Fresh H. erinaceus mushrooms (Houza 19 strain) were obtained from the Institute of Edible Fungus at Anhui Science and Technology University (Fengyang, China) at a mature stage. The mushrooms were sorted, cleaned, washed in cold sterilized water and drained. The fresh mushrooms were then lyophilized and powdered with a mixer (Zhejiang Ronghao Industry and Trade Co., Ltd., Zhejiang, China). The extractions and analyses were immediately conducted following lyophilization. All chemicals and reagents used in the present study were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) unless otherwise stated.

The powder was then subjected to extraction by the following eight solvents: n-Hexane, xylene, chloroform, anhydrous ether, ethyl acetate, acetone, anhydrous ethanol and distilled water. The optimized extraction conditions were used as follows: 20 g powder and 100 ml reagent solution (100%) added into a flask and extracted at room temperature for 24 h. This process was repeated twice more. The liquid was collected by centrifugation (2,500 × g, 5 min, room temperature), and then condensed by rotary evaporation at 50°C. The extracts were put into an evaporating dish and dried in a water bath at 50°C. The crude extracts were dissolved in methanol, yielding a series of sample solutions with different concentrations.

The reducing power was determined by the method of Jiang et al (12). The K₄Fe(CN)₆ was generated after the antioxidants reacted with K₃Fe(CN)₆. Then K₄Fe(CN)₆ reacted with FeCl₃ to produce Perl's Prussian blue (Fe₄[Fe(CN)₆]₃) which had the maximum absorbance at 700 nm. The reaction mixture containing 2.5 ml different concentrations of samples in phosphate-buffered saline (0.2 M, pH 6.6) was incubated with 2.5 ml K₃Fe (CN)₆ (1%, w/v) at 50°C for 20 min. The reaction was terminated by adding 2.5 ml trichloroacetic acid (10%) and the mixture was centrifuged at 1,400 × g for 10 min. The supernatant (2.5 ml) was mixed gently with 2.5 ml distilled water and 0.5 ml FeCl₃ (0.1%, w/v) and the absorbance was measured at 700 nm against a blank sample (V-1200 spectrophotometer; Shanghai Meipuda Instrument Co., Ltd., Shanghai, China). A higher absorbance indicated a higher reducing power. The experiments were performed in triplicate and the data was the average of all three.

The DPPH radical scavenging activity of the samples was measured according to the procedure described by Negro et al (13). The solution of DPPH (0.2 mM; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in methanol and was freshly prepared prior to the measurements. In brief, the extracted solutions (2 ml) were thoroughly mixed with 2 ml DPPH solution and allowed to stand for 30 min in the dark. The blank control solution contained an equal volume of distilled water instead of the sample solution. The absorbance was measured at 517 nm (V-1200 spectrophotometer). Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The experiments were performed in triplicate and the data were averaged. The scavenging rate was calculated according to the following equation: DPPH scavenging rate (%) = (A_c-A_s) × 100/A_c, in which A_c was the absorbance of the control, and A_s was the absorbance of the sample.

The superoxide radical scavenging activity of the samples was evaluated according to the method reported by Chen et al (14) with a slight modification. The extract produced by different solvents was dissolved in 2 ml methanol to form varying concentrations. The 2 ml extracts and 2 ml Tris-HCl buffer (50 mM, pH 8.2) were blended and incubated at 37°C for 20 min. Next, the solution was mixed with 1 ml 50 mM pyrogallol solution (preheated to 37°C) and the absorbance variation was detected within 2 min at 325 nm (UV762 ultraviolet and visible spectrophotometer; Shanghai Jingke Scientific Instrument Co., Ltd., Shanghai, China). For the blank control, the sample was replaced by Tris-HCl buffer. The scavenging rate was calculated within 2 min by the following equation: Scavenging rate (%) = (∆A_c-∆A_s) × 100/∆A_c, in which ∆A_s and ∆A_c were the absorbance variations of the extractions and the control.

The peroxide value (POV) was used to assess the inhibition ability of extracts against lipid peroxidation. POV was measured as described in the Standard Methods for the Analysis of Fats and Oils established by China (GB/T5538-2005) (15). Autoxidation caused the formation of peroxides, which in turn resulted in the release of iodine. The free iodine was then titrated with a sodium thiosulfate solution (15). To artificially induce a quick oxidation of the corn oil, all samples were marked and stored in an oven at 60°C for 120 h. During this process of oxidation, the POVs of samples were determined every 24 h using the Standard Methods for the Analysis of Fats and Oils established by China (GB/T5538-2005) (15). In brief, 2 g oil sample (the minimal effective concentration of extracts was 5%) was mixed with 50 ml isooctane and acetic acid mixture (2:3, v/v). Next, 0.5 ml saturated potassium iodide solution was added to the reaction system. The mixture was left in the darkness for 3 min and the supernatant was titrated with sodium thiosulfate solution (2 mM). The POV was expressed as milliequivalents of active oxygen per kilogram of oil (meq/kg). The experiment was performed in triplicate. The POV was calculated as follows: POV (meq/kg) = 1,000 × ∆V xc/w, in which the c was the molar concentration of sodium thiosulfate solution, and w was the mass of the oil sample. ∆V was the volume variation of the sodium thiosulfate solution that was consumed by the sample and the reagent blank control.

The antioxidant data were analyzed using analysis of variance in the SPSS statistics software version 18.0 (SPSS, Inc., Chicago, IL, USA) for significance. The data are expressed as the mean ± standard deviation. P<0.05 and P<0.01 were considered to indicate a statistical significant difference. All the graphs were prepared using Microsoft Excel 2003 (Microsoft, Redmond, WA, USA).

The antioxidants donate electrons and convert the oxidized form of iron (Fe³⁺) to the ferrous form (Fe²⁺), which can be monitored by measuring the formation of Perl's Prussian blue at 700 nm (16). The efficacy of certain antioxidants is associated with their reducing power (17). In the present assay, the results demonstrated that the absorbance, which reflects the reducing power, increased with the concentration of extractions (Fig. 1). The reducing powers at a maximum concentration of 2 mg/ml ranged between 0.254 for n-Hexane and 0.982 for distilled water. The reducing powers of the extractions that were produced by distilled water and anhydrous ethanol yielded the higehst absorbance levels. Thus, it may be deduced that the aqueous and alcoholic extracts possess significant levels of antioxidant compounds. The reducing power of the compounds after n-hexane extraction was the lowest.

The stable DPPH free radical has an absorption of 517 nm in methanol (18). It can be widely used to evaluate the free radical scavenging ability of natural compounds with the hydrogen-donating ability (16). The antioxidant donates protons to the DPPH radical and the absorption decreases with the discoloration of the initial purple compound. The radical scavenging activity increases as the absorption at 517 nm is decreased (19). The results demonstrated that the DPPH radical scavenging activities of all types of extractions were concentration-dependent (Fig. 2). The extractions at high doses exhibited significant scavenging effects on DPPH free radicals. The scavenging activity of different extracts at 2 mg/ml ranged from 28.2 to 93.4%. The distilled water and anhydrous ethanol presented the highest DPPH scavenging activities, whereas, the anhydrous ether extracts had the lowest DPPH scavenging activities, whereas, the extractions obtained from the four solvents with the higher polarity had a stronger scavenging rate than those from the four solvents with the lower polarity. The effective components in H. erinaceus with different polarities could be extracted by the solvents with the corresponding polarities. Furthermore, the biological activities of the effective components were different when the polarities were different. The results indicated that the scavenging activity of the extractions was associated with the polarities of the used reagents, and, the stronger the polarity of the solvent was, the higher the DPPH scavenging activity of its extraction.

Superoxide anions are the most common free radicals in vivo generated in a variety of biological systems. Higher levels of superoxide anion radicals are regarded as the beginning of ROS accumulation in cells. In addition, they have been implicated in a number of human diseases, including cancer and diabetes (20). In the present study, the superoxide anion radical scavenging effects of different extractions exhibited a dose-dependent association (Fig. 3). The scavenging rate of different extractions at 10 mg/ml was in the range of 13.4–97.9%. Furthermore, the extractions from anhydrous ethanol, n-hexane and chloroform had strong scavenging activities against superoxide anion radicals, whilst that of anhydrous ether was the weakest.

Edible oils can be autoxidized in the air. The peroxidation of lipids is a free radical driven chain reaction and various toxic secondary products are generated (21). In addition, these products are known to damage DNA and cell and organelle membranes, or cause cancers, such as liver cancer. Antioxidants terminate the lipid peroxidation chain reactions by removing free radical intermediates or inhibiting other oxidation reactions (22). Amongst the eight extractions, the anhydrous ethanol, chloroform and acetone extracts were capable of inhibiting lipid peroxidation in the corn oil system, as their POVs were lower than that of corn oil autoxidation (Fig. 4). The extractions did not significantly reduce the peroxide formation at the early stages (days 1–3) of the experiment. On the fourth day, the inhibition rates of anhydrous ethanol, chloroform and acetone extracts were 67.5, 50 and 10.2%, respectively, as compared with the POV of corn oil auto-oxidation. On the fifth day, anhydrous ethanol, chloroform and acetone extracts caused 62, 71.2 and 54.1% reductions in the formation of peroxides, respectively compared with oil autooxidation. Following statistical analysis, the three extractions on days 4 and 5 were observed to significantly inhibit lipid peroxidation at a 0.01 significance level.