Structural analysis and bioactivity of a polysaccharide from the roots of Astragalus membranaceus (Fisch) Bge. var. mongolicus (Bge.) Hsiao

Food Chemistry 128 (2011) 620–626

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Structural analysis and bioactivity of a polysaccharide from the roots of Astragalus membranaceus (Fisch) Bge. var. mongolicus (Bge.) Hsiao Yuge Niu a,b, Hengyu Wang a, Zhuohong Xie b, Monica Whent b, Xiangdong Gao a,⇑, Xian Zhang a, Shan Zou a, Wenbing Yao a,⇑, Liangli Yu b a b

School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, Jiangsu, China Department of Nutrition and Food Science, University of Maryland, College Park, 20742 MD, United States

a r t i c l e

i n f o

Article history: Received 6 January 2011 Received in revised form 2 March 2011 Accepted 9 March 2011 Available online 12 March 2011 Keywords: Astragalus polysaccharides Structural analysis NMR Antioxidant activity Bile acid binding

a b s t r a c t The Astragalus polysaccharide (APS) was extracted and purified from the roots of Astragalus membranaceus, and characterised for its chemical structure and potential health properties. The APS was composed of a-D-glc residues with the estimated equivalent dextran molecular weight of 2.07  104 Da. Periodate oxidation analysis, 1D and 2D NMR spectroscopy demonstrated that APS has repeating (1 ? 4)-linked backbone with a (1 ? 6)-linked branch every 10 residues. The APS possessed scavenging activities against hydroxyl radicals and hydrogen peroxide, and showed chelating effect on ferrous ions. The APS was also able to bind cholic and chenodeoxycholic acids in vitro. In addition, APS was able to stimulate activity of purified mouse B cells without promoting T cell proliferation. These data provided information for future development of APS as a nutraceutical. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Astragalus membranaceus (Fisch) Bge. var. mongolicus (Bge.) Hsiao, a specialty crop in China, has been used in preparing functional foods such as soups and teas. It has been recognised for potential detoxification, diuresis, antiperspirant, myogenic and antiageing effects (Qi et al., 2006; Wang, Shan, Wang, & Hu, 2006). Astragalus polysaccharides (APS) are considered as a group of possible bioactive components contributing to the beneficial effects of Astragalus. Several extractions of APS have been prepared and investigated for their monosaccharide compositions and molecular weights since the first one was purified from the roots of A. membranaceus in the 1980s (Fang & Wagner, 1988; Fang et al., 1982; Masashi, Noriko, & Naoko, 1992; Li, Chen, Wang, Tian, & Zhang, 2009). However, to our knowledge, its structure was not fully determined, and few studies have investigated APS for their antioxidant and bile acid-binding capacities. Reactive oxygen species (ROS), including superoxide anion  (O 2 ), hydroxyl radical ( OH) and hydrogen peroxide (H2O2), may be generated during the normal cell growth process. The involvement of ROS, in the pathogenesis of certain human diseases,

⇑ Corresponding authors. Tel./fax: +86 25 83271218 (W. Yao), tel.: +86 25 83271298; fax: +86 25 83271249 (X. Gao). E-mail addresses: [email protected] (X. Gao), [email protected] (W. Yao). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.03.055

including cancer, cardiovascular diseases, and other ageing associated health problems, is well recognised. For instance, the lipid peroxidation by ROS in the blood vessel wall is considered as one of the steps in atherogenesis. Dietary antioxidants have been shown to inhibit the activity of ROS, and are known for their potential in reducing the risk of cardiovascular diseases (CVD) (Rouanet et al., 2010; Tijburg, Wiseman, Meijer, & Weststrate, 1997). Recently, polysaccharides extracted from plants have provoked interest as sources of novel potential antioxidants, since published data indicate that plant polysaccharides in general have strong antioxidant activities (Wang & Luo, 2007; Xu et al., 2009; Zhong, Jin, Lai, Lin, & Jiang, 2010). The antioxidant ability may be related to the conformations of different polysaccharides. Liu et al. reported that two kinds of Ganoderma lucidum polysaccharides, a glucan and a heteropolysaccharide, exhibited antioxidant activities (Liu, Wang, Pang, Yao, & Gao, 2010). The glucan was more effective in free radical scavenging and Fe2+ chelating than the heteropolysaccharide. Additionally, polysaccharides with bile acid binding capacities have been recognised for their potential in reducing CVD risk since the binding may enhance the fecal elimination of bile acids, which may stimulate the conversion of cholesterol to bile acids and reduce the total and LDL cholesterol levels (Cheng, Blackford, Wang, & Yu, 2009; Kahlon & Smith, 2007; Liu, Zhang et al., 2010). In this study, the exact structural features of an APS were determined by combining the periodate oxidation analysis with 2D NMR analysis. Antioxidant activities and bile acid-binding activities of the APS were also evaluated.

Y. Niu et al. / Food Chemistry 128 (2011) 620–626

2. Experimental 2.1. Materials The roots of A. membranaceus Bge. var. mongolicus (Bge.) Hsiao were purchased from Shanxi Hunyuan Astragalus Co. (Shanxi, China). The DEAE-cellulose, Sephacryl S-400 and dextran standards of T-2000, T-500, T-70, T-40, and T-10 were obtained from Pharmacia Co. Ltd. (Uppsala, Sweden). Thirty percent hydrogen peroxide was purchased from Sinopharm chemical reagent Co. Ltd. Sodium salicylate, ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), and ascorbic acid were purchased from Sigma–Aldrich (St. Louis, USA). Cholestyramine, individual bile acids (cholic and chenodeoxycholic acids), diphorase, nicotinamide adenine dinucleotide, nitro blue tetrazolium, 3-R hydroxysterol dehydrogenase, concavalin A (ConA), lipopolysaccharide (LPS) from Escherichia coli and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO). RPMI-1640 medium dried powder and fetal calf serum were purchased from Invitrogen (New Zealand). Penicillin and streptomycin were ordered from Shenggong Co. (Shanghai, China). Nylon-wool column was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), while LAL chromogenic assay kit was from Zhanjiang A&C Biological Ltd. (Zhanjiang, China). Acid treated psyllium (Psy) was obtained from psyllium husks by acid hydrolysis as described previously (Cheng, Blackford, Wang, & Yu, 2009). All the other chemicals and reagents were of analytical grade.

621

phy (GLC) with a HP-5 capillary column (HP6820, Hewlett– Packard). The temperature program of the GC column was set as 110–280 °C at a rate of 5 °C/min with N2 as the carrier gas and myoinositol as the internal standard. A standard curve was prepared with standard monosaccharides derivatised and measured under the same procedure (Honda, Suzuki, Kakehi, Honda, & Takai, 1981). 2.5. Periodate oxidation of the APS APS (25 mg) was dissolved in 25 mL 0.015 mol/L NaIO4 solution and kept in dark at 4 °C. The reaction mixture was monitored for the absorption at 223 nm, and the reaction was completed when absorbance stopped to decrease after 72 h and terminated by addition of glycol (1 mL). The periodate product (5 mL) was sampled to calculate the yield of HCOOH by titration with 0.01 M NaOH. 2.6. NMR analysis of the APS For NMR analysis, H of the APS (50 mg) was replaced by deuterium in D2O. NMR measurement was recorded with a Bruker AV-500 spectrometer at 50 °C with TMS (dH = 0.00, dC = 0.00) in the inner tube. The 1H NMR, 13C NMR spectrum, 2D 1H–1H correlated spectroscopy (COSY), 1H–1H total correlation spectroscopy (TOCSY), 1H–13C heteronuclear single quantum coherence (HSQC), and 1H–13C heteronuclear multiple quantum coherence (HMBC) measurements were used to assign signals and determine the sequence of sugar residues.

2.2. Separation and purification of APS 2.7. In vitro antioxidant activities of APS The crude bioactive polysaccharide of A. membranaceus (APS) was extracted from the sliced rough roots (100 g) with hot water (600 mL) twice at 80 °C for 2 h. The combined supernatant was concentrated in a rotary evaporator under reduced pressure at 50 °C and filtered. The proteins in the filtrate were removed three times by Sevag reagent (Sevag, Lackman, & Smolens, 1983). After removal of the Sevag reagent, the extract was mixed with 3-fold volumes of 95% Et OH at 4 °C over 8 h and followed by centrifugation at 4000g for 10 min, and the crude polysaccharides were dissolved in distilled water, applied to a DEAE-cellulose column and eluted with water. Each 3 mL of eluate was collected at a flow rate of 20 mL/h and monitored for the presence of polysaccharides using the phenol–sulphuric acid method at 490 nm (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The collected fractions were concentrated and further loaded onto a Sephacryl-S400 column with water as the mobile phase, and the elutes were lyophilised to obtain the APS as a white powder. 2.3. Homogeneity and molecular weight of the APS The homogeneity and molecular weight of the purified APS was determined on an Agilent 1100 high-performance liquid chromatography (HPLC) system equipped with a Shodex SUGAR KS-805 column (8 mm ID  300 mm). The sample (20 lL, 0.2 wt.%) was injected into the column and eluted by distilled water at a flow rate of 1 mL/min. The molecular weight of the APS was estimated by the comparison to a calibration curve prepared with the T-series Dextran standards. 2.4. Monosaccharide composition analysis of the APS APS (5 mg) was hydrolysed in 1 mL 2 mol/L trifluoroacetic acid at 100 °C for 8 h. The product was reduced with NaBH4 at 65 °C for 1 h, and acetylated with mixture of pyridine and acetic anhydride (1:1, v/v) at 100 °C for 1 h, and then analysed by gas chromatogra-

2.7.1. Hydroxyl radical scavenging activity The hydroxyl radical scavenging capacity was examined using the method described previously (Smirnoff & Cumbes, 1989). For the control, sample was substituted by ascorbic acid. All values were determined in three replicates. The percentage of hydroxyl radical scavenging capacity was calculated according to the following equation:

Scavenging rate ¼ ½1  ðA1  A2 Þ=A0   100% where A0 was the absorbance of the control and A1 was the absorbance in the presence of the sample, A2 was the absorbance without sodium salicylate. 2.7.2. Hydrogen peroxide scavenging activity assay Hydrogen peroxide scavenging activity of the APS was measured according to a previously described procedure with minor modifications (Zhao, Xiang, Ye, Yuan, & Guo, 2006). The percentage of hydrogen peroxide scavenging was calculated as:

Scavenging rate ¼ ðV 0  V 1 Þ=V 0  100% where V0 was the volume of Na2S2O3 solution used to titrate the control in the presence of hydrogen peroxide (without sample), V1 was the volume of Na2S2O3 solution used in the presence of the sample. 2.7.3. Chelating effect against ferrous ions The chelating effect of APS was estimated using the method of Chua, Tung, and Chang (2008). For the control, sample was substituted with EDTA. The chelating activity was calculated as

Chelating rate ¼ ½1  ðA1  A2 Þ=A0   100% where A0 was the absorbance of the control (without sample) and A1 was the absorbance in the presence of the sample, A2 was the absorbance without ferrozine.

622

Y. Niu et al. / Food Chemistry 128 (2011) 620–626

2.8. Bile acid-binding capacity

3. Results and discussion

The bile acid-binding capacity of the APS was determined according to a previously reported laboratory protocol (Liu, Zhang et al., 2010). For simulating gastric conditions, 50 mg of each sample was treated with 0.5 mL HCl (0.01 mol/L), incubated at 37 °C for 1 h with continuous shaking (50 rpm). The pH of the mixture was adjusted to 7.0 using 0.05 mL NaOH (0.1 mol/L) and added 2.5 mL bile acid stock solution (400 lmol/L in 0.01 mol/L phosphate buffer, pH 7.0). The mixture was incubated for another hour at 37 °C, and centrifuged at 6000 rpm for 10 min. The supernatant was collected for bile acid determination. Quantification of the unbound bile acids was conducted using a commercial kit from Sigma–Aldrich (St. Louis, MO). The absorbance of each mixture was measured at 530 nm with cholestyramine as a positive control. The levels of unbound bile acids were determined using a standard curve prepared with each of the two standard bile acids, cholic and chenodeoxycholic acids. The bile acid-binding capacity (mg/g sample) was calculated against 0.01 mol/L phosphate buffer (pH 7.0). Duplicate tests were performed for each sample against each bile acid.

3.1. Isolation and characterisation of APS

2.9. In vitro immunomodulatory activity of the APS 2.9.1. Animals Female BALB/c mice between 6 and 8 weeks old (weight: 20.0 ± 2.0 g) were purchased from the Experimental Animal Center of Yangzhou University, China. The mice were fed at room temperature, with free access to standard rodent chow and water. 2.9.2. Preparation of mouse splenocytes, and spleen T and B cells Mouse splenocytes were prepared according to the method described previously (Shao, Dai, Xu, Lin, & Gao, 2004). Briefly, spleens from BALB/c mice were gently smashed. Red blood cells were depleted by treatment with Tris–NH4Cl buffer (Tris 7.47 mg/ml, NH4Cl 2.6 mg/ml). The cells were subsequently suspended in RPMI-1640 medium, plated in petri dishes, and incubated in a humidified 5% CO2 atmosphere at 37 °C for 6 h. The non-adherent cells were collected as splenocytes. The spleen T and B cells were selected from mouse splenocytes using nylon-wool column following the manufacturer’s instructions. According to the analysis of flow cytometry, the purities of T cells and B cells were greater than 90%. 2.9.3. Proliferation of splenocytes, and T and B cells Mouse splenocytes, and T and B cells were respectively cultured in 96-well microplate at a density of 2  106 cells/well in RPMI-1640 medium containing 10% FCS, supplemented with 60 mg/L penicillin, 100 mg/L streptomycin. Then the cells were stimulated with the APS at different concentrations (25–250 lg/mL) for 48 h in a 5% CO2 incubator, followed by incubation with MTT (5 mg/mL) for another 4 h. Hundred microliters DMSO was added to each well to fully dissolve the formazan. The absorbance at 570 nm was determined in a multiskan spectrum (Thermo, USA). ConA (2 lg/ml) and LPS (4 lg/ ml) were the positive control for T and B cells, respectively (Liu et al., 2008). 2.10. Statistical analysis

Isolated by the hot water, the crude polysaccharide precipitation was determined to be 8.81 ± 0.17 g glucose equivalents/ 100 g dry roots of A. membranaceus, of which the protein content ratio was 1.10 ± 0.11%. After being purified using DEAE-cellulose and Sephacryl S-400 columns, APS (0.131 g) was obtained from the crude polysaccharides (0.5 g) as the second major peak eluted by distilled water. HPLC analysis indicated that the APS was a homogeneous polysaccharide with a purity of 98.5%. The APS had no absorption at 280 or 260 nm in the UV spectrum, indicating that it contained no protein or nucleic acid. Its equivalent dextran molecular weight was estimated 2.07  104 Da based on the equation of the standard curve made with a group of dextran standards. According to GLC analysis of monosaccharide composition, the APS was composed of glucose as its only monosaccharide. 3.2. Structural analysis of the APS As the results of periodate oxidation, the ratio of the consumption of HIO4 at 0.72 mol/mol Glc and the production of HCOOH at a level of 0.093 mol/mol Glc suggested that (1 ? 6)-Glcp was 9.30% of the sugar residues. NMR spectroscopy was employed to confirm the structural characterisation of homogeneous polysaccharides. The 13C NMR spectrum of the APS contained three anomeric protons, which were assigned as residue A (102.6 ppm), residue B (102.4 ppm) and residue C (101.4 ppm), respectively. Accordingly, 1H spectrum had the three types of anomeric protons of the residues at 4.95– 5.40 ppm. Based on their chemical shifts and values of J1,2, residues A, B and C were supposed to have a-configurations (Cui et al., 2008; Roy, Maiti, Mondal, Das, & Islam, 2008; Yang & Zhang, 2009). The presence of a form glycosidic linkages was also supported by the relatively high positive value of optical rotation [a]D20 value of +141.4°. By the means of 2D NMR spectrums, the entire assignment of the 1H and 13C chemical shifts of the APS was achieved and presented in the Table 1. In addition, Fig. 1 reports the HMBC spectrum of the APS. Taking into account of the 13C NMR chemical shift data, the HMBC spectrum showed strong cross-peaks between 1H and 13C peak in different residues and could be assigned as follows: A H-1 (d 5.34) and B C-4 (d 74.0), A H-1 (d 5.34) and C C6 (d 76.1), BH-1 (d 5.38)and B C-4 (d 74.0). Taken together with the results from the periodate oxidation analysis, the chemical structure of the APS was determined as a (1 ? 4)-linked dextran backbone with a (1 ? 6)-linked branch every 10 residues as shown in Fig. 2. 3.3. The antioxidant activity of the APS 3.3.1. Hydroxyl radical scavenging activity Hydroxyl radical is the most reactive oxygen radical known in chemistry. It can severely damage almost any biological molecule Table 1 Chemical shifts (ppm) of 1H and Residue

Data were reported as mean ± SD for each experiment. ANOVA and Tukey’s tests were performed (SPSS for Windows, Version Rel. 10.0.5., 1999, SPSS Inc., Chicago, IL) to identify differences among means. When significant differences occurred, treatment groups were compared with the vehicle control using a Dunnett’s two-tailed t-test. Statistical significance was declared at P < 0.05.

A: a-D-Glcp-(1 ? B: ? 4)-a-D-Glcp-(1 ? C: ? 4,6)-a-D-Glcp-(1 ?

H: C: H: C: H: C:

13

C NMR signals of APS. 1

2

3

4

5

6

5.34 102.6 5.38 102.4 4.96 101.4

3.62 74.5 3.64 74.3 3.60 74.6

3.97 76.1 3.95 76.1 3.68 75.7

3.85 74.0 3.82 74.0 3.40 72.2

3.74 75.5 3.71 75.5 3.67 73.2

3.43 72.2 3.41 72.2 4.01 76.1

Y. Niu et al. / Food Chemistry 128 (2011) 620–626

623

Fig. 1. 2D 1H–13C HMBC spectra of APS.

Fig. 2. The repeating unit of APS.

it attacks in living cells such as proteins, nucleic acids, and polyunsaturated fatty acids. It is involved in ageing, and the development of cancer and several other diseases (Valentão et al., 2003). In the present study, the hydroxyl radical was generated by Fenton reaction. The scavenging rate of the APS against hydroxyl radicals was demonstrated in a dose-dependent manner (Fig. 3a). The APS at the final concentration of 20 mg/mL scavenged almost 100% radicals in the reaction mixture as that of ascorbic acid in 5 min, and the EC50 value was 0.63 mg/mL. Li et al. also reported that the hydroxyl radical scavenging activity of another Astragalus dextran increased with the increasing concentration, although it had a lower scavenging rate than APS at the same concentration (Li, Chen, Wang, Tian, & Zhang, 2010). It was suggested that polysaccharides with hydroxyl radical scavenging capacity had the same structure feature. Each of them had one or more hydroxyl groups in their monosaccharide block, and the scavenging ability was related to the number of active hydroxyl groups in the molecules (Li, Jiang, Xue, & Chen, 2002). According to the current results, the APS

may serve as a hydroxyl radical scavenger in biological or food systems. 3.3.2. Hydrogen peroxide scavenging activity Hydrogen peroxide can be formed in vivo by oxidising enzymes such as superoxide dismutase. It can cross membranes and slowly oxidise a number of cellular molecules. The ability of the APS to scavenge hydrogen peroxide is shown in Fig. 3b and compared with that of ascorbic acid. Hydrogen peroxide scavenging rates of the APS were dependent on its concentration. The scavenging rate of the APS at a concentration of 10 mg/mL was 46.2% while the rate of ascorbic acid at the same concentration was 70.5%, suggesting the hydrogen peroxide scavenging activity of the APS. Although hydrogen peroxide itself is not the most reactive oxygen species, it might be toxic in vivo because it is involved in hydroxyl radical formation in the cells. Addition of hydrogen peroxide to cells in culture can lead to transition metal ion-dependent OH radicals-mediated oxidative DNA damage. Levels of

624

Y. Niu et al. / Food Chemistry 128 (2011) 620–626

biological and food systems, the high chelating effects of APS would be beneficial. 3.4. Bile acid-binding properties of APS Binding to polymers may enhance the elimination of bile acids, which may promote the conversion of cholesterol to bile acids and reduce the total plasma and LDL cholesterol levels, and consequently reduce the risk of cardiovascular diseases (Cheng et al., 2009; Zhou, Xia, Zhang, & Yu, 2006). It has been reported that the cholesterol-lowering effect of psyllium, a dietary fibre, might be partially due to its ability to bind bile acids and stimulate the cholesterol metabolism. (Cheng et al., 2009; Trautwein, Kunath-Rau, & Erbersdobler, 1999). Cholestyramine resin, a quaternary ammonium anion exchange resin with a polystyrene backbone, is commercially used as an adjunctive therapy for reducing plasma cholesterol level through the bile acid-binding mechanism in the intestinal system (Shin, Lee, Lee, & Lee, 2005). As seen in Fig. 4, the bile acid-binding capacity of the APS was more than five times of that for psyllium on cholic acid (CA), and three times of that for psyllium against chenodeoxycholic acid (CDCA), on a per same weight basis. These are the two primary bile acids synthesised in the liver. The United States Food and Drug Administration allow a health claim for foods containing soluble fibre (including psyllium) due to their cholesterol-lowering benefits. Psyllium is already a widely used nutraceutical due to its cholesterol-lowering properties (Theuwissen & Mensink, 2008). The APS has demonstrated greater bile-acid binding properties per weight than psyllium, and could potentially be included in foods in a lower amount than psyllium. It may therefore be better tolerated than natural psyllium as a cholesterol-lowering nutraceutical. Additionally, the binding capacity of the APS was almost half of that of cholestyramine resin on a same per weight basis. This data suggested that APS has possible application as cholesterol-lowering adjunctive therapy. 3.5. The immunomodulatory activity of APS 3.5.1. Determination of endotoxin contamination in APS LPS is a typical thymus-independent antigen (TI-Ag) which can cause many immunomodulating effects on lymphocytes, such as B cell proliferation, cytokine production and Ig secretion. It was

Fig. 3. The antioxidant activity of APS in vitro. (a) The hydroxyl radical scavenging activities of APS; (b) the hydrogen peroxide scavenging activities of APS and (c) chelating abilities of APS to ferrous ions; data are presented as mean values (n = 3). Vertical bars represent the SD.

hydrogen peroxide at or below about 20–50 mg/cell seem to have limited cytotoxicity to many cell types (Gülcin, Huyut, Elmastas, & Aboul-Enein, 2010). Thus, removal of hydrogen peroxide is very important for protection of cells from oxidative damage. 3.3.3. Chelating effect on ferrous ions Transition metals, such as ferrous iron (Fe2+), can facilitate the generation of ROS. The chelating capacity of polysaccharides against Fe2+ can be an important approach for retarding metalcatalysed oxidation. The chelating effect of APS against Fe2+ was shown in a dose-dependent matter (Fig. 3c). At 10 mg/mL the chelating rate reached a plateau of 96.0%, and the EC50 value was about 1.15 mg/mL. Meanwhile, the chelating effect of EDTA at the same concentration was 99.7%, and that of ascorbic acid was only 42.1%. Since ferrous ions are the most effective prooxidants in both

Fig. 4. Bile acid-binding properties of the APS. CA and CDCA represent cholic and chenodeoxycholic acids respectively. Data are expressed as mean ± SD. Vertical bars represent the SD. Letters ‘‘a’’, ‘‘b’’ and ‘‘c’’ are used to designate the significant difference of CA binding capacities, while letters ‘‘z’’, ‘‘y’’ and ‘‘x’’ indicate the significant difference of CDCA binding capacities among the samples (P < 0.05).

Y. Niu et al. / Food Chemistry 128 (2011) 620–626

therefore important to check whether the APS preparations used in our study had any LPS contamination before further investigation of its immunomodulation activities. According to the manufacturer’s instruction of LAL chromogenic assay kit, no LPS contamination in APS was detected.

3.5.2. Effects of psyllium on proliferation of splenocytes, and T and B cells Some polysaccharides are known to stimulate immune function in both in vitro and animal models (Liu et al., 2008; Volman, Ramakers, & Plat, 2008; Zhang et al., 2010). From Fig. 5a, the APS was shown to increase the proliferation of splenocytes of BABL/c mouse significantly compared with the control (P < 0.01) in a dose-dependent manner at the concentrations from 0 to 200 lg/mL. Contrasting Fig. 5b with Fig. 5c, the APS had a strong effect on increasing proliferation of purified B cells with significant differences (P < 0.01) at the concentrations from 100 to 250 lg/mL,

625

while there was no effect on proliferation of T cells (Fig. 5c). The results confirmed that purified mouse splenic B cells were the main responders to the stimulation of APS in vitro. This may be valuable for research purposes when B cells are required in a greater quantity than T cells. A study by Zhang et al. (2010) demonstrated that a proteoglycan extract from G. lucidum also increased proliferation of mouse splenic B cells in a greater amount than T cells. Conversely, Liu et al. (2008) showed that polysaccharide extracted from sea urchin eggs stimulated higher proliferation of T cells compared with B cells. It can be inferred that various polysaccharides have different activity on splenic cells, but the mechanism for this stimulation is not yet well understood. 4. Conclusion On the basis of the previously mentioned results, it was concluded that APS, as a homogeneous bioactive polysaccharides extracted from the sliced roots of A. membranaceus by hot water and purified by DEAE-cellulose and Sephacryl-S400 columns, was confirmed to be composed of glucose with the particular structure of (1 ? 4)-linked backbone with every 10 residues a (1 ? 6)-linked branch. As a refined polysaccharide, APS was found to have antioxidant activity including hydroxyl radical scavenging activity, hydrogen peroxide scavenging activity, and chelating effect on ferrous ions. According to the Fenton-type reaction (Fe2+ + H2O2 ? Fe3+ + HO + OH), the APS can scavenge both hydroxyl radical and hydrogen peroxide severely, and also can efficiently bind ferrous iron in vitro. Moreover, APS has ability of bile acid-binding in vitro, which is another important parameter related to its potential for reducing the risk of cardiovascular diseases. As a refined polysaccharide, the APS also had a specific effect on proliferation of purified mouse B cells in splenocytes. Further study is warranted to investigate the health beneficial properties of APS in vivo on animal models. Acknowledgements The work was supported by National Department Public Benefit Research Foundation of China (No. 200707009). References

Fig. 5. The immunomodulatory activity of APS in vitro. (a) APS-induced proliferation of Mouse splenocytes; (b) APS-induced proliferation of splenic B cells and (c) APS-induced proliferation of splenic T cells. Values are means ± SD, n = 3. ⁄⁄indicates a significant difference from the negative control for each APS concentration (P < 0.01).

Cheng, Z. H., Blackford, J., Wang, Q., & Yu, L. L. (2009). Acid treatment to improve psyllium functionality. Journal of Functional Foods, 1, 44–49. Chua, M. T., Tung, Y. T., & Chang, S. T. (2008). Antioxidant activities of ethanolic extracts from the twigs of Cinnamomum osmophleum. Bioresource Technology, 99, 1918–1925. Cui, H. X., Liu, Q., Tao, Y. Z., Zhang, H. F., Zhang, L. N., & Ding, K. (2008). Structure and chain conformation of a (1 ? 6)-a-D-glucan from the root of Pueraria lobata (Willd.) Ohwi and the antioxidant activity of its sulfated derivative. Carbohydrate Polymers, 74, 771–778. 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, 28, 350–356. Fang, J. N., & Wagner, H. (1988). The chemical structure of polyglucosan of Astragalus membranaceus (in Chinese). ACTA CHINIC CINICA, 46, 1101–1104. Fang, S. D., Chen, Y., Xu, X. Y., Ye, C. Q., Zhai, S. K., & Shen, M. L. (1982). Studies of the active principles of Astragalus Mongholicus Bunge I. Isolation, characterization and biological effect of its polysaccharides (in Chinese, with English abstract). Organic Chemistry, 2, 26–31. Gülcin, I., Huyut, Z., Elmastas, M., & Aboul-Enein, H. Y. (2010). Radical scavenging and antioxidant activity of tannic acid. Arabian Journal of Chemistry, 3, 43–53. Honda, S., Suzuki, S., Kakehi, K., Honda, A., & Takai, T. (1981). Analysis of the monosaccharide compositions of total non-dialyzable urinary glycoconjugates by the dithioacetal method. Journal of Chromatography, 226, 341–350. Kahlon, T. S., & Smith, G. E. (2007). In vitro binding of bile acids by bananas, peaches, pineapple, grapes, pears, apricots and nectarines. Food Chemistry, 101, 1046–1051. Li, R., Chen, W. C., Wang, W. P., Tian, W. Y., & Zhang, X. G. (2009). Extraction, characterization of Astragalus polysaccharides and its immune modulating activities in rats with gastric cancer. Carbohydrate Polymers, 78, 738–742.

626

Y. Niu et al. / Food Chemistry 128 (2011) 620–626

Li, R., Chen, W. C., Wang, W. P., Tian, W. Y., & Zhang, X. G. (2010). Antioxidant activity of Astragalus polysaccharides and antitumour activity of the polysaccharides and siRNA. Carbohydrate Polymers, 82, 240–244. Li, W., Jiang, X., Xue, P., & Chen, S. (2002). Inhibitory effects of chitosan on superoxide anion radicals and lipid free radicals. Chinese Science Bulletin, 47, 887–889. Liu, C. H., Xi, T., Lin, Q. X., Xing, Y. Y., Ye, L., Luo, X. G., et al. (2008). Immunomodulatory activity of polysaccharides isolated from Strongylocentrotus nudus eggs. International Immunopharmacology, 8, 1835–1841. Liu, W., Wang, H. Y., Pang, X. B., Yao, W. B., & Gao, X. D. (2010). Characterization and antioxidant activity of two low-molecular-weight polysaccharides purified from the fruiting bodies of Ganoderma lucidum. International Journal of Biological Macromolecules, 46, 451–457. Liu, W., Zhang, B. C., Wang, Q., Xie, Z. H., Yao, W. B., Gao, X. D., et al. (2010). Effects of sulfation on the physicochemical and functional properties of psyllium. Journal of Agricultural Food Chemistry, 58, 172–179. Masashi, T., Noriko, S., & Naoko, O. (1992). A reticuloendothelial system-activating glycan from the roots of Astragalus membranaceus. Phytochemistry, 31, 63–66. Qi, L. W., Yu, Q. T., Li, P., Li, S. L., Wang, Y. X., Sheng, L. H., et al. (2006). Quality evaluation of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors. Journal of Chromatography A, 1134, 162–169. Rouanet, J. M., Décordé, K., Rio, D. D., Auger, C., Borges, G., Cristol, J. P., et al. (2010). Berry juices, teas, antioxidants and the prevention of atherosclerosis in hamsters. Food Chemistry, 118, 266–271. Roy, S. K., Maiti, D., Mondal, S., Das, D., & Islam, S. S. (2008). Structural analysis of a polysaccharide isolated from the aqueous extract of an edible mushroom, Pleurotus sajor-caju, cultivar Black Japan. Carbohydrate Research, 343, 1108–1113. Sevag, M. G., Lackman, D. B., & Smolens, J. (1983). The isolation the components of streptococcal nucleoproteins in nucleoproteins in serologic ally active form. Journal of Biological chemistry, 124, 425–436. Shao, B. M., Dai, H., Xu, W., Lin, Z. B., & Gao, X. M. (2004). Immune receptors for polysaccharides from Ganoderma lucidum. Biochemical and Biophysical Research Communications, 323, 133–141. Shin, M. S., Lee, S., Lee, K. Y., & Lee, H. G. (2005). Structural and biological characterization of aminated-derivatized oat b-glucan. Journal of Agricultural Food Chemistry, 53, 5554–5558. Smirnoff, N., & Cumbes, Q. J. (1989). Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry, 28(4), 1057–1060.

Tijburg, L. B. M., Wiseman, S. A., Meijer, G. W., & Weststrate, J. A. (1997). Effects of green tea, black tea and dietary lipophilic antioxidants on LDL oxidisability and atherosclerosis in hypercholesterolaemic rabbits. Atherosclerosis, 135, 37–47. Trautwein, E. A., Kunath-Rau, A., & Erbersdobler, H. F. (1999). Increased fecal bile acid excretion and changes in the circulating bile acid pool are involved in the hypocholesterolemic and gallstone preventive actions of psyllium in hamsters. Journal of Nutrition, 129, 896–902. Theuwissen, E., & Mensink, R. P. (2008). Water-soluble dietary fibers and cardiovascular disease. Physiology and Behavior, 94, 285–292. Valentão, P., Fernandes, E., Carvalho, F., Andrade, P. B., Seabra, R. M., & Bastos, M. L. (2003). Hydroxyl radical and hypochlorous acid scavenging activity of small Centaury (Centaurium erythraea) infusion. A comparative study with green tea (Camellia sinensis). Phytomedicine, 10, 517–522. Volman, J. J., Ramakers, J. D., & Plat, J. (2008). Dietary modulation of immune function by b-glucans. Physiology and Behavior, 94, 276–284. Wang, S. C., Shan, J. J., Wang, Z. T., & Hu, Z. B. (2006). Isolation and structural analysis of an acidic polysaccharide from Astragalus membranaceus (Fisch.) Bunge. Journal of Integrative Plant Biology, 48, 1379–1384. Wang, Z. J., & Luo, D. H. (2007). Antioxidant activities of different fractions of polysaccharide from Gynostemma pentaphyllum Makino. Carbohydrate Polymers, 68, 54–58. Xu, W. T., Zhang, F. F., Luo, Y. B., Ma, L. Y., Kou, X. H., & Huang, K. L. (2009). Antioxidant activity of a water-soluble polysaccharide purified from Pteridium aquilinum. Carbohydrate Research, 344, 217–222. Yang, L. Q., & Zhang, L. M. (2009). Chemical structural and chain conformational characterization of some bioactive polysaccharides isolated from natural sources. Carbohydrate Polymers, 76, 349–361. Zhang, J., Tang, Q., Zhou, C., Jia, W., DaSilva, L., & Nguygen, L. D. (2010). GLIS, a bioactive proteoglycan fraction from Ganoderma lucidum, displays anti-tumour activity by increasing both humoral and cellular immune response. Life Sciences, 87, 628–637. Zhao, G. R., Xiang, Z. J., Ye, T. X., Yuan, Y. J., & Guo, Z. X. (2006). Antioxidant activities of Salvia miltiorrhiza and Panax notoginseng. Food Chemistry, 9, 767–774. Zhong, X. K., Jin, X., Lai, F. Y., Lin, Q. S., & Jiang, J. G. (2010). Chemical analysis and antioxidant activities in vitro of polysaccharide extracted from Opuntia ficus indica Mill cultivated in China. Carbohydrate Polymers, 82, 722–727. Zhou, K. Q., Xia, W. S., Zhang, C., & Yu, L. L. (2006). In vitro binding of bile acids and triglycerides by selected chitosan preparations and their physico-chemical properties. LWT – Food Science and Technology, 39, 087–1092.