2.1. Phenolic Acid Compositions of Wheat Bran Extracts
Results of the free and bound phenolic acid compositions of the wheat bran extracts tested in this study are summarized in Table 1
. Free (soluble) and bound (insoluble) fractions were extracted from each bran sample, and total phenolic acid was calculated as the sum of the free and bound phenolic acids in each sample. Phenolic acids are hydroxylated compounds which are derived from benzoic acid or cinnamic acid [15
]. The results of this study indicated that hydroxycinnamic acid derivatives were more prevalent than hydroxybenzoic acid derivatives across all three samples. The hydroxycinnamic acids identified in the wheat bran samples were ferulic acid, p
-coumaric acid, sinapic acid, and caffeic acid. The hydroxybenzoic acids identified in the three samples included syringic acid, vanillic acid, and syringaldehyde.
As shown in Table 1
, free ferulic acid (the soluble fraction) significantly differed (p
< 0.01) across the three samples, ranging from 13.37 µg/g to 26.96 µg/g (Table 1
). Most (>99%) of the ferulic acid in the three samples was identified as bound ferulic acid (the insoluble fraction). The bound ferulic acids of MWB, IWB, and SIWB ranged from 1767–3071 µg/g. Although MWB (26.96 µg/g) contained more free ferulic acid than IWB (20.17 µg/g) or SIWB (13.37 µg/g), IWB (3071 µg/g) contained more bound ferulic acid than MWB (1767 µg/g) or SIWB (2532 µg/g). Ferulic acid was the predominant phenolic acid identified in the three samples, consistent with observations in previous wheat bran studies [6
]. As reported by Kim et al. [16
], total ferulic acid accounted for approximately 85% of the total phenolic acid compositions of bran samples, ranging from 1376 µg/g to 2020 µg/g. Lu et al. [6
] also studied the phenolic acid compositions of ten Maryland soft winter wheat bran samples, and their results demonstrated that bound ferulic acid, as the predominant phenolic acid found in bran, ranged from 1184–1725 µg/g. In this study, the amount of bound ferulic acid in MWB (1767 µg/g) was similar to the bound ferulic acid values reported by Lu et al. [6
]. However, the bound ferulic acid (3071 µg/g) identified in IWB was higher than the bound ferulic acid (1184–1725 µg/g) reported by Lu et al. [6
] for soft winter wheat bran, suggesting that IWB could be more beneficial to human health than MWB.
The second most abundant phenolic acid was sinapic acid. Significant differences in free sinapic acid were also observed between MWB, IWB, and SIWB (p
< 0.001), whereas no significant differences in bound sinapic acid were observed. In Table 1
, the total sinapic acid ranged from 107–124 µg/g. This range is comparable to the total sinapic acid content (110–207 µg/g) reported by Verma et al. [8
], who examined the sinapic acid content of acid- or alkaline-hydrolyzed bran of six wheat cultivars. In the present study, IWB contained more free sinapic acid (3.95 µg/g) than MWB (2.46 µg/g). These results suggested that IWB with high sinapic acid might have potential antioxidant activities.
In addition, free and bound p
-coumaric acid values differed significantly (both p
< 0.001) among the three samples and were in the range of 3.22–5.61 µg/g and 51.97–74.58 µg/g, respectively. In the report of Kim et al. [16
] and Verma et al. [8
-coumaric acid contents in brans of various wheat cultivars were in the range of 35–46 µg/g and 133–476 µg/g, and the amount of p
-coumaric acid varied depending on the cultivars. In the current study, the total p
-coumaric acid content was higher than that reported by Kim et al. [16
] but lower than that reported by Verma et al. [8
]. In addition, the free p
-coumaric acid (5.61 µg/g) and bound p
-coumaric acid (68.98 µg/g) of IWB were higher than the free (3.22 µg/g) and bound p
-coumaric acid (51.97 µg/g) of MWB.
Significant differences in free and bound syringaldehyde contents were also observed among the three samples (both p
< 0.001). Free syringaldehyde content ranged from 3.07 µg/g (MWB) to 12.32 µg/g (IWB), and bound syringaldehyde content ranged from 13.67 µg/g (MWB) to 18.70 µg/g (IWB). In addition, free and bound caffeic acids did not significantly differ among the three wheat bran samples. By contrast, MWB (vanillic acid: 62.52 µg/g; syringic acid: 26.11 µg/g) contained more bound vanillic acid and syringic acid than IWB (vanillic acid: 51.52 µg/g; syringic acid: 18.01 µg/g) or SIWB (vanillic acid: 47.07 µg/g; syringic acid: 19.08 µg/g). Overall, immature bran extracts contained more p
-coumaric acid, syringaldehyde, and ferulic acid than MWB (Table 1
2.2. Antioxidant Capacity of Bran Extract Samples
Antioxidant capacity is frequently used as an important index for health benefits in foods, and several methods are used to determine in vitro antioxidant properties [17
]. In this study, the antioxidant capacities of bran samples were measured using oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA). The results of antioxidant capacities are in Table 2
The free ORAC value significantly differed among the three samples (p
< 0.01) and ranged from 38–54 µM TE/g. Significant differences in bound ORAC values were also observed among the three samples (p
< 0.001). Recently, Lu et al. [6
] studied the antioxidant activities of bran fractions from ten Maryland-grown soft winter wheat cultivars and reported that the ORAC values of the wheat bran samples were in the range of 39.91–61.50 µM TE/g. The free ORAC values (38–54 µM TE/g) in the current study are comparable to the ORAC values reported by Lu et al. [6
]. According to Lu et al. [6
], 50% acetone was used for the extraction of phenolics to measure antioxidant activities, and only free phenolic fractions were extracted. In this study, the bound ORAC value was six times higher than the free ORAC values, indicating significant contributions to ORAC by the bound phenolics in bran. As reported by Hung [18
], the phenolic acids of wheat exist mostly in the bound form. Bound phenolics are considered to possess more health benefits than free phenolics because bound phenolics in wheat appear to serve as powerful antioxidants by radical scavenging. In the present study, the highest bound ORAC values were observed in the bran extract of the IWB (273 µM TE/g), followed by SIWB (252 µM TE/g) and MWB (237 µM TE/g). These results suggest that the health benefits from IWB might be stronger than those from SIWB or MWB.
Moore et al. [19
] reported that the total ORAC values of eight whole-wheat samples ranged from 32.9 µM TE/g to 47.7 µM TE/g, which were similar to free ORAC values (38–54 µM TE/g) in this study. Total ORAC values (281–327 µM TE/g) in this study were much higher than those reported by Moore et al. [19
]. Additionally, the free ORAC values (19.6–37.5 µM TE/g) of whole wheat reported by Okarter et al. [20
] was comparable to the free ORAC values of the current study, whereas the bound ORAC values (31.9–59.5 µM TE/g) reported by Okarter et al. [20
] were lower than the bound ORAC values (281–327 µM TE/g) of bran reported in this study. These results indicate that the bran fraction of wheat contains more antioxidant capacity than whole wheat, and the antioxidant capacity in the bound fractions of bran is higher than in the free fractions. Because phenolic compounds of wheat are concentrated in the outermost layers, brans obtained from milling may be used as a natural source of antioxidants [21
]. The results of this study suggest wheat bran is a functional food ingredient that may exert positive health effects [22
]. In addition, results of this study demonstrate that IWB exhibits higher antioxidant activity than MWB.
The CAA assay can evaluate the cellular-based antioxidant activity of foods more accurately than chemical methods [23
]. The CAA assay is more physiologically relevant to biological systems, which are complex in nature and different from chemical systems [17
]. In this study, the cellular antioxidant activities of the bran extracts were measured on HepG2 cells and expressed as µM QE/g of bran. Significant differences (p
< 0.001) in the cellular antioxidant activities of the bran extracts were observed among the three bran samples. IWB had the highest CAA value (4.59 µM QE/g), whereas MWB exhibited the lowest value, 0.63 µM QE/g (Table 2
). The CAA value measured in the IWB extract was higher than the reported levels from other grains [17
], suggesting that IWB can be utilized as an effective ingredient in functional foods to help prevent various cancers or chronic diseases.
2.3. Effect of IWB Extract on Cell Growth of Human Carcinoma Cells
The cell growth inhibition of Caco-2, HT-29, and HeLa cells treated with wheat bran extracts are illustrated in Figure 1
. Differences in cell growth inhibition of HT-29, Caco-2, and HeLa cells were observed among the three bran samples (p
In this study, IWB exhibited the highest inhibition of Caco-2, HT-29, and HeLa cells. For example, 30 mg/mL of IWB extract inhibited approximately 80%, 87%, and 75% of Caco-2, HT-29, and HeLa cell proliferation, respectively. By contrast, 30 mg/mL of MWB extract exhibited the lowest inhibition (66%, 48%, and 56% for Caco-2, HT-29, and HeLa cells, respectively). At 30 mg/mL of bran extract, IWB extract resulted in a two-fold reduction in proliferation of HT-29 cells compared with the MWB extract.
The median effective dose (EC50
) of the three wheat bran extracts for antiproliferative activity against Caco-2, HT-29, and HeLa cells are shown in Table 3
. Lower EC50
values indicate higher inhibition of cell proliferation. Among the three wheat bran extract samples, MWB exhibited the lowest inhibition of cell proliferation in Caco-2, HT-29, and HeLa cells. The EC50
of IWB for Caco-2 cells was 7.62 mg/mL, whereas those of SIWB and MWB were 17.25 mg/mL and 15.62 mg/mL, respectively, demonstrating increased inhibition of Caco-2 cells by IWB compared with SIWB and MWB. Overall, the EC50
values were the highest for HT-29 cells, indicating relatively high proliferative activities in these cells.
Previous studies have reported that cereals with antioxidant activities have cancer-protective effects [5
], suggesting that natural antioxidants from cereals can inhibit cancer cell growth. In this study, the IWB exhibited increased antioxidant capacity and antiproliferative activity compared with the other samples, suggesting potential for IWB as a functional food ingredient with antioxidant capacity and anticancer effects.
2.4. Induction of Apoptosis by Bran Extract
The levels of apoptotic cell death of HT-29 cells treated with the three bran extracts are presented in Figure 2
. Wang et al. [24
] reported that grain extracts exhibited antiproliferative properties in human cancer cells, and the mechanism was associated with human cancer cell apoptosis. To investigate whether bran extracts could induce apoptosis in HT-29 cells, relative apoptotic cell death was measured using a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit, and the results are presented in Figure 2
A. As indicated in Figure 2
A, the IWB extract induced more apoptotic cell death than did the MWB extract. PTEN functions as a tumor suppressor by negatively regulating the AKT/PKB signaling pathway [26
]. As a general mediator of survival signals, AKT/PKB is an important upstream negative regulator of p53 [26
]. Thus, pAKT might play a critical role in controlling survival and apoptosis. As shown in Figure 2
B, gene expression of p53 and PTEN increased in HT-29 cells when treated with IWB or SIWB extracts. By contrast, gene expression of pAKT decreased in HT-29 cells when treated with IWB or SIWB extracts, suggesting that IWB and SIWB extracts inhibit the survival of HT-29 cells. In addition, apoptotic cell properties induced by IWB and SIWB extracts were observed by increasing gene expression of Bax and decreasing gene expression of Bcl-XL and Mcl-1 (Figure 2
Previous studies have reported that Bcl-XL and Mcl-1, among the anti-apoptosis Bcl-2 family members, promoted cell survival by inhibiting apoptotic activity [27
]. However, Bax, from the pro-apoptosis Bcl-2 family, contributed to mitochondrial-mediated apoptosis, which is involved in the release of mitochondrial cytochrome c into the cytoplasm [28
]. These results were also confirmed by observations with Annexin V and propidium iodide (PI) staining after treatment for 24 h with 10 mg/mL bran extracts via fluorescent microscopy. A representative image of bran extract-treated and untreated HT-29 cells is provided in Figure 2
C. The untreated cells did not exhibit any staining, suggesting that they did not undergo significant apoptosis or necrosis. In Figure 2
C, green and red stained cells indicate apoptotic and necrotic cells, respectively. Yellow-stained cells represent late apoptotic cells. As shown in Figure 2
C, more green cells were observed after treatment with the IWB extract than after treatment with the MWB extract, indicating that the IWB extract effectively induced apoptosis.
2.5. Correlations between Phenolic Compositions and Antioxidant, Antiprolferative, and Apoptosis Cell Death Properties in Wheat Bran Samples
The correlation coefficients (r) between phenolic compositions and antioxidant, antiproliferative, and apoptotic cell death properties among the three wheat bran samples are summarized in Table 4
. ORAC values were correlated with bound ferulic acid (r = 0.815), free p
-coumaric acid (r = 0.823), bound p
-coumaric acid (r = 0.779), free syringaldehyde (r = 0.713), and bound syringaldehyde (r = 0.781). A strong correlation was observed between CAA values and bound ferulic acid (r = 0.988), free sinapic acid (r = 0.914), free p
-coumaric acid (r = 0.961), bound p
-coumaric acid (r = 0.950), free syringaldehyde (r = 0.940), and bound syringaldehyde (r = 0.961). Several in vitro and in vivo studies have demonstrated that phenolic acids function as antioxidants and the antioxidant properties of phenolic acids are mainly attributed to electron donation and hydrogen atom transfer to free radicals [7
As shown in Table 4
, bound ferulic acid, free sinapic acid, p
-coumaric acid, and syringaldehyde exhibited high correlations with the antioxidant properties of wheat bran extracts. Dai and Mumper [30
] suggested that a diet including a high consumption of antioxidant-rich foods significantly reduces the risk of many cancers. In this study, strong correlations were identified between antiproliferative activities against HT-29, Caco-2, and HeLa cells and bound ferulic acid (r = 0.863 in HT-29 cells; r = 0.939 in Caco-2 cells; r = 0.975 in HeLa cells) and free sinapic acid (r = 0.786 in HT-29 cells; r = 0.826 in Caco-2 cells; r = 0.927 in HeLa cells). In addition, antiproliferative activities against cancer cells used in this study were correlated with p
-coumaric acid and syringaldehyde. Apoptotic cell death was correlated with bound ferulic acid (r = 0.882), free sinapic acid (r = 0.828), free p
-coumaric acid (r = 0.795), bound p
-coumaric acid (r = 0.966), free syringaldehyde (r = 0.812), and bound syringaldehyde (r = 0.890). As indicated in Table 1
, IWB contained more bound ferulic acid, free sinapic acid, p
-coumaric acid, and syringaldehyde than MWB, suggesting effective antioxidant and anticancer properties in IWB. Overall, the results of this study demonstrated that IWB exhibited high antioxidant and anticancer properties, implying potential health benefits of IWB.