3.1. Isolation and Characterization of AXs
The yield of AXs extracted through different methods is shown in Table 1
. The yield of alkali-extracted AXs was higher than that of enzyme-extracted AXs and WEAX, which was consistent with previous reports [15
]. Therefore, efficiency of alkaline solution was higher than that of complex enzyme in AX extraction. Moreover, the total yield of CEAX and CEAX-1 was higher than that of AEAX, which indicated that alkali extraction can be more efficient after enzyme extraction, thereby elevating total AX yield.
Monosaccharide compositions of triticale-extracted AXs were measured by HPLC, and the results are shown in Table 2
. AXs extracted using different methods all contained nearly 90% of arabinose and xylose. Likewise, alkali-extracted AXs were highly substituted with an arabinose–xylose ratio of 1.14 to 1.52, which was higher than enzyme-extracted AXs with an arabinose–xylose ratio of 0.25. A previous study determined the monosaccharide compositions of AXs derived from wheat bran by enzyme, water, and alkali extraction [24
]. The results also demonstrated that the arabinose–xylose ratio of alkali-extracted AXs was higher than that of enzyme-extracted AXs. Other minor neutral sugars, such as galactose and mannose, were detected in AXs, which was consistent with previous research studies [25
MW distribution was determined (see Table 3
). The MWs of enzyme-extracted AXs were lower than those of alkali-extracted AXs, consistent with a previous report [15
]. The lower MW of enzyme-extracted AXs may be due to the ability of endo-1,4-β-xylanases to randomly cleave the xylan chain, which releases fractionated AXs and/or AXOS of various sizes [27
The content of free and esterified FA in AXs was determined, and the results are shown in Figure 1
. Enzyme-extracted and water-extracted AXs had significantly higher esterified FA content than free FA content (p
< 0.05). Malunga and Beta also observed that the amount of esterified FA was higher than free FA in enzyme-extracted AXs, which was in agreement with our result [3
]. Likewise, alkali-extracted AXs had significantly higher free FA content than esterified FA content (p
< 0.05). Alkali solutions are extremely violent and may break down ester linkage between FA and arabinofuranosyl residues; thus, a large amount of free FA was contained in alkali-extracted AXs. Furthermore, among all AXs extracted by different methods, CEAX-1 contained the highest amount of total FA and free FA, whereas CEAX contained the highest amount of esterified FA.
Interestingly, our results demonstrated that the arabinose–xylose ratio and MW of CEAX-1 was similar to AEAX, and the amount of total FA of CEAX-1 was even higher than of AEAX. These results were obtained probably because the enzyme treatment increased the efficiency of subsequent alkali extraction. Alkali treatment on residues after enzyme extraction allowed for the full use of raw materials.
The infrared absorption spectrum of AXs is shown in Figure 2
. A wide polysaccharide absorption peak in the range of 1200 cm−1
to 800 cm−1
, a stretch vibration peak of the glycosidic bond at 857 cm−1
, a stretch vibration peak of methyl C–H at 2928 cm−1
, and a bending vibration peak of C–H at 1409 cm−1
were observed. The wide peak at 3401 cm−1
was produced by the stretching vibration of the hydroxyl group; the strong peak at 1653 cm−1
resulted from the asymmetric stretching vibration of C–O in the carbonyl group; and the peak at 1039 cm−1
is attributed to the deformation vibration of alcohol hydroxyl O–H. The infrared absorption spectrum confirmed that the structural characteristics of the products extracted from triticale were consistent with AXs. Moreover, all absorption peaks had discrepancies in different AXs, which suggests that the structure of AXs extracted through different methods is inconsistent.
Based on the AX structural characteristics, antioxidant and hypoglycemic activities of AXs were estimated in further experiments, aiming to determine the relationship between AX structure and physiological functions.
3.2. Antioxidant Activity of AXs
Our results showed that along with the increased AX concentration, the hydroxyl radical, DPPH radical-scavenging ability, and metal-chelating activity of AXs increased; and these results confirmed the antioxidant activity of AXs. AXs are capable of donating electrons or hydrogen atoms to free radicals, which leads to the transformation from free radicals to stable products [28
]. Therefore, free radicals are eliminated, and oxidation reactions are interrupted. It has been reported that AX structure contributed to their antioxidant ability. Hydroxyl groups of AXs are the main structural features that affect their antioxidant capacity [30
]. The presence of electrophilic groups in AXs helps to release hydrogen from O–H bonds. Our findings showed that the structural characteristic of the AX moiety should not be ignored when evaluating the AX antioxidant. Figure 3
shows the results of the hydroxyl radical-scavenging assay, which is based on the reduction of hydroxyl radicals in the existence of an electron-donating substance [31
]. Based on the comparison between CEAX-1 and CEAX, CEAX exhibited higher hydroxyl radical-scavenging activity. Both CEAX and CEAX-1 contained similar amounts of FA, but CEAX had lower DS. Thus, lower DS contributes to higher antioxidant ability of CEAX. The unsubstituted xylose at O-2 and/or O-3 may have contributed to antioxidant capacity of AXs via donation of electrons or hydrogen atoms [30
]. Low DS increases the specific functional groups of xylan to elevate hydroxyl radical-scavenging activity through donation of electrons or hydrogen atoms [32
]; thus, the antioxidant activity of AX with low DS increased.
DPPH radical-scavenging activity was measured in Figure 4
, whose reaction mechanism involves the transfer of protons by the reducing agent to the DPPH radical [3
]. In contrast, the DPPH radical-scavenging ability of AXs elevated in line with the increase in DS. This finding was inconsistent with the results of hydroxyl radical-scavenging assay. AXs with higher DS were found to be easier to disperse into the reaction mixture because the steric hindrance caused by the presence of mono- or disubstituted xylose prevented intermolecular cross-linking, which leads to better participation of AXs in the redox reaction systems [4
]. Compared to the results of hydroxyl radicals with the DPPH radical-scavenging assay, we found that the structure of AXs would not only influence the amount of special functional groups, but also affect the interaction between AX molecules as well as between AXs and the solvent. In addition, both of these substances would exert an effect on antioxidant capacity.
The metal-chelating activity of AXs was determined in the present study (see Figure 5
). It has been reported that the specific functional groups such as –COOH and –OH of AXs demonstrated the ability to bind metal ions when those functional groups were deprotonated and carried negative charges [34
]. The metal-chelating activity of AX samples was elevated when the MW of the AXs was increased, despite the different FA content in the AXs extracted by different methods. Therefore, our results showed that AXs with higher MW lead to higher metal-chelating activity. High MW may elevate (1–4)-β-d
-xylopyranose linkages to some extent, which leads to a higher amount of specific functional groups (such as –COOH and –OH) of xylan with metal-binding activity [32
Moreover, we determined whether the FA content of AXs influenced their antioxidant ability. AEAX and CEAX-1 have similar MW and DS, but CEAX-1 performed higher hydroxyl radical- and DPPH radical-scavenging activity compared with AEAX. These results occurred because CEAX-1 contained a higher amount of FA, which suggested that FA content was the major determining factor for the antioxidant capacity of AXs [6
]. Furthermore, among AEAX, CEAX-1, and CEAX, CEAX had better hydroxyl radical-scavenging ability. Hydroxyl radical-scavenging assay was performed in aqueous medium, and FA solubility in aqueous medium increased when FA was esterified to arabinose. Thus, esterified FA had a higher antioxidant capacity when compared with a similar amount of free FA in aqueous medium [2
]. CEAX contained a higher amount of esterified FA than AEAX and CEAX-1, which may also be one of the reasons for higher antioxidant ability.
On the contrary, the result of DPPH radical-scavenging ability of AXs demonstrated that the antioxidant capacity of AEAX and CEAX-1 was higher than that of CEAX, although the latter contained higher esterified FA. It has been reported that in the presence of DPPH reagent, AXs with high esterified FA content may be oxidatively coupled in a phenomenon called oxidative gelation [4
]. This phenomenon has been brought up previously [35
]. Oxidative gelation decreases the potential of AXs to scavenge free radicals [4
]. Since CEAX contained the highest esterified FA content among all samples, the FA residue of CEAX probably underwent oxidative gelation via cross-linking, which then reduced the DPPH radical-scavenging ability of CEAX [36
]. Therefore, the results demonstrated that the reaction environment had significant impact on the antioxidant ability of esterified FA and free FA. Such findings indicated that the results of the experiment based on physiological condition can have different results compared with the results in the in vitro experiment. Since physiological condition was mostly aqueous medium, the condition of our study was close to physiological condition to some extent. However, to acquire a complete understanding of FA antioxidant ability, further study can focus on its antioxidant activity in vivo.
The reductive ability of AXs is important in the evaluation of their potential antioxidant capacity, in which the mechanism is totally based on electron transfer. However, no positive correlation was observed between the reductive ability of AXs and their concentration in our results (see Figure 6
), which is inconsistent with the results reported by Rivas et al. [32
] and Veenashri and Muralikrishna [37
]. It has been reported that not all antioxidants reduced Fe3+
at a fast rate as anticipated; in addition, the reductive ability of polyphenols such as FA, tannic acid, and caffeic acid was slowly increasing even after several hours of reaction time [38
]. Since FA is the major factor influencing the antioxidant capacity of AXs, the slow reaction rate may be one of the factors that limits the observation of the reductive ability of AXs.
Furthermore, the antioxidant capacity of FA has been reported to possibly inhibit the activity of α-glucosidase and α-amylase [9
]. Zhang et al. (2016) believed that the antioxidant capacity of AXs can reduce postprandial oxidative stress that is associated with lower risks of diabetes [11
]. Research studies indicated that AX antioxidant may contribute to their hypoglycemic activity. Thus, the present study further estimated the hypoglycemic activity of AXs.
3.3. Hypoglycemic Activity of AXs
α-Amylase has the ability to break down long-chain carbohydrates, which can divide amylose into maltotriose and maltose as well as decompose amylopectin into maltose, glucose, and amylodextrin. Thus, the inhibition of α-amylase activity will contribute to the delay of increase in postprandial blood sugar levels [39
]. The ability of AXs to inhibit α-amylase was determined, and the results are presented in Figure 7
. The inhibition of α-amylase activity increased along with the increase in AX concentration, which confirmed the α-amylase inhibition ability of AXs. Moreover, the inhibition rate of α-amylase activity of alkali-extracted AXs was higher than of water- and enzyme-extracted AXs. FA content in AXs was reported to contribute to the α-amylase activity inhibition [9
]. Malunga et al. also found that, compared with esterified FA, free FA had higher inhibition potency in rat intestine enzyme activity [10
]. Therefore, alkali-extracted AXs demonstrated better inhibition of α-amylase activity possibly because of the higher amount of free FA contained in alkali-extracted AXs.
The glucose absorption capacity of AXs was measured (see Figure 8
A) to evaluate the hypoglycemic activity of AXs because it can lower glucose concentration absorbed by the small intestine. Our results showed that the glucose absorption capacity of water- and enzyme-extracted AXs was significantly higher than that of alkali-extracted AXs. It has been reported that viscosity affected the glucose absorption capacity of polysaccharides [40
]. Esterified FA demonstrates a cross-linking effect that improves the gel-forming ability of AXs [41
]. The amount of esterified FA was higher in water- and enzyme-extracted AXs; such a characteristic probably caused better glucose absorption capacity.
The GDRI is an important in vitro index to infer the effect of AXs on the delay in glucose absorption in the gastrointestinal tract [42
]. The GDRI of AXs extracted through different methods is shown in Figure 8
B. The adsorption process could reach a dynamic equilibrium and the glucose adsorption capacity of AXs could be close to saturation with the prolongation of dialysis time; therefore, the GDRI of all AXs was decreased over time. Moreover, the GDRI of WEAX was notably higher than that of CEAX (p
< 0.05) because of the better solubility of WEAX [42
]. Higher solubility possibly makes WEAX easy to interact with glucose, which leads to a higher glucose absorption capacity.
The results demonstrated that FA content of AXs plays an important role in their hypoglycemic activity. Esterified FA also exhibited a higher ability to elevate AX viscosity and inhibit α-amylase activity compared with free FA. Moreover, the MW and DS of AXs were able to affect the viscosity of AXs, thereby exerting effects on their hypoglycemic activity [7