Identification and Antioxidant Capacity of Free and Bound Phenolics in Six Varieties of Mulberry Seeds Using UPLC-ESI-QTOF-MS/MS

Mulberry seeds are a byproduct of juice processing and may be an important resource for its abundant compounds. In this study, we analyzed the qualitative composition of free and bound phenolics from six varieties of mulberry seeds using UPLC-ESI-QTOF-MS/MS. Free phenolics (FPs) and bound phenolics (BPs) were measured using the Folin–Ciocalteu method; antioxidant capacity was determined by measuring 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, using the ferric reducing antioxidant power assay. A total of 28 free and 11 bound phenolics were extracted and identified, wherein five free phenolics were found in mulberry matrices for the first time. The six varieties of mulberry seeds exhibited higher content of FPs than BPs, and there was a correlation between the phenolic content and antioxidant capacity. Consequently, three varieties were selected for their high phenolic content and antioxidant capacity. This study might offer a theoretical basis for the utilization of mulberry seed.


Introduction
Mulberry (Morus alba L.) is an important plant from the Moraceae family, widely cultivated under different climatic conditions around the world, including China and India [1]. Studies on the various types and parts of the mulberry plant, including its fruits, leaves, branches, and Mori Cortex, have increased. Previous studies have shown that mulberry is abundant in bioactive compounds including flavonoids, carotenoids, anthocyanins, polysaccharides, alkaloids, stilbenes, and diels-alder type adducts [2][3][4], which provide the mulberry plant with a variety of biological properties including antioxidant, antibacterial, anti-inflammatory, hepatoprotective, antidiabetic, and anti-tumor activities [5,6].
The fruit of the mulberry contains a seed in every ovary [7]. Mulberry seeds may be obtained from ripe fruits and are a byproduct of juice processing. Each year, total mulberry production exceeds 6.5 million tons in China; therefore, mulberry seeds are available in tremendous quantities in the food industry [8]. However, compared with other mulberry matrices, mulberry seeds have attracted less attention. Gecgel et al. [9] found that 100 g of mulberry seeds were comprised of 27.5-33% crude oil, 20.2-22.5% crude protein, 3.5-6% ash, 42.4-46.6% carbohydrates, and 112.2-152.0 mg total phenolics, indicating that it is a rich source of bioactive substances. Given the high oil content (around 30-40%), there have been some studies on the composition [10] and antioxidant activity [11] of mulberry seed oil, in addition to the novel lipids it contains [8].
Phenolics in the leaves of mulberry fruits have been reported in several studies. Moreover, they have proven to be a vital component of mulberry seeds [9] and are usually

Samples
Six varieties of mulberry seeds were used for this study. Guiyou 12, Guiyou 62, and Teyou 2 were purchased from the Guangxi Nanning Tianlong Biological Technology Co., Ltd. (Guangxi, China). Yue 69851 and Yu 711 were purchased from Guangdong Siji Mulberry Garden Sericulture Technology Co., Ltd. (Guangdong, China). Shisheng was obtained from Zhejiang Haining Sericulture Technology Research Institute (Zhejiang, China). The mulberry seeds were ground to pass through a 60-mesh sieve and stored at −20 • C until further analysis.

Extraction of Free Phenolics (FPs)
Mulberry seed powder was defatted using n-hexane. The extraction process for FPs was optimized based on a previous procedure [15,16]. One gram of defatted mulberry seed powder was mixed at a ratio of 1:5 (m/v) with 80% methanol and extracted in an ultrasonic bath for 40 min. The supernatants were centrifuged at 3000 rpm for 7 min, collected, then 4 mL of 80% methanol was added, and the extraction procedure was repeated two times. The supernatants were collected and filtered through a 0.45 µm organic filter membrane. The methanol extracts were dissolved in water to obtain the sample solution after removing the organic solvent by vacuum rotary evaporation.
Solid phase extraction was used to separate the phenolics from the methanol extracts. C18 Sep-Pak cartridges (Agilent, Santa Clara, CA, USA) were preconditioned with 12 mL of methanol and 24 mL of ddH 2 O. The sample solution was passed through the cartridge, which was washed with 60 mL of ddH 2 O to remove impurities, such as sugar and acid. The absorbed phenolics were eluted with 24 mL of methanol. The methanol in the eluent was removed by vacuum rotary evaporation at 37 • C, and the residue was dissolved with a small amount of ddH 2 O. The methanol extract powder was obtained by vacuum freeze drying of the aqueous solution. The power was dissolved in methanol and the solution was filtered through a 0.45 µm organic filter membrane for UPLC-ESI-QTOF-MS/MS.

Extraction of Bound Phenolics (BPs)
The extraction process was based on the procedure by Singh et al. [17], with a few modifications. The residue after methanol extraction was hydrolyzed with 15 mL of 2 mol/L NaOH for 4 h in the dark. Then, the mixture was acidified to pH = 2.0 with 15 mL of 2 mol/L HCl. After centrifugation at 3000 rpm for 15 min, the supernatant was collected and 10 mL of n-hexane was added, thoroughly mixed, and extracted 3 times to ensure it was defatted. The aqueous layer was extracted 5 times with 10 mL of ethyl acetate. The ethyl acetate extracts were dried in a rotary evaporator. Finally, the dry powder was dissolved in 1 mL of methanol and stored at −80 • C.

Phenolic Content Measurement
The phenolic content of the FPs and BPs was determined using the Folin−Ciocalteu method [18] with some modifications. Briefly, 100 µL of sample was mixed with 800 µL ddH 2 O. Subsequently, 100 µL of 0.5 mol/L Folin−Ciocalteu was added and the mixture was incubated for 3 min. Next, 200 µL of 7% Na 2 CO 3 solution was added. After incubating in a water bath at 30 • C for 2 h in the dark, the absorbance at 760 nm was measured. The phenolic content was expressed as gallic acid equivalents (mg GAE/100 g DW).
DPPH radical scavenging activity assay: 0.1 mL of sample was added to 3.9 mL of 60 mmol/L DPPH solution. The mixture was maintained at 25 • C for 2 h in the dark and the absorbance was measured at 515 nm. The activity was expressed as milligrams Trolox per 100 g dry weight (mg Trolox/100 g DW).
FRAP assay: 0.1 mL of sample was mixed with FRAP solution at a ratio of 1:9. Then, the absorbance at 593 nm was recorded. Total FRAP was expressed as milligrams Trolox per 100 g dry weight (mg Trolox/100 g DW).

Statistical Analysis
Statistical analysis was performed for three independent replicates. The data were analyzed using Tukey's significance test and Pearson correlation coefficients were calculated to determine the relationship between phenolic content and antioxidant capacity. SPSS version 21.0 software was used and the data are presented as the mean ± standard deviation.

Results and Discussion
FPs and BPs were extracted by methanol and ethyl acetate, respectively and analyzed using UPLC-ESI-QTOF-MS/MS for the identification of the compounds. The phenolics were identified based on the retention time, molecular formula, m/z, and mass spectra fragmentation. A total of 39 phenolics, including 28 FPs and 11 BPs, were tentatively identified ( Figure 1) and their UPLC chromatograms can be found in Figures S1 and S2. Among these, five phenolics were previously not reported in the mulberry plant. Moreover, the phenolic content and antioxidant capacity of FPs and BPs were measured, which indicated the significant antioxidant capacity of the mulberry seeds and the potential for phenolics extraction.
the absorbance at 593 nm was recorded. Total FRAP was expressed as milligrams Trolox per 100 g dry weight (mg Trolox/100 g DW).

Statistical Analysis
Statistical analysis was performed for three independent replicates. The data were analyzed using Tukey's significance test and Pearson correlation coefficients were calculated to determine the relationship between phenolic content and antioxidant capacity. SPSS version 21.0 software was used and the data are presented as the mean ± standard deviation.

Results and Discussion
FPs and BPs were extracted by methanol and ethyl acetate, respectively and analyzed using UPLC-ESI-QTOF-MS/MS for the identification of the compounds. The phenolics were identified based on the retention time, molecular formula, m/z, and mass spectra fragmentation. A total of 39 phenolics, including 28 FPs and 11 BPs, were tentatively identified ( Figure 1) and their UPLC chromatograms can be found in Figure S1 and Figure S2. Among these, five phenolics were previously not reported in the mulberry plant. Moreover, the phenolic content and antioxidant capacity of FPs and BPs were measured, which indicated the significant antioxidant capacity of the mulberry seeds and the potential for phenolics extraction.  Table 1 lists the identification of 28 FPs in mulberry seeds, which were divided into the following groups: Flavonoids, phenolic acids and their derivatives, 2-arylbenzofurans, xanthone, stilbenes, coumarin derivatives, and other phenolics. Among these, five phenolics were reported for the first time including (E)-Caffeol 4-O-β-glucopyranoside, 2-  Table 1 lists the identification of 28 FPs in mulberry seeds, which were divided into the following groups: Flavonoids, phenolic acids and their derivatives, 2-arylbenzofurans, xanthone, stilbenes, coumarin derivatives, and other phenolics. Among these, five phenolics were reported for the first time including (E)-Caffeol 4-O-β-glucopyranoside, 2formyl-4-hydroxy-3-hydroxymethyl-6-methoxy-5-methyl-benzoic acid, Neolignan 2-O-(βapiofuranosyl)-β-glucopyranoside, Rubraxanthone, and Neophellamuretin.  18, both produced an MS 2 fragment at an m/z of 301, which corresponded to quercetin and resulted from the loss of rutinoside. Therefore, these two compounds were flavanols with quercetin as the mother nucleus. Compound 10 was tentatively identified as rutin and compound 19 was identified as hesperidin when compared with an authentic standard. For compound 14 (tR = 13.90 min), the MS yielded a molecular ion [M-H] − at m/z 353.10 and MS 2 yielded two fragments at m/z 338.08 and 279.06. The molecular formula was C 20 H 18 O 6 . According to studies from other groups, compound 14 was tentatively identified as Albanin A [19]. The fragment at m/z 285 was kaempferol, which is formed by the loss of glycoside, and the fragment at m/z 255 was a compound with a mother nucleus of flavonoid glycoside. Therefore, compound 16 was tentatively identified as kaempferol-3-O-rutinoside [20].  [21] and Desmodium caudatum [22]. This is the first time that Neophellamuretin was found to be present in mulberry seeds or other mulberry matrices.

Identification of FPs from Mulberry Seeds
Compound 25 (tR = 20.21 min) lost a hydroxyl to form m/z 339 and continued to lose two hydroxyls to form m/z 307. The remainder cracked into fragments at m/z 247 and 93. According to the MS fragments, compound 25 was tentatively identified as Leachianone G. Compound 27 (tR = 25.07 min) was a flavane which was first identified in mulberry leaves [23]. It yielded a molecular ion [M+H] + at m/z of 359.15. MS 2 = m/z 327.12 [M+H-OCH3] + and was obtained through the loss of a methoxyl group and continued to lose butyric acid to form m/z 240.08 [M+H-OCH3-C4H7O2] + . Therefore, compound 27 was tentatively identified as (2S)-2 , 4 -dihydroxyl-7-methoxy-8-butyricflavane.

Phenolic Acids and Their Derivatives
Phenolic acids are one group of aromatic secondary plant metabolites that exist widely in plants and exhibit a variety of physiological functions. Many phenolic acids and their derivatives, such as P-coumaroylquinic acid and isochlorogenic acid, have been reported in mulberry leaves and other mulberry matrices [24]. Two types of phenolic acid derivatives of benzoic acid and derivatives of cinnamic acid have been identified in mulberry seeds.

Identification of BPs in Mulberry Seeds
Numerous FPs in mulberry seeds have been reported; however, there have not been many studies focused on BPs from mulberry seeds. Therefore, our study will complement the existing knowledge of BPs in mulberry seeds. BPs accounted for an average of 24% of the total phenolics in foods, such as fruits and vegetables, and many bioactive properties have been reported including antioxidant, anti-inflammatory, and hepatoprotective activities [26]. As a result, BPs in food are of significant biological importance for their high content and biological activity. In this study, a total of 11 BPs in mulberry seeds were isolated (Table 1), which were primarily phenolic acids.

Phenolic Acids and Their Derivatives
Six phenolic acid derivatives were identified in the form of BPs from mulberry seeds. Compounds 2, 4, and 6 were identified as derivatives of benzoic acids and compounds 7, 9, and 10 were identified as derivatives of cinnamic acids with authentic standards. There was a similar structure of C6-C3 in compounds 7, 9, and 10; therefore, they may be classified as cinnamic acid derivatives.

Other Phenolics
Compound 1 (tR = 2.66 min) yielded a molecular ion [M-H] − at m/z 167.04 and MS 2 yielded a fragment of m/z 123.04 resulting from the loss of a carboxyl group. The compound was tentatively identified as 2,5-dihydroxyphenylacetic acid. The molecular ion [M-H] − of compound 3 (tR = 3.74 min) appeared at m/z 137.03 and its formula was C 7 H 6 O 3 , which was consistent with compound 4 (P-hydroxybenzoic acid). However, the fragments in the second-order mass spectrum were different, which suggested that they were isomers. The fragment of compound 3 at m/z 108 was formed by the loss of an aldehyde group from the precursor ion; thus, the compound was tentatively identified as 2,4-dihydroxybenzaldehyde. For compound 5 (tR = 5.37 min), there was a 29 Da difference between the molecular weights of the fragment at m/z 121 and the precursor ion [M-H] − at m/z 92.03, which corresponded to a loss of an aldehyde group. Therefore, compound 5 was tentatively identified as P-hydroxy benzaldehyde based on published reports. at m/z 137.06, which was consistent with compound 3 (2,4-dihydroxybenzaldehyde) and compound 4 (P-hydroxybenzoic acid). Their molecular formulas and fragments in the second-order mass spectrum were different, which indicated that compound 11 was not an isomer of compounds 3 and 4. On the basis of the two MS 2 fragments at an m/z of 93.06 and 77.04, compound 11 was tentatively identified as 4-hydroxyacetophenone.

Phenolic Content of FPs and BPs from Extracts
The phenolic content of FPs and BPs in mulberry seeds from six varieties was quantified using the Folin−Ciocalteu method and the results are presented in Table 2. The phenolic content of FPs ranged from 76.104 mg GAE/100 g DW to 109.107 mg GAE/100 g DW. The higher phenolic content of the FPs was observed in Guiyou 12 and Guiyou 62. The lower phenolic content of FPs was in Shisheng, Yue 69851 and Yu 711, which was at a significantly lower level compared with the other groups. For BPs, the phenolic content of ethyl acetate extracts ranged from 38.041 mg GAE/100 g DW to 44.973 mg GAE/100 g DW, which was much lower compared with the FP content. The lowest phenolic content of the ethyl acetate extracts was observed in Shisheng, whereas the others were above 40 mg GAE/100 g DW. Only the difference between Shisheng and the other five varieties was significant. BPs may be released by the presence of intestinal flora to provide large amounts of biological activities in vivo, thus more treatments should be developed to promote the release [27]. As a result, 63.4-70.8% of phenolics in mulberry seeds of different varieties existed in free form and 29.2-36.7% of phenolics existed in bound form. Therefore, phenolics in mulberry seeds were mainly in a free form as they contained approximately 67.5% FPs and 32.5% BPs on average. This finding was consistent with earlier studies that reported FPs accounted for the majority of phenolics in common fruits, vegetables, and seeds [28,29].
Mulberry matrices including fruits, leaves, and other parts are a valuable source of phenolics. Mulberry leaves are rich in phytochemical compounds, and the total phenolic content (TPC) ranges from 12.81 to 16.13 mg GAE/g DW [30]. Butkhup et al. [31] found that the average TPC range for white mulberry fruits was 104.78-213.53 mg GAE/100 g DW. It can be seen in Table 2 that free and bound phenolic content in mulberry seeds reached the lower limit for fruits. Seed coat consisted of epidermis, chlorenchyma, parenchyma, and other cells, and these cells all contain organs, such as vesicles and cell walls, which are the main locations of free and bound phenolics, respectively [32]. Therefore, seed contained high amounts of FPs and BPs. However, few studies have reported the TPC of mulberry seeds. Gómez-Mejía et al. [13] extracted phenolic compounds with a hydroethanolic solution (80:20 v/v ethanol-water), and total phenolic compounds in mulberry seed extracts were determined to be 8.02 mg/g by HPLC-DAD-MS analysis. However, Kim et al. [33] reported that TPC of the MeOH extract of Morus alba seeds was 367.26 mg GAE/100 g DW, which was considerably higher than our results. The variation of TPC in mulberry seeds as well as other mulberry matrices, was attributable to many factors, such as genetic differences in the varieties, physiological state, harvest time, and environmental parameters [34].
In this study, the variety and environment are the predominant factors. Shisheng is a wild mulberry variety without artificially breeding; therefore, it showed the lowest phenolic content of FPs and BPs, which was significantly lower compared with the elite hybrid varieties. Yu 711 is a diploid mulberry variety bred from Yu 54 and Yu 2, and was primarily planted in the lower reaches of the Yangtze River in a temperate climate. The remaining four, Guiyou 12, Guiyou 62, Teyou 2, and Yue 69851 are from the Guangdong and Guangxi Province in China, regions with a subtropical humid monsoon climate. Among these, three varieties from Guangxi Province exhibit a closer genetic relationship due to their parents: Guiyou 12 is a diploid bred from Sha 2 and Gui 7722, Guiyou 62 is a diploid bred from 7862 and Gui 7722, Teyou 2 is a triploid bred from 7862 and Guiyou P58. Consequently, the close genetic relationship, growth environment, and artificial selection have provided them with a higher and similar FP and BP content. Similar results were observed by Zou et al. [35] in which they reported that the phenolic content of mulberry leaves significantly varied between different cultivars and collection months. With respect to TPC, Guiyou 12, Guiyou 62, and Teyou 2 are present with the highest phenolic content of FPs and BPs, which enable them to produce more potential varieties.

Antioxidant Capacity of Extracts
The antioxidant capacities of FPs and BPs from six varieties of mulberry seeds were evaluated using two methods: DPPH and FRAP. The reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), can capture hydrogen ions and FRAP is based on the reduction of ferroin analogs [36]. The results are shown in Table 3.

DPPH Radical Scavenging Activity and FRAP
From the DPPH assay, the antioxidant activity of FPs (78.85-105.46 mg Trolox/100 g DW) from all varieties was considerably higher compared with the antioxidant activity of BPs (41.84-55.08 mg Trolox/100 g DW). The highest antioxidant activity of FPs was observed in three varieties: Teyou 2, Guiyou 62, and Yue 69851, while the results of other three varieties were below 95 mg Trolox/100 g DW. The higher antioxidant activity for the BPs was observed in Guiyou 12 and Guiyou 62, whereas the lowest activity for the BPs was observed in Shisheng. The activities of the other varieties were similar and showed significant differences. From the FRAP test, the antioxidant activity of FPs ranged from 60.31 mg Trolox/100 g DW to 75.87 mg Trolox/100 g DW. The highest antioxidant activity of FPs was observed in Guiyou 12, whereas the lowest activity was in Shisheng, and other varieties were slightly different and showed no significant differences. The antioxidant activity of the BPs was considerably lower compared with FPs. The highest antioxidant activity for the BPs was Yue 69851, followed by Guiyou 62 and Guiyou 12, and the activity of Shisheng and Teyou 2 was significantly lower than other varieties.
Overall, the FPs and BPs of Guiyou 12, Guiyou 62, and Teyou 2 exhibited higher DPPH radical scavenging activity and ferric reducing antioxidant power. The extracts of the mulberry seed may act as packaging materials to inhibit lipid peroxidation of food, given its high anti-oxidative effects [37].

Relationship between Antioxidant Capacity and TPC
Phenolic compounds contribute significantly to the antioxidant capacity of plants. The relationship between phenolic compound content and antioxidant function has attracted considerable attention and many researchers have confirmed a linear correlation between the two [38]. However, this is not applicable to all phenolic content and their associated antioxidant effects in mulberry seeds.
For example, as shown in Tables 2 and 3, the FP content of Guiyou 12 was highest, whereas the DPPH radical scavenging activity of Teyou 2 was highest, but the r 2 value of the Pearson correlation was 0.40, indicating a relatively poor correlation between them (Table 4). In contrast, there was a higher correlation coefficient between FPs and FRAP, BPs and DPPH, BPs and FRAP, and their high phenolic content was associated with a high antioxidant capacity. The corresponding r 2 values were 0.76 (p < 0.01), 0.07 (p < 0.01), and 0.58 (p < 0.05), respectively, which suggests that a significant correlation exists between phenolic content and antioxidant capacity. The results showing a weak or significant correlation was consistent with previous studies. Babbar et al. [39] reported that the r 2 value of TPC extracted from six kinds of fruit residues and their corresponding DPPH radical scavenging activity, ABTS radical scavenging activity, and reducing power was 0.36, 0.49, and 0.66, respectively. Karamać et al. [40] reported that there was no significant correlation between TPC of white lupin seed extracts and DPPH scavenging activity. These results demonstrated an insignificant relationship. Since high FP or BP content did not correspond with high antioxidant activity, there may be non-phenolic compounds including ascorbates, terpenes, and pigments that contributed to the antioxidant function; different compounds in FPs and BPs showed varying levels of antioxidants [39]. The antioxidant activity of phenolics was greatly influenced by chemical structures of compounds, especially the number and position of aromatic and hydroxyl groups, and it has been proven to be closely related to the degree of hydroxylation [41]. For example, flavonoids and phenolic acids with more hydroxyl groups tended to exhibit stronger antioxidant activity than others. Arruda et al. [42] found that flavonoids were the main source of antioxidant activity in the Araticum pulp and peel, while phenolic acids, such as ferulic acid, caffeic acid, and chlorogenic acid contributed the most to the antioxidant activity of seeds. Our results showed that there were eight kinds of flavonoids, six kinds of phenolic acids in FPs, and only six kinds of phenolic acids in BPs; therefore, different phenolics contribute to antioxidant activity differently and brought about the various correlation between phenolic content and antioxidant activity. In addition, the antagonistic and synergistic reactions between phenolics and other chemicals may result in the poor correlations [43]. As a result, the antioxidant capacity of non-phenolics and reactions between phytochemicals require further studies to better utilize plant byproducts, such as mulberry seeds.

Conclusions
Phenolic compounds from six varieties of mulberry seeds were extracted and identified in free and bound forms. The content of FPs and BPs was measured and their corresponding antioxidant capacity was determined. The results of UPLC-ESI-QTOF-MS/MS revealed 28 FPs, 11 BPs, and 5 FPs including (E)-caffeol 4-O-β-glucopyranoside, 2-formyl-4-hydroxy-3-hydroxymethyl-6-methoxy-5-methyl-benzoic acid, Neolignan 2-O-(β-apiofuranosyl)-β-glucopyranoside, Rubraxanthone, and Neophellamuretin, which were first reported in mulberry matrices compared with previous studies. For the phenolic content and antioxidant capacity, Guiyou 12, Guiyou 62, and Teyou 2 were rich in both FPs and BPs. FPs of Teyou 2 displayed the highest DPPH radical scavenging activity, whereas BPs of Guiyou 12 exhibited the highest DPPH radical scavenging activity. Guiyou 12 showed the highest FRAP in FPs, whereas Guiyou 62 contained the highest FRAP for the BPs. All three showed higher DPPH radical scavenging activity and FRAP compared with the other varieties. The results revealed that there was a correlation to a certain extent between the two; however, further studies are essential in this field. Consequently, our study was the first comprehensive analysis of FPs and BPs in mulberry seeds to date and five new free phenolics were identified in the mulberry seeds for the first time. The results provide essential information for the identification and antioxidant capacity of FPs and BPs from mulberry seeds. As a byproduct of the food manufacturing industry, mulberry seeds are expected to be a source of bioactive compounds and Guiyou 12, Guiyou 62, and Teyou 2 are specially recommended for consideration.