International Journal of Biological Macromolecules 62 (2013) 691–696
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Chemical analysis and antioxidant activity of polysaccharides extracted from Inonotus obliquus sclerotia XiuJu Du a,∗ , HongMei Mu b , Shuai Zhou c , Yang Zhang a , XiaoLi Zhu a a b c
College of Life Science, Liaocheng University, Liaocheng, Shandong 252059, PR China College of Agricultural Science, Liaocheng University, Liaocheng, Shandong 252059, PR China Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201106, PR China
a r t i c l e
i n f o
Article history: Received 25 August 2013 Received in revised form 8 October 2013 Accepted 13 October 2013 Available online 18 October 2013 Keywords: Inonotus obliquus polysaccharide Free radicals scavenging activity Monosaccharide composition Antioxidant activity
a b s t r a c t Three water-soluble polysaccharide fractions (IOP40, IOP60 and IOP80) were isolated by using different concentrations of alcohol precipitation from Inonotus obliquus sclerotia. Their physicochemical properties, including total sugar content, protein content, monosaccharide composition and percentage were analyzed. And their in vitro antioxidant capacities were investigated in terms of reducing power assay and scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, hydroxyl radicals, superoxide anion radicals and hydrogen peroxide (H2 O2 ). In general, three polysaccharide fractions exhibited increasing antioxidant activity with increasing concentration at the ranges of tested dosage. The orders of reducing power, DPPH-scavenging capacity, H2 O2 -scavenging capacity, and hydroxyl-scavenging activity were all IOP60 > IOP40 > IOP80. These findings demonstrated that three polysaccharide fractions extracted from I. obliquus, especially IOP60, could be employed as natural ingredients in functional food and pharmaceutical industry to alleviate the oxidative stress. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS), generated by triplet state oxygen reacting with other molecules, such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide (H2 O2 ), is the major sources of primary catalysts that initiate oxidation in vivo and in vitro [1]. ROS is also one of the main factors which cause the oxidation of polyunsaturated fatty acid in the surface of biomembrane, and it has been associated with the beginning of many diseases and degenerative processes in aging [2]. A growing number of reports indicated that the side effects of synthetic antioxidants might be responsible for some observed carcinogenesis and liver damage [3]. Thus, it was essential to discover new natural antioxidants that could protect the human body from free radicals without side effects. Recently, it had been reported that polysaccharides from fungi had exhibited strong antioxidant effects that related to their health-protecting functions [4,5]. Inonotus obliquus, a kind of traditional fungi belonging to the family of Hymenochaetaceae, Basidiomycetes, has been used as a folk remedy in Asian countries, such as China, Japan, Korea and Russia for five hundred years [6]. More and more studies have shown that polysaccharides from I. obliquus possessed clear antioxidant
activities [7–9]. However, up to now, no detailed investigation has been carried out on physicochemical characteristics and antioxidative capacity of different polysaccharide fractions isolated by precipitating with different alcohol concentration from the sclerotia of I. obliquus. In the present study, the purpose is to focus on the isolation and properties of three different polysaccharide fractions from the sclerotia of I. obliquus, and explored their antioxidant capabilities for seeking new natural functional ingredients used in food and pharmaceutical industry to alleviate the oxidative stress. Therefore, three water-soluble polysaccharide fractions (IOP40, IOP60 and IOP80) were obtained and their physicochemical properties, including total sugar content, protein content, monosaccharide composition and percentage were analyzed. And their in vitro antioxidant capacities were assayed in terms of reducing power and scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals, hydroxyl radicals, superoxide anion radicals and hydrogen peroxide (H2 O2 ).
2. Materials and methods 2.1. Materials and reagents
∗ Corresponding author at: College of Life Science, Liaocheng University, Hunan Road 1, Liaocheng 252059, PR China. Tel.: +86 635 8538917; fax: +86 635 8239908. E-mail addresses: [email protected], [email protected] (X. Du). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.016
The sclerotia of I. obliquus was purchased from the Northeast Natural Products Trading Company (Haerbin, China). The monosaccharide standards, d-Gal, d-Glc, d-Ara, l-Fuc, l-Rha, d-Fru, d-Man,
692
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
Small pieces of I. obliquus sclerotia Extracted twice with 10 volumes 95% EtoH, 24 h
Solid residues dried in air Extracted thrice with 10-fold volume (distilled water, 2 h), then centrifuge(1,776 g,10 min)
Aqueous extracts Concentrated 10-fold with vacuum rotary evaporator
Concentrated aqueous extracts Precipitated with 95% ethanol to 40% of final alcohol content, centrifugation(1,776 g,10 min)
Precipitate
Supernatant
Dialysis and
Precipitated with 95% ethanol to 60% of final
freeze-drying
alcohol content, centrifugation(1,776 g,10 min)
IOP40 Precipitate
Supernatant Precipitated with 95% ethanol to
Dialysis and
80% of final alcohol content
freeze-drying
IOP60
Precipitate Dialysis and freeze-drying
IOP80 Fig. 1. Scheme of extraction and fractionation of different polysaccharide from I. obliquus.
d-Xyl, d-GlcA and d-GalA were bought from Sigma–Aldrich (St. Louis, MO, USA). All other reagents were of analytical reagent grade and from Chinese sources. All aqueous solutions were prepared by using newly double-distilled water. 2.2. Extraction and fractionation of polysaccharide Three polysaccharide fractions IOP40, IOP60 and IOP80 were obtained according to the flow diagram shown in Fig. 1. Briefly, dried sclerotia of I. obliquus powder (1.5 kg) was immersed in 15 L of ethanol (95%) for 24 h, and the solid residue was collected by filtration and the procedure was repeated two times. Then the air-dried solid residue was extracted for three times with distilled water. The combined supernatant was concentrated to one-tenth
of the original volume. 95% of ethanol was added slowly to the concentrated supernatant to 40% of final alcohol content, then the mixture was set at 4 ◦ C for 24 h. After centrifugation, dialysis and freeze-drying, the precipitate was obtained and termed IOP40. Similarly, 95% of ethanol was added to the concentrated supernatant to the final alcohol content of 60% and 80%, accordingly the precipitate IOP60 and IOP80 were prepared. 2.3. General methods Total sugar contents were determined by the phenol-sulfuric acid method using d-glucose as standard [10]. The protein contents were measured by the method of Bradford using vine serum albumin as the standard [11]. Neutral sugar content and
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
uronic acid content were determined by using high-performance anion-exchange pulsed-amperometric detection chromatography (HPAEC-PAD) [12]. 2.4. Monosaccharide composition analysis Monosaccharide composition and component percentage were determined using HPAEC-PAD as reported by Du et al. [12] with minor modifications. Briefly, sample (10 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110 ◦ C for 5 h. A Dionex LC30 was equipped with a CarboPacTM PA20 column (3 mm × 150 mm), and the monosaccharide was monitored using a pulsed amperometric detector (Dionex). The column was eluted with 2 mM NaOH (0.45 mL/min). d-Gal, d-Glc, d-Ara, l-Fuc, l-Rha, d-Fru, d-Man, dXyl, d-Ribose, d-GlcA and d-GalA were employed as standards. 3. Antioxidant activity assays 3.1. Reducing power assay The reducing power was determined according to the method described by Wang et al. [13]. In brief, 1 mL samples (62.5–1000 g/mL), 2 mL phosphate buffer (0.2 M, pH 6.6) and 2 mL 1% (w/v) K3 Fe(CN)6 were incubated at 50 ◦ C for 20 min. After cooling rapidly, the reaction was terminated by adding 2 mL of trichloroacetic acid (TCA, 10%, w/v) and centrifuged at 1776 × g for 10 min. The supernatant (2.5 mL) mixed with 2.5 mL of distilled water and 1 mL of 0.1% (w/v) FeCl3 , and set for 10 min. The absorbance of the reaction mixture was read at 700 nm against a blank. Ascorbic acid was used as positive control. 3.2. DPPH-scavenging activity assay The DPPH radical scavenging activity of three polysaccharide fractions was measured according to the method by Ardestani et al. [14]. The sample solution (0.1 mL) with variable concentrations (62.5–1000 g/mL) was added to tube containing 2.9 mL of DPPH solution (0.1 mM in ethanol). The mixture was blended by vortex and then set for 30 min at 37 ◦ C in the dark. Then the absorbance was measured at 517 nm. In this work, distilled water and ascorbic acid served as the negative and positive controls respectively. The DPPH radical scavenging activities were calculated using the following formula: Scavenging rate (%) = (1 − A1 /A0 ) × 100, where A0 is the absorbance value of the control, A1 is the absorbance values of samples and ascorbic acid. 3.3. Superoxide anion-scavenging activity assay The superoxide anion radical assays of samples were measured according to the Pyrogallol-Chemiluminescence method by Guo et al. and Wang et al. [15,16]. Specific methods were as follows: 2 L of different test agents and 8 L of pyrogallic acid (0.625 mM) were added to each well of a 96-well microplate. In this work, PBS and ascorbic acid served as the negative and positive controls respectively. 150 L of Luminol-CBS (sodium carbonate buffer solution) mixture (pH 10.2) was pumped into each well of a 96-well microplate through external. Signal of each well was collected in every 0.6 s and kept for 30 s. Each sample was repeated 3 times. Luminescence values were measured using Clarity (BIO-TEK Company) auto-reader. The scavenging effect for superoxide anion radical was calculated according to the following formula: Scavenging rate (%) = (A0 − A1 )/A0 × 100, in which A0 was the luminescence value of the negative control without tested sample. A1 was the luminescence values of tested samples.
693
3.4. H2 O2 -scavenging activity assay The H2 O2 -scavenging activities were carried out by the method of Chemiluminescence [16,17]. 10 L of 1% H2 O2 and 40 L of different tested samples were added to each well of a 96-well microplate. Double distilled water and ascorbic acid were used as the negative and positive controls separately. The scavenging rates for H2 O2 were performed according to the method in superoxide anion-scavenging activity assay (Section 3.3). 3.5. Hydroxyl radical-scavenging assay The hydroxyl radical assay of sample was measured according to the salicylic acid method described by Qian et al. [18] with some modifications. Briefly, 8 mL of reaction mixture, containing 2 mL of tested samples with different concentrations [0 (control), 62.5, 125, 250, 500 and 1000 g/mL], 2 mL of FeSO4 aqueous solution (4 mM), 2 mL of salicylic acid ethanol solution (4 mM) and 2 mL of H2 O2 aqueous solution (4 mM), was shaken well, then incubated (37 ◦ C, 30 min), centrifuged (1776 × g, 10 min) and lastly the supernatant mixture was obtained. The absorbance of the reaction mixture was read at 510 nm. The Hydroxyl radical-scavenging rate (r) was calculated by the following formula: r(%) = (A0 − (AX − AX0 ))/A0 × 100, where A0 is the absorbance of blank control only, AX is the absorbance of sample and positive (ascorbic acid), and AX0 is the absorbance of sample and positive only without H2 O2 aqueous solution (replaced by 2 mL of distilled water). 3.6. Statistical analysis The data obtained were expressed as mean ± SD of three determinations and analyzed statistically by ANOVA method. Significance of any differences between groups was evaluated using the Student’s t-test. All computations were done by employing statistical software (STST). 4. Results and discussion 4.1. Characterization and composition of the three polysaccharide fractions Three water-soluble polysaccharide fractions IOP40, IOP60 and IOP80 were obtained successfully from I. obliquus sclerotia by boiling water and ethanol precipitation. The extraction yields of three fractions (IOP40, IOP60 and IOP80) were 2.2%, 11.6% and 0.84%, respectively. The properties of IOP40, IOP60 and IOP80, including total sugar content, protein content, neutral sugar content and uronic acid content, and monosaccharide components and percentage, were summarized and shown in Table 1. As we all know, it is important to know the monosaccharide composition and molar ratio of polysaccharides, which contribute to their bioactivity [19]. In this study, HPAEC-PAD was employed to identify and quantify the major compositional monosaccharide presented in IOP40, IOP60 and IOP80. As shown in Fig. 2A, 11 kinds of standard monosaccharide were baseline separated rapidly within 39 min. By matching their retention times with those of monosaccharide standards under the same analytical conditions, eleven peaks were identified in the order of l-fucose (l-Fuc), l-rhamnose (l-Rha), l-arabinose (l-Ara), d-galactose (dGal), d-glucose (d-Glc), d-xylose (d-Xyl), d-mannose (d-Man), d-fructose(d-Fru), d-ribose (d-Rib), d-galacturonic acid (d-GalA) and d-glucuronic acid (d-GluA). The chromatograms of the compositional monosaccharide of IOP40, IOP60 and IOP80 were also given in Fig. 2B–D, respectively. The results indicated that three polysaccharides all appeared to be the typical acidic hetero-polysaccharide.
694
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
Table 1 Monosaccharide components and properties of various polysaccharides from I. obliquus. Samples
IOP40
IOP60
IOP80
Yields (a W%) Total sugar content (W%) Neutral saccharide (W%) Uronic acid (W%) Protein content (W%)
2.2 35.1 30.1 5.0 2.4
11.6 24.6 22.4 2.2 3.2
0.8 23.2 21.7 1.5 4.6
Monosaccharide components (M%b ) l-Fucose (M%) l-Rhamnose (M%) d-Arabinose (M%) d-Galactose (M%) d-Glucose (M%) d-Xylose (M%) d-Mannose (M%) d-GalA (M%) d-GluA (M%)
3.3 4.4 4.2 14.5 40.0 9.6 9.8 4.6 9.7
c
nd 11.0 7.5 8.5 31.3 25.3 9.7 3.9 2.8
a b c
nd 9.7 5.6 12.6 32.2 22.9 8.0 4.4 4.7
W% is expressed as the weight percentage. M% is expressed as the molar percentage. nd: data were not detected.
As shown in Fig. 2A and B, IOP40 was composed of l-Fuc, l-Rha, l-Ara, d-Gal, d-Glc, d-Xyl, d-Man, d-GalA and d-GlcA in the molar ratio of 5.1:6.8:6.5:22.5:62.2:14.9:15.2:7.1:15.1. Similarly, both IOP60 and IOP80 consisted of l-Rha, l-Ara, d-Gal, d-Glc, d-Xyl, d-Man, d-GalA and d-GlcA, and the molar ratios of them were 5.5:3.2:7.1:18.1:12.9:4.5:2.5:2.6 and 5.0:3.4:3.9:14.2:11.4:4.4:1.8:1.3, respectively (shown in Fig. 2C and D). The monosaccharide percentage of IOP40, IOP60 and IOP80 were summarized in Table 1. However, the results also showed that one peak was associated with the retention time at 5.14 min in Fig. 2B and the some peaks were associated with the retention time at 9.62 min in Fig. 2C and D. These findings might imply new compounds might be contained in IOP40, IOP60 and IOP80, which would be the continuing work. 4.2. Reducing power assay The reducing properties were generally associated with the presence of electron-donating groups or hydrogen atoms, which could react with free radicals to stabilize and block radical chain reactions [20]. Accordingly, reducing power assay was an important method to test drugs of antioxidant activity. The data presented showed that increased absorbance of the reaction mixture indicated stronger reducing power. As shown in Fig. 3a, at the concentration range of 62.5–1000 g/mL, the reducing power of various polysaccharide fractions increased with increasing dosage, although all fractions showed lower activities than that of ascorbic acid at tested concentration. The RP0.5AU values (defined as the effective concentration at which the absorbance was 0.5 for reducing power) of ascorbic acid, IOP40, IOP60 and IOP80 were 58 g/mL, 354 g/mL, 154 g/mL and 411 g/mL, respectively. So the order of various polysaccharide fraction on reducing power was IOP60 > IOP40 > IOP80. 4.3. DPPH-scavenging activity assay The DPPH radical is a stable lipid free radical with absorption peak at 517 nm. The DPPH can capture either electrons or hydrogen atoms from antioxidant and make pair with its free radicals. Accordingly, the original purple-colored DPPH is partially disappeared and the stable yellow-colored diphenylpicrylhydrazine (DPPH-H) is simultaneously formed. Therefore, DPPH-scavenging activity assay is a useful method for determination free radical scavenging activities of antioxidant materials. The greater scavenging rate on DPPH radicals shows that antioxidant ability of materials is stronger [21]. As shown in Fig. 3b, the DPPH radical-scavenging rates of three polysaccharide fractions rose with increasing concentration at the dosage range of 62.5–1000 g/mL. The EC50 values (defined as the effective concentration at which the antioxidant activity was 50%) of IOP40, IOP60, IOP80 and ascorbic acid were 880 g/mL, 697 g/mL, 1190 g/mL and 107 g/mL, respectively (Table 2). Therefore, the DPPH radicals-scavenging effects exhibited the following order: IOP60 > IOP40 > IOP80, which was the same as the reducing power. 4.4. Superoxide anion-scavenging activity assay
Fig. 2. The spectrum of HPAEC-PAD of 11 standard monosaccharides (A) and component monosaccharides released from IOP40 (B), IOP60 (C) and IOP80 (D). Peaks: (1) l-fucose, (2) l-rhamnose, (3) l-arabinose, (4) d-galactose, (5) d-glucose, (6) d-xylose, (7) d-mannose, (8) d-fructose, (9) d-ribose, (10) d-GalA and (11) d-gluA.
Superoxide is generated in biological systems during the normal catalytic function [22]. Superoxide anion radical is one of harmful species to cellular component as a precursor of more reactive oxygen species (ROS). It can result in the formation of H2 O2 in dismulation reaction [23]. Therefore, superoxide anionscavenging activity is a significant part of the antioxidant activities. As shown in Fig. 3c, the superoxide radical scavenging effects of three polysaccharide fractions increased with increasing concentration at the tested dosage. The EC50 values of IOP40, IOP60
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
695
Fig. 3. Antioxidant activity of three polysaccharide fractions from I.·obliquus: (a) reducing power, (b) scavenging activities to DPPH-radical, (c) scavenging activities to superoxide anion, (d) scavenging activities to H2 O2 , and (e) scavenging activities to hydroxyl radical; data are presented as the mean values (n = 3).
and IOP80 were 425 g/mL, 473 g/mL, 2290 g/mL, respectively, which was higher than the positive control ascorbic acid (48 g/mL) (see Fig. 3c and Table 2). Thus, the order of three fractions on superoxide anion-scavenging activity was IOP40 > IOP60 > IOP80, which was different from that of the reducing power and the DPPH-scavenging activity. However, IOP40 and IOP60 exhibited remarkably stronger antioxidant capacities compared with IOP80 in the tested concentrations (P < 0.05). There was no markedly difference on superoxide anion-scavenging activity between IOP40 and IOP60 (P < 0.05).
4.5. H2 O2 -scavenging activity assay H2 O2 is one of representative ROS generated in the body [24]. As shown in the Fig. 3d, the H2 O2 -scavenging effects of three polysaccharide fractions increased with the increase of concentration. The polysaccharide fraction IOP60 exhibited strongest H2 O2 -scavenging capacity among them. The EC50 values of IOP40, IOP60 and IOP80 were 580 g/mL, 460 g/mL, 5000 g/mL respectively, which all showed higher capacities compared with the positive control ascorbic acid (226 g/mL) (Table 2). The order of
696
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
Table 2 EC50 values of various polysaccharide fraction from I. obliquus in antioxidant properties. Antioxidant activity
IOP40
IOP60
IOP80
Ascorbic acid
a
DPPH-scavenging activity Superoxide anion-scavenging activity H2 O2 -scavenging activity Hydroxyl-scavenging activity
EC50 value (g/mL) 880 697 1190 425 473 2290
107 48
580 458
460 368
5000 440
282 147
154
411
58
b
Reducing power
RP0.5AU 354
a
EC50 value: the effective concentration at which the antioxidant activity was 50%. b RP0.5AU value: the effective concentration at which the absorbance was 0.5 for reducing power.
H2 O2 -scavenging activity was IOP60 > IOP40 > IOP80, which was the same as the sequence of reducing power, as well as that of the DPPH-scavenging activity. 4.6. Hydroxyl-scavenging activity assay Hydroxyl free radical, one of the liveliest substances, may directly or indirectly cause tissue damage, induce many human diseases including cancer and promote aging [25]. Hydroxyl free radicals were generated when H2 O2 and Fe2+ were mixed together in salicylic acid method, which could be expressed as the reaction equation as follows: H2 O2 + Fe2+ → OH + H2 O + Fe3+ . Hydroxyl free radicals could be captured by salicylic acid and produce colored substances, which had maximum absorption at 510 nm. The absorbance value represented the content of hydroxyl free radicals. The greater scavenging rate implies that the antioxidant ability of materials is stronger. As shown in Fig. 3e, three polysaccharide fractions all have the abilities to scavenge hydroxyl free radicals at concentration from 62.5 g/mL to 1000 g/mL, and the scavenging abilities increased with the concentration increasing. The EC50 values of three polysaccharide fractions were 458 g/mL (IOP40), 368 g/mL (IOP60) and 458 g/mL (IOP80) (Table 2), so the order of the scavenging activity on hydroxyl free radicals was IOP60 > IOP80 > IOP40. The scavenging activity of IOP60 was the strongest among them, which was only less than the ascorbic acid (served as positive and its EC50 value was 147 g/mL). Moreover, at 1 mg/mL dose level, the scavenging activities of three crude polysaccharides were all stronger than 90% (Fig. 3e). Therefore, the polysaccharides from I. obliquus had significant effect on scavenging hydroxyl radical. 5. Conclusions In this study, three polysaccharide fractions (IOP40, IOP60 and IOP80) were isolated successfully from the sclerotia of I. obliquus. Not only their antioxidant capacities, but also their physicochemical properties were studied. The physicochemical properties included total sugar content, protein content, monosaccharide composition and percentage, which might be concerned
with the antioxidant capacities. The results of antioxidant assays showed that three crude polysaccharide fractions exhibited increasing antioxidant activities with increasing concentration at the ranges of tested dosages. The order of reducing power was IOP60 > IOP40 > IOP80, which was the same as DPPH-scavenging capacity, as well as the order of H2 O2 -scavenging capacity, and also similar to hydroxyl-scavenging activity. IOP60 and IOP40 exhibited distinctly stronger activities on superoxide-scavenging ability compared with IOP80 (P < 0.05). However, there was no markedly difference (P < 0.05) on superoxide anion-scavenging activity between IOP40 and IOP60. All these findings demonstrated that three fractions extracted from I. obliquus, especially IOP60, could be used as ingredients in healthy and functional food to alleviate the oxidative stress. The structural features of IOP60, especially the relationship between chemical characteristics and antioxidant activities, would be the subject of further research. Acknowledgements We thank Prof. Naizheng Guan, School of Foreign Language Education, Liaocheng University, China, for linguistic revision of the manuscript. The authors are grateful for financial sponsored by Natural Science Foundation of Shandong Province of China (no. ZR2010CL008) and Doctoral Research Startup Foundation of Liaocheng University (no. 31805). References [1] I.H. Park, S.K. Chung, K.B. Lee, Y.C. Yoo, S.K. Kim, G.S. Kim, K.S. Song, Arch. Pharm. Res. 27 (2004) 615–618. [2] A.A. Soares, C.G.M. de Souza, F.M. Daniel, G.P. Ferrari, S.M.G. da Costa, R.M. Peralta, Food Chem. 112 (2009) 775–781. [3] L. Soubra, D. Sarkis, C. Hilan, P. Verger, Regul. Toxicol. Pharm. 47 (2007) 68–77. [4] M.C. Tsai, T.Y. Song, P.H. Shih, G.C. Yen, Food Chem. 104 (2007) 1115–1122. [5] Y.H. Tseng, J.H. Yang, J.L. Mau, Food Chem. 107 (2008) 732–738. [6] X.Q. Xu, J.W. Zhu, Biochem. Eng. J. 58/59 (2011) 103–109. [7] L.S. Ma, H.X. Chen, Y. Zhang, N. Fu, L.L. Zhang, Carbohydr. Polym. 89 (2012) 371–378. [8] X.Q. Xu, Y.D. Wu, H. Chen, Food Chem. 127 (2011) 74–79. [9] H. Chen, M.C. Yan, J.W. Zhu, X.Q. Xu, J. Microbiol. Biotechnol. 38 (2011) 291–298. [10] M. DuBois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal. Chem. 28 (1956) 350–356. [11] J.M. Murphey, S.E. Spayd, J.R. Powers, Am. J. Enol. Viticult. 40 (1989) 199–207. [12] X.J. Du, J.S. Zhang, Y. Yang, L.B. Ye, Q.J. Tang, W. Jia, Y.F. Liu, S. Zhou, R.X. Hao, C.Y. Gong, Y.J. Pan, Carbohydr. Res. 344 (2009) 672–678. [13] Y.F. Wang, Z.W. Yang, X.L. Wei, Int. J. Biol. Macromol. 50 (2012) 558–564. [14] A. Ardestani, R. Yazdanparast, Food Chem. 104 (2007) 21–29. [15] A.G. Guo, Z.Y. Wang, Plant Physiol.Commun. 3 (1989) 54–57. [16] X.J. Du, J.S. Zhang, Y.F. Liu, Q.J. Tang, W. Jia, Y. Yang, Acta Agric. Shanghai 26 (2010) 49–52. [17] M.J. Qin, J. Liu, W.L. Ji, J. Zhao, J.Y. Ding, J. Pharm. Prac. 18 (2000) 304–306. [18] J.J. Qian, S. Ru, F. Zhang, X.J. Tian, L. Li, R.G. Jin, J. Beijing Univ. Chem. Technol. (Nat. Sci.) 5 (2010) 36–41. [19] Y. Lv, X.B. Yang, Y. Zhao, Y. Ruan, Y. Yang, Z.Z. Wang, Food Chem. 112 (2009) 742–746. [20] I.C.F.R. Ferreira, P. Baptista, M. Vilas-Boas, L. Barros, Food Chem. 100 (2007) 1511–1516. [21] W. Brand-Williams, M.E. Cuvelier, C. Berset, LWT-Food Sci. Technol. 28 (1995) 25–30. [22] Y.M.A. Naguib, Anal. Biochem. 284 (2000) 93–96. [23] B. Halliwell, J.M. Gutteridge, Biochem. J. 219 (1984) 1–14. [24] X.P. Chen, Y. Chen, S.B. Li, Y.G. Chen, J.Y. Lan, L.P. Liu, Carbohydr. Polym. 77 (2009) 389–393. [25] K. Tursun, R. Zhan, H. Zhang, S. Ababakri, Lett. Biol. 21 (2010) 406–411.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Chemical analysis and antioxidant activity of polysaccharides extracted from Inonotus obliquus sclerotia XiuJu Du a,∗ , HongMei Mu b , Shuai Zhou c , Yang Zhang a , XiaoLi Zhu a a b c
College of Life Science, Liaocheng University, Liaocheng, Shandong 252059, PR China College of Agricultural Science, Liaocheng University, Liaocheng, Shandong 252059, PR China Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201106, PR China
a r t i c l e
i n f o
Article history: Received 25 August 2013 Received in revised form 8 October 2013 Accepted 13 October 2013 Available online 18 October 2013 Keywords: Inonotus obliquus polysaccharide Free radicals scavenging activity Monosaccharide composition Antioxidant activity
a b s t r a c t Three water-soluble polysaccharide fractions (IOP40, IOP60 and IOP80) were isolated by using different concentrations of alcohol precipitation from Inonotus obliquus sclerotia. Their physicochemical properties, including total sugar content, protein content, monosaccharide composition and percentage were analyzed. And their in vitro antioxidant capacities were investigated in terms of reducing power assay and scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, hydroxyl radicals, superoxide anion radicals and hydrogen peroxide (H2 O2 ). In general, three polysaccharide fractions exhibited increasing antioxidant activity with increasing concentration at the ranges of tested dosage. The orders of reducing power, DPPH-scavenging capacity, H2 O2 -scavenging capacity, and hydroxyl-scavenging activity were all IOP60 > IOP40 > IOP80. These findings demonstrated that three polysaccharide fractions extracted from I. obliquus, especially IOP60, could be employed as natural ingredients in functional food and pharmaceutical industry to alleviate the oxidative stress. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS), generated by triplet state oxygen reacting with other molecules, such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide (H2 O2 ), is the major sources of primary catalysts that initiate oxidation in vivo and in vitro [1]. ROS is also one of the main factors which cause the oxidation of polyunsaturated fatty acid in the surface of biomembrane, and it has been associated with the beginning of many diseases and degenerative processes in aging [2]. A growing number of reports indicated that the side effects of synthetic antioxidants might be responsible for some observed carcinogenesis and liver damage [3]. Thus, it was essential to discover new natural antioxidants that could protect the human body from free radicals without side effects. Recently, it had been reported that polysaccharides from fungi had exhibited strong antioxidant effects that related to their health-protecting functions [4,5]. Inonotus obliquus, a kind of traditional fungi belonging to the family of Hymenochaetaceae, Basidiomycetes, has been used as a folk remedy in Asian countries, such as China, Japan, Korea and Russia for five hundred years [6]. More and more studies have shown that polysaccharides from I. obliquus possessed clear antioxidant
activities [7–9]. However, up to now, no detailed investigation has been carried out on physicochemical characteristics and antioxidative capacity of different polysaccharide fractions isolated by precipitating with different alcohol concentration from the sclerotia of I. obliquus. In the present study, the purpose is to focus on the isolation and properties of three different polysaccharide fractions from the sclerotia of I. obliquus, and explored their antioxidant capabilities for seeking new natural functional ingredients used in food and pharmaceutical industry to alleviate the oxidative stress. Therefore, three water-soluble polysaccharide fractions (IOP40, IOP60 and IOP80) were obtained and their physicochemical properties, including total sugar content, protein content, monosaccharide composition and percentage were analyzed. And their in vitro antioxidant capacities were assayed in terms of reducing power and scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals, hydroxyl radicals, superoxide anion radicals and hydrogen peroxide (H2 O2 ).
2. Materials and methods 2.1. Materials and reagents
∗ Corresponding author at: College of Life Science, Liaocheng University, Hunan Road 1, Liaocheng 252059, PR China. Tel.: +86 635 8538917; fax: +86 635 8239908. E-mail addresses: [email protected], [email protected] (X. Du). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.016
The sclerotia of I. obliquus was purchased from the Northeast Natural Products Trading Company (Haerbin, China). The monosaccharide standards, d-Gal, d-Glc, d-Ara, l-Fuc, l-Rha, d-Fru, d-Man,
692
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
Small pieces of I. obliquus sclerotia Extracted twice with 10 volumes 95% EtoH, 24 h
Solid residues dried in air Extracted thrice with 10-fold volume (distilled water, 2 h), then centrifuge(1,776 g,10 min)
Aqueous extracts Concentrated 10-fold with vacuum rotary evaporator
Concentrated aqueous extracts Precipitated with 95% ethanol to 40% of final alcohol content, centrifugation(1,776 g,10 min)
Precipitate
Supernatant
Dialysis and
Precipitated with 95% ethanol to 60% of final
freeze-drying
alcohol content, centrifugation(1,776 g,10 min)
IOP40 Precipitate
Supernatant Precipitated with 95% ethanol to
Dialysis and
80% of final alcohol content
freeze-drying
IOP60
Precipitate Dialysis and freeze-drying
IOP80 Fig. 1. Scheme of extraction and fractionation of different polysaccharide from I. obliquus.
d-Xyl, d-GlcA and d-GalA were bought from Sigma–Aldrich (St. Louis, MO, USA). All other reagents were of analytical reagent grade and from Chinese sources. All aqueous solutions were prepared by using newly double-distilled water. 2.2. Extraction and fractionation of polysaccharide Three polysaccharide fractions IOP40, IOP60 and IOP80 were obtained according to the flow diagram shown in Fig. 1. Briefly, dried sclerotia of I. obliquus powder (1.5 kg) was immersed in 15 L of ethanol (95%) for 24 h, and the solid residue was collected by filtration and the procedure was repeated two times. Then the air-dried solid residue was extracted for three times with distilled water. The combined supernatant was concentrated to one-tenth
of the original volume. 95% of ethanol was added slowly to the concentrated supernatant to 40% of final alcohol content, then the mixture was set at 4 ◦ C for 24 h. After centrifugation, dialysis and freeze-drying, the precipitate was obtained and termed IOP40. Similarly, 95% of ethanol was added to the concentrated supernatant to the final alcohol content of 60% and 80%, accordingly the precipitate IOP60 and IOP80 were prepared. 2.3. General methods Total sugar contents were determined by the phenol-sulfuric acid method using d-glucose as standard [10]. The protein contents were measured by the method of Bradford using vine serum albumin as the standard [11]. Neutral sugar content and
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
uronic acid content were determined by using high-performance anion-exchange pulsed-amperometric detection chromatography (HPAEC-PAD) [12]. 2.4. Monosaccharide composition analysis Monosaccharide composition and component percentage were determined using HPAEC-PAD as reported by Du et al. [12] with minor modifications. Briefly, sample (10 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110 ◦ C for 5 h. A Dionex LC30 was equipped with a CarboPacTM PA20 column (3 mm × 150 mm), and the monosaccharide was monitored using a pulsed amperometric detector (Dionex). The column was eluted with 2 mM NaOH (0.45 mL/min). d-Gal, d-Glc, d-Ara, l-Fuc, l-Rha, d-Fru, d-Man, dXyl, d-Ribose, d-GlcA and d-GalA were employed as standards. 3. Antioxidant activity assays 3.1. Reducing power assay The reducing power was determined according to the method described by Wang et al. [13]. In brief, 1 mL samples (62.5–1000 g/mL), 2 mL phosphate buffer (0.2 M, pH 6.6) and 2 mL 1% (w/v) K3 Fe(CN)6 were incubated at 50 ◦ C for 20 min. After cooling rapidly, the reaction was terminated by adding 2 mL of trichloroacetic acid (TCA, 10%, w/v) and centrifuged at 1776 × g for 10 min. The supernatant (2.5 mL) mixed with 2.5 mL of distilled water and 1 mL of 0.1% (w/v) FeCl3 , and set for 10 min. The absorbance of the reaction mixture was read at 700 nm against a blank. Ascorbic acid was used as positive control. 3.2. DPPH-scavenging activity assay The DPPH radical scavenging activity of three polysaccharide fractions was measured according to the method by Ardestani et al. [14]. The sample solution (0.1 mL) with variable concentrations (62.5–1000 g/mL) was added to tube containing 2.9 mL of DPPH solution (0.1 mM in ethanol). The mixture was blended by vortex and then set for 30 min at 37 ◦ C in the dark. Then the absorbance was measured at 517 nm. In this work, distilled water and ascorbic acid served as the negative and positive controls respectively. The DPPH radical scavenging activities were calculated using the following formula: Scavenging rate (%) = (1 − A1 /A0 ) × 100, where A0 is the absorbance value of the control, A1 is the absorbance values of samples and ascorbic acid. 3.3. Superoxide anion-scavenging activity assay The superoxide anion radical assays of samples were measured according to the Pyrogallol-Chemiluminescence method by Guo et al. and Wang et al. [15,16]. Specific methods were as follows: 2 L of different test agents and 8 L of pyrogallic acid (0.625 mM) were added to each well of a 96-well microplate. In this work, PBS and ascorbic acid served as the negative and positive controls respectively. 150 L of Luminol-CBS (sodium carbonate buffer solution) mixture (pH 10.2) was pumped into each well of a 96-well microplate through external. Signal of each well was collected in every 0.6 s and kept for 30 s. Each sample was repeated 3 times. Luminescence values were measured using Clarity (BIO-TEK Company) auto-reader. The scavenging effect for superoxide anion radical was calculated according to the following formula: Scavenging rate (%) = (A0 − A1 )/A0 × 100, in which A0 was the luminescence value of the negative control without tested sample. A1 was the luminescence values of tested samples.
693
3.4. H2 O2 -scavenging activity assay The H2 O2 -scavenging activities were carried out by the method of Chemiluminescence [16,17]. 10 L of 1% H2 O2 and 40 L of different tested samples were added to each well of a 96-well microplate. Double distilled water and ascorbic acid were used as the negative and positive controls separately. The scavenging rates for H2 O2 were performed according to the method in superoxide anion-scavenging activity assay (Section 3.3). 3.5. Hydroxyl radical-scavenging assay The hydroxyl radical assay of sample was measured according to the salicylic acid method described by Qian et al. [18] with some modifications. Briefly, 8 mL of reaction mixture, containing 2 mL of tested samples with different concentrations [0 (control), 62.5, 125, 250, 500 and 1000 g/mL], 2 mL of FeSO4 aqueous solution (4 mM), 2 mL of salicylic acid ethanol solution (4 mM) and 2 mL of H2 O2 aqueous solution (4 mM), was shaken well, then incubated (37 ◦ C, 30 min), centrifuged (1776 × g, 10 min) and lastly the supernatant mixture was obtained. The absorbance of the reaction mixture was read at 510 nm. The Hydroxyl radical-scavenging rate (r) was calculated by the following formula: r(%) = (A0 − (AX − AX0 ))/A0 × 100, where A0 is the absorbance of blank control only, AX is the absorbance of sample and positive (ascorbic acid), and AX0 is the absorbance of sample and positive only without H2 O2 aqueous solution (replaced by 2 mL of distilled water). 3.6. Statistical analysis The data obtained were expressed as mean ± SD of three determinations and analyzed statistically by ANOVA method. Significance of any differences between groups was evaluated using the Student’s t-test. All computations were done by employing statistical software (STST). 4. Results and discussion 4.1. Characterization and composition of the three polysaccharide fractions Three water-soluble polysaccharide fractions IOP40, IOP60 and IOP80 were obtained successfully from I. obliquus sclerotia by boiling water and ethanol precipitation. The extraction yields of three fractions (IOP40, IOP60 and IOP80) were 2.2%, 11.6% and 0.84%, respectively. The properties of IOP40, IOP60 and IOP80, including total sugar content, protein content, neutral sugar content and uronic acid content, and monosaccharide components and percentage, were summarized and shown in Table 1. As we all know, it is important to know the monosaccharide composition and molar ratio of polysaccharides, which contribute to their bioactivity [19]. In this study, HPAEC-PAD was employed to identify and quantify the major compositional monosaccharide presented in IOP40, IOP60 and IOP80. As shown in Fig. 2A, 11 kinds of standard monosaccharide were baseline separated rapidly within 39 min. By matching their retention times with those of monosaccharide standards under the same analytical conditions, eleven peaks were identified in the order of l-fucose (l-Fuc), l-rhamnose (l-Rha), l-arabinose (l-Ara), d-galactose (dGal), d-glucose (d-Glc), d-xylose (d-Xyl), d-mannose (d-Man), d-fructose(d-Fru), d-ribose (d-Rib), d-galacturonic acid (d-GalA) and d-glucuronic acid (d-GluA). The chromatograms of the compositional monosaccharide of IOP40, IOP60 and IOP80 were also given in Fig. 2B–D, respectively. The results indicated that three polysaccharides all appeared to be the typical acidic hetero-polysaccharide.
694
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
Table 1 Monosaccharide components and properties of various polysaccharides from I. obliquus. Samples
IOP40
IOP60
IOP80
Yields (a W%) Total sugar content (W%) Neutral saccharide (W%) Uronic acid (W%) Protein content (W%)
2.2 35.1 30.1 5.0 2.4
11.6 24.6 22.4 2.2 3.2
0.8 23.2 21.7 1.5 4.6
Monosaccharide components (M%b ) l-Fucose (M%) l-Rhamnose (M%) d-Arabinose (M%) d-Galactose (M%) d-Glucose (M%) d-Xylose (M%) d-Mannose (M%) d-GalA (M%) d-GluA (M%)
3.3 4.4 4.2 14.5 40.0 9.6 9.8 4.6 9.7
c
nd 11.0 7.5 8.5 31.3 25.3 9.7 3.9 2.8
a b c
nd 9.7 5.6 12.6 32.2 22.9 8.0 4.4 4.7
W% is expressed as the weight percentage. M% is expressed as the molar percentage. nd: data were not detected.
As shown in Fig. 2A and B, IOP40 was composed of l-Fuc, l-Rha, l-Ara, d-Gal, d-Glc, d-Xyl, d-Man, d-GalA and d-GlcA in the molar ratio of 5.1:6.8:6.5:22.5:62.2:14.9:15.2:7.1:15.1. Similarly, both IOP60 and IOP80 consisted of l-Rha, l-Ara, d-Gal, d-Glc, d-Xyl, d-Man, d-GalA and d-GlcA, and the molar ratios of them were 5.5:3.2:7.1:18.1:12.9:4.5:2.5:2.6 and 5.0:3.4:3.9:14.2:11.4:4.4:1.8:1.3, respectively (shown in Fig. 2C and D). The monosaccharide percentage of IOP40, IOP60 and IOP80 were summarized in Table 1. However, the results also showed that one peak was associated with the retention time at 5.14 min in Fig. 2B and the some peaks were associated with the retention time at 9.62 min in Fig. 2C and D. These findings might imply new compounds might be contained in IOP40, IOP60 and IOP80, which would be the continuing work. 4.2. Reducing power assay The reducing properties were generally associated with the presence of electron-donating groups or hydrogen atoms, which could react with free radicals to stabilize and block radical chain reactions [20]. Accordingly, reducing power assay was an important method to test drugs of antioxidant activity. The data presented showed that increased absorbance of the reaction mixture indicated stronger reducing power. As shown in Fig. 3a, at the concentration range of 62.5–1000 g/mL, the reducing power of various polysaccharide fractions increased with increasing dosage, although all fractions showed lower activities than that of ascorbic acid at tested concentration. The RP0.5AU values (defined as the effective concentration at which the absorbance was 0.5 for reducing power) of ascorbic acid, IOP40, IOP60 and IOP80 were 58 g/mL, 354 g/mL, 154 g/mL and 411 g/mL, respectively. So the order of various polysaccharide fraction on reducing power was IOP60 > IOP40 > IOP80. 4.3. DPPH-scavenging activity assay The DPPH radical is a stable lipid free radical with absorption peak at 517 nm. The DPPH can capture either electrons or hydrogen atoms from antioxidant and make pair with its free radicals. Accordingly, the original purple-colored DPPH is partially disappeared and the stable yellow-colored diphenylpicrylhydrazine (DPPH-H) is simultaneously formed. Therefore, DPPH-scavenging activity assay is a useful method for determination free radical scavenging activities of antioxidant materials. The greater scavenging rate on DPPH radicals shows that antioxidant ability of materials is stronger [21]. As shown in Fig. 3b, the DPPH radical-scavenging rates of three polysaccharide fractions rose with increasing concentration at the dosage range of 62.5–1000 g/mL. The EC50 values (defined as the effective concentration at which the antioxidant activity was 50%) of IOP40, IOP60, IOP80 and ascorbic acid were 880 g/mL, 697 g/mL, 1190 g/mL and 107 g/mL, respectively (Table 2). Therefore, the DPPH radicals-scavenging effects exhibited the following order: IOP60 > IOP40 > IOP80, which was the same as the reducing power. 4.4. Superoxide anion-scavenging activity assay
Fig. 2. The spectrum of HPAEC-PAD of 11 standard monosaccharides (A) and component monosaccharides released from IOP40 (B), IOP60 (C) and IOP80 (D). Peaks: (1) l-fucose, (2) l-rhamnose, (3) l-arabinose, (4) d-galactose, (5) d-glucose, (6) d-xylose, (7) d-mannose, (8) d-fructose, (9) d-ribose, (10) d-GalA and (11) d-gluA.
Superoxide is generated in biological systems during the normal catalytic function [22]. Superoxide anion radical is one of harmful species to cellular component as a precursor of more reactive oxygen species (ROS). It can result in the formation of H2 O2 in dismulation reaction [23]. Therefore, superoxide anionscavenging activity is a significant part of the antioxidant activities. As shown in Fig. 3c, the superoxide radical scavenging effects of three polysaccharide fractions increased with increasing concentration at the tested dosage. The EC50 values of IOP40, IOP60
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
695
Fig. 3. Antioxidant activity of three polysaccharide fractions from I.·obliquus: (a) reducing power, (b) scavenging activities to DPPH-radical, (c) scavenging activities to superoxide anion, (d) scavenging activities to H2 O2 , and (e) scavenging activities to hydroxyl radical; data are presented as the mean values (n = 3).
and IOP80 were 425 g/mL, 473 g/mL, 2290 g/mL, respectively, which was higher than the positive control ascorbic acid (48 g/mL) (see Fig. 3c and Table 2). Thus, the order of three fractions on superoxide anion-scavenging activity was IOP40 > IOP60 > IOP80, which was different from that of the reducing power and the DPPH-scavenging activity. However, IOP40 and IOP60 exhibited remarkably stronger antioxidant capacities compared with IOP80 in the tested concentrations (P < 0.05). There was no markedly difference on superoxide anion-scavenging activity between IOP40 and IOP60 (P < 0.05).
4.5. H2 O2 -scavenging activity assay H2 O2 is one of representative ROS generated in the body [24]. As shown in the Fig. 3d, the H2 O2 -scavenging effects of three polysaccharide fractions increased with the increase of concentration. The polysaccharide fraction IOP60 exhibited strongest H2 O2 -scavenging capacity among them. The EC50 values of IOP40, IOP60 and IOP80 were 580 g/mL, 460 g/mL, 5000 g/mL respectively, which all showed higher capacities compared with the positive control ascorbic acid (226 g/mL) (Table 2). The order of
696
X. Du et al. / International Journal of Biological Macromolecules 62 (2013) 691–696
Table 2 EC50 values of various polysaccharide fraction from I. obliquus in antioxidant properties. Antioxidant activity
IOP40
IOP60
IOP80
Ascorbic acid
a
DPPH-scavenging activity Superoxide anion-scavenging activity H2 O2 -scavenging activity Hydroxyl-scavenging activity
EC50 value (g/mL) 880 697 1190 425 473 2290
107 48
580 458
460 368
5000 440
282 147
154
411
58
b
Reducing power
RP0.5AU 354
a
EC50 value: the effective concentration at which the antioxidant activity was 50%. b RP0.5AU value: the effective concentration at which the absorbance was 0.5 for reducing power.
H2 O2 -scavenging activity was IOP60 > IOP40 > IOP80, which was the same as the sequence of reducing power, as well as that of the DPPH-scavenging activity. 4.6. Hydroxyl-scavenging activity assay Hydroxyl free radical, one of the liveliest substances, may directly or indirectly cause tissue damage, induce many human diseases including cancer and promote aging [25]. Hydroxyl free radicals were generated when H2 O2 and Fe2+ were mixed together in salicylic acid method, which could be expressed as the reaction equation as follows: H2 O2 + Fe2+ → OH + H2 O + Fe3+ . Hydroxyl free radicals could be captured by salicylic acid and produce colored substances, which had maximum absorption at 510 nm. The absorbance value represented the content of hydroxyl free radicals. The greater scavenging rate implies that the antioxidant ability of materials is stronger. As shown in Fig. 3e, three polysaccharide fractions all have the abilities to scavenge hydroxyl free radicals at concentration from 62.5 g/mL to 1000 g/mL, and the scavenging abilities increased with the concentration increasing. The EC50 values of three polysaccharide fractions were 458 g/mL (IOP40), 368 g/mL (IOP60) and 458 g/mL (IOP80) (Table 2), so the order of the scavenging activity on hydroxyl free radicals was IOP60 > IOP80 > IOP40. The scavenging activity of IOP60 was the strongest among them, which was only less than the ascorbic acid (served as positive and its EC50 value was 147 g/mL). Moreover, at 1 mg/mL dose level, the scavenging activities of three crude polysaccharides were all stronger than 90% (Fig. 3e). Therefore, the polysaccharides from I. obliquus had significant effect on scavenging hydroxyl radical. 5. Conclusions In this study, three polysaccharide fractions (IOP40, IOP60 and IOP80) were isolated successfully from the sclerotia of I. obliquus. Not only their antioxidant capacities, but also their physicochemical properties were studied. The physicochemical properties included total sugar content, protein content, monosaccharide composition and percentage, which might be concerned
with the antioxidant capacities. The results of antioxidant assays showed that three crude polysaccharide fractions exhibited increasing antioxidant activities with increasing concentration at the ranges of tested dosages. The order of reducing power was IOP60 > IOP40 > IOP80, which was the same as DPPH-scavenging capacity, as well as the order of H2 O2 -scavenging capacity, and also similar to hydroxyl-scavenging activity. IOP60 and IOP40 exhibited distinctly stronger activities on superoxide-scavenging ability compared with IOP80 (P < 0.05). However, there was no markedly difference (P < 0.05) on superoxide anion-scavenging activity between IOP40 and IOP60. All these findings demonstrated that three fractions extracted from I. obliquus, especially IOP60, could be used as ingredients in healthy and functional food to alleviate the oxidative stress. The structural features of IOP60, especially the relationship between chemical characteristics and antioxidant activities, would be the subject of further research. Acknowledgements We thank Prof. Naizheng Guan, School of Foreign Language Education, Liaocheng University, China, for linguistic revision of the manuscript. The authors are grateful for financial sponsored by Natural Science Foundation of Shandong Province of China (no. ZR2010CL008) and Doctoral Research Startup Foundation of Liaocheng University (no. 31805). References [1] I.H. Park, S.K. Chung, K.B. Lee, Y.C. Yoo, S.K. Kim, G.S. Kim, K.S. Song, Arch. Pharm. Res. 27 (2004) 615–618. [2] A.A. Soares, C.G.M. de Souza, F.M. Daniel, G.P. Ferrari, S.M.G. da Costa, R.M. Peralta, Food Chem. 112 (2009) 775–781. [3] L. Soubra, D. Sarkis, C. Hilan, P. Verger, Regul. Toxicol. Pharm. 47 (2007) 68–77. [4] M.C. Tsai, T.Y. Song, P.H. Shih, G.C. Yen, Food Chem. 104 (2007) 1115–1122. [5] Y.H. Tseng, J.H. Yang, J.L. Mau, Food Chem. 107 (2008) 732–738. [6] X.Q. Xu, J.W. Zhu, Biochem. Eng. J. 58/59 (2011) 103–109. [7] L.S. Ma, H.X. Chen, Y. Zhang, N. Fu, L.L. Zhang, Carbohydr. Polym. 89 (2012) 371–378. [8] X.Q. Xu, Y.D. Wu, H. Chen, Food Chem. 127 (2011) 74–79. [9] H. Chen, M.C. Yan, J.W. Zhu, X.Q. Xu, J. Microbiol. Biotechnol. 38 (2011) 291–298. [10] M. DuBois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal. Chem. 28 (1956) 350–356. [11] J.M. Murphey, S.E. Spayd, J.R. Powers, Am. J. Enol. Viticult. 40 (1989) 199–207. [12] X.J. Du, J.S. Zhang, Y. Yang, L.B. Ye, Q.J. Tang, W. Jia, Y.F. Liu, S. Zhou, R.X. Hao, C.Y. Gong, Y.J. Pan, Carbohydr. Res. 344 (2009) 672–678. [13] Y.F. Wang, Z.W. Yang, X.L. Wei, Int. J. Biol. Macromol. 50 (2012) 558–564. [14] A. Ardestani, R. Yazdanparast, Food Chem. 104 (2007) 21–29. [15] A.G. Guo, Z.Y. Wang, Plant Physiol.Commun. 3 (1989) 54–57. [16] X.J. Du, J.S. Zhang, Y.F. Liu, Q.J. Tang, W. Jia, Y. Yang, Acta Agric. Shanghai 26 (2010) 49–52. [17] M.J. Qin, J. Liu, W.L. Ji, J. Zhao, J.Y. Ding, J. Pharm. Prac. 18 (2000) 304–306. [18] J.J. Qian, S. Ru, F. Zhang, X.J. Tian, L. Li, R.G. Jin, J. Beijing Univ. Chem. Technol. (Nat. Sci.) 5 (2010) 36–41. [19] Y. Lv, X.B. Yang, Y. Zhao, Y. Ruan, Y. Yang, Z.Z. Wang, Food Chem. 112 (2009) 742–746. [20] I.C.F.R. Ferreira, P. Baptista, M. Vilas-Boas, L. Barros, Food Chem. 100 (2007) 1511–1516. [21] W. Brand-Williams, M.E. Cuvelier, C. Berset, LWT-Food Sci. Technol. 28 (1995) 25–30. [22] Y.M.A. Naguib, Anal. Biochem. 284 (2000) 93–96. [23] B. Halliwell, J.M. Gutteridge, Biochem. J. 219 (1984) 1–14. [24] X.P. Chen, Y. Chen, S.B. Li, Y.G. Chen, J.Y. Lan, L.P. Liu, Carbohydr. Polym. 77 (2009) 389–393. [25] K. Tursun, R. Zhan, H. Zhang, S. Ababakri, Lett. Biol. 21 (2010) 406–411.
