Structural characterization and antioxidant activity of a low-molecular polysaccharide from Dendrobium huoshanense

Fitoterapia 91 (2013) 247–255

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Structural characterization and antioxidant activity of a low-molecular polysaccharide from Dendrobium huoshanense Chang-Cheng Tian, Xue-Qiang Zha, Li-Hua Pan, Jian-Ping Luo ⁎ School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, PR China

a r t i c l e

i n f o

Article history: Received 27 July 2013 Accepted in revised form 24 September 2013 Available online 3 October 2013 Keywords: Dendrobium huoshanense Polysaccharide Structure Antioxidant activity

a b s t r a c t The present study aimed at investigating the structural features and antioxidant activities of a polysaccharide fraction (DHP1A) obtained from Dendrobium huoshanense, a precious herb medicine in China. DHP1A mainly consisted of mannose (Man), glucose (Glc) and a trace of galactose (Gal), with a molecular weight of 6700 Da. Its backbone contained (1 → 4)-linked α-D-Glcp, (1 → 6)-linked α-D-Glcp and (1 → 4)-linked β-D-Manp, with a branch of terminal β-D-Galp. The in vitro antioxidant evaluation revealed that DHP1A had a remarkable inhibition effect on the FeCl2-induced lipid peroxidation. Furthermore, DHP1A pretreatment decreased the production of malondialdehyde (MDA), and restored the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as the level of glutathione (GSH) in the livers of CCl4-treated mice. These results suggested that DHP1A was a potential antioxidant component in D. huoshanense. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Natural polysaccharides, existing in animals, plants, fungi and algae, are a type of abundant and useful resource, which are involved in materials, foods and medicines. It has been well documented that the polysaccharides possess potent and beneficial immunomodulating functions, such as antiinflammation, anti-tumor and innate immunity enhancement [1–3]. Some polysaccharides have been proven to be natural antioxidants as evidenced by their capacities of scavenging free radicals in vitro [4,5] and inhibition effects on the oxidative stress in vivo [6,7]. It is well known that the oxidative stress plays a key role in diverse pathogenesis, and the supplement of antioxidant polysaccharides may be an ideal alternative for preventing and curing some diseases [8–10]. In recent years, investigations focusing on the natural antioxidant polysaccharides have attracted more and more attention. Dendrobium huoshanense is a perennial epiphytic orchid species and mainly distributed in China, which has been used as a precious folk medicine since ancient times. Its stem, as ⁎ Corresponding author. Tel./fax: +86 551 62901539. E-mail address: [email protected] (J.-P. Luo). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.09.018

a functional beverage, exerts remarkable restorative effects, including nourishing the stomach, preventing the cataract, reliving the throat inflammation, promoting the secretion of body fluid and enhancing the body immunity [11]. Pharmacological researches have shown that there exist some active components in D. huoshanense, such as alkaloids, glycosides and polysaccharides [12]. Among them, the polysaccharides are increasingly regarded as the most important active component. Some polysaccharide fractions from D. huoshanense could activate the lymphocytes to secrete various cytokines in vitro, exhibiting the potent immunomodulating activities [13,14]. Besides, crude D. huoshanense polysaccharides (DHP) had been demonstrated to possess great potential as natural antioxidants by the in vitro experiments [15]. The oral administration of crude DHP could protect the diabetic cataract caused by streptozotocin through decreasing the expression of NO and iNOS [16], and restoring the activities of systematic antioxidant enzymes [17]. However, these investigations on the antioxidant activity were focused on the crude DHP, and our understanding on the specific antioxidant polysaccharide fraction was still limited [18]. In the present study, a lowmolecular polysaccharide fraction (DHP1A) was screened and purified from the crude DHP, and its structural characterization

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was carried out by high performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC–MS), Fourier transform-infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR). Moreover, the in vitro and in vivo antioxidant activities of DHP1A were evaluated. 2. Materials and methods

and G5000PWXL (7.8 × 300 mm) connected in series. The temperature of the column oven was set at 30 °C and the column was eluted with double distilled water at the flow rate of 0.5 mL/min. The concentration of sample was 1.0 mg/mL and each injection volume was 20 μL. A series of standard dextrans (5.0, 25.0, 80.0, 150.0, 470.0, and 610.0 kDa) were analyzed in the same condition and used for the calibration of standard curve.

2.1. Materials and reagents D. huoshanense was obtained by micropropagation in our lab as described in the previous methods [19]. DEAE-cellulose D-52 and Sephadex G-100 were purchased from Amersham Pharmacia Biotech (London, England) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Both dextrans (5.0, 25.0, 80.0, 150.0, 470.0, and 610.0 kDa) and monosaccharides (D-glucose, D-galactose, D-mannose, D-xylose, L-rhamnose, and D-arabinose) were purchased from Fluka (St. Louis, MO, USA). 2,2-Diphenyl1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GPx), malondialdehyde (MDA) and glutathione (GSH) kits were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.2. Preparation of DHP1A from D. huoshanense The dried D. huoshanense was ground to powder and immersed in 80% ethanol (1:100, g/mL) for three days to remove the pigments, lipids and small molecular compounds. The resulting material was extracted twice with distilled water (1:100, g/mL) for 2 h at 65 °C. The extract was concentrated using a vacuum rotary evaporator and centrifuged at 10,000 rpm for 10 min to give the supernatant, which was subsequently precipitated by adding four volumes of 95% ethanol for 24 h at room temperature. After centrifugation (10,000 rpm for 5 min), the precipitates were dissolved in distilled water again, and deproteinized six times by the Sevag method [20]. The polysaccharide solution was further dialyzed against tap water (molecular weight cutoff of 3500 Da) and freeze-dried to afford the crude polysaccharide, named DHP. The crude DHP, dissolving in distilled water at a concentration of 10 mg/mL, was loaded on a DEAE-cellulose anion exchange column (2.6 × 40 cm) and eluted with distilled water, 0.1, 0.2 and 0.3 M NaCl solution successively, to give four corresponding fractions of DHP1, DHP2, DHP3 and DHP4. Among these fractions, DHP1 was further fractionated using a Sephadex G-100 gel chromatographic column (2.6 × 60 cm), which was eluted with double distilled water at the flow rate of 0.2 mL/min. The eluate was collected by a fraction collector (2 mL/tube), and the total sugar content of each tube was measured by the phenol–sulfuric acid methods [21]. Following the selective collection, concentration and freeze-drying, a purified fraction (DHP1A) was prepared. 2.3. Structural characterization of DHP1A 2.3.1. Homogeneity and molecular weight of DHP1A The molecular weight of DHP1A was determined by a HPLC system (1260 infinity, Agilent Technologies) equipped with a refractive index detector (RID). The separation action was performed with TSK-GEL column G4000PWXL (7.8 × 300 mm)

2.3.2. Monosaccharide compositions DHP1A (5 mg) was dissolved in 2 M trifluoroacetic acid (TFA, 3 mL) and hydrolyzed at 120 °C for 4 h. Then, the superfluous TFA in the hydrolyzate was removed via co-evaporation with methanol (3 mL) on a rotary evaporator for at least five times. The resulting hydrolyzate was converted into alditol acetates through reaction with NaBH4 (30 mg) at room temperature (RT) for 3 h and anhydride–pyridine (1:1, v/v; 3 mL) at 100 °C for 1 h successively. After drying, the derivatives were dissolved in chloroform and analyzed by GC (GC–MS QP2010 system, Shimadzu) equipped with a flame ionization detector (FID) and a HP-5 capillary column (30 m × 0.32 mm × 0.25 μm, Agilent). The injector and detector temperatures were 270 and 300 °C, respectively. The flow rate of carrier gas (N2) was set at 0.3 mL/min. The temperature of the column oven was programmed as follows: (1) 150 °C for 1 min; (2) increasing to 180 °C at 10 °C/min; and (3) increasing to 250 °C at 4 °C/min.

2.3.3. Infrared spectrum analysis The dried polysaccharide sample was pressed into the pellets with KBr powder, and then scanned with a Fourier transform infrared (FT-IR) spectrometer (Nicolet 67, Thermo Nicolet) in the range of 4000–400 cm−1.

2.3.4. Methylation analysis DHP1A (5 mg) was methylated with anhydrous sodium hydroxide and methyl iodide in dimethyl sulfoxide for six times according to the previous method [22]. The resulting products were hydrolyzed with 2 M TFA (3 mL) for 4 h at 110 °C, and further converted into the partially methylated alditol acetates via reaction with NaBH4 and anhydride–pyridine successively. GC–MS (QP2010 system, Shimadzu) equipped with a HP-5 capillary column (0.25 μm × 0.32 mm × 30 m) was used for identification of the resulting derivatives. The temperature of the column oven was initially cooled to 50 °C for 1 min, and then raised to 250 °C at 10 °C/min. The partially methylated alditol acetates were identified by the fragment ions, and their molar ratios were calculated by the corresponding peak areas.

2.3.5. Nuclear magnetic resonance (NMR) analysis DHP1A (30 mg) was dried in a vacuum oven (60 °C) for 8 h and then dissolved in D2O. The 1H, 13C, heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) spectra of DHP1A were performed and recorded on a nuclear magnetic resonance spectrometer (400.13 MHz, Bruker, Switzerland) at 50 °C. The chemical shift of HOD (δ 4.70) was used as the internal reference for 1 H NMR spectrum.

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Fig. 1. Spectral analysis of DHP1A. High performance liquid chromatography (A); infrared spectrum (B); gas chromatograms of standard monosaccharides (C) and DHP1A derivatives (D). 1) Rhamnose; 2) arabinose; 3) xylose; 4) mannose; 5) glucose; 6) galactose.

2.4. Antioxidant activities of DHP1A in vitro 2.4.1. DPPH radical scavenging activity of DHP1A The DPPH radical scavenging activity of DHP1A was determined according to the previous method [23] with some modifications. The reaction mixture contained 2.0 mL of DPPH methanol solution (0.1 mM) and 1.0 mL of DHP1A solution (dissolving in double water at 0.3, 0.6, 1.5, 3.0 and 6.0 mg/mL). After shaking well, the mixture was kept at room temperature for 30 min, and the absorbance at 517 nm was measured. Double distilled water was used in the control group. Vitamin C and dextran (20.0 kDa, Shanghai, China) were used as positive and negative reference substances, respectively. The measurement of each sample was carried out in triplicate. The scavenging rate was calculated by the following formula:   Scavenging rateð% Þ ¼ Acontrol −Asample =Acontrol  100 where Acontrol is the absorbance value of the control group, and Asample is the absorbance value of the group treated with sample. 2.4.2. Inhibition activity of DHP1A on lipid peroxidation The inhibition activity of DHP1A on lipid peroxidation was evaluated according to thiobarbituric acid method as described in the literature [24] with some modifications. The reaction mixture, including 1.0 mL of DHP1A solution (0.5– 10.0 mg/mL), 1.0 mL of liver homogenate (1%, w/v), 50 μL of

FeCl2 solution (0.5 mM) and 50 μL of H2O2 (0.5 mM), was incubated at 37 °C for 60 min, and then was terminated by adding 1.5 mL of trichloroacetic acid (20%, w/v). The resulting mixture was mixed with 1.5 mL of thiobarbituric acid (0.8%, w/v) and incubated for another 60 min at 95 °C. After centrifugation at 4000 rpm for 10 min, the supernatant was measured at 532 nm. The inhibition effect was calculated by the formula as described in Section 2.4.1. 2.5. Effects of DHP1A on oxidative stress in vivo 2.5.1. Experiment design Sixty male Kunming mice (23 ± 2 g) were purchased from the Experimental Animal Center of Anhui Medical University, China. They were housed in an air-conditioned room (25 ± 2 °C) with a normal day/night light cycle, and fed with rodent chow and tap water. Animal care and procedure were carried out in accordance with the Guideline for animal experimentation of Hefei University of Technology (Hefei, China). After acclimatizing for a week, the mice were divided into 6 groups randomly (10 mice/group): group 1, control group; group 2, CCl4 group; group 3, CCl4 plus silymarin (25 mg/kg body weight, BW); group 4, CCl4 plus dextran (200 mg/kg BW); group 5, CCl4 plus DHP1A (100 mg/kg BW) and group 6, CCl4 + DHP1A (200 mg/kg BW). All samples including DHP1A, silymarin and dextran were dissolved in distilled water. Groups 1 and 2 were administered orally with distilled water at

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2.5.2. Effects of DHP1A on MDA, T-SOD, CAT, GPx and GSH The supernatant obtained from the liver tissue homogenate was used to evaluate the levels of T-SOD, MDA, CAT, GPx and GSH by the corresponding commercial clinical kits according to the manufacturer's instructions.

Table 1 Methylation analysis of DHP1A from Dendrobium huoshanense. Methylated sugar

Mass fraction

Molar Linkage type ratio

0.9 2,3,4,6-Me4-Galp 43, 71, 87, 101, 117, 129, 145, 161, 205 43, 58, 71, 87, 101, 117, 12.50 2,3,6-Me3-Glcp 127, 143, 161, 173, 233 43, 58, 71, 87, 99, 101, 2.00 2,3,4-Me2-Glcp 117, 129, 161, 189, 233 43, 85, 87, 101, 117, 127, 1.10 2,3-Me3-Glcp 142, 159, 161, 201, 261 2.60 2,3,6-Me3-Manp 43, 45, 71, 87, 99, 101, 113, 117, 129, 161, 173, 233

Terminal

2.6. Data analysis

→4)-Glcp-(1→ →6)-Glcp-(1→

The results were expressed as means ± standard deviations (SD). Statistical difference among the groups was evaluated by one-way analysis of variance. The difference was considered as significant when p value b 0.01.

→4,6)-Glcp-(1→ →4)-Manp-(1→

3. Results 3.1. Isolation and purification of DHP1A from D. huoshanense 10 mL/kg BW. Group 3 was administered with silymarin at the dose of 25 mg/kg BW. Group 4 was administered with dextran at the dose of 200 mg/kg BW and the other groups were administered with DHP1A (100 and 200 mg/kg BW). All mice were treated once daily for 14 days. Two hours after the last treatment, the mice in control group were injected intraperitoneally with the olive oil at the dose of 10 mL/kg BW, while the rest of the mice were injected intraperitoneally with 0.15% CCl4 solution (dissolving in the olive oil, w/v) at the same dose. Twenty-four hours after the CCl4 treatment, the blood was collected from inner canthus and centrifuged at 3000 rpm for 10 min to give the serum, which was stored at − 80 °C until analysis. After that, the mice were sacrificed by cervical dislocation and the livers were removed quickly. A part of the liver tissue (0.2 g) was homogenized with nine volumes of 0.9% NaCl solution (1.8 mL) using a homogenizer. After centrifugation at 3000 rpm for 10 min, the generated supernatant was collected for the subsequent analysis.

Fig. 2. 1H and

13

The crude DHP was isolated from the dried D. huoshanense by water extraction and ethanol precipitation, and its yield was about 5.1% of the dried material. After that, DHP was fractionated on the DEAE-cellulose column to give four subfractions, DHP1 (78% of the crude DHP), DHP2 (12% of the crude DHP), DHP3 (5% of the crude DHP) and DHP4 (3% of the crude DHP). Among them, DHP1 was further purified by a gel filtration chromatography (Sephadex G-100) to afford a polysaccharide fraction of DHP1A (yield: 50% of DHP1). Dried DHP1A was white powder, and no absorbance peaks were found in the UV spectrum. According to the phenol–sulfuric acid methods, its carbohydrate content reached to 98.9%, and no proteins were detected based on the Lowry method [25]. 3.2. Structural characterization of DHP1A As shown in Fig. 1A, the HPLC profile of DHP1A presented a narrow and symmetrical peak, suggesting that it was a

C NMR spectra of DHP1A.

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Fig. 3. HSQC and HMBC spectra of DHP1A.

homogenous polysaccharide fraction. Its molecular weight was about 6700 Da according to the calibration of standard dextrans. The FT-IR spectrum of DHP1A was shown in Fig. 1B. The broad characteristic peak at 3388 cm−1 was attributed to the stretching vibration of hydroxyl group. The peaks at 2929 cm−1 and 1644 cm−1 were assigned to the vibration of C\H and bound water, respectively. The peak at 930 cm−1 suggested the existence of α-configuration glucose, and the peak at 870 cm−1 was the characteristic absorption peak of mannose [26]. The monosaccharide composition analysis showed that DHP1A was mainly composed of mannose, glucose and a little of galactose in a molar ratio of 2.5:16.0:1.0 (Fig. 1C, D). According to the results of methylation analysis, five methylated alditol acetates were found in DHP1A as listed in Table 1. Among them, (1 → 4)-linked D-Glcp and (1 → 4)-linked β-Manp were relatively predominant. Besides, the branch structure was found in DHP1A due to the existence of (1 → 4, 6)-linked Glcp, which was possibly substituted by a single D-Galp at C-6 position. The NMR spectra, including 1H, 13C, HSQC and HMBC, were used to delineate the structural features of DHP1A further. In

the 1H NMR spectrum (Fig. 2A), the strong anomeric signals around δ 5.37–5.29 were assigned to (1 → 4)-linked and (1 → 4, 6)-linked α-D-Glcp. The signals at δ 4.95, 4.73 and 4.49 were assigned to (1 → 6)-linked α-D-Glcp, (1 → 4)-linked β-D-Manp and terminal β-D-Galp, respectively. Moreover, the signal at δ 2.16 showed the presence of acetyl group. The 13C NMR spectrum of DHP1A presented five anomeric signals at δ 103.56–99.50. The signals at δ 103.39, 100.82, 100.58 and 99.5 belonged to terminal β-D-Galp, (1 → 4)-linked α-D-Glcp, (1 → 4, 6)-linked α-D-Glcp and (1 → 6)-linked α-D-Glcp, respectively. The anomeric signal of (1 → 4)-linked β-D-Manp was at δ 100.47, which was overlapped with the signal of (1 → 4)-linked α-D-Glcp. The signal at δ 77.80 indicated the substitution of C-4 of α-D-Glcp. Presence of acetyl groups in DHP1A was further confirmed by the signals at δ 173.94 and 21.48 (Fig. 2B). The HSQC spectrum of DHP1A showed four dominant coupling signals at δ 5.37/100.82, 4.95/99.50, 4.73/100.58 and 4.49/103.39 (Fig. 3A), which were assigned to (1 → 4)-linked Glcp, (1 → 6)-linked Glcp, (1 → 4)-linked Manp and terminal

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Galp, respectively. In the HMBC spectrum of DHP1A (Fig. 3B), the glycosidic linkages of the sugar residues were proposed according to the coupling between proton and carbon. The coupling signals at δ 5.37/77.80 indicated a (1 → 4) linkage existing among the glucopyranose. H-1 of Glcp linking to C-6 of Glcp showed a weak coupling signal at δ 4.95/71.47. Moreover, the signal at δ 4.73/76.89 represented the linkage between H-1 of β-Manp and C-4 of α-Glcp [13,14,26–30]. Based on the chemical and spectral analysis above, a possible structural unit of DHP1A was proposed as shown in Fig. 4. 3.3. In vitro antioxidant activities of DHP1A 3.3.1. Scavenging activity on DPPH radical of DHP1A The DPPH scavenging effect of DHP1A was shown in Fig. 5A, and its scavenging rates were increased from 31.4% to 38.7% with increasing the polysaccharide concentration from 0.5 to 2.0 mg/mL. As compared with vitamin C, a well-known low molecular anti-oxidant, the scavenging rate of DHP1A was lower. However, its effect was proven to be better than dextran (p b 0.01), a reference polysaccharide. 3.3.2. Inhibition effect on lipid peroxidation of DHP1A As shown in Fig. 5B, DHP1A showed a significant inhibition effect on the FeCl2-induced lipid peroxidation. There was an obvious dose-dependent relationship as the concentrations of DHP1A ranging from 0.1 to 1.0 mg/mL. At 2.0 mg/mL, the inhibition rate of DHP1A was increased to 56.5%, which was higher than that of dextran (p b 0.01), and close to that of vitamin C. 3.4. Effect of DHP1A on oxidative stress caused by CCl4 in mice 3.4.1. Effect of DHP1A on level of MDA The in vivo anti-lipid peroxidation of DHP1A was also evaluated in the CCl4-induced liver injury model (Fig. 6A). It was confirmed that CCl4 administration significantly increased the level of MDA (2.71 nmol/mg prot) as compared to the control group (1.42 nmol/mg prot), which suggested the occurrence of lipid peroxidation. The mice pretreated with DHP1A showed an effective inhibition effect on the increase of MDA. At 200 mg/kg BW, the level of MDA in the liver was

Fig. 4. Predicted structural unit of DHP1A from D. huoshanense.

Fig. 5. Antioxidant activities of DHP1A in vitro. Scavenging capacity for DPPH radical (A); inhibition effect on the lipid peroxidation (B). Values are means ± SD (n = 3).

decreased to 1.56 nmol/mg prot, which was close to the normal level. 3.4.2. Effect of DHP1A on levels of T-SOD, CAT, GPx and GSH To evaluate the in vivo anti-oxidative stress, the effects of DHP1A on the systematic antioxidant enzymes (substance) were investigated (Fig. 6, B–E). Silymarin, a traditional hepatoprotective drug, was used as a positive agent in this study [31], while dextran (20.0 kDa, Shanghai, China) was regarded as a parallel negative control. Following CCl4 treatment, the level of GSH was decreased to 4.33 nmol/mg prot as compared with that in the normal mice (7.8 nmol/mg prot). Pretreatment with DHP1A (100 or 200 mg/kg BW) remarkably alleviated the depletion of GSH as compared with the mice treated with CCl4 alone (p b 0.01). Silymarin pretreatment also decreased the depletion of GSH, but dextran seemed to be invalid. Moreover, the activities of T-SOD, CAT and GPx were significantly decreased in the CCl4-treated mice as compared with the control group (p b 0.01), while DHP1A pretreatment showed the significant protective effects on these antioxidant enzymes. In particular, the activities of T-SOD, CAT and GPx in the mice pretreated with DHP1A at 200 mg/kg BW were increased to 248.18 U/mg prot, 140.08 U/g prot and 340.13 U/mg prot, respectively, which were significantly higher than the mice treated with CCl4 alone (p b 0.01).

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Fig. 6. Effects of DHP1A on CCl4-induced oxidative stress in liver. MDA (A); GSH (B); T-SOD (C); CAT (D); GPx (E). Data were expressed as mean ± SD (n = 9–10). *, p b 0.01 as compared to the model group.

Silymarin pretreatment also exhibited obvious protective effects on T-SOD and GPx, but the effects of dextran were not significant. 4. Discussion During the past years, the antioxidant activities of natural polysaccharides had been reported in many literatures [32–34]. Unlike the synthetic antioxidants, most of natural polysaccharides were low toxicity, therefore they might be explored as ideal antioxidant agents. However, there also existed a few opposite views. Wang et al. indicated that the radical scavenging potential of the crude tea polysaccharides (TPS) was attributed to tea polyphenols rather than tea polysaccharides [35]. In the current study, our aim was to elucidate whether a purified

polysaccharide fraction (DHP1A) obtained from D. huoshanense possessed the antioxidant activities. Composition analysis indicated that the total sugar content in DHP1A was about 98.9%, and no protein existed, so its antioxidant activity should be attributed to the polysaccharide component. Moreover, DHP1A was proven to be a novel low-molecular polysaccharide according to its structural characterization, which was distinguished from the polysaccharides found in D. huoshanense previously. It is well known that the antioxidant activity of polysaccharide is dependent on its structural features, such as molecular weight, glycosyl linkage, monosaccharide composition and configuration. Among them, the molecular weight was proven to be an important parameter, and the polysaccharide with a relatively lower molecular weight usually showed the higher

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antioxidant activity [36]. Besides, some substituted groups, such as sulfate, acetyl and phosphate groups, could improve the antioxidant activities of polysaccharides in vitro [37]. On the other hand, a polysaccharide usually showed the different antioxidant effects when evaluated in different experimental models. Luo et al. found that a water-soluble polysaccharide derived from Dendrobium nobile lindl. (DNP) had a high scavenging effect on ABTS radicals and an appropriate scavenging effect on hydroxyl radicals, but a weak scavenging effect on DPPH radicals [38]. This phenomenon possibly reflected its special antioxidant mechanism. In the present study, the in vitro antioxidant activities of DHP1A were significantly higher than dextran in the same conditions, possibly due to its special structural features. Moreover, the scavenging effect of DHP1A on DPPH radicals was relatively weak as compared with vitamin C, but it had a remarkable inhibition effect on the FeCl2-induced lipid peroxidation. These results implied that the anti-lipid peroxidation played a crucial role in the antioxidant mechanism of DHP1A. The CCl4-induced tissue injury is a representative oxidative model that can be used to evaluate the antioxidant activity of natural products [6,7]. In the pathogenesis of CCl4, the excessive production of free radicals, such as the trichloromethyl radical (•CCl3) and trichloromethyl peroxyl radical (•CCl3O2•) derived from CCl4 by the cytochrome P450 system, leads to a rapid lipid peroxidation and disturbs the systemic redox balance [39]. In this study, MDA, a type of important lipid peroxide, obviously increased in the livers following CCl4 administration. Meanwhile, CCl4 injection lowered the activities of SOD, CAT and GPx, as well as the level of GSH, indicating the occurrence of oxidative stress. Previous studies had demonstrated that some polysaccharides could alleviate the CCl4-induced oxidative stress as natural antioxidants. However, the underlying mechanism, so far, was still not clear. Xiao et al. found that the pretreatment with Lycium barbarum polysaccharides (LBP) could restore the mRNA expression levels of GPx, CAT and Cu/Zn SOD in the CCl4-treated mice [7]. Panax ginseng polysaccharides (Ginsan) showed a short-term potentiation effect on the activity of heme oxygenase (HO), which was an important antioxidant enzyme in livers [40]. Shim et al. also demonstrated that the pretreatment with ginsan enhanced the level of GSH, and the protein expressions of Mn-SOD, CAT and GPx in livers [6]. Therefore, the anti-oxidative stress of polysaccharides may be associated with their modulation effects on the systemic antioxidant defense. In this study, the mice pretreated with DHP1A showed a significant resistance against the oxidative stress caused by CCl4, in which the antioxidant enzymes (substance) remained in a relatively higher level. Based on these results, DHP1A, we supposed, should be considered as a polysaccharide fraction with antioxidant activities, and it possibly plays an important role in the therapeutic effects of D. huoshanense. Furthermore, it is of great interest for us to improve the isolation yield of DHP1A via optimizing the extraction process, and investigate its hepatoprotective effects in our next researches. 5. Conclusion In summary, a purified polysaccharide fraction (DHP1A) was obtained from D. huoshanense, and its structural characteristic was delineated by the diverse spectral methods. DHP1A

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