- Email: [email protected]
Polysaccharides purified from Cordyceps cicadae protects PC12 cells against glutamate-induced oxidative damage
Carbohydrate Polymers 153 (2016) 187–195
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Polysaccharides purified from Cordyceps cicadae protects PC12 cells against glutamate-induced oxidative damage Opeyemi J. Olatunji a,1 , Yan Feng a,1 , Oyenike O. Olatunji b , Jian Tang a , Yuan Wei a , Zhen Ouyang a,∗ , Zhaoliang Su c a
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, China Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences Prince of Songkla University, Hat Yai, 90112, Thailand c School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, 212013, China b
a r t i c l e
i n f o
Article history: Received 6 January 2016 Received in revised form 22 June 2016 Accepted 28 June 2016 Available online 7 July 2016 Keywords: Cordyceps cicadae Polysaccharide Glutamate Neuroprotection PC12 cells
a b s t r a c t Two polysaccharides CPA-1 and CPB-2 were isolated purified from Cordyceps cicadae by hot water extraction, ethanol precipitation and purification using anion exchange and gel filtration chromatography. Preliminary structural characterization of CPA-1 and CPB-2 were performed. The protective effect of CPA-1 and CPB-2 against glutamate-induced oxidative toxicity in PC12 cells was analyzed. The results indicated that pretreatment of PC12 cells with CPA-1 and CPB-2 significantly increased cell survival, Ca2+ overload and ROS generation. CPA-1 and CPB-2 also markedly up-regulated the antioxidant status of pretreated PC12 cells. Our results suggested that Cordyceps cicadae polysaccharides can protect PC12 cells against glutamate excitotoxicity and might serve as therapeutic agents for neuronal disorders. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Polysaccharides belong to a class of polymeric carbohydrate consisting of monosaccharides that are connected through glycosidic bonds as branched or unbranched chains (Zong, Cao, & Wang, 2012). Fungal-derived polysaccharides have gained popularity in recent years due to the wide range of chemical diversity and bioactivities they display, including antioxidant, immunomodulatory, hepatoprotective, anti-inflammatory, antitumor, steroidogenic, hypolipidemic and hypocholesterolemic properties (Cheung, 1996; Cheung et al., 2009; Fisher & Yang, 2002; Gondim et al., 2012; Muller et al., 1996; Rashid et al., 2011; Sun et al., 2009; Wu et al., 2012; Yu et al., 2004; Zhang et al., 2012; Zhang, Cui, Cheung, & Wang, 2007). Oxidative stress plays an important role in several neuronal disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) (Halliwell, 2006), and it occurs when there is an imbalance between the oxidative and antioxidant system within the cell leading to an increase in the generation of reactive oxygen species
∗ Corresponding author at: School of Pharmacy, Jiangsu University 301 Xuefu road, Zhenjiang, 212013, China. E-mail address: [email protected] (Z. Ouyang). 1 These two authors contributed equally to the work. http://dx.doi.org/10.1016/j.carbpol.2016.06.108 0144-8617/© 2016 Elsevier Ltd. All rights reserved.
(ROS) and peroxyl radicals. Increase in the level of ROS causes damages to the DNA, proteins and mitochondrial of cells (Surendran & Rajasankar, 2010). Therefore, the use of antioxidants to eradicate ROS may be an effective means in the treatment of neurodegenerative diseases (Soto-Otero, Méndez-Alvarez, Hermida-Ameijeiras, ˜ ˜ & Labandeira-Garcia, 2000). Munoz-Pati no, Glutamate is a neurotransmitter mediating excitatory synaptic responses. It plays a pivotal role during the development of neurons through the activation of glutamic receptors present in the central nervous system (Jin, Horning, Mayer, & Gouaux, 2002; Molnar & Saac, 2002). However, excessive quantities of extracellular glutamate have been implicated in the pathogenesis of neuronal disorders (Tokarski, Bobula, Wabno, & Hess, 2008; Tzschentke, 2002). Two main mechanisms are involved in glutamate neurotoxicity; oxidative stress and glutamate receptor-mediated neurotoxicity. The first one is associated with glutamate-induced oxidative stress as a result of the prevention of cystine uptake. The reduced uptake of cystine, a precursor of glutathione leads to a decrease in the antioxidant defense resulting in oxidative stress (Albrecht et al., 2010; Pereira & Oliveira, 2000). Neuronal cell death induced by oxidative stress is mediated through the production of reactive oxygen species (ROS), mitochondrial dysfunction and the stimulation of several apoptosis-related deaths signaling pathways (Penugonda, Mare, Goldstein, Banks, & Ercal, 2005). The glutamate
188
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
receptor mediated excitotoxicity occurs through the activation of glutamate receptors (NMDA receptors), leading to the massive influx of Ca2+ and cell death (Lipton & Rosenberg, 1994; Michaels & Rothman, 1990). High concentrations of glutamate can lead to PC12 cell toxicity, and antioxidants have been proven to show protective effects against the cytotoxicity induced by glutamate in PC12 cells (Kawakami, Kanno, Ikarashi, & Kase, 2011; Li, Liu, Dluzen, & Jin, 2007; Penugonda et al., 2005). The exposure of PC12 cells to lofty concentrations of glutamate can be used as a simple but efficient experimental model for studying the neuroprotective effects as well as exploring the molecular mechanistic pathways involved. Cordyceps cicadae is an entomopathogenic fungi which belongs to the family Clavicipitaceae in the order Hypocreales (Tang & Eisenbrand, 1992). It is a well-known traditional Chinese medicine which has been in use for thousands of years as treatment for fatigue, night sweat, fever, infantile convulsion, palpitation and dizziness (Kuo et al., 2002). Previous researchers have demonstrated that polysaccharides isolated from Cordyceps cicadae displayed potent pharmacological properties such as immunomodulatory, renal protective, anti-oxidative properties (Kim et al., 2011, 2012; Ren, He, Cheng, & Chang, 2014). Recently, pilot studies conducted in our laboratory indicated that the crude polysaccharide extracts from Cordyceps cicadae displayed antioxidant and neuroprotective effects. Therefore, in this present study, we report the isolation, purification as well as the neuroprotective activity of two biological polysaccharides obtained from Cordyceps cicadae. 2. Materials and methods 2.1. Chemical and reagent Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Industries Inc. (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), l-glutamate, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Missouri, USA). DCFH-DA ROS assay kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Lactase dehydrogenase (LDH), glutathione (GSHPx), superoxide dismutase (SOD), malondialdehyde (MDA) assay kits were procured from NanJing Jiancheng Bioengineering Institute (Jiangsu, China). DEAE-52 cellulose and Sephadex G-100 were purchased from GE Healthcare (Waukesha, WI, USA). All other chemicals and reagents used were of analytical grade. PC12 cells were obtained from the Institute of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, China.
2015). The sample was loaded on a DEAE-cellulose column and successively eluted with distilled water, and a gradient of 0.1–0.2 M NaCl at 1.0 mL/min. Each fraction was monitored and assayed for its carbohydrate content using the phenol-sulfuric acid method. The eluates obtained from distilled water and 0.1 M NaCl was subjected to further individual purification using Sephadex-G100 to obtain two polysaccharide’s fraction (CPA-1 and CPB-2) were collected, dialyzed and lyophilized. 2.4. Analysis of monosaccharide composition The monosaccharide composition of the purified CPA-1 and CPB-2 was performed by gas chromatography (GC). The polysaccharides (10 mg each) were hydrolyzed in a sealed glass tube with 2 M trifluoroacetic acid at 100 ◦ C for 4 h. After removing the excess acid, the hydrolysate was acetylated with 5 mg of hydroxyl-amine hydrochloride in 1 mL of pyridine for 1 h at 90 ◦ C. After cooling, acetic anhydride (1 mL) was added and further incubated for 1 h at 90 ◦ C. The derivative was then converted into their corresponding aldononitrile acetate derivatives and analyzed with GC. Composition identification was done by comparison with reference standard (rhamnose, arabinose, xylose, mannose, glucose and galactose). 2.5. FT-IR spectral analysis CPA-1 and CPB-2 (5.0 mg/each) was ground with KBr and pressed into a pellet. The FT-IR spectra were analyzed on a PerkinElmer spectrometer from 4000 to 400 cm-1. 2.6. Cell culture and treatment PC12 cells were routinely maintained in DMEM containing 10% (v/v) FBS, 100 U/ml penicillin and 100 g/ml streptomycin at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 . The culture medium was changed every other day. PC12 cells were pretreated with different concentrations of CPA-1 and CPB-2 (25, 50, 100 and 200 g/ml) for 24 h, and then incubated with 5 mM glutamate for an additional 24 h. The control group was administered with the same amount of DMEM. CPA-1 and CPB-2 were dissolved in DMSO with the final concentration of DMSO less than 0.1% (v/v). 2.7. Determination of cell viability
2.2. Plant material Cordyceps cicadae were collected from Jurong, Jiangsu Province, China and authenticated by Professor Zhen Ouyang. A voucher specimen (CC-0015) was deposited in the herbarium of the School of Pharmacy, Jiangsu University. 2.3. Extraction and isolation Dried powder (500 g) of Cordyceps cicadae was extracted under reflux with distilled water (2 L × 3) at 100 ◦ C for 2 h. The resulting supernatant were pulled together and concentrated to a specific volume under reduced pressure using a rotary evaporator. The concentrated supernatant was precipitated by adding 4 vol. of cold 50% ethanol (v/v) overnight at 4 ◦ C. The precipitate obtained was centrifuged, washed twice with 95% ethanol and dissolved in distilled water. The solution was lyophilized to obtain the crude polysaccharide. The crude polysaccharide was deproteinized using Sevag method and decolorized using macro-porous resin D101 (Li et al.,
Cell viability was performed using MTT colorimetric assay. PC12 cells (1.0 × 104 cells/well) were seeded in a 96-well plate. After cell treatment, the medium was removed, and the cells were incubated with 20 L of 5 mg/ml MTT solution for 4 h at 37 ◦ C. The medium was carefully removed, and the dark-blue formazan was dissolved with 150 L of DMSO. The absorbance was determined at 490 nm with a microplate reader. Cell viability was expressed as a percentage of control cells. 2.8. LDH activity determination LDH leakage into the culture medium indicates cell injury. The LDH activity was measured according to the manufacturer’s instructions in the assay kit. After the treatment, the media was collected and the supernatant was used in the assay of extracellular LDH activity. The absorbance was measured at 450 nm using a microplate reader. Data represents the percentage of LDH released relative to control cells.
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
189
Fig. 1. (A) Elution profiles of crude polysaccharide separated from Cordyceps cicadae on DEAE-52 column and (B) Gel filtration chromatography column by Sephadex G-100.
2.9. Determination of ROS level The level of intracellular ROS was determined using the DCFHDA protocol. In brief, after cell treatment, cells were incubated with 10 M of DCFH-DA at 37 ◦ C for 30 min in the dark and then washed thrice with PBS. The fluorescence of DCF was analyzed with flow cytometry (Guava easyCyteTM, Merck Millipore, USA) with excitation at 488 nm and emission at 530 nm.
2.10. Determination of GSH-Px, SOD and MDA levels After the cell treatment, the medium was removed and the cells were washed thrice with PBS. Cells were collected, centrifuged, resuspended in PBS (500 L), dissociated using cell lysis buffer, and centrifuged for 6 min (12,000 rpm). The supernatant obtained after centrifuging was used to measure the GSH-Px, SOD activities and MDA levels using assay kit based on the specified manufacturer’s instructions. 2.11. Determination of intracellular calcium (Ca2+ ) level Cells were seeded in 96-well plates. After the cell treatment, cells were collected, washed with D-Hanks and incubated with Fura 2-AM (5 mol/L) at 37 ◦ C for 45 min in the dark. After incubation, cells were washed and re-suspended in cold salt solution buffer containing 0.2% bovine serum albumin. The cells were incubated at 37 ◦ C for 5 min and the fluorescence intensity measured at an
emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm on a microplate reader.
2.12. Statistical analysis Statistical analysis was performed using SPSS (version 16.0, SPSS Inc, Chicago, IL, USA). All data were expressed as mean ± SD. Statistical analysis was performed one way Analysis of Variance (ANOVA) followed by Post hoc analysis using Tukey test. Differences were considered significant at P < 0.05.
3. Results and discussion 3.1. Extraction, isolation and purification of polysaccharides The crude polysaccharide was isolated from Cordyceps cicadae through hot-water extraction, ethanol precipitation and lyophilization. It was fractionated using DEAE-52 cellulose eluting with distilled water and 0.1–0.2 N NaCl to obtain two major fractions (Fig. 1A). The two main fractions were further purified on Sephadex G-100 gel filtration eluting with 0.1 N NaCl and the resulting eluent were collected. Two polysaccharides designated CPA-1 and CPB2 were obtained with the yield of 3.11% and 5.07% respectively (Fig. 1B).
190
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
Fig. 2. Monosaccharide composition of analysis by GC; standard sugars (A), CPA-1 (B) and CPB-2 (C). Rhamnose (1); arabinose (2); xylose (3); mannose (4); glucose (5); galactose (6).
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
191
Fig. 3. FT-IR spectrum of CPA-1 and CPB-2.
3.2. Sugar composition of CPA-1 and CPB-2 As shown in the GC chromatogram (Fig. 2B–C), the result indicated that glucose was the main monosaccharide present in both CPA-1 and CPB-2 (47.7% and 79.7%, respectively). In addition CPA1 had mannose and galactose with a percentage composition of 22.9% and 29.4%. Likewise, CPB-2 had mannose (11.1%) and galactose (9.1%). 3.3. FT-IR spectra of CPA-1 and CPB-2 As shown in Fig. 3, the FT-IR spectra of CPA-1 and CPB-2 exhibited the characteristic strong and broad absorption peak at 3455 cm−1 and 3379 cm−1 respectively due to the stretching vibration of the hydroxyl group (Santhiya, Subramanian, & Natarajan, 2002). A weak absorption band at 2928 cm−1 (CPA-1) as well as 2929 cm−1 in CPB-2 was attributed to a CH stretching. The absorption band at 1652 cm−1 in CPA-1 and 1656 cm−1 in CPB2 corresponds to the CO stretching of the carbonyl. The signal at 1543 cm−1 (CPA-1) and 1539 (CPB-2) was attributable to the vibration of C O, while the signal at 1465 cm−1 (CPA-1) and 1410 cm−1 (CPB-2) is distinctive of CH bending. The characteristic bands at 1200–1000 cm−1 are attributed to the stretching vibrations of C O C and C OH in sugar rings (Zhao, Kan, Li, & Chen, 2005). A weak absorption band at 860 cm−1 (CPA-1) and 855 cm−1 (CPB2) indicated the presence of anomeric configuration in CPA-1 and CPB-2 (Coimbra, Goncalves, Barros, & Delgadillo, 2002). 3.4. Effect of CPA-1 and CPB-2 on glutamate-induced PC12 cell viability The increase in glutamate neurotransmission has been widely reported to be associated with the pathophysiology of neuronal disorders. High level of extracellular glutamate serves as a neurotoxin leading to programmed cell death (Mattson, 2000; Siegel & Sanacora, 2012). The survival rate of PC12 cells exposed to 5 mM glutamate for 24 h was decreased to 43.2% compared with the control group. However, in cells pretreated with CPA-1 and CPB-2, the viability was observed to be increased in a dose dependent manner. The viability of treated cells was found to increase to 53.2% (25 g/ml), 66.0% (50 g/ml), 76.8% (100 g/ml) and 84.8% (200 g/ml) for CPA-1, while CPB-2 increased cell viability by 57.3%, 70.8%, 80.6% and 89.4%, respectively (Fig. 4A). These results indicated that CPA-1 and CPB-2 could protect PC12 cells from glutamate induced cell death with CPB-2 displaying the maximum protective effect in all the experimental doses. Furthermore, the cytotoxicity of CPA-1 and CPB-2 on PC12 cells was accessed. CPA-1 and CPB-
Fig. 4. (A) Effect of CPA-1 and CPB-2 on PC12 cell viability. PC12 cells were incubated with CPA-1 and CPB-2 for 24 h, and then exposure to 5 mM glutamate for 24 h. Data are expressed as mean ± SD (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group. (B) Effect of CPA-1 and CPB-2 on cell viability. Cells were treated with CPA-1 and CPB-2 for 24 h. Results are expressed as% viability of the control. Data are expressed as mean ± SD (n = 5). **P < 0.01 as compared to control.
2 did not display any cytotoxic effect on the cells at the highest concentration (200 g/ml), instead it was observed that CPA-1 and CPB-2 promoted the proliferation of PC12 cells in a concentration
192
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
Fig. 5. Effect of CPA-1 and CPB-2 on glutamate-induced LDH leakage. Cells were administrated with CPA-1 and CPB-2 (25–200 g/ml) for 24 h and then treated with 5 mM glutamate for 24 h. The data are represented as means ± SD (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group.
Fig. 6. Effects of CPA-1 and CPB-2 on intracellular production of ROS in glutamatetreated cells. The data are represented as means ± SD (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group.
dependent manner. This effect was observed to be significant at 50, 100 and 200 g/ml (Fig. 4B).
3.5. Effect of CPA-1 and CPB-2 on glutamate-induced LDH leakage in PC12 cells LDH (lactate dehydrogenase) is an enzyme that is found in cells, and it is responsible for catalyzing the oxidation of lactate to pyruvate. Upon cell injury or damage, LDH is released from the cells. As such the quantification of LDH leakage from cells is one of the main tools for the assessment of cell death. As observed in Fig. 5, pretreatment with CPA-1 and CPB-2 significantly reduced LDH leakage by 68.2%, 58.0%, 46.8%, 32.8% in CPA-1 treated group and 64.2%, 54.8%, 42.6% and 28.4% in CPB-2 treated group, respectively when compared to the glutamate-treated group (86.8%).
3.6. Effect of CPA-1 and CPB-2 on glutamate-induced ROS production Oxidative stress results due to an imbalance between free radical generation and the antioxidant defense system. The increased production of free radicals initiates the generation of reactive oxygen species (ROS) leading to oxidative stress (Ko, Kim, & Jeon, 2012; Ko, Lee, Samarakoon, Kim, & Jeon, 2013). Oxidative stress has been implicated in the pathogenesis and progression of several diseases, including neurodegenerative diseases. Glutamate inflicts cell injury and apoptosis by causing an increase in ROS generation which leads to oxidative stress (Uttara, Singh, Zamboni, & Mahajan, 2009). Glutamate excitotoxicity has been widely reported to be associated with mitochondrial dysfunction and oxidative damage in cell culture experimental studies (Pereira & Oliveira, 2000; Schubert & Piasecki, 2001). After the incubation of PC12 cells with 5 mM glutamate, the level of intracellular ROS was observed to be significantly increased to approximately 4 folds as compared to the control group. However, CPA-1 and CPB-2 obviously decreased ROS generated as a result of glutamate toxicity to 251.4% and 246.1% (25 g/ml), 225.6% and 211.3% (50 g/ml), 185.4% and 175.1% (100 g/ml), 149.1% and 134.3% (200 g/ml), respectively (Fig. 6).
Fig. 7. Effect of CPA-1 and CPB-2 on glutamate-induced intracellular Ca2+ influx. CPA-1 and CPB-2 (25–200 g/ml) could ameliorate the increased levels of Ca2+ induced by glutamate. The data were expressed as means ± S.D (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group.
3.7. Effect of CPA-1 and CPB-2 on glutamate-induced intracellular Ca2+ level in PC12 cells Several studies have reported that glutamate toxicity can lead to excessive Ca2+ overload by the activation of NMDA receptors (Baptisite et al., 2004; Vazhappilly, Wee, Sucher, & Low, 2010). The increase in intracellular Ca2+ concentration can also results in the formation of ROS (Randall & Thayer, 1992). Furthermore, high levels of intracellular Ca2+ concentration can lead to the activation of lipases, proteases, nitric oxide synthases and oxidative stress which damages the mitochondria (Oster et al., 2014). In this present study, it was observed that the exposure of PC12 cells to glutamate led to an increase in the level of Ca2+ concentration in the untreated cells as compared to the control group. The concentration of Ca2+ in the untreated group was increased to 397.3 mmol/L. However, pretreatment of cells with CPA-1 (25, 50, 100 and 200 g/ml) decreased the intracellular Ca2+ concentration to 310.3, 250.0, 195.6 and 153.5 mmol/L, respectively. While CPB-2 dose dependently reduced Ca2+ concentration to 299.8, 246.6, 184.5 and 148.2 mmol/L respectively (Fig. 7).
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
193
Widmann, Trapp, & Holsboer, 1995; Sun, Zhang, Zhu, Chen, & Mei, 2010). Antioxidants have the ability to prevent glutamaterelated toxicity, thus affirming the hypothesis which stipulates that glutamate-induced cell death is mediated via the increase in free radicals and ROS generation (Pereira & Oliveira, 1997, 2000). Antioxidant enzymes SOD and GSH-Px are an integral protective mechanism that cells utilize in preventing oxidative stress and cell injury. Thus, the enhancement of their effects can protect cells against oxidative-induced damages (Han et al., 2014; Shen, Song, Wu, & Zhang, 2011). As indicated in Fig. 8A and B, the prior treatment of PC12 cells with CPA-1 and CPB-2 caused a marked increase in the activities of the GSH-Px from 76.2 U/mg protein in glutamate treated cells to 91.2, 110, 122.2, 137.9 U/mg protein (25, 50, 100 and 200 g/ml of CPA-1 respectively) and 93.6, 118.1, 129.1 and 140.1 U/mg protein (25, 50, 100 and 200 g/ml of CPB-2 respectively). Likewise, the activity of SOD was significant increased in all pretreated cell groups when compared with the glutamate treated group. Furthermore, a significant increase was observed in the level of MDA in cells exposed to glutamate only (254.6%). However, pretreatment with CPA-1 and CPB-2 significantly decreased the up-regulated levels of MDA in a dose-dependent manner (Fig. 8C). This result suggests that CPA-1 and CPB-2 have the ability to protect cells against glutamate induced oxidative stress by reducing lipid peroxidation product and enhancing the antioxidant enzyme activities. Several reports have demonstrated the emerging potentials of polysaccharides in the treatment for experimental models of neurodegenerative disorders (Cui et al., 2016; Deng & Yang, 2014; Jia, Rao, Xue, & Lei, 2015; Zhang, Wang, Jin, & Zhang, 2013). The results obtained from this study thus add to the already existing evidence that supports polysaccharides as safe therapeutic options for the treatment of neuronal disorders.
4. Conclusion
Fig. 8. Effects of CPA-1 and CPB-2 (A) GSH-Px activity (B) SOD activity and (C) MDA levels in glutamate-treated cells. Cells were pretreated with different concentrations of CPA-1 and CPB-2 for 24 h and then exposed to 5 mM glutamate for 24 h. Data were presented as means ± S.D. (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group.
3.8. Effect of CPA-1 and CPB-2 on GSH-Px, SOD activities and MDA levels Cell death which results from ROS overproduction is accompanied by an increase in lipid peroxide and a decrease in the activity of enzymes and membrane fluids (Park et al., 2002). Glutamate toxicity leads to elevation of malondialdehyde content, reduction in the activities of glutathione and superoxide dismutase (Behl,
In this study the isolation and neuroprotective property of two polysaccharides from Cordyceps cicadae were investigated. Two polysaccharides CPA-1 and CPB-2 were purified and preliminary structural characterizations were conducted. Monosaccharides analysis indicated that CPA-1 and CPB-2 were heterogeneous polysaccharides and mainly composed of mannose, glucose and galactose. CPA-1 and CPB-2 exhibited protective effects against glutamate induced oxidative toxicity in PC12 cells by increasing cell viability, reducing LDH leakage, ROS production and Ca2+ overload in the pretreated cells. Furthermore, this present work also demonstrated that glutamate markedly inhibited the activities of SOD and GSH-Px in PC12 cells, but the pretreatment of PC12 cells with CPA-1 and CPB-2 reversed these changes. This suggests that the protective effects of CPA-1 and CPB-2 may be linked to its antioxidant properties. Previous researches have also reported the isolation of polysaccharides from Cordyceps species with neuroprotection through the increase in antioxidant enzymes activities as well as decreasing MDA levels (Li et al., 2003; Shen, et al., 2011). In conclusion, CPA-1 and CPB-2 possesses protective effects in PC12 cells against glutamate-induced oxidative damage and polysaccharides from Cordyceps cicadae may be applied as potential health and functional food source especially in the management of neuronal disorders
Conflict of interest The authors hereby declare no conflict of interest associated with this article.
194
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
Acknowledgement This work was supported by National Natural Science Foundation of China (Nos. 81072985, 81373480, 81573529). References Albrecht, P., Lewerenz, J., Dittmer, S., Noack, R., Maher, P., & Methner, A. (2010). Mechanisms of oxidative glutamate toxicity: the glutamate/cystine antiporter system xc- as a neuroprotective drug target. CNS & Neurological Disorders—Drug Targets, 9, 373–382. Baptisite, D. C., Hartwick, A. T., Jollimore, C. A., Baldridge, W. H., Seigel, G. M., & Kelly, M. E. (2004). An investigation of the neuroprotective effects of tetracycline derivatives in experimental models of retinal cell death. Molecular Pharmacology, 66, 1113–1122. Behl, C., Widmann, M., Trapp, T., & Holsboer, F. (1995). 17-beta estradiol protects neurons from oxidative stress-induced cell death in vitro. Biochemical and Biophysical Research Communication, 216, 473–482. Cheung, P. C. K. (1996). The hypocholesterolemic effect of extracellular polysaccharide from the submerged fermentation of mushroom. Nutrition Research, 16, 1953–1957. Cheung, J. K., Li, J., Cheung, A. W., Zhu, Y., Zheng, K. Y., Bi, C. W., et al. (2009). Cordysinocan, a polysaccharide isolated from cultured Cordyceps: activates immune responses in cultured T-lymphocytes and macrophages: signaling cascade and induction of cytokines. Journal of Ethnopharmacology, 124, 61–68. Coimbra, M. A., Goncalves, F., Barros, A. S., & Delgadillo, I. (2002). Fourier transform infrared spectroscopy and chemometric analysis of white wine polysaccharide extracts. Journal of Agriculture and Food Chemistry, 50, 3405–3411. Cui, C., Cui, N., Wang, P., Song, S., Liang, H., & Ji, A. (2016). Neuroprotective effect of sulfated polysaccharide isolated from sea cucumber Stichopus japonicus on 6-OHDA-induced death in SH-SY5Y through inhibition of MAPK and NF-(B and activation of PI3 K/Akt signaling pathways. Biochemical and Biophysical Research Communications, 470, 375–383. Deng, Q., & Yang, X. (2014). Protective effects of Gynostemma pentaphyllum polysaccharides on PC12 cells impaired by MPP+ . International Journal of Biological Macromolecules, 69, 171–175. Fisher, M., & Yang, L. X. (2002). Anticancer effects and mechanism of polysaccharide-K (PSK): implications of cancer immunotherapy. Anticancer Research, 22, 1737–1754. Gondim, D. V., Costa, J. L., Rocha, S. S., Brito, G. A. D., Ribeiro, R. D., & Vale, M. L. (2012). Antinociceptive and anti-inflammatory effects of electroacupuncture on experimental arthritis of the rat temporomandibular joint. Canadian Journal of Physiology and Pharmacology, 904, 395–405. Halliwell, B. (2006). Oxidative stress and neurodegeneration: where are we now? Journal of Neurochemistry, 97, 1634–1658. Han, X., Zhu, S., Wang, B., Chen, L., Li, R., Yao, W., et al. (2014). Antioxidant action of 7,8-dihydroxyflavone protects PC12 cells against 6-hydroxydopamine-induced cytotoxicity. Neurochemistry International, 64, 18–23. Jia, D., Rao, C., Xue, S., & Lei, J. (2015). Purification: characterization and neuroprotective effects of a polysaccharide from Gynostemma pentaphyllum. Carbohydrate Polymers, 22, 93–100. Jin, R., Horning, M., Mayer, M. L., & Gouaux, E. (2002). Mechanism of activation and selectivity in a ligand-gated ion channel: structural and functional studies of GluR2 and quisqualate. Biochemistry, 41, 15635–15643. Kawakami, Z., Kanno, H., Ikarashi, Y., & Kase, Y. (2011). Yokukansan, a kampo medicine: protects against glutamate cytotoxicity due to oxidative stress in PC12 cells. Journal of Ethnopharmacology, 134, 74–81. Kim, H. S., Kim, Y. J., Lee, H. K., Ryu, H. S., Kim, J. S., Yoon, M. J., et al. (2012). Activation of macrophages by polysaccharide isolated from Paecilomyces cicadae through toll-like receptor 4. Food and Chemical Toxicology, 50, 3190–3197. Kim, H. S., Kim, J. Y., Ryu, H. S., Shin, B. R., Kang, J. S., Kim, H. M., et al. (2011). Phenotypic and functional maturation of dendritic cells induced by polysaccharide isolated from Paecilomyces cicadae. Journal of Medicinal Food, 14, 847–856. Ko, J. Y., Lee, J. H., Samarakoon, K., Kim, J. S., & Jeon, Y. J. (2013). Purification and determination of two novel antioxidant peptides from flounder fish (Paralichthys olivaceus) using digestive proteases. Food and Chemical Toxicology, 52, 113–120. Ko, S. C., Kim, D. K., & Jeon, Y. J. (2012). Protective effect of a novel antioxidant peptide purified from a marine Chlorella elthpsoidea protein against free radical-induced oxidative stress. Food and Chemical Toxicology, 50, 2294–2302. Kuo, Y. C., Lin, L. C., Don, M. J., Liao, H. F., Tsai, Y. P., Lee, G. H., et al. (2002). Cyclodepsipeptide and dioxomorpholine derivatives isolated from the insect-body portion of the fungus Cordyceps cicadae. Journal of Chinese Medicine, 13, 209–219. Li, C., Huang, Q., Fua, X., Yue, X.-J., Liu, R. H., & You, L.-J. (2015). Characterization: antioxidant and immunomodulatory activities of polysaccharides from Prunella vulgaris Linn. International Journal of Biological Macromolecules, 75, 298–305. Li, N., Liu, B., Dluzen, D. E., & Jin, Y. (2007). Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 cells. Journal of Ethnopharmacology, 111, 458–463. Li, S. P., Zhao, K. J., Ji, Z. N., Dong, T. T., Lo, C. K., Cheung, J. K., et al. (2003). A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese
medicine: protects PC12 cells against hydrogen peroxide-induced injury. Life Sciences, 73, 2503–2513. Lipton, S. A., & Rosenberg, P. A. (1994). Excitatory amino acids as a final common pathway for neurologic disorders. New England Journal of Medicine, 330, 613–622. Mattson, M. P. (2000). Apoptosis in neurodegenerative disorders. Nature Reviews Molecular Cell Biology, 1, 120–129. Michaels, R. L., & Rothman, S. M. (1990). Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations. Journal of Neuroscience, 10, 283–292. Molnar, E., & Saac, J. T. (2002). Developmental and activity dependent regulation of ionotropic glutamate receptors at synapses. The Scientific World Journal, 2, 27–47. Muller, A., Rice, P. J., Ensley, H. E., Coogan, P. S., Kalbfleish, J. H., Kelley, J. L., et al. (1996). Receptor binding and internalization of a water-soluble (1→3)-beta-d-glucan biologic response modifier in two monocyte/macrophage cell lines. Journal of Immunology, 156, 3418–3425. Oster, S., Radad, K., Scheller, D., Hesse, M., Balanzew, W., Reichmann, H., et al. (2014). Rotigotine protects against glutamate toxicity in primary dopaminergic cell culture. European Journal of Pharmacology, 724, 31–42. Park, J. E., Yang, J. H., Yoon, S. J., Lee, J. H., Yang, E. S., & Park, J. W. (2002). Lipid peroxidation-mediated cytotoxicity and DNA damage in U937 cells. Biochimie, 84, 1199–1205. Penugonda, S., Mare, S., Goldstein, G., Banks, W. A., & Ercal, N. (2005). Effects of N-acetylcysteine amide (NACA), a novel thiol antioxidant against glutamate-induced cytotoxicity in neuronal cell line PC12. Brain Research, 1056, 132–138. Pereira, C. M., & Oliveira, C. R. (1997). Glutamate toxicity on a PC12 cell line involves glutathione (GSH) depletion and oxidative stress. Free Radical Biology Medicine, 23, 637–647. Pereira, C. F., & Oliveira, C. R. D. (2000). Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis. Neuroscience Research, 37, 227–236. Randall, R. D., & Thayer, S. A. (1992). Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons. Journal of Neuroscience, 12, 1881–1895. Rashid, S., Unyayar, A., Mazmanci, M. A., McKeown, S. R., Banat, I. M., & Worthington, J. (2011). A study of anti-cancer effects of Funalia trogii in vitro and in vivo. Food and Chemical Toxicology, 49, 1477–1483. Ren, X., He, L., Cheng, J., & Chang, J. (2014). Optimization of the solid-state fermentation and properties of a polysaccharide from Paecilomyces cicadae (Miquel) Samson and its antioxidant activities in vitro. PLosOne, 9, e87578. Santhiya, D., Subramanian, S., & Natarajan, K. (2002). Surface chemical studies on sphalerite and galena using extracellular polysaccharides isolated from Bacillus polymyxa. Journal of Colloid and Interface Science, 256, 237–248. Schubert, D., & Piasecki, D. (2001). Oxidative glutamate toxicity can be a component of the excitotoxicity cascade. Journal of Neuroscience, 21, 7455–7462. Shen, W., Song, D., Wu, J., & Zhang, W. (2011). Protective effect of a polysaccharide isolated from a cultivated Cordyceps mycelia on hydrogen peroxide-induced oxidative damage in PC12 cells. Phytotherapy Research, 25, 675–680. Siegel, S., & Sanacora, G. (2012). The roles of glutamate receptors across major neurological and psychiatric disorders. Pharmacology Biochemistry and Behavior, 100, 653–655. ˜ ˜ A. M., & Soto-Otero, R., Méndez-Alvarez, E., Hermida-Ameijeiras, A., Munoz-Pati no, Labandeira-Garcia, J. L. (2000). Autoxidation and neurotoxicity of 6hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson’s disease. Journal of Neurochemistry, 74, 1605–1612. Sun, H. H., Mao, W. J., Chen, Y., Guo, S. D., Li, H. Y., & Qi, X. H. (2009). Isolation: chemical characteristics and antioxidant properties of the polysaccharides from marine fungus Penicillium sp. F23-2. Carbohydrate Polymers, 78, 117–124. Sun, Z. W., Zhang, L., Zhu, S. J., Chen, W. C., & Mei, B. (2010). Excitotoxicity effects of glutamate on human neuroblastoma SH-SY5Y cells via oxidative damage. Neuroscience Bulletin, 26, 8–16. Surendran, S., & Rajasankar, S. (2010). Parkinson’s disease: oxidative stress and therapeutic approaches. Neurological Sciences, 31, 531–540. Tang, W., & Eisenbrand, G. (1992). Chinese drugs of plant origin. GmbH & Co. Berlin Heidelberg KG: Springer-Verlag. Tokarski, K., Bobula, B., Wabno, J., & Hess, G. (2008). Repeated administration of imipramine attenuates glutamatergic transmission in rat frontal cortex. Neuroscience, 153, 789–795. Tzschentke, T. M. (2002). Glutamatergic mechanisms in different disease states: overview and therapeutic implications—an introduction. Amino Acids, 23, 147–152. Uttara, B., Singh, A. V., Zamboni, P., & Mahajan, R. T. (2009). Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Current Neuropharmacology, 7, 65–74. Vazhappilly, R., Wee, K. S., Sucher, N. J., & Low, C. M. (2010). A non-muscle myosin II motor links NR1 to retrograde trafficking and proteasomal degradation in PC12 cells. Neurochemistry International, 56, 569–576. Wu, F. Y., Yan, H., Ma, X. N., Jia, J. Q., Zhang, G. Z., Guo, X. J., et al. (2012). Comparison of the structural characterization and biological activity of acidic polysaccharides from Cordyceps militaris cultured with different media. World Journal of Microbiology and Biotechnology, 28, 2029–2038.
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195 Yu, R., Song, L., Zhao, Y., Bin, W., Wang, L., Zhang, H., et al. (2004). Isolation and biological properties of polysaccharide CPS-1 from cultured Cordyceps militaris. Fitoterapia, 75, 465–472. Zhang, M., Cui, S. W., Cheung, P. C. K., & Wang, Q. (2007). Antitumor polysaccharides from mushrooms: a review on their isolation process: structural characteristics and antitumor activity. Trends in Food Science & Technology, 18, 4–19. Zhang, Z. F., Lv, G. Y., Pan, H. J., Pandey, A., He, W. Q., & Fan, L. F. (2012). Antioxidant and hepatoprotective potential of endo-polysaccharides from Hericium erinaceus grown on tofu whey. International Journal Biological Macromolecules, 51, 1140–1146.
195
Zhang, W., Wang, J., Jin, W., & Zhang, Q. (2013). The antioxidant activities and neuroprotective effect of polysaccharides from the starfish Asterias rollestoni. Carbohydrate Polymers, 95, 9–15. Zhao, G., Kan, J., Li, Z., & Chen, Z. (2005). Structural features and immunological activity of a polysaccharide from Dioscorea opposita Thunb roots. Carbohydrate Polymers, 61, 125–131. Zong, A. Z., Cao, H. Z., & Wang, F. S. (2012). Anticancer polysaccharides from natural resources: a review of recent research. Carbohydrate Polymers, 90, 1395–1410.
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Polysaccharides purified from Cordyceps cicadae protects PC12 cells against glutamate-induced oxidative damage Opeyemi J. Olatunji a,1 , Yan Feng a,1 , Oyenike O. Olatunji b , Jian Tang a , Yuan Wei a , Zhen Ouyang a,∗ , Zhaoliang Su c a
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, China Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences Prince of Songkla University, Hat Yai, 90112, Thailand c School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, 212013, China b
a r t i c l e
i n f o
Article history: Received 6 January 2016 Received in revised form 22 June 2016 Accepted 28 June 2016 Available online 7 July 2016 Keywords: Cordyceps cicadae Polysaccharide Glutamate Neuroprotection PC12 cells
a b s t r a c t Two polysaccharides CPA-1 and CPB-2 were isolated purified from Cordyceps cicadae by hot water extraction, ethanol precipitation and purification using anion exchange and gel filtration chromatography. Preliminary structural characterization of CPA-1 and CPB-2 were performed. The protective effect of CPA-1 and CPB-2 against glutamate-induced oxidative toxicity in PC12 cells was analyzed. The results indicated that pretreatment of PC12 cells with CPA-1 and CPB-2 significantly increased cell survival, Ca2+ overload and ROS generation. CPA-1 and CPB-2 also markedly up-regulated the antioxidant status of pretreated PC12 cells. Our results suggested that Cordyceps cicadae polysaccharides can protect PC12 cells against glutamate excitotoxicity and might serve as therapeutic agents for neuronal disorders. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Polysaccharides belong to a class of polymeric carbohydrate consisting of monosaccharides that are connected through glycosidic bonds as branched or unbranched chains (Zong, Cao, & Wang, 2012). Fungal-derived polysaccharides have gained popularity in recent years due to the wide range of chemical diversity and bioactivities they display, including antioxidant, immunomodulatory, hepatoprotective, anti-inflammatory, antitumor, steroidogenic, hypolipidemic and hypocholesterolemic properties (Cheung, 1996; Cheung et al., 2009; Fisher & Yang, 2002; Gondim et al., 2012; Muller et al., 1996; Rashid et al., 2011; Sun et al., 2009; Wu et al., 2012; Yu et al., 2004; Zhang et al., 2012; Zhang, Cui, Cheung, & Wang, 2007). Oxidative stress plays an important role in several neuronal disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) (Halliwell, 2006), and it occurs when there is an imbalance between the oxidative and antioxidant system within the cell leading to an increase in the generation of reactive oxygen species
∗ Corresponding author at: School of Pharmacy, Jiangsu University 301 Xuefu road, Zhenjiang, 212013, China. E-mail address: [email protected] (Z. Ouyang). 1 These two authors contributed equally to the work. http://dx.doi.org/10.1016/j.carbpol.2016.06.108 0144-8617/© 2016 Elsevier Ltd. All rights reserved.
(ROS) and peroxyl radicals. Increase in the level of ROS causes damages to the DNA, proteins and mitochondrial of cells (Surendran & Rajasankar, 2010). Therefore, the use of antioxidants to eradicate ROS may be an effective means in the treatment of neurodegenerative diseases (Soto-Otero, Méndez-Alvarez, Hermida-Ameijeiras, ˜ ˜ & Labandeira-Garcia, 2000). Munoz-Pati no, Glutamate is a neurotransmitter mediating excitatory synaptic responses. It plays a pivotal role during the development of neurons through the activation of glutamic receptors present in the central nervous system (Jin, Horning, Mayer, & Gouaux, 2002; Molnar & Saac, 2002). However, excessive quantities of extracellular glutamate have been implicated in the pathogenesis of neuronal disorders (Tokarski, Bobula, Wabno, & Hess, 2008; Tzschentke, 2002). Two main mechanisms are involved in glutamate neurotoxicity; oxidative stress and glutamate receptor-mediated neurotoxicity. The first one is associated with glutamate-induced oxidative stress as a result of the prevention of cystine uptake. The reduced uptake of cystine, a precursor of glutathione leads to a decrease in the antioxidant defense resulting in oxidative stress (Albrecht et al., 2010; Pereira & Oliveira, 2000). Neuronal cell death induced by oxidative stress is mediated through the production of reactive oxygen species (ROS), mitochondrial dysfunction and the stimulation of several apoptosis-related deaths signaling pathways (Penugonda, Mare, Goldstein, Banks, & Ercal, 2005). The glutamate
188
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
receptor mediated excitotoxicity occurs through the activation of glutamate receptors (NMDA receptors), leading to the massive influx of Ca2+ and cell death (Lipton & Rosenberg, 1994; Michaels & Rothman, 1990). High concentrations of glutamate can lead to PC12 cell toxicity, and antioxidants have been proven to show protective effects against the cytotoxicity induced by glutamate in PC12 cells (Kawakami, Kanno, Ikarashi, & Kase, 2011; Li, Liu, Dluzen, & Jin, 2007; Penugonda et al., 2005). The exposure of PC12 cells to lofty concentrations of glutamate can be used as a simple but efficient experimental model for studying the neuroprotective effects as well as exploring the molecular mechanistic pathways involved. Cordyceps cicadae is an entomopathogenic fungi which belongs to the family Clavicipitaceae in the order Hypocreales (Tang & Eisenbrand, 1992). It is a well-known traditional Chinese medicine which has been in use for thousands of years as treatment for fatigue, night sweat, fever, infantile convulsion, palpitation and dizziness (Kuo et al., 2002). Previous researchers have demonstrated that polysaccharides isolated from Cordyceps cicadae displayed potent pharmacological properties such as immunomodulatory, renal protective, anti-oxidative properties (Kim et al., 2011, 2012; Ren, He, Cheng, & Chang, 2014). Recently, pilot studies conducted in our laboratory indicated that the crude polysaccharide extracts from Cordyceps cicadae displayed antioxidant and neuroprotective effects. Therefore, in this present study, we report the isolation, purification as well as the neuroprotective activity of two biological polysaccharides obtained from Cordyceps cicadae. 2. Materials and methods 2.1. Chemical and reagent Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Industries Inc. (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), l-glutamate, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Missouri, USA). DCFH-DA ROS assay kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Lactase dehydrogenase (LDH), glutathione (GSHPx), superoxide dismutase (SOD), malondialdehyde (MDA) assay kits were procured from NanJing Jiancheng Bioengineering Institute (Jiangsu, China). DEAE-52 cellulose and Sephadex G-100 were purchased from GE Healthcare (Waukesha, WI, USA). All other chemicals and reagents used were of analytical grade. PC12 cells were obtained from the Institute of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, China.
2015). The sample was loaded on a DEAE-cellulose column and successively eluted with distilled water, and a gradient of 0.1–0.2 M NaCl at 1.0 mL/min. Each fraction was monitored and assayed for its carbohydrate content using the phenol-sulfuric acid method. The eluates obtained from distilled water and 0.1 M NaCl was subjected to further individual purification using Sephadex-G100 to obtain two polysaccharide’s fraction (CPA-1 and CPB-2) were collected, dialyzed and lyophilized. 2.4. Analysis of monosaccharide composition The monosaccharide composition of the purified CPA-1 and CPB-2 was performed by gas chromatography (GC). The polysaccharides (10 mg each) were hydrolyzed in a sealed glass tube with 2 M trifluoroacetic acid at 100 ◦ C for 4 h. After removing the excess acid, the hydrolysate was acetylated with 5 mg of hydroxyl-amine hydrochloride in 1 mL of pyridine for 1 h at 90 ◦ C. After cooling, acetic anhydride (1 mL) was added and further incubated for 1 h at 90 ◦ C. The derivative was then converted into their corresponding aldononitrile acetate derivatives and analyzed with GC. Composition identification was done by comparison with reference standard (rhamnose, arabinose, xylose, mannose, glucose and galactose). 2.5. FT-IR spectral analysis CPA-1 and CPB-2 (5.0 mg/each) was ground with KBr and pressed into a pellet. The FT-IR spectra were analyzed on a PerkinElmer spectrometer from 4000 to 400 cm-1. 2.6. Cell culture and treatment PC12 cells were routinely maintained in DMEM containing 10% (v/v) FBS, 100 U/ml penicillin and 100 g/ml streptomycin at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 . The culture medium was changed every other day. PC12 cells were pretreated with different concentrations of CPA-1 and CPB-2 (25, 50, 100 and 200 g/ml) for 24 h, and then incubated with 5 mM glutamate for an additional 24 h. The control group was administered with the same amount of DMEM. CPA-1 and CPB-2 were dissolved in DMSO with the final concentration of DMSO less than 0.1% (v/v). 2.7. Determination of cell viability
2.2. Plant material Cordyceps cicadae were collected from Jurong, Jiangsu Province, China and authenticated by Professor Zhen Ouyang. A voucher specimen (CC-0015) was deposited in the herbarium of the School of Pharmacy, Jiangsu University. 2.3. Extraction and isolation Dried powder (500 g) of Cordyceps cicadae was extracted under reflux with distilled water (2 L × 3) at 100 ◦ C for 2 h. The resulting supernatant were pulled together and concentrated to a specific volume under reduced pressure using a rotary evaporator. The concentrated supernatant was precipitated by adding 4 vol. of cold 50% ethanol (v/v) overnight at 4 ◦ C. The precipitate obtained was centrifuged, washed twice with 95% ethanol and dissolved in distilled water. The solution was lyophilized to obtain the crude polysaccharide. The crude polysaccharide was deproteinized using Sevag method and decolorized using macro-porous resin D101 (Li et al.,
Cell viability was performed using MTT colorimetric assay. PC12 cells (1.0 × 104 cells/well) were seeded in a 96-well plate. After cell treatment, the medium was removed, and the cells were incubated with 20 L of 5 mg/ml MTT solution for 4 h at 37 ◦ C. The medium was carefully removed, and the dark-blue formazan was dissolved with 150 L of DMSO. The absorbance was determined at 490 nm with a microplate reader. Cell viability was expressed as a percentage of control cells. 2.8. LDH activity determination LDH leakage into the culture medium indicates cell injury. The LDH activity was measured according to the manufacturer’s instructions in the assay kit. After the treatment, the media was collected and the supernatant was used in the assay of extracellular LDH activity. The absorbance was measured at 450 nm using a microplate reader. Data represents the percentage of LDH released relative to control cells.
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
189
Fig. 1. (A) Elution profiles of crude polysaccharide separated from Cordyceps cicadae on DEAE-52 column and (B) Gel filtration chromatography column by Sephadex G-100.
2.9. Determination of ROS level The level of intracellular ROS was determined using the DCFHDA protocol. In brief, after cell treatment, cells were incubated with 10 M of DCFH-DA at 37 ◦ C for 30 min in the dark and then washed thrice with PBS. The fluorescence of DCF was analyzed with flow cytometry (Guava easyCyteTM, Merck Millipore, USA) with excitation at 488 nm and emission at 530 nm.
2.10. Determination of GSH-Px, SOD and MDA levels After the cell treatment, the medium was removed and the cells were washed thrice with PBS. Cells were collected, centrifuged, resuspended in PBS (500 L), dissociated using cell lysis buffer, and centrifuged for 6 min (12,000 rpm). The supernatant obtained after centrifuging was used to measure the GSH-Px, SOD activities and MDA levels using assay kit based on the specified manufacturer’s instructions. 2.11. Determination of intracellular calcium (Ca2+ ) level Cells were seeded in 96-well plates. After the cell treatment, cells were collected, washed with D-Hanks and incubated with Fura 2-AM (5 mol/L) at 37 ◦ C for 45 min in the dark. After incubation, cells were washed and re-suspended in cold salt solution buffer containing 0.2% bovine serum albumin. The cells were incubated at 37 ◦ C for 5 min and the fluorescence intensity measured at an
emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm on a microplate reader.
2.12. Statistical analysis Statistical analysis was performed using SPSS (version 16.0, SPSS Inc, Chicago, IL, USA). All data were expressed as mean ± SD. Statistical analysis was performed one way Analysis of Variance (ANOVA) followed by Post hoc analysis using Tukey test. Differences were considered significant at P < 0.05.
3. Results and discussion 3.1. Extraction, isolation and purification of polysaccharides The crude polysaccharide was isolated from Cordyceps cicadae through hot-water extraction, ethanol precipitation and lyophilization. It was fractionated using DEAE-52 cellulose eluting with distilled water and 0.1–0.2 N NaCl to obtain two major fractions (Fig. 1A). The two main fractions were further purified on Sephadex G-100 gel filtration eluting with 0.1 N NaCl and the resulting eluent were collected. Two polysaccharides designated CPA-1 and CPB2 were obtained with the yield of 3.11% and 5.07% respectively (Fig. 1B).
190
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
Fig. 2. Monosaccharide composition of analysis by GC; standard sugars (A), CPA-1 (B) and CPB-2 (C). Rhamnose (1); arabinose (2); xylose (3); mannose (4); glucose (5); galactose (6).
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
191
Fig. 3. FT-IR spectrum of CPA-1 and CPB-2.
3.2. Sugar composition of CPA-1 and CPB-2 As shown in the GC chromatogram (Fig. 2B–C), the result indicated that glucose was the main monosaccharide present in both CPA-1 and CPB-2 (47.7% and 79.7%, respectively). In addition CPA1 had mannose and galactose with a percentage composition of 22.9% and 29.4%. Likewise, CPB-2 had mannose (11.1%) and galactose (9.1%). 3.3. FT-IR spectra of CPA-1 and CPB-2 As shown in Fig. 3, the FT-IR spectra of CPA-1 and CPB-2 exhibited the characteristic strong and broad absorption peak at 3455 cm−1 and 3379 cm−1 respectively due to the stretching vibration of the hydroxyl group (Santhiya, Subramanian, & Natarajan, 2002). A weak absorption band at 2928 cm−1 (CPA-1) as well as 2929 cm−1 in CPB-2 was attributed to a CH stretching. The absorption band at 1652 cm−1 in CPA-1 and 1656 cm−1 in CPB2 corresponds to the CO stretching of the carbonyl. The signal at 1543 cm−1 (CPA-1) and 1539 (CPB-2) was attributable to the vibration of C O, while the signal at 1465 cm−1 (CPA-1) and 1410 cm−1 (CPB-2) is distinctive of CH bending. The characteristic bands at 1200–1000 cm−1 are attributed to the stretching vibrations of C O C and C OH in sugar rings (Zhao, Kan, Li, & Chen, 2005). A weak absorption band at 860 cm−1 (CPA-1) and 855 cm−1 (CPB2) indicated the presence of anomeric configuration in CPA-1 and CPB-2 (Coimbra, Goncalves, Barros, & Delgadillo, 2002). 3.4. Effect of CPA-1 and CPB-2 on glutamate-induced PC12 cell viability The increase in glutamate neurotransmission has been widely reported to be associated with the pathophysiology of neuronal disorders. High level of extracellular glutamate serves as a neurotoxin leading to programmed cell death (Mattson, 2000; Siegel & Sanacora, 2012). The survival rate of PC12 cells exposed to 5 mM glutamate for 24 h was decreased to 43.2% compared with the control group. However, in cells pretreated with CPA-1 and CPB-2, the viability was observed to be increased in a dose dependent manner. The viability of treated cells was found to increase to 53.2% (25 g/ml), 66.0% (50 g/ml), 76.8% (100 g/ml) and 84.8% (200 g/ml) for CPA-1, while CPB-2 increased cell viability by 57.3%, 70.8%, 80.6% and 89.4%, respectively (Fig. 4A). These results indicated that CPA-1 and CPB-2 could protect PC12 cells from glutamate induced cell death with CPB-2 displaying the maximum protective effect in all the experimental doses. Furthermore, the cytotoxicity of CPA-1 and CPB-2 on PC12 cells was accessed. CPA-1 and CPB-
Fig. 4. (A) Effect of CPA-1 and CPB-2 on PC12 cell viability. PC12 cells were incubated with CPA-1 and CPB-2 for 24 h, and then exposure to 5 mM glutamate for 24 h. Data are expressed as mean ± SD (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group. (B) Effect of CPA-1 and CPB-2 on cell viability. Cells were treated with CPA-1 and CPB-2 for 24 h. Results are expressed as% viability of the control. Data are expressed as mean ± SD (n = 5). **P < 0.01 as compared to control.
2 did not display any cytotoxic effect on the cells at the highest concentration (200 g/ml), instead it was observed that CPA-1 and CPB-2 promoted the proliferation of PC12 cells in a concentration
192
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
Fig. 5. Effect of CPA-1 and CPB-2 on glutamate-induced LDH leakage. Cells were administrated with CPA-1 and CPB-2 (25–200 g/ml) for 24 h and then treated with 5 mM glutamate for 24 h. The data are represented as means ± SD (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group.
Fig. 6. Effects of CPA-1 and CPB-2 on intracellular production of ROS in glutamatetreated cells. The data are represented as means ± SD (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group.
dependent manner. This effect was observed to be significant at 50, 100 and 200 g/ml (Fig. 4B).
3.5. Effect of CPA-1 and CPB-2 on glutamate-induced LDH leakage in PC12 cells LDH (lactate dehydrogenase) is an enzyme that is found in cells, and it is responsible for catalyzing the oxidation of lactate to pyruvate. Upon cell injury or damage, LDH is released from the cells. As such the quantification of LDH leakage from cells is one of the main tools for the assessment of cell death. As observed in Fig. 5, pretreatment with CPA-1 and CPB-2 significantly reduced LDH leakage by 68.2%, 58.0%, 46.8%, 32.8% in CPA-1 treated group and 64.2%, 54.8%, 42.6% and 28.4% in CPB-2 treated group, respectively when compared to the glutamate-treated group (86.8%).
3.6. Effect of CPA-1 and CPB-2 on glutamate-induced ROS production Oxidative stress results due to an imbalance between free radical generation and the antioxidant defense system. The increased production of free radicals initiates the generation of reactive oxygen species (ROS) leading to oxidative stress (Ko, Kim, & Jeon, 2012; Ko, Lee, Samarakoon, Kim, & Jeon, 2013). Oxidative stress has been implicated in the pathogenesis and progression of several diseases, including neurodegenerative diseases. Glutamate inflicts cell injury and apoptosis by causing an increase in ROS generation which leads to oxidative stress (Uttara, Singh, Zamboni, & Mahajan, 2009). Glutamate excitotoxicity has been widely reported to be associated with mitochondrial dysfunction and oxidative damage in cell culture experimental studies (Pereira & Oliveira, 2000; Schubert & Piasecki, 2001). After the incubation of PC12 cells with 5 mM glutamate, the level of intracellular ROS was observed to be significantly increased to approximately 4 folds as compared to the control group. However, CPA-1 and CPB-2 obviously decreased ROS generated as a result of glutamate toxicity to 251.4% and 246.1% (25 g/ml), 225.6% and 211.3% (50 g/ml), 185.4% and 175.1% (100 g/ml), 149.1% and 134.3% (200 g/ml), respectively (Fig. 6).
Fig. 7. Effect of CPA-1 and CPB-2 on glutamate-induced intracellular Ca2+ influx. CPA-1 and CPB-2 (25–200 g/ml) could ameliorate the increased levels of Ca2+ induced by glutamate. The data were expressed as means ± S.D (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group.
3.7. Effect of CPA-1 and CPB-2 on glutamate-induced intracellular Ca2+ level in PC12 cells Several studies have reported that glutamate toxicity can lead to excessive Ca2+ overload by the activation of NMDA receptors (Baptisite et al., 2004; Vazhappilly, Wee, Sucher, & Low, 2010). The increase in intracellular Ca2+ concentration can also results in the formation of ROS (Randall & Thayer, 1992). Furthermore, high levels of intracellular Ca2+ concentration can lead to the activation of lipases, proteases, nitric oxide synthases and oxidative stress which damages the mitochondria (Oster et al., 2014). In this present study, it was observed that the exposure of PC12 cells to glutamate led to an increase in the level of Ca2+ concentration in the untreated cells as compared to the control group. The concentration of Ca2+ in the untreated group was increased to 397.3 mmol/L. However, pretreatment of cells with CPA-1 (25, 50, 100 and 200 g/ml) decreased the intracellular Ca2+ concentration to 310.3, 250.0, 195.6 and 153.5 mmol/L, respectively. While CPB-2 dose dependently reduced Ca2+ concentration to 299.8, 246.6, 184.5 and 148.2 mmol/L respectively (Fig. 7).
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
193
Widmann, Trapp, & Holsboer, 1995; Sun, Zhang, Zhu, Chen, & Mei, 2010). Antioxidants have the ability to prevent glutamaterelated toxicity, thus affirming the hypothesis which stipulates that glutamate-induced cell death is mediated via the increase in free radicals and ROS generation (Pereira & Oliveira, 1997, 2000). Antioxidant enzymes SOD and GSH-Px are an integral protective mechanism that cells utilize in preventing oxidative stress and cell injury. Thus, the enhancement of their effects can protect cells against oxidative-induced damages (Han et al., 2014; Shen, Song, Wu, & Zhang, 2011). As indicated in Fig. 8A and B, the prior treatment of PC12 cells with CPA-1 and CPB-2 caused a marked increase in the activities of the GSH-Px from 76.2 U/mg protein in glutamate treated cells to 91.2, 110, 122.2, 137.9 U/mg protein (25, 50, 100 and 200 g/ml of CPA-1 respectively) and 93.6, 118.1, 129.1 and 140.1 U/mg protein (25, 50, 100 and 200 g/ml of CPB-2 respectively). Likewise, the activity of SOD was significant increased in all pretreated cell groups when compared with the glutamate treated group. Furthermore, a significant increase was observed in the level of MDA in cells exposed to glutamate only (254.6%). However, pretreatment with CPA-1 and CPB-2 significantly decreased the up-regulated levels of MDA in a dose-dependent manner (Fig. 8C). This result suggests that CPA-1 and CPB-2 have the ability to protect cells against glutamate induced oxidative stress by reducing lipid peroxidation product and enhancing the antioxidant enzyme activities. Several reports have demonstrated the emerging potentials of polysaccharides in the treatment for experimental models of neurodegenerative disorders (Cui et al., 2016; Deng & Yang, 2014; Jia, Rao, Xue, & Lei, 2015; Zhang, Wang, Jin, & Zhang, 2013). The results obtained from this study thus add to the already existing evidence that supports polysaccharides as safe therapeutic options for the treatment of neuronal disorders.
4. Conclusion
Fig. 8. Effects of CPA-1 and CPB-2 (A) GSH-Px activity (B) SOD activity and (C) MDA levels in glutamate-treated cells. Cells were pretreated with different concentrations of CPA-1 and CPB-2 for 24 h and then exposed to 5 mM glutamate for 24 h. Data were presented as means ± S.D. (n = 5). ##P < 0.01 as compared to control; **P < 0.01 as compared to the glutamate treated group.
3.8. Effect of CPA-1 and CPB-2 on GSH-Px, SOD activities and MDA levels Cell death which results from ROS overproduction is accompanied by an increase in lipid peroxide and a decrease in the activity of enzymes and membrane fluids (Park et al., 2002). Glutamate toxicity leads to elevation of malondialdehyde content, reduction in the activities of glutathione and superoxide dismutase (Behl,
In this study the isolation and neuroprotective property of two polysaccharides from Cordyceps cicadae were investigated. Two polysaccharides CPA-1 and CPB-2 were purified and preliminary structural characterizations were conducted. Monosaccharides analysis indicated that CPA-1 and CPB-2 were heterogeneous polysaccharides and mainly composed of mannose, glucose and galactose. CPA-1 and CPB-2 exhibited protective effects against glutamate induced oxidative toxicity in PC12 cells by increasing cell viability, reducing LDH leakage, ROS production and Ca2+ overload in the pretreated cells. Furthermore, this present work also demonstrated that glutamate markedly inhibited the activities of SOD and GSH-Px in PC12 cells, but the pretreatment of PC12 cells with CPA-1 and CPB-2 reversed these changes. This suggests that the protective effects of CPA-1 and CPB-2 may be linked to its antioxidant properties. Previous researches have also reported the isolation of polysaccharides from Cordyceps species with neuroprotection through the increase in antioxidant enzymes activities as well as decreasing MDA levels (Li et al., 2003; Shen, et al., 2011). In conclusion, CPA-1 and CPB-2 possesses protective effects in PC12 cells against glutamate-induced oxidative damage and polysaccharides from Cordyceps cicadae may be applied as potential health and functional food source especially in the management of neuronal disorders
Conflict of interest The authors hereby declare no conflict of interest associated with this article.
194
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195
Acknowledgement This work was supported by National Natural Science Foundation of China (Nos. 81072985, 81373480, 81573529). References Albrecht, P., Lewerenz, J., Dittmer, S., Noack, R., Maher, P., & Methner, A. (2010). Mechanisms of oxidative glutamate toxicity: the glutamate/cystine antiporter system xc- as a neuroprotective drug target. CNS & Neurological Disorders—Drug Targets, 9, 373–382. Baptisite, D. C., Hartwick, A. T., Jollimore, C. A., Baldridge, W. H., Seigel, G. M., & Kelly, M. E. (2004). An investigation of the neuroprotective effects of tetracycline derivatives in experimental models of retinal cell death. Molecular Pharmacology, 66, 1113–1122. Behl, C., Widmann, M., Trapp, T., & Holsboer, F. (1995). 17-beta estradiol protects neurons from oxidative stress-induced cell death in vitro. Biochemical and Biophysical Research Communication, 216, 473–482. Cheung, P. C. K. (1996). The hypocholesterolemic effect of extracellular polysaccharide from the submerged fermentation of mushroom. Nutrition Research, 16, 1953–1957. Cheung, J. K., Li, J., Cheung, A. W., Zhu, Y., Zheng, K. Y., Bi, C. W., et al. (2009). Cordysinocan, a polysaccharide isolated from cultured Cordyceps: activates immune responses in cultured T-lymphocytes and macrophages: signaling cascade and induction of cytokines. Journal of Ethnopharmacology, 124, 61–68. Coimbra, M. A., Goncalves, F., Barros, A. S., & Delgadillo, I. (2002). Fourier transform infrared spectroscopy and chemometric analysis of white wine polysaccharide extracts. Journal of Agriculture and Food Chemistry, 50, 3405–3411. Cui, C., Cui, N., Wang, P., Song, S., Liang, H., & Ji, A. (2016). Neuroprotective effect of sulfated polysaccharide isolated from sea cucumber Stichopus japonicus on 6-OHDA-induced death in SH-SY5Y through inhibition of MAPK and NF-(B and activation of PI3 K/Akt signaling pathways. Biochemical and Biophysical Research Communications, 470, 375–383. Deng, Q., & Yang, X. (2014). Protective effects of Gynostemma pentaphyllum polysaccharides on PC12 cells impaired by MPP+ . International Journal of Biological Macromolecules, 69, 171–175. Fisher, M., & Yang, L. X. (2002). Anticancer effects and mechanism of polysaccharide-K (PSK): implications of cancer immunotherapy. Anticancer Research, 22, 1737–1754. Gondim, D. V., Costa, J. L., Rocha, S. S., Brito, G. A. D., Ribeiro, R. D., & Vale, M. L. (2012). Antinociceptive and anti-inflammatory effects of electroacupuncture on experimental arthritis of the rat temporomandibular joint. Canadian Journal of Physiology and Pharmacology, 904, 395–405. Halliwell, B. (2006). Oxidative stress and neurodegeneration: where are we now? Journal of Neurochemistry, 97, 1634–1658. Han, X., Zhu, S., Wang, B., Chen, L., Li, R., Yao, W., et al. (2014). Antioxidant action of 7,8-dihydroxyflavone protects PC12 cells against 6-hydroxydopamine-induced cytotoxicity. Neurochemistry International, 64, 18–23. Jia, D., Rao, C., Xue, S., & Lei, J. (2015). Purification: characterization and neuroprotective effects of a polysaccharide from Gynostemma pentaphyllum. Carbohydrate Polymers, 22, 93–100. Jin, R., Horning, M., Mayer, M. L., & Gouaux, E. (2002). Mechanism of activation and selectivity in a ligand-gated ion channel: structural and functional studies of GluR2 and quisqualate. Biochemistry, 41, 15635–15643. Kawakami, Z., Kanno, H., Ikarashi, Y., & Kase, Y. (2011). Yokukansan, a kampo medicine: protects against glutamate cytotoxicity due to oxidative stress in PC12 cells. Journal of Ethnopharmacology, 134, 74–81. Kim, H. S., Kim, Y. J., Lee, H. K., Ryu, H. S., Kim, J. S., Yoon, M. J., et al. (2012). Activation of macrophages by polysaccharide isolated from Paecilomyces cicadae through toll-like receptor 4. Food and Chemical Toxicology, 50, 3190–3197. Kim, H. S., Kim, J. Y., Ryu, H. S., Shin, B. R., Kang, J. S., Kim, H. M., et al. (2011). Phenotypic and functional maturation of dendritic cells induced by polysaccharide isolated from Paecilomyces cicadae. Journal of Medicinal Food, 14, 847–856. Ko, J. Y., Lee, J. H., Samarakoon, K., Kim, J. S., & Jeon, Y. J. (2013). Purification and determination of two novel antioxidant peptides from flounder fish (Paralichthys olivaceus) using digestive proteases. Food and Chemical Toxicology, 52, 113–120. Ko, S. C., Kim, D. K., & Jeon, Y. J. (2012). Protective effect of a novel antioxidant peptide purified from a marine Chlorella elthpsoidea protein against free radical-induced oxidative stress. Food and Chemical Toxicology, 50, 2294–2302. Kuo, Y. C., Lin, L. C., Don, M. J., Liao, H. F., Tsai, Y. P., Lee, G. H., et al. (2002). Cyclodepsipeptide and dioxomorpholine derivatives isolated from the insect-body portion of the fungus Cordyceps cicadae. Journal of Chinese Medicine, 13, 209–219. Li, C., Huang, Q., Fua, X., Yue, X.-J., Liu, R. H., & You, L.-J. (2015). Characterization: antioxidant and immunomodulatory activities of polysaccharides from Prunella vulgaris Linn. International Journal of Biological Macromolecules, 75, 298–305. Li, N., Liu, B., Dluzen, D. E., & Jin, Y. (2007). Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 cells. Journal of Ethnopharmacology, 111, 458–463. Li, S. P., Zhao, K. J., Ji, Z. N., Dong, T. T., Lo, C. K., Cheung, J. K., et al. (2003). A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese
medicine: protects PC12 cells against hydrogen peroxide-induced injury. Life Sciences, 73, 2503–2513. Lipton, S. A., & Rosenberg, P. A. (1994). Excitatory amino acids as a final common pathway for neurologic disorders. New England Journal of Medicine, 330, 613–622. Mattson, M. P. (2000). Apoptosis in neurodegenerative disorders. Nature Reviews Molecular Cell Biology, 1, 120–129. Michaels, R. L., & Rothman, S. M. (1990). Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations. Journal of Neuroscience, 10, 283–292. Molnar, E., & Saac, J. T. (2002). Developmental and activity dependent regulation of ionotropic glutamate receptors at synapses. The Scientific World Journal, 2, 27–47. Muller, A., Rice, P. J., Ensley, H. E., Coogan, P. S., Kalbfleish, J. H., Kelley, J. L., et al. (1996). Receptor binding and internalization of a water-soluble (1→3)-beta-d-glucan biologic response modifier in two monocyte/macrophage cell lines. Journal of Immunology, 156, 3418–3425. Oster, S., Radad, K., Scheller, D., Hesse, M., Balanzew, W., Reichmann, H., et al. (2014). Rotigotine protects against glutamate toxicity in primary dopaminergic cell culture. European Journal of Pharmacology, 724, 31–42. Park, J. E., Yang, J. H., Yoon, S. J., Lee, J. H., Yang, E. S., & Park, J. W. (2002). Lipid peroxidation-mediated cytotoxicity and DNA damage in U937 cells. Biochimie, 84, 1199–1205. Penugonda, S., Mare, S., Goldstein, G., Banks, W. A., & Ercal, N. (2005). Effects of N-acetylcysteine amide (NACA), a novel thiol antioxidant against glutamate-induced cytotoxicity in neuronal cell line PC12. Brain Research, 1056, 132–138. Pereira, C. M., & Oliveira, C. R. (1997). Glutamate toxicity on a PC12 cell line involves glutathione (GSH) depletion and oxidative stress. Free Radical Biology Medicine, 23, 637–647. Pereira, C. F., & Oliveira, C. R. D. (2000). Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis. Neuroscience Research, 37, 227–236. Randall, R. D., & Thayer, S. A. (1992). Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons. Journal of Neuroscience, 12, 1881–1895. Rashid, S., Unyayar, A., Mazmanci, M. A., McKeown, S. R., Banat, I. M., & Worthington, J. (2011). A study of anti-cancer effects of Funalia trogii in vitro and in vivo. Food and Chemical Toxicology, 49, 1477–1483. Ren, X., He, L., Cheng, J., & Chang, J. (2014). Optimization of the solid-state fermentation and properties of a polysaccharide from Paecilomyces cicadae (Miquel) Samson and its antioxidant activities in vitro. PLosOne, 9, e87578. Santhiya, D., Subramanian, S., & Natarajan, K. (2002). Surface chemical studies on sphalerite and galena using extracellular polysaccharides isolated from Bacillus polymyxa. Journal of Colloid and Interface Science, 256, 237–248. Schubert, D., & Piasecki, D. (2001). Oxidative glutamate toxicity can be a component of the excitotoxicity cascade. Journal of Neuroscience, 21, 7455–7462. Shen, W., Song, D., Wu, J., & Zhang, W. (2011). Protective effect of a polysaccharide isolated from a cultivated Cordyceps mycelia on hydrogen peroxide-induced oxidative damage in PC12 cells. Phytotherapy Research, 25, 675–680. Siegel, S., & Sanacora, G. (2012). The roles of glutamate receptors across major neurological and psychiatric disorders. Pharmacology Biochemistry and Behavior, 100, 653–655. ˜ ˜ A. M., & Soto-Otero, R., Méndez-Alvarez, E., Hermida-Ameijeiras, A., Munoz-Pati no, Labandeira-Garcia, J. L. (2000). Autoxidation and neurotoxicity of 6hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson’s disease. Journal of Neurochemistry, 74, 1605–1612. Sun, H. H., Mao, W. J., Chen, Y., Guo, S. D., Li, H. Y., & Qi, X. H. (2009). Isolation: chemical characteristics and antioxidant properties of the polysaccharides from marine fungus Penicillium sp. F23-2. Carbohydrate Polymers, 78, 117–124. Sun, Z. W., Zhang, L., Zhu, S. J., Chen, W. C., & Mei, B. (2010). Excitotoxicity effects of glutamate on human neuroblastoma SH-SY5Y cells via oxidative damage. Neuroscience Bulletin, 26, 8–16. Surendran, S., & Rajasankar, S. (2010). Parkinson’s disease: oxidative stress and therapeutic approaches. Neurological Sciences, 31, 531–540. Tang, W., & Eisenbrand, G. (1992). Chinese drugs of plant origin. GmbH & Co. Berlin Heidelberg KG: Springer-Verlag. Tokarski, K., Bobula, B., Wabno, J., & Hess, G. (2008). Repeated administration of imipramine attenuates glutamatergic transmission in rat frontal cortex. Neuroscience, 153, 789–795. Tzschentke, T. M. (2002). Glutamatergic mechanisms in different disease states: overview and therapeutic implications—an introduction. Amino Acids, 23, 147–152. Uttara, B., Singh, A. V., Zamboni, P., & Mahajan, R. T. (2009). Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Current Neuropharmacology, 7, 65–74. Vazhappilly, R., Wee, K. S., Sucher, N. J., & Low, C. M. (2010). A non-muscle myosin II motor links NR1 to retrograde trafficking and proteasomal degradation in PC12 cells. Neurochemistry International, 56, 569–576. Wu, F. Y., Yan, H., Ma, X. N., Jia, J. Q., Zhang, G. Z., Guo, X. J., et al. (2012). Comparison of the structural characterization and biological activity of acidic polysaccharides from Cordyceps militaris cultured with different media. World Journal of Microbiology and Biotechnology, 28, 2029–2038.
O.J. Olatunji et al. / Carbohydrate Polymers 153 (2016) 187–195 Yu, R., Song, L., Zhao, Y., Bin, W., Wang, L., Zhang, H., et al. (2004). Isolation and biological properties of polysaccharide CPS-1 from cultured Cordyceps militaris. Fitoterapia, 75, 465–472. Zhang, M., Cui, S. W., Cheung, P. C. K., & Wang, Q. (2007). Antitumor polysaccharides from mushrooms: a review on their isolation process: structural characteristics and antitumor activity. Trends in Food Science & Technology, 18, 4–19. Zhang, Z. F., Lv, G. Y., Pan, H. J., Pandey, A., He, W. Q., & Fan, L. F. (2012). Antioxidant and hepatoprotective potential of endo-polysaccharides from Hericium erinaceus grown on tofu whey. International Journal Biological Macromolecules, 51, 1140–1146.
195
Zhang, W., Wang, J., Jin, W., & Zhang, Q. (2013). The antioxidant activities and neuroprotective effect of polysaccharides from the starfish Asterias rollestoni. Carbohydrate Polymers, 95, 9–15. Zhao, G., Kan, J., Li, Z., & Chen, Z. (2005). Structural features and immunological activity of a polysaccharide from Dioscorea opposita Thunb roots. Carbohydrate Polymers, 61, 125–131. Zong, A. Z., Cao, H. Z., & Wang, F. S. (2012). Anticancer polysaccharides from natural resources: a review of recent research. Carbohydrate Polymers, 90, 1395–1410.