Abstract
The extraction parameters of intracellular polysaccharide (IPS) from Hypsizigus marmoreus SK-01 mycelia were optimized, and the antioxidant activities of IPS were investigated. The optimum conditions of IPS extraction were predicted to be: an ultrasonic treatment time of 618.07 s, an extraction temperature of 84.53 °C, a pH of 7.57, and an IPS yield of ca. 6.84%. The in vitro inhibition effects of IPS on hydroxyl, superoxide anion, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were 52.63 ± 3.19%, 68.21 ± 5.09%, and 63.43 ± 5.27%, respectively, and the EC50 values of IPS were 0.85 ± 0.05 g/L, 0.47 ± 0.03 g/L, and 0.62 ± 0.04 g/L, respectively. The activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in mice blood were 2.52 ± 0.02 U/mL, 453.19 ± 38.43 U/mL, and 118.59 ± 9.64 U/mL, respectively. The results provide a reference for large-scale extraction of IPS by H. marmoreus SK-01 in industrial fermentation, suggesting that IPS can be used as a potential antioxidant that enhances adaptive immune responses.
Download PDF
Full Article
PRODUCTION AND ANTIOXIDANT ACTIVITY OF INTRACELLULAR POLYSACCHARIDE BY HYPSIZIGUS MARMOREUS SK-01
Honghong Liu,a,† Liqin Wang,a Zhaogang Sun,b,† Lifei Pang,c Mengshi Jia,d Suqian Wu,a Xinyi Sun,a Long Hao,a Shanglong Gao,a Shouhua Jia,c,* and Le Jia a,*
The extraction parameters of intracellular polysaccharide (IPS) from Hypsizigus marmoreus SK-01 mycelia were optimized, and the antioxidant activities of IPS were investigated. The optimum conditions of IPS extraction were predicted to be: an ultrasonic treatment time of 618.07 s, an extraction temperature of 84.53 °C, a pH of 7.57, and an IPS yield of ca. 6.84%. The in vitro inhibition effects of IPS on hydroxyl, superoxide anion, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were 52.63 ± 3.19%, 68.21 ± 5.09%, and 63.43 ± 5.27%, respectively, and the EC50 values of IPS were 0.85 ± 0.05 g/L, 0.47 ± 0.03 g/L, and 0.62 ± 0.04 g/L, respectively. The activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in mice blood were 2.52 ± 0.02 U/mL, 453.19 ± 38.43 U/mL, and 118.59 ± 9.64 U/mL, respectively. The results provide a reference for large-scale extraction of IPS by H. marmoreus SK-01 in industrial fermentation, suggesting that IPS can be used as a potential antioxidant that enhances adaptive immune responses.
Keywords: Hypsizigus marmoreus SK-01; Intracellular polysaccharide; Central composite design; Antioxidant activity; In vitro; In vivo
Contact information: a: College of Life Science, Shandong Agricultural University, Daizong Street 61, Taian, Shandong 271018, PR China; b: The Central Hospital of Taian, Longtan Street 29,Taian, Shandong 271000, PR China; c: College of Chemistry and Material Science, Shandong Agricultural University, Daizong Street 61, Taian, Shandong 271018, PR China; d: The Second High School of Taian, Hushan Street 6, Taian, Shandong 271000, PR China; * Corresponding authors: shjia@sdau.edu.cn (Shouhua. Jia) and jiale9015@163.com (Le Jia); †Equal contributors
INTRODUCTION
Hypsizigus marmoreus (Peck) Bigelow, an edible and nutrient mushroom with high medicinal and economic values, is widely distributed and has been artificially cultivated in China for the past few years. It contains many biological active materials, such as carbohydrates, protein, trace elements, essential amino acids, lectins, terpenoids, tocopherols, ascorbic acid, enzymes, dietary fiber, vitamins, etc. (Lee et al. 2009; Chang et al. 2009). The polysaccharides from H. marmoreus fruiting bodies or fermentation broths have potential antioxidant, antitumor, antivirus, and immunomodulating properties (Zhang et al. 2012).
Many reports on polysaccharide content of mushrooms have focused on the exopolysaccharide (EPS) of fermentation broth and polysaccharides of fruiting bodies by Cordyceps sinensis(Yan et al. 2009), Shiraia bambusicola (Cai et al. 2010), Pleurotus sajor-caju (Telles et al. 2011), Agaricus brasiliensis (Lima et al. 2008), Gomphidius rutilus (Gao et al. 2012), Lentinus edodes (Feng et al. 2010), A. bisporus (Tian et al. 2012), Tricholoma lobayense (Wang et al. 2012), Ganoderma lucidum (Zhao et al. 2012) and Dictyophora indusiata (Hua et al. 2012). Moreover, the intracellular polysaccharide (IPS) extracted from mycelia of P. nebrodensis (Liu et al. 2010), G. applanatum (Li and Wu 2009), Phellinus igniarius (Huang et al. 2010), Antrodia camphorate (Zhang et al. 2008) and Boletus edulis (Chen et al. 2012) have been reported. However, there are fewer reports about the extraction parameters of IPS from H. marmoreus mycelia in submerged culture and its antioxidant activities.
In this work, factors that affect the extraction of IPS from H. marmoreus SK-01 mycelia were analyzed by Plackett–Burman (PB) tests, and three significant variables (ultrasonic treatment time, extraction temperature, and pH) were chosen to optimize the extraction conditions using central composite design (CCD). In addition, the in vitro and in vivo experiments were performed to investigate the antioxidant activities of IPS.
EXPERIMENTAL
Chemicals
Butylated hydroxytoluene (BHT), nitroblue tetrazolium (NBT), methionine (MET), riboflavin (RF), and 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) were obtained from Sigma Chemicals Co. (St. Louis, USA). All other chemicals used in this study were analytical reagent grade and purchased from local chemical suppliers in China.
Strain and Liquid Culture
A strain of H. marmoreus SK-01 used in this experiment was from our laboratory and maintained on potato dextrose agar (PDA) slant. The culture was incubated at 25 °C for 7 days, stored at 4 °C, and subcultured every two months.
Cultivation in liquid media was carried out in 250-mL Erlenmeyer flasks containing 100 mL of (g/L): potato, 200; glucose, 20; KH2PO4, 1.5; and MgSO4·7H2O, with natural pH. Each flask was inoculated with a 0.5-cm2 mycelial block of H. marmoreus SK-01 from the solid media, incubated at 25 °C without shaking for 24 h, and then shaken on a rotary shaker (Anting, Shanghai, China) at 140 rpm for 7 days.
Measurement and Preparation of IPS
The mycelia precipitates of H. marmoreus SK-01 were obtained by filtration and dried to a constant weight at 60 °C for 24 h. The mycelia power was dissolved in distilled water for 3 h and then fragmented with an ultrasonic processor (Sonics & Materials, USA) at 600 W for 15 min. The supernatant was mixed with 3 volumes of ethanol (95%, v/v), stirred vigorously, and kept at 4 °C for 24 h. After centrifugation (3,000 g, 15 min), the precipitated IPS was dissolved in distilled water (60 °C), and the IPS content was determined by the phenol–sulfuric acid method, using glucose as the standard (Chaplin and Kennedy 1994).
IPS powder was obtained by quick prefreezing at -35 °C for 1 h and then by vacuum freeze drying (Labconco, USA) for 8 h, and applied to evaluate the in vitro and in vivo antioxidant activities. The extraction rate of IPS was expressed as a percentage of IPS to dried mycelia (w/w).
PB Experiments for IPS Extraction
Ten variables, including water multiple, pH, ultrasonic power, ultrasonic treatment time, extraction temperature, extraction time, ethanol concentration, ethanol multiple, precipitation temperature, and precipitation time, were initially screened by PB design as reported by Plackett and Burman (1946). In addition, five center points were added for the variables that could be assigned numerical values. The experimental design with the name and symbol code is shown in Table 1.
Table 1. Levels and Codes of Variables for Plackett–Burman Design
CCD Optimization for IPS Extraction
Three parameters that significantly affect IPS extraction of H. marmoreus SK-01, ultrasonic treatment time, extraction temperature, and pH, were selected by PB tests and taken into consideration for optimization of IPS extraction by CCD design. The experimental design with name and symbol code is shown in Table 2. The test factors were coded according to the following equation,
xi = (Xi – X0) / Xi i = 1, 2, 3, …, k (1)
where xi is the coded value of an independent variable, Xi is the real value of an independent variable, X0 is the real value of the independent variable at the center point, and Δ Xi is the step change value. To correlate the response variable to the independent variable, the following quadratic polynomial equation was applied to fit the response variable to a quadratic model,
Y = β0 + ∑ βi xi + ∑ βii xi2 + ∑ βij xi xj (2)
where Y is the predicted response value, β0 is the intercept term, βi is the linear term, βii is the squared term, βij is the interaction term, and Xi and Xj are the coded level of independent variables.
Table 2. Levels and Codes of Variables for Central Composite Design
Antioxidant Capacity of IPS in vitro
The hydroxyl radical-scavenging assay was measured according to the method of Winterbourn and Sutton (1984) with slight modification. The reaction mixture (10 mL) contained 4 mL of phosphate buffer saline (0.2 M, pH 7.4), 1.5 mL of 1,10-phenanthroline (7.5 mM), 1 mL of FeSO4 (0.75 mM), 1 mL of H2O2 (1%, v/v), and 2.5 mL of the IPS (0.1 to 1 g/L). After incubating at 37 °C for 30 min, the absorbance of the IPS was measured at 560 nm, using BHT as a positive control. The EC50 value (g/L) of IPS is the effective concentration at which the hydroxyl radicals were scavenged by 50%. The hydroxyl radical scavenging activity was expressed as,
Scavenging rate (%) = [(A0 – A1) /A0] × 100 (3)
where A0 is the absorbance of the blank and A1 is the absorbance of IPS or BHT.
The superoxide anion scavenging activity of IPS was determined according to the method of Stewar and Beewley (1980) with slight modification. The reaction mixture contained 0.2 M phosphate buffer saline (pH 7.8), 10 mM riboflavin (RF), 13 mM methionine, 0.25 mL of 0.51 mM NBT, and the IPS (0.1 to 1 g/L). After illuminating the reaction mixture with a fluorescent lamp at 25 °C for 20 min, the absorbance of the IPS was measured at 560 nm, using BHT for a positive control. The EC50 value (g/L) of IPS is the effective concentration at which the superoxide anion radicals were scavenged by 50%. The scavenging rate was calculated using the following formula,
Scavenging rate (%) = [(A0 – A1)/A0] × 100 (4)
where A0 is the absorbance of the blank and A1 is the absorbance of the IPS or BHT.
The DPPH scavenging activity of IPS was measured according to the method of Shimada et al. (1992). The reaction mixture contained 2 mL of 95% ethanol, 0.1 μM DPPH, and 2 mL of the IPS (0.1 to 1 g/L). The solution was incubated at 25 °C for 30 min, and the absorbance of IPS was determined at 517 nm. The EC50 value (g/L) of IPS is the effective concentration at which the DPPH radicals were scavenged by 50%. The antioxidant activity of IPS was evaluated according to the following formula,
Scavenging rate (%) = [(A0 – A1) /A0)] ×100 (5)
where A0 was the absorbance of the DPPH solution and A1 was absorbance of IPS or BHT. The reducing power of IPS was evaluated according to the method of Oyaizu (1980) with slight modification. The reaction mixtures contained 2.5 mL phosphate buffer (pH 6.6, 0.2 M), 2.5 mL potassium ferricyanide (1%, w/v), and the IPS (0.1 to 1 g/L). After incubating at 50 °C for 20 min, 2.5 mL of trichloroacetic acid (10%, w/v) was added to the mixture for terminating the reaction, and then centrifuged at 1,200 g for 10 min.
An aliquot of 2.5 mL supernatant was collected and mixed with 2.5 mL deionized water and 0.5 mL FeCl3 (0.1%, w/v). After incubating at 25 °C for 15 min, the absorbance of the IPS was measured at 700 nm. The EC50 value (g/L) of IPS is the effective concentration at which the absorbance at 700 nm was 0.5.
Animal Experiments
Sixty male mice (Kunming strain), weighing 20 ± 2 g, were purchased from Taibang Biological Products Ltd. Co. (Taian, China) and housed in stainless steel cages under controlled conditions (temperature 22 ± 1°C, humidity 60 to 65%, lights on 12 h every day) with free access to standard food. After a 3-day acclimatization period, all animals were randomly divided into six groups (10 in each group). The five experiment groups received doses of 25, 50, 100, 200, 400 mg/kg body weight of mice by filling the stomach (0.01 mL/g body weight) with a syringe. The control group received the same volume of saline. The mice were allowed to have free access to water and food for 28 days. Experiments were performed in accordance with the institutional ethical guidelines and under guidance of Shandong Agricultural University Committee. At the end of the experiment, all animals were sacrificed under ether anesthesia, and blood samples were taken from the retrobulbar vein with a vacutainer and anticoagulated by heparin (stored at -80 °C). The hearts, livers, spleens, and kidneys were rapidly removed, weighed, and homogenized (1:9, w/v) immediately in 0.2 M phosphate buffer (4 °C, pH 7.4), respectively. The homogenates were centrifuged (6,000 g) at 4 °C for 20 min and the supernatants were stored at -20 °C for further biochemical analysis.
Measurement of Biochemical Parameters in vivo
The activity of GSH-Px was measured as described by Rong et al. (1994). The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM EDTA, 1 mM NaN3, 0.15 mM NADPH, 1 mM GSH, and 2.4 U/mL of glutathione reductase (GR). The reaction was initiated by adding 0.15 mM H2O2. The rate of NADPH consumption was recorded at 340 nm. The activity of GSH-Px was expressed as mM of NADPH oxidized per minute per milligram of tissue or micromoles per minute per milliliter of blood.
The superoxide dismutase (SOD) activities were measured according to the method of Bayer and Fridovich (1987). Fifty microliters of the samples were mixed with 1 mL of physiological saline, centrifuged at 2,500 g for 3 min, and the pellet was used for the enzyme reaction. Ice-cold water (0.2 mL), 0.2 mL of 95% ethanol, and 0.1 mL of trichloromethane were added to the pellet and mixed thoroughly with a homogenizer (Bagmixer 400, France). The mixture presented three layers after centrifugation (3,500 g, 5 min): SOD extract (top layer), hemoglobin precipitation (middle layer), and trichloromethane (bottom layer). The top layer was removed by micropipettor, and the mixture, including 0.05 mL of SOD extract, 50 mM potassium phosphate buffer (pH 7.0), 13 mM methionine, 0.075 mM NBT, 0.01 mM EDTA, and 0.002 mM riboflavin, was reacted for 20 min. The enzyme activity was expressed as relative units per milligram protein or micromoles per minute per milliliter of blood. The unit of SOD activity was expressed as U (50% inhibition of photochemical reduction of NBT as 1 U).
The activity of catalase (CAT) was determined by the method of Schlorff et al. (1999) with a slight modification. The mixture, including 2 mL sodium-potassium phosphate, 65 μM of H2O2, and 0.5 mL of blood or tissue homogenate, was reacted at 37 °C for 10 min. Ammonium molybdate (2 mL, 32.4 mM) was added for termination reaction at 25 °C. The activity of CAT was expressed as 1 μM of H2O2 degraded per minute per milligram of tissue or per minute per milliliter of blood.
The content of malondialdehyde (MDA) was measured by the method of Zhao et al. (2002) with a slight modification. The mixture, including 0.2 mL sample and 2 mL of 0.6% thiobarbituric acid (TBA, w/v), was heated in boiling water for 15 min. After cooling rapidly, the mixture was centrifuged at 3,000 g for 10 min, and the supernatant was used for the determination of MDA level.
Statistical Analysis
All the data obtained were expressed as means ± SD (standard deviation) of three replicated measurements. Design Expert software (Version 7.1.3, Stat-Ease. Inc., Minneapolis, USA) including ANOVA was applied for Statistical analysis, and P < 0.05 were considered statistically significant.
RESULTS AND DISCUSSION
Determination of Parameters of IPS Extraction
As showed in Table 3, the maximum yield of IPS extraction reached 5.56 ± 0.15%, while the optimal conditions were: water multiple 20, pH 9; ultrasonic power, 400 W; ultrasonic treatment time, 800 s; extraction temperature, 90 °C; extraction time, 1 h; ethanol concentration, 95%; ethanol multiple, 4; precipitation temperature, 4 °C; and precipitation time, 12 h . Ultrasonic treatment time, extraction temperature, and pH had a highly significant influence on IPS extraction at the 1% level, and the influence of other parameters was at the 5% level or not significant (P > 0.05) (Table 1). Therefore, these three important variables were chosen to optimize the process of IPS extraction using CCD.
Table 3. Results of Plackett–Burman for IPS Extraction by H. marmoreus SK-01
* Significant at 5 % level.
** Significant at 1 % level.
CCD Optimization of IPS Extraction
The CCD matrix together with the experimental and predicted IPS data is shown in Table 4, while adequacy and fitness were evaluated by ANOVA (Table 5). By using multiple regression analysis, the polynomial model for an empirical relationship between the extraction rate of IPS and test variables in coded units was expressed by Eq. (6),
YIPS = 6.44 + 1.29x1 + 0.5x2 + 0.3x3 − 0.035x1x2 + 0.085x1x3 + 0.29x2x3 − 1.2x12 − 0.43x22 − 0.33x32 (6)
where YIPS is the predicted response for IPS yield (%), and x1, x2, and x3 are the coded test variables for pH, ultrasonic treatment time (s), and extraction temperature (°C), respectively.
Table 4. Experimental and Predicted Values of IPS Based on Central Composite Design
Table 5. ANOVA for the Evaluation of the Quadratic Model
R2 = 0.9996. R = 0.9998. * Significant at 5% level. ** Significant at 1% level.
adj-R2 = 0.9990.
It can be seen from Table 5 that the linear term regression coefficients (x1, x2, x3) , quadratic coefficients (x12, x22, x32), and the interaction coefficient (x1x3, x2x3) were significant at the 1% level, indicating that the precipitation time, ultrasonic treatment time, and pH are all significantly correlated with the yield of IPS extraction. The model was also significant (P < 0.0001) with a very high F-value (1822.48). The value of correlation coefficient (R = 0.9998) indicated good agreement between the experimental and predicted values of IPS, and R2(coefficient of determination) was 0.9996, showing a good agreement between experimental and predicted values which can explain 99.96% variability of the responses. The value of adjusted determinant coefficient (adj-R2) was 0.9990, suggesting that the total variation of 99.9% for IPS can be attributed to the independent variables and only 0.1% of the total variation cannot be explained by the model. The F-value (0.29) and P-value (0.8344) of lack-of-fit implied that it was not significant relative to the pure error, which indicated that the model equation was appropriate to predict the yield of IPS extraction under any combination of values.
By solving the inverse matrix (from Eq. (6)), the optimal values of the variables affecting IPS yield were ultrasonic treatment time 618.07 s, extraction temperature 84.53 °C, and pH 7.57. Under these optimal conditions, the model gave the maximum predicted values of IPS extraction (6.84%). In view of the operating convenience, the optimal extraction parameters were determined to be ultrasonic treatment time 620 s, extraction temperature 85 °C, and pH 7.6, while the predicted value of IPS extraction was 6.85%.
Triplicate experiments were performed under the determined conditions and the value of IPS extraction (6.93 ± 0.22%) in agreement with the predicted value (6.84%) was obtained, indicating that the model was adequate for the process of IPS extraction.
Antioxidant Capacities of IPS in vitro
Antioxidant activities also have been attributed to various reactions and mechanisms, such as radical scavenging, reductive capacity, prevention of chain initiation, binding of transition metal ion catalysts, etc. (Frankel and Meyer 2000). In this experiment, the in vitro antioxidative capacities of IPS from H. marmoreus SK-01 were evaluated using different biochemical methods of hydroxyl, superoxide anion, and DPPH radical scavenging assay and reducing power analysis.
Hydroxyl radicals are main reactive oxygen free radicals in living organisms, which are the important reasons for causing the general processes of aging and tissue damage, and could influence the evolution of many degenerative diseases (In et al. 2002). As shown in Fig. 1A, the scavenging rate of IPS of H. marmoreus SK-01 reached 52.63 ± 3.19% (P < 0.01) at a dosage of 1 g/L, which was about 27% higher than that of BHT (41.51 ± 2.86%, P < 0.05). It was higher than 44.1% of P. nebrodensis (Li et al. 2003), 28.3% of C. militaris (Zhu et al. 2011), 47.3% of Pholiota adiposa (Yang et al. 2009), 39.8% of G. applanatum (Li et al. 2010), 35.9% of G. lucidum (Yi et al. 2010), 38.1% of T. matsutake (Wang et al. 2009), 20.9% of C. sinensis (Li et al. 2009), and 51.2% of Inonotus obliquus (Xu et al. 2011), respectively. The EC50 value of IPS (0.85 ± 0.05 g/L, P < 0.01) was lower than 1.6 g/L of P. nebrodensis (Li et al. 2003), 2.7 g/L of C. militaris (Zhu et al. 2011), 1.2 g/L of P. adiposa (Yang et al. 2009), 1.8 g/L of G. applanatum (Li et al. 2010), 1.3 g/L of G. lucidum (Yi et al. 2010), and 7.6 g/L of T. matsutake (Wang et al. 2009), respectively. The results showed that the IPS of H. marmoreus SK-01 significantly affects the scavenging of hydroxyl radical.
Superoxide anion is one of the precursors of the singlet oxygen and hydroxyl radicals; therefore, it indirectly initiates lipid peroxidation. Apart from that, the presence of superoxide anion can magnify cellular damage because it produces other kinds of free radicals and oxidizing agents (Athukorala et al. 2006). The results of superoxide anion radical scavenging assay are shown in Fig. 1B, and the inhibition activity of IPS was concentration-dependent at a dosage range of 0.1 to 1 g/L. The scavenging rate of IPS of H. marmoreus SK-01 at 1 g/L was 68.21 ± 5.09% (P < 0.01), about 34% higher than that of BHT (50.81 ± 4.35%, P < 0.01). It was also higher than 21.4% of C. militaris (Zhu et al. 2011), 26.6% of P. adiposa (Yang et al. 2009), 56.6% of Morchella esculenta (Yang et al. 2010), 36.4% of G. applanatum (Li et al. 2010), and 43.8% of C. sinensis (Li et al. 2009), respectively. The EC50 value of IPS was 0.47 ± 0.03 g/L (P < 0.01), which was about 106% lower than that of BHT (0.97 ± 0.05 g/L, P < 0.05), indicating that the IPS significantly affects the scavenging of superoxide anion radical.
Fig. 1. Antioxidant activities of IPS in vitro. (A) Scavenging effect of IPS on hydroxyl radical, (B) Scavenging effect of IPS on superoxide anion radical, (C) Scavenging effect of IPS on DPPH, and (D) Reducing power of IPS
DPPH is a stable free radical that shows maximum absorbance at 517 nm in ethanol. When DPPH encounters a proton-donating substance such as an antioxidant, the radical would be scavenged and the absorbance is reduced (Shimada et al. 1992). It can be seen from Fig. 1C that the DPPH scavenging ability of IPS at 1 g/L reached 63.43 ± 5.27% (P < 0.01), which was higher than 44.79 ± 3.82% of BHT (P < 0.05), 30.8% of C. militaris (Zhu et al. 2011), 27.3% of P. baumii (Xie et al. 2011), 45.3% of G. applanatum (Li et al. 2010), 17.8% of C. sinensis(Li et al. 2009), and 22.4% of Lentinula edodes (Turło et al. 2010), respectively. The EC50 value of IPS was 0.62 ± 0.04 g/L (P < 0.05), which was much lower than 2.1 g/L of C. militaris(Zhu et al., 2011), 1.8 g/L of P. baumii (Xie et al. 2011), 1.2 g/L of G. applanatum (Li et al. 2010), and 2.7 g/L of I. obliquus (Xu et al. 2011), respectively.
Figure 1D shows that the reducing power of IPS at 1 g/L was 0.74 ± 0.05 (P < 0.01), which was not only 12.12 ± 1.07% higher than that of BHT (0.66 ± 0.04, P < 0.05), but also higher than 0.1 of P. baumii (Xie et al. 2011), 0.4 of G. applanatum (Li et al. 2010), and 0.5 of L. edodes (Turło et al. 2010), respectively. The EC50 value of IPS (0.54 ± 0.02 g/L, P < 0.01) was about 31% lower than that of BHT (0.71 ± 0.05 g/L, P < 0.05). These results indicated that the IPS of H. marmoreus SK-01 in this study has potential antioxidant capacities in vitro.
Antioxidant Activity of IPS in vivo
GSH-Px catalyzes the reduction of hydrogen peroxide that derived from oxidative metabolism as well as peroxides from oxidation of lipids and is considered to be the most effective enzyme against lipid peroxidation (Zhang et al. 2009). As shown in Fig. 2A, the GSH-Px activities of 400 mg/kg group in blood, heart, liver, spleen, and kidney of mice were 2.52 ± 0.02 U/mL, 1.04 ± 0.01 U/mg, 1.81 ± 0.02 U/mg, 0.52 ± 0.01 U/mg, and 0.65 ± 0.03 U/mg, respectively, which were 51.81 ± 3.64%, 40.54 ± 3.29%, 110.47 ± 9.05%, 79.31 ± 6.17%, and 66.67 ± 5.64% higher than that of the control, respectively. The ANOVA showed that GSH-Px activities of blood, heart, liver, spleen, and kidney of mice treated with IPS were significantly higher (P < 0.01). These data showed that the IPS tested has a noticeable effect on improving GSH-Px activity.
Fig. 2. Effects of IPS on biochemical parameters in blood, heart, liver, spleen, and kidney of mice. (A) GSH-Px activity, (B) SOD activity, (C) CAT activity, and (D) MDA content. The units of GSH-Px, SOD, and CAT activity are expressed as U/mg protein in heart, liver, spleen, and kidney, and U/mL in blood. The unit of MDA content is expressed as nmoL/mg protein in heart, liver, spleen, and kidney, and nmoL/mL in blood
SOD catalyzes the dismutation of superoxide radicals into oxygen and hydrogen peroxide plus, thus participating with other antioxidant enzymes in the enzymatic defense against oxidative injury (Luo et al. 2010). It can be seen from Fig. 2B that the SOD activities of 400 mg/kg group in blood, heart, liver, spleen, and kidney of mice reached 453.19 ± 38.43 U/mL, 76.47 ± 5.62 U/mg, 195.83 ± 17.05 U/mg, 104.21 ± 9.25 U/mg, and 125.87 ± 11.48 U/mg, respectively, which were about 91%, 62%, 92%, 57%, and 47% higher than that of the control, respectively. The ANOVA showed that the activity of SOD at a dosage of 400 mg/kg was significant compared to that of the control group (P < 0.01).
CAT is the most important enzyme to provide a homeostasis for hydrogen peroxide. Chance et al. (1979) reported that the physiological variation of CAT concentration in different organs and tissues leads to different steady-state levels of hydrogen peroxide concentration for the same rate of hydrogen peroxide generation. Figure 2C showed that the CAT activities of 400 mg/kg group in blood, heart, liver, spleen, and kidney of mice reached 118.59 ± 9.64 U/mL, 105.01 ± 8.67 U/mg, 152.15 ± 13.47 U/mg, 99.05 ± 8.25 U/mg, and 156.73 ± 12.08 U/mg, respectively, which were about 71%, 58%, 83%, 69%, and 96% higher than that of the control, respectively.
MDA, a secondary product of lipid peroxidation, is used as an indicator of oxidative stress, and the value of MDA can effectively reflect the content of free radical produced by lipid peroxidation (Jayakumar et al. 2011). As shown in Fig. 2D, the MDA contents at 400 mg/kg group in blood, heart, liver, spleen, and kidney of mice were 1.96 ± 0.02 nmoL/mL, 0.82 ± 0.01 nmoL/mg, 1.03 ± 0.01 nmoL/mg, 1.81 ± 0.02 nmoL/mg, and 1.59 ± 0.02 nmoL/mg, respectively, which were about 109%, 122%, 154%, 83%, and 91% lower than that of control, respectively.
These data indicated that the IPS of H. marmoreus SK-01 can increase antioxidant abilities by improving SOD, GSH-Px, and CAT activities, and reducing MDA content in blood, heart, liver, spleen, and kidney of mice. The reason for the strong ability of antioxidation may be due to the molecular biochemical composition of IPS and the higher extraction rate of IPS. The antioxidant mechanism of IPS is an area for future studies.
CONCLUSIONS
- A three-factor-three-level central composite design was a successful tool for extraction optimization of intracellular polysaccharide (IPS) produced by H. marmoreus SK-01 in submerged culture.
- The IPS showed antioxidant activities in vitro and in vivo.
- The results provide a reference for large-scale extraction of IPS by H. marmoreus SK-01 in industrial fermentation, suggesting that IPS can be used as a potential antioxidant which enhances adaptive immune responses.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial supports by Natural Science Fund Program of Shandong (Y2006D08) and Doctoral Fund Program of Shandong (2007BS02021).
REFERENCES CITED
Athukorala, Y., Kim, K. N., and Jeon, Y. J. (2006). “Antiproliferative and antioxidant properties of an enzymatic hydrolysate from brown alga, Ecklonia cava,” Food Chem. Toxicol. 44, 1065-1070.
Bayer, W. F., and Fridovich, J. L. (1987). “Assaying for superoxide dismutase activity: Some large consequences of minor changes in conditions,” Anal. Biochem. 161, 559-567.
Cai, Y. J., Wei, Z. Y., Liao, X. R., Ding, Y. R., and Sun, J. (2010). “Characterization of three extracellular polysaccharides from Shiraia sp. Super-H168 under submerged fermentation,” Carbohyd. Polym. 82, 34-38.
Chance, B., Sies, H., and Boveris, A. (1979). “Hydroperoxide metabolism in mammalian organs,” Physiol. Rev. 59, 527-605.
Chang, J. S., Bae, J. T., Oh, E. J., Kim, J. Y., Park, S. H., and Lee, K. R. (2009). “Cancer preventive potential of methanol extracts of Hypsizigus marmoreus,” J. Med. Food 12, 493-500.
Chaplin, M. F., and Kennedy, J. F. (1994). Carbohydrate Analysis: A Practical Approach, IRL Press Ltd, New York.
Chen, W., Wang, W. P., Zhang, H. S., and Huang, Q. (2012). “Optimization of ultrasonic-assisted extraction of water-soluble polysaccharides from Boletus edulis mycelia using response surface methodology,” Carbohyd. Polym. 87, 614-619.
Feng, Y. L., Li, W. Q., Wu, X. Q., Cheng, J. W., and Ma, S. Y. (2010). “Statistical optimization of media for mycelial growth and exo-polysaccharide production by Lentinus edodes and a kinetic model study of two growth morphologies,” Biochem. En. J. 49, 104-112.
Frankel, E. N., and Meyer, A. S. (2000). “The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants,” J. Sci. Food Agr. 80, 1925-1941.
Gao, C. J., Wang, Z. Y., Su, T. T., Zhang, J., and Yang, X. H. (2012). “Optimisation of exopolysaccharide production by Gomphidius rutilus and its antioxidant activities in vitro,” Carbohyd. Polym. 87, 2299-2305.
Hua, Y. L., Gao, Q., Wen, L. R., Yang, B., Tang, J., You, L. J., and Zhao, M. M. (2012). “Structural characterisation of acid- and alkali-soluble polysaccharides in the fruiting body of Dictyophora indusiata and their immunomodulatory activities,” Food Chem. 132, 739-743.
Huang, Z. W., Hu, J. X., Peng, Z. M., Zhou, H. S., and Cui, Y. H. (2010). “Optimization of processing parameters for extraction of intracellular soluble Phellinus igniarius polysaccharides by response surface methodology,” Journal of Yangzhou University 31, 90-94.
In, G. K., Il, L. J., Tae, J. O., Kug, C. K., and Hae, W. S. (2002). “Polysaccharide-enriched fraction isolated from Duchesnea chrysantha protects against oxidative damage,” Biotechnol. Lett. 24, 1299-1305.
Jayakumar, T., Thomas, P. A., Sheu, J. R., and Geraldine, P. (2011). “In-vitro and in-vivo antioxidant effects of the oyster mushroom Pleurotus ostreatus,” Food Res. Int. 44, 851-861.
Lee, Y. L., Jian, S. Y., and Mau, J. L. (2009). “Composition and non-volatile taste components of Hypsizigus marmoreus,” LWT – Food Sci. Technol. 42, 594-598.
Li, X. L., Zhao, J., Li, D., and Gao, C. C. (2009). “Comparative study on antioxidant activities of mycelium from submerged fermentation of Cordyceps sinensis (Berk.) and fruit body from cultured Cordyceps militaris (L.) Link,” Food Science and Technology 34, 75-78.
Li, Y. Q., Wu, J., Hua, L. M., Wu, Y. W., Yang, Q. Z., and Zhang, Y. K. (2003). “Antioxidative activity of polysacthe antiocharide from Pleurotus nebrodensis mycelium,” Journal of Lanzhou University 39, 70-73.
Li, Z. P., and Wu, P. (2009). “Study on extraction technology of polysaccharide from medicinal Ganoderma applanatum mycelium,” Journal of Anhui Science and Technology University23, 19-22.
Li, Z. P., Wu, P., and Wu, S. Q. (2010). “Study on antioxidant activity of Ganoderma applanatum intracellular polysaccharides,” Science and Technology of Food Industry 6, 108-110.
Lima, L. F. O., Habu, S., Gern, J. C., Nascimento, B. M., Parada, J. L., Noseda, M. D., Gonçalves, A. G., Nisha, V. R., Pandey, A., Soccol, V. T., and Soccol, C. R. (2008). “Production and characterization of the exopolysaccharides produced by Agaricus brasiliensis in submerged fermentation,” Appl. Biochem. Biotechnol. 151, 283-294.
Liu, X. N., Zhou, B., Lin, R. S., Jia, L., Deng, P., Fan, K. M., Wang, G. Y., Wang, L., amd Zhang, J. J. (2010). “Extraction and antioxidant activities of intracellular polysaccharide fromPleurotus sp. Mycelium,” Int. J. Biol. Macromol. 47, 116-119.
Luo, J. G., Liu, J., Sun, Y., Ye, H., Zhou, C. H., and Zeng, X. X. (2010). “Medium optimization, preliminary characterization and antioxidant activity in vivo of mycelial polysaccharide from Phellinus baumii Pilát,” Carbohyd. Polym. 81, 533-540.
Oyaizu, M. (1980). “Studies on products of browning reaction: antioxidative activities of products of browning reaction prepared from glucosamine,” Japanese Journal of Nutrition 44, 307-315.
Plackett, R. L., and Burman, J. P. (1946). The design of optimum multifactorial experiments. Biometrika 33, 305-325.
Rong, Z. X., Liu, H. Z., and Bao, J. Q. (1994). “Micromethod of glutathione peroxidase activity of the blood in mice,” Advance in Biochemistry and Biophysics 21, 62-68.
Schlorff, E. C., Husain, K., and Somani, S. M. (1999). “Dose- and time-dependent effects of ethanol on plasma antioxidant system in rat,” Alcohol 2, 97-105.
Shimada, K., Fujikawa, K., Yahara, K., and Nakamura, T. (1992). “Antioxidative properties of xanthan on the antioxidation of soybean oil in cyclodextrin emulsion,” J. Agr. Food Chem.40, 945-952.
Stewar, R. C., and Beewley, J. D. (1980). “Lipid peroxidation associated with accelerated aging of soybean areas,” Plant Physiol. 65, 245-248.
Telles, C. B. S., Sabry, D. A., Almedia-Lima, J., Costa, M. S. S. P., Melo-Silveria, R. F., Trindade, E. S., Sassaki, G. L., Wisbeck, E., Furlan, S. A., Leite, E. L., and Rocha, H. A. O. (2011). “Sulfation of the extracellular polysaccharide produced by the edible mushroom Pleurotus sajor-caju alters its antioxidant, anticoagulant and antiproliferative properties in vitro,” Carbohyd. Polym. 85, 514-521.
Tian, Y. T., Zeng, H. L., Xu, Z. B., Zheng, B. D., Lin, Y. X., Gan, C. J., and Lo, Y. M. (2012). “Ultrasonic-assisted extraction and antioxidant activity of polysaccharides recovered from white button mushroom (Agaricus bisporus),” Carbohyd. Polym. 88, 522-529.
Turło, J., Gutkowska, B., and Herold, F. (2010). “Effect of selenium enrichment on antioxidant activities and chemical composition of Lentinula edodes (Berk.) Pegl. mycelial extracts,” Food Chem. Toxicol. 48, 1085-1091.
Wang, C., Chen, Y., Hu, M. L., Ding, J. N., Xu, C. J., and Wang, R. J. (2012). “In vitro antioxidant activities of the polysaccharides from Tricholoma lobayense,” Int. J. Biol. Macromol. 50, 534-539.
Wang, D., Wang, W., Mou, H. J., Wang, J., Teng, L. R., Zhang, Y. B., and Liu, H. S. (2009). “Optimization of ultrasound-assisted extraction of Tricholoma matsutake mycelium polysaccharide using response surface methology and its antioxidative effect,” Chemistry and Industry of Forest Products 29, 109-114.
Winterbourn, C. C., and Sutton, H. C. (1984). “Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence,” Arch. Biochem. Biophys. 235, 116-126.
Xie, L. Y., Zhang, Y., Peng, W. H., and Gan, B. C. (2011). “Immune function and antioxidant activity of intracellular polysaccharides from Phellinus baumii,” Food Science 32, 276-281.
Xu, X. Q., Wu, Y. D., and Chen, H. (2011). “Comparative antioxidative characteristics of polysaccharide-enriched extracts from natural sclerotia and cultured mycelia in submerged fermentation of Inonotus obliquus,” Food Chem. 127, 74-79.
Yan, J. K., Li, L., Wang, Z. M., Leung, P. H., Wang, W. Q., and Wu, J. Y. (2009). “Acidic degradation and enhanced antioxidant activities of exopolysaccharides from Cordyceps sinensismycelial culture,” Food Chem. 117, 641-646.
Yang, F., Wang, X. F., Weng, L., Ge, Q. M., and Chen, S. S. (2010). “Antioxidant activity of intracellular polysaccharides (IPS) from Morchella esculenta,” Food Science 31, 76-78.
Yang, L. H., Huang, Q. R., Feng, P. Y., Cai, D. H., and Geng, H. L. (2009). “Isolation, identification and anti-oxidant activity of polysaccharides from Pholiota adiposa mycelium,” Food Science 30, 131-134.
Yi, Y. L., Wang, J. R., Hu, Y. S., Liu, N., and Wang, F. Z. (2010). “Study on the extraction of Lentinus edodes and Ganoderma lucidum mycelia polysaccharides and the compound antioxidative activity,” Journal of Anhui Agricultural Science 38, 9521-9522.
Zhang, B. Z., Yan, P. S., Chen, H., and He, J. (2012). “Optimization of production conditions for mushroom polysaccharides with high yield and antitumor activity,” Carbohyd. Polym. 87, 2569-2575.
Zhang, H., Mu, Z. B., Xu, L. M., Xu, G. F., Liu, M., and Shan, A. S. (2009). “Dietary lipid level induced antioxidant response in Manchurian Trout, Brachymystax lenok (Pallas) larvae,” Lipids 44, 643-654.
Zhang, Z. X., Zhang, H. H., and Ma, H. L. (2008). “Studies on extraction process of polysaccharides from Antrodia camphorata,” Food Research and Development 29, 17-20.
Zhao, S. J., Shi, G. A., and Dong, X. C. (2002). Experiment Instruction of Plant Physiology, China Agricultural Press, Beijing.
Zhao, W., Jiang, X. J., Deng, W. W., Lai, Y. H., Wu, M., and Zhang, Z. Z. (2012). “Antioxidant activities of Ganoderma lucidum polysaccharides and their role on DNA damage in mice induced by cobalt-60 gamma-irradiation,” Food Chem. Toxicol. 50, 303-309.
Zhu, Y. L., Chen, H. W., and Zhang, C. (2011). “Scavenging function of intracellular polysaccharide of enriched selenium Cordyceps militaris on radical,” Agricultural Engineering 1, 53-57.
Article submitted: August 18, 2012; Peer review completed: October 20, 2012; Revised version received and accepted: October 22, 2012: Published: October 24, 2012.