ANTIOXIDANT ACTIVITY OF POLYSACCHARIDES EXTRACTED FROM Lentinus edodes MYCELIA AND THEIR PROTECTIVE EFFECTS ON INS-1 CELLS

The disruption of the delicate balance between the generation of reactive oxygen species (ROS) and antioxidant scavenging systems can lead to many health problems. Polysaccharides extracted from fungi fruit body and myceliums have several potential health benefits. In this study, the antioxidant capacity and cell protective effects of a polysaccharide isolated from Lentinus edodes mycelia (LMP) were investigated. The antioxidant properties of LMP were screened using radical scavenging (1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and superoxide anion), reducing power and inhibition of lipid peroxidation assays. Intracellular reactive oxygen species (ROS), in response to H2O2, was determined by the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Rat insulinoma (INS-1) cells apoptosis was detected using flow cytometry analysis. The results showed that LMP exhibited significantly strong radical scavenging activity. At the concentration of 2.0 mg mL, DPPH radical, hydroxyl radical, and superoxide anion radical scavenging rates are 80.32 ± 2.58%, 92.56 ± 3.11% and 93.73 ± 2.82%, respectively. The reducing power and inhibition of lipid peroxidation activity of LMP were as strong as the positive control. LMP alleviated the intracellular reactive oxygen species level and inhibited islet cell apoptosis significantly. The results open perspectives for studies of LMP in the treatment of diabetes.


INTRODUCTION
The disruption of the delicate balance between the generation of reactive oxygen species (ROS) and antioxidant scavenging systems can lead to health problems (XIA et al., 2011).A large amount of evidence has suggested that the production of oxygen derived free radicals is associated with many diseases, such as diabetes, Alzheimer's and Parkinson's disease, cancer as well as being involved in the degenerative process of aging (DURACKOVÁ, 2010).To alleviate damage to the human body, many antioxidants have been synthesized industrially.However, these synthetic antioxidants are suspected of being responsible for liver damage and certain types of cancer (LI et al., 2011).Therefore, it is important to develop and use effective and non-poisonous antioxidants that are able to scavenge free radicals in human bodies and maintain health.
In recent years, there has been a growing trend of finding natural antioxidants from edible mushrooms.Lentinus edodes is a mushroom widely consumed as a nutritious dietary and medicinal food in China.Many bioactive materials have been found in this mushroom, for example, polyphenolic compounds, polysaccharides, and dietary fiber (CHEN et al., 2012).Polysaccharides are biopolymers comprised of monosaccharides linked together through glycosidic bonds (ZHANG et al., 2007).Several recent studies have indicated that Lentinus edodes polysaccharides exhibit significant antioxidant activity by scavenging free radicals (CHEN et al., 2012;TSRIMUANG et al., 2011;CHEN et al., 2008).Lentinus edodes polysaccharides can be obtained from both the fruiting body and the mycelia.Generally, mature Lentinus edodes are harvested from plastic bags about 2 months after maturity, whereas the mycelia can be cultivated through submerged cultures.Thus, the mycelia of Lentinus edodes are substitute products for the mature fruiting bodies, which can be used for the production of polysaccharides.The polysaccharides extracted from Lentinus edodes mycelia are expected to become a new biologically active substance.
Type 2 diabetes is a serious metabolic disorder characterized by defects in the control of blood glucose (RAINS et al., 2011).Pancreatic beta cell dysfunction is closely related to the development and progression of type 2 diabetes, which is related to the loss of beta cells leading to a reduced beta cell mass (LEE et al., 2011).There is growing evidence that beta cell loss is largely because of apoptosis induced by oxidative stress (LEE et al., 2013;LIN et al., 2012;MEHMETI, et al., 2011).Although there has been a study on the antioxidant capacity of polysaccharides from the Lentinus edodes fruiting body carried out by Tsrimuang et al (TSRIMUANG, et al., 2011), there is still relatively little information concerning the antioxidant activities of Lentinus edodes mycelia polysaccharides and their function in reducing oxidative stress in islet cells.To fully develop this polysaccharide resource and extend the potential application of Lentinus edodes mycelia, the antioxidant activities of polysaccharides extracted from Lentinus edodes mycelia harvested from submerged cultures were analyzed to screen for natural antioxidants which could be used to protect islet cells.

Preparation of Lentinus edodes mycelia polysaccharide (LMP)
The origin of the Lentinus edodes samples is Lentinus edodes 973 stored in the School of Life Science, Liaoning University, China.The procedures for the preparation and purification of the Lentinus edodes mycelia polysaccharide have been described previously (CAO et al., 2013).Briefly, the dried fungal biomass was boiled for 4 h.After centrifugation at 8000 × g for 30 min, the crude polysaccharide was extracted from the supernatant using four volumes of absolute ethanol (4:1 v/v) at 4 °C for 10 h.The supernatant was discarded after centrifugation at 8000 × g for 20 min.The pellet was washed, redissolved in distilled water, applied to a Sephadex G-200 column (2.0 × 60 cm) and eluted with H 2 O at a flow rate of 1.0 mL min -1 .Fractions were further purified on a diethylaminoethyl (DEAE)-32 column (2.5 × 40 cm), with phosphate buffer solution-water used as the mobile phase and a gradient mode elution.A polysaccharide fraction identified as LMP was obtained.

DPPH radical scavenging assay
The DPPH radical scavenging assay was conducted according to the procedure of Song et al with minor modifications (SONG et al., 2010).DPPH solution (0.1 mM, in 95% ethanol solution) was prepared and used fresh on the day of each test.Two mL of DPPH solution was incubated with 2.0 mL of the LMP solution at varying concentrations (0.25, 0.5, 1.0, 1.5, and 2.0 mg mL -1 ).The reaction mixture was mixed well and incubated for 30 min at room temperature.The absorbance was then measured at 517 nm against a blank.The DPPH radical scavenging activity was measured and calculated using the following equation: Scavenging effect (%)=(1− A Sample / A Control )×100%

Hydroxyl radical scavenging assay
The hydroxyl radical scavenging effect of LMP was measured according to the procedure of He et al (2012) with slight modifications (He et al., 2012).1.0 mL of sample solution at different concentrations (0.25, 0.5, 1.0, 1.5, and 2.0 mg mL -1 ) was mixed with 1.0 mL of 6 mM FeSO 4 .Then 1.0 mL of 6 mM H 2 O 2 was added to start the reaction.1.0 mL of 6 mM salicylic acid was added after 10 min.Finally, the reaction mixture was incubated for 30 min at room temperature and the absorbance was recorded at 510 nm with a visible spectrometer (details of machine manufacturer, location).Ascorbic acid was used as the positive control.The scavenging activity of the polysaccharides against the hydroxyl radicals was calculated according to the following equation: HO radical scavenging activity (%) = (1 − (A sample − A blank ) / A control ) × 100%

Superoxide anion radical scavenging assay
The measurement of superoxide anion radical scavenging activity was investigated according to the method reported by Zha et al (ZHA et al., 2009).1.0 mL of 86 µM nitroblue tetrazolium (NBT), 1.0 mL of 469 µM nicotinamide adenine dinucleotide (NADH), 1.0 mL of LMP at different concentrations (0.25, 0.5, 1.0, 1.5, and 2.0 mg mL -1 ) and 0.4 mL phenazine methosulfate (PMS) were mixed in test tubes to initiate the reaction.The reaction mixture was then incubated at ambient temperature for 5 min and the absorbance of the mixture solution was determined at 560 nm with a visible spectrometer (details of machine manufacturer, location).Ascorbic acid was used as the positive control.The superoxide anion radical scavenging capability of LMP was calculated as follows: Scavenging activity

Reducing power assay
The measurement of reducing power was conducted according to the method reported by Soares et al (SOARES et al., 2009).1.0 mL of LMP solution at Different concentrations (0.25, 0.5, 1.0, 1.5, and 2.0 mg mL -1 ) were mixed with 2.5 mL of phosphate buffer (50 mM, pH 7.0) and 2.5 mL of 1% potassium ferricyanide.The mixture was then incubated at 50 °C for 20 min.Then, 2.5 mL of 10% trichloroacetic acid was added to the mixture to terminate the reaction, followed by centrifuging at 3000 × g for 10 min.Finally, 1.25 mL of the supernatant was mixed with 1.25 mL of distilled water and 0.25 mL of a 0.1% FeCl 3 solution.The absorbance was immediately measured at 700 nm by using a spectrometer.Ascorbic acid was used as the positive control.A higher absorbance of the reaction mixture indicated a high reducing power.

Assay for inhibition of liver lipid peroxidation
The assay for inhibition of liver lipid peroxidation was performed using the method described by Ma et al (MA et al., 2012) with some modifications.Healthy Rat livers were washed with 0.15 M NaCl and homogenized in 10 volumes (v/w) of normal saline at 4 °C.Reaction mixtures contained 2.5 mL of liver homogenate, 1.4 mL of 0.2 mM PBS (pH 7.4), 1.0 mL of 10 µM FeSO 4 •7H 2 O, 2.5 mL of 100 mM ascorbic acid and 1.0 mL of various concentrations of LMP (0.25, 0.5, 1.0, 1.5, and 2.0 mg mL -1 ).The mixtures were incubated at 37 °C for 30 min, followed by centrifugation (3000 ×g, 10 min).After the addition of 1.0 mL of the TBA reagent to the supernatant, the tubes were placed in a boiling water bath for 15 min.Absorbance was then measured at 530 nm by a spectrophotometer.The percentage of lipid peroxidation inhibition by the samples was then calculated.Ascorbic acid was used as the positive control.

Measurement of intracellular ROS
Intracellular reactive oxygen species (ROS) were measured by a fluorescent dye technique as described by Lu et al (LU et al., 2008).1×10 5 INS-1 cells were seeded on plate overnight，then treated with 0.5 mM H 2 O 2 , and then incubated with or without 2.0 mg mL -1 LMP 3 h.INS-1 cells were incubated for 30 min with 10 µM DCFH-DA at 37 °C in the dark in phosphate buffered saline (PBS).Images were obtained with a fluorescence microscope with excitation at 488 nm and emission at >500 nm.The fluorescent intensities of individual cells were analyzed using the InterVision 2D program (Noran Instruments Inc, WI, USA).

Flow cytometry analysis
Flow cytometry analysis was performed to determine this event using the Annexin V-FITC Apoptosis Detection Kit (KeyGen, Nanjing, China).
Cells were seeded and treated with 0.5 mM H 2 O 2 followed by incubating with or without 2.0 mg mL -1 LMP 3 h.Cells were harvested by trypsinization, then washed twice with cold PBS and centrifuged at 1000 × g.About 1-5×10 5 cells were resuspended in 500 µL of binding buffer and transferred to a flow cytometry glass tube.Five microliters of Annexin V-FITC and 5 µL of propidium iodide were added and the tube was incubated in the dark for 15 min at room temperature.Apoptotic cells were measured using a FACScan flow cytometer.

Data analysis
Statistical analysis was performed using SPSS 18.0 (IBM Corp., Armonk, NY, USA).All data were expressed as mean ± standard deviation (SD).Statistical comparisons were carried out using one-way analysis of variance (ANOVA) and randomized block analysis of variance.The Students-Newman-Keuls (SNK) method was employed for intergroup comparison.Associations between categorical variables were verified by the Chi-squared test.Statistical significance was determined as p < 0.05.

DPPH radical scavenging activity
DPPH (1,1-diphenyl-2-picrylhydrazyl) is a compound that has been widely used to determine the free-radical scavenging ability of different materials (CHEN et al., 2011).Alcoholic solutions of DPPH have a characteristic maximum absorption at 517 nm.When an antioxidant that can provide electrons or hydrogen atoms is added to DPPH, the absorbance at 517 nm disappears because the DPPH radical is scavenged (LI et al., 2011).The scavenging ability of LMP on the DPPH radical is shown in Figure 1A.The results indicated that LMP showed evident scavenging activities on the DPPH radical in a concentration-dependent manner.High levels of scavenging abilities were found for all samples used in the test.The scavenging ability of LMP on DPPH radical was 36.69 ± 1.98% at 0.25 mg mL -1 and increased to 80.32 ± 2.58% at 2.0 mg mL -1 which is close to that of ascorbic acid.The scavenging ability of ascorbic acid on DPPH radical was 92.3± 3.18%.The EC 50 value of LMP on DPPH is 0.39 mg mL -1 , which is higher than the result of Tsrimuang et al (TSRIMUANG et al., 2011).These results indicated that LMP is a strong free radicalscavenging polysaccharide that could help prevent oxidative stress.

Hydroxyl radical scavenging activity
The hydroxyl radical has a very short in vivo half-life of approximately 10 −9 s and a high reactivity.This makes it a very dangerous compound in adjacent bimolecular reactions (CHEN et al., 2012).These radicals can attack biological molecules such as DNA, proteins and lipids.Thus, the removal of hydroxyl radicals is important for the protection of living systems (LIU et al., 2012).As shown in Fig. 1B, the results indicated effective hydroxyl radical scavenging activities at all tested concentrations of LMP.The scavenging ratios were improved with the increase of polysaccharide concentration.The highest scavenging rate was 92.56 ± 3.11% at the concentration of 2.0 mg mL -1 , which was similar to that of ascorbic acid (The scavenging rate was 95.21 ± 4.56%).These results show that LMP is a good scavenger for the hydroxyl radical.The scavenging activity of LMP on the hydroxyl radical is also higher than that of polysaccharides from the fruiting body of Lentinus edodes (CHEN et al., 2012).This indicates that Lentinus edodes mycelium could be a new resource of bioactive polysaccharides.

Superoxide radical scavenging activity
The superoxide radical is normally formed first in cellular oxidation reactions, and then produces other types of cell damaging free radicals and oxidizing agents (LIU et al., 2012).Although the superoxide radical is a weak oxidant in most organisms, it could produce hydrogen peroxide and hydroxyl radicals through dismutation and other types of reaction in vivo (ZHA et al., 2009).Superoxide radical is also known to indirectly initiate lipid peroxidation as a result of the formation of hydrogen peroxide.Therefore, scavenging the superoxide anion is extremely important in antioxidant work.In the present study, the scavenging ability of LMP on the superoxide radical was exhibited in a concentration-dependent manner as shown in Figure 1C.The highest scavenging ability of 2.0 mg mL -1 LMP was 93.73 ± 2.82%, which was better than that of ascorbic acid (The scavenging rate was 69.58 ± 2.36%).Chen et al (2012) reported that the polysaccharides from the fruiting body of Lentinus edodes could scavenge the superoxide radical, with a scavenging rate of about 90% (CHEN et al., 2012).Our results showed that LMP had a higher scavenging rate.However the mechanism and the basis of the LMP structure should be investigated in future studies.

Reducing power
The reducing power of a compound may serve as an indicator of its potential antioxidant activity.Higher absorbance values indicate stronger reducing power (LIU et al., 2012).The reducing power of LMP was investigated and showed in Figure 2A.As shown in the figure, the reducing power of the samples correlated well with increasing concentrations.At the lower concentration, the reducing power was far lower than that of ascorbic acid.However, at the higher concentration, the reducing power of LMP was close to that of ascorbic acid.The reducing power of LMP in our data suggested that it was likely to contribute to the observed antioxidant effect.

Activity of inhibition on liver lipid peroxidation
The liver peroxidation inhibition activity of LMP is shown in Figure 2B, The data revealed that LMP demonstrated great capacity for the inhibition of lipid peroxidation.The inhibition rate of LMP increased from 12.53 ± 1.22% to 61.76 ± 3.38% as the concentration of LMP increased from 0.25 to 2.0 mg mL -1 , suggesting that LMP possesses relatively a strong liver peroxidation capacity and the inhibitory effects were concentration dependent.In the concentration range tested, the inhibiting effect of LMP was lower than that of ascorbic acid.It is generally admitted that the initiation of a peroxidation sequence in a cell or polyunsaturated fatty acid is because of the abstraction of a hydrogen atom from the double bond in the fatty acid (MA et al., 2012).Many studies showed that lipid peroxidation is associated with various pathological events, such as insulin resistance, inflammation, postischemic reperfusion injury, atherosclerosis, ethanol toxicity, and even cancer.(MIRMIRAN P et al., 2014;WANG et al., 2012).Therefore, it is very important for health to prevent the lipid peroxidation.

LMP attenuate intracellular ROS
The antioxidant capacity in β cells is very low because of the weak expression of antioxidant enzymes such as glutathione peroxidase and superoxide dismutase in pancreatic islets compared with that in various other tissues (FUJIMOTO et al., 2011).It is believed that the generation of ROS ultimately culminates in β cell dysfunction and death (MCCABE et al., 2006).As shown in Figure 3A, many INS-1 cells were detached within 3 h of H 2 O 2 loading.Most of the LMP loaded cells treated with H 2 O 2 remained adherent.The data demonstrated that LMP prevented cell death induced by H 2 O 2 .The number of coherent cells after LMP loading for 3 h was significantly higher than that after 3 h loading of H 2 O 2 .Statistical analysis demonstrated that the decrease in the adherent cell number induced by H 2 O 2 loading were significant (Figure 3B).To determine whether the cell death of INS-1 cells was because of oxidative stress, the DCFH-DA assay was applied to evaluate intracellular ROS, as shown in Figure 4A.The intracellular ROS decreased significantly after LMP loading in INS-1 cells in the presence of H 2 O 2 for 3h.Dichlorofluorescein fluorescence was assayed using confocal microscopy.The ROS content of H 2 O 2 -treated INS-1 cells was more than nine fold compared with the control cells (Figure 4B).The loading of LMP significantly decreased the level of ROS.

LMP inhibit cell apoptosis induced by H 2 O 2
Many studies have shown that reduced β cell mass is one of important determining risk factor for diabetes.(XU et al., 2009) Pancreatic β cells are sensitive to ROS damage which can induce cell apoptosis in many kinds of cell lines.It has been proposed that treatment using antioxidants can prevent pancreatic β cells apoptosis.Our results indicated that 0.25 mM H 2 O 2 can induce the apoptosis of pancreatic β cell with a 10.23 ± 0.87% apoptosis rate, whereas LMP can reduce the pancreatic β cell apoptosis to a 3.26 ± 0.28% apoptosis rate (Figure 5).The results indicated that LMP can inhibit pancreatic β cell apoptosis by reducing oxidative stress, which suggests that LMP has the potential to be used as an antioxidant for diabetes prevention.

CONCLUSIONS
LMP exhibited significantly strong radical scavenging activity.
The radical scavenging rate of the DPPH radical, hydroxyl radical and superoxide anion radical were 80.32 ± 2.58%, 92.56 ± 3.11% and 93.73 ± 2.82%, respectively.The reducing power and inhibition of lipid peroxidation activity were as strong as the positive control.

Figure 1 .
Figure 1.Free radical scavenging capacity of LMP.(A) DPPH radical scavenging activity of LMP.(B)Hydroxyl radical scavenging activity of LMP.(C) Superoxide anion scavenging activity of LMP.Vitamin C (Vc) was used as positive control.Data were mean ± SD of three parallel measurements.

Figure 2 .
Figure 2. Reducing powers and Inhibitory activity on liver lipid peroxidation of LMP.(A) Reducing power of LMP.(B) Inhibitory activity on liver lipid peroxidation of LMP.Data were mean ± SD of three parallel measurements.

Figure 3 . 2 Figure 4
Figure 3. LMP protected cell viability after H 2 O 2 exposure.(A) The cell images were obtained with a phasecontrast microscope at 3 h after treated with H 2 O 2 in the absence or presence of 2.0 mg mL -1 LMP.(B) The numbers of adherent cells were expressed as percentage of the number of control cells, Data were mean ± SD of three parallel measurements.*p<0.05 vs. the levels of control.#p<0.05 vs. the levels of cells untreated with H 2 O 2

Figure
Figure 5． ． ． ．LMP inhibited INS-1 cell apoptosis induced by H 2 O 2. (A) Control group.(B) Measurement of INS-1 cell apoptosis with H 2 O 2 treatment.(C) Measurement of INS-1 cell apoptosis induced by H 2 O 2 in the presence of 2.0 mg mL -1 LMP.(D) Statistical analysis of apoptosis rate.Data were mean ± SD of three parallel measurements *p<0.05 vs. the levels of control.#p<0.05 vs. the levels of cells untreated with LMP.