 3 min, 20% B, 10 min, 100% B. Mass spectrometric analysis was performed in a negative ion mode with the spectrometer settings as follows: capillary, 2 kV, sample cone voltage, 40 V; desolvation gas temperature, 450 V, flow rate of desolvation gas, 900 L/h, flow rate of cone gas, 50 L/h, source temperature, 115 • C, acquisition range, 50-1000 m/z, scan rate, 0.2 s, collision energy, and 6 eV/20-45 eV. To ensure accuracy and reproducibility, all analyses were conducted using an independent reference spray via the Lock Spray interface with the setting as follows: lock mass, Tyr-Gly-Gly-Phe-Leu (leucine-encephalin, 250 pg/µL), flow rate, 10 µL/min, interval, 0.5 s, sample time, 0.5 s, and collision energy, 6 eV. The mass data were processed with MassLynx 4.1 software (Waters, Milford, MA, USA). The exact elemental composition of each parent and generated fragments were calculated with a molecular formula calculator. All compounds were identified or tentatively identified by comparing the retention time, parent ion, and mass fragments with those in references and the mass spectrometry database.

Statistical Analysis
All the measurements were performed in triplicate and the results were expressed as mean ± standard deviation (SD), with a p-value less than 0.05 defined as statistically significant. The statistical software GraphPad Prism (Graphpad Software, Inc., San Diego, CA, USA) was used for statistical analysis.

Changes of TPC and TFC Induced by Germination
The changes occurring in germinated peanut extracts were investigated with respect to their TPC and TFC values ( Figure 1). TPC in soaked peanuts varied from 132.5 to 248.8 mg GAE/100 g DW in all tested cultivars, among which the soaked seeds of Qicai cultivar had the highest TPC value of 248.8 mg GAE/100 g DW ( Figure 1A). The TPC observed here were higher than those in Korean-grown peanuts [8], which may be due to different extraction conditions, growth conditions, and genotypes. On the other hand, the TPC in germinated peanuts ranged from 403.4 to 596.2 mg GAE/100 g DW, which was significantly higher than that of the soaked seeds. This indicates that germination could significantly accumulate phenolic compounds. The germinated peanuts from Xiaosilihong and Xiaosuangli cultivars had the highest TPC value among all the tested cultivars, which is even higher than that of most germinated edible seeds and sprouts [1]. 248.8 mg GAE/100 g DW ( Figure 1A). The TPC observed here were higher than those in Koreangrown peanuts [8], which may be due to different extraction conditions, growth conditions, and genotypes. On the other hand, the TPC in germinated peanuts ranged from 403.4 to 596.2 mg GAE/100 g DW, which was significantly higher than that of the soaked seeds. This indicates that germination could significantly accumulate phenolic compounds. The germinated peanuts from Xiaosilihong and Xiaosuangli cultivars had the highest TPC value among all the tested cultivars, which is even higher than that of most germinated edible seeds and sprouts [1]. The TFC in soaked peanut seeds ranged from 36.6 to 63.4 mg CE/100 g DW with the highest TFC value in peanut seeds of Xiaosuangli cultivar ( Figure 1B), which was less than that of the peanut skins [17]. The TFC in germinated peanuts were 48.6-101.3 mg CE/100 g DW. This was significantly higher than that in soaked seeds. However, the TFC in germinated peanuts of Sanhua-NO.11 cultivar was lower than that in soaked seeds. Previous publications on TFC of germinated peanuts were very limited, but it was reported that germinated peanuts contained 63.0 to 730.8 μg quercetin equivalent per gram samples [8]. Generally, germination can significantly accumulate TPC and TFC, which is in agreement with the previous conclusion [1,18]. The TFC in soaked peanut seeds ranged from 36.6 to 63.4 mg CE/100 g DW with the highest TFC value in peanut seeds of Xiaosuangli cultivar ( Figure 1B), which was less than that of the peanut skins [17]. The TFC in germinated peanuts were 48.6-101.3 mg CE/100 g DW. This was significantly higher than that in soaked seeds. However, the TFC in germinated peanuts of Sanhua-NO.11 cultivar was lower than that in soaked seeds. Previous publications on TFC of germinated peanuts were very limited, but it was reported that germinated peanuts contained 63.0 to 730.8 µg quercetin equivalent per gram samples [8]. Generally, germination can significantly accumulate TPC and TFC, which is in agreement with the previous conclusion [1,18].

Changes of Antioxidant Activities Induced by Germination
It has been widely accepted that the antioxidant ability should be evaluated by at least two assays because the experimental results obtained in different assays may be inconsistent [19]. In this study, antioxidant activity was evaluated by a DPPH free radical scavenging assay and a ferric-reducing antioxidant power (FRAP) assay. The results are shown in Figure 1C,D.
The DPPH and FRAP values of soaked peanut extracts, respectively, ranged from 2.65 to 4.80 mmol Trolox/g DW and 4.98 to 8.09 mmol Fe (II)/g DW. Soaked peanut extracts of Qicai cultivar exhibited the strongest antioxidant activity measured by the two assays. Despite its relatively strong antioxidant capability, it is still less effective than germinated peanut extracts ( Figure 1C,D), which showed DPPH values ranged from 5.66 to 11.09 mmol Trolox/g DW and FRAP values ranged from 12.56 to 28.98 mmol Fe (II)/g DW. All of them were significantly higher than the corresponding soaked peanuts, which indicates that germination can significantly enhance antioxidant capacity of peanuts. In addition, in both assays, germinated peanut extracts of Xiaosilihong and Xiaosuangli cultivars had the highest antioxidant activity. We speculate that this phenomenon is related to phenolic contents because it has been demonstrated that there is a positive correlation between antioxidant capacity and TPC in fruits [20], vegetables [21], flowers [22], red rice [23], spices [24], and medicinal plants [25]. Therefore, we further confirmed this conclusion by generating the correlation coefficients of TPC, TFC, DPPH, and FRAP data. From Figure 2, we can see that TPC had a high correlation coefficient with the DPPH value (r = 0.9832, p < 0.0001) and FRAP value (r = 0.9474, p = 0.0012), which indicates that the antioxidant capacity of germinated peanuts might be mainly attributed to phenolics in germinated peanuts. Additionally, TFC also had a positive correlation with the DPPH value (r = 0.9885, p < 0.0001) and the FRAP value (r = 0.9521, p = 0.0009), which further demonstrated that the antioxidant capacity was closely related to flavonoid compounds in germinated peanuts. There was a positive correlation between DPPH and FRAP values (r = 0.9804, p = 0.0001), which indicates that antioxidants in germinated peanuts can not only scavenge free radicals, but also reduce oxidants. These results also confirmed that both antioxidant assays are consistent with each other. In addition, TPC and TFC also exhibited positive correlation (r = 0.9368, p = 0.0019), which suggests that flavonoids can be the main phenolics in germinated peanuts.

Characterization of the Phenolic Profile of Peanut Extracts by UPLC-QTOF/MS
According to Figure 1A, germinated Xiaosilihong extracts had relatively higher TPC. In order to find out the changes of the phenolic profile induced by germination, we further analyzed the phenolic profile of soaked and germinated Xiaosilihong extracts by UPLC-QTOF/MS. Corresponding base peak ion chromatograms (BPI) were shown in Figure 3. All tentatively identified compounds were elucidated by their retention time (min), molecular ions, fragment ions, calculated molecular formula

Characterization of the Phenolic Profile of Peanut Extracts by UPLC-QTOF/MS
According to Figure 1A, germinated Xiaosilihong extracts had relatively higher TPC. In order to find out the changes of the phenolic profile induced by germination, we further analyzed the phenolic profile of soaked and germinated Xiaosilihong extracts by UPLC-QTOF/MS. Corresponding base peak ion chromatograms (BPI) were shown in Figure 3. All tentatively identified compounds were elucidated by their retention time (min), molecular ions, fragment ions, calculated molecular formula of each peak, and by matching the above information with relevant references and internal mass spectrometry database. Notably, we have tentatively identified 36 compounds, including three flavanols, four flavanones, three flavones, eleven flavonols, one isoflavonoid, one anthocyanin, four coumarins, and nine others. Previous studies found that many phenolic compounds mainly exist in the peanut skins [26][27][28][29]. However, our study demonstrates that they can also exist in soaked and germinated peanut extracts without skins. The backbones of flavonoids and coumarins are shown in Figure 4, and their potential cleavage bonds are marked with numbers.    This compound was also reported in a previous study [39]. The fragment ion at m/z 577.15302 was obtained by losing a methylbut-2-enyl and a hydroxymethyl from the parental ion, while the fragment ion at m/z 519.14986 was obtained by losing ring B, a hydroxymethyl, and a molecular water from the parental ion. Other fragments could further confirm the structure of icariin and the potential cleavage methods were illustrated in bracket following its corresponding m/z molecules, including m/ which has also been reported in peanut skin extracts [10]. Compared to the profile of soaked peanut extract, peaks 2-6 newly emerged in germinated peanut extract, which potentially contributed to the enhanced antioxidant activity of germinated peanuts. Peak 2 and 4 had the same molecular ion [M-H] − at m/z 581.1835 and similar fragment ions, and they were tentatively assigned as phenylethyl-trihydroxy-tetrahydro-phenylethyl-hydroxychromone-oxy-chromone. The chromone derivatives were also detected in peanut pods [30]. The fragment ion at m/z 423.09024 was generated by losing three molecules of water and one molecule phenylethyl from the parental ion, while m/z 314.04595 was obtained by losing [ 1,4 B], one molecule of water, and phenylmethyl from the parental ion.

Conclusions
In conclusion, peanut germination induced compositions of phenolic compounds that varied markedly. Meanwhile, the total phenolics and total flavonoids were observed to significantly increase. These variations of phenolic compounds were responsible for the antioxidant activities of germinated peanuts. The positive correlation between phenolic contents and antioxidant activity has profound guiding significance for improving the efficacy of natural antioxidants. In addition, this study found that germinated peanuts of Xiaosilihong and Xiaosuangli cultivars overall had the highest TPC, TFC, and antioxidant activities, which indicates that these two varieties can be selected to produce peanut sprouts rich in antioxidant phenolics.