Antioxidant and Immunomodulating Activities of Exo- and ...

International Journal of Medicinal Mushrooms, 15(3): 251–266 (2013)

Antioxidant and Immunomodulating Activities of Exoand Endopolysaccharide Fractions from Submerged Mycelia Cultures of Culinary-Medicinal Mushrooms Sang Chul Jeong,1,2 Sundar Rao Koyyalamudi,1,* J. Margaret Hughes,3 Cheang Khoo,1 Trevor Bailey,1 Karthik Marripudi,4 Jong Pil Park,2 Jin Hee Kim,5 & Chi Hyun Song6 School of Science and Health, University of Western Sydney, Penrith New South Wales, Australia; 2Department of Pharmaceutical Engineering, Daegu Haany University, Gyeongsan, Gyeongbuk, Korea; 3Faculty of Pharmacy, University of Sydney, Sydney, New South Wales, Australia; 4Gosford Hospital, Gosford, New South Wales, Australia; 5 Department of Herbal Skin Care, Daegu Haany University, Gyeongsan, Gyeongbuk, Korea; 6Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk, Korea. 1

*Address all correspondence to: Sundar Rao Koyyalamudi, School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith South DC NSW 1797, Australia; Tel.: +61 2 9685 9987; Fax: + 61 2 9685 9915; [email protected].

ABSTRACT: A number of mushrooms are known to possess pharmacological activities. In this study, the phenolic and flavonoid contents of extracts of exo- and endopolysaccharide fractions obtained from submerged mycelia cultures of 7 edible or medicinal mushroom species, as well as their antioxidant and immunomodulatory properties, were evaluated. The exo- and endopolysaccharide yields were 0.576–1.950 and 0.438–0.933 g/L, respectively. The sugar and protein contents of these fractions were analyzed and contained predominantly sugars (52.3–87.6%). The exo- and endopolysaccharide fractions contained appreciable amounts of phenolics and flavonoids. The highest flavonoid contents were found in Cryptosporus volvatus (349.6 mg/g), followed by Cordyceps militaris (312.6 mg/g). The antioxidant activities were evaluated by 4 assays: biological assay using Saccharomyces cerevisiae, DPPH radical scavenging activity, chelating ability for ferrous ions and ferric reducing antioxidant power. The mycelia polysaccharide fractions had more ferric reducing antioxidant power than other antioxidant activities. Both exo- and endo polysaccharides of C. volvatus inhibited production of the T lymphocyte Th1 cytokines interferon (IFN)-γ and interleukin (IL)-2, the Th2 cytokines IL-4 and IL-5, and macrophage enzyme activity. Although those from C. militaris had similar inhibitory effects on cytokine production, the exopolysaccharides stimulated macrophage enzyme activity. The other exopolysaccharides (Pleurotus citrinopileatus, P. australis, and P. pulmonarius) inhibited IFN-γ and IL-5 production, but they had varying effects on IL-2 and IL-4 production. Only 3 exopolysaccharides (P. pulmonarius, Tremella mesenterica, and Cordyceps sinensis) also stimulated macrophage enzyme activity to the same extent as lipopolysaccharides. All of them reduced IL-5 production, but those from T. mesenterica also inhibited IFN-γ, IL-2, and IL-4 production. Thus the polysaccharide fractions from the mushrooms studied have antioxidant activities and general immunomodulating effects in vitro. KEYWORDS: mycelia, medicinal mushrooms, polysaccharides, antioxidant activities, immunomodulating effects ABBREVIATIONS: DMEM, Dulbecco’s modified Eagle’s medium; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ENP, endopolysaccharide; EXPs, exopolysaccharide; FBS, fetal bovine serum; F-C, Folin-Ciocalteu; FRAP, ferricreducing antioxidant power; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; OD600, optical density at 600 nm; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin-L; Th1, T helper lymphocyte 1; Th2, T helper lymphocyte 2

I. INTRODUCTION For hundreds of years, mushrooms, especially those belonging to higher Basidiomycetes, have been used as a food and therapeutically because they exhibit a wide spectrum of antioxidant, immunomodulatory, 1045-4403/13/$35.00 © 2013 Begell House, Inc. www.begellhouse.com

and therapeutic properties.1–10 There recently has been more research focused on mycelia as a substitute for mushrooms for use as a food or in pharmaceutical preparations. Their polysaccharides generally are thought to have antioxidant activities11,12 and antitumor or immunomodulatory properties through 251

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the regulation of cytokine production.8 Oxidation is essential for the energy production of biological processes but generates free radicals and highly reactive oxygen species. Excess generation of free radicals and reactive oxygen species damages essential biological compounds such as DNA, proteins, and lipids.13 This can lead to a range of diseases including age-related disorders, cancer, atherosclerosis, neurodegenerative diseases, and inflammation.14,15 Many synthetic antioxidant agents have been developed and used to remediate oxidative stress. However, factors such as their limited availability and side effects remain major setbacks in combating oxidative stress. In this direction, there is evidence from many reports that mushroom polysaccharides are good sources of natural antioxidants and warrant further investigation as potential natural antioxidants.1–3 Cytokines are proteins produced by immune cells and structural cells in tissues that regulate immunity, inflammation, and tissue remodeling from early development onward. Those produced by T helper lymphocytes (Th1 and Th2 cells) coordinate the cell-mediated immune/inflammatory responses that follow a tissue insult. Interferon (IFN)-γ is one of the major cytokines produced by Th1 cells in response to infections, whereas interleukin (IL)4 and IL-5 are secreted by Th2 cells and mediate many of the responses observed in response to Helminth infections and in inflammatory and allergic diseases.16 IL-4 is central to B-cell switching to immunoglobulin E antibody production,17 which underlies allergic inflammatory diseases.18,19 The cytokine IL-5 induces eosinophilia20 and so has been a target in asthma.21 Macrophages play an important role in the host defense system because they can phagocytose and eliminate invading organisms/foreign material, including tumor cells. It is important to note that they process and present those foreign antigens to other immune cells to activate a cell-mediated immune response. They can be activated by cytokines from lymphocytes, bacterial endotoxins, and various other cell mediators. Once activated, macrophages produce tumor ne-

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crosis factor, reactive nitrogen intermediates, and lysosomal enzymes to aid in the elimination of the foreign material.22 There seem to be few reports of the effects of medicinal mushrooms on cytokine production. The objective of this study was to evaluate and compare the antioxidant activities and immunomodulatory effects of exopolysaccharides (EXPs) and endopolysaccharides (ENPs) of 7 submerged cultures of mushroom mycelia. The EXPs and ENPs studied were from Pleurotus citrinopileatus (M1), P. australis (M2), P. pulmonarius (M3), Tremella mesenterica (M4), Cryptoporus volvatus (M5), Cordyceps militaris (M6) and C. sinensis (M7). II. MATERIALS AND METHODS A. Strains and Culture Medium The strains of P. citrinopileatus and C. sinensis were from the Institute of Botany, Kunming, China. The cultures of P. australis, P. pulmonarius, T. mesenterica, and C. volvatus were purchased from the Korean Collection for Type Cultures, Daejeon, South Korea. C. militaris was obtained from the Rural Development Administration, Suwon, South Korea. The strains were grown in a potato/dextrose broth on a rotary shaker (120 rpm) at 25°C. After incubation for 10 days, 100 mL of culture broth was homogenized aseptically and inoculated at 1% (v/v) into a mushroom complete medium with the following composition: glucose 20 g/L, magnesium sulfate 0.5 g/L, monopotassium phosphate 1 g/L, yeast extract 2 g/L, and peptone 2 g/L. The pH was adjusted to 5 before sterilization23 and cultures were grown for 7–10 days. B. Preparing Exo- and Endopolysaccharides The EXPs and ENPs from the culture broth were prepared and isolated as described previously24; the process is summarized in Fig. 1.

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Antioxidant and Immunomodulating Activities of Exo- and Endopolysaccharides from Mycelia of Mushrooms

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FIGURE 1. Schematic diagram for the process of exopolysaccharide (EXP) and endopolysaccharide (ENP) fractions from submerged mycelial cultures of mushrooms. DIW, deionized water; EtOH, ethanol.

C. Reagents and Chemicals Gallic acid, quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), dimethyl sulfoxide, sodium carbonate, aluminium chloride, sodium nitrate, sodium hydroxide, hydrogen peroxide, Folin-Ciocalteu (FC) reagent, ascorbic acid, and 95% ethanol were purchased from Sigma (Australia) and Lomb Scientific Pty Ltd. (Australia). Ficoll-paque was from Pharmacia Biotech AMRAD (Uppsala, Sweden) and fetal bovine serum (FBS) from Hyclone (Logan, UT); Roswell Park Memorial Institute-1640 medium containing 25 mM HEPES, phosphatebuffered saline, and gentamycin were from Thermo Electron (Melbourne, Australia). Phytohemagglutinin-L (PHA), HEPES, Hank’s balanced salts solution, and Triton X-100, 20 were from Sigma (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, and amphotericin B were from GIBCO BRL. Dialysis tubing (molecular weight cut-off; 6000–8000 Da) was from Spectrum Laboratories, Inc. All other chemicals were of analytical reagent grade. Enzyme-linked immunosorbant assay kits for ILVolume 15, Number 3, 2013

2, IL-4, IL5, and IFN-γ were from R&D Systems (Minneapolis, MN). Tetramethylbenzidine solution was obtained from KPL (Gaithersburg, MD). D. Determining Total Phenolic Content The total phenolic content was determined using the F-C colorimetric method.25 Briefly, 50 µL of sample and 50 µL of F-C reagent were pipetted into an Eppendorf tube. The contents were vortexed for 10 seconds and then left to stand at room temperature for 2 minutes. After 2 minutes, the reaction was stopped by adding 500 µL of 5% (w/v) sodium carbonate solution, and the volume was made up to 1 mL with distilled water. The vortexed reaction mixture was incubated at 45°C for 30 minutes and then cooled rapidly in ice. Absorbance was measured at 760 nm. Gallic acid concentrations ranging from 0 to 300 µg/mL were prepared and the calibration curve was obtained using a linear fit (r2 = 0.9961).26 The samples were analyzed in duplicate.

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E. Determining Total Flavonoid Content Total flavonoids were estimated using the aluminium chloride method.27 Briefly, 0.5 mL of sample and 300 µL of sodium nitrate (1:20 w/v) were pipetted into a test tube. The contents were vortexed for 10 seconds and then left at room temperature for 5 minutes. Then 300 µL of aluminium chloride (1:10 w/v), 2 mL of 1 M sodium hydroxide, and 1.9 mL of distilled water were added to the reaction mixture. The mixture was vortexed for 10 seconds and absorbance was measured at 510 nm. Quercetin concentrations ranging from 0 to 1200 µg/mL were prepared and the standard calibration curve was obtained using a linear fit (r2 = 0.9980). The samples were analyzed in duplicate. F. Free Radical DPPH Scavenging Assay The DPPH assay was carried out according to the procedure described by Brand-William et al.,28 with minor modifications. In this study, different volumes (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μL) of polysaccharides were mixed with a methanolic solution of DPPH radical (2.2 mg/L, 200 μL) in a 96-well microplate. The final volume of each well was made up to 300 μL by adding appropriate amounts of methanol. The mixture was shaken gently on a microplate reader, and the absorbance at 515 nm was taken every 2 minutes for 30 minutes or until the absorbance reached its maximum value. The DPPH concentration in the reaction medium was calculated on the basis of a calibration curve derived from serial dilution of the DPPH standard. The control (containing all reagents except the test compound) and standards were subject to the same procedure. The free radical scavenging activity was expressed as the percentage inhibition of free radicals by the sample and was calculated using the formula: % of DPPH radical scavenging effect = [(Acontrol – Asample)/Acontrol] × 100, where Acontrol is the absorbance of control and Asamis the absorbance of sample at 515 nm. Samples ple were analyzed in triplicate.

G. Assay for Screening Scavenging Activity in a 96-Well Microplate Using Saccharomyces cerevisiae The antioxidant capacity of the polysaccharide also was measured using a Saccharomyces cerevisiae–based high throughput assay.29 S. cerevisiae BY4743 was cultured overnight in a 50-mL volume by inoculation of a single colony. The culture then was diluted in media to an optical density at 600 nm (OD600) of 0.2, and 180 µL of each broth was added in a 96-well microtiter plate, where 10 µL per well of each polysaccharide also were added to duplicate wells. Hydrogen peroxide (10 µL) was added to give a final concentration of 4 mM. The initial OD600 reading was taken using a microplate reader (Multiskan EX; Thermo Electron), and the plates then were incubated in a 30°C incubator and shaken at 750 rpm. Yeast growth was monitored by reading OD600 until 20 hours. Ascorbate was used as a positive control. The samples were analyzed in duplicate. H. Ferrous Ion-Chelating Effect The ferrous ion-chelating effect of the polysaccharide was estimated by the method described by Chua et al.30 Briefly, 740 μL of methanol and the samples (200 μL) were incubated with 20 μL of 2 mM iron(II) chloride solution. The reaction was initiated by the addition of 40 μL of 5 mM ferrozine into the mixture, which was left standing at ambient temperature for 10 minutes. The absorbance of the reaction mixture was measured at 562 nm. Distilled water instead of ferrozine solution was used as a blank and used for error correction because of the different colors of the sample solutions. The ferrous ion-chelating ability was calculated as follows: Ferrous ion-chelating ability (%) = [Acontrol – (Asample – Ablank)] /Acontrol × 100, where Acontrol is the absorbance of control, Asample is the absorbance of sample or standard, and Ablank is the absorbance of the blank.



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I. Ferric-Reducing Antioxidant Power Assay

K. Measuring PBMC IFN-γ, IL-2, IL-4, and IL-5 Production

The ferric-reducing antioxidant power (FRAP) of the polysaccharide was tested using the assay described by Oyaizu.31 One milliliter of different concentrations of the samples, as well as chlorogenic acid as reference for comparative purposes, was added to 2.5 mL of phosphate buffer (0.1 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1% w/v). Later, the mixtures were incubated at 50oC for 20 minutes and then 2.5 mL of 10% trichloroacetic acid were added. After the mixtures were shaken vigorously, 2.5 mL of the solutions were mixed with 2.5 mL distilled water and 0.5 mL iron(III) chloride (0.1% w/v). After a 30-minute incubation, absorbances were read at a wavelength of 700 nm. Analyses were achieved in duplicate. Increased absorbance of the reaction meant increased reducing power.

Total PMBC synthesis of IFN-γ, IL-2, IL-4, and IL-5 in the cell cultures was independently assessed by enzyme-linked immunosorbant assay using commercial kits and following the protocols provided by the manufacturer. The absorbance of the solutions in the wells was measured at 450 nm on a microplate reader within 20 minutes. The concentrations of cytokines in each sample were calculated from a standard curve prepared using known concentrations of recombinant cytokines.

J. Peripheral Blood Mononuclear Cell Isolation, Culture, and Treatment To assess the effects of polysaccharides on cytokine production, human peripheral blood mononuclear cells (PBMCs) from healthy adult donors were purified from anticoagulated blood by discontinuous density gradient centrifugation with Ficoll-Plaque solution. The PBMCs were plated into 24-well plates at 1 × 106 cells/mL in Roswell Park Memorial Institute 1640 supplemented with 5% heat-inactivated FBS, 25 mM HEPES, 2 mM l-glutamine, and gentamycin 20 mg/mL. PBMC cultures were left untreated or treated in triplicate with polysaccharides at 500 µg/mL. They also were activated with PHA (1 µg/mL) or left unstimulated and cultured in a humidified 5% carbon dioxide in air at 37°C. After 44 hours of incubation the cultures were harvested and stored at -80°C before determining their cytokine content.

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L. Experimental Animals and Breeding Conditions Six-week-old C57BL/6 mice weighing approximately 25 g were purchased from Daehan Biolink Co., Ltd., and were housed in plastic cages. The mice were maintained in a room with a constant temperature (22 ± 2°C), humidity (55 ± 5%), and a 12-hour cycle of light and dark. Mice were fed with a commercial pellet diet (Sam Yang Co., Korea) throughout the experimental period. M. Preparing and Treating Mouse Macrophages The effects of the polysaccharides on macrophage lysosomal enzyme activity was assessed in macrophages harvested from 5 mice 3 days after an IP injection of 3 mL of 10% thioglycolate medium. Cell density was adjusted to 1 × 106 cells/mL with DMEM, supplemented with 10% FBS. Thereafter, each well of a 96-well microplate was inoculated with 200 μL of the cell suspension (2 × 105 cells/ well). Adherent macrophages were isolated by incubating the cells for 2 hours at 37°C with 5% carbon dioxide in a humidified incubator followed by vigorous shaking and washing the plate to remove nonadherent cells. Macrophage cultures then were covered with 200 μL DMEM with 10% FBS and incubated with or without the addition of test polysaccharide in the absence or presence of IFN-γ (20 U/mL) or lipopolysaccharide (LPS) as above

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TABLE 1. Yield of Exo- and Endopolysaccharide Fractions (Dry Weight) in Submerged Mycelial Cultures Mushrooms Exopolysaccharides (g/L) Endopolysaccharides (g/L) Pleurotus citrinopileatus (M1)

0.576

0.438

Pleurotus australis (M2)

0.592

0.754

Pleurotus pulmonarius (M3)

0.884

0.590

Tremella mesenterica (M4)

1.950

0.885

Cryptoporus volvatus (M5)

1.233

0.462

Cordyceps militaris (M6)

0.892

0.922

Cordyceps sinensis (M7)

0.611

0.676

for a further 24 hours to determine the polysaccharide’s effects on lysosomal enzyme activity. All treatments were set up in triplicate. N. Determining Macrophage Cellular Lysosomal Enzyme Activity (Acid Phosphatase) The lysosomal enzyme activity of the macrophage cultures was assayed, as described previously,32 in 96-well, flat-bottomed tissue culture plates.33 The macrophages in wells were washed with phosphate-buffered saline and solubilized with 0.1% Triton X-100 (25 mL). The substrate for acid phosphatase 10 mM p-nitrophenyl phosphate solution (150 mL) was added followed by 0.1 M citrate buffer (50 mL). After incubation for 1 hour at 37°C, 25 mL of 0.2 M borate buffer (pH 9.8) was added to the reaction mixture, and the optical density was measured at 405 nm. All treatments were set up in triplicate. O. Chemical Analysis of Polysaccharides Total protein content of the polysaccharide was determined using the method described by Lowry et al.34 with bovine serum albumin as a standard, and total sugar was determined by the phenol sulphuric acid method35 using a mixture of glucose and galactose (1:1) as a standard.

P. Data Analysis Average results for cytokine release or acid phosphatase activity were determined for the triplicate cultures/wells. Mean cytokine release data then were obtained using the average results for each treatment from all the blood donors. Any treatment-induced differences were analyzed to determine whether they were statistically significant using Duncan’s multiple-range test. Significance was defined as P < 0.05. III. RESULTS A. Isolation of Exo- and Endopolysaccharides from Submerged Mycelial Cultures The amounts of EXPs and ENPs extracted from mycelia are presented in Table 1. The EXPs and ENPs were present in the range of 0.57–1.95 and 0.43–0.92 g/L, respectively. The yield of EXPs in T. mesenterica and C. volvatus mycelia were high compared with the other submerged mycelia cultures. The sugar and protein contents were determined and the results are presented in Table 1. The amount of sugar content was present in higher proportions (52.3–87.6%) than protein content (5.9–20.3%) in all EXP and ENP fractions.



B. Total Phenolic and Flavonoid Content in the Exo- and Endopolysaccharides from Submerged Mycelial Cultures The total phenolic and flavonoid content of EXPs and ENPs obtained from submerged mycelia cultures are given in Table 2. The mean values of total phenolic contents varied from 2.3 to 10.0 QE mg/g, while the flavonoid contents ranged from 4.2 to 349.6 QE mg/g. The amounts of phenolics were more in EXPs compared to ENPs. Among all the polysaccharides, high flavonoid content was found in EXP of C. volvatus (349.6 QE mg/g), followed by C. militaris (312.6 QE mg/g). The flavonoid concentration was relatively high compared with that in phenols in the majority of EXPs and ENPs. The proportional relation (percentage) of flavonoids content to phenolic content in EXPs and ENPs was shown in Fig. 2. The total combined EXP and ENP phenolic and flavonoid contents of the 7 mycelia decreased in the following order: C. volvatus > C. militaris > P. pulmonarius > P. citrinopileatus > T. mesenterica > P. australis > C. sinensis. C. Antioxidant Activities of Exo- and Endopolysaccharides from Submerged Mycelial Cultures The antioxidant activities of polysaccharides were evaluated by 4 methods, namely, yeast-based antioxidant screening, DPPH free radical scavenging, chelating ability for ferrous ions, and FRAP assays. The results are presented in Table 2. The polysaccharides exhibited antioxidant activities in either one of the yeast model or DPPH or, in some samples, in both assays. Only 3 samples (EXP of C. sinensis and ENPs of P. australis and C. militaris) did not show any activity. The extracted EXPs and ENPs had different patterns of chelating ability, ranging from 0.0% to 64.8%. The highest ferrous ion-chelating ability of 64.8% was detected in the EXP of C. sinensis. The FRAP results of polysaccharides are depicted in Table 2; these varied among EXPs and ENPs, and the values are in the range of 1.2–8.2 CPE mg/g dry weight. In


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the FRAP assay, the presence of antioxidants in the polysaccharides would result in the reduction of Fe3+ to Fe2+. The increasing absorbance in the FRAP assay suggests an increase in reducing power.36 The strongest antioxidant properties measured by FRAP assay were P. australis followed by C. volvatus, P. citrinopileatus, P. pulmonarius, and C. sinensis, while T. mesenterica had a very low FRAP value. The overall antioxidant activities of submerged cultures of mycelia were appreciable. D. Effects of Exo- and Endopolysaccharides of Submerged Mycelial Cultures on Production of Inflammatory Cytokines by Human PBMC The effects of EXPs on Th1 and Th2 cell cytokine production by are shown in Fig. 2. We tested the in vitro effects of polysaccharide on inflammatory cytokine secretion by human PBMC following activation with the T-lymphocyte mitogen PHA.37 Detectable IL-2, IFN-γ, and IL-5 were produced by the PHA-stimulated PBMC from all 3 donors. IL-4 production was detectable in PBMC from 2 of the 3 donors (Fig. 3). Many of the EXPs affected the production of the Th1 cytokines by PHA-stimulated PBMC. P. citrinopileatus (M1), P. australis (M2), P. pulmonarius (M3), C. volvatus (M5), C. militaris (M6), and C. sinensis (M7) EXPs all inhibited the production of IFN-γ by >40% (Fig. 3A), but in the latter the difference did not achieve statistical significance. T. mesenterica (M4) EXP had no significant effects (Fig. 3A). PHA-induced IL-2 production also was affected by the EXPs. The C. sinensis (M7) EXP enhanced its production more than 2-fold (Fig. 3B). In contrast to this, P. citrinopileatus (M1), P. pulmonarius (M3), and C. volvatus (M5) EXPs significantly reduced IL-2 production (Fig. 3B), whereas the reductions in IL-2 production observed with the 4 other EXPs tested were not significant. The EXPs also modulated the production of Th2 cytokines by the PHA-stimulated PBMC. Interesting trends were observed for IL-4 production in EXP-treated PBMC from the 2 donors in which

87.6 ± 6.6 79.9 ± 1.1 75.3 ± 6.4 52.3 ± 3.7 60.5 ± 6.2 83.1 ± 9.9

M2

M3

M4

M5

M6

M7

84.5 ± 3.7 71.8 ± 3.7 70.5 ± 9.6 77.2 ± 5.7 75.9 ± 4.6

M2

M3

M4

M5

M6 20.3 ± 4.0

14.6 ± 1.6

19.2 ± 3.5

20.1 ± 1.8

5.9 ± 3.1

11.4 ± 1.3

12.3 ± 1.7

6.4 ± 0.6

7.6 ± 1.8

16.9 ± 2.8

9.5 ± 4.7

7.9 ± 1.4

11.3 ± 2.8

4.98 ± 0.04

6.83 ± 0.07

3.25 ± 0.04

2.36 ± 0.08

3.89 ± 0.03

4.36 ± 0.05

7.16 ± 0.04

4.27 ± 0.04

8.58 ± 0.05

3.99 ± 0.04

9.44 ± 0.04

10.00 ± 0.03

7.63 ± 0.00

17.76 ± 0.14

46.60 ± 2.83

7.76 ± 0.42

24.26 ± 0.28

9.46 ± 0.28

8.66 ± 0.28

15.66 ± 0.28

312.60 ± 2.83

349.60 ± 1.41

35.46 ± 0.28

29.06 ± 0.57

12.76 ± 0.14

40.26 ± 0.28

–

–

–

–

–

+

–

+

+

– e

+

+

+d

—

19.52 ± 0.48

20.55 ± 1.94

0.68 ± 0.00

—

—

—

9.59 ± 0.97

—

10.63 ± 5.91

—

17.86 ± 7.27-

—

24.13 ± 0.37

64.81 ± 0.74

—

12.02 ± 8.87-

-

17.12 ± 0.97 9.93 ± 10.17

21.60 ± 1.72

34.23 ± 0.62

37.98b ± 0.99

—

—

—

3.08 ± 0.46

4.66 ± 0.28

1.26 ± 0.21

2.73 ± 0.88

6.26 ± 0.21

4.23 ± 0.32

4.61 ± 0.49

3.63 ± 0.25

3.48 ± 0.32

1.33 ± 0.88

4.53 ± 0.25

8.23 ± 1.73

3.66 ± 0.99

86.5 ± 5.2 10.9 ± 2.3 5.73 ± 0.05 4.26 ± 0.28 – 2.05 ± 0.00 30.92 ± 0.62 3.26 ± 0.21 Data are mean ± standard deviation values of triplicate samples from one representative experiment. Yeast oxidative stress was measured on the basis of survival of yeast cells (yeast growth) after treatment with hydrogen peroxide. a The ratio of inhibition of free radical by percentage of DPPH (I(%)) was calculated as: I (%) = [(Acontrol - Asample)/Acontrol] × 100. b Ferrous ion-chelating ability was calculated as % = [Acontrol-(Asample-Ablank)] /Acontrol × 100 . c Ferric-reducing antioxidant power was expressed in milligrams of chlorogenic acid power (CPE) per grams of dry weight. d Active. e No activity.

M7

78.5 ± 1.6

M1

ENP

80.1 ±5.9

M1

EXP

TABLE 2. Antioxidant Activities of Polysaccharide Fractions Obtained from Mycelial Culture of Edible Mushrooms Ferrous IonFerric-Reducing Mushrooms Sugar Protein Total Phenol Total FlavoScavenging Chelating Antioxidant (Mycelial Content Content Content noid Content Activity on Percentage Ability Power (CPEc A Culture) (%) (%) (GAE mg/g) (QE mg/g) Yeast DPPH(I(%)) (%) mg/g)

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FIGURE 2. Proportional relation (percentage) of flavonoid content to phenolic content in submerged mycelia cultures of mushrooms. A: Exopolysaccharide. B: Endopolysaccharide.

FIGURE 3. Influence of exopolysaccharide fractions from submerged cultures of mycelia on cytokine production by activated human T lymphocytes. Data shown are mean ± standard deviation and expressed as a percentage of phytohemaglutinin (PHA) control. A: interferon (IFN)-γ; B: interleukin (IL)-2; C: IL-4; D: IL-5. M1, Pleurotus citrinopileatus; M2, P. australis; M3, P. pulmonarius; M4, Tremella mesenterica; M5, Cryptoporus volvatus; M6, Cordyceps militaris; M7, C. sinensis; UT, untreated. *Significant difference from control (PHA-treated group) (n = 3), P < 0.05.


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FIGURE 4. Influence of endopolysaccharides fractions from submerged cultures of mycelia on cytokine production by activated human T lymphocytes. A: interferon (IFN)-γ; B: interleukin (IL)-2; C: IL-4; D: IL-5. Data shown are mean ± standard deviation and expressed as a percentage of phytohemaglutinin (PHA) control. M1, Pleurotus citrinopileatus; M2, P. australis; M3, P. pulmonarius; M4, Tremella mesenterica; M5, Cryptoporus volvatus; M6, Cordyceps militaris; M7, C. sinensis; UT, untreated. *Significant difference from control (PHA-treated group) (n = 3), P < 0.05.

IL-4 was detectable. IL-4 production in P. pulmonarius (M3), and C. sinensis (M7)–treated samples increased compared with the PHA control. However, production was decreased by treatment with P. australis (M2), C. volvatus (M5), and C. militaris (M6). Production of the proeosinophilic Th2 cytokine IL-5 by PHA-stimulated PBMC also was affected by treatment with the EXPs. All EXPs significantly reduced production except for T. mesenterica (M4) and C. sinensis (M7). The effects of ENPs extracted from submerged cultures of mycelia on cytokine production by activated T lymphocytes are presented in Fig. 4. The production of the Th1 cytokines IFN-γ and IL-2 was decreased in the presence of T. mes-

enterica (M4) and C. volvatus (M5) ENPs. All the other ENPs tested had no significant effect on the production of these cytokines. Treatment with ENPs from T. mesenterica (M4), C. volvatus (M5), and C militaris (M6) caused marked reductions in production of the Th2 cytokine IL-4 by the PBMC from the 2 donors with detectable production, whereas increased levels of IL-4 were found in C. sinensis (M7)–treated samples. IL-5 production was significantly inhibited by all the ENPs except P. australis (M2) and C. sinensis (M7).



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FIGURE 5. Effects of exopolysaccharide fractions from submerged cultures of mycelia on the cellular lysosomal enzyme activity of mouse peritoneal macrophages. A: Exopolysaccharide. B: Endopolysaccharide. Data shown are mean ± standard deviation and expressed as a percentage of saline. Macrophage concentration was 1 × 106 cells/ mL. LPS, lipopolysaccharide (positive control; from Escherichia coli 0127: B8); M1, Pleurotus citrinopileatus; M2, P. australis; M3, P. pulmonarius; M4, Tremella mesenterica; M5, Cryptoporus volvatus; M6, Cordyceps militaris; M7, C. sinensis

E. Effects of Exo- and Endopolysaccharides of Submerged Mycelial Cultures on Murine Macrophage Lysosomal Enzyme Activity We have examined the effects of the EXPs isolated from the mycelia on the lysosomal enzyme activity of murine peritoneal macrophages and found some interesting trends. The lysosomal enzyme activities of macrophages treated with the EXPs (M1 to M7) at various concentrations (10, 100, and 1000 mg/mL) are shown in Fig. 5A. The acid phosphatase enzyme activity of macrophages treated with


the EXPs of P. pulmonarius (M3), T. mesenterica (M4), C. volvatus (M5), and C. sinensis (M7) were comparable to the positive control (LPS from Escherichia coli) at the same concentration of 10 µg/mL. In samples treated with T. mesenterica (M4) and C. militaris (M6) EXPs, the enzyme activity was greater than for the positive control at the highest concentration (1000 μg/mL) used. Enzyme activities in samples treated with the highest concentration of C. volvatus (M5) were remarkably decreased. The lysosomal enzyme activities of macrophages treated with the ENPs extracted from the

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FIGURE 6. Correlation between flavonoid content exopolysaccharide (EXP) and endopolysaccharide (ENP) fractions (A) and ferrous ion-chelating capacity of EXP and ENP fractions (B).

mycelial cultures are shown in Fig. 5B. The enzyme activities in samples treated with P. australis (M2) and P. pulmonarius (M3) ENPs were increased and similar to those in the positive control (LPS). The remaining ENPs either had no effect or caused only very slight increases in enzyme activity. To understand the antioxidant activities of the selected mushrooms in terms of their flavonoid content and ferrous ion-chelating capacity, correlation plots were developed (Fig. 6). The flavonoid content of EXPs showed significant correlation with flavonoid content of ENPs (0.5584; P < 0.05) (Fig. 6A), and ferrous ion-chelating capacity between EXPs and ENPs showed significant correlation (0.9473; P < 0.05) (Fig. 6B). IV. DISCUSSION The macromolecules of polysaccharides from mushrooms are a new source of natural antioxidant compounds with less toxicity.38 The preliminary screen of the EXPs and ENPs produced by submerged mycelial cultures of medicinal mushrooms M1 through M7 has provided evidence that these mushrooms do have antioxidant and immu-

nomodulatory activities in vitro. The scavenging DPPH radical activity of the polysaccharide samples was low compared to vitamin C. In general, the antioxidant activity depends on the structure and substitution of hydroxyl groups.39 All fractions (EXPs and ENPs) were found to have more potent chelating ability on Fe2+ (10.63–64.8%); however, M2 (ENP), M4 (EXP and END), and M6 (EXP and ENP) fractions showed no activity. The molecular masses of the EXP and ENP polysaccharide fractions are important for the chelating ability. Reportedly, the compounds containing the following functional groups: -OH, -COOH,C=O, -SH, -NR2 are in favor of metal chelating activity.40 As mentioned earlier, all samples (EXPs and ENPs) showed positive FRAP and exhibited higher capacities to reduce ferric ion (Fe3+) to ferrous ion (Fe2+) than to scavenged free radicals. The FRAP assay is commonly used to estimate the total concentration of redox-active compounds and measure the reducing capacity based on the ferric ion.41 Another study reported similar activities in the polysaccharide produced from mushroom as well.42 The antioxidant activity of mushroom-derived polysaccharides (EXPs and ENPs) contained higher proportions of sugar (range, 52.3–87.6%)



than protein (5.9–20.3%). It is difficult to compare the antioxidant and immunomodulatory activities of these fractions based on sugar and protein contents. Some phenolic compounds such as tyrosine and ferulic acid are linked covalently to polysaccharides and can contribute to antioxidant and immunomodulatory activities.10,43,44 Mushroom-produced polysaccharides activate the immune system via stimulation of macrophages, T cells, B cells, and natural killer cells.10 The polysaccharides of C. volvatus (M5) inhibited production of each of the cytokines and enzyme activity. Although those from C. militaris (M6) had similar inhibitory effects on cytokine production, the EXP stimulated macrophage enzyme activity. The other EXPs (P. citrinopileatus, P. australis, and P. pulmonarius) inhibited IFN-γ and IL-5 production, but they had varying effects on IL-2 and IL-4 production, and only 3 EXPs (P. pulmonarius, T. mesenterica, and C. sinensis) also stimulated macrophage enzyme activity to the same extent as LPS. However, the group treated with ENPs generally were less active compared with EXPs. Reportedly, ENP produced from mushroom mycelial culture of Inonotus obliquus did not affect the IL-2 expression of Th1 cells and the IL-4 expression of Th2 cells.45 All of them reduced IL-5 production, but those from T. mesenterica (M4) also inhibited IFN-γ, IL-2, and IL-4 production. Macrophages are key cells in the innate immune response and protect mucosal surfaces and organs. The production of lysosomal acid phosphatases (lysosomal enzymes) by macrophages occurs in response to numerous exogenous stimuli.46 It is crucial to their function and so it is often used in the assessment of their function.5 Stimulation of macrophage lysosomal enzyme activity could lead to more efficient clearance of dying cells, invading organisms, or foreign material such as tumor antigens/cells. Thus it was of interest to find that macrophage lysosomal enzyme activities were enhanced by P. pulmonarius, T. mesenterica, C. Militaris, and C. sinensis EXPs and P. australis and P. pulmonarius ENPs to the same extent as the bacterial cell wall component LPS and in a concentration-related manner. Their activities were compa-


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rable with those reported for polysaccharides from some other mushroom fruiting bodies.47,48 Their macrophage-stimulating activity was attributed to their polysaccharides.49,50 Further testing of these polysaccharides is warranted to establish their specific effects on macrophage functions. The effects of these mushrooms on the production of Th1 and Th2 cytokines by PBMC were of interest to study because of the putative roles of these cytokines in health and disease. The Th1 cytokine IFN-γ negatively regulates IL-4 and IL-5 production.51–53 Thus it can prevent the IL-4-dependent switch of B-lymphocytes to immunoglobulin E production that underlies allergic reactions, as well as IL-5-induced maturation, recruitment, and activation of eosinophils.54 Eosinophilic inflammation is a characteristic feature of asthma and is thought to result from an imbalance in the level of these Th1 and Th2 cytokines. Other medicinal mushrooms previously have been reported to contain significant immunomodulating activities. IL-1, IL-2, IL-6, tumor necrosis factor-α, and INF-γ synthesis by human PBMCs were stimulated by partially purified shiitake mushroom (Lentinus edodes) lectin.55 A protein was isolated from Volvariela volvacea56 and Flammulina velutipes57 that stimulated gene expression of IL-2, IL-4, tumor necrosis factor-α, and INF-γ. Mizuno et al.58 isolated macrophage-stimulating polysaccharides from Agaricus brasiliensis fruiting bodies. Coriolus versicolor,32,59 Ganoderma lucidum,60 Phellinus linteus,61 Paecilomyces japonica,50 Tricholoma mongolicum,62 and several other mushrooms9 all have been reported to be immunomodulatory. In this initial in vitro screen, none of the polysaccharides promoted Th1 cytokine production or specifically inhibited IL-4 and IL-5 production, and so they are unlikely to have an impact on the balance during production of these cytokines. Rather, the data presented here are consistent with the polysaccharides from the mushrooms with a general suppressive effect on cytokine production. Thus they are more likely to have any beneficial effects through reducing the magnitude of any immune/inflammatory response. Further testing is required in vitro and in vivo to establish this.

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V. CONCLUSION The EXPs and ENPs of mycelia of M1 to M7 were shown to possess significant antioxidant and immunomodulatory activities. The mycelia are a good source of polysaccharides for the development of natural antioxidants. Further research is warranted to establish their effects on innate and cell-mediated immune responses in detail and to elucidate the structure of the active constituents in the mycelia.

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