International Journal of Biological Macromolecules 73 (2015) 236–244
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Enzymatic and acidic degradation effect on intracellular polysaccharide of Flammulina velutipes SF-08 Zhao Ma a,1 , Chen Zhang a , Xia Gao b,1 , Fangyuan Cui a , Jianjun Zhang a , Mengshi Jia c , Shouhua Jia d,∗ , Le Jia a,∗ a
College of Life Science, Shandong Agricultural University, Taian, Shandong 271018, PR China Shandong Agricultural Technology Extending Station, Jinan, Shandong 250100, PR China c The Second High School of Taian, Taian, Shandong 271000, PR China d College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, PR China b
a r t i c l e
i n f o
Article history: Received 4 October 2014 Received in revised form 23 November 2014 Accepted 27 November 2014 Available online 4 December 2014 Keywords: Flammulina velutipes SF-08 Intracellular polysaccharide Enzymolysis and acidolysis
a b s t r a c t The intracellular polysaccharide (IPS) from Flammulina velutipes SF-08 mycelia was isolated and degraded by enzyme and acid. IPS and its derivative were purified by DEAE-52 cellulose chromatography, and five fractions were obtained. The structural features and antioxidant activities in vitro of the isolated fractions were evaluated. On the basis of chemical composition and antioxidant ability analyses, rhamnose as the main monosaccharide might contribute to the strongest antioxidant capacity. The in vivo results showed that IPS significantly enhanced the activities of anti-aging enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and reduced the content of lipid peroxidantion (LPO). These results suggested that IPS should be a potent natural polymer and can be developed to be novel functional food. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS), including superoxide anion radical, hydroxyl radical, hydrogen peroxide, reactive oxygen, and nitrogen species, play important roles in cell signaling and homeostasis of organism [1]. However, uncontrolled ROS can induce physiological effects related to the pathogenesis of several human diseases such as heart diseases, gastric problems, aging and certain types of cancer [2]. Antioxidants, widely discussed in clinical and nutritional literature, can reduce the oxidative stress to prevent the formation of these free radicals and protect the body from oxidative damage [3]. Some commonly used synthetic antioxidants, such as BHA and BHT, are suspected to have some toxic effects and as possible carcinogens [4]. Therefore, new interest has been developed to employ non-toxic antioxidants from natural sources by consumer preference for natural products. Many dietary compounds have been suggested to be important antioxidants. Polysaccharides, obtained from many edible mushrooms,
∗ Corresponding authors. Tel.: +86 0538 8249958/1570; fax: +86 0538 8248696/1570. E-mail addresses: [email protected] (S. Jia), [email protected] (L. Jia). 1 Equal contributors. http://dx.doi.org/10.1016/j.ijbiomac.2014.11.028 0141-8130/© 2014 Elsevier B.V. All rights reserved.
have exhibited strong antioxidant activities and can be acted as novel potential antioxidants [5,6]. Flammulina velutipes, an edible mushroom that was widely distributed and artificially cultivated in oriental countries, has been proved to have high nutritional value and attractive taste [7]. According to the previous literature, F. velutipes contains biologically active components such as protein, dietary fiber, and polysaccharides [8]. It has been showed that bioactive polysaccharides obtained from fruiting bodies of F. velutipes have beneficial immunomodulatory and biological activity [9]. In addition, previous studies indicate antioxidant activities of polysaccharides can be affected by many factors including chemical components, uronic acid contents, and even the extraction and isolation methods [10,11]. However, there are few reports of the correlation of structural characterization and antioxidant activities of various polysaccharide fractions obtained from mycelia of F. velutipes. In this work, enzyme and acid hydrolysis productions of the intracellular polysaccharide (IPS) of F. velutipes SF-08 mycelia were isolated and purified by DEAE-52 cellulose column chromatography. The monosaccharide compositions of the purified polysaccharides were elucidated by the methods of gas chromatography (GC) and their antioxidant activities were evaluated. Based on the analysis of antioxidant and monosaccharides, we preliminary investigated the correlation of the monosaccharide composition
Z. Ma et al. / International Journal of Biological Macromolecules 73 (2015) 236–244
and antioxidant activity in vitro. In addition, the in vivo anti-aging properties of IPS were evaluated. 2. Materials and methods 2.1. Chemicals and reagents DEAE-52 cellulose was purchased from Whatman 4057200. Hydrogen peroxide (H2 O2 ), ferrozine and 1,1-diphenyl-2picrylhydrazyl (DPPH) were purchased from Sigma Chemicals Company (St. Louis, USA). The standard monosaccharides were provided by Merck Company (Darmstadt, Germany) and Sigma Chemical Company (St. Louis, USA). All the other chemicals and reagents were analytical grade and purchased from local chemical suppliers in China. 2.2. Culture media and conditions The F. velutipes SF-08 strain, initially incubated at 26 ◦ C on potato dextrose agar (PDA) slants by our laboratory, was used in this experiment. A 10 mm2 portion of the agar plate culture was sliced from the slant for inoculating the seed culture medium. Seeding cultivation in liquid media was cultured in a 1 L filter flask containing 500 mL of potato dextrose broth at 25 ◦ C on a rotary shaker (160 rpm, Anting, Shanghai, China) for 7 d and then the cultured broth was gradually homogenized. Batch fermentation experiment was performed in a 100 L fermenter (Xianmin, Luoyang, China) with air agitation at 25 ◦ C for 7 d. The mycelium of F. velutipes SF-08 collected from fermenter was centrifuged at 3000 × g for 15 min, washed several times with distilled water and dried by a lyophilizer in vacuum to constant weight. 2.3. Extraction of IPS The F. velutipes SF-08 mycelia was extracted twice with distilled water at 90 ◦ C for 2 h each, and centrifuged at 3000 × g for 15 min, the supernatants were concentrated and precipitated by addition of 3 volumes of 95% ethanol (v/v), stirred vigorously, and kept at 4 ◦ C for 24 h. The precipitate was collected by centrifugation at 3000 × g for 15 min, dissolved in distilled water at 60 ◦ C, and measured according to the phenol–sulfuric acid method using glucose as the standard [12]. The IPS was deproteined by Sevag reagent, dialyzed and lyophilized by vacuum freeze drying (Labconco, USA) [13]. 2.4. Enzymatic and acidic degradation According to the method of Yang et al. and Li et al. with some modifications, the dried polysaccharide powder (0.5 g) and snailase (0.1 g) were dissolved in 100 mL of 1% acetic acid [14,15]. The mixture system was subjected to chemical treatment under following conditions with temperature of 40 ◦ C, time 5 h, pH 6, and enzyme to the polysaccharide sample ratio of 1:5. The enzymatic hydrolysis polysaccharide (EIPS) was concentrated at low pressure by a rotary evaporator, and then lyophilized. Tests on the acid degradation of the polysaccharide were performed by the method of Zhang et al. with minor modifications [16]. The IPS (0.5 g) in test tube (18 mm × 180 mm) with stopper was dissolved in 10 mL of 1 mol/L H2 SO4 solution and kept in boiled water for 8 h. After acid hydrolysis termination, hydrolysate was centrifuged at 6000 × g for 10 min. The supernatant (4.5 mL) was neutralized with 2 mol/L NaOH solution and then diluted using distilled water to obtain a final volume of 10 mL. After centrifuged (6000 × g, 10 min), the liquid supernatant was concentrated at low
237
pressure by a rotary evaporator and lyophilized, and acid hydrolysis polysaccharide (AIPS) was obtained.
2.5. Purification of IPS, EIPS and AIPS The samples (IPS, EIPS and AIPS) were purified by DEAE-52 cellulose chromatography according to the reported method with sight modification [17,18]. The sample (1.0 g) dissolved in 15 mL of distilled water at 60 ◦ C was filtered through 0.22 m organic membrane filter. The sample was applied to a DEAE-52 cellulose column (26 mm × 400 mm) and the column was stepwise eluted with 0, 0.2, 0.5, and 1.0 mol/L sodium chloride solutions at a flow rate of 1.0 mL/min. Eluate was collected automatically (2 mL/tube) though fraction collector (BSZ-100, Huxi, Shanghai, China) and the carbohydrates in each tube were determined spectrophotometrically by the phenol–sulfuric acid method [12]. After dialysis of the distilled water, the obtained eluates were concentrated and lyophilized for further studies.
2.6. Analysis of monosaccharide composition The monosaccharide composition was analyzed using the method reported by Xu et al. with a slight modification [19]. The purified fractions (5.0 mg) were hydrolyzed with 2 mL of 2 mol/L trifluoroacetic acid in an oven at 120 ◦ C for 2 h. The hydrolyzate was repeatedly co-concentrated with methanol to dryness and acetylated by the addition of a mixture of methanol, pyridine and acetic anhydride. The monosaccharide standards including rhamnose, ribose, arabinose, xylose, inositol, glucose, mannose and galactose were acetylated in the same way. All acetylated derivatives were analyzed using a 7890N GC (Agilent Technologies, Santa Clara, CA, USA) equipped with flame ionization detector and an HP5 fused silica capillary column (30 m × 0.32 mm × 0.25 mm). The temperatures of oven, detector and inlet were set at 210, 280 and 250 ◦ C, respectively. The initial oven temperature was maintained at 120 ◦ C for 3 min, and then increased gradually to 210 ◦ C at a rate of 3 ◦ C/min. The flow rates of nitrogen, hydrogen and air were 25, 30 and 400 mL/min, respectively and the injection volume was 1 L aliquot for each run.
2.7. Evaluation of antioxidant activity in vitro 2.7.1. Reducing power assay The reducing power of the polysaccharide samples was determined according to the reported method with slight modification [20]. One milliliter polysaccharide samples at different concentrations (100–1000 mg/L) were added to 2.5 mL of phosphate buffer (pH 6.6, 0.2 mol/L) and 1.0 mL of potassium ferricyanide (1%, w/v). The mixture was shaken well and incubated for 20 min at 50 ◦ C, and then the reaction was terminated via adding 2.0 mL of trichloroacetic acid (10%, w/v). After centrifugation at 1200 × g for 10 min, the supernatant was collected and incubated with 1.2 mL of ferric trichloride (0.1%, w/v) for 15 min at room temperature. The absorbance of the mixture was determined at 700 nm.
2.7.2. Hydroxyl radical assay The hydroxyl radical scavenging was measured by the method of Smironff and Cumbes with slight modifications [21]. The hydroxyl radical was generated in the mixture of ferrous sulfate (1 mL, 9 mmol/L), salicylic acid (1 mL, 9 mmol/L) and hydrogen peroxide (1 mL, 0.03%, v/v). After addition of 1 mL polysaccharide samples (100–1000 mg/L), the mixture was incubated at 37 ◦ C for 30 min.
238
Z. Ma et al. / International Journal of Biological Macromolecules 73 (2015) 236–244
The absorbance of the mixture was measured at 510 nm. The scavenging rate of hydroxyl radical was calculated as follows: Scavenging rate (%) =
1−
A A0
× 100%
(1)
where A is the absorbance of the polysaccharide samples and A0 is the absorbance of the blank. 2.7.3. Superoxide radical assay The superoxide radical scavenging activity of the polysaccharide samples was determined in accordance with the reported method of Stewar and Beewley with a slight modification [22]. Each 0.5 mL of phosphate buffer saline (0.2 mol/L, pH 7.8), 0.3 mL of riboflavin (10 mmol/L), 0.25 mL of methionine (13 mmol/L), were mixed with 1 mL the polysaccharide samples solution with different concentrations (100–1000 mg/L). The mixture was shaken well and illuminated with a fluorescent lamp at 25 ◦ C for 5 min. The absorbance of the complex was measured at 560 nm. The scavenging activity on superoxide radical was calculated according to the following formula: Scavenging rate (%) =
1−
A A0
× 100%
(2)
where A is the absorbance of the polysaccharide samples and A0 is the absorbance of the blank. 2.7.4. DPPH scavenging assay The scavenging effects of the polysaccharide samples on DPPH radicals were determined by the method of Brand-Williams et al. and Kong et al. with some modifications [23,24]. A mixture containing 2 mL of polysaccharide samples (100–1000 mg/L) and 2 mL of DPPH (0.1 mol/L) or ethanol (95%, w/v) was incubated for 15 min at room temperature. The absorbance of the mixture was determined at 517 nm. The ability to scavenge DPPH radical was calculated using the following formula: Scavenging rate (%) =
1−
A A0
× 100%
(3)
2.8.2. Superoxide dismutase (SOD) The activity of SOD was measured according to the method of Winterbourn et al. with slight modification [25]. The reaction mixture, containing 0.05 mL supernatant, 1.5 mL of potassium phosphate buffer (0.05 mol/L, pH 7.8), 0.3 mL of methionine (130 mmol/L), 0.3 mL of nitroblue tetrazolium (NBT, 750 mol/L), 0.3 mL of EDTA (100 mol/L) and 0.3 mL of riboflavin (20 mol/L), was reacted for 20 min under the illumination (4000 lx). The absorbance was measured at 560 nm against the reaction mixture in dark as a blank. The SOD activity was expressed in relative units per milligram protein or micromoles per minute per liter of blood. One unit of SOD was expressed as the quantity required inhibiting the photochemical reduction of NBT reduction by 50%. 2.8.3. GSH peroxidase (GSH-Px) Glutathione peroxidase (GSH-Px) activity was analyzed by the reported method of Flohé and Günzler with slight modification [26]. The reaction mixture contained 50 mmol/L of potassium phosphate buffer (pH 7.0), 0.5 mmol/L of EDTA, 1 mmol/L of NaN3 , 0.15 mmol/L of NADPH, 1 mmol/L of GSH, and 2.4 U/mL of glutathione reductase (GR). Then 0.15 mmol/L of H2 O2 was added and incubated at 37 ◦ C for further 5 min and the rate of NADPH consumption was recorded at 340 nm. 2.8.4. Lipid peroxidase (LPO) The assay of Zuo with slight modifications was used to measure the content of LPO [27]. The reaction mixture consisting of 0.05 mL supernatant and 2.5 mL TBA (2.68 g/L) was incubated in boiling water at 5 min. The reaction was cooled to termination immediately, and then 7.5 mL n-butyl alcohol was added and incubated at room temperature for further 5 min. The absorbance was measured at 532 nm, using distilled water for blank. And the positive control was performed with 1,1,3,3-tetraethoxypropane (TEP, 40 nmol/L) instead of supernatant. The content of LPO was calculated as follows: Content (nmol/L) =
A × 40 A0
(4)
where A was the absorbance of the tested sample and A0 was the absorbance of the blank.
where A is the absorbance of samples, and A0 is the absorbance of TEP.
2.8. Anti-aging in vivo
2.9. Statistical analysis
2.8.1. Animals and treatment Fifty male mice (Kunming strain) weighing 20 ± 2 g were purchased from the Center for Animal Testing of Shandong Lukang Drugs Group Limited (Jining, China) and animal experiments were in accordance with the institutional ethical guidelines and under guidance of Shandong Agricultural University Committee. Animals were housed in a well-ventilated room with free access to water and standard food at 22 ± 1 ◦ C with a 12 h light–dark cycle. The animals were divided into the test group (n = 30), the model control group (MC, n = 10), and the negative control group (NC, n = 10). The test group was further divided into low dose of IPS group (LIPS, n = 10) middle dose of IPS group (MIPS, n = 10), and high dose of IPS group (HIPS, n = 10). Mice in LIPS group, MIPS group and HIPS group were orally fed with doses of 200, 400 and 800 mg/kg body weight of mice by filling the stomach (0.01 mL/g body weight) with a syringe and d-galactose intraperitoneal injection (1000 mg/kg body weight) every day, respectively. Mice were treated with the same volume of saline instead of IPS in NC group and were treated with the same volume of saline instead of IPS and d-galactose in MC group. After 20 d of treatment, mice were anaesthetized and blood and tissues were collected immediately and stored for further analysis.
All results were carried out in triplicates and presented as means ± standard deviation (S.D.). Data were analyzed by one-way analysis of variance (ANOVA) using SPSS 16.0 and Dunnett’s test. P values
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Enzymatic and acidic degradation effect on intracellular polysaccharide of Flammulina velutipes SF-08 Zhao Ma a,1 , Chen Zhang a , Xia Gao b,1 , Fangyuan Cui a , Jianjun Zhang a , Mengshi Jia c , Shouhua Jia d,∗ , Le Jia a,∗ a
College of Life Science, Shandong Agricultural University, Taian, Shandong 271018, PR China Shandong Agricultural Technology Extending Station, Jinan, Shandong 250100, PR China c The Second High School of Taian, Taian, Shandong 271000, PR China d College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, PR China b
a r t i c l e
i n f o
Article history: Received 4 October 2014 Received in revised form 23 November 2014 Accepted 27 November 2014 Available online 4 December 2014 Keywords: Flammulina velutipes SF-08 Intracellular polysaccharide Enzymolysis and acidolysis
a b s t r a c t The intracellular polysaccharide (IPS) from Flammulina velutipes SF-08 mycelia was isolated and degraded by enzyme and acid. IPS and its derivative were purified by DEAE-52 cellulose chromatography, and five fractions were obtained. The structural features and antioxidant activities in vitro of the isolated fractions were evaluated. On the basis of chemical composition and antioxidant ability analyses, rhamnose as the main monosaccharide might contribute to the strongest antioxidant capacity. The in vivo results showed that IPS significantly enhanced the activities of anti-aging enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and reduced the content of lipid peroxidantion (LPO). These results suggested that IPS should be a potent natural polymer and can be developed to be novel functional food. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS), including superoxide anion radical, hydroxyl radical, hydrogen peroxide, reactive oxygen, and nitrogen species, play important roles in cell signaling and homeostasis of organism [1]. However, uncontrolled ROS can induce physiological effects related to the pathogenesis of several human diseases such as heart diseases, gastric problems, aging and certain types of cancer [2]. Antioxidants, widely discussed in clinical and nutritional literature, can reduce the oxidative stress to prevent the formation of these free radicals and protect the body from oxidative damage [3]. Some commonly used synthetic antioxidants, such as BHA and BHT, are suspected to have some toxic effects and as possible carcinogens [4]. Therefore, new interest has been developed to employ non-toxic antioxidants from natural sources by consumer preference for natural products. Many dietary compounds have been suggested to be important antioxidants. Polysaccharides, obtained from many edible mushrooms,
∗ Corresponding authors. Tel.: +86 0538 8249958/1570; fax: +86 0538 8248696/1570. E-mail addresses: [email protected] (S. Jia), [email protected] (L. Jia). 1 Equal contributors. http://dx.doi.org/10.1016/j.ijbiomac.2014.11.028 0141-8130/© 2014 Elsevier B.V. All rights reserved.
have exhibited strong antioxidant activities and can be acted as novel potential antioxidants [5,6]. Flammulina velutipes, an edible mushroom that was widely distributed and artificially cultivated in oriental countries, has been proved to have high nutritional value and attractive taste [7]. According to the previous literature, F. velutipes contains biologically active components such as protein, dietary fiber, and polysaccharides [8]. It has been showed that bioactive polysaccharides obtained from fruiting bodies of F. velutipes have beneficial immunomodulatory and biological activity [9]. In addition, previous studies indicate antioxidant activities of polysaccharides can be affected by many factors including chemical components, uronic acid contents, and even the extraction and isolation methods [10,11]. However, there are few reports of the correlation of structural characterization and antioxidant activities of various polysaccharide fractions obtained from mycelia of F. velutipes. In this work, enzyme and acid hydrolysis productions of the intracellular polysaccharide (IPS) of F. velutipes SF-08 mycelia were isolated and purified by DEAE-52 cellulose column chromatography. The monosaccharide compositions of the purified polysaccharides were elucidated by the methods of gas chromatography (GC) and their antioxidant activities were evaluated. Based on the analysis of antioxidant and monosaccharides, we preliminary investigated the correlation of the monosaccharide composition
Z. Ma et al. / International Journal of Biological Macromolecules 73 (2015) 236–244
and antioxidant activity in vitro. In addition, the in vivo anti-aging properties of IPS were evaluated. 2. Materials and methods 2.1. Chemicals and reagents DEAE-52 cellulose was purchased from Whatman 4057200. Hydrogen peroxide (H2 O2 ), ferrozine and 1,1-diphenyl-2picrylhydrazyl (DPPH) were purchased from Sigma Chemicals Company (St. Louis, USA). The standard monosaccharides were provided by Merck Company (Darmstadt, Germany) and Sigma Chemical Company (St. Louis, USA). All the other chemicals and reagents were analytical grade and purchased from local chemical suppliers in China. 2.2. Culture media and conditions The F. velutipes SF-08 strain, initially incubated at 26 ◦ C on potato dextrose agar (PDA) slants by our laboratory, was used in this experiment. A 10 mm2 portion of the agar plate culture was sliced from the slant for inoculating the seed culture medium. Seeding cultivation in liquid media was cultured in a 1 L filter flask containing 500 mL of potato dextrose broth at 25 ◦ C on a rotary shaker (160 rpm, Anting, Shanghai, China) for 7 d and then the cultured broth was gradually homogenized. Batch fermentation experiment was performed in a 100 L fermenter (Xianmin, Luoyang, China) with air agitation at 25 ◦ C for 7 d. The mycelium of F. velutipes SF-08 collected from fermenter was centrifuged at 3000 × g for 15 min, washed several times with distilled water and dried by a lyophilizer in vacuum to constant weight. 2.3. Extraction of IPS The F. velutipes SF-08 mycelia was extracted twice with distilled water at 90 ◦ C for 2 h each, and centrifuged at 3000 × g for 15 min, the supernatants were concentrated and precipitated by addition of 3 volumes of 95% ethanol (v/v), stirred vigorously, and kept at 4 ◦ C for 24 h. The precipitate was collected by centrifugation at 3000 × g for 15 min, dissolved in distilled water at 60 ◦ C, and measured according to the phenol–sulfuric acid method using glucose as the standard [12]. The IPS was deproteined by Sevag reagent, dialyzed and lyophilized by vacuum freeze drying (Labconco, USA) [13]. 2.4. Enzymatic and acidic degradation According to the method of Yang et al. and Li et al. with some modifications, the dried polysaccharide powder (0.5 g) and snailase (0.1 g) were dissolved in 100 mL of 1% acetic acid [14,15]. The mixture system was subjected to chemical treatment under following conditions with temperature of 40 ◦ C, time 5 h, pH 6, and enzyme to the polysaccharide sample ratio of 1:5. The enzymatic hydrolysis polysaccharide (EIPS) was concentrated at low pressure by a rotary evaporator, and then lyophilized. Tests on the acid degradation of the polysaccharide were performed by the method of Zhang et al. with minor modifications [16]. The IPS (0.5 g) in test tube (18 mm × 180 mm) with stopper was dissolved in 10 mL of 1 mol/L H2 SO4 solution and kept in boiled water for 8 h. After acid hydrolysis termination, hydrolysate was centrifuged at 6000 × g for 10 min. The supernatant (4.5 mL) was neutralized with 2 mol/L NaOH solution and then diluted using distilled water to obtain a final volume of 10 mL. After centrifuged (6000 × g, 10 min), the liquid supernatant was concentrated at low
237
pressure by a rotary evaporator and lyophilized, and acid hydrolysis polysaccharide (AIPS) was obtained.
2.5. Purification of IPS, EIPS and AIPS The samples (IPS, EIPS and AIPS) were purified by DEAE-52 cellulose chromatography according to the reported method with sight modification [17,18]. The sample (1.0 g) dissolved in 15 mL of distilled water at 60 ◦ C was filtered through 0.22 m organic membrane filter. The sample was applied to a DEAE-52 cellulose column (26 mm × 400 mm) and the column was stepwise eluted with 0, 0.2, 0.5, and 1.0 mol/L sodium chloride solutions at a flow rate of 1.0 mL/min. Eluate was collected automatically (2 mL/tube) though fraction collector (BSZ-100, Huxi, Shanghai, China) and the carbohydrates in each tube were determined spectrophotometrically by the phenol–sulfuric acid method [12]. After dialysis of the distilled water, the obtained eluates were concentrated and lyophilized for further studies.
2.6. Analysis of monosaccharide composition The monosaccharide composition was analyzed using the method reported by Xu et al. with a slight modification [19]. The purified fractions (5.0 mg) were hydrolyzed with 2 mL of 2 mol/L trifluoroacetic acid in an oven at 120 ◦ C for 2 h. The hydrolyzate was repeatedly co-concentrated with methanol to dryness and acetylated by the addition of a mixture of methanol, pyridine and acetic anhydride. The monosaccharide standards including rhamnose, ribose, arabinose, xylose, inositol, glucose, mannose and galactose were acetylated in the same way. All acetylated derivatives were analyzed using a 7890N GC (Agilent Technologies, Santa Clara, CA, USA) equipped with flame ionization detector and an HP5 fused silica capillary column (30 m × 0.32 mm × 0.25 mm). The temperatures of oven, detector and inlet were set at 210, 280 and 250 ◦ C, respectively. The initial oven temperature was maintained at 120 ◦ C for 3 min, and then increased gradually to 210 ◦ C at a rate of 3 ◦ C/min. The flow rates of nitrogen, hydrogen and air were 25, 30 and 400 mL/min, respectively and the injection volume was 1 L aliquot for each run.
2.7. Evaluation of antioxidant activity in vitro 2.7.1. Reducing power assay The reducing power of the polysaccharide samples was determined according to the reported method with slight modification [20]. One milliliter polysaccharide samples at different concentrations (100–1000 mg/L) were added to 2.5 mL of phosphate buffer (pH 6.6, 0.2 mol/L) and 1.0 mL of potassium ferricyanide (1%, w/v). The mixture was shaken well and incubated for 20 min at 50 ◦ C, and then the reaction was terminated via adding 2.0 mL of trichloroacetic acid (10%, w/v). After centrifugation at 1200 × g for 10 min, the supernatant was collected and incubated with 1.2 mL of ferric trichloride (0.1%, w/v) for 15 min at room temperature. The absorbance of the mixture was determined at 700 nm.
2.7.2. Hydroxyl radical assay The hydroxyl radical scavenging was measured by the method of Smironff and Cumbes with slight modifications [21]. The hydroxyl radical was generated in the mixture of ferrous sulfate (1 mL, 9 mmol/L), salicylic acid (1 mL, 9 mmol/L) and hydrogen peroxide (1 mL, 0.03%, v/v). After addition of 1 mL polysaccharide samples (100–1000 mg/L), the mixture was incubated at 37 ◦ C for 30 min.
238
Z. Ma et al. / International Journal of Biological Macromolecules 73 (2015) 236–244
The absorbance of the mixture was measured at 510 nm. The scavenging rate of hydroxyl radical was calculated as follows: Scavenging rate (%) =
1−
A A0
× 100%
(1)
where A is the absorbance of the polysaccharide samples and A0 is the absorbance of the blank. 2.7.3. Superoxide radical assay The superoxide radical scavenging activity of the polysaccharide samples was determined in accordance with the reported method of Stewar and Beewley with a slight modification [22]. Each 0.5 mL of phosphate buffer saline (0.2 mol/L, pH 7.8), 0.3 mL of riboflavin (10 mmol/L), 0.25 mL of methionine (13 mmol/L), were mixed with 1 mL the polysaccharide samples solution with different concentrations (100–1000 mg/L). The mixture was shaken well and illuminated with a fluorescent lamp at 25 ◦ C for 5 min. The absorbance of the complex was measured at 560 nm. The scavenging activity on superoxide radical was calculated according to the following formula: Scavenging rate (%) =
1−
A A0
× 100%
(2)
where A is the absorbance of the polysaccharide samples and A0 is the absorbance of the blank. 2.7.4. DPPH scavenging assay The scavenging effects of the polysaccharide samples on DPPH radicals were determined by the method of Brand-Williams et al. and Kong et al. with some modifications [23,24]. A mixture containing 2 mL of polysaccharide samples (100–1000 mg/L) and 2 mL of DPPH (0.1 mol/L) or ethanol (95%, w/v) was incubated for 15 min at room temperature. The absorbance of the mixture was determined at 517 nm. The ability to scavenge DPPH radical was calculated using the following formula: Scavenging rate (%) =
1−
A A0
× 100%
(3)
2.8.2. Superoxide dismutase (SOD) The activity of SOD was measured according to the method of Winterbourn et al. with slight modification [25]. The reaction mixture, containing 0.05 mL supernatant, 1.5 mL of potassium phosphate buffer (0.05 mol/L, pH 7.8), 0.3 mL of methionine (130 mmol/L), 0.3 mL of nitroblue tetrazolium (NBT, 750 mol/L), 0.3 mL of EDTA (100 mol/L) and 0.3 mL of riboflavin (20 mol/L), was reacted for 20 min under the illumination (4000 lx). The absorbance was measured at 560 nm against the reaction mixture in dark as a blank. The SOD activity was expressed in relative units per milligram protein or micromoles per minute per liter of blood. One unit of SOD was expressed as the quantity required inhibiting the photochemical reduction of NBT reduction by 50%. 2.8.3. GSH peroxidase (GSH-Px) Glutathione peroxidase (GSH-Px) activity was analyzed by the reported method of Flohé and Günzler with slight modification [26]. The reaction mixture contained 50 mmol/L of potassium phosphate buffer (pH 7.0), 0.5 mmol/L of EDTA, 1 mmol/L of NaN3 , 0.15 mmol/L of NADPH, 1 mmol/L of GSH, and 2.4 U/mL of glutathione reductase (GR). Then 0.15 mmol/L of H2 O2 was added and incubated at 37 ◦ C for further 5 min and the rate of NADPH consumption was recorded at 340 nm. 2.8.4. Lipid peroxidase (LPO) The assay of Zuo with slight modifications was used to measure the content of LPO [27]. The reaction mixture consisting of 0.05 mL supernatant and 2.5 mL TBA (2.68 g/L) was incubated in boiling water at 5 min. The reaction was cooled to termination immediately, and then 7.5 mL n-butyl alcohol was added and incubated at room temperature for further 5 min. The absorbance was measured at 532 nm, using distilled water for blank. And the positive control was performed with 1,1,3,3-tetraethoxypropane (TEP, 40 nmol/L) instead of supernatant. The content of LPO was calculated as follows: Content (nmol/L) =
A × 40 A0
(4)
where A was the absorbance of the tested sample and A0 was the absorbance of the blank.
where A is the absorbance of samples, and A0 is the absorbance of TEP.
2.8. Anti-aging in vivo
2.9. Statistical analysis
2.8.1. Animals and treatment Fifty male mice (Kunming strain) weighing 20 ± 2 g were purchased from the Center for Animal Testing of Shandong Lukang Drugs Group Limited (Jining, China) and animal experiments were in accordance with the institutional ethical guidelines and under guidance of Shandong Agricultural University Committee. Animals were housed in a well-ventilated room with free access to water and standard food at 22 ± 1 ◦ C with a 12 h light–dark cycle. The animals were divided into the test group (n = 30), the model control group (MC, n = 10), and the negative control group (NC, n = 10). The test group was further divided into low dose of IPS group (LIPS, n = 10) middle dose of IPS group (MIPS, n = 10), and high dose of IPS group (HIPS, n = 10). Mice in LIPS group, MIPS group and HIPS group were orally fed with doses of 200, 400 and 800 mg/kg body weight of mice by filling the stomach (0.01 mL/g body weight) with a syringe and d-galactose intraperitoneal injection (1000 mg/kg body weight) every day, respectively. Mice were treated with the same volume of saline instead of IPS in NC group and were treated with the same volume of saline instead of IPS and d-galactose in MC group. After 20 d of treatment, mice were anaesthetized and blood and tissues were collected immediately and stored for further analysis.
All results were carried out in triplicates and presented as means ± standard deviation (S.D.). Data were analyzed by one-way analysis of variance (ANOVA) using SPSS 16.0 and Dunnett’s test. P values
