A water soluble β-glucan of an edible mushroom Termitomyces heimii: Structural and biological investigation

Carbohydrate Polymers 134 (2015) 375–384

Contents lists available at ScienceDirect

Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

A water soluble ␤-glucan of an edible mushroom Termitomyces heimii: Structural and biological investigation Dilip K. Manna a , Ashis K. Nandi a , Manabendra Pattanayak a , Prasenjit Maity a , Satyajit Tripathy b , Amit K. Mandal c , Somenath Roy b , Sushri S. Tripathy d , Nibha Gupta d , Syed S. Islam a,∗ a

Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, India Department of Human Physiology with Community Health, Vidyasagar University, Midnapore 721 102, West Bengal, India c Department of Microbiology, Vidyasagar University, Midnapore 721102, West Bengal, India d Division of Plant Pathology and Microbiology, Regional Plant Resource Centre, Bhubaneswar 751 015, Odisha, India b

a r t i c l e

i n f o

Article history: Received 23 April 2015 Received in revised form 29 July 2015 Accepted 30 July 2015 Available online 6 August 2015 Keywords: Termitomyces heimii ␤-Glucan NMR studies Biological properties

a b s t r a c t A water soluble ␤-glucan (PS-I) with an average molecular weight ∼1.48 × 105 Da was isolated from the alkaline extract of an edible mushroom Termitomyces heimii. PS-I contained (1 → 3)-, (1 → 6)-, (1 → 3, 6)linked and terminal ␤-d-glucopyranosyl moieties in a ratio of nearly 2:1:1:1. Based on the total hydrolysis, methylation analysis, periodate oxidation, Smith degradation, partial hydrolysis and 1D/2D NMR experiments the structure of the PS-I was elucidated. On the basis of these experiments, the repeating unit of the polysaccharide was found to consist of a backbone chain of two (1 → 6)-␤-d-glucopyranosyl residues, one of which was branched at O-3 position with the side chain consisting of two (1 → 3)-␤-d-glucopyranosyl and a terminal ␤-d-glucopyranosyl residue. Cytotoxic effect of PS-I on human blood lymphocytes at varied concentrations was studied. Moreover, it also exhibited potent antioxidant activities by diminishing the ROS and NO in the nicotine stimulated lymphocytes up to 200 ␮g/ml. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Termitomyces heimii, an agaric fungus belongs to the family Lyophyllaceae, also known as “termite mushroom” because of its growth in symbiotic relationship with termites (Rahmad et al., 2014). It is a wild, edible and delicious mushroom that grows in association with the termite’s guts of the laterite forest soil in southwest Bengal and Odisha, India and other parts of the world (Kumari, Atri, & Upadhyay, 2012; Malek, Kanagasabapathy, Sabaratnam, Abdullah, & Yaacob, 2012; Tripathy et al., 2014a; Tripathy, Rajoriya, & Gupta, 2014). Several polysaccharides like glucans and heteroglycans isolated from the genus Termitomyces, namely Termitomyces eurhizus (Mondal, Chakraborty, Pramanik, Rout, & Islam, 2004), T. striatus (Mondal, Chakraborty, Rout, & Islam, 2006), T. robustus (Chandra et al., 2007), T. microcarpus (Mondal et al., 2008), and T. clypeatus (Pattanayak et al., 2015) have been reported by our group. ␤-Glucans are recognized as biological response modifier (BRM) and used for the treatment of cancer and various infectious

∗ Corresponding author. Tel.: +91 03222 276558x437/+91 fax: +91 03222 275329. E-mail address: sirajul [email protected] (S.S. Islam). http://dx.doi.org/10.1016/j.carbpol.2015.07.099 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

9932629971;

diseases (Chan, Chan, & Sze, 2009; Kidd, 2000). These are also useful as immunomodulator (Wasser & Weis, 1999), anti-tumor (Wasser, 2002) as well as antioxidant agents (Blokhina, Virolainen, & Fagerstedt, 2003; Kozarski et al., 2011; Maity et al., 2014a–c; Patra et al., 2013). It is well known that reactive oxygen species (ROS) have the potential to damage lipids, proteins, carbohydrates, and nucleic acids (Papas, 1999) and are responsible for developing diseases like cancer, Alzheimer, and Parkinson (Halliwell & Gutteridge, 1989; Papas, 1999). ␤-Glucans from the mushrooms are well-known antioxidant material (Kofuji et al., 2012) which can counter the adverse effect of ROS like superoxide anion, hydrogen peroxide and hydroxyl radical by terminating the chain reaction with the donation of hydrogen to the free radicals. In this way, it can minimize the ROS generation as well as tissue damage due to oxidative stress (Aruoma, Laughton, & Halliwell, 1989; Wade, Jackson, Highton, & Van Rij, 1987). Several linear (1 → 3) and branched (1 → 3)-, (1 → 6)-␤-d-glucans (Bhanja et al., 2013, 2014; Chakraborty, Mondal, Rout, & Islam, 2006; Maji et al., 2012; Rout, Mondal, Chakraborty, Pramanik, & Islam, 2005) were reported as immunoactive materials. Some immune-stimulating water soluble ␤-d-glucans have also been reported (Chakraborty, Mondal, Pramanik, Rout, & Islam, 2004; Maity et al., 2013, 2014a–c; Nandi et al., 2014; Sen et al., 2013) by our group. Two water soluble

376

D.K. Manna et al. / Carbohydrate Polymers 134 (2015) 375–384

polysaccharides (PS-I & PS-II) have been isolated from the alkaline extract of this mushroom. The detailed structural investigation and biological properties of PS-I were carried out in the present investigation and reported herein. 2. Materials and methods 2.1. Collection of Termitomyces heimii Fruit bodies of mushroom T. heimii, was collected from the laterite forest of Moyurbhonj area of Odisha in the month of August (Tripathy et al., 2014a,b) and identified by Prof. Krishnendu Acharya, Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, West Bengal, India. 2.2. Isolation and purification of the polysaccharide The isolation of crude polysaccharide was done from alkaline (4% NaOH solution) extract of dried fruit bodies of mushroom T. heimii and purification of a portion (30 mg) of this crude polysaccharide was performed by gel permeation chromatography (GPC) on Sepharose 6B column using water as eluant by the methods described earlier (Maity et al., 2014a–c; Nandi et al., 2014; Sen et al., 2013). A set of 95 test tubes were collected using Redifrac fraction collector and monitored by the phenol–sulfuric acid procedure (York, Darvill, McNeil, Stevenson, & Albersheim, 1985) at 490 nm using Shimadzu UV–vis spectrophotometer, Model-1601. Two homogeneous fractions, PS-I and PS-II were collected and freeze-dried. The same procedure was repeated in several lots to yield more purified PS-I. The first fraction, PS-I was further purified on GPC by previously described method which produced only a single homogeneous fraction. 2.3. Optical rotation measurement Optical rotation was measured using a Jasco Polarimeter model P-1020 at 31 ◦ C. 2.4. Determination of molecular weight The average molecular weight of pure PS-I was determined by gel-permeation chromatographic technique. Standard dextrans T200, T-70 and T-40 were passed through a Sepharose 6B column, and the elution volumes were plotted against the logarithms of their respective molecular weights (Hara, Kiho, Tanaka, & Ukai, 1982). The elution volume of PS-I was then plotted in the same graph, and molecular weight of PS-I was determined. 2.5. Monosaccharide and methylation analysis The pure PS-I (3.0 mg) was hydrolyzed with trifluoro-acetic acid (CF3 COOH; TFA) (2 M, 2 ml) in a boiling water bath for 18 h. The excess acid was completely removed by co-distillation with distilled water. The hydrolyzed product was then reduced with NaBH4 (7 mg) followed by addition of dilute AcOH and co-distillation with pure CH3 OH to remove excess boric acid. The reduced sugars were acetylated with pyridine–acetic anhydride (1:1) in a boiling water bath for 1 h to give the alditol acetates (Lindahl, 1970), which were analyzed by gas chromatography (GC). HewlettPackard model 5730 A was used with flame ionization detector and glass columns (1.8 m × 6 mm) packed with 3% ECNSS-M (A) on Gas Chrom Q (100–120 mesh) and 1% OV-225 (B) on Gas Chrom Q (100–120 mesh). All GC analyses were performed at 170 ◦ C. PS-I (3 mg) was methylated using the procedure described by method reported earlier (Ciucanu & Kerek, 1984) and the

product was isolated by making a partition between CHCl3 and water (5:2, v/v). The organic layer containing desire product was washed with water for several times and dried. The methylated PS-I was hydrolyzed with 90% HCOOH (1 ml) at 100 ◦ C for 1 h and by co-distillation with distilled water the excess HCOOH was evaporated. After that the hydrolyzed product was reduced using NaBH4 and acetylated with pyridine and Ac2 O (1:1). The alditol acetates of the methylated sugars were analyzed by GC–MS. GC–MS analysis was performed on Shimadzu GC–MS Model QP-2010 Plus automatic system, using ZB-5MS capillary column (30 m × 0.25 mm). The program was isothermal at 150 ◦ C; hold time of 5 min, with a temperature gradient of 2 ◦ C/min up to a final temperature of 200 ◦ C. 2.6. Periodate oxidation and Smith degradation study PS-I (35 mg) was treated with 0.1 M sodium metaperiodate solution (20 ml) for 72 h in the dark at 25 ◦ C. The excess periodate was destroyed by adding ethylene glycol and dialyzed against distilled water for 2 h. The dialyzed and concentrated material was reduced with NaBH4 and kept for 12 h and neutralized with 50% AcOH, and again dialyzed with distilled water and finally freeze dried (16 mg). The periodate-reduced material was divided into three portions. First portion (2 mg) was hydrolyzed with 2 M TFA (1 ml) at 100 ◦ C for 18 h and used for alditol acetate preparation and analyzed by GC. The second portion (2 mg) was methylated by the method of Ciucanu and Kerek (1984), followed by preparation of alditol acetates which were analyzed by GC–MS. Smith degradation experiment was performed with the third portion (12 mg). The mild hydrolysis of the periodate oxidizedreduced material was performed by the addition of 0.5 M TFA for 15 h at 25 ◦ C to destroy the residual part of the oxidized sugars attached to the polysaccharide chain. The excess acid was removed by repeated freeze drying. The material was further purified by passing through a Sephadex G-25 column, freeze-dried and kept over P2 O5 in vacuum for 13 C NMR analysis. 2.7. Absolute configuration of the monosaccharides The absolute configuration of monosaccharide was determined by the method of Gerwig, Kamerling, and Vliegenthart (1978). PS-I (1.0 mg) was hydrolyzed with TFA and then the excess acid was removed by co-distillation with distilled water. The hydrolyzed product was treated with a volume of 250 ␮l of HCl (0.625 M) solution in R-(+)-2-butanol and heated at 80 ◦ C for 16 h. Then the TMS-derivatives of dry reactants were prepared with N,Obis(trimethylsilyl) trifluroacetamide (BSTFA). The products were analyzed by GC using a capillary column SPB-1 (30 m × 0.26 mm) with a temperature program (3 ◦ C/min) from 150 to 210 ◦ C. The (+)-2-butyl-2,3,4,6-tetra-O-TMS-glycosides were identified by comparison with those prepared from the d- and l-enantiomers of different monosaccharides. 2.8. Partial acid hydrolysis The PS-I (30 mg) was hydrolyzed in 0.1 M TFA (6 ml) at 100 ◦ C for 1 h. The precipitate obtain after addition of ethanol solution (1:3 v/v) to the acid free aqueous solution of hydrolyzed product was separated, washed with ethanol and then freeze-dried (P1), for methylation and 13 C NMR analysis. The supernatant from ethanol precipitation was dried by evaporation and residue was dissolved in water, and reduced with NaBH4 at 25 ◦ C for 2 h. After neutralization with AcOH (1 M), it was desalted by passing through a Sephadex G-25 column. The carbohydrate containing eluate was collected, freeze-dried and subjected to methylation analysis.

D.K. Manna et al. / Carbohydrate Polymers 134 (2015) 375–384

2.9. NMR studies The dried PS-I over P2 O5 in vacuum was subjected to deuterium ˜ et al., exchange with D2 O (99.96% atom 2 H, Aldrich) (Duenas-Chaso 1997) followed by lyophilizing and the same process were repeated four times. The 1 H and 13 C NMR experiments were carried out at 500 MHz and 125 MHz, respectively with a Bruker Avance DPX-500 spectrometer. The 1 H, 13 C, TOCSY, DQF-COSY, ROESY, and HSQC NMR spectra were recorded in D2 O at 30 ◦ C. The 1 H NMR spectrum was recorded by suppressing the HOD signal (fixed at ı 4.70) using presaturation method.

2.10. Biological activities 2.10.1. Isolation of lymphocytes from peripheral blood mononuclear cells (PBMCs) Fresh blood samples were collected from all groups of individuals (total 30 blood samples, 6 samples for each group) satisfying the Helsinki protocol. The lymphocytes were isolated from the heparinized blood samples according to the method described earlier (Mandal et al., 2015). Blood was diluted in phosphate buffered saline (PBS) (pH 7.0) and layered carefully in a ratio of 1:2 on the basis of density gradient (histopaque 1077), centrifuged at 1400 rpm for 45 min. The white milky layer of mononuclear cells were carefully isolated and cultured in RPMI 1640 medium for 2 h under 5% CO2 and 95% humidified atmosphere at 37 ◦ C (Chattopadhyay et al., 2013). The lymphocytes were obtained from the non adherent layer of the cultured cells by washing with PBS and centrifugation at 2000 rpm for 10 min. The depletion of the macrophages and B cells in PBMC were performed by passing through a nylon wool column as described earlier (Saxena, Mezey, & Adler, 1980).

2.10.2. Cell viability of PS-1isolated from T. heimii The in-vitro cellular toxicity of PS-I was studied on PBMC which were seeded into 96 wells of culture plates having 180 ␮l of complete media and incubated for 48 h. PS-I were added to the cells at different concentrations (D1: 25 ␮g/ml, D2: 50 ␮g/ml, D3: 100 ␮g/ml, D4: 200 ␮g/ml, and D5: 400 ␮g/ml) and incubated for 24 h at 37 ◦ C in a humidified CO2 -incubator. The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay as reported earlier (Chattopadhyay et al., 2012).

2.10.3. Cell lysate preparation The cell suspension was centrifuged at 1500 rpm for 5 min and the supernatants were collected and stored at −20 ◦ C. The cell pellets were suspended in ice cold PBS and subjected to freeze–thaw at least four cycles followed by sonication for 20 s (Ultrasonic Processor, Tekmar, Cincinnati, OH, USA). The cellular debris was removed from the lysates by centrifugation at 12,000 rpm for 20 min at 4 ◦ C. Protein content of lysate was measured as described previously using bovine serum albumin as standard (Lowry, Rosenbrough, Farr, & Randall, 1951).

2.10.4. Determination of reduced glutathione (GSH) and oxidized glutathione (GSSG) level Estimation of reduced glutathione of the cell lysates were carried out by the method applied earlier (Tripathy et al., 2013). The levels of GSH were expressed as ␮g of GSH/mg protein. The oxidized glutathione level was measured after derivatization of GSH with 2vinylpyidine according to the method reported earlier (Mahapatra, Chakraborty, Majumdar, Bag, & Roy, 2009).

377

2.10.5. Determination of lipid peroxidation (MDA) Lipid peroxidation was estimated adopting the method of Ohkawa, Ohishi, and Yagi (1979) using cell lysates. The levels of lipid peroxidation were expressed in terms of nmol/mg protein. 2.10.6. Protective role on cell viability Human lymphocytes (4 × 106 ) were seeded into 96 wells plate and treated with 10 mM nicotine (lethal dose) for 6 h at 37 ◦ C (Mahapatra et al., 2009). After incubation, cells were washed with PBS (50 mM) for 3 times and incubated with D1, D2, and D3 (as described in Section 2.10.2) for 24 h at 37 ◦ C. The cell viability was estimated by 3-(4,5-dimethylthiazol)-2-diphenyltetrazolium bromide (DNTB) as reported earlier (Chattopadhyay et al., 2013). 2.10.7. Effect on ROS generation The generation of intracellular reactive oxygen species (ROS) from cells has been investigated using the DCFH2 -DA (2 ,7 dichlorodihydrofluorescein diacetate) to detect and quantify the ROS (Tripathy et al., 2013). The trapped fluorescent dye (DCF) inside the cells was used to evaluate and detection of ROS. Flow cytometry (FACS CALIBUR, Becton Dickinson, USA) assay was performed to investigate the antioxidant effect of PS-I on nicotine-induced lymphocytes using the protocol described earlier (Maity et al., 2014a–c) and the data were analysed by CellQuest software. 2.10.8. Role on nitric oxide (NO) generation The NO concentration was measured by a microplate assay method using Griess reagent (1% sulfanilamide, 0.3% naphthylethylenediaminedihydrochloride, 7.5% H3 PO4 ) according to the method described previously (Maity et al., 2014a–c). 2.10.9. Statistical analysis The data were expressed as mean ± the standard error of the mean (SEM), n = 4. Comparisons of the means of control, and experimental groups were made by one way ANNOVA tests (using a statistical package, Origin 6.1, Northampton, MA 01060 USA), p < 0.05 as a limit of significance. The correlation analysis was performed using Statistica software version 8.0. 3. Results and discussion 3.1. Isolation, purification and chemical analysis of the PS-I 600 mg of crude water soluble polysaccharide was obtained from the alkaline (4% NaOH) extract of the dried fruit bodies of the edible mushroom T. heimii (120 g) followed by EtOH (1:5 v/v) precipitation, dialysis and freeze drying. Two homogeneous fractions, PS-I (test tube 16–26) and PS-II (test tube 33–39) (Fig. 1a) were obtained from 30 mg crude polysaccharide on fractionation through Sepharose 6B column. These are collected and freeze-dried, yielding pure PS-I (12 mg) and PS-II (2 mg). The same procedure was repeated for 10 times to yield 120 mg of pure polysaccharide (PS-I). Further purification of the PS-I produced a symmetrical peak which established the homogeneous nature of PS-I and the average molecular weight of PS-I was found to be as ∼1.48 × 105 Da (Fig. 1b). GC analysis of the alditol acetates of PS-I showed the presence of only glucose as monomeric sugar unit. The PS-I showed specific rotation [␣]D 31 –19.5 (c 0.2, water). The negative value of optical rotation indicates the presence of ␤-anomeric configuration in glucosyl residues (Dong, Yao, Yang, & Fang, 2002). The monosaccharide unit, glucose had d-configuration (Gerwig et al., 1978). The polysaccharide was methylated according to the method of Ciucanu and Kerek (1984) followed by hydrolysis and then conversion into their alditol acetates. The result of GC–MS analysis of the partially methylated alditol acetates depicted in Table 1 indicated the linkage pattern and their ratios present in the polysaccharide.

378

D.K. Manna et al. / Carbohydrate Polymers 134 (2015) 375–384

Fig. 1. (a) Chromatogram of crude polysaccharide isolated from an edible mushroom T. heimii using Sepharose 6B column. (b) Chromatogram showing the symmetrical homogeneous peak of PS-I and molecular weight.

According to these results there are possibilities of three types backbone as, a (1 → 6)-linked backbone, a (1 → 3)-linked backbone or an alternatively (1 → 3)-, (1 → 6)-linked backbone. So, for confirmation of the backbone chain present in the polysaccharide periodate oxidation and mild hydrolysis were performed. The GC analysis of the alditol acetates of the periodate-oxidized (Goldstein, Hay, Lewis, & Smith, 1965), reduced PS-I showed the presence of d-glucose along with glycerol and GC–MS analysis of periodate-oxidised, reduced, methylated (Akher & Smith, 1950) showed the presence of 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-d-glucitol; 1,3,5,6-tetraO-acetyl-2,4-di-O-methyl-d-glucitol in a ratio of nearly 2:1. Mild hydrolysis was carried out with the periodate-oxidised, reduced polysaccharide to get Smith degradation product (SDPS). The GC analysis of the alditol acetates of Smith degraded hydrolyzed product showed the presence of d-glucose and d-glycerol. The GC–MS analysis of the of methylated SDPS shows the presence of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-d-glucitol and 1,3,5-triO-acetyl-2,4,6-tri-O-methyl-d-glucitol in a ratio of nearly 1:2. Partial acid hydrolysis (Dong et al., 2002; Nandi et al., 2014) of the ␤-glucan was performed with 0.1 M TFA to identify the backbone chain of the ␤-glucan present in the repeating unit. Two fractions were obtained after hydrolysis i.e. a polysaccharide (P1) and an oligosaccharide (O2). The methylation analysis of P1 showed the presence of 1,5,6-tri-O-acetyl-2,3,4tri-O-methyl-d-glucitol only which indicates the presence of (1 → 6)-linked backbone of the PS, but the other fraction O2 showed the presence of 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyld-glucitol and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-d-glucitol indicating the presence of (1 → 3)-linked and terminal glucopyranosyl moieties as oligosaccharide side chain. Hence, these studies established that the backbone PS-I consisted of two (1 → 6)-␤d-glucopyranosyl residues as repeating unit, one of which was branched at O-3 position with the side chain consisting of two (1 → 3)-␤-d-glucopyranosyl and one terminal ␤-d-glucopyranosyl residues.

3.2. NMR and structural analysis of ˇ-glucan Five signals in the anomeric region at ı 4.72, 4.71, 4.47, 4.45 and 4.44 were observed in the 1 H NMR (500 MHz) spectrum (Fig. 1a, Table 2a) at 30 ◦ C. They were designated as AI , AII , B, C and D residues according to their decreasing proton chemical shifts. In the 13 C (125 MHz) spectrum (Fig. 2b) at 30 ◦ C five anomeric signals appeared at ı 102.8, 102.7, 102.5, 102.36 and 102.2. Based on HSQC experiment (Fig. 2c, Table 2a), the anomeric carbon signals at ı 102.8 corresponded to AI , 102.7 corresponded to AII , the signal at ı 102.5 corresponded to D, 102.36 corresponded to C and the peak at ı 102.2 was correlated to B residues of the anomeric proton signals, respectively. All the 1 H and 13 C signals (Table 2a) were assigned using DQF-COSY, TOCSY, and HSQC experiments. Coupling constants were measured from DQF-COSY spectrum. The large JH-2, H-3 and JH-3, H-4 coupling constant values ∼8 Hz of A–D residues and JC-1, H-1 ∼160 Hz indicated the presence of ␤-configuration of the d-glucopyranosyl (Glcp) residues in the polysaccharide. The downfield shift of C-3 (ı 84.3) of residues A (AI and AII ) with respect to standard value of methyl glycosides (Agrawal, 1992) indicated that they were (1 → 3)-linked ␤-d-Glcp. All chemical shifts of residue B were very close to the standard values of methyl glycoside (Agrawal, 1992) of ␤-d-glucose, evidently indicated that the residue B was non-reducing end. In residue C, the chemical shift values of C-3 (ı 84.0) and C-6 (ı 68.86) shifted downfield, indicating the presence of (1 → 3,6)-linked ␤-d-Glcp. Since, the residue D is glycosidically linked to the rigid part C, the C-6 value would show downfield shift (ı 68.94) in comparison to C residue (ı 68.86) due to the neighboring effect (Nandi et al., 2014; Yoshioka, Tabeta, Saito, Uehara, & Fukuoka, 1985) of rigid part C. The DEPT-135 spectrum (Fig. 2b) further confirmed the linkage at C-6 of the residues C and D. The ROESY experiment showed the inter-residual contacts AI H-1/CH-3; AII H-1/AI H-3; BH-1/AII H-3; CH-1/DH-6a,DH-6b; DH1/CH-6a,CH-6b along with other intra-residual contacts AI H-1/AI H3; AI H-1/AI H-5; BH-1/BH-2; BH-1/BH-3; BH-1/BH-5; CH-1/CH-2;

Table 1 GC–MS analysis of methylated PS-I isolated from T. heimii. Methylated sugars

Molar ratio

Linkage type

Major mass fragments (m/z)

2,3,4,6-Me4 -Glc 2,4,6-Me3 -Glc 2,3,4,-Me3 -Glc 2,4-Me2 -Glc

1 2 1 1

␤-d-Glcp-(1→ →3)-␤-d-Glcp-(1→ →6)-␤-d-Glcp-(1→ →3,6)-␤-d-Glcp-(1→

41,43,59,71,87,101,117,129,145,161,173,205 41,43,58,71,87,101,117,129,143,161,173,189,203.217,233 41,43,58,71,87,101,117,129,145,161,173,189,203,217,233 40,43,58,74,87,101,117,129,143,159,173,189,201,233,245,305

D.K. Manna et al. / Carbohydrate Polymers 134 (2015) 375–384

379

Table 2a The 1 H NMRa and 13 C NMRb chemical shifts of the polysaccharide (PS-I) isolated from the edible mushroom T. heimii. Glucosyl residue →3)-␤-d-Glcp-(1→

H-1/C-1 4.72x ,4.71y

H-2/C-2

H-3/C-3

H-4/C-4

H-5/C-5

3.34

3.73

3.40

3.49

H-6a,H-6b/C-6 3.89c ,3.73d

A (AI , AII )

102.8x 102.7y

73.0

84.3

68.2

75.7

60.81

␤-d-Glcp-(1→ B →3,6)-␤-d-Glcp-(1→ C →6)-␤-d-Glcp-(1→ D

4.47 102.2 4.45 102.36 4.44 102.5

3.51 73.6 3.50 73.0 3.29 73.2

3.44 75.7 3.71 84.0 3.43 75.7

3.36 69.6 3.49 68.2 3.46 69.6

3.51 75.7 3.60 75.0 3.62 75.0

3.87c ,3.70d 60.81 4.18c ,3.85d 68.86 4.16c ,3.83d 68.94

a b c d x y

The values of chemical shifts were recorded keeping HOD signal fixed at ı 4.70 at 30 ◦ C. The values of chemical shifts were recorded with reference to acetone as internal standard and fixed at ı 31.05 at 30 ◦ C. Interchangeable. Interchangeable. for residue AI . for residue AII .

CH-1/CH-3; CH-1/CH-5; DH-1/DH-2; DH-1/DH-3 and DH-1/DH-5 (Fig. 2d and Table 2b). Hence, the following sequences in PS-I were established as: AI (1 → 3) C; AII (1 → 3) AI ; B (1 → 3) AII ; C (1 → 6) D and D (1 → 6) C. The Smith degraded material (SDPS) of PS-I was prepared and 13 C NMR experiment (125 MHz) (Fig. 2e) carried out at 30 ◦ C for further confirmation of the sequence of linkages (Table 3). The 13 C NMR spectrum showed two anomeric carbon signals at ı 102.6 and 102.8 with a ratio of nearly 2:1 corresponding to → 3)-␤-d-Glcp-(1 → (F) and ␤-d-Glcp-(1 → (E) residues,

respectively. The carbon signals C-1, C-2, and C-3 of the glycerol moiety were assigned at ı 68.15, 72.0, and 62.57, respectively. The non-reducing ␤-d-Glcp (E) was produced from (1 → 3)-␤-dGlcp (AII ) after complete oxidation of the ␤-d-Glcp (B) of the intact PS-I. One (1 → 3)-␤-d-Glcp (F) was retained from (1 → 3)␤-d-Glcp (AI ) and the other (1 → 3)-␤-d-Glcp (F) was produced from the (1 → 3,6)-␤-d-Glcp (C) due to oxidation followed by Smith degradation. The glycerol (G) moiety was produced from (1 → 6)-␤-d-Glcp (D) after periodate oxidation followed by Smith degradation and be attached to (1 → 3)-␤-d-Glcp moiety (F). Hence,

Fig. 2. (a) 1 H NMR spectrum (500 MHz, D2 O, 30 ◦ C) (b) 13 C NMR spectrum (125 MHz, D2 O, 30 ◦ C) and part of DEPT-135 spectrum (D2 O, 30 ◦ C) (inset) (c) Part of HSQC spectrum (d) Part of ROESY spectrum The ROESY mixing time was 300 ms. (e) 13 C NMR spectrum (125 MHz, D2 O, 30 ◦ C) of Smith-degraded glycerol-containing disaccharide of PS-I isolated from the mushroom T. heimii.

380

D.K. Manna et al. / Carbohydrate Polymers 134 (2015) 375–384

Table 2b ROESY data for the polysaccharide isolated from the mushroom T. heimii. Glycosyl residue

Anomeric proton

3.3. Biological properties

ROE contact proton

ı

ı

Residue

Atom

→3)-␤-d-Glcp-(1 → AI

4.72

3.71 3.73 3.49

C AI AI

H-3 H-3 H-5

→3)-␤-d-Glcp-(1 → AII

4.71

3.73 3.73 3.49

AI AII AII

H-3 H-3 H-5

→␤-d-Glcp-(1 → B

4.47

3.73 3.51 3.44 3.51

AII B B B

H-3 H-2 H-3 H-5

→3,6)-␤-d-Glcp-(1 → C

4.45

4.16 3.82 3.50 3.71 3.60

D D C C C

H-6a H-6b H-2 H-3 H-5

→6)-␤-d-Glcp-(1 → D

4.44

4.18 3.85 3.29 3.43 3.62

C C D D D

H-6a H-6b H-2 H-3 H-5

Smith degradation had resulted an oligosaccharide from polysaccharide and the structure was established as OH

E

HO

O

OH HO

F

OH O

HO O

OH

HO

F G

O

O

OH

OH

O

2 OH

3 1 OH

Therefore, the above discussions indicated that the backbone chain of the polysaccharide consisted of only (1 → 6)-linked ␤-dglucose residues neither (1 → 3)-linked nor alternative (1 → 3)-, (1 → 6)-linked ␤-d-glucose. On the basis of all these above experiments, the ␤-glucan possessed a backbone of two (1 → 6)-linked ␤-d-glucopyronosyl residues, one of which was branched at O3positions with the side chain consisting of two (1 → 3)- linked and one terminal ␤-d-glucopyranosyl residues. Hence, the structure of the repeating unit of the polysaccharide was established as

C

O

O O

H

H OH H H

H HO

AI

O

H

O

OH

HO

H

H OH

AII

O

H

O

OH

H

HO

OH O

B HO

HO HO

O OH

H

HO HO

D O O OH

H

n

The cytotoxic effect of the PS-I isolated from T. heimii was studied on human blood lymphocytes at varied concentrations ranging from 25 ␮g/ml to 400 ␮g/ml. The 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay suggested that the polysaccharide have no considerable cytotoxic effect on normal lymphocytes. Cell proliferative activity was observed significantly (p < 0.05) up to 200 ␮g/ml but at the dose of 400 ␮g/ml the PS-I showed mild toxicity towards the lymphocytes (Fig. 3a). Earlier reports showed that polysaccharides extracted from mushroom Entoloma lividoalbum and bacterium Klebsiella pneumoniae PB12 showed cellular toxicity above 100 ␮g/ml of PS dosage (Maity et al., 2014a–c; Mandal et al., 2015). Glutathione, an important antioxidant, cells were measured both in its reduced and oxidized forms to know its intensity in cellular system (Fig. 3b and c). The reduced glutathione (GSH) level significantly increased up to 200 ␮g/ml of PS-I, whereas at 400 ␮g/ml, it was moderately decreased exhibited slight increase in GSSG level. It was clearly observed that the alteration of redox ratio (GSH/GSSG) is fully correlated with alteration in PS-I concentrations (Pearson co-efficient r = 0.951, Pearson correlation p < 0.05). Lipid peroxidation is one of the important determinants to assess the cellular damage. Several toxic by-products especially malondialdehyde (MDA) is released due to lipid peroxidation in lymphocytes cells. Lipid peroxidation in lymphocytes was measured in terms of malondialdehyde (MDA). Our result showed slight increase of MDA at the dose of 400 ␮g/ml of PS-I (Fig. 3d). To establish the protective role of PS-I against nicotine toxicity, lymphocytes were stimulated with nicotine (10 mM) as positive control and different concentrations of PS-I along with nicotine were treated for 24 h in culture media. The significant (p < 0.05) increase of cell viability were observed up to 200 ␮g/ml (Fig. 3e). Results showed that nicotine stimulated lymphocytes secreted several factors like ROS and NO. The treatment of the polysaccharides (D1–D4) in the nicotine stimulated lymphocytes diminishes the NO (Fig. 3f) and ROS (Fig. 4), respectively, but at 400 ␮g/ml (p < 0.05) there is an increase in both ROS and NO production in PS-I treated lymphocytes. The fluorescence images revealed that the PS-I (at the dose of 200 ␮g/ml) was able to scavenge the ROS generated due to nicotine, but when the dose was increased to 400 ␮g/ml, the polysaccharide lost their protective roles which establish the results (Fig. 4). The biological studies evidently showed that in vitro application of PS-I isolated from T. heimii has good effects up to certain level. These results indicate that the polysaccharide possesses antioxidant property as well as beneficial role on cellular toxicity. The ROS scavenging property of PS-I was established using flow cytometry study. It is well known that ROS and lipid peroxidation is interrelated (Farmer & Mueller, 2013). Lipid peroxidation damages cell membrane and ultimately causes disruption of membrane integrity. FACS analysis revealed that in nicotine treated lymphocytes the fluorescence intensity of propidium iodide (PI) increases compared to that of control (Fig. 4ix–xi). Whereas, when treated with 200 ␮g/ml of PS-I along with nicotine there is a decrease in fluorescence intensity indicating the possible ROS scavenging property of PS-I. Moreover, this study revealed that the treatment of PS-I on normal lymphocyte showed no adverse effect in lipid peroxidation and reduction in GSSG level up to 200 ␮g/ml indicating the protective role of PS-I against oxidative stress. Various antioxidants of synthetic origin are available commercially, although the recent studies revealed its adverse side effects (Hazra, Biswas & Mandal, 2008). Hence, natural antioxidants having lowest side effects and acceptable biocompatibility are in recent demand. These studies establish the antioxidant property of PS-I isolated from T. heimii.

D.K. Manna et al. / Carbohydrate Polymers 134 (2015) 375–384

381

Fig. 3. (a) MTT assay showing in-vitro cytotoxicity of PS-I on peripheral blood lymphocytes. (b) Concentration of reduced glutathione (GSH) and (c) oxidized glutathione (GSSG) level in normal human lymphocytes treated with different concentrations of PS-I. (d) Concentration of MDA level of PS-I treated normal human lymphocytes to evaluate lipid peroxidation (e) MTT assay showing in-vitro cell viability of PS-I on nicotine stimulated peripheral blood lymphocytes (f) Concentration of nitric oxide release in PS-I treated normal human lymphocytes (n = 6, values are expressed as mean ± SEM. * Indicates the significant difference as compared to control group).

Table 3 The 13 C NMRn chemical shifts of Smith-degraded glycerol-containing disaccharide of the mushroom T. heimii in D2 O at 30 ◦ C. Sugar residue

C-1

C-2

C-3

C-4

C-5

C-6

␤-d-Glcp-(1 → E → 3)-␤-d-Glcp-(1 → F Gro-(3 → G

102.8 102.6 68.15

73.58 73.34 72.0

76.0 84.18 62.57

70.70 69.70

75.67 75.67

60.75 60.75

n

The values of chemical shifts were recorded with reference to acetone as internal standard and fixed at ı 31.05 at 30 ◦ C.

382

D.K. Manna et al. / Carbohydrate Polymers 134 (2015) 375–384

Fig. 4. Fluorescence microscopic images (100× magnifications) showing intracellular ROS generation in lymphocytes. The scale bar represents 20 ␮m (i) control; (ii) nicotine treated; (iii) nicotine + 25␮g/ml of PS-I; (iv) nicotine + 50 ␮g/ml of PS-I; (v) nicotine + 100 ␮g/ml of PS-I; (vi) nicotine + 200 ␮g/ml of PS-I; (vii) nicotine + 400 ␮g/ml of PS-I; (viii) histogram showing the mean fluorescent intensity (n = 6, values are expressed as mean ± SEM. * Indicates the significant difference as compared to control group). (ix) Flow cytometry of human lymphocytes: without any treatment (control); (x) lymphocytes treated with 10 mM of Nicotine; and (xi) lymphocytes treated with 10 mM of nicotine +200 ␮g/ml of PS-I. The propidium iodide was used for analysis of cell death.

D.K. Manna et al. / Carbohydrate Polymers 134 (2015) 375–384

4. Conclusion A water soluble ␤-glucan (PS-I), with an average molecular weight ∼1.48 × 105 Da, was isolated from the alkaline extract of an edible mushroom, T. heimii and the following structure was characterized by chemical analysis and 1D/2D NMR studies:

C D →6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→ 3 ↑ [1)- β-D-Glcp-(3]2←1)-β-D-Glcp B A (AI/AII) The polysaccharide showed no efficacy to induce lipid peroxidation up to 200 ␮g/ml. These results indicated that PS-I does not exhibit any cellular toxicity and play a beneficial role on cell proliferation and its protection against nicotine stimulated toxicity. Acknowledgements The authors are grateful to Mr. Barun Majumdar, Bose Institute, Kolkata, for preparing NMR spectra. D. K. Manna (one of the authors) is thankful to the CSIR for offering senior research fellowship (CSIR-09/599(0056)/2011-EMR-I). References Abdel-Akher, M., & Smith, F. (1950). Use of lithium aluminium hydride in the study of carbohydrates. Nature, 166, 1037–1038. Agrawal, P. K. (1992). NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry, 31, 3307–3330. Aruoma, O. I., Laughton, M. J., & Halliwell, B. (1989). Carnosine, homocarnosine and anserine: Could they act as antioxidants in vivo? Biochemistry Journal, 264, 863–869. Bhanja, S. K., Rout, D., Patra, P., Nandan, C. K., Behera, B., Maiti, T. K., et al. (2013). Structural studies of animmuno enhancing glucan of an ectomycorrhizal fungus Ramaria botrytis. Carbohydrate Research, 374, 59–66. Bhanja, S. K., Rout, D., Patra, P., Sen, I. K., Nandan, C. K., & Islam, S. S. (2014). Water-insoluble glucans from the edible fungus, Ramaria botrytis. Bioactive Carbohydrates and Dietary Fibre, 3, 52–58. Blokhina, O., Virolainen, E., & Fagerstedt, V. K. (2003). Antioxidants, oxidative damage and oxygen deprivation stress: A review. Annals of Botany, 91, 179–194. Chakraborty, I., Mondal, S., Pramanik, M., Rout, D., & Islam, S. S. (2004). Structural investigation of a water-soluble glucan from an edible mushroom, Astraeus hygrometricus. Carbohydrate Research, 339, 2249–2254. Chakraborty, I., Mondal, S., Rout, D., & Islam, S. S. (2006). A water-insoluble (1 → 3)-␤-d-glucan from the alkaline extract of an edible mushroom Termitomyces eurhizus. Carbohydrate Research, 341, 2990–2993. Chan, G. C., Chan, W. K., & Sze, D. M. (2009). The effects of ␤-glucan on human immune and cancer cells. Journal of Hematology & Oncology, 2, 25. Chandra, K., Ghosh, K., Roy, S. K., Mondal, S., Maiti, D., Ojha, A. K., et al. (2007). A water-soluble glucan isolated from an edible mushroom Termitomyces microcarpus. Carbohydrate Research, 342, 2484–2489. Chattopadhyay, S., Chakraborty, S. P., Laha, D., Baral, R., Pramanik, P., & Roy, S. (2012). Surface-modified cobalt oxide nanoparticles: New opportunities for anti-cancer drug development. Cancer Nanotechnology, 3, 13–23. Chattopadhyay, S., Das, S. K., Ghosh, T., Das, S., Tripathy, S., Mandal, D., et al. (2013). Anticancer and immunostimulatory role of encapsulated tumor antigen containing cobalt oxide nanoparticles. Journal of Biological Inorganic Chemistry, 18, 957–973. Ciucanu, I., & Kerek, F. (1984). Simple and rapid method for the permethylation of carbohydrates. Carbohydrate Research, 131, 209–217. Dong, Q., Yao, J., Yang, X., & Fang, J. (2002). Structural characterization of a water-soluble ␤-d-glucan from fruiting bodies of Agaricus blazei Murr. Carbohydrate Research, 337, 1417–1421. ˜ Duenas-Chaso, M. T., Rodriguez-Carvajal, M. A., Mateo, P. T., Franco-Rodríguez, G., Espartero, J. L., Irastorza-Iribas, A., et al. (1997). Structural analysis of the exo-polysaccharide produced by Pediococcus damnosus. Carbohydrate Research, 303, 453–458. Gerwig, G. J., Kamerling, J. P., & Vliegenthart, J. F. G. (1978). Determination of the d and l configuration of neutral monosaccharides by high-resolution capillary g.l.c. Carbohydrate Research, 62, 349–357. Goldstein, I. J., Hay, G. W., Lewis, B. A., & Smith, F. (1965). Controlled degradation of polysaccharides by periodate oxidation, reduction and hydrolysis. Methods in Carbohydrate Chemistry, 5, 361–370.

383

Farmer, E. E., & Mueller, M. J. (2013). ROS-mediated lipid peroxidation and RES-activated signaling. Annual Review Plant Biology, 64, 429–450. Halliwell, B., & Gutteridge, J. M. C. (1989). Free radical in biology and medicine (2nd ed., pp. 1–225). Oxford, UK: Oxford University Press. Hara, C., Kiho, T., Tanaka, Y., & Ukai, S. (1982). Anti-inflammatory activity and conformational behavior of a branched (1 → 3)-␤-d-glucan from an alkaline extract of Dictyophoraindusiata Fisch. Carbohydrate Research, 110, 77–87. Hazra, B., Biswas, S., & Mandal, N. (2008). Antioxidant and free radical scavenging activity of Pondias pinnata. BMC Complementary and Alternative Medicine, 8, 63–72. Kidd, P. M. (2000). The use of mushroom glucans and proteoglycans in cancer treatment. Alternative Medicine Review, 5, 4–27. Kofuji, K., Aoki, A., Tsubaki, K., Konishi, M., Isobe, T., & Murata, Y. (2012). Antioxidant activity of ␤-glucan. ISRN Pharmaceutics, http://dx.doi.org/10. 5402/2012/125864 Kozarski, M., Klaus, A., Niksic, M., Jakovljevic, D., Helsper, J. P. F. G., & Van Griensven, L. J. L. D. (2011). Antioxidative and immunomodulating activities of polysaccharide extracts of the medicinal mushrooms Agaricus bisporus, Agaricus brasiliensis, Ganoderma lucidum and Phellinus linteus. Food Chemistry, 129, 1667–1675. Kumari, B., Atri, N. S., & Upadhyay, R. C. (2012). Culinary status and sociobiology of termitophilous and lepiotoid mushrooms of north west India. World Journal of Agricultural Sciences, 8(4), 415–420. Lindahl, U. (1970). Attempted isolation of a heparin proteoglycan from bovine liver Capsule. Biochemistry Journal, 116, 27–34. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, 193, 255–275. Mahapatra, S. K., Chakraborty, S. P., Majumdar, S., Bag, B. G., & Roy, S. (2009). Eugenol protects nicotine-induced superoxide mediated oxidative damage in murine peritoneal macrophages in-vitro. European Journal of Pharmacology, 623, 132–140. Maity, P., Samanta, S., Nandi, A. K., Sen, I. K., Paloi, S., Acharia, K., et al. (2014). Structure elucidation and antioxidant properties of a soluble ␤-d-glucan from mushroom Entoloma lividoalbum. International Journal of Biological Macromolecules, 63, 140–149. Maity, P., Nandi, A. K., Sen, I. K., Pattanayak, M., Chattopadhyay, S., Dash, S. K., et al. (2014). Heteroglycan of an edible mushroom Entoloma lividoalbum: Structural characterization and study of its protective role for human lymphocytes. Carbohydrate Polymers, 114, 157–165. Maity, K. K., Patra, S., Dey, B., Bhunia, S. K., Mandal, S., Behera, B., et al. (2013). A ␤-glucan from the alkaline extract of asomatic hybrid (PfloVv5FB) of Pleurotus florida and Volvariella volvacea: Structural characterization and study of immunoactivation. Carbohydrate Research, 370, 13–18. Maity, P., Samanta, S., Nandi, A. K., Sen, I. K., Paloi, S., Acharya, K., et al. (2014). Structure elucidation and antioxidant properties of a soluble ␤-d-glucan from mushroom Entoloma lividoalbum. International Journal of Biological Macromolecules, 63, 140–149. Maji, P. K., Sen, I. K., Behera, B., Maiti, T. K., Mallick, P., Sikdar, S. R., et al. (2012). Structural characterization and study of immune enhancing properties of a glucan isolated from a hybrid mushroom of Pleurotus florida and Lentinula edodes. Carbohydrate Research, 358, 110–115. Malek, N. A., Kanagasabapathy, G., Sabaratnam, V., Abdullah, N., & Yaacob, H. (2012). Lipid components of a Malaysian edible mushroom, Termitomyces heimii Natarajan. International Journal of Food Properties, 15, 809–814. Mandal, A. K., Sen, I. K., Maity, P., Chattopadhyay, S., Roy Chakraborty, R., & Islam, S. S. (2015). Structural elucidation and biological studies of a novel exopolysaccaride from Klebsiella pneumoniae PB12. International Journal of Biological Macromolecules, 79, 413–422. Mondal, S., Chakraborty, I., Pramanik, M., Rout, D., & Islam, S. S. (2004). Structural studies of water-soluble polysaccharides of an edible mushroom, Termitomyces eurhizus. A re-investigation. Carbohydrate Research, 339, 1135–1140. Mondal, S., Chakraborty, I., Rout, D., & Islam, S. S. (2006). Isolation and structural elucidation of a water-soluble polysaccharide (PS-I) of a wild edible mushroom, Termitomyces striatus. Carbohydrate Research, 341, 878–886. Mondal, S., Chandra, K., Maiti, D., Ojha, A. K., Das, D., Roy, S. K., et al. (2008). Chemical analysis of a new fucoglucan isolated from an edible mushroom, Termitomyces robustus. Carbohydrate Research, 343, 1062–1070. Nandi, A. K., Samanta, S., Maity, S., Sen, I. K., Khatua, S., Sanjana, K., et al. (2014). Antioxidant and immune-stimulant ␤-glucanfrom edible mushroom Russula albonigra (Krombh.) Fr. Carbohydrate Polymers, 99, 774–782. Ohkawa, H., Ohishi, N., & Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95, 351–358. Papas, A. M. (1999). Antioxidant status, diet, nutrition and health. United Kingdom: CRC Press. Patra, S., Patra, P., Maity, K. K., Mandal, S., Bhunia, S. K., Dey, B., et al. (2013). A heteroglycan from the mycelia of Pleurotus ostreatus: Structure determination and study of antioxidant properties. Carbohydrate Research, 368, 16–21. Pattanayak, M., Samanta, S., Maity, P., Sen, I. K., Nandi, A. K., Manna, D. K., et al. (2015). Heteroglycan of an edible mushroom Termitomyces clypeatus: Structure elucidation and antioxidant properties. Carbohydrate Research, 413, 30–36. Rahmad, N., Al-Obaidi, J. R., Rashid, N. M. N., Zean, N. B., Yusoff, M. H. Y. M., Shaharuddin, N. S., et al. (2014). Comparative proteomic analysis of different developmental stages of the edible mushroom Termitomyces heimii. Biological Research, 47, 30.

384

D.K. Manna et al. / Carbohydrate Polymers 134 (2015) 375–384

Rout, D., Mondal, S., Chakraborty, I., Pramanik, M., & Islam, S. S. (2005). Chemical analysis of a new (1 → 3)-, (1 → 6)-branched glucan from an edible mushroom, Pleurotusflorida. Carbohydrate Research, 340, 2533–2539. Sen, I. K., Maji, P. K., Behera, B., Maiti, T. K., Mallick, P., Sikdar, S. R., et al. (2013). Glucan of a somatic hybrid mushroom, pfls1 h: Structural characterization and study of immunological activities. International Journal of Biological Macromolecules, 53, 127–132. Saxena, Q. B., Mezey, E., & Adler, W. A. (1980). Regulation of natural killer activity in vivo. II. The effect of alcohol consumption on human peripheral blood natural killer activity. International Journal of Cancer, 26, 413–417. Tripathy, S., Das, S., Dash, S. K., Mahapatra, S. K., Chattopadhyay, S., Majumder, S., et al. (2014). A prospective strategy to restore the tissue damage in malaria infection: Approach with chitosan-trypolyphosphate conjugated nanochloroquine in Swiss mice. European Journal of Pharmacology, 737, 11–21. Tripathy, S., Mahapatra, S. K., Chattopadhyay, S., Das, S., Dash, S. K., Majumder, S., et al. (2013). A novel chitosan based antimalarial drug delivery against Plasmodium berghei infection. Acta Tropica, 128, 494–503.

Tripathy, S. S., Rajoriya, A., & Gupta, N. (2014). Wild indegenous mushrooms as a source of food from different forest divisions of Odisha. Journal of Pharmaceutical Biology, 4(3), 138–147. Wade, C. R., Jackson, P. G., Highton, j., & Van Rij, A. M. (1987). Lipid peroxidation and malondialdehyde in the synovial fluid and plasma of patients with rheumatoid arthritis. Clinica Chimica Acta, 164(3), 245–250. Wasser, S. P. (2002). Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Applied Microbiology and Biotechnology, 60, 258–274. Wasser, S. P., & Weis, A. L. (1999). Medicinal properties of substances occurring in higher Basidiomycetes mushrooms: Current perspectives. International Journal of Medicinal Mushrooms, 1, 31–62 (Review). York, W. S., Darvill, A. G., McNeil, M., Stevenson, T. T., & Albersheim, P. (1985). Methods in Enzymology, 118, 33–40. Yoshioka, Y., Tabeta, R., Saito, H., Uehara, N., & Fukuoka, F. (1985). Antitumor ´ Isolation and structure of a polysaccharides from P. Ostreatus (Fr.) Quel.: ␤-glucan. Carbohydrate Research, 140, 93–100.