Phenol contents of different varieties of fresh waxy corn
The polyphenol content of fresh waxy corn differed significantly (p < 0.05) among the varieties. The flavonoid content was relatively close to that of phenolic acids, whereas anthocyanin content was considerably lower than both (Table 1). The free phenolic acid and free anthocyanin contents ranged from 744.36 to 1907.70 GAE μg/g DW and from 21.12 to 291.50 AE μg/g DW, respectively, which was in accordance with the ranges measured by Hu and Xu [6]. In terms of color, the colored varieties contained more phenols than the common varieties (white and/or yellow), except that the lowest flavonoid content of multicolor corn maybe due to the large number of white and yellow kernel (Fig. 1). The varieties with the highest content of phenolic acids, flavonoids, and anthocyanins were ‘Huanuo 1’, ‘Caizhen 100’, and ‘Yuheinuo 600’, respectively.
Table 1 Polyphenol content of different varieties of fresh waxy corn (μg/g DW).
Sample
|
WC
|
YC
|
RC
|
YWC
|
MC
|
BC
|
Phenolic acid
|
|
|
|
|
|
|
Free form
|
1031.21±33.70bA
|
744.36±30.01dA
|
1165.73±55.25bA
|
882.36±17.91cB
|
906.37±39.58cB
|
1907.70±29.76aA
|
Bound form
|
1114.60±59.56bA
|
415.65±42.13cB
|
1190.60±89.28bA
|
980.60±21.30bA
|
2157.09±65.97aA
|
418.11±13.23cB
|
Subtotal
|
2145.81±75.65bc
|
1160.01±60.18d
|
2356.33±145.99b
|
1862.96±45.23c
|
3063.97±116.39a
|
2325.81±56.77b
|
Flavonoid
|
|
|
|
|
|
|
Free form
|
872.89±25.63bcA
|
1260.70±47.58aA
|
1194.52±51.20aB
|
685.47±20.32dA
|
860.66±29.55cA
|
975.85±34.53bA
|
Bound form
|
833.11±29.60bA
|
572.91±6.59cB
|
1564.48±89.22aA
|
624.99±12.15cA
|
344.27±7.10dB
|
934.76±20.34bB
|
Subtotal
|
1706.95±38.49c
|
1833.61±47.97bc
|
2756.98±153.24a
|
1310.46±94.36d
|
1204.93±38.52d
|
1910.61±49.32b
|
Anthocyanin
|
|
|
|
|
|
|
Free form
|
3.56±0.53d
|
2.23±0.31d
|
21.12±0.88b
|
12.46±0.89c
|
19.44±1.57b
|
291.50±8.233a
|
Bound form
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
Subtotal
|
3.56±0.53d
|
2.23±0.31d
|
21.12±0.88b
|
12.46±0.89c
|
19.44±1.57c
|
291.50±8.233a
|
Polyphenol form
|
|
|
|
|
|
|
Free form
|
1907.55±45.56cA
|
2006.12±39.42cA
|
2382.23±88.31bB
|
1578.93±33.72eA
|
1788.32±66.87dB
|
3174.11±23.96aA
|
Bound form
|
1947.21±77.30cA
|
988.39±22.21fB
|
2754.79±122.94aA
|
1605.52±33.61dA
|
2498.73±49.38bA
|
1351.84±42.70eB
|
Total
|
3856.14±111.72c
|
2994.56±70.11d
|
5136.67±168.75a
|
3184.49±115.67d
|
4286.38±125.32b
|
4524.44±79.81b
|
All values are means of replicate determinations ± standard deviation (n=3). nd indicates none detected. Different lowercase letters in the same row indicate statistical differences (p < 0.05) in the LSD test. Different uppercase letters in the same column of phenolic acid, flavonoid, or polyphenol indicate statistical differences (p < 0.05) in LSD test.
The major portion of phenolics in grains existed in the bound form; for example, corn contains 85% bound phenolics [4, 12]. However, in the present study, the total free phenolic content of the six varieties was 6637.73 μg/g DW, which was slightly higher (p > 0.05) than that of the total bound form (6276.65 μg/g DW). The total free flavonoid content was 5850.09 μg/g DW, which was higher (p < 0.05) than that of the total bound form (4874.51 μg/g DW). No bound anthocyanin was detected in the methanol extracts of the tested varieties (Table 1). Therefore, we concluded that milk stage corn contained higher free phenols than the physiological mature corn. The abundance of free phenols in fresh waxy corn suggested that its functional activities were different from those of mature corn.
In terms of free phenol content, the order of the six varieties was YWC<MC<WC<YC<RC <BC. The free phenol content ranged from 1578.93 to 3174.11 μg/g DW. In terms of bound phenol content, the order of the six varieties was: YC<BC<YWC<WC<MC<RC. The bound phenol content ranged from 988.39 to 2754.79 μg/g DW. The differences among varieties in the bound form content were larger than those of the free forms. ‘Yuheinuo 600’ and ‘Caizhen 100’ had the highest levels of the free and bound forms, respectively.
In vitro chemical antioxidant activity of polyphenol extracts of different varieties of fresh waxy corn
Corn possesses higher antioxidant activity than wheat, barley, rice, and oats [13]. In the present study, the differences in the antioxidant activity of different varieties and forms of polyphenols were evaluated using the DPPH and hydroxyl radical scavenging abilities, together with ferric reducing antioxidant power (FRAP). The free phenolics of different varieties showed higher (p < 0.05) antioxidant activity than the bound form (Fig. 2). No significant differences (p > 0.05) in DPPH scavenging rate was observed among the different varieties for the free form of polyphenols; the hydroxyl radical scavenging ability showed similar characteristics, except that YWC exhibited lower (p < 0.05) value than the other varieties. There was apparently no correlation between the radical scavenging activity and polyphenol content, which can possibly be because all radicals were scavenged by the different phenolic extracts despite differences in phenolic content among the varieties [5]. YWC contained the lowest free polyphenol content (Table 1), which suggested that it had the worst hydroxyl radical scavenging ability. The FRAP of free polyphenols differed from the radical scavenging ability, and significant differences among varieties were observed. The FRAP ranged from 0 to 21.23 μmol FeSO4/g DW, and BC showed the strongest ability than others, while YC showed the lowest ability without any ferric ion reducing power. Analysis revealed a significant (p < 0.05) positive correlation between FRAP and free anthocyanin content (R2=0.9876). This was because the experiment was conducted under acidic conditions, which stabilizes anthocyanins [15].
The bound form of polyphenols in different varieties showed significant differences (p < 0.05) in chemical antioxidant activity. Most varieties showed higher hydroxyl radical scavenging ability than that DPPH scavenging ability for bound polyophenols, whereas the free forms showed completely opposite results. RC and MC contained the highest amount of polyphenol (Table 1), and correspondingly, both showed higher antioxidant activity (Fig. 2a, 2b). Apart from this, significant correlation between phenolic contents and radical scavenging capacity was not observed.
In summary, free polyphenols contributed 86-100% and 70-78% of the DPPH and hydroxyl radical scavenging abilities, respectively. The free polyphenols were responsible for 100% of the FRAP activity. This was considerably different from the results obtained with mature corn [4, 12], indicating that the difference between milk stage and mature period are due to differences in the polyphenol composition. Xu et al. [5] suggested that bound phenolics might contain highly reactive antioxidative substances. Das and Singh [12] considered free phenolics to be capable of participating in the FRAP reaction as much as bound phenolics owing to an electron transfer-based mechanism. However, the present results do not support these viewpoints.
Some studies have reported a correlation between polyphenol content and antioxidant activity [6, 12]. However, apart from the significant correlation between free anthocyanin content and FRAP, our results did not show a clear correlation between antioxidant activity and polyphenol content for any variety. Such inconsistencies have been reported previously [5, 19, 20]. Hu and Xu [6] indicated that these differences in correlation might be related to the types of materials, methods of assessing antioxidation, solvent extraction systems, and the complications associated with extracts containing two or more antioxidants.
In vitro hypoglycemic effect of phenol extract of different varieties of fresh waxy corn
Corn is considered as a potential value-added functional food ingredient that can reduce the risk for type 2 diabetes. Phenolic compounds have been considered another bioactive phytochemical that can control postprandial serum glucose levels and the incidence of type 2 diabetes [14, 21]. Phenolics, as inhibitors of pancreatic α-amylase and intestinal α-glucosidase, play important roles in inhibiting the rapid rise of postprandial blood glucose [2, 21, 22]. The free phenolics of fresh waxy corn showed significantly better (p < 0.05) hypoglycemic effect than the bound form in terms of α-amylase activity inhibition, irrespective of variety (Fig. 3a). The inhibition rate of the free form was 5~160 fold higher than that of the bound form. Oboh et al. [23] reported similar results with jute leaf methanol extracts. On the contrary, different varieties showed considerable differences in the inhibition of α-glucosidase activity by the bound and free forms of phenolics. Compared to the bound forms, free phenolics of only two varieties, MC and BC, showed better inhibition of α-glucosidase activity. The bound phenolics of other varieties showed higher inhibitory activity than the free forms.
In terms of corn varieties, the free phenolic compounds of YC showed the highest α-amylase inhibitory activity, followed by WC, whereas RC showed the lowest α-amylase inhibitory activity. The other varieties exhibited similar activity. The bound phenolic compounds also showed significant differences among different varieties. YC showed strong α-amylase inhibitory activity, with a value of 72.82±2.18 %/100g DW for the free form and 11.18±0.69%/100g DW for the bound form, whereas RC exhibited the lowest activity. The bound phenolic compounds also showed large differences in α-glucosidase inhibitory activity. YC and YWC possessed remarkable inhibitory activity, followed by WC and RC. In contrast, the α-glucosidase inhibitory activity of free phenolic compounds did not vary significantly among the varieties, with the exception of BC.
Oboh et al. [23] concluded that the α-glucosidase inhibitory activity of plant food was a function of their phenolic acid content, whereas Tadera et al. [24] indicated that flavonoids were stronger α-amylase inhibitors than other phenolic compounds. However, as shown in Table 1, the content of the free form of phenolic acids was not significantly higher than that of the bound form for most varieties except BC. At the same time, the content of the bound form of flavonoids was not significantly higher than that of the free form for most varieties except RC. Therefore, profile changes and some unidentified phenolic compounds might be responsible for our observations.
In vitro hypocholesterolemic effect of phenol extracts of different varieties of fresh waxy corn
Polyphenols can prevent reabsorption of bile acids in the small intestine by binding to them and triggering cholesterol destruction [25, 26]. Bansode et al. [27] concluded that low molecular weight polyphenols inhibited the intestinal transport of dietary cholesterol because of the decomposition of the bile acid-emulsified micellar structure in the intestines. In vitro bile acid binding activity is potentially related to the reduction in cholesterol content [18]. The methanol extracts of fresh waxy corn showed strong bile acid binding activity for all tested materials. The extracts showed the highest binding capacity for sodium glycocholate, with values above 130 μmol/100mg DW (Fig. 4a), while the binding ability for sodium cholate was the lowest (Fig. 4b). The ability of the extract to bind to sodium taurocholate ranged from 66.59±5.36 to 135.93±1.48 μmol/100mg DW (Fig. 4c). Different forms of polyphenols showed considerable difference in bile acid binding capacity. The free phenolics showed significantly better (p < 0.05) sodium glycocholate binding effect than the bound form for all varieties (Fig. 4a), while the bound polyphenols showed better or equivalent sodium cholate binding capacity than the free form, with the exception of YWC (Fig. 4b). The free forms in most varieties exhibited higher or equivalent sodium taurocholate binding activity than the bound form, with the exception of WC and YC (Fig. 4c).
Previous studies have comprehensively determined bile acid binding capacity of some vegetables, fruits, cereal bran, and legumes [25]. Karataş and Sayar [26] observed that the bile acid binding value of fava bean seed coat was 37.50 ± 3.08 μmol/100mg DW, which was lower than that obtained in the present study. However, most studies have used whole plants instead of extracts, and focused on dietary fiber instead of polyphenols. Studies on the bile acid binding ability of different forms of polyphenols are still limited.
Overall, MC showed the best bile acid binding activity for free and bound forms, whereas WC and YWC showed lower bile acid binding capacity. However, there was no significant correlation (p > 0.05) between the polyphenol content and the bile acid binding activity. Mäkynen et al. [17] obtained similar results in pomelo extracts. Condensed tannins [26, 28], insoluble dietary fiber [29], flavonoids [28], and lignin [30] or non-lignin components [31] were believed to be responsible for the bile acid binding activity. In fact, bile acid binding may be related to phytochemical, anionic, cationic, physical, and chemical structures, metabolite composition, and interaction with active binding sites [28, 32].