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Synthesis, characterization, antioxidant activity and neuroprotective effects of selenium polysaccharide from Radix hedysari
Carbohydrate Polymers 125 (2015) 161–168
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Synthesis, characterization, antioxidant activity and neuroprotective effects of selenium polysaccharide from Radix hedysari Dongfeng Wei a,b,1 , Tong Chen c,1 , Mingfei Yan d , Wanghong Zhao e , Fei Li f , Weidong Cheng a,∗ , Lixia Yuan a,∗ a
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, PR China Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, PR China c State Key Laboratory of Food Additive and Condiment Testing, Zhenjiang Entry-Exit Inspection Quarantine Bureau, Zhenjiang 212008, PR China d Medical Experiment Center of Shaanxi University of Chinese Medicine, 712046, PR China e Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China f School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, PR China b
a r t i c l e
i n f o
Article history: Received 18 December 2014 Received in revised form 10 February 2015 Accepted 13 February 2015 Available online 21 February 2015 Keywords: Alzheimer’s disease Radix hedysari Polysaccharide Selenylation modification Antioxidant activity Neuroprotection
a b s t r a c t Beta-amyloid (A) peptide, the hallmark of Alzheimer’s disease, invokes oxidative damage to neurons and eventually leads to neuronal death. Selenylation modification of polysaccharide obtained from Radix hedysari (RHP) was studied to access antioxidant activities and neuroprotective effects against oxidative stress and apoptosis induced by A25–35 in vitro. A series of the selenylation derivatives of RHP (Se-RHP) was synthesized using nitric acid–sodium selenite (HNO3 –Na2 SeO3 ) method. The organic selenium content of Se-RHP increased from 1.04 to 3.29 mg/g. However, compared with the weight-average molecular mass (Mw) of RHP, Mw of Se-RHP showed a significant decrease, and varied from 27.7 kDa to 62.7 kDa. FT-IR spectra and 13 C NMR spectra indicated the selenite groups had been introduced mainly at the C-6 positions of RHP. Compared with RHP, Se-RHP showed greater antioxidant activities in vitro. Furthermore, both RHP and Se-RHP3 had neuroprotective effects against A25–35 -induced oxidative stress and apoptosis in SH-SY5Y human neuroblastoma cells, which might be a potential therapeutic agent for preventing or treating neurodegenerative diseases. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is the most common type of dementia in the elderly and is an progressive neurodegenerative disorder clinically characterized by the cognitive decline including impairment of learning, episodic memory, decision making and orientation (Blennow, de Leon, & Zetterberg, 2006). One of the major histopathological features of AD is the extracellular amyloid plaques that are composed principally of fibrillar -amyloid (A) peptide (Wilquet & De Strooper, 2004). As the hallmark of AD, A neurotoxic effects have been reported in the researches of in vitro and in vivo. A invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death in AD pathology (Zhang et al., 2010). AD brain proteins are apt to be more oxidized
∗ Corresponding authors. Tel.: +86 2061647420; fax: +86 2061647420. E-mail addresses: [email protected] (W. Cheng), [email protected] (L. Yuan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.carbpol.2015.02.029 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
than that of control subjects. Previous study demonstrated that A can generate free radicals with potent lipid peroxidation effects on the synaptosomal membranes in the neocortex after fragmentizing (Hensley et al., 1995). It has been shown that free radical overproduction directly induce DNA damage and cause apoptosis (Amoroso, Gioielli, Cataldi, Di Renzo, & Annunziato, 1999). The generation of reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radicals induced by A is a key molecular mechanism underlying the pathogenesis of AD (Tabner, Mayes, & Allsop, 2010). Zhang et al. indicated that the levels of intracellular ROS markedly increased in AD cellular model induced by A25–35 (Zhang et al., 2010). ROS can cause damage to a wide range of essential bio-molecules, and which have been associated with neurodegenerative, cardiovascular diseases and many other health problems related to advancing age (Blander, de Oliveira, Conboy, Haigis, & Guarente, 2003; Hu, Liu, Chen, Wu, & Wang, 2010). A number of antioxidants are known to delay or prevent the oxidation of cellular oxidizable substrates, including protecting against A-induced neurotoxicity (Li, 2007). Therefore, dietary or pharmacological
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intake of antioxidants that can scavenge free radicals may be considered as one of the therapeutic strategies to treat A-induced neurotoxicity in AD. Recently, plant polysaccharide with low toxicity has been demonstrated to play an important role as free radical scavengers in vitro and antioxidants in the prevention of oxidative damage in living organisms (Kim et al., 2007; Souza et al., 2012). Furthermore, some studies have confirmed that antioxidant activities of polysaccharide can be improved by the molecular modification and structure improvement (Chen et al., 2011; Zhang et al., 2009; Zou et al., 2008). The phosphorylated modification or selenylation modification of polysaccharide showed greater antioxidant activities compared to native polysaccharide (Che, Xu, Zhang, & Fanbo, 2009; Guo et al., 2013). Selenium (Se) is an essential element for nutrition of a capital importance in the human biology (Beckett & Arthur, 2005; Chen, Wong, Zheng, Bai, & Huang, 2008). This element is a cofactor of a large number of selenium-dependent enzymes such as an antioxidant enzyme, which is involved in cellular protection from severe oxidation by free radicals (El-Sayed, Aboul-Fadl, Lamb, Roberts, & Franklin, 2006; Zapletal, Heyne, Breitkreutz, Gebhard, & Golling, 2008). Compared with inorganic selenium, it is known that organic selenium can be better absorbed and has less toxicity. Selenium polysaccharide, including natural selenium polysaccharides extracted from plants or synthesized derivatives with selenium and polysaccharide, belongs to organic selenium compound and exhibits significantly stronger antioxidant activities than seleniumfree polysaccharide in vitro and in vivo (Malinowska et al., 2009; Wang et al., 2011; Yu et al., 2009). Therefore, the selenylation modification becomes a hot spot in polysaccharides research field. Radix hedysari (RH), known as Hongqi (HQ) in China, is the dried root of Hedysarum polybotrys Hand.-Mazz., which belongs to the Fabaceae family. RH is a well-known Chinese herbal medicine used for the treatment of various diseases as a tonic medicine for thousands of years (Li, Wang, & Hu, 1987). Polysaccharide isolated from RH (RHP) was the main effective ingredient with a wide array of biological activities, such as antioxidant properties, antitumor activities, immunomodulatory effect and antiviral activities (Shon, Kim, & Nam, 2002; Wei, Wei, Cheng, & Zhang, 2012). Hui reported that RHP was composed of rhamnose, xylose, arabinose, glucose and galactose, and their molar ratio was 0.3:0.2:2.7:16.1:2.0. The glucosidicbond configuration of RHP was mainly -configuration (Hui, Feng, Zhao, Shi, & Liu, 2010). In this study, a series of the selenylation derivatives of RHP (Se-RHP) was synthesized using nitric acid–sodium selenite (HNO3 –Na2 SeO3 ) method with HNO3 and BaCl2 as catalyst. SeRHP was studied including Se content, weight-average molecular mass (Mw) and chain conformation. The chemical structure of Se-RHP was analyzed by UV spectra, FT-IR spectra, and 13 C NMR spectroscopy. Their antioxidant activities of both RHP and Se-RHP in vitro were investigated, including superoxide, hydroxyl, DPPH radicals-scavenging effects and reducing power. The effects of chain conformation and Se content on the antioxidant activities of SeRHP were discussed. Furthermore, neuroprotective effects of both RHP and Se-RHP3 against oxidative stress and apoptosis induced by A25–35 were investigated in vitro in SH-SY5Y human neuroblastoma cells.
2. Materials and methods 2.1. Materials and reagents The root of RH was collected from the mountain area in Wudu City, Gansu Province, China. The crude RHP was obtained from the laboratory of School of Basic Medical Science at Lanzhou University.
Sephadex G-100 was from Pharmacia (Sweden). Papain was purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). HNO3 , Na2 SeO3 and BaCl2 were the product of Xiya Chemical Reagent Co., Ltd. (Chengdu, China). Nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), ethylene diamine tetra-acetic acid (EDTA), phenazine methosulfate (PMS), butylated hydroxyanisole (BHA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), trichloroacetic acid (TCA), thiobarbituric acid (TBA) and deoxyribose were purchased from Sigma–Aldrich. Ascorbic acid (VC ), H2 O2 and potassium ferricyanide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). SH-SY5Y human neuroblastoma cell lines were purchased from ATCC (Manassas, USA). Eagle’s Minimum Essential Medium/F12 medium and penicillin–streptomycin liquid were purchased from Gibco (Gaithersburg, USA). Fetal bovine serum (FBS) was purchased from PAA Laboratories GmbH (Pasching, Austria). A25–35 , thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and Hoechst 33258 were purchased from Sigma–Aldrich (St. Louis, USA). 2.2. Purification of RHP The crude RHP was purified as follows: the protein was removed by the Sevage method (Miu, Yang, & Zhou, 2007), combined with neutral enzyme (papain, 150 U/mL). After centrifugation, the supernatant was dialyzed with distilled water for 48 h. Through Sephadex G-100 column, the purified RHP was obtained after lyophilizing. Mw of RHP was 84.7 kDa. The content of RHP was measured by Vitriol–Phenol taking anhydrous glucose as standard control. 2.3. Selenylation modification of RHP The RHP powder (400 mg) was dissolved in 40 mL of HNO3 (0.6%, v/v) at room temperature with stirring for 30 min. Then Na2 SeO3 and BaCl2 was added and the mixture was stirred for 8 h at 65 ◦ C. After the reaction, the mixture was cooled to room temperature and the pH value was adjusted to 7–8 with 1 mol/L NaOH solution. Sodium sulfate was added to remove the Ba2+ . After centrifugation, the supernatant were dialyzed (molecular weight cutoff 3.0 kDa) by using distilled water for 24 h. The polysaccharide solutions were concentrated and precipitated with ethanol. Se-RHP with different Se content was obtained after lyophilizing with vacuum freezedrying machine. 2.4. Characterization of Se-RHP Mw of Se-RHP were determined by high-performance gelfiltration chromatography (HPGFC) on a Waters 2695 instrument equipped with a Waters 2414 Refractive Index Detector (RID). 0.5 mg/mL sample solution (Se-RHP solution) was injected in each run with using 0.05 mol/L Na2 SO4 aqueous solution as the mobile phase. The analysis was performed at 25 ◦ C and a flow rate of 0.7 mL/min. The HPGFC system was calibrated with T-series Dextran standards. The Se contents of Se-RHP were determined on an inductively coupled plasma atomic emission spectrophotometer (ICP-AES) IRIS ER/S (TJA Company, USA). Absorbance spectra and measurements were carried out using a Puxi UV-1810 visible spectrophotometer (Beijing, China). FTIR spectra of RHP and Se-RHP were recorded with a NICOLET iS10 FT-IR spectrophotometer (Thermo Fisher, USA) in the range 4000–500 cm−1 . The resolution of FT-IR was 4 (data spacing: 0.482 cm−1 ), and number of scans were 16. 13 C NMR spectra of 40 mg/mL solutions in D2 O were recorded on a Bruker Avance
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400 MHz spectrometer (Germany) at 40 ◦ C with 2,2-dimethyl-2silapentane-5-sulfonate (DSS) as internal standard. 2.5. Assay for antioxidant activities The superoxide radical scavenging ability of RHP and Se-RHP was assessed by the method of Nishikimi, Appaji, and Yagi (1972). Hydroxyl radical assay was measured using a method of Ghiselli, Nardini, Baldi, and Scaccini (1998). The free-radical scavenging capacity of RHP and Se-RHP was analyzed using the DPPH radical according to the method of Shimada, Fujikawa, Yahara, and Nakamura (1992). The reducing power of all samples was investigated according to the method reported by Li, Li, and Zhou (2007). 2.6. Cell culture and treatment SH-SY5Y cells were cultured in D-MEM/F12 supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 g/mL streptomycin. The cultures were maintained in a 5% CO2 /95% air humidified atmosphere at 37 ◦ C, and the culture medium was changed every 2–3 days. SH-SY5Y cells were seeded into 96- or 6well plates, which were then allowed to equilibrate for 24 h prior to experiments. A25–35 was dissolved in deionized water at a concentration of 2 mM. The stock solution was diluted to desired concentrations immediately before use. In the establishment of oxidative stress experiments, cells were induced by different concentrations (15, 25, 35 M) of A25–35 for 48 h. In protective experiments of RHP or Se-RHP3, cells were pretreated with various concentrations (5, 10, 20, 40 g/mL) of RHP or Se-RHP3 dissolved in serum-free media for 4 h respectively, followed by exposed to 35 M (final concentration) of A25–35 in the presence of RHP or Se-RHP3 for another 48 h. The control cells were added with the same medium without A25–35 . All experiments were repeated three times for each treatment condition. 2.7. Determination of cell viability Cell viability was determined by using the thiazolyl blue tetrazolium bromide (MTT) assay, which was based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases. After incubation with the indicated drugs, 96-well plates had all media removed and replaced with serum-free media containing 0.5 mg/mL of MTT. The plate was further incubated for 3 h at 37 ◦ C with 5% CO2 , then MTT solution was removed and cells were lysed with 150 L DMSO. The plate was then placed on an orbital shaker for 10 min before determining absorbance at 570 nm using a microplate reader (Bio-Rad, Hercules, USA). The absorbance values were expressed as a percentage of untreated control cells (control = 100%). 2.8. Nuclear staining for assessment of apoptosis The cell nuclear morphology and number of cells were detected using Hoechst 33258 staining and fluorescence microscopy, as previously described (Zhang et al., 2012). Briefly, after treatment with 35 M A25–35 and RHP or Se-RHP3 for 48 h, SH-SY5Y cells were washed twice with PBS and incubated for 15 min with Hoechst 33258 (5 g/mL) at room temperature in the dark. After a rinse in PBS, the nuclear morphology of SH-SY5Y cells was observed and photographed using a fluorescence microscope (Olympus IX71, Japan). The cells were counted from four random fields of view in four different slides from each treatment, and the number of apoptotic cells was expressed as a percentage of the total number of cells counted.
Fig. 1. UV–vis absorption spectra of RHP and Se-RHP3.
2.9. Statistical analysis All data was presented as mean ± standard error (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by the Dunnett’s t-test. Statistical significance was set at P < 0.05. 3. Results and discussion 3.1. Chemical Analysis of Se-RHP The RHP and Se-RHP were confirmed by UV–vis spectroscopy analysis, as shown in Fig. 1. There was no absorption at 280 or 260 nm in the UV spectrum, which indicated the absence of proteins and nucleic acids in the purified RHP. Compared with the UV spectrum of RHP, it was found that the 336 nm wavelength showed the characteristic peak of selenium. The result indicated the existence of selenium and the formation of Se-RHP (Zhao, 2006). Se content and Mw of four derivatives of RHP were investigated in Table 1. The result showed that the organic selenium content of the samples increased from 1.04 to 3.29 mg/g. Se-RHP3 had the highest Se content that was 3.29 mg/g at 10 h with the yield of 345 mg. However, Se content had no obvious influence when it was more than 10 h. It implied that hydroxyl groups were substituted successfully by selenite groups on the polysaccharide. Compared with Mw of RHP, Mw of four derivatives of RHP showed a significant decrease, and varied from 27.7 kDa to 62.7 kDa. It was reported that polysaccharide in the strong acid circumstance would be depolymerized severely due to longer reaction time (Chen et al., 2010). In the present study, Mw of Se-RHP also decreased with the increase of reaction time. The decrease of Mw was mainly due to the hydrolysis of polysaccharide, and higher concentration of acid and temperature and longer reaction time could accelerate the hydrolysis rate.
Table 1 Characterization of RHP and Se-RHP. Samples
Reaction time (h)
Mw (kDa)
Yield (mg)
Se content (mg/g)
Se-RHP1 Se-RHP2 Se-RHP3 Se-RHP4 RHP
3 5 10 15 –
62.7 50.5 29.6 27.7
347 379 345 365 –
1.04 1.96 3.29 3.17 0
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Fig. 2. FT-IR spectra of RHP (A) and Se-RHP3 (B).
The FT-IR spectra of the native RHP and Se-RHP were shown in Fig. 2. The IR spectrum of the RHP showed a strong band at 3290 cm−1 attributed to the hydroxyl stretching vibration of the polysaccharide. The bands in the region of 2927 cm−1 and 1590 cm−1 were due to C H stretching vibration and OH bending vibrations. This amalgamation indicated that the RHP was polysaccharides. Compared with the native RHP, two new strong absorption peaks appeared at 925 cm−1 and 840 cm−1 for SeRHP, assigned to the C O Se stretching vibration and the Se O asymmetric stretching, respectively. These absorptions indicated that the selenylation modification in the samples had actually occurred. The selenyl position of polysaccharide was usually determined by 13 C NMR spectra. The 13 C NMR spectra of the native RHP and its derivative (Se-RHP3) were shown in Fig. 3. As shown in Fig. 3A, the signals of RHP at 96.1, 75.9, 74.1, 72.7, 69.5, and 60.5 ppm were assigned to C1 , C3 , C5 , C2 , C4 , and C6 , respectively. The new peak at 62.8 ppm of Se-RHP3 was assigned to the O-6 substituted carbons, suggesting reaction of O-6 (Melo, Feitosa, Feitosa, Freitas, & Paula, 2002). However, C-6 peaks still remained at 60.5 ppm for Se-RHP3, and had not been decreased obviously, suggesting that the primary OH groups on the internal side were not selenized. From the results of 13 C NMR, the non-selective reaction of RHP occurred, and the intensity of the signals of the O-substitution carbons denoted that C-6 substitution was predominant.
3.2. Antioxidant activity analysis 3.2.1. Scavenging activity of superoxide radical The scavenging ability of superoxide radicals was extremely important to antioxidant work. Fig. 4A showed that the inhibitory ability of Se-RHP on superoxide radical was significant in a concentration-dependent fashion. At a concentration of 3.2 mg/mL, the scavenging effect was 39.5%, 40.9%, 54.1%, 63.2% and 60.5% for RHP, Se-RHP1, Se-RHP2, Se-RHP3 and Se-RHP4, respectively. At the same concentration, the scavenging effect of Vc was 80.3%. Compared with RHP, Se-RHP exhibited more effective antioxidant activity. We also found that polysaccharide with higher selenium content exhibited stronger scavenging activity of superoxide radical. In the study, antioxidation mechanism may be that the presence of selenyl group ( SeH) or seleno-acid ester could change the three-dimensional structure of polysaccharide, and increasing hydroxyl groups would emerge, which affected the antioxidant ability.
Fig. 3. 13 C NMR (400 MHz) spectra of RHP and its selenylation derivatives. (A) RHP; (B) Se-RHP3.
3.2.2. Scavenging activity of hydroxyl radical. Hydroxyl radical, known to be generated through the Fenton reaction, can easily cross cell membranes, and readily react with biomolecules including carbohydrates, proteins, lipids, and DNA in cells, and cause tissue damage or cell death (Liu et al., 2010). The scavenging ability of all samples on hydroxyl radical was shown in Fig. 4B. Compared with the native RHP, Se-RHP showed higher scavenging ability. Furthermore, Se-RHP4 showed the strongest scavenging ability, and the scavenging ability was 43.5% at a dose of 3.2 mg/mL, but lower than Vc (88.2%). It was reported that the scavenging activity of hydroxyl radical was due to the inhibition of hydroxyl radical generation by chelating ions such as Fe2+ and Cu2+ . The contribution of the selenium moiety in the Se-RHP samples to antioxidant activity probably stemmed from, in part, the ability to complex the free metal ion. It was proposed that selenium possibly existed in the polysaccharides containing Se in the form of selenyl group ( SeH) or seleno-acid ester (Huang, 1994). The new groups may be able to complex Fe2+ or Cu2+ , there by inhibiting the generation of hydroxyl radicals (HO• ). However, the real mechanism of Se-RHP protecting ability from oxidative damage was beyond the scope of the present study and need further testifying. 3.2.3. Scavenging activity of DPPH radicals In the DPPH free radical scavenging test the stable yellowcolored diphenylpicrylhydrazine (DPPH-H) was formed in the presence of an antioxidant. As shown in Fig. 4C, all the samples were found to exhibit DPPH radical scavenging activity. Compared with RHP, Se-RHP exhibited more effective scavenging activity. Scavenging activity of Se-RHP (from Se-RHP1 to Se-RHP4) on DPPH radicals increased with increasing concentrations and reached a stable level of 44.9–52.6% at 3.2 mg/mL. No significant differences
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Fig. 4. Antioxidant effect of RHP and Se-RHP: (A) scavenging activity of superoxide radicals; (B) scavenging activity of hydroxyl radicals; (C) scavenging activity of DPPH radicals; (D) reducing power.
were observed between radical scavenging activity of polysaccharides containing different content of Se. However, the DPPH radical scavenging ability was more increased significantly for Vc than that of RHP and Se-RHP. It has been reported that the higher scavenging rate was for DPPH free radical due to the hydrogen-donating ability to form a stable DPPH-H molecule when accepted an electron or hydrogen radical. The presence of selenyl group ( SeH) or seleno-acid ester groups in the Se-RHP molecule could activate the hydrogen atom of the anomeric carbon. The higher activated capacity of the group, the stronger hydrogen atom-donating capacity.
3.2.4. Reducing power The reducing capacity of a compound might serve as a significant indicator of its potential antioxidant activity. The reducing properties were associated with the presence of reductones, which had been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom. As shown in Fig. 4D, all the samples showed high reducing power, which correlated well with increasing concentrations. At a concentration of 3.2 mg/mL, the reducing power was 0.31, 0.40, 0.58 and 0.54 for Se-RHP1, SeRHP2, Se-RHP3 and Se-RHP4, respectively, whereas the activity of polysaccharide from control medium (without Se) was significantly
Fig. 5. Protective effects of RHP or Se-RHP3 on A25–35 induced cytotoxicity in SH-SY5Y cells. All data were expressed as mean ±SEM of three experiments. # P < 0.05, ## P < 0.01 vs control; *P < 0.05, **P < 0.01 vs A25–35 alone.
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Fig. 6. Ameliorative effects of RHP or Se-RHP3 on A25–35 induced morphological alterations in SH-SY5Y cells (original magnification, ×100).
Fig. 7. RHP or Se-RHP3 protected SH-SY5Y cells against A25–35 induced apoptosis. Morphological apoptosis was determined by Hoechst 33258 staining. Arrow heads indicated apoptosis cells (original magnification, ×400).
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weaker and reached a maximum of 0.29 at 3.2 mg/mL. However, ascorbic acid showed significantly stronger reducing power (1.03 at 3.2 mg/mL) than the polysaccharide examined. The present studies confirmed the result that Se-RHP showed higher reducing power than the native RHP. 3.3. Neuroprotective effects of RHP or Se-RHP3 3.3.1. RHP or Se-RHP3 prevented SH-SY5Y cells from oxidative stress induced by Aˇ25–35 in vitro A25–35 invokes a cascade of oxidative stress and eventually leads to neuronal toxicity, which is characterized by the decrease of cell viability. Antioxidant can inhibit oxidative stress and prevents A25–35 -induced neuronal toxicity in cell culture. In addition, Se-RHP3 showed greater antioxidant activity than others in vitro and therefore was selected for further neuroprotective study. In the study, SH-SY5Y cells were exposed to different concentrations (15, 25, 35 M) of A25–35 for 48 h and cell viability was determined by MTT assay. As shown in Fig. 5A, 15–35 M of A25–35 induced significant decrease of cell survival in a dosedependent manner. Compared with the control group, treatment group using 35 M of A25–35 for 48 h reduced cell survival to 52.53 ± 3.65%, which was used to induce SH-SY5Y cell oxidative stress injury in the subsequent experiments. As shown in Fig. 5B, viability of the SH-SY5Y cells was significantly decreased to 50.02 ± 3.81% with the concentration of 35 M A25–35 for 48 h. However, compared with the A25–35 treatment group, cell viability rate of group pretreated with 5, 10, 20, and 40 g/mL of RHP or Se-RHP3 restored in a dose-dependent manner, increasing to 53.10 ± 2.70%, 64.07 ± 2.75%, 69.70 ± 3.74%, 77.84 ± 2.65%, and 54.82 ± 3.31%, 68.23 ± 4.81%, 75.65 ± 5.12%, 86.45 ± 4.73%, respectively. Furthermore, the cell morphological changes were observed with inverted phase contrast microscope. Compared with the control group, treatment of A25–35 at 35 M for 48 h resulted in obviously cells loss, synapse injury, shrinkage, and membrane blebbing. However, pretreatment with 40 g/mL of RHP or Se-RHP3 for 4 h before treatment with A25–35 obviously ameliorated such morphological alterations of cell injury (Fig. 6). 3.3.2. RHP or Se-RHP3 protected SH-SY5Y cells from apoptosis induced by Aˇ25–35 Oxidative stress induced by A25–35 is one of the major stimuli for apoptotic signal transduction in SH-SY5Y cells. Hoechst 33258, a fluorescent DNA binding dye, can display typical morphological features of apoptosis with chromatin agglutination and karyopyknosis. In this study, the nuclear staining with Hoechst 33258 was used to quantify the rate of apoptosis in each group using fluorescence microscope. As shown in Fig. 7, compared to the control group, the treatment with 35 M of A25–35 for 48 h showed typical characteristics of apoptosis, including the chromatin agglutination, karyopyknosis, and the appearance of apoptotic bodies and increased the percentage of apoptotic cells from 2.01% to 16.25%. However, pretreatment with 40 g/mL of RHP or Se-RHP3 for 4 h before treatment with A25–35 markedly decreased the number of cells with karyopyknosis and nuclear fragmentation and the percentage of apoptotic cells was reduced to 9.63% and 6.82%, respectively. 4. Conclusion In the present study, Se-RHP was synthesized by Na2 SeO3 /HNO3 with the highest Se content of 3.29 mg/g. Mw of Se-RHP had a sharp decrease after the selenylation modification. UV spectrum and FTIR spectra indicated that the selenized reaction had occurred. From the results of 13 C NMR, the signals of the O-substitution carbons denoted that C-6 substitution was predominant in Se-RHP. The
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results indicated that selenylation modification of RHP could significantly enhance the antioxidant activity in vitro. Furthermore, both RHP and Se-RHP3 could ameliorate A25–35 -induced oxidative stress and apoptosis in SH-SY5Y cells. The protective effects of both RHP and Se-RHP3 may help to provide the pharmacological basis of its clinical usage in the preventing or treatment of neurodegeneration in AD. Meanwhile, Se-RHP3 may serve as a good source of selenium as a supplement and could be used as an antioxidant for the food and pharmaceutical industries. Acknowledgments This work is supported in part by the General Program of National Natural Science Foundation of China (Grant No. 81373806), the General Project of Administration of Traditional Chinese Medicine of Gansu Province (Grant No. GZK-2012-43), and the Science and Technology Planning project of Jiangsu Entry-Exit Inspection Quarantine Bureau, China (2014KJ33). References Amoroso, S., Gioielli, A., Cataldi, M., Di Renzo, G., & Annunziato, L. (1999). In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca(2+) increase. Biochimica et Biophysica Acta, 1452(2), 151–160. Beckett, G. J., & Arthur, J. R. (2005). Selenium and endocrine systems. Journal of Endocrinology, 184(3), 455–465. Blander, G., de Oliveira, R. M., Conboy, C. M., Haigis, M., & Guarente, L. (2003). Superoxide dismutase 1 knock-down induces senescence in human fibroblasts. Journal of Biological Chemistry, 278(40), 38966–38969. Blennow, K., de Leon, M. J., & Zetterberg, H. (2006). Alzheimer’s disease. Lancet, 368(9533), 387–403. Che, X. Y., Xu, X. J., Zhang, L. N., & Fanbo, Z. (2009). Chain conformation and anti-tumor activities of phosphorylated (1-3)--d-glucan from Poria cocos. Carbohydrate Polymers, 78(3), 581–587. Chen, T., Li, B., Li, Y., Zhao, C., Shen, J., & Zhang, H. (2011). Catalytic synthesis and antitumor activities of sulfated polysaccharide from Gynostemma pentaphyllum Makino. Carbohydrate Polymers, 83(2), 554–560. Chen, T., Wang, J., Li, Y., Shen, J., Zhao, T., & Zhang, H. (2010). Sulfated modification and cytotoxicity of Portulaca oleracea L. polysaccharides. Glycoconjugate Journal, 27(6), 635–642. Chen, T., Wong, Y. S., Zheng, W., Bai, Y., & Huang, L. (2008). Selenium nanoparticles fabricated in Undaria pinnatifida polysaccharide solutions induce mitochondriamediated apoptosis in A375 human melanoma cells. Colloids Surface B: Biointerfaces, 67(1), 26–31. El-Sayed, W. M., Aboul-Fadl, T., Lamb, J. G., Roberts, J. C., & Franklin, M. R. (2006). Effect of selenium-containing compounds on hepatic chemoprotective enzymes in mice. Toxicology, 220(2-3), 179–188. Ghiselli, A., Nardini, M., Baldi, A., & Scaccini, C. (1998). Antioxidant activity of different phenolic fractions separated from an Italian red wine. Journal of Agricultural and Food Chemistry, 46(2), 361–367. Guo, Y., Pan, D., Li, H., Sun, Y., Zeng, X., & Yan, B. (2013). Antioxidant and immunomodulatory activity of selenium exopolysaccharide produced by Lactococcus lactis subsp. lactis. Food Chemistry, 138(1), 84–89. Hensley, K., Hall, N., Subramaniam, R., Cole, P., Harris, M., Aksenov, M., et al. (1995). Brain regional correspondence between Alzheimer’s disease histopathology and biomarkers of protein oxidation. Journal of Neurochemistry, 65(5), 2146–2156. Hu, T., Liu, D., Chen, Y., Wu, J., & Wang, S. (2010). Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro. International Journal of Biological Macromolecules, 46(2), 193–198. Huang, K. (1994). In H. B. Xu (Ed.), Selenium: Its chemistry, biochemistry and application in life science. Wuhan: Huazhong Science and Technology University Press. Hui, H. P., Feng, S. L., Zhao, L. G., Shi, Y. K., & Liu, X. H. (2010). Purification of the Radix hedysari polysaccharide and analysis of its constitution. Lishizhen Medicine and Materia Medica Research, 21(9), 2302–2303. Kim, S., Choi, D., Athukorala, Y., Jeon, Y., Senevirathne, M., & Rha, C. (2007). Antioxidant activity of sulfated polysaccharides isolated from Sargassum fulvellu. Journal of Food Science and Nutrition, 12(2), 65–73. Li, G. M., Wang, W. N., & Hu, M. S. (1987). Pharmacognosy of Radix hedysari. Zhong Yao Tong Bao, 12(8), 5–8. Li, X. M. (2007). Protective effect of Lycium barbarum polysaccharides on streptozotocin-induced oxidative stress in rats. International Journal of Biological Macromolecules, 40(5), 461–465. Li, X. M., Li, X. L., & Zhou, A. G. (2007). Evaluation of antioxidant activity of the polysaccharides extracted from Lycium barbarum fruits in vitro. European Polymer, 43(2), 488–497. Liu, X., Zhou, B., Lin, R., Jia, L., Deng, P., Fan, K., et al. (2010). Extraction and antioxidant activities of intracellular polysaccharide from Pleurotus sp. mycelium. International Journal of Biological Macromolecules, 47(2), 116–119.
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Synthesis, characterization, antioxidant activity and neuroprotective effects of selenium polysaccharide from Radix hedysari Dongfeng Wei a,b,1 , Tong Chen c,1 , Mingfei Yan d , Wanghong Zhao e , Fei Li f , Weidong Cheng a,∗ , Lixia Yuan a,∗ a
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, PR China Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, PR China c State Key Laboratory of Food Additive and Condiment Testing, Zhenjiang Entry-Exit Inspection Quarantine Bureau, Zhenjiang 212008, PR China d Medical Experiment Center of Shaanxi University of Chinese Medicine, 712046, PR China e Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China f School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, PR China b
a r t i c l e
i n f o
Article history: Received 18 December 2014 Received in revised form 10 February 2015 Accepted 13 February 2015 Available online 21 February 2015 Keywords: Alzheimer’s disease Radix hedysari Polysaccharide Selenylation modification Antioxidant activity Neuroprotection
a b s t r a c t Beta-amyloid (A) peptide, the hallmark of Alzheimer’s disease, invokes oxidative damage to neurons and eventually leads to neuronal death. Selenylation modification of polysaccharide obtained from Radix hedysari (RHP) was studied to access antioxidant activities and neuroprotective effects against oxidative stress and apoptosis induced by A25–35 in vitro. A series of the selenylation derivatives of RHP (Se-RHP) was synthesized using nitric acid–sodium selenite (HNO3 –Na2 SeO3 ) method. The organic selenium content of Se-RHP increased from 1.04 to 3.29 mg/g. However, compared with the weight-average molecular mass (Mw) of RHP, Mw of Se-RHP showed a significant decrease, and varied from 27.7 kDa to 62.7 kDa. FT-IR spectra and 13 C NMR spectra indicated the selenite groups had been introduced mainly at the C-6 positions of RHP. Compared with RHP, Se-RHP showed greater antioxidant activities in vitro. Furthermore, both RHP and Se-RHP3 had neuroprotective effects against A25–35 -induced oxidative stress and apoptosis in SH-SY5Y human neuroblastoma cells, which might be a potential therapeutic agent for preventing or treating neurodegenerative diseases. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is the most common type of dementia in the elderly and is an progressive neurodegenerative disorder clinically characterized by the cognitive decline including impairment of learning, episodic memory, decision making and orientation (Blennow, de Leon, & Zetterberg, 2006). One of the major histopathological features of AD is the extracellular amyloid plaques that are composed principally of fibrillar -amyloid (A) peptide (Wilquet & De Strooper, 2004). As the hallmark of AD, A neurotoxic effects have been reported in the researches of in vitro and in vivo. A invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death in AD pathology (Zhang et al., 2010). AD brain proteins are apt to be more oxidized
∗ Corresponding authors. Tel.: +86 2061647420; fax: +86 2061647420. E-mail addresses: [email protected] (W. Cheng), [email protected] (L. Yuan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.carbpol.2015.02.029 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
than that of control subjects. Previous study demonstrated that A can generate free radicals with potent lipid peroxidation effects on the synaptosomal membranes in the neocortex after fragmentizing (Hensley et al., 1995). It has been shown that free radical overproduction directly induce DNA damage and cause apoptosis (Amoroso, Gioielli, Cataldi, Di Renzo, & Annunziato, 1999). The generation of reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radicals induced by A is a key molecular mechanism underlying the pathogenesis of AD (Tabner, Mayes, & Allsop, 2010). Zhang et al. indicated that the levels of intracellular ROS markedly increased in AD cellular model induced by A25–35 (Zhang et al., 2010). ROS can cause damage to a wide range of essential bio-molecules, and which have been associated with neurodegenerative, cardiovascular diseases and many other health problems related to advancing age (Blander, de Oliveira, Conboy, Haigis, & Guarente, 2003; Hu, Liu, Chen, Wu, & Wang, 2010). A number of antioxidants are known to delay or prevent the oxidation of cellular oxidizable substrates, including protecting against A-induced neurotoxicity (Li, 2007). Therefore, dietary or pharmacological
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intake of antioxidants that can scavenge free radicals may be considered as one of the therapeutic strategies to treat A-induced neurotoxicity in AD. Recently, plant polysaccharide with low toxicity has been demonstrated to play an important role as free radical scavengers in vitro and antioxidants in the prevention of oxidative damage in living organisms (Kim et al., 2007; Souza et al., 2012). Furthermore, some studies have confirmed that antioxidant activities of polysaccharide can be improved by the molecular modification and structure improvement (Chen et al., 2011; Zhang et al., 2009; Zou et al., 2008). The phosphorylated modification or selenylation modification of polysaccharide showed greater antioxidant activities compared to native polysaccharide (Che, Xu, Zhang, & Fanbo, 2009; Guo et al., 2013). Selenium (Se) is an essential element for nutrition of a capital importance in the human biology (Beckett & Arthur, 2005; Chen, Wong, Zheng, Bai, & Huang, 2008). This element is a cofactor of a large number of selenium-dependent enzymes such as an antioxidant enzyme, which is involved in cellular protection from severe oxidation by free radicals (El-Sayed, Aboul-Fadl, Lamb, Roberts, & Franklin, 2006; Zapletal, Heyne, Breitkreutz, Gebhard, & Golling, 2008). Compared with inorganic selenium, it is known that organic selenium can be better absorbed and has less toxicity. Selenium polysaccharide, including natural selenium polysaccharides extracted from plants or synthesized derivatives with selenium and polysaccharide, belongs to organic selenium compound and exhibits significantly stronger antioxidant activities than seleniumfree polysaccharide in vitro and in vivo (Malinowska et al., 2009; Wang et al., 2011; Yu et al., 2009). Therefore, the selenylation modification becomes a hot spot in polysaccharides research field. Radix hedysari (RH), known as Hongqi (HQ) in China, is the dried root of Hedysarum polybotrys Hand.-Mazz., which belongs to the Fabaceae family. RH is a well-known Chinese herbal medicine used for the treatment of various diseases as a tonic medicine for thousands of years (Li, Wang, & Hu, 1987). Polysaccharide isolated from RH (RHP) was the main effective ingredient with a wide array of biological activities, such as antioxidant properties, antitumor activities, immunomodulatory effect and antiviral activities (Shon, Kim, & Nam, 2002; Wei, Wei, Cheng, & Zhang, 2012). Hui reported that RHP was composed of rhamnose, xylose, arabinose, glucose and galactose, and their molar ratio was 0.3:0.2:2.7:16.1:2.0. The glucosidicbond configuration of RHP was mainly -configuration (Hui, Feng, Zhao, Shi, & Liu, 2010). In this study, a series of the selenylation derivatives of RHP (Se-RHP) was synthesized using nitric acid–sodium selenite (HNO3 –Na2 SeO3 ) method with HNO3 and BaCl2 as catalyst. SeRHP was studied including Se content, weight-average molecular mass (Mw) and chain conformation. The chemical structure of Se-RHP was analyzed by UV spectra, FT-IR spectra, and 13 C NMR spectroscopy. Their antioxidant activities of both RHP and Se-RHP in vitro were investigated, including superoxide, hydroxyl, DPPH radicals-scavenging effects and reducing power. The effects of chain conformation and Se content on the antioxidant activities of SeRHP were discussed. Furthermore, neuroprotective effects of both RHP and Se-RHP3 against oxidative stress and apoptosis induced by A25–35 were investigated in vitro in SH-SY5Y human neuroblastoma cells.
2. Materials and methods 2.1. Materials and reagents The root of RH was collected from the mountain area in Wudu City, Gansu Province, China. The crude RHP was obtained from the laboratory of School of Basic Medical Science at Lanzhou University.
Sephadex G-100 was from Pharmacia (Sweden). Papain was purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). HNO3 , Na2 SeO3 and BaCl2 were the product of Xiya Chemical Reagent Co., Ltd. (Chengdu, China). Nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), ethylene diamine tetra-acetic acid (EDTA), phenazine methosulfate (PMS), butylated hydroxyanisole (BHA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), trichloroacetic acid (TCA), thiobarbituric acid (TBA) and deoxyribose were purchased from Sigma–Aldrich. Ascorbic acid (VC ), H2 O2 and potassium ferricyanide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). SH-SY5Y human neuroblastoma cell lines were purchased from ATCC (Manassas, USA). Eagle’s Minimum Essential Medium/F12 medium and penicillin–streptomycin liquid were purchased from Gibco (Gaithersburg, USA). Fetal bovine serum (FBS) was purchased from PAA Laboratories GmbH (Pasching, Austria). A25–35 , thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and Hoechst 33258 were purchased from Sigma–Aldrich (St. Louis, USA). 2.2. Purification of RHP The crude RHP was purified as follows: the protein was removed by the Sevage method (Miu, Yang, & Zhou, 2007), combined with neutral enzyme (papain, 150 U/mL). After centrifugation, the supernatant was dialyzed with distilled water for 48 h. Through Sephadex G-100 column, the purified RHP was obtained after lyophilizing. Mw of RHP was 84.7 kDa. The content of RHP was measured by Vitriol–Phenol taking anhydrous glucose as standard control. 2.3. Selenylation modification of RHP The RHP powder (400 mg) was dissolved in 40 mL of HNO3 (0.6%, v/v) at room temperature with stirring for 30 min. Then Na2 SeO3 and BaCl2 was added and the mixture was stirred for 8 h at 65 ◦ C. After the reaction, the mixture was cooled to room temperature and the pH value was adjusted to 7–8 with 1 mol/L NaOH solution. Sodium sulfate was added to remove the Ba2+ . After centrifugation, the supernatant were dialyzed (molecular weight cutoff 3.0 kDa) by using distilled water for 24 h. The polysaccharide solutions were concentrated and precipitated with ethanol. Se-RHP with different Se content was obtained after lyophilizing with vacuum freezedrying machine. 2.4. Characterization of Se-RHP Mw of Se-RHP were determined by high-performance gelfiltration chromatography (HPGFC) on a Waters 2695 instrument equipped with a Waters 2414 Refractive Index Detector (RID). 0.5 mg/mL sample solution (Se-RHP solution) was injected in each run with using 0.05 mol/L Na2 SO4 aqueous solution as the mobile phase. The analysis was performed at 25 ◦ C and a flow rate of 0.7 mL/min. The HPGFC system was calibrated with T-series Dextran standards. The Se contents of Se-RHP were determined on an inductively coupled plasma atomic emission spectrophotometer (ICP-AES) IRIS ER/S (TJA Company, USA). Absorbance spectra and measurements were carried out using a Puxi UV-1810 visible spectrophotometer (Beijing, China). FTIR spectra of RHP and Se-RHP were recorded with a NICOLET iS10 FT-IR spectrophotometer (Thermo Fisher, USA) in the range 4000–500 cm−1 . The resolution of FT-IR was 4 (data spacing: 0.482 cm−1 ), and number of scans were 16. 13 C NMR spectra of 40 mg/mL solutions in D2 O were recorded on a Bruker Avance
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400 MHz spectrometer (Germany) at 40 ◦ C with 2,2-dimethyl-2silapentane-5-sulfonate (DSS) as internal standard. 2.5. Assay for antioxidant activities The superoxide radical scavenging ability of RHP and Se-RHP was assessed by the method of Nishikimi, Appaji, and Yagi (1972). Hydroxyl radical assay was measured using a method of Ghiselli, Nardini, Baldi, and Scaccini (1998). The free-radical scavenging capacity of RHP and Se-RHP was analyzed using the DPPH radical according to the method of Shimada, Fujikawa, Yahara, and Nakamura (1992). The reducing power of all samples was investigated according to the method reported by Li, Li, and Zhou (2007). 2.6. Cell culture and treatment SH-SY5Y cells were cultured in D-MEM/F12 supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 g/mL streptomycin. The cultures were maintained in a 5% CO2 /95% air humidified atmosphere at 37 ◦ C, and the culture medium was changed every 2–3 days. SH-SY5Y cells were seeded into 96- or 6well plates, which were then allowed to equilibrate for 24 h prior to experiments. A25–35 was dissolved in deionized water at a concentration of 2 mM. The stock solution was diluted to desired concentrations immediately before use. In the establishment of oxidative stress experiments, cells were induced by different concentrations (15, 25, 35 M) of A25–35 for 48 h. In protective experiments of RHP or Se-RHP3, cells were pretreated with various concentrations (5, 10, 20, 40 g/mL) of RHP or Se-RHP3 dissolved in serum-free media for 4 h respectively, followed by exposed to 35 M (final concentration) of A25–35 in the presence of RHP or Se-RHP3 for another 48 h. The control cells were added with the same medium without A25–35 . All experiments were repeated three times for each treatment condition. 2.7. Determination of cell viability Cell viability was determined by using the thiazolyl blue tetrazolium bromide (MTT) assay, which was based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases. After incubation with the indicated drugs, 96-well plates had all media removed and replaced with serum-free media containing 0.5 mg/mL of MTT. The plate was further incubated for 3 h at 37 ◦ C with 5% CO2 , then MTT solution was removed and cells were lysed with 150 L DMSO. The plate was then placed on an orbital shaker for 10 min before determining absorbance at 570 nm using a microplate reader (Bio-Rad, Hercules, USA). The absorbance values were expressed as a percentage of untreated control cells (control = 100%). 2.8. Nuclear staining for assessment of apoptosis The cell nuclear morphology and number of cells were detected using Hoechst 33258 staining and fluorescence microscopy, as previously described (Zhang et al., 2012). Briefly, after treatment with 35 M A25–35 and RHP or Se-RHP3 for 48 h, SH-SY5Y cells were washed twice with PBS and incubated for 15 min with Hoechst 33258 (5 g/mL) at room temperature in the dark. After a rinse in PBS, the nuclear morphology of SH-SY5Y cells was observed and photographed using a fluorescence microscope (Olympus IX71, Japan). The cells were counted from four random fields of view in four different slides from each treatment, and the number of apoptotic cells was expressed as a percentage of the total number of cells counted.
Fig. 1. UV–vis absorption spectra of RHP and Se-RHP3.
2.9. Statistical analysis All data was presented as mean ± standard error (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by the Dunnett’s t-test. Statistical significance was set at P < 0.05. 3. Results and discussion 3.1. Chemical Analysis of Se-RHP The RHP and Se-RHP were confirmed by UV–vis spectroscopy analysis, as shown in Fig. 1. There was no absorption at 280 or 260 nm in the UV spectrum, which indicated the absence of proteins and nucleic acids in the purified RHP. Compared with the UV spectrum of RHP, it was found that the 336 nm wavelength showed the characteristic peak of selenium. The result indicated the existence of selenium and the formation of Se-RHP (Zhao, 2006). Se content and Mw of four derivatives of RHP were investigated in Table 1. The result showed that the organic selenium content of the samples increased from 1.04 to 3.29 mg/g. Se-RHP3 had the highest Se content that was 3.29 mg/g at 10 h with the yield of 345 mg. However, Se content had no obvious influence when it was more than 10 h. It implied that hydroxyl groups were substituted successfully by selenite groups on the polysaccharide. Compared with Mw of RHP, Mw of four derivatives of RHP showed a significant decrease, and varied from 27.7 kDa to 62.7 kDa. It was reported that polysaccharide in the strong acid circumstance would be depolymerized severely due to longer reaction time (Chen et al., 2010). In the present study, Mw of Se-RHP also decreased with the increase of reaction time. The decrease of Mw was mainly due to the hydrolysis of polysaccharide, and higher concentration of acid and temperature and longer reaction time could accelerate the hydrolysis rate.
Table 1 Characterization of RHP and Se-RHP. Samples
Reaction time (h)
Mw (kDa)
Yield (mg)
Se content (mg/g)
Se-RHP1 Se-RHP2 Se-RHP3 Se-RHP4 RHP
3 5 10 15 –
62.7 50.5 29.6 27.7
347 379 345 365 –
1.04 1.96 3.29 3.17 0
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Fig. 2. FT-IR spectra of RHP (A) and Se-RHP3 (B).
The FT-IR spectra of the native RHP and Se-RHP were shown in Fig. 2. The IR spectrum of the RHP showed a strong band at 3290 cm−1 attributed to the hydroxyl stretching vibration of the polysaccharide. The bands in the region of 2927 cm−1 and 1590 cm−1 were due to C H stretching vibration and OH bending vibrations. This amalgamation indicated that the RHP was polysaccharides. Compared with the native RHP, two new strong absorption peaks appeared at 925 cm−1 and 840 cm−1 for SeRHP, assigned to the C O Se stretching vibration and the Se O asymmetric stretching, respectively. These absorptions indicated that the selenylation modification in the samples had actually occurred. The selenyl position of polysaccharide was usually determined by 13 C NMR spectra. The 13 C NMR spectra of the native RHP and its derivative (Se-RHP3) were shown in Fig. 3. As shown in Fig. 3A, the signals of RHP at 96.1, 75.9, 74.1, 72.7, 69.5, and 60.5 ppm were assigned to C1 , C3 , C5 , C2 , C4 , and C6 , respectively. The new peak at 62.8 ppm of Se-RHP3 was assigned to the O-6 substituted carbons, suggesting reaction of O-6 (Melo, Feitosa, Feitosa, Freitas, & Paula, 2002). However, C-6 peaks still remained at 60.5 ppm for Se-RHP3, and had not been decreased obviously, suggesting that the primary OH groups on the internal side were not selenized. From the results of 13 C NMR, the non-selective reaction of RHP occurred, and the intensity of the signals of the O-substitution carbons denoted that C-6 substitution was predominant.
3.2. Antioxidant activity analysis 3.2.1. Scavenging activity of superoxide radical The scavenging ability of superoxide radicals was extremely important to antioxidant work. Fig. 4A showed that the inhibitory ability of Se-RHP on superoxide radical was significant in a concentration-dependent fashion. At a concentration of 3.2 mg/mL, the scavenging effect was 39.5%, 40.9%, 54.1%, 63.2% and 60.5% for RHP, Se-RHP1, Se-RHP2, Se-RHP3 and Se-RHP4, respectively. At the same concentration, the scavenging effect of Vc was 80.3%. Compared with RHP, Se-RHP exhibited more effective antioxidant activity. We also found that polysaccharide with higher selenium content exhibited stronger scavenging activity of superoxide radical. In the study, antioxidation mechanism may be that the presence of selenyl group ( SeH) or seleno-acid ester could change the three-dimensional structure of polysaccharide, and increasing hydroxyl groups would emerge, which affected the antioxidant ability.
Fig. 3. 13 C NMR (400 MHz) spectra of RHP and its selenylation derivatives. (A) RHP; (B) Se-RHP3.
3.2.2. Scavenging activity of hydroxyl radical. Hydroxyl radical, known to be generated through the Fenton reaction, can easily cross cell membranes, and readily react with biomolecules including carbohydrates, proteins, lipids, and DNA in cells, and cause tissue damage or cell death (Liu et al., 2010). The scavenging ability of all samples on hydroxyl radical was shown in Fig. 4B. Compared with the native RHP, Se-RHP showed higher scavenging ability. Furthermore, Se-RHP4 showed the strongest scavenging ability, and the scavenging ability was 43.5% at a dose of 3.2 mg/mL, but lower than Vc (88.2%). It was reported that the scavenging activity of hydroxyl radical was due to the inhibition of hydroxyl radical generation by chelating ions such as Fe2+ and Cu2+ . The contribution of the selenium moiety in the Se-RHP samples to antioxidant activity probably stemmed from, in part, the ability to complex the free metal ion. It was proposed that selenium possibly existed in the polysaccharides containing Se in the form of selenyl group ( SeH) or seleno-acid ester (Huang, 1994). The new groups may be able to complex Fe2+ or Cu2+ , there by inhibiting the generation of hydroxyl radicals (HO• ). However, the real mechanism of Se-RHP protecting ability from oxidative damage was beyond the scope of the present study and need further testifying. 3.2.3. Scavenging activity of DPPH radicals In the DPPH free radical scavenging test the stable yellowcolored diphenylpicrylhydrazine (DPPH-H) was formed in the presence of an antioxidant. As shown in Fig. 4C, all the samples were found to exhibit DPPH radical scavenging activity. Compared with RHP, Se-RHP exhibited more effective scavenging activity. Scavenging activity of Se-RHP (from Se-RHP1 to Se-RHP4) on DPPH radicals increased with increasing concentrations and reached a stable level of 44.9–52.6% at 3.2 mg/mL. No significant differences
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Fig. 4. Antioxidant effect of RHP and Se-RHP: (A) scavenging activity of superoxide radicals; (B) scavenging activity of hydroxyl radicals; (C) scavenging activity of DPPH radicals; (D) reducing power.
were observed between radical scavenging activity of polysaccharides containing different content of Se. However, the DPPH radical scavenging ability was more increased significantly for Vc than that of RHP and Se-RHP. It has been reported that the higher scavenging rate was for DPPH free radical due to the hydrogen-donating ability to form a stable DPPH-H molecule when accepted an electron or hydrogen radical. The presence of selenyl group ( SeH) or seleno-acid ester groups in the Se-RHP molecule could activate the hydrogen atom of the anomeric carbon. The higher activated capacity of the group, the stronger hydrogen atom-donating capacity.
3.2.4. Reducing power The reducing capacity of a compound might serve as a significant indicator of its potential antioxidant activity. The reducing properties were associated with the presence of reductones, which had been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom. As shown in Fig. 4D, all the samples showed high reducing power, which correlated well with increasing concentrations. At a concentration of 3.2 mg/mL, the reducing power was 0.31, 0.40, 0.58 and 0.54 for Se-RHP1, SeRHP2, Se-RHP3 and Se-RHP4, respectively, whereas the activity of polysaccharide from control medium (without Se) was significantly
Fig. 5. Protective effects of RHP or Se-RHP3 on A25–35 induced cytotoxicity in SH-SY5Y cells. All data were expressed as mean ±SEM of three experiments. # P < 0.05, ## P < 0.01 vs control; *P < 0.05, **P < 0.01 vs A25–35 alone.
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Fig. 6. Ameliorative effects of RHP or Se-RHP3 on A25–35 induced morphological alterations in SH-SY5Y cells (original magnification, ×100).
Fig. 7. RHP or Se-RHP3 protected SH-SY5Y cells against A25–35 induced apoptosis. Morphological apoptosis was determined by Hoechst 33258 staining. Arrow heads indicated apoptosis cells (original magnification, ×400).
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weaker and reached a maximum of 0.29 at 3.2 mg/mL. However, ascorbic acid showed significantly stronger reducing power (1.03 at 3.2 mg/mL) than the polysaccharide examined. The present studies confirmed the result that Se-RHP showed higher reducing power than the native RHP. 3.3. Neuroprotective effects of RHP or Se-RHP3 3.3.1. RHP or Se-RHP3 prevented SH-SY5Y cells from oxidative stress induced by Aˇ25–35 in vitro A25–35 invokes a cascade of oxidative stress and eventually leads to neuronal toxicity, which is characterized by the decrease of cell viability. Antioxidant can inhibit oxidative stress and prevents A25–35 -induced neuronal toxicity in cell culture. In addition, Se-RHP3 showed greater antioxidant activity than others in vitro and therefore was selected for further neuroprotective study. In the study, SH-SY5Y cells were exposed to different concentrations (15, 25, 35 M) of A25–35 for 48 h and cell viability was determined by MTT assay. As shown in Fig. 5A, 15–35 M of A25–35 induced significant decrease of cell survival in a dosedependent manner. Compared with the control group, treatment group using 35 M of A25–35 for 48 h reduced cell survival to 52.53 ± 3.65%, which was used to induce SH-SY5Y cell oxidative stress injury in the subsequent experiments. As shown in Fig. 5B, viability of the SH-SY5Y cells was significantly decreased to 50.02 ± 3.81% with the concentration of 35 M A25–35 for 48 h. However, compared with the A25–35 treatment group, cell viability rate of group pretreated with 5, 10, 20, and 40 g/mL of RHP or Se-RHP3 restored in a dose-dependent manner, increasing to 53.10 ± 2.70%, 64.07 ± 2.75%, 69.70 ± 3.74%, 77.84 ± 2.65%, and 54.82 ± 3.31%, 68.23 ± 4.81%, 75.65 ± 5.12%, 86.45 ± 4.73%, respectively. Furthermore, the cell morphological changes were observed with inverted phase contrast microscope. Compared with the control group, treatment of A25–35 at 35 M for 48 h resulted in obviously cells loss, synapse injury, shrinkage, and membrane blebbing. However, pretreatment with 40 g/mL of RHP or Se-RHP3 for 4 h before treatment with A25–35 obviously ameliorated such morphological alterations of cell injury (Fig. 6). 3.3.2. RHP or Se-RHP3 protected SH-SY5Y cells from apoptosis induced by Aˇ25–35 Oxidative stress induced by A25–35 is one of the major stimuli for apoptotic signal transduction in SH-SY5Y cells. Hoechst 33258, a fluorescent DNA binding dye, can display typical morphological features of apoptosis with chromatin agglutination and karyopyknosis. In this study, the nuclear staining with Hoechst 33258 was used to quantify the rate of apoptosis in each group using fluorescence microscope. As shown in Fig. 7, compared to the control group, the treatment with 35 M of A25–35 for 48 h showed typical characteristics of apoptosis, including the chromatin agglutination, karyopyknosis, and the appearance of apoptotic bodies and increased the percentage of apoptotic cells from 2.01% to 16.25%. However, pretreatment with 40 g/mL of RHP or Se-RHP3 for 4 h before treatment with A25–35 markedly decreased the number of cells with karyopyknosis and nuclear fragmentation and the percentage of apoptotic cells was reduced to 9.63% and 6.82%, respectively. 4. Conclusion In the present study, Se-RHP was synthesized by Na2 SeO3 /HNO3 with the highest Se content of 3.29 mg/g. Mw of Se-RHP had a sharp decrease after the selenylation modification. UV spectrum and FTIR spectra indicated that the selenized reaction had occurred. From the results of 13 C NMR, the signals of the O-substitution carbons denoted that C-6 substitution was predominant in Se-RHP. The
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results indicated that selenylation modification of RHP could significantly enhance the antioxidant activity in vitro. Furthermore, both RHP and Se-RHP3 could ameliorate A25–35 -induced oxidative stress and apoptosis in SH-SY5Y cells. The protective effects of both RHP and Se-RHP3 may help to provide the pharmacological basis of its clinical usage in the preventing or treatment of neurodegeneration in AD. Meanwhile, Se-RHP3 may serve as a good source of selenium as a supplement and could be used as an antioxidant for the food and pharmaceutical industries. Acknowledgments This work is supported in part by the General Program of National Natural Science Foundation of China (Grant No. 81373806), the General Project of Administration of Traditional Chinese Medicine of Gansu Province (Grant No. GZK-2012-43), and the Science and Technology Planning project of Jiangsu Entry-Exit Inspection Quarantine Bureau, China (2014KJ33). References Amoroso, S., Gioielli, A., Cataldi, M., Di Renzo, G., & Annunziato, L. (1999). In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca(2+) increase. Biochimica et Biophysica Acta, 1452(2), 151–160. Beckett, G. J., & Arthur, J. R. (2005). Selenium and endocrine systems. Journal of Endocrinology, 184(3), 455–465. Blander, G., de Oliveira, R. M., Conboy, C. M., Haigis, M., & Guarente, L. (2003). Superoxide dismutase 1 knock-down induces senescence in human fibroblasts. Journal of Biological Chemistry, 278(40), 38966–38969. Blennow, K., de Leon, M. J., & Zetterberg, H. (2006). Alzheimer’s disease. Lancet, 368(9533), 387–403. Che, X. Y., Xu, X. J., Zhang, L. N., & Fanbo, Z. (2009). Chain conformation and anti-tumor activities of phosphorylated (1-3)--d-glucan from Poria cocos. Carbohydrate Polymers, 78(3), 581–587. Chen, T., Li, B., Li, Y., Zhao, C., Shen, J., & Zhang, H. (2011). Catalytic synthesis and antitumor activities of sulfated polysaccharide from Gynostemma pentaphyllum Makino. Carbohydrate Polymers, 83(2), 554–560. Chen, T., Wang, J., Li, Y., Shen, J., Zhao, T., & Zhang, H. (2010). Sulfated modification and cytotoxicity of Portulaca oleracea L. polysaccharides. Glycoconjugate Journal, 27(6), 635–642. Chen, T., Wong, Y. S., Zheng, W., Bai, Y., & Huang, L. (2008). Selenium nanoparticles fabricated in Undaria pinnatifida polysaccharide solutions induce mitochondriamediated apoptosis in A375 human melanoma cells. Colloids Surface B: Biointerfaces, 67(1), 26–31. El-Sayed, W. M., Aboul-Fadl, T., Lamb, J. G., Roberts, J. C., & Franklin, M. R. (2006). Effect of selenium-containing compounds on hepatic chemoprotective enzymes in mice. Toxicology, 220(2-3), 179–188. Ghiselli, A., Nardini, M., Baldi, A., & Scaccini, C. (1998). Antioxidant activity of different phenolic fractions separated from an Italian red wine. Journal of Agricultural and Food Chemistry, 46(2), 361–367. Guo, Y., Pan, D., Li, H., Sun, Y., Zeng, X., & Yan, B. (2013). Antioxidant and immunomodulatory activity of selenium exopolysaccharide produced by Lactococcus lactis subsp. lactis. Food Chemistry, 138(1), 84–89. Hensley, K., Hall, N., Subramaniam, R., Cole, P., Harris, M., Aksenov, M., et al. (1995). Brain regional correspondence between Alzheimer’s disease histopathology and biomarkers of protein oxidation. Journal of Neurochemistry, 65(5), 2146–2156. Hu, T., Liu, D., Chen, Y., Wu, J., & Wang, S. (2010). Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro. International Journal of Biological Macromolecules, 46(2), 193–198. Huang, K. (1994). In H. B. Xu (Ed.), Selenium: Its chemistry, biochemistry and application in life science. Wuhan: Huazhong Science and Technology University Press. Hui, H. P., Feng, S. L., Zhao, L. G., Shi, Y. K., & Liu, X. H. (2010). Purification of the Radix hedysari polysaccharide and analysis of its constitution. Lishizhen Medicine and Materia Medica Research, 21(9), 2302–2303. Kim, S., Choi, D., Athukorala, Y., Jeon, Y., Senevirathne, M., & Rha, C. (2007). Antioxidant activity of sulfated polysaccharides isolated from Sargassum fulvellu. Journal of Food Science and Nutrition, 12(2), 65–73. Li, G. M., Wang, W. N., & Hu, M. S. (1987). Pharmacognosy of Radix hedysari. Zhong Yao Tong Bao, 12(8), 5–8. Li, X. M. (2007). Protective effect of Lycium barbarum polysaccharides on streptozotocin-induced oxidative stress in rats. International Journal of Biological Macromolecules, 40(5), 461–465. Li, X. M., Li, X. L., & Zhou, A. G. (2007). Evaluation of antioxidant activity of the polysaccharides extracted from Lycium barbarum fruits in vitro. European Polymer, 43(2), 488–497. Liu, X., Zhou, B., Lin, R., Jia, L., Deng, P., Fan, K., et al. (2010). Extraction and antioxidant activities of intracellular polysaccharide from Pleurotus sp. mycelium. International Journal of Biological Macromolecules, 47(2), 116–119.
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