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Selenium-containing polysaccharides from Lentinula edodes—Biological activity
Carbohydrate Polymers 223 (2019) 115078
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Selenium-containing polysaccharides from Lentinula edodes—Biological activity
T
Beata Kaletac, Andrzej Górskib,c, Radosław Zagożdżonc,e, Marcin Cieślakd, Julia Kaźmierczak-Barańskad, Barbara Nawrotd, Marzenna Klimaszewskaa, Eliza Malinowskaa, ⁎ Sandra Górskaa, Jadwiga Turłoa, a
Department of Drug Technology and Pharmaceutical Biotechnology, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland Bacteriophage Laboratory, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland c Department of Clinical Immunology, Medical University of Warsaw, Nowogrodzka 59, 02-006 Warsaw, Poland d Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland e Department of Immunology, Transplant Medicine and Internal Diseases, Medical University of Warsaw, Nowogrodzka 59, 02-006 Warsaw, Poland b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lentinula edodes Selenium Se-polysaccharides Immunosuppressants Lymphocytes T
We hypothesized that selenium(Se)-enriched polysaccharides would possess superior biological activity when compared to those non-enriched. To verify this hypothesis, we obtained by biotechnological methods a Seenriched analog of Japanese anticancer drug lentinan and, as a reference, the non-Se-enriched fraction. We tested the effects of the obtained fractions on the proliferation of human peripheral blood mononuclear cells. The results suggested a selective immunosuppressive activity, non-typical for mushroom derived polysaccharides. Both fractions caused significant inhibition of human T lymphocyte proliferation induced by mitogens, without significant effects on B lymphocytes. The inhibitory effect was not due to the toxicity of the examined polysaccharides. In normal (HUVEC) or malignant (HeLa) cells tested fractions significantly enhanced cell viability and protected the cells from oxidative stress conditions. However, we observed no effect of the polysaccharide fractions on the production of reactive oxygen species by granulocytes in vitro. The selenium content increased the biological activity of the tested polysaccharide fractions.
1. Introduction
immune cells, including macrophages, natural killer (NK) cells, and T and B lymphocytes (Zheng, Jie, Hanchuan, & Moucheng, 2005). Recent studies have demonstrated the structure – activity relationship of influence of polysaccharides on the immune system (polysaccharides with immunomodulatory properties differ greatly in their chemical structures). There are reports suggesting that various immunomodulatory effects of fungal polysaccharides are associated with their different monosaccharide composition, molecular weight, branching degrees, and triple helical conformation (Ferreira, Passos, Madureira, Vilanova, & Coimbra, 2015; Zhang, Cui, Cheung, & Wang, 2007). Lentinan, branched 1-6,1-3-β-D-glucan, is a highly purified polysaccharide
The anticancer activity of mushroom-derived polysaccharides has been associated with their immunomodulating properties (Bohn & Miller, 1995; Chihara, Maeda, Taguchi, & Hamuro, 1989; Chihara, 1992; Yu, Shen, Song, & Xie, 2018). Polysaccharides could affect both innate and adaptive immunity, including cellular and humoral responses (Guo, Savelkoul, Kwakkel, Williams, & Verstegen, 2003). Based on the mechanisms of pharmacological activity, mushroom-derived polysaccharides are classified as biological response modifiers (BRMs) (Friedman, 2016). These polysaccharides can affect various kinds of
Abbreviations: ATCC, American Type Culture Collection; BHT, butylated hydroxytoluene; BRM, biological response modifiers; BSA, bovine serum albumin; DETBA, 1,3-diethyl-2-thiobarbituric acid; ECGF, endothelial cell growth factor; FBS, fetal bovine serum; HUVECs, human umbilical vein endothelial cells; mAb, monoclonal antibody; NK, natural killer cells; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PHA, phytohaemagglutinin; PMA, phorbol 12myristate 13-acetate; SAC, Staphylococcus aureus Cowan; SOD, superoxide dismutase ⁎ Corresponding author. E-mail addresses: [email protected] (B. Kaleta), [email protected] (A. Górski), [email protected] (R. Zagożdżon), [email protected] (M. Cieślak), [email protected] (J. Kaźmierczak-Barańska), [email protected] (B. Nawrot), [email protected] (M. Klimaszewska), [email protected] (E. Malinowska), [email protected] (S. Górska), [email protected] (J. Turło). https://doi.org/10.1016/j.carbpol.2019.115078 Received 16 March 2019; Received in revised form 10 July 2019; Accepted 11 July 2019 Available online 13 July 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. The effects of the mycelial polysaccharide fractions (selenated – Se-L and not selenated L) on HUVEC and HeLa cells viability: A. HUVEC cells after 24 h of incubation, B. HeLa cells after 24 h of incubation, C. HUVEC cells after 48 h of incubation, D. HeLa cells after 48 h of incubation. The viability of tested cells nontreated with polysaccharide fractions was taken as a 100%. The error bars correspond to the standard deviation. Fig. 2. The protective effect of polysaccharides (selenated fraction Se-L, not selenated fraction L and lentinan in concentrations of 25 μg/ml) on exogenous oxidative stress induced by hydrogen peroxide in concentrations of 100 and 300 μM. KK- control; H2O2 - control cells H2O2treated; * - p < 0.05 versus control cells H2O2treated (H2O2). The error bars correspond to the standard deviation.
from the Se-enriched mycelium of L. edodes (Malinowska et al., 2018). Nowadays, the most important issue to be investigated is the examination whether the incorporation of selenium affects the biological activity of the polysaccharide fractions. We examined the impact of polysaccharides on normal and malignant cells viability, and antioxidant activity of the polysaccharide fractions, as well as their effects on human peripheral blood mononuclear cells (PBMCs) proliferation. This line of inquiry was based on the results of our previous studies, which have shown a selective cytotoxic (Klimaszewska et al., 2017) and strong antioxidant activity (Turło, Gutkowska, & Herold, 2010) of the Se-polysaccharide-containing water extracts from the L. edodes mycelium. Thus, the objective of the current research was to study the effects of the polysaccharide fractions isolated from the mycelial cultures (selenium-enriched and non-enriched) on a range of human cells,
fraction, extracted from Lentinula edodes (shiitake mushroom) fruiting bodies. This substance is approved for use in cancer treatment as an adjunct to conventional therapy (Boon & Wong, 2004; Sullivan, Smith, & Rowan, 2006; Yeung & Gubili, 2008) It appears that selenium supplementation stimulates the immune response (Brozmanová, Mániková, Vlčková, & Chovanec, 2010; Rayman, 2005, 2008, 2012), therefore we hypothesized that insertion of selenium into mushroom-derived polysaccharides would enhance their immunomodulatory activity. To verify these assumptions, we obtained by biotechnological methods a selenium-containing polysaccharide – an analog of the Japanese anticancer drug lentinan (Malinowska et al., 2018). In the previous report, we described an isolation and structural analysis of a Se-containing polysaccharide-protein complex isolated 2
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Fig. 3. Antioxidant activity of fractions Se-L and L determined by the DETBA method. * - p < 0.05 versus control. The error bars correspond to the standard deviation.
Nachman, Becker, & Minick, 1973) and cultured in plastic dishes coated with gelatin, in RPMI 1640 medium (Thermo Fischer Scientific) supplemented with 20% fetal bovine serum (FBS, Sigma-Aldrich), 90 U/ml heparin (Sigma-Aldrich), 150 μg/ml endothelial cell growth factor (ECGF, Roche Diagnostics, Mannheim, Germany) and antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin, Thermo Fischer Scientific). Cells were grown in a monolayer at 37 °C in an atmosphere of 5% CO2. The HeLa cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with antibiotics and 10% fetal calf serum (FCS, Sigma-Aldrich), in a 5% CO2. Cells (5 × 103) were seeded in each well on a 96-well plate (Nunc). After 24 h, without changing the medium, cells were exposed to the tested polysaccharides for another 24 or 48 h. Stock solutions of polysaccharides from L. edodes were freshly prepared in water. Then, four serial dilutions (from 0.5 mg/ml to 0.06 mg/ml) were prepared in saline (0.9% NaCl) and subsequently used in cell cultures. The concentrations of polysaccharide in the wells were 25, 12.5, 6.25 and 3.15 μg/ml. The cytotoxicity of all compounds was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO] assay as described (Maszewska et al., 2003). Briefly, after 24 h or 48 h of incubation with polysaccharides, the cells were treated with the MTT reagent and incubation was continued for 2 h. MTT-formazan crystals were dissolved in 20% SDS and 50% DMF at pH 4.7 and absorbance was read at 562 and 630 nm on an ELISA-PLATE READER (ELX800, Bio-Tek, USA). Cultured cells treated with vehicle (saline) served as a control and represented 100% viability.
including the immune cells. 2. Materials and methods 2.1. Biosynthesis and isolation of mycelial Se-polysaccharides 2.1.1. Mushroom strain and cultivation conditions The Lentinula edodes (Berk.) Pegler strain used in this study was ATCC 48085. The mycelial cultures were grown under the conditions described in our previous reports (Malinowska et al., 2018; Turło et al., 2010, 2011). The mycelial raw materials used for extraction of polysaccharides were cultivated under submerged conditions in a 10-L fermenter (BioTec FL 110, Stockholm, Sweden) in the culture media either in the presence or absence of selenium at a concentration of 20 μg/ml by the addition of sodium selenite (Na2SeO3, Sigma, Cell Culture Tested). 2.1.2. Extraction and isolation of Se-enriched polysaccharide fraction According to our previous report (Malinowska et al., 2018), the selenium-polysaccharide fraction (fraction Se-L) that corresponded to lentinan, was isolated from the Se-enriched mycelium by use of the modified approach (Malinowska et al., 2018; Yap & Ng, 2001). The reference polysaccharide fraction was extracted from mycelium of L. edodes cultivated in medium not enriched in Se (fraction L) (Malinowska et al., 2018). 2.2. Biological activity of Se-enriched and reference fractions In the present study, the biological activities of the Se-polysaccharide fraction, the non-selenium-enriched fraction and the commercial drug lentinan (CheMall, Mundelein, CAS 37339-90-5, Concentration 98,3%) were studied. The use of these fractions permitted us to compare the differences between the activity of the commercial drug isolated from fruiting bodies of L. edodes (lentinan), the analogous fraction isolated from the mycelial cultures.
2.2.2. Antioxidant effects of polysaccharides 2.2.2.1. Protective effect on exogenous oxidative stress. The HeLa cells (cultured as described above) were pre-incubated with polysaccharides isolated from L. edodes (at a final concentration of 25 μg/ml) for 30 min. Following this incubation, H2O2 was added to the cells (final concentration 100 μM or 300 μM) for 24 h. Cell viability was determined using the MTT assay.
2.2.1. The effects of polysaccharides on cell viability (MTT assay) Human umbilical vein endothelial cells (HUVECs) and human cervix carcinoma (HeLa) cell lines were used. HUVEC were isolated from freshly collected umbilical cords as previously described (Jaffe,
2.2.2.2. In vitro determination of antioxidant activity (DETBA method). The antioxidant activity was determined by the DETBA (1,3diethyl-2-thiobarbituric acid) method, according to our previous paper (Turło et al., 2010). Various concentrations of polysaccharides Se-Land 3
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Fig. 4. The effects of polysaccharides (Se-L, L and lentinan in concentrations of 1, 10 and 100 μg/ml) on the production of superoxide anions (O2−). The auto- (A0) and phorbol 12-myristate 13-acetate (APMA) stimulation of granulocytes was expressed as a level of nmols O2− determined by measurement of cytochrome c reduction rate.
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Fig. 5. The effects of polysaccharides (Se-L, L and lentinan in concentrations of 1, 10 and 100 μg/ml) on proliferation of peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3 monoclonal antibody (OKT3), phytohemagglutinin (PHA) and suspension of Staphylococcus aureus Cowan strain (SAC). The results are presented as a level of radioactivity as ‘Corrected Counts per Minute’ (CCPM). * p < 0.05 versus control (K). The error bars correspond to the standard deviation.
L water solutions (50 μl) were mixed with 50 μl of linoleic acid emulsion (2 mg/ml in 95% ethanol), resulting in a final concentration of polysaccharides of 6.25, 12.5 and 25 μg/ml. The mixtures were incubated at 80 °C for 60 min, and cooled in an ice bath. The following were then sequentially added: 200 μl of 20 mM butylated hydroxytoluene (BHT, Sigma), 200 μl of 8% sodium dodecyl sulphate, 400 μl of deionized water, and 3.2 ml of 12.5 mM DETBA in sodium phosphate buffer (pH 3.0). The mixture was stirred, placed in an oven at 95 °C for 15 min, and then cooled in an ice bath. After 4 ml of ethyl acetate was added, the mixture was stirred and centrifuged at 2000 rpm for 15 min. Ethyl acetate was separated and its absorbance was measured in a spectrofluorometer with fluorescence excitation at 515 nm and emission at 555 nm. The antioxidant activity was expressed as the percentage of lipid peroxidation, with a control containing no sample set as 100%. A higher percentage indicated a lower antioxidant activity.
2.2.4. The effects of polysaccharides on human peripheral blood mononuclear cells (PBMCs) proliferation PBMCs from healthy donors were isolated by density-gradient centrifugation on Histopaque-1077 (Sigma). Blood was commercially obtained from the Regional Blood Centre in Warsaw. PBMCs were cultured in Parker medium (Biomed) supplemented with 2 mM L-glutamine (Sigma), 0.1 mg/ml gentamycin (KRKA), βmercaptoethanol (Sigma), 0.23% Hepes (Sigma) and 10% heat inactivated fetal bovine serum (FBS, Gibco). Fractions of L. edodes (Se-L, reference fraction L or lentinan) were diluted in 0.9% NaCl (Fresenius Kabi) to achieve concentrations of 0.1–0.001 mg/ml. PBMCs cultures were established in 96-well flat–bottom microplates (at density 1 × 106 cells/well) and induced with specific T and B cells mitogens: anti-CD3 mAb (OKT3, 1 μg/ml, BD Pharmingen, T cell mitogen), phytohemagglutinin (PHA, 20 μg/ml, Sigma, T cell mitogen), and a suspension of Staphylococcus aureus Cowan strain (SAC, 0.004% w/v, Calbiochem, B cel mitogen). For stimulation with anti-CD3 mAb, culture plates were first coated overnight with anti-CD3 mAb followed by washing with sterile phosphate buffered saline (PBS). Polysaccharides were added to cell cultures in aliquots (100 μl) of the prepared dilution per well. Control cultures contained an equivalent amount of 0.9% NaCl. PBMCs were cultured for 72 h at 37 °C in a humidified atmosphere with 5% CO2. After 72 h cells were pulsed with 1 μCi/well of [3H]-thymidine (113 Ci/nmol, NEN) for the last 18 h of the incubation and then harvested with an automated cell harvester (Skatron). The amount of [3H]-thymidine incorporated into the cells was measured using a Wallac Microbeta scintillation counter (Wallac), giving the level of radioactivity as ‘Corrected Counts per Minute’ (CCPM). As an internal control for evaluation of results, an analogous test without any mitogens (Autostimulation) was carried out. All experiments were performed in triplicates. The study was approved by the Local Ethics Committee and all subjects provided written informed consent. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000.
2.2.3. The effects of polysaccharides on superoxide production by granulocytes Granulocytes were obtained by Histopaque-1119 (Sigma) separation from blood from healthy donors, commercially obtained from the Regional Blood Centre in Warsaw. The granulocyte pellet was resuspended in sterile phosphate buffered saline (PBS, Biomed) supplemented with 6 mM glucose (Sigma) and 1% bovine serum albumin (BSA, Biowest). Granulocytes cultures were established in 96-well round–bottom microplate (concentration 2.5 × 105 cells/well). Granulocytes activated by phorbol 12-myristate 13-acetate (PMA, Sigma) as well as nonactivated samples were incubated with cytochrome c (Sigma) and polysaccharide fractions (concentrations 1, 10, 100 μg/ml) for 30 min at 37 °C in a humidified atmosphere with 5% CO2. The auto- and PMA stimulation of granulocytes was expressed as a level of superoxide anions (nmols O2−) and calculated using the Lambert–Beer law. Control cultures contained an equivalent amount of medium. Additional controls containing superoxide dismutase (SOD, Sigma, 30 mg/ml) were also prepared in order to provide correction for the O2– independent reduction of cytochrome c. The level of O2– was determined by measurement of cytochrome c reduction rate at room temperature using microplates reader at the wavelength 550 nm (Björquist, Palmer, & Ek, 1994). Experiments were performed in triplicates.
2.3. Statistical analysis Mann – Whitney U-test and Spearman correlation were applied using the Statistica 9.0 (StatSoft Inc). Differences from control cultures 5
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3.2. Antioxidant activity of polysaccharides in cell cultures 3.2.1. Protective effect on exogenous oxidative stress Antioxidant activity of the polysaccharides Se-L and L in HeLa cell cultures was confirmed by the higher cells viability after H2O2 exposure. All tested polysaccharide fractions display antioxidant activity. As shown in Fig. 2, cells viability in the presence of the selenated polysaccharides was higher as compared to non-selenated polysaccharides and significantly higher as compared to the control H2O2treated cells. This was particularly evident for cells exposed to 300 μM H2O2, for which Se-L enhanced viability of cells two-fold compared to L fraction, and nearly six-fold compared to the control cells (treated with H2O2 only). Both mycelial fractions (Se-L and L) express much higher activity than lentinan (Fig. 2). 3.2.2. In vitro determination of antioxidant activity (DETBA method) In DETBA test the fractions Se-L and L show statistically significant antioxidant activity (Fig. 3). At a concentration of 25 μg/ml lipid peroxidation was inhibited by 32 and 12%, respectively. 3.3. The effects of polysaccharides on the production of reactive oxygen species by human granulocytes The analysis of O2− concentration [nmol] in granulocytes supernatants revealed that none of the polysaccharides has significant effect on the reactive oxygen species generation by these cells (Fig. 4A–C). 3.4. The effects of polysaccharides on the proliferation of human peripheral blood mononuclear cells The effect of polysaccharide fractions (Se-L, L and lentinan) on PHA and OKT3-stimulated PBMC proliferation is presented in Fig. 4B and C and supplementary Table S.1. In the autostimulation and, non-significantly, in SAC-stimulation settings, both S-Le and L fractions showed some tendency to decrease proliferation of PMBC. It remains to be elucidated what type of cells within the PMBC pool was inhibited in these groups. However, this effect was very moderate in comparison the T cell mitogen-stimulated settings. Indeed, OKT3-stimulated proliferation was significantly inhibited by all examined fractions at the doses of 100 μg/ml, 10 μg/ml and 1 μg/ml, in a dose-dependent manner. For the Se-L fraction the degree of inhibition at 100 and 10 μg/ml was approximately 89% and for fraction L, 86%. The difference between selenated and non-selenated fractions was significant. The relatively high values of standard deviations (Fig. 5, Table S.1) were due to the diverse response of the lymphocyte cultures from different donors to the mitogen. No statistically significant inhibitory effect of polysaccharide fractions on SACstimulated PBMC was observed. For the commercial preparation of lentinan, used as reference, no impact on the proliferation of T or B lymphocytes was observed (Fig. 5A–D). The cell viability assessment by the trypan blue exclusion method confirmed that the inhibitory effect on OKT3- and PHA-stimulated PBMC proliferation was not due to the toxicity of the examined polysaccharides.
Fig. 6. A. Correlation between the HeLa cells viability, non-treated with polysaccharide fraction (1), treated by the fractions Se-L (2), L (3) or lentinan (4), in HeLa cells after 24 h exposure (determined in MTT test) and the protective effect of the same fractions on exogenous oxidative stress induced in HeLa cells by hydrogen peroxide in concentrations of 100 and 300 μM (determined in H2O2 test). B. Correlation between the results of determination the polysaccharide antioxidant activity in Se-L and L 25 μg/mL solutions by two different methods: as protective effect on exogenous oxidative stress (H2O2 test) and as inhibition of lipid peroxidation (DETBA method).
were considered statistically significant at a p value < 0.05.
3. Results 3.1. The effects of polysaccharides on HUVEC and HeLa cells viability In general, the mycelial polysaccharide fractions (selenated and not selenated) display no cytotoxic activity (Fig. 1). The study demonstrated that all tested polysaccharides do not adversely affect the viability of HUVEC, as well as HeLa cells. Surprisingly, as shown in Fig. 1A–D, cells viability in the presence of selenated and non-selenated fractions was higher as compared to the control, non-treated cells. This effect was significantly stronger for Se-L fraction than for fraction L in higher concentrations, however, especially in HUVEC cells, increased viability was noticed for lower concentrations of L fractions. Interestingly, enhancement of cells viability was more pronounced in normal endothelial cells than in cancer cells. We hypothesized that the enhancement of the cell viability by polysaccharide fractions could be a result of their antioxidant activity. To clarify this issue we analyzed the protective effects of polysaccharides on exogenous oxidative stress.
4. Discussion In recent years, there has been increasing interest in searching drugs of natural origin to be safely and efficiently used as a primary or adjuvant therapy of many disorders. Medicinal mushrooms are considered to be a potential source of biologically active substances with significant pharmacological properties (Ganeshpurkar, Rai, & Jain, 2010). Nowadays, the most valuable medicinal mushroom is Lentinula edodes (shiitake mushroom), which is a source of multiple active compounds, including polysaccharides (Hobbs, 2000). Some studies demonstrated 6
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a result of relatively low content of polysaccharides in previously tested mycelial extracts (96 and 288 mg/g in selenated and non-selenated extract, respectively) (Turło et al., 2010). Importantly, the results of determination of antioxidant activity of Se-L and L fractions by two different methods: as inhibition of lipid peroxydation (DETBA method) and as protective effect on exogenous oxidative stress (H2O2 test in HeLa cells) are compatible (Fig. 6B). We have shown, that incorporation of selenium at the –II oxidation state into the polysaccharide structure resulted in a significant increase of cell viability and enhanced antioxidant activity. In turn, the mycelial polysaccharide fraction analogues of lentinan were much more active as antioxidants than was commercially prepared lentinan. Moreover, we evaluated the effects of polysaccharide fractions on O2− production by human granulocytes but we revealed that none of the polysaccharides affects the reactive oxygen species. It suggests that the antioxidant activities of fractions studied are not related to inhibition of early stages ROS production. Additionally, we tested the effects of the Se-enriched and reference fractions on the proliferation of human PBMCs. Lymphocyte proliferation induced by anti-CD3 mAb (OKT3) and phytohemagglutynin (PHA) may be used as a method to evaluate T proliferative response. The two mitogens use different mechanisms to promote T cell effector functions. PHA stimulates T cell proliferation by interactions with the N-acetylgalactosamine glycoprotein present on these cells (Lindahl-Kiessling & Peterson, 1969). OKT3 stimulates T cells via CD3-mediated signaling (Van Wauwe, De Mey, & Goossens, 1980). Lymphocyte proliferation induced by Staphylococcus aureus Cowan strain (SAC) may be used to evaluate B cell responsiveness. We observed that both samples isolated from the submerged cultures of L. edodes fractions – Se-containing analog of lentinan (fraction Se-L) and non-selenium enriched fraction L significantly inhibited proliferation of the T lymphocytes, most likely via CD3 receptor, but a mechanism of interactions with the N-acetylgalactosamine glycoprotein has not been excluded. In PHA-stimulated PBMC, the suppression was also seen with fractions L and Se-L, although the effect was significantly (three times) weaker (Table S.1). The suppression of the T lymphocyte proliferation by mycelial polysaccharides was distinctly different from the effect with lentinan, which showed no such activity (Fig. 5). This outcome suggested that in contrast to lentinan, fractions Se-L and L were selectively immunosuppressive. The suppression of T cell proliferation in both the OKT3 and PHA tests did not permit us to determine at this stage of the research the exact mechanism of action of the examined fractions. Most likely both processes play a role, via the CD3 receptors and by interactions with the N-acetylgalactosamine glycoprotein present on the T lymphocyte. The polysaccharide fractions isolated from the mycelial culture of L. edodes significantly differed in biological activity from the material isolated by the same method from mycelial fruiting bodies, i.e., the Japanese drug lentinan. Most likely it is a result of significant differences in the structure of the tested fractions and of lentinan. As previously reported (Malinowska et al., 2018) the selenium-containing fraction Se-L occurred as a mixture of 1,4-α- and 1,3-1,6-β-glucans, whereas lentinan is a 1,6-1,3-β-glucan. The proportion of 1,6 and 1,3-βglycosidic linkages in the mycelial fraction is also different from lentinan (Malinowska et al., 2018). The molecular weight of the mycelial polysaccharide is very high, much higher than that of lentinan (nearly 4 × 106 Da versus 5 × 105 Da). These variations suggest a different composition of the cell walls of the mycelium cultivated under submerged conditions versus the L. edodes fruiting bodies. Incorporation of selenium strongly enhanced the antioxidant activity, protection of cells against oxidative stress, and selective immunosuppressive activity, characteristics which are not typical for mushroom polysaccharides. Further structural and biological activity tests are required.
that polysaccharides isolated from L. edodes can modulate functions of immune cells, however, the polysaccharide fractions isolated from the mycelial cultures of L. edodes may significantly differ in biological activity (Kojima, Akaki, Nakajima, Kamei, & Tamesada, 2010). In the present study, we evaluated the immunomodulatory properties of polysaccharides isolated from mycelial culture of L. edodes with the Yap and Ng method (Yap & Ng, 2001). We used a Se-enriched polysaccharide fraction that corresponds to lentinan (fraction Se-L). A reference fraction (fraction L, which corresponds to lentinan) was isolated from the non-selenium enriched mycelium. Our previous study (Malinowska et al., 2018) demonstrated the selenium concentration in Se-enriched mycelium was equal to 1045 μg/g; nearly 3.4% (w/w) of this amount was combined with the crude polysaccharide fraction Se-C (1090.7 μg/g of the crude polysaccharide fraction). The selenium polysaccharide (fraction Se-L) contained proteoglycans with molecular weights of 3.9 × 106 Da and 2.6 × 105 Da, and were composed of glucose and mannose. This fraction also contained nearly 8% protein. The IR spectra confirmed the presence of β-glucans, however α-glycosidic bonds were also detected. The NMR spectral analysis confirmed that the main types of detected glycosidic bonds were β-1,3-glycosidic linkages, α-1,4-glycosidic linkages, and β-1,6-glycosidic linkages. X-ray absorption fine structure (XAFS) spectra analysis in the near edge region (XANES) showed that selenium in the Se-polysaccharides structure is present at the minus II oxidation state and is organically bound. The simulation analysis in the EXAFS region suggested that selenium is most likely bound by a glycosidic-linkage in a β-1,3 or α-1,4-glycosidic bond (Malinowska et al., 2018). Recently, we revealed that Se-polysaccharide-containing water extracts from the L. edodes mycelium had a selective cytotoxic (Klimaszewska et al., 2017) and strong antioxidant activity (Turło et al., 2010). The mycelial L. edodes extracts containing Se-polysaccharides showed weak cytotoxic activity in HeLa cells, while strongly increased HMEC1 cells viability (Klimaszewska et al., 2017). Thus, in the present study, we investigated if these effects were caused by the Se-polysaccharides content. The results of the current study showed that both selenated and non-selenated polysaccharide fractions did not adversely affect cells viability. This result suggests that selenium compounds other than the Se-polysaccharides were responsible for the previously observed cytotoxic activity of L. edodes mycelial extracts. Moreover, our current study revealed that the Se-polysaccharides were responsible for a strong increase in cell viability, significantly higher for normal cells (by 54–86%) rather than cancer cells (by 7–62%) (Fig. 1A–D). In our previous report (Klimaszewska et al., 2017), we speculated that effects of selenated fractions on cell viability were associated with the counteracting oxidative stress, as is apparent from data on other compounds (Aherne, Kerry, & O’Brien, 2007; Hadjaz et al., 2011). Our present study, which demonstrated that cell viability correlate well with the antioxidant activities of the polysaccharide fractions supported our hypothesis. A correlation analysis of viability of HeLa cells, non-treated with polysaccharide fraction, treated by the fractions Se-L, L or lentinan (determined in MTT test, Section 3.1) and the protective effect of the same fractions on exogenous oxidative stress induced in HeLa cells by hydrogen peroxide (H2O2 test, Section 3.2.1) was linear (R2 = 0.82 and 0.84, for hydrogen peroxide concentrations of 100 and 300 μM in H2O2 test, respectively) (Fig. 6A). The antioxidant activity of polysaccharide-containing water extracts from L. edodes mycelia (selenated and non-selenated) was described in our previous work (Turło et al., 2010). The chemical composition of the extracts was precisely determined. However, unclear was which of the components were responsible for antioxidant activity. Current studies have shown, the activity was mainly due to the polysaccharide content. The fractions Se-L and L in DETBA test show similar antioxidant activity to mushroom extracts (when it comes to the % protection against lipid peroxidation), however, in much lower concentrations (6.25–25 μg/mL versus 75–250 μg/mL) (Turło et al., 2010). The difference most likely is 7
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5. Conclusion
5(4), 80. Ganeshpurkar, A., Rai, G., & Jain, A. P. (2010). Medicinal mushrooms: Towards a new horizon. Pharmacognosy Reviews, 4(8), 127–135. Guo, F. C., Savelkoul, H. F. J., Kwakkel, R. P., Williams, B. A., & Verstegen, M. W. A. (2003). Immunoactive, medicinal properties of mushroom and herb polysaccharides and their potential use in chicken diets. World’s Poultry Science Journal, 59(04), 427–440. Hadjaz, F., Besret, S., Martin-Nizard, F., Yous, S., Dilly, S., Lebegue, N., et al. (2011). Antioxydant activity of β-carboline derivatives in the LDL oxidation model. European Journal of Medicinal Chemistry, 46(6), 2575–2585. Hobbs, C. H. (2000). Medicinal value of Lentinus edodes (Berk.) Sing. A literature review. International Journal of Mededicinal Mushrooms, 2, 287–302. Jaffe, E. A., Nachman, R. L., Becker, C. G., & Minick, C. R. (1973). Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. Journal of Clinical Investigation, 52(11), 2745–2756. Klimaszewska, M., Górska, S., Dawidowski, M., Podsadni, P., Szczepanska, A., Orzechowska, E., et al. (2017). Selective cytotoxic activity of Se-methyl-seleno-Lcysteine–and Se-polysaccharide–containing extracts from shiitake medicinal mushroom, Lentinus edodes (Agaricomycetes). International Journal of Medicinal Mushrooms, 19(8), 709–716. Kojima, H., Akaki, J., Nakajima, S., Kamei, K., & Tamesada, M. (2010). Structural analysis of glycogen-like polysaccharides having macrophage-activating activity in extracts of Lentinula edodes mycelia. Journal of Natural Medicines, 64(1), 16–23. Lindahl-Kiessling, K., & Peterson, R. D. A. (1969). The mechanism of phytohemagglutinin (PHA) action. Experimental Cell Research, 55(1), 85–87. Malinowska, E., Klimaszewska, M., Strączek, T., Schneider, K., Kapusta, C., Podsadni, P., et al. (2018). Selenized polysaccharides–biosynthesis and structural analysis. Carbohydrate Polymers, 198, 407–417. Maszewska, M., Leclaire, J., Cieslak, M., Nawrot, B., Okruszek, A., Caminade, A.-M., et al. (2003). Water-soluble polycationic dendrimers with a phosphoramidothioate backbone—Preliminary studies of cytotoxicity and oligonucleotide/plasmid delivery in cell culture. Oligonucleotides, 13(4), 193–205. Rayman, M. P. (2005). Selenium in cancer prevention: A review of the evidence and mechanism of action. Proceedings of the Nutrition Society, 64(4), 527–542. Rayman, M. P. (2008). Food-chain selenium and human health: Emphasis on intake. British Journal of Nutrition, 100(2), 254–268. Rayman, M. P. (2012). Selenium and human health. Lancet, 379(9822), 1256–1268. Sullivan, R., Smith, J. E., & Rowan, N. J. (2006). Medicinal mushrooms and cancer therapy: Translating a traditional practice into Western medicine. Perspectives in Biology and Medicine, 49(2), 159–170. Turło, J., Gutkowska, B., & Herold, F. (2010). Effect of selenium enrichment on antioxidant activities and chemical composition of Lentinula edodes (Berk.) Pegl. mycelial extracts. Food and Chemical Toxicology, 48(4), 1085–1091. Turło, J., Gutkowska, B., Herold, F., Gajzlerska, W., Dawidowski, M., Dorociak, A., et al. (2011). Biological availability and preliminary selenium speciation in selenium-enriched mycelium of Lentinula edodes (Berk.). Food Biotechnology, 25(1), 16–29. Van Wauwe, J. P., De Mey, J. R., & Goossens, J. G. (1980). OKT3: A monoclonal antihuman T lymphocyte antibody with potent mitogenic properties. Journal of Immunology, 124(6), 2708–2713. Yap, A.-T., & Ng, M.-L. (2001). An improved method for the isolation of lentinan from the edible and medicinal shiitake mushroom, Lentinus edodes (Berk.) Sing. (Agaricomycetideae). International Journal of Medicinal Mushrooms, 3(1), 9–19. Yeung, K., & Gubili, J. (2008). Shiitake mushroom (Lentinula edodes). Journal of the Society for Integrative Oncology, 6(3), 134–135. Yu, Y., Shen, M. Y., Song, Q. Q., & Xie, J. H. (2018). Biological activities and pharmaceutical applications of polysaccharide from natural resources: A review. Carbohydrate Polymers, 183, 91–101. 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(1), 4–19. Zheng, R., Jie, S., Hanchuan, D., & Moucheng, W. (2005). Characterization and immunomodulating activities of polysaccharide from Lentinus edodes. International Immunopharmacology, 5(5), 811–820.
The aim of the present study was to obtain a selenium-containing polysaccharide, the analog of the immunostimulatory polysaccharide drug lentinan. We expected enhanced immunostimulating activity by the selenated fraction versus lentinan. However, the results of the preliminary biological tests of the obtained fractions were unexpected and remarkable. We obtained a selective immunosuppressant with a strong antioxidant activity, which was non-toxic and even enhanced cell viability. This biological effect was completely different from lentinan and depended in-part on the selenium incorporation into the polysaccharide molecule, but also on the dissimilar structure of the fractions isolated from mycelial cultures versus lentinan. The potential use of the Secontaining mycelial polysaccharides as selective immunosuppressants has been covered by the patent (PL402082). However, there are many questions regarding both the Se-polysaccharides exact structure and their mechanism of action. We intend to investigate these issues in our ongoing further research. Funding This work was supported by grant from the National Science Centre, Poland, grant number DEC-2013/09/B/NZ7/03978. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115078. References Aherne, A., Kerry, J. P., & O’Brien, N. M. (2007). Effects of plant extracts on antioxidant status and oxidant-induced stress in Caco-2 cells. British Journal of Nutrition, 97(2), 321–328. Björquist, P., Palmer, M., & Ek, B. (1994). Measurement of superoxide anion production using maximal rate of cytochrome (III) C reduction in phorbol ester stimulated neutrophils, immobilised to microtiter plates. Biochemical Pharmacology, 48(10), 1967–1972. Bohn, J. A., & Miller, N. B. (1995). (1-3)-β-D-glucans as biological response modifiers: a review of structure-functional activity relationships. Carbohydrate Polymers, 28, 3–14. Boon, H., & Wong, J. (2004). Botanical medicine and cancer: A review of the safety and efficacy. Expert Opinion on Pharmacotherapy, 5(12), 2485–2501. Brozmanová, J., Mániková, D., Vlčková, V., & Chovanec, M. (2010). Selenium: A doubleedged sword for defense and offence in cancer. Archives of Toxicology, 84(12), 919–938. Chihara, G., Maeda, Y. Y., Taguchi, T., & Hamuro, J. (1989). Lentinan as a host defence potentiator (HDP). Inernational Journal of Immunotherapy, 5, 145–150. Chihara, G. (1992). Immunopharmacology of Lentinan, a polysaccharide isolated from Lentinus edodes: Its applications as a host defense potentiator. International Journal of Oriental Medicine, 17, 57–77. Ferreira, S. S., Passos, C. P., Madureira, P., Vilanova, M., & Coimbra, M. A. (2015). Structure–Function relationships of immunostimulatory polysaccharides: A review. Carbohydrate Polymers, 132, 378–396. Friedman, M. (2016). Mushroom polysaccharides: Chemistry and antiobesity, antidiabetes, anticancer, and antibiotic properties in cells, rodents, and humans. Foods,
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Selenium-containing polysaccharides from Lentinula edodes—Biological activity
T
Beata Kaletac, Andrzej Górskib,c, Radosław Zagożdżonc,e, Marcin Cieślakd, Julia Kaźmierczak-Barańskad, Barbara Nawrotd, Marzenna Klimaszewskaa, Eliza Malinowskaa, ⁎ Sandra Górskaa, Jadwiga Turłoa, a
Department of Drug Technology and Pharmaceutical Biotechnology, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland Bacteriophage Laboratory, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland c Department of Clinical Immunology, Medical University of Warsaw, Nowogrodzka 59, 02-006 Warsaw, Poland d Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland e Department of Immunology, Transplant Medicine and Internal Diseases, Medical University of Warsaw, Nowogrodzka 59, 02-006 Warsaw, Poland b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lentinula edodes Selenium Se-polysaccharides Immunosuppressants Lymphocytes T
We hypothesized that selenium(Se)-enriched polysaccharides would possess superior biological activity when compared to those non-enriched. To verify this hypothesis, we obtained by biotechnological methods a Seenriched analog of Japanese anticancer drug lentinan and, as a reference, the non-Se-enriched fraction. We tested the effects of the obtained fractions on the proliferation of human peripheral blood mononuclear cells. The results suggested a selective immunosuppressive activity, non-typical for mushroom derived polysaccharides. Both fractions caused significant inhibition of human T lymphocyte proliferation induced by mitogens, without significant effects on B lymphocytes. The inhibitory effect was not due to the toxicity of the examined polysaccharides. In normal (HUVEC) or malignant (HeLa) cells tested fractions significantly enhanced cell viability and protected the cells from oxidative stress conditions. However, we observed no effect of the polysaccharide fractions on the production of reactive oxygen species by granulocytes in vitro. The selenium content increased the biological activity of the tested polysaccharide fractions.
1. Introduction
immune cells, including macrophages, natural killer (NK) cells, and T and B lymphocytes (Zheng, Jie, Hanchuan, & Moucheng, 2005). Recent studies have demonstrated the structure – activity relationship of influence of polysaccharides on the immune system (polysaccharides with immunomodulatory properties differ greatly in their chemical structures). There are reports suggesting that various immunomodulatory effects of fungal polysaccharides are associated with their different monosaccharide composition, molecular weight, branching degrees, and triple helical conformation (Ferreira, Passos, Madureira, Vilanova, & Coimbra, 2015; Zhang, Cui, Cheung, & Wang, 2007). Lentinan, branched 1-6,1-3-β-D-glucan, is a highly purified polysaccharide
The anticancer activity of mushroom-derived polysaccharides has been associated with their immunomodulating properties (Bohn & Miller, 1995; Chihara, Maeda, Taguchi, & Hamuro, 1989; Chihara, 1992; Yu, Shen, Song, & Xie, 2018). Polysaccharides could affect both innate and adaptive immunity, including cellular and humoral responses (Guo, Savelkoul, Kwakkel, Williams, & Verstegen, 2003). Based on the mechanisms of pharmacological activity, mushroom-derived polysaccharides are classified as biological response modifiers (BRMs) (Friedman, 2016). These polysaccharides can affect various kinds of
Abbreviations: ATCC, American Type Culture Collection; BHT, butylated hydroxytoluene; BRM, biological response modifiers; BSA, bovine serum albumin; DETBA, 1,3-diethyl-2-thiobarbituric acid; ECGF, endothelial cell growth factor; FBS, fetal bovine serum; HUVECs, human umbilical vein endothelial cells; mAb, monoclonal antibody; NK, natural killer cells; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PHA, phytohaemagglutinin; PMA, phorbol 12myristate 13-acetate; SAC, Staphylococcus aureus Cowan; SOD, superoxide dismutase ⁎ Corresponding author. E-mail addresses: [email protected] (B. Kaleta), [email protected] (A. Górski), [email protected] (R. Zagożdżon), [email protected] (M. Cieślak), [email protected] (J. Kaźmierczak-Barańska), [email protected] (B. Nawrot), [email protected] (M. Klimaszewska), [email protected] (E. Malinowska), [email protected] (S. Górska), [email protected] (J. Turło). https://doi.org/10.1016/j.carbpol.2019.115078 Received 16 March 2019; Received in revised form 10 July 2019; Accepted 11 July 2019 Available online 13 July 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. The effects of the mycelial polysaccharide fractions (selenated – Se-L and not selenated L) on HUVEC and HeLa cells viability: A. HUVEC cells after 24 h of incubation, B. HeLa cells after 24 h of incubation, C. HUVEC cells after 48 h of incubation, D. HeLa cells after 48 h of incubation. The viability of tested cells nontreated with polysaccharide fractions was taken as a 100%. The error bars correspond to the standard deviation. Fig. 2. The protective effect of polysaccharides (selenated fraction Se-L, not selenated fraction L and lentinan in concentrations of 25 μg/ml) on exogenous oxidative stress induced by hydrogen peroxide in concentrations of 100 and 300 μM. KK- control; H2O2 - control cells H2O2treated; * - p < 0.05 versus control cells H2O2treated (H2O2). The error bars correspond to the standard deviation.
from the Se-enriched mycelium of L. edodes (Malinowska et al., 2018). Nowadays, the most important issue to be investigated is the examination whether the incorporation of selenium affects the biological activity of the polysaccharide fractions. We examined the impact of polysaccharides on normal and malignant cells viability, and antioxidant activity of the polysaccharide fractions, as well as their effects on human peripheral blood mononuclear cells (PBMCs) proliferation. This line of inquiry was based on the results of our previous studies, which have shown a selective cytotoxic (Klimaszewska et al., 2017) and strong antioxidant activity (Turło, Gutkowska, & Herold, 2010) of the Se-polysaccharide-containing water extracts from the L. edodes mycelium. Thus, the objective of the current research was to study the effects of the polysaccharide fractions isolated from the mycelial cultures (selenium-enriched and non-enriched) on a range of human cells,
fraction, extracted from Lentinula edodes (shiitake mushroom) fruiting bodies. This substance is approved for use in cancer treatment as an adjunct to conventional therapy (Boon & Wong, 2004; Sullivan, Smith, & Rowan, 2006; Yeung & Gubili, 2008) It appears that selenium supplementation stimulates the immune response (Brozmanová, Mániková, Vlčková, & Chovanec, 2010; Rayman, 2005, 2008, 2012), therefore we hypothesized that insertion of selenium into mushroom-derived polysaccharides would enhance their immunomodulatory activity. To verify these assumptions, we obtained by biotechnological methods a selenium-containing polysaccharide – an analog of the Japanese anticancer drug lentinan (Malinowska et al., 2018). In the previous report, we described an isolation and structural analysis of a Se-containing polysaccharide-protein complex isolated 2
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Fig. 3. Antioxidant activity of fractions Se-L and L determined by the DETBA method. * - p < 0.05 versus control. The error bars correspond to the standard deviation.
Nachman, Becker, & Minick, 1973) and cultured in plastic dishes coated with gelatin, in RPMI 1640 medium (Thermo Fischer Scientific) supplemented with 20% fetal bovine serum (FBS, Sigma-Aldrich), 90 U/ml heparin (Sigma-Aldrich), 150 μg/ml endothelial cell growth factor (ECGF, Roche Diagnostics, Mannheim, Germany) and antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin, Thermo Fischer Scientific). Cells were grown in a monolayer at 37 °C in an atmosphere of 5% CO2. The HeLa cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with antibiotics and 10% fetal calf serum (FCS, Sigma-Aldrich), in a 5% CO2. Cells (5 × 103) were seeded in each well on a 96-well plate (Nunc). After 24 h, without changing the medium, cells were exposed to the tested polysaccharides for another 24 or 48 h. Stock solutions of polysaccharides from L. edodes were freshly prepared in water. Then, four serial dilutions (from 0.5 mg/ml to 0.06 mg/ml) were prepared in saline (0.9% NaCl) and subsequently used in cell cultures. The concentrations of polysaccharide in the wells were 25, 12.5, 6.25 and 3.15 μg/ml. The cytotoxicity of all compounds was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO] assay as described (Maszewska et al., 2003). Briefly, after 24 h or 48 h of incubation with polysaccharides, the cells were treated with the MTT reagent and incubation was continued for 2 h. MTT-formazan crystals were dissolved in 20% SDS and 50% DMF at pH 4.7 and absorbance was read at 562 and 630 nm on an ELISA-PLATE READER (ELX800, Bio-Tek, USA). Cultured cells treated with vehicle (saline) served as a control and represented 100% viability.
including the immune cells. 2. Materials and methods 2.1. Biosynthesis and isolation of mycelial Se-polysaccharides 2.1.1. Mushroom strain and cultivation conditions The Lentinula edodes (Berk.) Pegler strain used in this study was ATCC 48085. The mycelial cultures were grown under the conditions described in our previous reports (Malinowska et al., 2018; Turło et al., 2010, 2011). The mycelial raw materials used for extraction of polysaccharides were cultivated under submerged conditions in a 10-L fermenter (BioTec FL 110, Stockholm, Sweden) in the culture media either in the presence or absence of selenium at a concentration of 20 μg/ml by the addition of sodium selenite (Na2SeO3, Sigma, Cell Culture Tested). 2.1.2. Extraction and isolation of Se-enriched polysaccharide fraction According to our previous report (Malinowska et al., 2018), the selenium-polysaccharide fraction (fraction Se-L) that corresponded to lentinan, was isolated from the Se-enriched mycelium by use of the modified approach (Malinowska et al., 2018; Yap & Ng, 2001). The reference polysaccharide fraction was extracted from mycelium of L. edodes cultivated in medium not enriched in Se (fraction L) (Malinowska et al., 2018). 2.2. Biological activity of Se-enriched and reference fractions In the present study, the biological activities of the Se-polysaccharide fraction, the non-selenium-enriched fraction and the commercial drug lentinan (CheMall, Mundelein, CAS 37339-90-5, Concentration 98,3%) were studied. The use of these fractions permitted us to compare the differences between the activity of the commercial drug isolated from fruiting bodies of L. edodes (lentinan), the analogous fraction isolated from the mycelial cultures.
2.2.2. Antioxidant effects of polysaccharides 2.2.2.1. Protective effect on exogenous oxidative stress. The HeLa cells (cultured as described above) were pre-incubated with polysaccharides isolated from L. edodes (at a final concentration of 25 μg/ml) for 30 min. Following this incubation, H2O2 was added to the cells (final concentration 100 μM or 300 μM) for 24 h. Cell viability was determined using the MTT assay.
2.2.1. The effects of polysaccharides on cell viability (MTT assay) Human umbilical vein endothelial cells (HUVECs) and human cervix carcinoma (HeLa) cell lines were used. HUVEC were isolated from freshly collected umbilical cords as previously described (Jaffe,
2.2.2.2. In vitro determination of antioxidant activity (DETBA method). The antioxidant activity was determined by the DETBA (1,3diethyl-2-thiobarbituric acid) method, according to our previous paper (Turło et al., 2010). Various concentrations of polysaccharides Se-Land 3
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Fig. 4. The effects of polysaccharides (Se-L, L and lentinan in concentrations of 1, 10 and 100 μg/ml) on the production of superoxide anions (O2−). The auto- (A0) and phorbol 12-myristate 13-acetate (APMA) stimulation of granulocytes was expressed as a level of nmols O2− determined by measurement of cytochrome c reduction rate.
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Fig. 5. The effects of polysaccharides (Se-L, L and lentinan in concentrations of 1, 10 and 100 μg/ml) on proliferation of peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3 monoclonal antibody (OKT3), phytohemagglutinin (PHA) and suspension of Staphylococcus aureus Cowan strain (SAC). The results are presented as a level of radioactivity as ‘Corrected Counts per Minute’ (CCPM). * p < 0.05 versus control (K). The error bars correspond to the standard deviation.
L water solutions (50 μl) were mixed with 50 μl of linoleic acid emulsion (2 mg/ml in 95% ethanol), resulting in a final concentration of polysaccharides of 6.25, 12.5 and 25 μg/ml. The mixtures were incubated at 80 °C for 60 min, and cooled in an ice bath. The following were then sequentially added: 200 μl of 20 mM butylated hydroxytoluene (BHT, Sigma), 200 μl of 8% sodium dodecyl sulphate, 400 μl of deionized water, and 3.2 ml of 12.5 mM DETBA in sodium phosphate buffer (pH 3.0). The mixture was stirred, placed in an oven at 95 °C for 15 min, and then cooled in an ice bath. After 4 ml of ethyl acetate was added, the mixture was stirred and centrifuged at 2000 rpm for 15 min. Ethyl acetate was separated and its absorbance was measured in a spectrofluorometer with fluorescence excitation at 515 nm and emission at 555 nm. The antioxidant activity was expressed as the percentage of lipid peroxidation, with a control containing no sample set as 100%. A higher percentage indicated a lower antioxidant activity.
2.2.4. The effects of polysaccharides on human peripheral blood mononuclear cells (PBMCs) proliferation PBMCs from healthy donors were isolated by density-gradient centrifugation on Histopaque-1077 (Sigma). Blood was commercially obtained from the Regional Blood Centre in Warsaw. PBMCs were cultured in Parker medium (Biomed) supplemented with 2 mM L-glutamine (Sigma), 0.1 mg/ml gentamycin (KRKA), βmercaptoethanol (Sigma), 0.23% Hepes (Sigma) and 10% heat inactivated fetal bovine serum (FBS, Gibco). Fractions of L. edodes (Se-L, reference fraction L or lentinan) were diluted in 0.9% NaCl (Fresenius Kabi) to achieve concentrations of 0.1–0.001 mg/ml. PBMCs cultures were established in 96-well flat–bottom microplates (at density 1 × 106 cells/well) and induced with specific T and B cells mitogens: anti-CD3 mAb (OKT3, 1 μg/ml, BD Pharmingen, T cell mitogen), phytohemagglutinin (PHA, 20 μg/ml, Sigma, T cell mitogen), and a suspension of Staphylococcus aureus Cowan strain (SAC, 0.004% w/v, Calbiochem, B cel mitogen). For stimulation with anti-CD3 mAb, culture plates were first coated overnight with anti-CD3 mAb followed by washing with sterile phosphate buffered saline (PBS). Polysaccharides were added to cell cultures in aliquots (100 μl) of the prepared dilution per well. Control cultures contained an equivalent amount of 0.9% NaCl. PBMCs were cultured for 72 h at 37 °C in a humidified atmosphere with 5% CO2. After 72 h cells were pulsed with 1 μCi/well of [3H]-thymidine (113 Ci/nmol, NEN) for the last 18 h of the incubation and then harvested with an automated cell harvester (Skatron). The amount of [3H]-thymidine incorporated into the cells was measured using a Wallac Microbeta scintillation counter (Wallac), giving the level of radioactivity as ‘Corrected Counts per Minute’ (CCPM). As an internal control for evaluation of results, an analogous test without any mitogens (Autostimulation) was carried out. All experiments were performed in triplicates. The study was approved by the Local Ethics Committee and all subjects provided written informed consent. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000.
2.2.3. The effects of polysaccharides on superoxide production by granulocytes Granulocytes were obtained by Histopaque-1119 (Sigma) separation from blood from healthy donors, commercially obtained from the Regional Blood Centre in Warsaw. The granulocyte pellet was resuspended in sterile phosphate buffered saline (PBS, Biomed) supplemented with 6 mM glucose (Sigma) and 1% bovine serum albumin (BSA, Biowest). Granulocytes cultures were established in 96-well round–bottom microplate (concentration 2.5 × 105 cells/well). Granulocytes activated by phorbol 12-myristate 13-acetate (PMA, Sigma) as well as nonactivated samples were incubated with cytochrome c (Sigma) and polysaccharide fractions (concentrations 1, 10, 100 μg/ml) for 30 min at 37 °C in a humidified atmosphere with 5% CO2. The auto- and PMA stimulation of granulocytes was expressed as a level of superoxide anions (nmols O2−) and calculated using the Lambert–Beer law. Control cultures contained an equivalent amount of medium. Additional controls containing superoxide dismutase (SOD, Sigma, 30 mg/ml) were also prepared in order to provide correction for the O2– independent reduction of cytochrome c. The level of O2– was determined by measurement of cytochrome c reduction rate at room temperature using microplates reader at the wavelength 550 nm (Björquist, Palmer, & Ek, 1994). Experiments were performed in triplicates.
2.3. Statistical analysis Mann – Whitney U-test and Spearman correlation were applied using the Statistica 9.0 (StatSoft Inc). Differences from control cultures 5
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3.2. Antioxidant activity of polysaccharides in cell cultures 3.2.1. Protective effect on exogenous oxidative stress Antioxidant activity of the polysaccharides Se-L and L in HeLa cell cultures was confirmed by the higher cells viability after H2O2 exposure. All tested polysaccharide fractions display antioxidant activity. As shown in Fig. 2, cells viability in the presence of the selenated polysaccharides was higher as compared to non-selenated polysaccharides and significantly higher as compared to the control H2O2treated cells. This was particularly evident for cells exposed to 300 μM H2O2, for which Se-L enhanced viability of cells two-fold compared to L fraction, and nearly six-fold compared to the control cells (treated with H2O2 only). Both mycelial fractions (Se-L and L) express much higher activity than lentinan (Fig. 2). 3.2.2. In vitro determination of antioxidant activity (DETBA method) In DETBA test the fractions Se-L and L show statistically significant antioxidant activity (Fig. 3). At a concentration of 25 μg/ml lipid peroxidation was inhibited by 32 and 12%, respectively. 3.3. The effects of polysaccharides on the production of reactive oxygen species by human granulocytes The analysis of O2− concentration [nmol] in granulocytes supernatants revealed that none of the polysaccharides has significant effect on the reactive oxygen species generation by these cells (Fig. 4A–C). 3.4. The effects of polysaccharides on the proliferation of human peripheral blood mononuclear cells The effect of polysaccharide fractions (Se-L, L and lentinan) on PHA and OKT3-stimulated PBMC proliferation is presented in Fig. 4B and C and supplementary Table S.1. In the autostimulation and, non-significantly, in SAC-stimulation settings, both S-Le and L fractions showed some tendency to decrease proliferation of PMBC. It remains to be elucidated what type of cells within the PMBC pool was inhibited in these groups. However, this effect was very moderate in comparison the T cell mitogen-stimulated settings. Indeed, OKT3-stimulated proliferation was significantly inhibited by all examined fractions at the doses of 100 μg/ml, 10 μg/ml and 1 μg/ml, in a dose-dependent manner. For the Se-L fraction the degree of inhibition at 100 and 10 μg/ml was approximately 89% and for fraction L, 86%. The difference between selenated and non-selenated fractions was significant. The relatively high values of standard deviations (Fig. 5, Table S.1) were due to the diverse response of the lymphocyte cultures from different donors to the mitogen. No statistically significant inhibitory effect of polysaccharide fractions on SACstimulated PBMC was observed. For the commercial preparation of lentinan, used as reference, no impact on the proliferation of T or B lymphocytes was observed (Fig. 5A–D). The cell viability assessment by the trypan blue exclusion method confirmed that the inhibitory effect on OKT3- and PHA-stimulated PBMC proliferation was not due to the toxicity of the examined polysaccharides.
Fig. 6. A. Correlation between the HeLa cells viability, non-treated with polysaccharide fraction (1), treated by the fractions Se-L (2), L (3) or lentinan (4), in HeLa cells after 24 h exposure (determined in MTT test) and the protective effect of the same fractions on exogenous oxidative stress induced in HeLa cells by hydrogen peroxide in concentrations of 100 and 300 μM (determined in H2O2 test). B. Correlation between the results of determination the polysaccharide antioxidant activity in Se-L and L 25 μg/mL solutions by two different methods: as protective effect on exogenous oxidative stress (H2O2 test) and as inhibition of lipid peroxidation (DETBA method).
were considered statistically significant at a p value < 0.05.
3. Results 3.1. The effects of polysaccharides on HUVEC and HeLa cells viability In general, the mycelial polysaccharide fractions (selenated and not selenated) display no cytotoxic activity (Fig. 1). The study demonstrated that all tested polysaccharides do not adversely affect the viability of HUVEC, as well as HeLa cells. Surprisingly, as shown in Fig. 1A–D, cells viability in the presence of selenated and non-selenated fractions was higher as compared to the control, non-treated cells. This effect was significantly stronger for Se-L fraction than for fraction L in higher concentrations, however, especially in HUVEC cells, increased viability was noticed for lower concentrations of L fractions. Interestingly, enhancement of cells viability was more pronounced in normal endothelial cells than in cancer cells. We hypothesized that the enhancement of the cell viability by polysaccharide fractions could be a result of their antioxidant activity. To clarify this issue we analyzed the protective effects of polysaccharides on exogenous oxidative stress.
4. Discussion In recent years, there has been increasing interest in searching drugs of natural origin to be safely and efficiently used as a primary or adjuvant therapy of many disorders. Medicinal mushrooms are considered to be a potential source of biologically active substances with significant pharmacological properties (Ganeshpurkar, Rai, & Jain, 2010). Nowadays, the most valuable medicinal mushroom is Lentinula edodes (shiitake mushroom), which is a source of multiple active compounds, including polysaccharides (Hobbs, 2000). Some studies demonstrated 6
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a result of relatively low content of polysaccharides in previously tested mycelial extracts (96 and 288 mg/g in selenated and non-selenated extract, respectively) (Turło et al., 2010). Importantly, the results of determination of antioxidant activity of Se-L and L fractions by two different methods: as inhibition of lipid peroxydation (DETBA method) and as protective effect on exogenous oxidative stress (H2O2 test in HeLa cells) are compatible (Fig. 6B). We have shown, that incorporation of selenium at the –II oxidation state into the polysaccharide structure resulted in a significant increase of cell viability and enhanced antioxidant activity. In turn, the mycelial polysaccharide fraction analogues of lentinan were much more active as antioxidants than was commercially prepared lentinan. Moreover, we evaluated the effects of polysaccharide fractions on O2− production by human granulocytes but we revealed that none of the polysaccharides affects the reactive oxygen species. It suggests that the antioxidant activities of fractions studied are not related to inhibition of early stages ROS production. Additionally, we tested the effects of the Se-enriched and reference fractions on the proliferation of human PBMCs. Lymphocyte proliferation induced by anti-CD3 mAb (OKT3) and phytohemagglutynin (PHA) may be used as a method to evaluate T proliferative response. The two mitogens use different mechanisms to promote T cell effector functions. PHA stimulates T cell proliferation by interactions with the N-acetylgalactosamine glycoprotein present on these cells (Lindahl-Kiessling & Peterson, 1969). OKT3 stimulates T cells via CD3-mediated signaling (Van Wauwe, De Mey, & Goossens, 1980). Lymphocyte proliferation induced by Staphylococcus aureus Cowan strain (SAC) may be used to evaluate B cell responsiveness. We observed that both samples isolated from the submerged cultures of L. edodes fractions – Se-containing analog of lentinan (fraction Se-L) and non-selenium enriched fraction L significantly inhibited proliferation of the T lymphocytes, most likely via CD3 receptor, but a mechanism of interactions with the N-acetylgalactosamine glycoprotein has not been excluded. In PHA-stimulated PBMC, the suppression was also seen with fractions L and Se-L, although the effect was significantly (three times) weaker (Table S.1). The suppression of the T lymphocyte proliferation by mycelial polysaccharides was distinctly different from the effect with lentinan, which showed no such activity (Fig. 5). This outcome suggested that in contrast to lentinan, fractions Se-L and L were selectively immunosuppressive. The suppression of T cell proliferation in both the OKT3 and PHA tests did not permit us to determine at this stage of the research the exact mechanism of action of the examined fractions. Most likely both processes play a role, via the CD3 receptors and by interactions with the N-acetylgalactosamine glycoprotein present on the T lymphocyte. The polysaccharide fractions isolated from the mycelial culture of L. edodes significantly differed in biological activity from the material isolated by the same method from mycelial fruiting bodies, i.e., the Japanese drug lentinan. Most likely it is a result of significant differences in the structure of the tested fractions and of lentinan. As previously reported (Malinowska et al., 2018) the selenium-containing fraction Se-L occurred as a mixture of 1,4-α- and 1,3-1,6-β-glucans, whereas lentinan is a 1,6-1,3-β-glucan. The proportion of 1,6 and 1,3-βglycosidic linkages in the mycelial fraction is also different from lentinan (Malinowska et al., 2018). The molecular weight of the mycelial polysaccharide is very high, much higher than that of lentinan (nearly 4 × 106 Da versus 5 × 105 Da). These variations suggest a different composition of the cell walls of the mycelium cultivated under submerged conditions versus the L. edodes fruiting bodies. Incorporation of selenium strongly enhanced the antioxidant activity, protection of cells against oxidative stress, and selective immunosuppressive activity, characteristics which are not typical for mushroom polysaccharides. Further structural and biological activity tests are required.
that polysaccharides isolated from L. edodes can modulate functions of immune cells, however, the polysaccharide fractions isolated from the mycelial cultures of L. edodes may significantly differ in biological activity (Kojima, Akaki, Nakajima, Kamei, & Tamesada, 2010). In the present study, we evaluated the immunomodulatory properties of polysaccharides isolated from mycelial culture of L. edodes with the Yap and Ng method (Yap & Ng, 2001). We used a Se-enriched polysaccharide fraction that corresponds to lentinan (fraction Se-L). A reference fraction (fraction L, which corresponds to lentinan) was isolated from the non-selenium enriched mycelium. Our previous study (Malinowska et al., 2018) demonstrated the selenium concentration in Se-enriched mycelium was equal to 1045 μg/g; nearly 3.4% (w/w) of this amount was combined with the crude polysaccharide fraction Se-C (1090.7 μg/g of the crude polysaccharide fraction). The selenium polysaccharide (fraction Se-L) contained proteoglycans with molecular weights of 3.9 × 106 Da and 2.6 × 105 Da, and were composed of glucose and mannose. This fraction also contained nearly 8% protein. The IR spectra confirmed the presence of β-glucans, however α-glycosidic bonds were also detected. The NMR spectral analysis confirmed that the main types of detected glycosidic bonds were β-1,3-glycosidic linkages, α-1,4-glycosidic linkages, and β-1,6-glycosidic linkages. X-ray absorption fine structure (XAFS) spectra analysis in the near edge region (XANES) showed that selenium in the Se-polysaccharides structure is present at the minus II oxidation state and is organically bound. The simulation analysis in the EXAFS region suggested that selenium is most likely bound by a glycosidic-linkage in a β-1,3 or α-1,4-glycosidic bond (Malinowska et al., 2018). Recently, we revealed that Se-polysaccharide-containing water extracts from the L. edodes mycelium had a selective cytotoxic (Klimaszewska et al., 2017) and strong antioxidant activity (Turło et al., 2010). The mycelial L. edodes extracts containing Se-polysaccharides showed weak cytotoxic activity in HeLa cells, while strongly increased HMEC1 cells viability (Klimaszewska et al., 2017). Thus, in the present study, we investigated if these effects were caused by the Se-polysaccharides content. The results of the current study showed that both selenated and non-selenated polysaccharide fractions did not adversely affect cells viability. This result suggests that selenium compounds other than the Se-polysaccharides were responsible for the previously observed cytotoxic activity of L. edodes mycelial extracts. Moreover, our current study revealed that the Se-polysaccharides were responsible for a strong increase in cell viability, significantly higher for normal cells (by 54–86%) rather than cancer cells (by 7–62%) (Fig. 1A–D). In our previous report (Klimaszewska et al., 2017), we speculated that effects of selenated fractions on cell viability were associated with the counteracting oxidative stress, as is apparent from data on other compounds (Aherne, Kerry, & O’Brien, 2007; Hadjaz et al., 2011). Our present study, which demonstrated that cell viability correlate well with the antioxidant activities of the polysaccharide fractions supported our hypothesis. A correlation analysis of viability of HeLa cells, non-treated with polysaccharide fraction, treated by the fractions Se-L, L or lentinan (determined in MTT test, Section 3.1) and the protective effect of the same fractions on exogenous oxidative stress induced in HeLa cells by hydrogen peroxide (H2O2 test, Section 3.2.1) was linear (R2 = 0.82 and 0.84, for hydrogen peroxide concentrations of 100 and 300 μM in H2O2 test, respectively) (Fig. 6A). The antioxidant activity of polysaccharide-containing water extracts from L. edodes mycelia (selenated and non-selenated) was described in our previous work (Turło et al., 2010). The chemical composition of the extracts was precisely determined. However, unclear was which of the components were responsible for antioxidant activity. Current studies have shown, the activity was mainly due to the polysaccharide content. The fractions Se-L and L in DETBA test show similar antioxidant activity to mushroom extracts (when it comes to the % protection against lipid peroxidation), however, in much lower concentrations (6.25–25 μg/mL versus 75–250 μg/mL) (Turło et al., 2010). The difference most likely is 7
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5. Conclusion
5(4), 80. Ganeshpurkar, A., Rai, G., & Jain, A. P. (2010). Medicinal mushrooms: Towards a new horizon. Pharmacognosy Reviews, 4(8), 127–135. Guo, F. C., Savelkoul, H. F. J., Kwakkel, R. P., Williams, B. A., & Verstegen, M. W. A. (2003). Immunoactive, medicinal properties of mushroom and herb polysaccharides and their potential use in chicken diets. World’s Poultry Science Journal, 59(04), 427–440. Hadjaz, F., Besret, S., Martin-Nizard, F., Yous, S., Dilly, S., Lebegue, N., et al. (2011). Antioxydant activity of β-carboline derivatives in the LDL oxidation model. European Journal of Medicinal Chemistry, 46(6), 2575–2585. Hobbs, C. H. (2000). Medicinal value of Lentinus edodes (Berk.) Sing. A literature review. International Journal of Mededicinal Mushrooms, 2, 287–302. Jaffe, E. A., Nachman, R. L., Becker, C. G., & Minick, C. R. (1973). Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. Journal of Clinical Investigation, 52(11), 2745–2756. Klimaszewska, M., Górska, S., Dawidowski, M., Podsadni, P., Szczepanska, A., Orzechowska, E., et al. (2017). Selective cytotoxic activity of Se-methyl-seleno-Lcysteine–and Se-polysaccharide–containing extracts from shiitake medicinal mushroom, Lentinus edodes (Agaricomycetes). International Journal of Medicinal Mushrooms, 19(8), 709–716. Kojima, H., Akaki, J., Nakajima, S., Kamei, K., & Tamesada, M. (2010). Structural analysis of glycogen-like polysaccharides having macrophage-activating activity in extracts of Lentinula edodes mycelia. Journal of Natural Medicines, 64(1), 16–23. Lindahl-Kiessling, K., & Peterson, R. D. A. (1969). The mechanism of phytohemagglutinin (PHA) action. Experimental Cell Research, 55(1), 85–87. Malinowska, E., Klimaszewska, M., Strączek, T., Schneider, K., Kapusta, C., Podsadni, P., et al. (2018). Selenized polysaccharides–biosynthesis and structural analysis. Carbohydrate Polymers, 198, 407–417. Maszewska, M., Leclaire, J., Cieslak, M., Nawrot, B., Okruszek, A., Caminade, A.-M., et al. (2003). Water-soluble polycationic dendrimers with a phosphoramidothioate backbone—Preliminary studies of cytotoxicity and oligonucleotide/plasmid delivery in cell culture. Oligonucleotides, 13(4), 193–205. Rayman, M. P. (2005). Selenium in cancer prevention: A review of the evidence and mechanism of action. Proceedings of the Nutrition Society, 64(4), 527–542. Rayman, M. P. (2008). Food-chain selenium and human health: Emphasis on intake. British Journal of Nutrition, 100(2), 254–268. Rayman, M. P. (2012). Selenium and human health. Lancet, 379(9822), 1256–1268. Sullivan, R., Smith, J. E., & Rowan, N. J. (2006). Medicinal mushrooms and cancer therapy: Translating a traditional practice into Western medicine. Perspectives in Biology and Medicine, 49(2), 159–170. Turło, J., Gutkowska, B., & Herold, F. (2010). Effect of selenium enrichment on antioxidant activities and chemical composition of Lentinula edodes (Berk.) Pegl. mycelial extracts. Food and Chemical Toxicology, 48(4), 1085–1091. Turło, J., Gutkowska, B., Herold, F., Gajzlerska, W., Dawidowski, M., Dorociak, A., et al. (2011). Biological availability and preliminary selenium speciation in selenium-enriched mycelium of Lentinula edodes (Berk.). Food Biotechnology, 25(1), 16–29. Van Wauwe, J. P., De Mey, J. R., & Goossens, J. G. (1980). OKT3: A monoclonal antihuman T lymphocyte antibody with potent mitogenic properties. Journal of Immunology, 124(6), 2708–2713. Yap, A.-T., & Ng, M.-L. (2001). An improved method for the isolation of lentinan from the edible and medicinal shiitake mushroom, Lentinus edodes (Berk.) Sing. (Agaricomycetideae). International Journal of Medicinal Mushrooms, 3(1), 9–19. Yeung, K., & Gubili, J. (2008). Shiitake mushroom (Lentinula edodes). Journal of the Society for Integrative Oncology, 6(3), 134–135. Yu, Y., Shen, M. Y., Song, Q. Q., & Xie, J. H. (2018). Biological activities and pharmaceutical applications of polysaccharide from natural resources: A review. Carbohydrate Polymers, 183, 91–101. 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(1), 4–19. Zheng, R., Jie, S., Hanchuan, D., & Moucheng, W. (2005). Characterization and immunomodulating activities of polysaccharide from Lentinus edodes. International Immunopharmacology, 5(5), 811–820.
The aim of the present study was to obtain a selenium-containing polysaccharide, the analog of the immunostimulatory polysaccharide drug lentinan. We expected enhanced immunostimulating activity by the selenated fraction versus lentinan. However, the results of the preliminary biological tests of the obtained fractions were unexpected and remarkable. We obtained a selective immunosuppressant with a strong antioxidant activity, which was non-toxic and even enhanced cell viability. This biological effect was completely different from lentinan and depended in-part on the selenium incorporation into the polysaccharide molecule, but also on the dissimilar structure of the fractions isolated from mycelial cultures versus lentinan. The potential use of the Secontaining mycelial polysaccharides as selective immunosuppressants has been covered by the patent (PL402082). However, there are many questions regarding both the Se-polysaccharides exact structure and their mechanism of action. We intend to investigate these issues in our ongoing further research. Funding This work was supported by grant from the National Science Centre, Poland, grant number DEC-2013/09/B/NZ7/03978. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115078. References Aherne, A., Kerry, J. P., & O’Brien, N. M. (2007). Effects of plant extracts on antioxidant status and oxidant-induced stress in Caco-2 cells. British Journal of Nutrition, 97(2), 321–328. Björquist, P., Palmer, M., & Ek, B. (1994). Measurement of superoxide anion production using maximal rate of cytochrome (III) C reduction in phorbol ester stimulated neutrophils, immobilised to microtiter plates. Biochemical Pharmacology, 48(10), 1967–1972. Bohn, J. A., & Miller, N. B. (1995). (1-3)-β-D-glucans as biological response modifiers: a review of structure-functional activity relationships. Carbohydrate Polymers, 28, 3–14. Boon, H., & Wong, J. (2004). Botanical medicine and cancer: A review of the safety and efficacy. Expert Opinion on Pharmacotherapy, 5(12), 2485–2501. Brozmanová, J., Mániková, D., Vlčková, V., & Chovanec, M. (2010). Selenium: A doubleedged sword for defense and offence in cancer. Archives of Toxicology, 84(12), 919–938. Chihara, G., Maeda, Y. Y., Taguchi, T., & Hamuro, J. (1989). Lentinan as a host defence potentiator (HDP). Inernational Journal of Immunotherapy, 5, 145–150. Chihara, G. (1992). Immunopharmacology of Lentinan, a polysaccharide isolated from Lentinus edodes: Its applications as a host defense potentiator. International Journal of Oriental Medicine, 17, 57–77. Ferreira, S. S., Passos, C. P., Madureira, P., Vilanova, M., & Coimbra, M. A. (2015). Structure–Function relationships of immunostimulatory polysaccharides: A review. Carbohydrate Polymers, 132, 378–396. Friedman, M. (2016). Mushroom polysaccharides: Chemistry and antiobesity, antidiabetes, anticancer, and antibiotic properties in cells, rodents, and humans. Foods,
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