The antioxidant functions of two feruloyl oligosaccharide (FO1 and FO2) were investigated in vivo and in vitro. Effects of FO1 and FO2 on hemolysis of rat red blood cell (RBC) and malondialdehyde (MDA) formation in rat liver homogenate and rat liver mitochondria in vitro were studied. Hemolysis of rat RBC and MDA formation in rat liver homogenate and rat liver mitochondria were inhibited in a dosage-dependent manner by FO1 and FO2 in the tested concentration range of 0.5 to 10 mg/mL. The results showed that FO1 and FO2 had antioxidative activity in vitro, and the effect of FO2 was better than that of FO1. With increasing dosage, FO1 and FO2 could increase the activity of SOD and GSH-Px in serum of S180 tumor-bearing mice, reduce the level of MDA, and thus improve the activity of the antioxidant in vivo. When the dosage reached 250 mg/kg/d, FO2 was more likely to improve the capabilities of the antioxidants of tumor-burdened mice than were 5-FU and FO1 in vivo.Thus, these oligosaccharides may be used as functional biological materials produced from fermented lignocellulose of WB.
Antioxidant Activity In Vivo and In Vitro of Two Feruloyl Oligosaccharides Preparations Produced from Wheat Bran and Fermented by Aureobasidium pullulans
Xiaohong Yu,a,b,* Xuemei Zhu,a Zhenxin Gu,b and Shaojuan Lai c
The antioxidant functions of two feruloyl oligosaccharide (FO1 and FO2) were investigated in vivo and in vitro. Effects of FO1 and FO2 on hemolysis of rat red blood cell (RBC) and malondialdehyde (MDA) formation in rat liver homogenate and rat liver mitochondria in vitro were studied. Hemolysis of rat RBC and MDA formation in rat liver homogenate and rat liver mitochondria were inhibited in a dosage-dependent manner by FO1 and FO2 in the tested concentration range of 0.5 to 10 mg/mL. The results showed that FO1 and FO2 had antioxidative activity in vitro, and the effect of FO2 was better than that of FO1. With increasing dosage, FO1 and FO2 could increase the activity of SOD and GSH-Px in serum of S180 tumor-bearing mice, reduce the level of MDA, and thus improve the activity of the antioxidant in vivo. When the dosage reached 250 mg/kg/d, FO2 was more likely to improve the capabilities of the antioxidants of tumor-burdened mice than were 5-FU and FO1 in vivo. Thus, these oligosaccharides may be used as functional biological materials produced from fermented lignocellulose of WB.
Keywords: Wheat bran; A. pullulans; Feruloyl oligosaccharide; Antioxidant activity; In vivo and in vitro
Contact information: a: College of Chemistry and Biological Engineering, Yan Cheng Institute of Technology, Yancheng 224003, China; b: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; c: College of Biological Engineering, Henan University of Technology, Zhengzhou, 450001, China; *Corresponding author: firstname.lastname@example.org
Wheat bran (WB) is a good source of dietary fiber and is produced worldwide in enormous quantities as an important by-product of the cereal industry. Many studies have dealt with the isolation of feruloylated oligosaccharides (FOs) from WB (Ishii 1997; Yuan et al. 2006; Xie 2010; Yu et al. 2013; Yu and Gu 2014a,b). The interest in these oligosaccharides is motivated by their biological activities and their technological applications. A high value is placed on FOs, as they may be used as natural antioxidant materials. The antioxidant activity of FOs is higher than that of ferulic acid (FA) and vitamin C, as they exhibit strong inhibition of the hemolysis of mice red blood cells as well as elimination of Fe2+, H2O2, and hydroxyl radicals (Wang et al. 2010, 2011). FOs have also been reported to display significant antioxidant capacity in 1-1-diphenyl-2-picrylhydrazyl (DPPH) and lipid peroxidation systems (Wang et al. 2008, 2009).
Based on the fact that FOs produced from A. pullulans-fermented WB displayed significant antioxidant capacity in vivo (Yu and Gu 2014a), there is a motivation to further carry out studies of this topic. However, the manner in which the component purity and degree of polymerization of FOs affect their antioxidant capacity in vivo and in vitro is still unknown. In addition, the antioxidant capacity invitro of FOs produced from A. pullulans-fermented WB has not been reported.
In our previous work (Yu et al. 2013; Yu and Gu 2014a,b), two components (FO1 and FO2) were produced from WB fermented by A. pullulans via one- and two-stage pH and temperature controlling processes, respectively. In this study, their antioxidant capacities were investigated in vivo and in vitro. The influences of different purities and degrees of polymerization of FOs prepared under the two fermentation conditions on their antioxidant capacity were determined. Because FOs can be produced easily and abundantly from A. pullulans-fermented WB, it is feasible to use FOs as potential antioxidant functional foods from WB. Therefore, this study may provide both a theoretical basis and an application prospect to develop FOs as a health-promoting food.
Raw materials and reagents
FO1 and FO2 were prepared by one-stage and two-stage pH and temperature processes for control of WB fermentation by A. pullulans 2012, respectively (Yu et al. 2013; Yu and Gu 2014a,b). FO1 consisted of feruloyl arabinosyl xylopentose (FAX5, Mw986), feruloyl arabinosyl xylotetraose (FAX4, Mw854), feruloyl arabinosyl xylotriose (FAX3, Mw722), and feruloyl arabinosyl xylobiose (FAX2, Mw590), whereas FO2 contained feruloyl arabinosyl xylohexose (FAX6, Mw 1118), FAX5, and FAX4 (Yu et al. 2014).
The fluorouracil injection (5-FU) was from Hengrui Medicine Co. (Lianyungang, Jiangsu, China; batch number: 1111262; specifications: 10 mL, 0.25 g). Fetal bovine serum was purchased from Sigma Chemical Co. (St. Louis, MO, USA). MDA, SOD and GSH-Px kits were purchased from Jiancheng Biotech Co. Ltd. (Nanjing, Jiangsu, China). Vitamin C (Vc) was purchased from Xian Ruilin Biotech Co. Ltd. (Xian, Shanxi, China). Phosphate buffered saline (PBS), H2O2 and FeSO4 were purchased from Luke Chemical Product Co., Ltd. (Shouguang, Shandong, China). S180 was provided by Jiangsu Provincial Institute of Cancer Research (Nanjing, Jiangsu, China).
The tumor-bearing mice were purchased from the Comparative Medicine Centre of Yangzhou University (Yangzhou, Jiangsu, China; Certificate No. SCXK-2007-0001).
In Vitro Antioxidant Activity of FOs
Effect of FOs on mice erythrocytic osmotic fragility
Male Sprague-Dawley mice (280 to 300 g) were housed for seven days at 18 to 24 °C and a relative humidity of 70% on a 12 h light/12 h dark cycle. Blood (8 mL/rat) from the abdominal aorta was collected into a heparinized tube. Red blood cells (RBCs) were separated from plasma by centrifugation at 10,000 x g for 10 min. The crude RBCs were then washed three times with five volumes of phosphate buffered saline (PBS, pH = 7.4), and suspended in PBS to obtain a 0.5% RBC suspension. In the present study, the method described by Yuan et al. (2005) was used to determine the hemolysis of RBC. Five milliliters of RBC suspension was mixed with 2 mL of PBS solution containing varying amounts of FOs, and incubated at 37 °C for 24 h. The resulting solutions were centrifuged at 10,000 x g for 10 min. The absorbance (A) of the supernatant at 540 nm was recorded in a spectrophotometer. Percentage inhibition was calculated as the formula of (%) = (1-T/C)×100, and hemolysis rate (%) = (T/C)×100, where T and C are the averages of the sampling groups and control group (containing no FOs) , respectively.
Effect of FOs on mice erythrocyte hemolysis induced by H2O2
The method described by Zhang et al. (2002) was used to determine the hemolysis of RBC induced by H2O2.
Preparation of sampling group: One milliliter of 0.5% RBC suspension was mixed with 0.2 mL PBS solution containing varying amounts of FOs and 0.1 mL of 100 mM H2O2. The incubation mixture was shaken gently in a water bath at 37 °C for 1 h. After incubation, 7.8 mL of PBS solution was added into the reaction mixture, followed by centrifugation at 10,000 x g for 10 min. The absorbance (A) of the supernatant at 540 nm was recorded in a spectrophotometer.
Preparation of normal control group: The sampling solution (PBS solution containing varying amounts of FOs) and H2O2 were replaced with an equal volume of PBS solution, and the others were kept the same as mentioned above.
Preparation of induced control group: The sampling solution was replaced with an equal volume of PBS solution, and the others were same as mentioned above.
The hemolysis extent of FOs was expressed as the average A value of the sampling group relative to the average A of the induced control group. The inhibition rate of FOs was calculated as the formula of (%) = (1-T/C)×100, where T and C are the average As of the sampling groups and induced control group, respectively.
Effect of FOs on MDA formation in mice liver homogenate
The effect of FOs on MDA formation in mice liver homogenate was determined by the method of Huang et al. (2003) with a slight modification. Livers were excised immediately after the animals were sacrificed. The liver was frozen quickly and stored at 4 °C. The thawed tissue samples were homogenized in nine volumes of ice cold PBS and centrifuged at 10,000 x g for 10 min at 4 °C. The supernatant (10% liver homogenate) was used for determination of MDA using commercially available diagnostic kits. The reaction mixture, for the analysis of MDA in mice liver homogenate, containing 0.4 mL of 10% liver homogenate and 0.2 mL of sampling solution, was incubated for 1 h at 37 °C. An equal volume of PBS was added as the control group. The absorbance of the mixture was measured at 532 nm against the blank. The levels of MDA were normalized with protein. The reaction mixture for the analysis of MDA in induced mice liver homogenate was 0.2 mL of 10% liver homogenate mixed with 0.2 mL of sampling solution and 0.2 mL of 1 mM FeSO4 (or H2O2), and what followed was similar to the methods for the MDA as mentioned above.
Effect of FOs on MDA formation in mice liver mitochondria
The effect of FOs on MDA formation in mice liver mitochondria was determined by the method of You and Lin (2003). The washed fresh liver was homogenized in nine volumes of PBS and centrifuged at 10,000 x g for 10 min at 4 °C. The supernatant was collected and centrifuged repeatedly. The collected precipitation was washed with PBS twice, and then dissolved in PBS to prepare for 0.5 mg/mL mice liver mitochondria suspension. The sampling group reaction mixture, containing 1 mL of the rat mitochondria suspension (0.5 mg/mL), 0.4 mL of sampling solution, 0.4 mL of 0.5 mM FeSO4,and 0.4 mL of 0.5 mM Vc, was incubated for 1 h at 37 °C. Both the sampling solutions, FeSO4 and Vc, were replaced with an equal volume of PBS, which was used for the normal group. The sampling solution was replaced with an equal volume of PBS, and it was used for the control group. The content of MDA was measured according to the kit method.
In vivo Antioxidant Activity of FOs
In vivo antioxidant activity of FOs was measured according to the method of Yu and Gu (2014a).
Experimental data are expressed as the mean ± SD. All the data are at least triplicate and the results were reproducible within 10% deviation. SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used for ANOVA statistical analysis. When P < 0.05, the difference was considered statistically significant.
RESULTS AND DISCUSSION
Antioxidant Activity of FOs in vitro
Impact of FOs on mice erythrocytic osmotic fragility
The in vitro effect of FO1 and FO2 on the mice erythrocytic osmotic fragility is shown in Table 1. Incubation of an erythrocyte suspension with both FO1 and FO2 (0.5, 2.5, 5.0, 7.5, and 10 mg/mL) significantly decreased the hemolysis rate in a dose-dependent manner as compared to the normal control group (P < 0.05). This indicated that FOs had inhibitory functions against mice erythrocytic osmotic fragility. The inhibition rate showed an ascendant trend with an increment of FOs concentration. When the dose was 10 mg/mL, FO1 and FO2 inhibited 41.742% and 52.853% of erythrocytic osmotic fragility, respectively. FO2 showed a significantly higher inhibition rate than FO1 did. This indicated that FO2 was more efficient than FO1 in the inhibitory functions against mouse erythrocytic osmotic fragility.
Table 1. Effects of FOs on Mice Erythrocytic Osmotic Fragility
Impact of FOs on mice erythrocyte hemolysis induced by H2O2
Table 2 shows the effect of different concentrations of FOs on H2O2-induced changes in mice erythrocyte hemolysis. Each dose of FO1 and FO2 showed significant differences from the normal control group and the induced control group (P < 0.05). When the erythrocyte suspension was incubated with FOs, a significant dose-dependent decrease in hemolysis rate and increase in inhibition rate of erythrocytes was observed. When the dose of FO1 and FO2 reached 10 mg/mL the inhibition rates against erythrocyte hemolysis were 46.520 ± 5.715% and 62.800 ± 1.361%, respectively. All five doses (0.5, 2.5, 5, 7.5 and 10 mg/mL) of FO2 had significant differences from A540. As a result, FO2 showed more efficient inhibitory functions against erythrocyte hemolysis than FO1 did.
Table 2. Effects of FOs on Mice Erythrocyte Hemolysis Induced by H2O2
Impact of FOs on MDA formation in mice liver homogenate
The effects of FO1 and FO2 on MDA formation in mice liver homogenate in the absence of inducer, and induced by H2O2 and FeSO4, are shown in Table 3. Among the three systems, all five doses (0.5, 2.5, 5, 7.5, and 10 mg/mL) of FO1 and FO2 had significant inhibitory functions against MDA formation in mice liver homogenate from the blank control group, and both FO1 and FO2 improved the inhibitory functions as the dosage increased.
In addition, increasing FOs dose to 5 mg/mL, the inhibitory rate with an inducer was higher than that without an inducer. When the dose was 10 mg/mL in the absence of an inducer and induced by FeSO4, the MDA content of the FO2 controlling group was significantly less than that of the FO1 controlling group. When the dose was 7.5 mg/mL induced by H2O2, the MDA content of the FO2 controlling group was significantly less than that of the FO1 controlling group. It was therefore concluded that FO2 had a better effect on the inhibitory functions against MDA formation than FO1 did.
Table 3. Effects of FOs on MDA Formation in Mice Liver Homogenate
Impact of FOs on MDA formation in mice liver mitochondria
The effects of FOs on MDA formation in mice liver mitochondria are shown in Table 4. MDA formation of mice liver mitochondria was inhibited in a dose-dependent manner by FO1 and FO2 and showed significant differences from blank control group. The inhibitory rates of FO2 were significantly higher (P < 0.05) than those of FO1 at the same dosage. When the dosage was 10 mg/mL, the inhibitory rate of FO2 was 47.34%, while that of FO1 was 41.30%. It was therefore concluded that FO2 had a better effect on inhibitory functions against oxidative damage than FO1 did.
Antioxidant Activity of FOs in vivo
As shown in Table 5, with increasing the dosage of FO1 (Yu and Gu 2014a) and FO2, the activities of SOD and GSH-Px in S180 tumor-bearing mice sera were improved and the content of MDA was reduced, thereby the antioxidant activity of tumor-bearing mice was enhanced. When the dose was 250 mg/kg/d, the enzyme activity of the FOs group hasn’t significant difference with that of the 5-FU positive control group, therefore, it indicated that FOs had an excellent in vivo antioxidant effect. When the dosage reached 250 mg/kg/d, FO2 improved the antioxidant capability of tumor-burdened mice more than 5-FU and FO1 (Yu and Gu 2014a) in vivo.
Table 4. Effects of FOs on MDA Formation in Mice Liver Mitochondria
Table 5. Effects of FOs on SOD, GSH-Px and MDA of SCR Mice and S180 Tumor-bearing Mice Sera in vivo
The results showed that both FO1 and FO2 presented significant antioxidant capacity in vivo and in vitro. Yuan et al. (2005) indicated that the FOs could efficiently protect normal rat erythrocytes against hemolysis induced by free radicals under in vitro conditions. According to the literature, the membrane of erythrocytes is rich in polyunsaturated fatty acids that are very susceptible to free radical-mediated peroxidation (Yuan et al. 2005). It may be deduced that scavenging free radicals may be directly depended on hydrogen atoms and, thus, that the provision of hydrogen atoms enhances the antioxidant capacity. There is a phenolic hydroxyl group in the parent nucleus for the existence of the FA structure in FOs, therefore, the provision of hydrogen atoms as well as the ability to scavenge free radicals were enhanced (Wang et al. 2010). Thereby, the result in our work is similar to the results of other studies (Ohta et al. 1994; Katapodis et al. 2003; Wang et al. 2008, 2011).
It has been reported that the antioxidant activity of oligosaccharides can be affected significantly by the polymerization degree (Chen and Yan 2005). To date, data have revealed that a polymerization degree of six is beneficial to the improvement of antioxidant activity. In the present study, the component purity and polymerization degree of FO2 were higher than those of FO1; therefore, the antioxidant activity in vivo of FO2 would be expected to be better. It may be presumed that the antioxidant activity of FOs is closely related to their purity and structure, and this will be further investigated. Thus, the results in our work are similar to the results of other studies (Wang et al. 2008, 2011).
- FO1 and FO2 inhibited the hemolysis of mice RBCs and the MDA formation of mice liver homogenate and mice liver mitochondria in a dosage-dependent manner in the tested concentration, showing that FOs had an excellent in vitro antioxidant effect.
- As the doses of FO1 and FO2 increased, the activities of SOD and GSH-Px in tumor S180-bearing mice serum were promoted with decreasing MDA content, thus enhancing the in vivo antioxidant activity of tumor-bearing mice.
- FO2 exhibits more efficient antioxidative functions than FO1 does. The structure of FOs affects the antioxidative functions. These oligosaccharides can thus be used as antioxidative agents in the future functional food industry from fermented hemicellulose of WB.
The authors are grateful for the support of the Natural Science Foundation of Jiangsu Province, Grant. Nos. BK2012249 and BK20141220, the Jiangsu Science & Technology Department of China, Grant. No. BY2013057-03, BE2011728, the Ministry of Science and Technology of China, Grant. No. 2013GA690132, the Natural Science Fund for Colleges and Universities in Jiangsu Province, Grant. No. JHB2012-5, and the Joint Innovation Fund Project of Yancheng Science and Technology Bureau, Grant. No. YKN2012026. Projects 31200801, 31471605, 31371851 and 31071617 supported by NSFC are also acknowledged.
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Article submitted: October 20, 2014; Peer review completed: December 15, 2014; Revised version received and accepted: February 7, 2015; Published: February 12, 2015.