2.1. Chemical Analysis
Xylan, the most common hemicellulose, accounts for more than 60% of polysaccharides present in the cell walls of corn cobs [16
]. Using a methodology that combined alkaline extraction and ultrasound, we obtained water soluble xylan from the corn cobs, yielding 40 ± 5% (w/w). When only alkali solution is used as extractant, xylan extraction yield is about 15% [17
]. The beneficial sonication effect on xylan extractability can be explained by both the mechanical disruption of the cell walls and breaking of inter- and intramolecular xylan linkages, enhanced in the presence of hot alkali [18
]. As a result, accessibility, solubility and diffusion of dissolved cell wall molecules increased.
We used a method for xylan extraction from corn cobs proposed by Wang and Zhang [14
]. However, these authors did not analyze the molecular weight of xylan in their article. The xylan solution obtained in this work was submitted to gel permeation chromatography, showing a peak of 130 ± 20 kDa heteroxylan. This material was pooled and lyophilized and its chemical (Table 1
) and biological features analyzed. Three additional peaks composed of small oligosaccharides were also found.
Chemical analysis of xylan is summarized in Table 1
. Phenolic and protein content for the corn cob sample was very low, whereas sugar content was elevated, indicating the efficiency of the extraction method. With respect to total sugar, phenolic compounds and protein contents, the sum of the three components found in the xylan does not approach 100%. This is due to the fact that this polymer is highly hygroscopic, absorbing water from the atmosphere very quickly after lyophilization. Furthermore, because of the negative charges of glucuronic acids, metals are not eliminated from xylan structures, even after dialysis. Another important point is the conformation exhibited by this polymer in aqueous solutions, which may capture cations within their structures.
The xylan obtained here is composed mainly of xylose, arabinose, galactose and glucose and trace of mannose and glucuronic acid. These monosaccharides were also described in other studies on xylan extracted from corn cobs [15
The FT-IR spectrum of corn cob xylan is shown in Figure 1
. Typical signals of heteroxylan at 3422, 2924, 1637, 1125, 1044, and 895 cm−1
were clearly observed in the sample. Xylan exhibited a broad stretching intense characteristic peak at around 3444 cm−1
for the hydroxyl group, and a weak band at 2929 cm−1
due to CH2
]. A large peak around 1637 cm−1
, which overlaps the residual water at 1645 cm−1
, was assigned to the carboxyl group of glucuronic acid [21
]. Xylan has a band in the 1200–1000 cm−1
region, which is dominated by ring vibrations overlapped with stretching vibrations of C–OH side groups and C–O–C glycosidic band vibration [22
]. A specific signal at 1044 cm−1
was assigned to the stretching vibration of COH. This signal dominates the xylan spectrum with (1–4)-backbone, moreover, the anomeric region of (1–4)-xylan was assigned at 895 cm−1
in accordance with the IR results in xylobioside models [21
2.2. Antioxidant Activity
Total antioxidant activity (TAC) was determined using phosphomolybdenum blue complex. This method is based on the reduction of Mo+6
by antioxidant compounds and the formation of green Mo+5
complexes with a maximum absorption at 695 nm. The green complex is quite stable for several days and is not affected by various organic solvents used for polysaccharide extraction [23
]. Corn cob xylan showed a TAC corresponding to 48.5 mg of ascorbic acid equivalent/g of xylan. This activity was similar to that observed for sulfated polysaccharides from the edible red seaweed Gracilaria caudata
] and higher than the activity of tamarind seed polysaccharide [24
]. Due to the higher TAC, corn cob xylan was selected for further detailed analysis of its antioxidative properties, using superoxide radical scavenging activity assay, hydroxyl radical scavenging activity assay, iron-chelating capacity, and reducing power.
Superoxide anion radicals act as an oxidant but are highly unstable, immediately dismutating in the intracellular environment either spontaneously or enzymatically. Although superoxide is a relatively weak oxidant, it decomposes to form stronger reactive oxidative hydroxyl radical species. Among reactive oxygen species (ROS), the hydroxyl radical is the most reactive in chemistry. It can abstract hydrogen atoms from biological thiol molecules and form sulfur radicals capable of combining with oxygen to generate oxysulfur radicals and damage biological molecules [25
]. Corn cob xylan (from 0.05 to 2.00 mg/mL) did not show scavenging activity against these ROS. Similar data were observed with commercial xyloglucans, which showed no superoxide or hydroxyl radical scavenging activity (from 0.1 to 1.6 mg/mL). However, when two types of xyloglucan derivatives (xyloglucan selenious ester and sulfated xyloglucan) were prepared from that commercial xyloglucan, they showed stronger superoxide and hydroxyl radical scavenging activities [26
]. In fact, fucoidans (sulfated polysaccharides) from the seaweed Laminaria japonica
] and ulvans (sulfated polysaccharides) from the seaweed Ulva pertusa
] exhibited much stronger scavenging activity in the superoxide radical than vitamin C. In addition, the last two studies proposed that scavenging ability depends on sulfate content. Moreover, when chitosan was sulfated, it exhibited high scavenging activity as compared to the original compound [29
]. The xylan corn cob also did not show any activity in the reducing power tests at all concentrations used (from 0.05 to 2.00 mg/mL). The presence of sulfate in the polysaccharide structure is also related to reducing power. Several studies show high reducing power activity of sulfated polysaccharides extracted from different sources [27
]. In fact, the presence of the sulfated group decreases the bond energy of the C–H in the vicinity of the glycosidic bond, and then increases the hydrogen atom donating capability. Therefore, the introduction of sulfate group might enhance the electron cloud density of active hydroxyl groups and enhance the molecular electron-withdrawing activity, which can eliminate free radicals and terminate radical-mediated oxidative chain reactions [30
]. However, several authors showed that scavenging activity and reducing power of sulfated polysaccharides is depend on the degree and position of sulfation [8
]. Therefore, further to this study the xylan from corn cob have been selected for further sulfation, including sulfation at different positions of the monosaccharide chain, in order to improve its scavenging activity and reducing power.
Antioxidants inhibit interaction between metals and lipids through the formation of insoluble metal complexes with ferrous ion or the generation of steric hindrance. The iron-chelating capacity test measures the ability of antioxidants to compete with ferrozine in chelating ferrous ion. Activity is measured as absorbance of the red Fe2+
/ferrozine complex decreases. The plot of iron-chelating capacity as a function of sample concentration is shown in Figure 2
. Corn cob xylan showed dose-dependent activity, the highest occurring at 2.0 mg/mL (70%). Xylan activity was actually only 1.47 times lower than EDTA activity at the same concentration under the same experimental conditions (Data not shown).
Ferrous ions are considered to be the most effective pro-oxidants present in food systems. The high chelating effect of this polysaccharide would also be beneficial if it were formulated into foods. The metal-chelating property of this polymer displayed that it might be applied in adsorption, metal ions separation or waste water treatment. Some polysaccharides extracted from seaweed showed lower activity compared with those extracted from corn cobs. The most active compounds were from green seaweeds Caulerpa prolifera
and Caulerpa sertularioides
with 69.9 and 57.8% of ferrous chelating, respectively, at 2 mg/mL. In fact, the activity of sulfated polysaccharides from C. prolifera
was only 1.36 times lower than EDTA activity at the same concentration under the same experimental conditions [8
]. Several articles have shown that polysaccharide iron chelating activity depends mainly on the presence of groups such as sulfate, alcohol hydroxyl or carboxyl groups. Even when there is a small amount of uronic acid in polysaccharide composition it may have chelating activity, such as Zizyphus jujuba
polysaccharide, which, despite containing 5.7% of uronic acid, showed a chelating effect about 75% at a concentration of 2000 μg/mL [31
]. However, sulfated polysaccharides showed a weak chelating effect even in high concentration (2000 μg/mL) [8
]. In addition, when sulfate groups were added to neutral monogalactoglucan [32
] or neutral glucan [33
] they did not improve the polysaccharide chelating activity, which indicates that polysaccharide chelating activity is stereo-specific, is depending on spatial patterns of negative groups, and it is not merely a consequence of polysaccharide charge density. It was clear that xylan iron-chelating capacity is dependent on its structural features. Additional investigation will help our understanding of the complete xylan structure, including sequence of monosaccharides, configuration and position of glycosidic linkages, the position of branching points and the structure–function relationship.
2.3. Antiproliferative Activity
Antiproliferative studies of antioxidant compounds have been reported in recent years. The development of compounds that inhibit or delay tumor cell proliferation and do not affect normal cells is one of the main challenges in the search for antitumor compounds. In this work, corn cob xylan was effective against HeLa tumor cells in a dose-dependent manner, with maximum antiproliferative activity of 50% for about 2 mg/mL. In addition, no activity was found against normal fibroblast cells (3T3) (Figure 3
). To the best of our knowledge, there are no data about the antiproliferative activity of xylan from corn cobs. However, polysaccharides from other sources inhibit tumor cell proliferation. Ryu et al.
reported that 1 mg/mL of purified polysaccharides from Salicornia herbacea
can inhibit 50% of human colon cancer cell proliferation [34
]. The antiproliferative activity of polysaccharides may be attributed to the presence of charged groups attached to the molecule. This was reported by Costa and colleges [8
] who showed a relationship between the number of sulfate groups and the antiproliferative action of polysaccharides extracted from seaweed. Moreover, commercial derivatives of xyloglucans (esters of xyloglucans selenium) showed an antiproliferative effect against tumor cells (HepG2) with a 30% inhibition of proliferation with a concentration of 1 mg/mL [26
]. However, neutral polysaccharides, containing mainly xylose extracted from Japanese bamboo, manifest selective cytotoxicity against acute lymphoblastic leukemia cells with 60% antiproliferative activity of around 200 μg/mL [35
]. This indicates that the antiproliferative activity of polysaccharides, including xylan, is not merely a consequence of their charge density but a set of structural factors of the polysaccharide. No relationship was found between antiproliferative activity and antioxidant content (R2
= 0.398, p
> 0.05). Similar results were obtained when antioxidant and anticoagulant activities of aqueous extracts from strawberry were analyzed [36
2.4. Anticoagulant Activity
Assessment of anticoagulant activity is always considered when working with polysaccharides. On the other hand, anticoagulant activity of plant polysaccharides is commonly not evaluated, because they are mostly neutral. No study to date has assessed the anticoagulant activity potential of xylans from corn cobs. It was therefore decided to evaluate the anticoagulant activity of this compound extracted from corn cob flour. To that end, xylan was submitted to anticoagulant tests such as those described in the Experimental Section. Commercial aPTT and PT kits were used to determine if the anticoagulant action of xylan could influence intrinsic and extrinsic coagulation pathways, respectively.
Clotting time of the extrinsic pathway (PT test) showed no increase at any of the conditions used (100, 200, 400, 600 μg). However, a dose-dependent prolongation of clotting time was observed when the intrinsic coagulation pathway was analyzed (aPTT test). It should be noted that with 100 μg of xylan plasma clotting time doubled in relation to the control (Table 2
Anticoagulant polysaccharides exhibit this activity due to the presence of charges, primarily sulfate groups. Therefore, sulfate concentration was measured, as described in Methods. The sample contained 3.4% sulfate (w/w), which albeit low, might be sufficient for it to exhibit anticoagulant activity. However, the sulfate identified in the sample may not be covalently linked to xylan, but rather to the three dimensional polymer structure. This hypothesis corroborates the fact that the same infrared peaks found in the 800–900 cm−1
region of sulfated polysaccharides were not observed. To confirm the link of sulfated groups to the xylan molecule, the latter was submitted to agarose gel electrophoresis. Figure 4
is the digitization of a gel electrophoresis slide after toluidine blue staining. This compound complex with the sulfate groups, forming a purple dye complex, as can be observed with the sulfate polysaccharides used (heparin sulfate, chondroitin sulfate, dermatan sulfate, and sulfate fucans A and B from the brown seaweed Spatoglossum schroederi
). On the other hand, xylan did not stain purple with toluidine blue, suggesting the absence of sulfate in this molecule.
Another charged group may be involved in the anticoagulant activity of xylan, such as carboxyl groups of glucuronic acid residues present in its structure. Yoon and colleges showed that 23 glucuronic acid-containing polysaccharides extracted from 23 different vascular plants have anticoagulant activity. These authors promote the reduction of the glucuronic acid carboxyl groups of the most potent anticoagulant polysaccharide and this abolished its activity. These data showed that glucuronic acid residues are essential for activity since after reduction of its carboxyl groups the anticoagulant activity disappears [37
]. Therefore, xylan was submitted to a carboxy-reduction process to evaluate the involvement of carboxyl groups in anticoagulant activity. Analyses indicated a 70% loss of the carboxyls present in the xylan. As a result, this new compound was also submitted to aPTT and PT tests. Evaluation of anticoagulant activity of xylan after carboxy-reduction showed a drastic reduction (Figure 5
), indicating the importance of these groups in this activity. These results are in agreement with those obtained by the authors cited above.
2.5. Antimicrobial Activity
The microbial resistance to antibiotics is a wide problem of medical importance. Each year, many microbial isolates have been reported as resistant to antibiotics usually used in clinical therapy [10
]. This worrisome problem costs annually millions of dollars and is a threat for the human life. Thus, global efforts to understand the biology and biochemistry, and search for new compounds with antibiotic action have been forward in university and research centers around the world. Plant extracts have been used for thousand years to cure a diversity of human illness, including bacterial infection [38
]. Therefore, new antibiotic compounds have been discovery and developed from the plant extracts.
In this study, the xylan from corn cob did not inhibit the growth of Staphylococcus epidermidis
(ATCC 35984). However, the polysaccharide was able to inhibit the growth of one clinical isolate of KPC producing Klebsiella pneumoniae
in approximately 25%, and consequently the biofilm formation was decreased in a similar proportion (Table 3
). KPC isolates are resistant to most antibiotics used to treat clinical infections, in particular, carbapenems. This apparent low activity observed against KPC might be further increased by fractionation or chemical modification of the active compounds, serving the xylan as scaffold molecule. Gul et al.
have shown that different plant extracts present distinct antimicrobial actions against various different species of bacteria [39
]. The difference in this result could possibly be due to the different compilation of receptors in the each species of bacteria that recognize distinct patterns of polysaccharides and thus the way of answers and signaling is unique.