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Chemical properties and antioxidant activity of exopolysaccharides fractions from mycelial culture of Inonotus obliquus in a ground corn stover medium
Food Chemistry 134 (2012) 1899–1905
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Chemical properties and antioxidant activity of exopolysaccharides fractions from mycelial culture of Inonotus obliquus in a ground corn stover medium Yuling Xiang, Xiangqun Xu ⇑, Juan Li Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, China
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Article history: Received 18 October 2011 Received in revised form 12 March 2012 Accepted 24 March 2012 Available online 13 April 2012 Keywords: Inonotus obliquus Exopolysaccharides Antioxidant activity Submerged fermentation Lignocellulose decomposition Composition Molecular weight
a b s t r a c t The medicinal mushroom Inonotus obliquus has been a folk remedy for a long time in East-European and Asian countries. We first reported the enhancement in production and antioxidant activity of exopolysaccharides by I. obliquus culture under lignocellulose decomposition. In this study, the two different sources of exopolysaccharides from the control medium and the lignocellulose (corn stover) containing medium by I. obliquus in submerged fermentation were fractionated and purified by chromatography. The exopolysaccharides from the corn stover-containing medium presented significantly stronger hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity than the control. Three fractions from the control medium and the corn stover-containing medium were isolated respectively. The fraction of DEPL3 from the corn stover-containing medium with the highest protein content (38.3%), mannose content (49.6%), and the lowest molecular weight (29 kDa) had the highest antioxidant activity with the lowest IC50 values. In conclusion, lignocellulose decomposition changed the chemical characterisation and significantly enhanced the antioxidant activity of exopolysaccharide fractions. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Mushrooms such as Ganoderma lucidum (Ling Zhi or Reishi), Lentinus edodes (Shiitake), Inonotus obliquus (Pers.: Fr.) Pilát (Chaga) and many others have been collected and used for hundreds of years in China, Japan, Korea, and eastern Russia. Mushrooms have also played an important role in the treatment of ailments affecting rural populations of eastern European countries (Wasser, 2011). I. obliquus is a rare edible and medicinal mushroom. It belongs to the family Hymenochaetaceae, Basidiomycetes. The pharmacological importance of the mushrooms is very high in the Far East as a traditional medicine for treating cancer, heart, liver, and stomach diseases, and tuberculosis (Saar, 1991). Chemical investigations show that I. obliquus produces a diverse range of bioactive metabolites, including lanostane-type triterpenoids, steroids, phenolic compounds and polysaccharides (Zheng et al., 2010). For almost 40 years, medicinal mushrooms have been intensively investigated for medicinal effects in vivo and in vitro model systems, and many new antitumour and immunomodulating polysaccharides have been identified and put into practical use (Ikekawa, 2001; Mizuno, 1999). In the last few years an increasing number of studies have been published concerning the biological
⇑ Corresponding author. Address: Department of Chemistry, School of Science, Zhejiang Sci-Tech University, Hangzhou 310018, China. Tel.: +86 571 86843228; fax: +86 571 87055836. E-mail address: [email protected] (X. Xu). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.03.121
activities of polysaccharides from the I. obliquus fruit bodies, cultured mycelium, and culture broth such as antitumour, immunomodulating, antioxidant, radical scavenging, and anti-caducity (Kim et al., 2005; Mizuno et al., 1999; Rhee, Cho, Kim, Cha, & Park, 2008; Shamtsyan et al., 2004; Tseng, Yang, & Mau, 2008; Xu, Wu, & Chen, 2011). The limited natural resource and difficult artificial cultivation of I. obliquus to obtain fruit body make it impossible to obtain large quantity of bioactive molecules. Submerged fermentation is an effective process for the production of mycelial biomass and bioactive compounds, especially polysaccharides (Pokhrel & Ohga, 2007; Zhang & Cheung, 2011). Recently, we reported a submerged fermentation optimisation for bioactive polysaccharide production from I. obliquus using the response surface methodology (RSM) method combined with hydroxyl radical-scavenging activity screening (Chen, Xu, & Zhu, 2010). With the RSM optimised medium, the hydroxyl radical-scavenging activity per unit of the exopolysaccharides (EP) was significantly enhanced compared to that from either the basal fermentation medium or the single variable optimisation of fermentation medium (Chen et al., 2010; Xu et al., 2011). In our previous study, we first found that hydroxyl radicals played the same important role when I. obliquus in submerged fermentation decomposed the lignocellulose in corn stover added in a liquid medium (Chen, Yan, Zhu, & Xu, 2011) as in nature where I. obliquus degraded wood polymers as a kind of white rot fungi (Gao & Gu, 2007). Correspondingly, the production and antioxidant
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activity of the EP as a defense response by I. obliquus were further enhanced in a corn stover-containing submerged fermentation with lignocellulose decomposition (Chen et al., 2011). Polysaccharides with biological activity differ greatly in their chemical compositions, configurations, and physical properties. Although it is difficult to correlate polysaccharide structure with antioxidant activity, immuno-stimulating effect, and anti-tumour activity, some relationships can be inferred. In medicinal mushrooms, the biological activities of polysaccharides are closely related to molecular mass, sugar content, monosaccharide compositions. The purified endo-polysaccharide from the mycelia of I. obliquus that was an a-linked fucoglucomannan composed primarily of mannose with small amounts of glucose, fucose, and glucosamine could inhibit tumour growth in vivo (Kim et al., 2006). The water-soluble polysaccharide from the sclerotia of I. obliquus was a heteropolysaccharide of xylogalactoglucan composed of glucose, mannose, galactose, xylose, arabinose, and fucose (Mizuno et al., 1999). Although the corn stover-containing submerged fermentation with lignocellulose was proven to enhance the production and antioxidant activity of the EP in our previous studies (Chen et al., 2011), there have been no reports regarding a relationship of the sugar content, monosaccharide compositions, and molecular mass with biological activity in exopolysaccharides from the mycelia of I. obliquus to the culture process in the two mediums. The aim of this work was to examine the characterisation and antioxidant effects of the two kinds of purified EP from the control medium optimised with the RSM (Chen et al., 2010) and the medium supplemented with ground corn stover (Chen et al., 2011). The two different sources of EP obtained from I. obliquus mycelial liquid culture were fractionated and purified by chromatography on DEAE-cellulose and Sephadex G-200 columns. The chemical properties and antioxidant activity of the EP fractions were compared between different culture processes.
2. Materials and methods 2.1. Cultivation method I. obliquus (CBS314.39) was purchased from the Centraal Bureau voor Schimmelcultuur, Utrecht, Netherlands. It was maintained on malt extract agar slants containing (% w/v) malt extract 3, peptone 0.3 and agar 1.5 at pH 5.6 ± 0.2. The slants were cultivated at 25 °C for about 2 weeks. When the mycelia overgrew the slants, they were stored at 4 °C and sub-cultured every 3 months. The seed culture was prepared by incubating mycelia on a malt extract agar slant into a 250-mL Erlenmeyer flask with 100 mL medium. One cm2 of malt extract agar with mycelia was chipped off and then transferred into the Erlenmeyer flask. The medium contained (% w/v) glucose 2, peptone 0.3, yeast extract 0.2, KH2PO4 0.1, MgSO4 0.15, and CaCl2 0.01. Cultures were incubated for 4–5 days in a rotary shaker (150 rpm) at 28 °C. The harvested seed culture was added into 250-mL Erlenmeyer flasks containing 100-mL control medium or corn stover-containing medium and incubated at a rate of 10% (v/v) and at 28 °C in a rotary shaker at 150 rpm. The culture was incubated for 9 days to produce mycelia (Xu et al., 2011). The mycelia and the culture broth were harvested on day 9. The two kinds of medium used in this study were as below. Control medium (% w/v): corn flour 5.3, peptone 0.3, KH2PO4 0.1, ZnSO42H2O 0.001, K2HPO4 0.04, FeSO47H2O 0.005, MgSO47H2O 0.05, CuSO45H2O 0.002, CoCl2 0.001, MnSO4H2O 0.008. pH = 6.0. The medium was optimised by the response surface methodology (RSM) based on the basal medium in our previous work (Chen et al., 2010).
Corn stover-containing medium (% w/v): corn flour 3.5, ground corn stover 3%, and all of the other components were the same as the control medium. The content of corn flour in the control medium was lower because corn stover provided reducing sugars when the reducing sugars from the corn flour hydrolysis exhausted with the lignocellulose decompositions by I. obliquus (Chen et al., 2011). 2.2. Extraction of EP The I. obliquus mycelial cells were removed from culture broths using a filter paper. The filtrate was mixed with four volumes of chilled 95% (v/v) ethanol, then stirred vigorously and left at 4 °C overnight. The precipitate was collected, then centrifuged at 6500g for 6 min and lyophilised. The lyophilised samples were termed as EPC from the control medium and EPL from the corn stover-containing medium. The Sevage method was employed to remove protein after neutrase treatment with some modification (Yan, Han, & Jiang, 2004). The EPC and EPL aqueous solution were further mixed with the Sevage reagent [chloroform: butanol = 5:1 (v/v)] at the proportion 5:1 (v/v) and oscillated intensively. The supernatant was concentrated and the aqueous solution was dialysed against distilled water for 48 h then lyophilised. The deproteinated EPC from the control medium and EPL from the corn stovercontaining medium were named as DEPC and DEPL (Xu et al., 2011). 2.3. Fractionation and purification of EP DEPC and DEPL (200 mg in 10 ml buffer) were subjected to a DEAE-cellulose anion-exchange column (50 2.4 cm, i.d.), which was equilibrated with 0.01 M sodium phosphate buffer (PBS, pH 7.8) before. Then the fractions were eluted with PBS for 200 ml, 0.1 M NaCl–PBS for 150 ml, 0.2 M NaCl–PBS for 150 ml, 0.3 M NaCl–PBS for 200 ml and at a flow rate of 72 ml/h, respectively. Four fractions from both DEPC and DEPL were obtained. The first three fractions of DEPC1, DEPC2, DEPC3 and DEPL1, DEPL2, DEPL3 were concentrated, dialysed against distilled water for 48 h and then lyophilised. To make sure a complete isolation of each fraction for molecular weight determination, the six fractions were fractionated and purified further by a gel permeation chromatography technique over a Sephadex G-200 (50 2.4 cm) and eluted at 0.3 ml/min with 0.1 M NaCl. A single fraction from each sample was collected and coded as PDEPC1, PDEPC2, PDEPC3 and PDEPL1, PDEPL2, PDEPL3 (purified fractions), respectively (Asker, Ahmed, & Ramadan, 2009). 2.4. Compositional analysis of EP The carbohydrate and protein content of all the EP samples obtained above were analysed by the phenol–sulphuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) and the Bradford’s method with bovine serum albumin as a standard (Bradford, 1976). Monosaccharides composition was analysed by gas chromatography (Varian Inc., Palo ALto, CA, USA) with a Varian CP-Sil 5 CB capillary chromatography column (25 m 0.53 mm, 0.25 lm film thickness) and a flame-ionisation detector for analysis (Chen et al., 2011; Benhura & Chidewe, 2002). 2.5. Determination of molecular weight Molecular weights of the EP fractions were determined by size exclusion chromatography with laser light scattering (SEC-LLS). The EP fractions (1 mg) were dissolved in 0.2 M NaCl (1 ml), and optical clarification were achieved by filtration through a sand filter followed by a 0.2 lm pore-size filter (Whatman, England) into
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the scattering cell (Fused Silica). Molecular mass parameters of the EP fractions were measured with a eight-angle laser light scattering instrument equipped with a He–Ne laser (k = 658 nm, HELEOS 8, Wyatt Technology Co., USA) at 25 °C. The value of 0.125 ml/g was used as the specific refractive index increment of the EP fractions in 0.2 M NaCl (Tamura, Wada, & Isogai, 2009). Data acquisition and processing was performed with the ASTRA software (Wyatt Technologies). 2.6. Scavenging effect on hydroxyl radicals Hydroxyl radical-scavenging activity of each extract solution was measured according to the salicylic method with some modifications (Smirnoff & Cumbes, 1989). Reaction mixtures in a final volume of 4 ml contained: 1 ml H2O2 (8.8 mM/l), 1 ml FeSO4 (9 mM/l), 1 ml salicylic (9 mM/l), and 1 ml sample solution in a concentration gradient. The H2O2 was added to the mixture and the reaction was started up at last. The reaction mixture was incubated at 37 °C for 60 min and then centrifuged at 10,000g for 8 min. The absorbance (A) of the reaction solutions at 510 nm was measured. The hydroxyl radical-scavenging ratio was calculated by the following formula:
Scavenging rate ð%Þ ¼ ½A0 ðAx Ax0 Þ 100=A0
ð1Þ
A control contained all the reaction reagents except the samples was prepared and measured as A0, Ax was the result of samples and Ax0 is the absorbance for background, i.e., the reaction mixture without H2O2. 2.7. Scavenging effect on DPPH radicals A 2.4 ml extract solution was mixed with 0.8 ml of 0.4 mM 2,2diphenyl-1-picrylhydrazyl (DPPH) in methanol, to produce a final DPPH concentration of 0.1 mM. The mixture was vigorously shaken and left to stand for 30 min in the dark, and its absorbance was measured at 517 nm as Ax. A control sample containing the same amount of methanol and DPPH radicals was measured as A0. The absorbance of samples containing the same amount of methanol and the extract solution was recorded as C (Yang et al., 2007). The DPPH radical-scavenging ratio was calculated by the following formula:
Scavenging rate ð%Þ ¼ ½A0 ðAx CÞ 100=A0
ð2Þ
2.8. Statistical analysis Experimental results recorded were the means ± standard deviation (SD) of triple determinations. The obtained data were subjected to One-way ANOVA and the differences between means were at the 5% probability level using Duncan’s new multiple range tests. The IC50 values were calculated by using median-effect analysis and origin 7.5 software (OriginLab Corp., Northampton, MA). 3. Result 3.1. Isolation, purification and composition Fig. 1 shows the elution curves of DEPC (Fig. 1a) and DEPL (Fig. 1b) from I. obliquus in the control medium and the corn stover containing medium over DEAE-cellulose column. DEPC1, DEPC2, DEPC3 from DEPC and DEPL1, DEPL2, DEPL3 from DEPL were eluted. The yield of the three fractions from DEPC were 25.1%, 18.5%, 8.78% and the three fractions from DEPL were 26.4%, 17.8%, 9.67% (w/w, fraction/deproteinated extract), respectively.
Fig. 1. Elution curves of DEPC and DEPL over DEAE–cellulose column.
After purification on Sephadex G-200, the single corresponding peak of PDEPC1, PDEPC2, PDEPC3 from DEPC and PDEPL1, PDEPL2, PDEPL3 from DEPL were obtained and used for molecular weight measurements. Table 1 is a summary of carbohydrate, protein content and monosaccharide components of the EP samples from the control medium and the lignocellulose medium. DEPC3 had a lower sugar content of 64.5% than DEPC1 (71.3%) and DEPC3 (68.1%) but a higher protein content of 16.6%. DEPL3 had a lower sugar content of 60.2% as well than DEPL1 (89.7%) and DEPL3 (74.1%) but a higher protein content of 38.3% than DEPL1 and DEPL2. DEPC was composed of rhamnose (Rha), arabinose (Ara), xylose (Xyl), Mannose (Man), glucose (Glu), galactose (Gal) with molar ratios of 2.64:5.09:3.03:24.8:10.3:54.1. There was no Xyl detected in DEPL composed of the other five with the ratios of 4.43:4.56:38.1:27.2:25.7. Gal was the dominant component in DEPC1 and DEPC2, which was over two times the amount of Man. An inverse result was found for DPEC3. Man was the dominant component in DEPC3. Glu was the least among the three major components in the all three fractions. Differently, DEPL1, DEPL2 and DEPL3 were all composed of only three monosaccharides: Man, Glu and Gal. The monosaccharide components of the three fractions were not completely the same as those in DEPL. The two other components i.e. Rha and Ara might be in the fourth fraction. Contrary to the fractions from DEPC, the fractions from DEPL had a higher content of Man and Glu and lower content of Gal. Especially, DEPL3 contained
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Table 1 Major chemical content and monosaccharide components of the exopolysaccharide extracts. Sugar content
EPC DEPC DEPC1 DEPC2 DEPC3 EPL DEPL DEPL1 DEPL2 DEPL3
Protein content
Sugar component (mol%)
(wt.%)
(wt.%)
Rhamnose
Arabinose
Xylose
Glucose
Mannose
Galactose
44.3 70.5 71.4 68.1 64.5 49.5 66.3 89.7 74.1 60.2
13.3 16.5 13.6 18.2 16.6 23.5 31.2 7.12 22.1 38.3
2.64 – 3.86 –
5.09 – 3.59 –
3.03 – –
10.3 18.9 15.8 16.5
24.8 24.2 23.8 49.8
54.1 56.9 52.9 33.7
4.43 – – –
4.56 – – –
– – – –
27.2 15.6 33.1 33.2
38.1 43.2 29.2 49.6
25.7 41.2 37.7 17.2
–: Not detected.
49.6% Man and 33.2% Glu but 17.2% Gal while DEPC3 contained 49.8% Man and 33.7% Gal but 16.5% Glu. The result showed that there was evidently different in chemical components between the two different sources of EP. The average molecular weights (MW) of PDEPC1, PDEPC2, PDEPC3 and PDEPL1, PDEPL2, PDEPL3 were determined as 41, 35, 35 kDa and 44, 33, 29 kDa by gel permeation chromatography technique (Table 2). The molecular size (MN) of the six fractions was between 36 and 19 kDa. The MW/MN values of PDEPL1, PDEPL2, PDEPL3 were much greater than those of PDEPC1, PDEPC2, PDEPC3. The value of PDEPC1 was closer to 1 than the others, indicating more homogeneous. 3.2. Scavenging effect on hydroxyl radicals As is shown in Fig. 2a, the EP samples obtained from the two kinds of fermentation media exhibited a hydroxyl radical-scavenging activity in a dose-dependent manner (0.25–3.0 mg/ml). The scavenging activity of the EPL from the lignocellulose medium was significantly stronger than that from the control medium in the same concentrations. The crude extracts of EPC and EPL had a higher activity than the deproteinated extracts of DEPC and DEPL. The activity of DEPL was higher than DEPC. The highest scavenging rates of EPC, DEPC, EPL, and DEPL were 54.4%, 41.2%, 68.0%, and 52.5%, respectively. The three polysaccharide conjugate fractions from DEPC (Fig. 2b) or DEPL (Fig. 2c) were found to have the ability to scavenge hydroxyl radicals at concentrations from 0.1 to 2 mg/ml. For the fractions from the control medium, DEPC3 had a higher scavenging effect than DEPC1 and DEPC2 in the same concentrations. For the fractions from the corn stover-containing medium, the scavenging activity of DEPL3 was stronger than the other two fractions. The scavenging ability of all fractions increased with concentration up to 1.0 mg/ml and then leveled off. The highest scavenging rates of DEPC1, DEPC2, DEPC3, DEPL1, DEPL2, and DEPL3 were 24.4%, 32.7%, 40.2%, 24.8%, 33.1% and 52.6%, respectively. Table 2 Molecular weight (MW) and molecular size (MN) of the exopolysaccharide fractions.
PDEPC1 PDEPC2 PDEPC3 PDEPL1 PDEPL2 PDEPL3
MN
MW
MW/MN
36,000 29,000 29,000 29,000 21,000 19,000
41,000 35,000 35,000 44,000 33,000 29,000
1.14 1.21 1.21 1.52 1.57 1.52
MW: Molecular weight. MN: molecular size. MW/MN: molecular dispersion degree.
3.3. Scavenging activity of DPPH radicals Both EPC and EPL showed a DPPH radical-scavenging activity in a dose-dependent manner (0.25–3.0 mg/ml). Like the hydroxyl radical-scavenging activity, EPL had a significantly stronger ability than EPC in the same concentrations. For the deproteinated extracts, DEPL had a higher activity than DEPC. Different from the precede results on hydroxyl radical scavenging, DEPL and EPL showed almost the same scavenging effect against DPPH radicals. The scavenging ability of the four extracts increased with concentration up to 2.5 mg/ml and then leveled off (Fig. 3a). The highest scavenging rates of EPC, DEPC, EPL, and DEPL were 26.7%, 33.6%, 57.1%, and 59.6%, respectively. The three polysaccharide conjugate fractions from DEPC (Fig. 3b) or DEPL (Fig. 3c) were found to have the ability to scavenge DPPH radicals at concentrations from 0.1 to 2 mg/ml. The order of scavenging ability was DEPC2 > DEPC3 > DEPC1, and DEPL3 > DEPL2 > DEPL1. DEPC1 and DEPL1 had the lowest scavenging ability. There was no significant different between DEPC2 and DEPC3 although DEPC2 had a slightly higher scavenging effect. DEPL3 had a significantly stronger scavenging activity than DEPL2 and DEPL3. The highest scavenging rates of DEPC1, DEPC2, DEPC3, DEPL1, DEPL2, and DEPL3 were 21.6%, 38.6%, 35.6%, 21.7%, 42.2% and 58.1%, respectively. 3.4. IC50 value in antioxidant properties As shown in Table 3, the effective concentrations corresponding to IC50 values of EPL were lower than EPC, and the IC50 values of DEPL were lower than DEPC in the hydroxyl and DPPH radicalscavenging assay. The IC50 value of DEPC was lower than that of EPC, and DEPL had a lower IC50 value than EPL against the DPPH radical scavenging. Opposite results were found regarding the hydroxyl radical scavenging activity. As shown in Table 3, the order of IC50 values of the three fractions from DEPC in the hydroxyl radical scavenging assay was DEPC1 > DEPC2 > DEPC3, and DEPC1 > DEPC3 > DEPC2 in the DPPH scavenging radical assay. For the fractions from DEPL, the order of IC50 values was DEPL1 > DEPL2 > DEPL3 in both hydroxyl and DPPH radical-scavenging assay. DEPL3 was the most effective antioxidant among the six purified fractions. 4. Discussion Hydroxyl radicals can readily react with most biomolecules including carbohydrates, proteins, lipids, and DNA in cells, thus causing tissue damage or cell death. Removing hydroxyl radicals is important for the protection of living systems. EPC and EPL had a stronger antioxidant effect against hydroxyl radicals than
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Fig. 2. Hydroxyl radical-scavenging rates of EPC, DEPC, EPL and DEPL (a), the fractions from DEPC (b) and DEPL (c). Each point is the mean ± SD of triplicates.
the deproteinated extracts of DEPC and DEPL, respectively. It might be due to a little amount of small reducing molecules in EPC and EPL. The result was consistent with our previous report (Xu et al., 2011). The scavenging activity on DPPH radical is a widely used index and a quick method to evaluate antioxidant activity. DPPH scavenging activity of all extracts was moderate (Cui, Kim, & Park, 2005). The results were very different from those against the hydroxyl radical scavenging. DEPC had an obviously stronger scav-
Fig. 3. DPPH radical-scavenging rates of EPC, DEPC, EPL and DEPL (a), the fractions from DEPC (b) and DEPL (c). Each point is the mean ± SD of triplicates.
enging activity than EPC, and DEPL had a slightly stronger scavenging activity than EPL (Fig. 3a). The contradictory results on hydroxyl radical and DPPH radical scavenging activity in between the EPC and EPL with their respective deproteinated counterparts might be attributed to the different solubility in the two reaction systems, i.e. the reaction mixture for hydroxyl radical as-
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Y. Xiang et al. / Food Chemistry 134 (2012) 1899–1905 Table 3 IC50 values of the exopolysaccharide extracts in antioxidant property. IC50 (mg/ml)
EPC DEPC DEPC1 DEPC2 DEPC3 EPL DEPL DEPL1 DEPL2 DEPL3
Hydroxyl radicals
DPPH radicals
1.822c 3.223e 5.604g 3.472e 2.681d 0.653a 2.456d 5.431g 4.460f 1.394b
4.351f 3.762e 3.093d 2.264c 2.344c 2.042b 1.936b 3.185d 2.114b 1.329a
IC50 value is the effective concentration at which hydroxyl radicals and DPPH radicals are inhibited by 50%. Data with different alphabet letters in the same column are statistically significantly different according to Turkey–HSD (P < 0.05).
say was aqueous solution but for DPPH radical assay was methanol solution. DPPH is a stable free radical and exhibits a strong purple colour in methanol solution with a characteristic absorption at 517 nm. It might be because the deproteinated extracts have a better solubility in methanol than the untreated samples, which boosted the quenching ability (Xu et al., 2011). Besides the solubility, stereo selectivity of radicals and structure of the extract compound should be taken into account as well (Tang, Hu, & Chen, 2007). The EP samples from the corn stover-containing medium (lignocellulose medium) demonstrated a much stronger antioxidant effect than those from the control medium. The order of antioxidant effect of the extracts was EPL > EPC and DEPL > DEPC. The higher scavenging ability of EPL and DEPL might be because lignocellulose decomposition by I. obliquus stimulated the mycelia to produce more exopolysaccharides with higher antioxidant effect to protect themselves (Chen et al., 2011). The bioactivities of polysaccharides and their conjugates can be affected by many factors including chemical components, molecular mass, structure, conformation, even the extraction and isolation methods (Asker et al., 2009; Kim et al., 2006). DEPC2 and DEPC3 with a MW of 35 kDa had a higher activity than DEPC1 of 41 kDa, and DEPL3 of 29 kDa had a higher activity than DEPL2 of 33 kDa and DEPL1 of 44 kDa. The results seemed likely to demonstrate that the lower MW, the higher antioxidant activity. Polysaccharides with low molecular weight might bind radicals more easily (Asker et al., 2009). Unlike b-(1 ? 3)-glucans with medicinal properties that are strongly dependent on high molecular weight, ranging from 500 to 2000 kDa (Mizuno, Yeohlui, Kinoshita, Zhuang, Ito, & Mayuzumi, 1996), a-(1 ? 3)-glucuronoxylomannans, which are characteristic of Jelly mushrooms, are not strongly dependent on molecular weight. Differences in molecular weight had no obvious influence on the activity of the heteroglycans; the activity may be due to the common structure of the a-(1 ? 3)-mannan backbone (Gao, Seljelid, Chen, & Jiang, 1996). The exopolysaccharides from submerged fermentation do not always exist by oneself but conjugate with other components, including protein, lipids and nucleic acids, etc. The non-saccharide components are always removed by chemical or enzymatic methods for EP isolation. Small organic and inorganic molecular materials can be removed by dialysis. Sevage reagent can get rid of the protein and so on. However, sometimes the polysaccharide conjugates act as a whole in isolation (Chen, Zhang, Qu, & Xie, 2008). In the present study, all the deproteinated EP extracts by Sevage reagent were polysaccharide-protein conjugates (Table 1). The pres-
ence of protein has been reported to be important for biological activities and especially important for the biological activities of the hetero-polysaccharides (Mizuno et al., 1992). Our previous results demonstrated that with increasing of the protein content, the antioxidant activities of the three polysaccharide-protein conjugates by I. obliquus culture increased (Chen et al., 2008). In this study, we found consistently that EPL (DEPL) with higher protein content of 23.5% (31.2%) had a stronger antioxidant activity than EPC (DEPC) with a protein content of 13.3% (16.5%), respectively. The deproteinated extracts (DEPC and DEPL) were found having higher protein content as compared to their untreated samples (EPC and EPL). The results may be explained that the untreated samples (EPC and EPL) contain other compounds such as small molecular sugar and non-saccharide components except exopolysaccharide and protein. The deproteinated process and the following dialysis process removed not only free protein but also small molecular impurities. Thus, the remaining protein complex (glycoprotein) in DEPC and DEPL was purified, resulting in higher protein content in deproteinated extract (DEPC and DEPL). For the fractions of DEPL, the protein content was very different from each other. With increasing of the protein content, the antioxidant activities of the fractions increased In other words, DEPL3 with the highest protein content had the strongest antioxidant effect against the hydroxyl and DPPH radicals (Fig. 2c and 3c). The existence of protein might affect the physico-chemical properties of the polysaccharides and hence their bioactivities (Chen et al., 2008). The main monosaccharides of the EP obtained from the control medium were Gal and Man, while those from the lignocellulose medium were Man and Glu. DEPL with a high molar proportion of Man (38.1%) and Glu (27.2%) had a higher antioxidant activity than DEPC (Table 1). In addition, Man was the main monosaccharide of DEPC3 (49.8%), DEPL1 (43.2%) and DEPL3 (49.6%) among the six fractions. Correspondingly, DEPC3 had a higher activity than the two others from the control medium and DEPL3 had a higher activity than the two others from the lignocellulose medium. The high molar proportion of mannose may contribute to the enhancement of antioxidant activity (Chen et al., 2011). Kim et al. reported that endo-polysaccharide fractions from cultivated mycelia of I. obliquus with mannose as the major component had an anti-cancer effect (Kim et al., 2006). Wasser reported that mannan was the major component of some active hetero-polysaccharides, e.g., a-(1 ? 3)-mannans from Dictyophora indusiata, glucuronoxylomannan from Tremella fuciformis, glucomannan from Agaricus blazei, and galactoglucomannan from L. edodes (Wasser, 2002). Moreover, the antioxidant capacity of polysaccharide molecules also depends strongly on the organisation of sugar monomers, the linkage pattern of the main chain (a or b) and the branching configuration (Liu, Wang, Xu, & Wang, 2007). In recent years, some bioactive polysaccharides isolated from natural sources have attracted much attention in the field of biochemistry and pharmacology. They exhibit various biological activities affected by different chemical structures (Yang & Zhang, 2009). Further researches show that the activities of polysaccharides are not only dependent on their chemical structures, but also are related to their chain conformations (Tao, Zhang, Yan, & Wu, 2007). In general, it is interesting and important to elucidate the relation among chemical structures, chain conformations of polysaccharides and their biological activities. However, polysaccharides are usually composed of various monosaccharides linked with different glucosidic bonds. Some polysaccharides have hyperbranched structures. Moreover, polysaccharides often have high molecular weights, and tend to form aggregates in solution that can mask the behaviour of individual macromolecules. In consequence, to characterise the chemical structures and chain conformations of polysaccharides is not an easy task (Yang & Zhang, 2009). The complexity of poly-
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saccharide molecular structure and chemical composition of polysaccharide fractions isolated from medicinal fungi makes it difficult to elucidate their structure and activity relationships (Leung, Zhao, Ho, & Wu, 2009). In conclusion, the EP fractions from the lignocellulose medium had the stronger antioxidant effects than those from the control medium. Lignocellulose decomposition resulted in a change in protein content, monosaccharide composition, and MW/MN, which may lead to the difference in antioxidant activity. More analysis and evaluation of chemical structure and chain conformation of the EP fractions are required for further understanding the EP difference between the two media. Acknowledgements This research was supported by research Grants from the Science and Technology Department of Zhejiang Province, China (2012C33G2060004) and Zhejiang Provincial Natural Science Foundation (Z12B020006). References Asker, M. M. S., Ahmed, Y. M., & Ramadan, M. F. (2009). Chemical characteristics and antioxidant activity of exopolysaccharide fractions from Microbacterium terregens. Carbohydrate Polymers, 77, 563–567. Benhura, M. A. N., & Chidewe, C. (2002). Some properties of a polysaccharide preparation that is isolated from the fruit of Cordia abyssinica. Food Chemistry, 76, 343–347. Bradford, M. M. (1976). A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry, 72, 248–254. Chen, H., Xu, X., & Zhu, Y. (2010). Optimization of hydroxyl radical scavenging activity of exo-polysaccharides from Inonotus obliquus in submerged fermentation using response surface methodology. Journal of Microbiology and Biotechnology, 20, 835–843. Chen, H., Yan, M., Zhu, J., & Xu, X. (2011). Enhancement of exo-polysaccharide production and antioxidant activity in submerged cultures of Inonotus obliquus by lignocellulose decomposition. Journal of Industrial Microbiology and Biotechnology, 38, 291–298. Chen, H., Zhang, M., Qu, Z., & Xie, B. (2008). Antioxidant activities of different fractions of polysaccharide conjugates from green tea (Camellia sinensis). Food Chemistry, 106, 559–563. Cui, Y., Kim, D. S. K., & Park, K. C. (2005). Antioxidant effect of Inonotus obliquus. Journal of Ethnopharmacology, 96, 79–85. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 29, 350–356. Gao, H., & Gu, W. (2007). Optimization of polysaccharides production from Agarieus brasilienses by fermentation process. Biochemical Engineering Journal, 33, 202–210. Gao, Q. P., Seljelid, R., Chen, H. Q., & Jiang, R. (1996). Characterization of acidic heteroglycans from Tremella fuciformis Berk. with cytokine stimulating activity. Carbohydrate Research, 288, 135–142. Ikekawa, T. (2001). Beneficial effects of edible and medicinal mushrooms in health care. International Journal of Medicinal Mushrooms, 3, 291–298. Kim, Y. O., Han, S. B., Lee, H. W., Ahn, H. J., Yoon, Y. D., Jung, J. K., Kim, H. M., & Shin, C. S. (2005). Immuno-stimulating effect of the endo-polysaccharides produced by submerged culture of Inonotus obliquus. Life Science, 77, 2438–2456. Kim, Y. O., Park, H. W., Kim, J. H., Lee, J. Y., Moon, S. H., & Shin, C. S. (2006). Anticancer effect and structural characterization of endo-polysaccharide from cultivated mycelia of Inonotus obliquus. Life Science, 79, 72–80. Leung, P. H., Zhao, S., Ho, K. P., & Wu, J. Y. (2009). Chemical properties and antioxidant activity of exopolysaccharides from mycelial culture of Cordyceps sinensis fungus Cs-HK1. Food Chemistry, 114, 1251–1256.
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Chemical properties and antioxidant activity of exopolysaccharides fractions from mycelial culture of Inonotus obliquus in a ground corn stover medium Yuling Xiang, Xiangqun Xu ⇑, Juan Li Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, China
a r t i c l e
i n f o
Article history: Received 18 October 2011 Received in revised form 12 March 2012 Accepted 24 March 2012 Available online 13 April 2012 Keywords: Inonotus obliquus Exopolysaccharides Antioxidant activity Submerged fermentation Lignocellulose decomposition Composition Molecular weight
a b s t r a c t The medicinal mushroom Inonotus obliquus has been a folk remedy for a long time in East-European and Asian countries. We first reported the enhancement in production and antioxidant activity of exopolysaccharides by I. obliquus culture under lignocellulose decomposition. In this study, the two different sources of exopolysaccharides from the control medium and the lignocellulose (corn stover) containing medium by I. obliquus in submerged fermentation were fractionated and purified by chromatography. The exopolysaccharides from the corn stover-containing medium presented significantly stronger hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity than the control. Three fractions from the control medium and the corn stover-containing medium were isolated respectively. The fraction of DEPL3 from the corn stover-containing medium with the highest protein content (38.3%), mannose content (49.6%), and the lowest molecular weight (29 kDa) had the highest antioxidant activity with the lowest IC50 values. In conclusion, lignocellulose decomposition changed the chemical characterisation and significantly enhanced the antioxidant activity of exopolysaccharide fractions. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Mushrooms such as Ganoderma lucidum (Ling Zhi or Reishi), Lentinus edodes (Shiitake), Inonotus obliquus (Pers.: Fr.) Pilát (Chaga) and many others have been collected and used for hundreds of years in China, Japan, Korea, and eastern Russia. Mushrooms have also played an important role in the treatment of ailments affecting rural populations of eastern European countries (Wasser, 2011). I. obliquus is a rare edible and medicinal mushroom. It belongs to the family Hymenochaetaceae, Basidiomycetes. The pharmacological importance of the mushrooms is very high in the Far East as a traditional medicine for treating cancer, heart, liver, and stomach diseases, and tuberculosis (Saar, 1991). Chemical investigations show that I. obliquus produces a diverse range of bioactive metabolites, including lanostane-type triterpenoids, steroids, phenolic compounds and polysaccharides (Zheng et al., 2010). For almost 40 years, medicinal mushrooms have been intensively investigated for medicinal effects in vivo and in vitro model systems, and many new antitumour and immunomodulating polysaccharides have been identified and put into practical use (Ikekawa, 2001; Mizuno, 1999). In the last few years an increasing number of studies have been published concerning the biological
⇑ Corresponding author. Address: Department of Chemistry, School of Science, Zhejiang Sci-Tech University, Hangzhou 310018, China. Tel.: +86 571 86843228; fax: +86 571 87055836. E-mail address: [email protected] (X. Xu). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.03.121
activities of polysaccharides from the I. obliquus fruit bodies, cultured mycelium, and culture broth such as antitumour, immunomodulating, antioxidant, radical scavenging, and anti-caducity (Kim et al., 2005; Mizuno et al., 1999; Rhee, Cho, Kim, Cha, & Park, 2008; Shamtsyan et al., 2004; Tseng, Yang, & Mau, 2008; Xu, Wu, & Chen, 2011). The limited natural resource and difficult artificial cultivation of I. obliquus to obtain fruit body make it impossible to obtain large quantity of bioactive molecules. Submerged fermentation is an effective process for the production of mycelial biomass and bioactive compounds, especially polysaccharides (Pokhrel & Ohga, 2007; Zhang & Cheung, 2011). Recently, we reported a submerged fermentation optimisation for bioactive polysaccharide production from I. obliquus using the response surface methodology (RSM) method combined with hydroxyl radical-scavenging activity screening (Chen, Xu, & Zhu, 2010). With the RSM optimised medium, the hydroxyl radical-scavenging activity per unit of the exopolysaccharides (EP) was significantly enhanced compared to that from either the basal fermentation medium or the single variable optimisation of fermentation medium (Chen et al., 2010; Xu et al., 2011). In our previous study, we first found that hydroxyl radicals played the same important role when I. obliquus in submerged fermentation decomposed the lignocellulose in corn stover added in a liquid medium (Chen, Yan, Zhu, & Xu, 2011) as in nature where I. obliquus degraded wood polymers as a kind of white rot fungi (Gao & Gu, 2007). Correspondingly, the production and antioxidant
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activity of the EP as a defense response by I. obliquus were further enhanced in a corn stover-containing submerged fermentation with lignocellulose decomposition (Chen et al., 2011). Polysaccharides with biological activity differ greatly in their chemical compositions, configurations, and physical properties. Although it is difficult to correlate polysaccharide structure with antioxidant activity, immuno-stimulating effect, and anti-tumour activity, some relationships can be inferred. In medicinal mushrooms, the biological activities of polysaccharides are closely related to molecular mass, sugar content, monosaccharide compositions. The purified endo-polysaccharide from the mycelia of I. obliquus that was an a-linked fucoglucomannan composed primarily of mannose with small amounts of glucose, fucose, and glucosamine could inhibit tumour growth in vivo (Kim et al., 2006). The water-soluble polysaccharide from the sclerotia of I. obliquus was a heteropolysaccharide of xylogalactoglucan composed of glucose, mannose, galactose, xylose, arabinose, and fucose (Mizuno et al., 1999). Although the corn stover-containing submerged fermentation with lignocellulose was proven to enhance the production and antioxidant activity of the EP in our previous studies (Chen et al., 2011), there have been no reports regarding a relationship of the sugar content, monosaccharide compositions, and molecular mass with biological activity in exopolysaccharides from the mycelia of I. obliquus to the culture process in the two mediums. The aim of this work was to examine the characterisation and antioxidant effects of the two kinds of purified EP from the control medium optimised with the RSM (Chen et al., 2010) and the medium supplemented with ground corn stover (Chen et al., 2011). The two different sources of EP obtained from I. obliquus mycelial liquid culture were fractionated and purified by chromatography on DEAE-cellulose and Sephadex G-200 columns. The chemical properties and antioxidant activity of the EP fractions were compared between different culture processes.
2. Materials and methods 2.1. Cultivation method I. obliquus (CBS314.39) was purchased from the Centraal Bureau voor Schimmelcultuur, Utrecht, Netherlands. It was maintained on malt extract agar slants containing (% w/v) malt extract 3, peptone 0.3 and agar 1.5 at pH 5.6 ± 0.2. The slants were cultivated at 25 °C for about 2 weeks. When the mycelia overgrew the slants, they were stored at 4 °C and sub-cultured every 3 months. The seed culture was prepared by incubating mycelia on a malt extract agar slant into a 250-mL Erlenmeyer flask with 100 mL medium. One cm2 of malt extract agar with mycelia was chipped off and then transferred into the Erlenmeyer flask. The medium contained (% w/v) glucose 2, peptone 0.3, yeast extract 0.2, KH2PO4 0.1, MgSO4 0.15, and CaCl2 0.01. Cultures were incubated for 4–5 days in a rotary shaker (150 rpm) at 28 °C. The harvested seed culture was added into 250-mL Erlenmeyer flasks containing 100-mL control medium or corn stover-containing medium and incubated at a rate of 10% (v/v) and at 28 °C in a rotary shaker at 150 rpm. The culture was incubated for 9 days to produce mycelia (Xu et al., 2011). The mycelia and the culture broth were harvested on day 9. The two kinds of medium used in this study were as below. Control medium (% w/v): corn flour 5.3, peptone 0.3, KH2PO4 0.1, ZnSO42H2O 0.001, K2HPO4 0.04, FeSO47H2O 0.005, MgSO47H2O 0.05, CuSO45H2O 0.002, CoCl2 0.001, MnSO4H2O 0.008. pH = 6.0. The medium was optimised by the response surface methodology (RSM) based on the basal medium in our previous work (Chen et al., 2010).
Corn stover-containing medium (% w/v): corn flour 3.5, ground corn stover 3%, and all of the other components were the same as the control medium. The content of corn flour in the control medium was lower because corn stover provided reducing sugars when the reducing sugars from the corn flour hydrolysis exhausted with the lignocellulose decompositions by I. obliquus (Chen et al., 2011). 2.2. Extraction of EP The I. obliquus mycelial cells were removed from culture broths using a filter paper. The filtrate was mixed with four volumes of chilled 95% (v/v) ethanol, then stirred vigorously and left at 4 °C overnight. The precipitate was collected, then centrifuged at 6500g for 6 min and lyophilised. The lyophilised samples were termed as EPC from the control medium and EPL from the corn stover-containing medium. The Sevage method was employed to remove protein after neutrase treatment with some modification (Yan, Han, & Jiang, 2004). The EPC and EPL aqueous solution were further mixed with the Sevage reagent [chloroform: butanol = 5:1 (v/v)] at the proportion 5:1 (v/v) and oscillated intensively. The supernatant was concentrated and the aqueous solution was dialysed against distilled water for 48 h then lyophilised. The deproteinated EPC from the control medium and EPL from the corn stovercontaining medium were named as DEPC and DEPL (Xu et al., 2011). 2.3. Fractionation and purification of EP DEPC and DEPL (200 mg in 10 ml buffer) were subjected to a DEAE-cellulose anion-exchange column (50 2.4 cm, i.d.), which was equilibrated with 0.01 M sodium phosphate buffer (PBS, pH 7.8) before. Then the fractions were eluted with PBS for 200 ml, 0.1 M NaCl–PBS for 150 ml, 0.2 M NaCl–PBS for 150 ml, 0.3 M NaCl–PBS for 200 ml and at a flow rate of 72 ml/h, respectively. Four fractions from both DEPC and DEPL were obtained. The first three fractions of DEPC1, DEPC2, DEPC3 and DEPL1, DEPL2, DEPL3 were concentrated, dialysed against distilled water for 48 h and then lyophilised. To make sure a complete isolation of each fraction for molecular weight determination, the six fractions were fractionated and purified further by a gel permeation chromatography technique over a Sephadex G-200 (50 2.4 cm) and eluted at 0.3 ml/min with 0.1 M NaCl. A single fraction from each sample was collected and coded as PDEPC1, PDEPC2, PDEPC3 and PDEPL1, PDEPL2, PDEPL3 (purified fractions), respectively (Asker, Ahmed, & Ramadan, 2009). 2.4. Compositional analysis of EP The carbohydrate and protein content of all the EP samples obtained above were analysed by the phenol–sulphuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) and the Bradford’s method with bovine serum albumin as a standard (Bradford, 1976). Monosaccharides composition was analysed by gas chromatography (Varian Inc., Palo ALto, CA, USA) with a Varian CP-Sil 5 CB capillary chromatography column (25 m 0.53 mm, 0.25 lm film thickness) and a flame-ionisation detector for analysis (Chen et al., 2011; Benhura & Chidewe, 2002). 2.5. Determination of molecular weight Molecular weights of the EP fractions were determined by size exclusion chromatography with laser light scattering (SEC-LLS). The EP fractions (1 mg) were dissolved in 0.2 M NaCl (1 ml), and optical clarification were achieved by filtration through a sand filter followed by a 0.2 lm pore-size filter (Whatman, England) into
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the scattering cell (Fused Silica). Molecular mass parameters of the EP fractions were measured with a eight-angle laser light scattering instrument equipped with a He–Ne laser (k = 658 nm, HELEOS 8, Wyatt Technology Co., USA) at 25 °C. The value of 0.125 ml/g was used as the specific refractive index increment of the EP fractions in 0.2 M NaCl (Tamura, Wada, & Isogai, 2009). Data acquisition and processing was performed with the ASTRA software (Wyatt Technologies). 2.6. Scavenging effect on hydroxyl radicals Hydroxyl radical-scavenging activity of each extract solution was measured according to the salicylic method with some modifications (Smirnoff & Cumbes, 1989). Reaction mixtures in a final volume of 4 ml contained: 1 ml H2O2 (8.8 mM/l), 1 ml FeSO4 (9 mM/l), 1 ml salicylic (9 mM/l), and 1 ml sample solution in a concentration gradient. The H2O2 was added to the mixture and the reaction was started up at last. The reaction mixture was incubated at 37 °C for 60 min and then centrifuged at 10,000g for 8 min. The absorbance (A) of the reaction solutions at 510 nm was measured. The hydroxyl radical-scavenging ratio was calculated by the following formula:
Scavenging rate ð%Þ ¼ ½A0 ðAx Ax0 Þ 100=A0
ð1Þ
A control contained all the reaction reagents except the samples was prepared and measured as A0, Ax was the result of samples and Ax0 is the absorbance for background, i.e., the reaction mixture without H2O2. 2.7. Scavenging effect on DPPH radicals A 2.4 ml extract solution was mixed with 0.8 ml of 0.4 mM 2,2diphenyl-1-picrylhydrazyl (DPPH) in methanol, to produce a final DPPH concentration of 0.1 mM. The mixture was vigorously shaken and left to stand for 30 min in the dark, and its absorbance was measured at 517 nm as Ax. A control sample containing the same amount of methanol and DPPH radicals was measured as A0. The absorbance of samples containing the same amount of methanol and the extract solution was recorded as C (Yang et al., 2007). The DPPH radical-scavenging ratio was calculated by the following formula:
Scavenging rate ð%Þ ¼ ½A0 ðAx CÞ 100=A0
ð2Þ
2.8. Statistical analysis Experimental results recorded were the means ± standard deviation (SD) of triple determinations. The obtained data were subjected to One-way ANOVA and the differences between means were at the 5% probability level using Duncan’s new multiple range tests. The IC50 values were calculated by using median-effect analysis and origin 7.5 software (OriginLab Corp., Northampton, MA). 3. Result 3.1. Isolation, purification and composition Fig. 1 shows the elution curves of DEPC (Fig. 1a) and DEPL (Fig. 1b) from I. obliquus in the control medium and the corn stover containing medium over DEAE-cellulose column. DEPC1, DEPC2, DEPC3 from DEPC and DEPL1, DEPL2, DEPL3 from DEPL were eluted. The yield of the three fractions from DEPC were 25.1%, 18.5%, 8.78% and the three fractions from DEPL were 26.4%, 17.8%, 9.67% (w/w, fraction/deproteinated extract), respectively.
Fig. 1. Elution curves of DEPC and DEPL over DEAE–cellulose column.
After purification on Sephadex G-200, the single corresponding peak of PDEPC1, PDEPC2, PDEPC3 from DEPC and PDEPL1, PDEPL2, PDEPL3 from DEPL were obtained and used for molecular weight measurements. Table 1 is a summary of carbohydrate, protein content and monosaccharide components of the EP samples from the control medium and the lignocellulose medium. DEPC3 had a lower sugar content of 64.5% than DEPC1 (71.3%) and DEPC3 (68.1%) but a higher protein content of 16.6%. DEPL3 had a lower sugar content of 60.2% as well than DEPL1 (89.7%) and DEPL3 (74.1%) but a higher protein content of 38.3% than DEPL1 and DEPL2. DEPC was composed of rhamnose (Rha), arabinose (Ara), xylose (Xyl), Mannose (Man), glucose (Glu), galactose (Gal) with molar ratios of 2.64:5.09:3.03:24.8:10.3:54.1. There was no Xyl detected in DEPL composed of the other five with the ratios of 4.43:4.56:38.1:27.2:25.7. Gal was the dominant component in DEPC1 and DEPC2, which was over two times the amount of Man. An inverse result was found for DPEC3. Man was the dominant component in DEPC3. Glu was the least among the three major components in the all three fractions. Differently, DEPL1, DEPL2 and DEPL3 were all composed of only three monosaccharides: Man, Glu and Gal. The monosaccharide components of the three fractions were not completely the same as those in DEPL. The two other components i.e. Rha and Ara might be in the fourth fraction. Contrary to the fractions from DEPC, the fractions from DEPL had a higher content of Man and Glu and lower content of Gal. Especially, DEPL3 contained
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Table 1 Major chemical content and monosaccharide components of the exopolysaccharide extracts. Sugar content
EPC DEPC DEPC1 DEPC2 DEPC3 EPL DEPL DEPL1 DEPL2 DEPL3
Protein content
Sugar component (mol%)
(wt.%)
(wt.%)
Rhamnose
Arabinose
Xylose
Glucose
Mannose
Galactose
44.3 70.5 71.4 68.1 64.5 49.5 66.3 89.7 74.1 60.2
13.3 16.5 13.6 18.2 16.6 23.5 31.2 7.12 22.1 38.3
2.64 – 3.86 –
5.09 – 3.59 –
3.03 – –
10.3 18.9 15.8 16.5
24.8 24.2 23.8 49.8
54.1 56.9 52.9 33.7
4.43 – – –
4.56 – – –
– – – –
27.2 15.6 33.1 33.2
38.1 43.2 29.2 49.6
25.7 41.2 37.7 17.2
–: Not detected.
49.6% Man and 33.2% Glu but 17.2% Gal while DEPC3 contained 49.8% Man and 33.7% Gal but 16.5% Glu. The result showed that there was evidently different in chemical components between the two different sources of EP. The average molecular weights (MW) of PDEPC1, PDEPC2, PDEPC3 and PDEPL1, PDEPL2, PDEPL3 were determined as 41, 35, 35 kDa and 44, 33, 29 kDa by gel permeation chromatography technique (Table 2). The molecular size (MN) of the six fractions was between 36 and 19 kDa. The MW/MN values of PDEPL1, PDEPL2, PDEPL3 were much greater than those of PDEPC1, PDEPC2, PDEPC3. The value of PDEPC1 was closer to 1 than the others, indicating more homogeneous. 3.2. Scavenging effect on hydroxyl radicals As is shown in Fig. 2a, the EP samples obtained from the two kinds of fermentation media exhibited a hydroxyl radical-scavenging activity in a dose-dependent manner (0.25–3.0 mg/ml). The scavenging activity of the EPL from the lignocellulose medium was significantly stronger than that from the control medium in the same concentrations. The crude extracts of EPC and EPL had a higher activity than the deproteinated extracts of DEPC and DEPL. The activity of DEPL was higher than DEPC. The highest scavenging rates of EPC, DEPC, EPL, and DEPL were 54.4%, 41.2%, 68.0%, and 52.5%, respectively. The three polysaccharide conjugate fractions from DEPC (Fig. 2b) or DEPL (Fig. 2c) were found to have the ability to scavenge hydroxyl radicals at concentrations from 0.1 to 2 mg/ml. For the fractions from the control medium, DEPC3 had a higher scavenging effect than DEPC1 and DEPC2 in the same concentrations. For the fractions from the corn stover-containing medium, the scavenging activity of DEPL3 was stronger than the other two fractions. The scavenging ability of all fractions increased with concentration up to 1.0 mg/ml and then leveled off. The highest scavenging rates of DEPC1, DEPC2, DEPC3, DEPL1, DEPL2, and DEPL3 were 24.4%, 32.7%, 40.2%, 24.8%, 33.1% and 52.6%, respectively. Table 2 Molecular weight (MW) and molecular size (MN) of the exopolysaccharide fractions.
PDEPC1 PDEPC2 PDEPC3 PDEPL1 PDEPL2 PDEPL3
MN
MW
MW/MN
36,000 29,000 29,000 29,000 21,000 19,000
41,000 35,000 35,000 44,000 33,000 29,000
1.14 1.21 1.21 1.52 1.57 1.52
MW: Molecular weight. MN: molecular size. MW/MN: molecular dispersion degree.
3.3. Scavenging activity of DPPH radicals Both EPC and EPL showed a DPPH radical-scavenging activity in a dose-dependent manner (0.25–3.0 mg/ml). Like the hydroxyl radical-scavenging activity, EPL had a significantly stronger ability than EPC in the same concentrations. For the deproteinated extracts, DEPL had a higher activity than DEPC. Different from the precede results on hydroxyl radical scavenging, DEPL and EPL showed almost the same scavenging effect against DPPH radicals. The scavenging ability of the four extracts increased with concentration up to 2.5 mg/ml and then leveled off (Fig. 3a). The highest scavenging rates of EPC, DEPC, EPL, and DEPL were 26.7%, 33.6%, 57.1%, and 59.6%, respectively. The three polysaccharide conjugate fractions from DEPC (Fig. 3b) or DEPL (Fig. 3c) were found to have the ability to scavenge DPPH radicals at concentrations from 0.1 to 2 mg/ml. The order of scavenging ability was DEPC2 > DEPC3 > DEPC1, and DEPL3 > DEPL2 > DEPL1. DEPC1 and DEPL1 had the lowest scavenging ability. There was no significant different between DEPC2 and DEPC3 although DEPC2 had a slightly higher scavenging effect. DEPL3 had a significantly stronger scavenging activity than DEPL2 and DEPL3. The highest scavenging rates of DEPC1, DEPC2, DEPC3, DEPL1, DEPL2, and DEPL3 were 21.6%, 38.6%, 35.6%, 21.7%, 42.2% and 58.1%, respectively. 3.4. IC50 value in antioxidant properties As shown in Table 3, the effective concentrations corresponding to IC50 values of EPL were lower than EPC, and the IC50 values of DEPL were lower than DEPC in the hydroxyl and DPPH radicalscavenging assay. The IC50 value of DEPC was lower than that of EPC, and DEPL had a lower IC50 value than EPL against the DPPH radical scavenging. Opposite results were found regarding the hydroxyl radical scavenging activity. As shown in Table 3, the order of IC50 values of the three fractions from DEPC in the hydroxyl radical scavenging assay was DEPC1 > DEPC2 > DEPC3, and DEPC1 > DEPC3 > DEPC2 in the DPPH scavenging radical assay. For the fractions from DEPL, the order of IC50 values was DEPL1 > DEPL2 > DEPL3 in both hydroxyl and DPPH radical-scavenging assay. DEPL3 was the most effective antioxidant among the six purified fractions. 4. Discussion Hydroxyl radicals can readily react with most biomolecules including carbohydrates, proteins, lipids, and DNA in cells, thus causing tissue damage or cell death. Removing hydroxyl radicals is important for the protection of living systems. EPC and EPL had a stronger antioxidant effect against hydroxyl radicals than
Y. Xiang et al. / Food Chemistry 134 (2012) 1899–1905
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Fig. 2. Hydroxyl radical-scavenging rates of EPC, DEPC, EPL and DEPL (a), the fractions from DEPC (b) and DEPL (c). Each point is the mean ± SD of triplicates.
the deproteinated extracts of DEPC and DEPL, respectively. It might be due to a little amount of small reducing molecules in EPC and EPL. The result was consistent with our previous report (Xu et al., 2011). The scavenging activity on DPPH radical is a widely used index and a quick method to evaluate antioxidant activity. DPPH scavenging activity of all extracts was moderate (Cui, Kim, & Park, 2005). The results were very different from those against the hydroxyl radical scavenging. DEPC had an obviously stronger scav-
Fig. 3. DPPH radical-scavenging rates of EPC, DEPC, EPL and DEPL (a), the fractions from DEPC (b) and DEPL (c). Each point is the mean ± SD of triplicates.
enging activity than EPC, and DEPL had a slightly stronger scavenging activity than EPL (Fig. 3a). The contradictory results on hydroxyl radical and DPPH radical scavenging activity in between the EPC and EPL with their respective deproteinated counterparts might be attributed to the different solubility in the two reaction systems, i.e. the reaction mixture for hydroxyl radical as-
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Y. Xiang et al. / Food Chemistry 134 (2012) 1899–1905 Table 3 IC50 values of the exopolysaccharide extracts in antioxidant property. IC50 (mg/ml)
EPC DEPC DEPC1 DEPC2 DEPC3 EPL DEPL DEPL1 DEPL2 DEPL3
Hydroxyl radicals
DPPH radicals
1.822c 3.223e 5.604g 3.472e 2.681d 0.653a 2.456d 5.431g 4.460f 1.394b
4.351f 3.762e 3.093d 2.264c 2.344c 2.042b 1.936b 3.185d 2.114b 1.329a
IC50 value is the effective concentration at which hydroxyl radicals and DPPH radicals are inhibited by 50%. Data with different alphabet letters in the same column are statistically significantly different according to Turkey–HSD (P < 0.05).
say was aqueous solution but for DPPH radical assay was methanol solution. DPPH is a stable free radical and exhibits a strong purple colour in methanol solution with a characteristic absorption at 517 nm. It might be because the deproteinated extracts have a better solubility in methanol than the untreated samples, which boosted the quenching ability (Xu et al., 2011). Besides the solubility, stereo selectivity of radicals and structure of the extract compound should be taken into account as well (Tang, Hu, & Chen, 2007). The EP samples from the corn stover-containing medium (lignocellulose medium) demonstrated a much stronger antioxidant effect than those from the control medium. The order of antioxidant effect of the extracts was EPL > EPC and DEPL > DEPC. The higher scavenging ability of EPL and DEPL might be because lignocellulose decomposition by I. obliquus stimulated the mycelia to produce more exopolysaccharides with higher antioxidant effect to protect themselves (Chen et al., 2011). The bioactivities of polysaccharides and their conjugates can be affected by many factors including chemical components, molecular mass, structure, conformation, even the extraction and isolation methods (Asker et al., 2009; Kim et al., 2006). DEPC2 and DEPC3 with a MW of 35 kDa had a higher activity than DEPC1 of 41 kDa, and DEPL3 of 29 kDa had a higher activity than DEPL2 of 33 kDa and DEPL1 of 44 kDa. The results seemed likely to demonstrate that the lower MW, the higher antioxidant activity. Polysaccharides with low molecular weight might bind radicals more easily (Asker et al., 2009). Unlike b-(1 ? 3)-glucans with medicinal properties that are strongly dependent on high molecular weight, ranging from 500 to 2000 kDa (Mizuno, Yeohlui, Kinoshita, Zhuang, Ito, & Mayuzumi, 1996), a-(1 ? 3)-glucuronoxylomannans, which are characteristic of Jelly mushrooms, are not strongly dependent on molecular weight. Differences in molecular weight had no obvious influence on the activity of the heteroglycans; the activity may be due to the common structure of the a-(1 ? 3)-mannan backbone (Gao, Seljelid, Chen, & Jiang, 1996). The exopolysaccharides from submerged fermentation do not always exist by oneself but conjugate with other components, including protein, lipids and nucleic acids, etc. The non-saccharide components are always removed by chemical or enzymatic methods for EP isolation. Small organic and inorganic molecular materials can be removed by dialysis. Sevage reagent can get rid of the protein and so on. However, sometimes the polysaccharide conjugates act as a whole in isolation (Chen, Zhang, Qu, & Xie, 2008). In the present study, all the deproteinated EP extracts by Sevage reagent were polysaccharide-protein conjugates (Table 1). The pres-
ence of protein has been reported to be important for biological activities and especially important for the biological activities of the hetero-polysaccharides (Mizuno et al., 1992). Our previous results demonstrated that with increasing of the protein content, the antioxidant activities of the three polysaccharide-protein conjugates by I. obliquus culture increased (Chen et al., 2008). In this study, we found consistently that EPL (DEPL) with higher protein content of 23.5% (31.2%) had a stronger antioxidant activity than EPC (DEPC) with a protein content of 13.3% (16.5%), respectively. The deproteinated extracts (DEPC and DEPL) were found having higher protein content as compared to their untreated samples (EPC and EPL). The results may be explained that the untreated samples (EPC and EPL) contain other compounds such as small molecular sugar and non-saccharide components except exopolysaccharide and protein. The deproteinated process and the following dialysis process removed not only free protein but also small molecular impurities. Thus, the remaining protein complex (glycoprotein) in DEPC and DEPL was purified, resulting in higher protein content in deproteinated extract (DEPC and DEPL). For the fractions of DEPL, the protein content was very different from each other. With increasing of the protein content, the antioxidant activities of the fractions increased In other words, DEPL3 with the highest protein content had the strongest antioxidant effect against the hydroxyl and DPPH radicals (Fig. 2c and 3c). The existence of protein might affect the physico-chemical properties of the polysaccharides and hence their bioactivities (Chen et al., 2008). The main monosaccharides of the EP obtained from the control medium were Gal and Man, while those from the lignocellulose medium were Man and Glu. DEPL with a high molar proportion of Man (38.1%) and Glu (27.2%) had a higher antioxidant activity than DEPC (Table 1). In addition, Man was the main monosaccharide of DEPC3 (49.8%), DEPL1 (43.2%) and DEPL3 (49.6%) among the six fractions. Correspondingly, DEPC3 had a higher activity than the two others from the control medium and DEPL3 had a higher activity than the two others from the lignocellulose medium. The high molar proportion of mannose may contribute to the enhancement of antioxidant activity (Chen et al., 2011). Kim et al. reported that endo-polysaccharide fractions from cultivated mycelia of I. obliquus with mannose as the major component had an anti-cancer effect (Kim et al., 2006). Wasser reported that mannan was the major component of some active hetero-polysaccharides, e.g., a-(1 ? 3)-mannans from Dictyophora indusiata, glucuronoxylomannan from Tremella fuciformis, glucomannan from Agaricus blazei, and galactoglucomannan from L. edodes (Wasser, 2002). Moreover, the antioxidant capacity of polysaccharide molecules also depends strongly on the organisation of sugar monomers, the linkage pattern of the main chain (a or b) and the branching configuration (Liu, Wang, Xu, & Wang, 2007). In recent years, some bioactive polysaccharides isolated from natural sources have attracted much attention in the field of biochemistry and pharmacology. They exhibit various biological activities affected by different chemical structures (Yang & Zhang, 2009). Further researches show that the activities of polysaccharides are not only dependent on their chemical structures, but also are related to their chain conformations (Tao, Zhang, Yan, & Wu, 2007). In general, it is interesting and important to elucidate the relation among chemical structures, chain conformations of polysaccharides and their biological activities. However, polysaccharides are usually composed of various monosaccharides linked with different glucosidic bonds. Some polysaccharides have hyperbranched structures. Moreover, polysaccharides often have high molecular weights, and tend to form aggregates in solution that can mask the behaviour of individual macromolecules. In consequence, to characterise the chemical structures and chain conformations of polysaccharides is not an easy task (Yang & Zhang, 2009). The complexity of poly-
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