Removing hydroxyl radicals is important for the protection of living systems because hydroxyl radicals are considered to be a highly potent oxidant, which can react with most biomacromolecules functioning in living cells and induce severe damage to the adjacent biomolecules. Scavenging abilities of the four polysaccharides isolated from four species of edible mushrooms on hydroxyl radicals were depicted in Figure 2. In this work, the scavenging abilities of CABP and CAAP on hydroxyl radicals were concentration-dependent, however, the scavenging abilities on hydroxyl radicals of CFVP and CLDP were not very concentration dependent, at the tested concentration range of 0.125–2.0 mg/mL. At 1.0 mg/mL, the scavenging abilities of CABP, CAAP, CFVP and CLDP on hydroxyl radicals were 37.63%, 16.68%, 16.74% and 30.89%, respectively. At 2.0 mg/mL, the scavenging abilities of CABP, CAAP, CFVP and CLDP were 70.68%, 31.84%, 20.84% and 51.16%, respectively. Moreover, at 2.0 mg/mL, CABP was found to have a higher scavenging activity on hydroxyl radicals than CAAP, CFVP and CLDP (p < 0.05). Effectiveness in antioxidant properties is inversely correlated with EC₅₀ values. It is important to note the effectiveness of polysaccharides in reacting with free radicals under different conditions as a lower EC₅₀ value corresponds to higher antioxidant activity of the edible mushroom’s polysaccharides. The EC₅₀ values of scavenging ability on hydroxyl radicals for CABP and CLDP were 1.05 mg/mL and 1.90 mg/mL, respectively (Table 3). However, the EC₅₀ values of scavenging ability on hydroxyl radicals were both >2 mg/mL for CAAP and CFVP (Table 3). The present results proved that CABP was a good scavenger for hydroxyl radicals. The polysaccharides minimized the concentration of Fe²⁺ in the Fenton reaction, and the scavenging abilities of the polysaccharides might be due to the active hydrogen donating ability of hydroxyl substitutions of polysaccharides. Therefore, CABP had a better potency to donate hydrogen to reactive hydroxyl radicals than CAAP, CFVP and CLDP. However, the scavenging abilities of the four polysaccharides on hydroxyl radicals were all relatively lower than that of Vc at the same concentrations. The EC₅₀ values for hydroxyl radical-scavenging ability of CABP and CLDP were lower than that of the polysaccharide from another common edible mushroom, Auricularia polytricha, which was about 2.4 mg/mL [8]. However, the value of EC₅₀ of the intracellular polysaccharide from one precious edible mushroom, Pholiota adipose, was 0.042 mg/mL [11], remarkably much lower than that of the four crude water soluble polysaccharides in this experiment.

Superoxide anions are a precursor of active free radicals react with biological macromolecules and induce tissue damage and play important roles in the formation of other reactive oxygen species such as hydrogen peroxide, hydroxyl radicals, and singlet oxygen, which induce oxidative damage in lipids, proteins and DNA [14]. Therefore, the superoxide radical-scavenging ability is of great importance to its potential antioxidant activity. Figure 3 reveals the scavenging abilities of the four crude polysaccharides on superoxide anion radicals. An increase of the scavenging activity on hydroxyl anion radicals was observed with the increasing tested concentration at the concentration range (0.125–2.0 mg/mL) of the polysaccharides, but didn’t exhibit a visible concentration-dependent manner. The scavenging effects of CABP, CAAP, CFVP and CLDP on superoxide anion radicals were 46.84%, 27.29%, 22.2% and 31.77%, respectively, at 1.0 mg/mL. The scavenging effects of CABP, CAAP, CFVP and CLDP on superoxide anion radicals were 65.78%, 45.21%, 27.9% and 43.99% at 2.0 mg/mL, respectively. CABP revealed a better antioxidant activity because the EC₅₀ values of scavenging ability on superoxide anion radicals for CABP and the other three polysaccharides were 1.17 mg/mL and >2 mg/mL, respectively (Table 3). These results suggested that CABP was an effective scavenger for superoxide anion radicals, and it might be advantageous for preventing injury induced by superoxide radicals in pathological conditions. However, the four studied polysaccharides showed lower scavenging activities on superoxide radicals than Vc. The EC₅₀ value of scavenging ability on superoxide anion radicals for the intracellular polysaccharide from P. adiposa was 0.0196 mg/mL [11], indicating much stronger superoxide anion radical-scavenging ability than the four crude water soluble polysaccharides in this experiment.

The model of scavenging the stable DPPH radicals has been widely accepted as a tool to evaluate the free radical-scavenging activities of materials [15]. Figure 4 describes the scavenging abilities of different polysaccharides from four species of edible mushrooms on DPPH radicals. At all concentrations tested, CABP, CAAP and CLDP exhibited a dose-dependent DPPH radical-scavenging activity. Nevertheless, for CFVP, the scavenging ability on DPPH radicals slightly increased with increasing sample concentration. In the lower concentration ranged from 0.125 to 1.0 mg/mL, the scavenging abilities on DPPH radicals for CABP and CLDP were increased with increasing sample concentration, which were 64.56% and 67.39%, respectively, at 1.0 mg/mL. However, at 2.0 mg/mL, the scavenging abilities on DPPH radicals for CABP and CLDP were 72.24% and 71.16%, respectively, indicating no significantly increasing ability (p < 0.05). With regard to scavenging ability on DPPH radicals, CABP and CLDP showed very good scavenging abilities as evidenced by their particularly low EC₅₀ values (≈0.5 mg/mL) (Table 3). For CAAP and CFVP, the EC₅₀ values of scavenging ability on DPPH radicals were 1.62 and >2.0 mg/mL, respectively (Table 3). These results suggested that CABP and CLDP had higher scavenging abilities than CAAP and CFVP on DPPH radicals. It might be that CABP and CLDP could be better advantageous than CAAP and CFVP for reacting with DPPH radicals to convert them to more stable products and thereby terminate radical chain reactions. However, the scavenging abilities on DPPH radicals of the four polysaccharides were lower than that of Vc. The EC₅₀ value of the polysaccharide from A. bisporus for DPPH radical-scavenging ability was 2 mg/mL [6], much higher than that of CABP. Moreover, the EC₅₀ values of the polysaccharides from wild edible mushrooms of Armillaria mellea, Calocybe gambosa, Clitocybe odora and Coprinus comatus for DPPH radical-scavenging ability were 3.95 mg/mL, 7.08 mg/mL, 3.56 mg/mL and 7.31 mg/mL, respectively [7], which were remarkably much higher than that of CABP, CAAP and CLDP. It was worthy of noting that the EC₅₀ value for DPPH radical-scavenging ability of the polysaccharide from another edible mushroom, Agaricus brasiliensis, was 0.27 mg/mL [6], much lower than that of the four polysaccharides in this experiment.

The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity [16]. In this assay, the concentration-dependent profile of reducing power was obvious for all the tested polysaccharides (Figure 5). At 1.0 mg/mL, the reducing powers were 0.388, 0.128, 0.076 and 0.270 for CABP, CAAP, CFVP and CLDP, respectively. At 2.0 mg/mL, the reducing powers were 0.752, 0.218, 0.135 and 0.452 for CABP, CAAP, CFVP and CLDP, respectively. With regard to reducing power, CABP showed the best antioxidant property because of its lowest EC₅₀ value (1.35 mg/mL), indicating that CABP was good effective as an antioxidant (Table 3). High reducing power of CABP suggested a high potential in hydrogen-donating ability which could react with free radicals to convert them to more stable products and thereby terminate radical chain reactions [17]. CABP showed a obviously higher ability than that of the other three polysaccharides, but less than that of Vc. Kozarski et al. [6] and Vaz et al. [7] reported that the EC₅₀ values for reducing power of the polysaccharides from A. brasiliensis, Calocybe gambosa and Coprinus comatus were 3.13 mg/mL, 2.38 mg/mL and 4.67 mg/mL, respectively, which were higher than that of CABP. Furthermore, for reducing power of the polysaccharide from A. bisporus, EC₅₀ value was found of 14.83 mg/mL [6], remarkably higher than that of CABP studied in our experiment. However, the polysaccharides of A. mellea and C. odora showed the lower EC₅₀ values for reducing power (0.98 mg/mL and 0.94 mg/mL, respectively) than CABP [7].

In general, it is interesting and important to elucidate the relation among chemical structures and chain conformations of polysaccharides and their biological activities [18]. The antioxidant activities of the polysaccharides might be related to monosaccharide component, molecular size, structure and conformation [19]. Tsiapali et al. [20] stated that the antioxidant abilities were partially related to monosaccharide composition. Moreover, Lo et al. [9] reported that the monosaccharide composition and the type of glycosyl linkage modulated the antioxidant properties of the polysaccharides. The antioxidant properties of the polysaccharides were dependent on the ratio of different monosaccharides in the composition. Among the monosaccharides, rhamnose was the most significant determinant factor associated with antioxidant properties. The glycosyl linkage of the monosaccharides also affected the antioxidation characteristics of the polysaccharides. Specifically, the arabinose 1→4 and mannose 1→2 linkages of the side-chain were significantly related to the reducing power, whereas the glucose 1→6 linkage and arabinose 1→4 linkage were related to the scavenging on DPPH radicals. In addition, a correlation was found between EC₅₀ value of the reducing power ability and the amount of total glucans content in polysaccharides [6]. Furthermore, Luo et al. [18] figured out that the antioxidant activities of polysaccharides were supposed to relate to the structural characteristics of the polysaccharides, including molecular weight, monosaccharide composition and configuration. Polysaccharides owned smaller molecular weight exhibited stronger antioxidant activities. Polysaccharides with β-configuration in pyranose form sugars also exhibited stronger antioxidant activities. Besides, the high content of rhamnose was supposed to relate to the strong antioxidant effects.

In our experiment, the four crude polysaccharides showed different antioxidant activities. However, it is hard to clear the correlation between chemical characteristics and antioxidant properties of crude water soluble polysaccharides because using crude polysaccharides may cause many factors of chemical characteristics to influence their antioxidant properties. The objective of our study was to evaluate and compare the chemical characteristics and antioxidant properties of crude water soluble polysaccharides from four species of common edible mushrooms. Our results can give guidance for well-balanced diets in our daily life and a source of antioxidant compounds. Meanwhile, the correlation between chemical characteristics and antioxidant properties for purified polysaccharide fractions is undergoing in our lab.

Hydroxyl radical-scavenging activity was determined based on the method described by Smirnoff and Cumbes [22] with some modifications. The reaction mixture contained polysaccharide sample with different concentrations (0–2.0 mg/mL, 1 mL) was incubated with a solution containing orthophenanthroline (5 mM, 1 mL), phosphate buffer (7.5 mM, pH 7.4, 0.8 mL) and FeSO₄ (7.5 mM, 0.5 mL). Finally, H₂O₂ (8.8 mM, 0.5 mL) was added, and the reaction mixture was then incubated at 37 °C for 1 h. The absorbance of the resulting solution was measured spectrophotometrically at 532 nm. The scavenging ability of hydroxyl radicals was calculated according to the equation: scavenging ability (%) = (1 − A_sample/A_control) × 100, where A_control is the absorbance of control without the polysaccharide sample, and A_sample is the absorbance in the presence of the polysaccharide sample. The EC₅₀ value (mg/mL) is the effective concentration at which the hydroxyl radicals are scavenged by 50%. Vc was used for comparison because it is a standard antioxidant.

The scavenging activity of superoxide anion radicals was assessed referring to the reference [23] with several modifications. A tube containing Tris-HCl buffer (50.0 mM, pH 8.2, 3 mL) and polysaccharide sample (0–2.0 mg/mL, 1 mL) was incubated in a water bath at 25 °C for 20 min, then pyrogallic acid (5.0 mM, 0.4 mL) at the same temperature was added and proceed at 25 °C. HCl solution (8.0 M, 0.1 mL) was used to terminate the reaction after 4 min. The absorbance of the mixture was measured at 320 nm. The scavenging ability of superoxide anion radicals was calculated using the following formula: scavenging ability (%) = (1 − A_sample/A_control) × 100, where A_control is the absorbance of control without the polysaccharide sample, and A_sample is the absorbance in the presence of the polysaccharide sample. The EC₅₀ value (mg/mL) is the effective concentration at which the superoxide anion radicals are scavenged by 50%. For comparison, Vc was used as positive control.

The DPPH radical-scavenging activity was measured according to the method of Braca et al. [24]. Polysaccharide sample with different concentrations (0–2.0 mg/mL, 1 mL) was mixed with methanol solution (2 mL) containing DPPH radicals (0.2 mM). The mixture was shaken vigorously and incubated for 30 min in darkness at room temperature, and then absorbance at 517 nm was measured. The scavenging ability of DPPH radicals was calculated using the following formula: scavenging ability (%) = (1 − A_sample/A_control) × 100, where A_control is the absorbance of control without the polysaccharide sample, and A_sample is the absorbance in the presence of the polysaccharide sample. The EC₅₀ value (mg/mL) is the effective concentration at which the DPPH radicals are scavenged by 50%. Vc was used as standard antioxidant and positive control.

The reducing power was determined referring to the method described in literature [25] with slight modifications. Polysaccharide sample with different concentrations (0–2.0 mg/mL, 1 mL) was mixed with phosphate buffer (0.2 M, pH 6.6, 2.5 mL) and K₃Fe(CN)₆ (1%, w/v, 2.5 mL). The mixture was incubated at 50 °C for 20 min, then trichloroacetic acid (10%, w/v, 2.5 mL) was added to the mixture, subsequently centrifuged at 3,000 × g for 10 min. An aliquot of supernatant (5 mL) was mixed with deionized water (4 mL) and FeCl₃ (0.1%, w/v, 1 mL). After incubating at room temperature for 10 min, the absorbance of the mixture was measured at 700 nm. A higher absorbance in the reaction mixture indicates greater reducing power ability. Vc was used as positive control for comparison.