Antiinflammatory, antioxidant and cytotoxic actions of β-glucan-richextract from Geastrum saccatum mushroom

Celina M.P. Guerra Dore a, Tarciana C.G. Azevedo a, Micheline C.R. de Souza a,Leonardo A. Rego a, Julio C.M. de Dantas a, Fernando R.F. Silva b, Hugo A.O. Rocha a,

Iuri G. Baseia c, Edda L. Leite a,⁎

a Laboratório de Glicobiologia, Departamento de Bioquímica, Universidade Federal do Rio Grande do Norte-UFRN, Natal-RN, Brazilb Laboratório de Biologia, Universidade Estadual do Ceará, UECE, Iguatú-CE, Brazil

c Laboratório de Micologia, Departamento de Botânica, Ecologia e Zoologia, Universidade Federal do Rio Grande do Norte-UFRN,Natal-RN, Brazil

Received 4 August 2006; received in revised form 25 April 2007; accepted 25 April 2007

Abstract

The Geastrum saccatum a mushroom, native to Brazil, is produced under natural conditions in the unexplored reserve of Matada Estrela-RN. This species has curative properties for eye infections and diseases such as asthma. The tissues of this mushroomcontain carbohydrates, proteins, lipids, moisture and ashes in amounts of 42.3%, 37.05%, 9.01, 1.4% and 10.2%, respectively. Anextract from this mushroom was characterized by chemical analyses and 13C and 1H NMR spectroscopy. It contains high amount ofglucose and traces of galactose. The signal appearing at 103.5 ppmwas assigned to C1 ofβ-glucose. The signals observed between 20and 40 ppm suggest the presence of a glucan–protein compound. This glucan inhibited the lipid peroxidation at the dose of 0.27 mg/mL (59.1%) and it can protect cells against oxidative stress by scavenging of the hydroxyl (77%) and superoxide (88.4%) radicals at0.27mg/mL. The glucan (30mg/kg) reduces the polymorphonuclear cell migration (57.6%). The ear edema induced by croton oil wasinhibited by glucan (60.4% at 10 mg/kg) and by its association with diclofenac (5 mg/kg) (89.2%) or L-NAME (60 mg/kg) (86.23%).Histological analyses of the ear edema induced by croton oil in the presence of glucan (10, 30 or 50 mg/kg) showed a reduced degreeof the polymorphonuclear cell migration.We concluded that the glucan has antioxidant, and antiinflammatory properties as well as itsantiinflammatory effect are mediated by inhibition of both nitric oxide synthase (NOS) and cyclooxygenase (COX).© 2007 Elsevier B.V. All rights reserved.

Keywords: Mushroom; Antioxidant activity; Antiinflammatory; Nitric oxide; Geastrum saccatum

1. Introduction

According to Chang [1], the world trade in medicinalproducts and their by-products amounted to US$ 6billion. In Brazil, the largest barriers to commercializing

mushrooms are related to the widespread belief in theirpoisonous nature, eating habits and low productivity.However, the consumption of edible fungi is growingsignificantly because of their high nutritional value andincreased availability. The main commercial mush-rooms are the Agaricus bisporus Lange (champignon),Lentinula edodes Berk (shiitake) and species of thegender Pleurotus [2]. The interest in these organisms is

International Immunopharmacology 7 (2007) 1160–1169www.elsevier.com/locate/intimp

⁎ Corresponding author.E-mail address: eddaleite@cb.ufrn.br (E.L. Leite).

1567-5769/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.intimp.2007.04.010

also attributed to their antimutagenic, antitumoral,antiviral, antithrombotic, hypocholesterolemic, hypoli-pidemic and antioxidant properties. These activities arerelated to a wide range of substances such as esters,linoleic and oleic acids, proteins, enzymes, vitamins andpolysaccharides [3].

The Basidiomycete Geastrum saccatum is a saprobicfungus and it is well adapted to tropical regions [4].Although there are more than 100 species of fungi des-cribed as this type [5], only 27 species ofGeastrum havebeen catalogued in Brazil [6]. This mushroom, popularlyknown as star of the land, is used in popular medicine byobstetricians and healers and has curative properties foreye infections and diseases, such as asthma [7,8].

It has long been recognized that many naturallyoccurring substances in plants and fungi have antioxi-dant activities. Several species of mushroom contain agreat variety of molecules, scavenger-free radicals orreactive oxygen species such as polysaccharides andphenolic compounds [9,10]. This explains why mush-rooms have recently become attractive as nutritionallybeneficial foods and as a source material for the deve-lopment of drugs. Several types of inflammatory tissueinjury are mediated by reactive oxygen metabolites.The most likely sources of these oxidizing agents arethe phagocytic leukocytes (e.g., neutrophils, mono-cytes, macrophages and eosinophils) that invade thetissue. It is becoming increasingly apparent that inaddition to promoting cytotoxicity, reactive oxygenmetabolites may also initiate and/or increase inflamma-tion [11]. In this work, chemical features of a β-glucan–protein complex from G. saccatum as well as its anti-inflammatory, antioxidant and cytotoxic activities wereanalyzed.

2. Materials and methods

2.1. Mushroom

Fresh mushroom fruiting bodies of G. saccatum, FiloBasidiomycota, class Basydiomicete, subclass Agaricomyce-tidae, order Phallales, family Geastraceae were obtained froma private Atlantic Forest reserve in the state of Rio Grande doNorte, Brazil. The mushroom was identified by Prof. IuriGoulart Baseia, Department of Botany, Ecology and Zoologyof UFRN.

2.2. Chemicals

Thiobarbituric acid, D2O, sodium salicylate, nitrobluetetrazolium, phenazine methosulfate, EDTA, H2O2, indo-methacin, croton oil, NADPH, Phycoll isopaque, carrageenan,NG-nitro-L-arginine methyl ester (L-NAME), diclofenac and

mercaptoethanol were purchased from Sigma Aldrich, EUA(St. Louis, MO, USA). The RPMI 1640 medium waspurchased from Gibco BRL (Grand Island, NY, USA).

2.3. Animals

Male BALBc mice around 25 g and male Wistar rats (150–180 g) were housed in temperature-controlled rooms (22–23 °C) until use. Food and water were supplied ad libitum.Each experimental group included 7 animals. The mice wereused in croton oil-induced ear edema tests and the rats inpleurisy. The animal experiments were conducted in accor-dance with international norms.

2.4. Extract preparation

Fruiting bodies of G. saccatum were dried at 40 °C andpowdered. The powder was delipidated with acetone andplaced in solution in a proportion of 10 g of powder to 100 mLof distilled water at 100 °C.We have usedwater because it is themost adequate solvent for extraction of proteins and carbohy-drates. After 3 h, dialysis and centrifugation (10000×g, 4 °C)were performed. To obtain mainly polysaccharides andproteins, 3 volumes of ethanol was added to the supernatant(see Fig. 1). After centrifugation (10000×g, 4 °C), theprecipitate was evaporated in vacuum to obtain a solid glucanextract.

2.5. Chemical analysis

Total protein of the extract was determined by Spector's [9]method. Total sugar content was determined by phenol sulfuricacidmethod [10]. The chemical composition was determined byAOAC [11]. The glucan extract was hydrolyzed with 2 M HCl.The resulting monosaccharides were converted to their alditolacetate derivates and analyzed by gas–liquid chromatography(GLC).

2.6. 13C and 1H nuclear magnetic resonancespectroscopy (NMR)

13C and 1H NMR spectroscopy experiments wereperformed using a 200-MHz Varian Mercury 200 MagnetoOxford spectrometer at 60 °C, with the sample dissolved inD2O.

2.7. In vitro antioxidant tests

Superoxide radicals [12] were generated in 3 mLTris–HClbuffer (16 mM, pH 8.0), which contained 78 μM NADH(reduced form), 50 μM nitroblue tetrazolium, 10 μM phena-zine methosulfate and varying concentrations of polysacchar-ide extract. The color reaction of superoxide radicals andnitroblue tetrazolium was detected by monitoring the absor-bance of 560 nm. In the control, NADH was substituted withTris–HCl buffer.

1161C.M.P. Guerra Dore et al. / International Immunopharmacology 7 (2007) 1160–1169

Hydroxyl radicals were generated by [13] in sodiumphosphate buffer (150 mM, pH 7.4), which contained0.15 mM FeSO4–EDTA, 2 mM sodium salicylate, 6 mMH2O2 and varying concentrations of polysaccharide extract. Inthe control, sodium phosphate buffer replaced H2O2. Thesolutions were incubated at 37 °C for 1 h and detected bymonitoring the absorbance at 510 nm.

Lipid peroxidation was performed by preparing livermicrosomes from male Wistar rats according to Liu et al.[14]. The microsomes (200–300 μg mL−1) were incubated at37 °C for 1 h with varying concentrations of polysaccharideextract, 10 μM FeSO4 and 0.1 mM ascorbic acid in 1.0 mLpotassium phosphate buffer solution (0.2 M, pH 7.4). Thereaction was stopped by 20% trichloroacetic acid (1.0 mL)and 0.67% 2-thiobarbituric acid (1.5 mL) in succession, andthe solution was then heated at 100 °C for 15 min. The colorreaction of malondialdehyde–thiobarbituric acid complex wasdetected by monitoring the absorbance at 532 nm. Thecontrol was performed without FeSO4 and ascorbic acid. Thepercentage of antioxidant activity of the samples wasevaluated according to the following formula: Inhibitionrate (%)=(A0−A) / (A0−Ae)×100%, where A0 is the absor-bance of the free radical generation system, A is theabsorbance of the test sample and Ae is the absorbance ofthe control [15].

2.8. Croton oil-induced ear edema test

In order to test whether glucan G. saccatum extractpossesses a plausible antiinflammatory property, the crotonoil-induced ear edema test was performed as previously

described [16–18]. Mice were anesthetized with ketaminehydrochloride and xylazine hydrochloride in a proportion of1:1 (v/v) at a concentration of 1 mL/kg of animal weight. Atotal of 20 μL 0.4 μg of croton oil in acetone was applied tothe inner surface of the right ear of each mouse. After 24 h theglucan extract was injected intravenously in concentrations of10, 20 and 50 mg/kg. The left ear remained untreated. Controlanimals received only the irritant. The oil-induced edema ofthe other groups of animals was also pretreated with L-NAMEat 60 mg/kg or diclofenac at 5 mg/kg (i.p.). After 30 minglucan G. saccatum extract (10 mg/kg) was injectedintravenously. The animals were sacrificed by cervicaldislocation 24 h after. The difference in thickness betweenthe two plugs before and after treatment was taken as ameasure of edematous response.

2.9. Action of extract in carrageenan-induced pleurisy

The rats were anesthetized with ketamine hydrochloride andxylazine hydrochloride in the proportion of 1:1 (v/v) at aconcentration of 1 mL/kg. The animals had been previouslytreated with 30 or 50 mg/kg i.p. of the glucan extract with theexception of the control group. After 30 min the animals weresubmitted to a skin incision at the level of the sixth intercostalspace. The muscles were dissected and 0.2 mL saline solutioncontaining 1% (w/v) carrageenan was injected into the pleuralcavity. The skin incision was closed with a suture and theanimals were allowed to recover. The animals were sacrificed4 h after carrageenan injection. The pleural cavity was carefullyopened and washed with 2 mL of saline solution with heparin(5 U/mL) and indomethacin (10 μg/mL). The exudate was

Fig. 1. Extraction of the glucans from G. saccatum.

1162 C.M.P. Guerra Dore et al. / International Immunopharmacology 7 (2007) 1160–1169

removed by aspiration. The number of exudate leukocytes wascounted by optical microscope after fixation with Türk'ssolution.

2.10. Measurement of nitrite-nitrate concentration in pleuralexudates

The total nitrite in exudates, an indicator of NO synthesis,was measured as previously described by Cuzzocrea et al. [19].The nitrate in the samples was first reduced to nitrite byincubation with nitrate reductase (670 mU/mL) and NADPH(160 μM) at room temperature for 3 h. The total nitriteconcentration in the samples was then measured using theGriess reaction, by adding 100 μL of Griess reagent to the 100-μL sample. Optical density was measured at 550 nm. Nitriteconcentration was calculated by comparing OD550 withstandard solutions of sodium nitrite prepared in H2O.

2.11. Colorimetric MTT (tetrazolium) assay

Extract cytotoxicity was measured as previously describedby Mosmann [20]. The leukocytes were obtained from humanperipheral blood, incremented with heparin and centrifugedwith Phycoll isopaque (Histopaque-1077). The cells werewashed successively with RPMI 1640 medium and supple-mented with 50 μM 2-mercaptoethanol and 5–10% fetalbovine serum, in a 5% CO2 atmosphere. A total of 100 μL ofthe solution was added to the cells in a ratio of 1×106 cells/well. These cells had been incubated for 4 h with differentconcentrations (0.5, 1.0 and 1.5 μg/mL) of glucanG. saccatumextract and stock MTT solution (10 μL/ 100 μL mediumRPMI) was added to all assay wells, and the plates wereincubated at 37 °C for 4 h. Acid-isopropanol (100 μL of 0.04 NHCl) was added to all the wells and mixed thoroughly todissolve the dark blue crystals. The plates were read at 570 nm.

2.12. Histological examination

The mice were sacrificed for histological examination andear biopsies were taken 48 h after croton oil-induced edema.The tissue slices were fixed in 10% neutral-bufferedformaldehyde, embedded in paraffin and sectioned. Thesections were stained with hematoxylin and eosin.

2.13. Statistical analysis

Values are presented as mean±SEM. Analysis of variance(ANOVA) and Tukey–Kramer test were used for dataevaluation with pb0.05 accepted as statistically significant.

3. Results

3.1. Chemical analysis

The results of centesimal composition analyses performedaccording to AOAC [11], the official tissue analysis methods

for the native mushroom G. saccatum, showed a percentage ofcarbohydrates, proteins, lipids, moisture and ashes of 42.3%,37.05%, 1.4%, 9.01% and 10.5%, respectively (Table 1).Furthermore, the chemical analyses of the extracts showed thepresence mainly carbohydrates (84%) and proteins (7.7%).High amounts of glucose and traces of galactose were foundby nmol Thus, the extract of G. saccatum contains high levelsof polysaccharides, composed mainly of a glucan.

3.2. 13C and 1H NMR spectroscopy of the extract fromG. saccatum

The 13C NMR spectroscopy analysis of extracts of thismushroom revealed a relative simplicity. This is due to thehomopolysaccharide type present in these organisms. Fig. 2shows that the 13C NMR spectrum of the G. saccatum glucanextract, the absence of signals at 180–120 ppm in this figureshows that this spectrum was not contaminated by phenoliccompounds. The presence of glucose was observed through asignal at 103.5 ppm, characteristic of the β-configuration.Other important signals in the present spectra are those in thearea of 60–80 ppm, where they are related to C2, C3, C4, C5and C6 of that carbohydrate. This spectrum displays signals inthe area between 28.5 and 33.0 ppm. According to Gonzaga etal. [21], the presence of signals between 20 and 40 ppmsuggests characteristic glucan–protein compound structure.

The 1H spectra (Fig. 3) showed a chemical shift in theanomeric region at 4–6 ppm. Signals of 4.1 and 4.6 wereobtained in this spectrum, corresponding to the ppm of β-glucan [21,22]. We also observed signals in the 1.4- to 2.5-ppm region that are related to the glucan–protein structure.

3.3. Antioxidant activity

The superoxide radicals were generated in a phenazine–NADH methosulfate system [23] with different extractconcentrations (0.07, 0.11, 0.20 and 0.27 mg/mL). The resultsshowed high inhibition of superoxide radicals with 88.4±0.48% at 0.27 mg/mL and 51.2±0.73% at 0.07 mg/mL ofextract. Thus, it can be observed (see Table 2) that the inhibitionrate of the superoxide radicals of the sample tested isproportional to the increased concentration in a dose-dependentway.

The formation of radical hydroxyls (HO·) in biologicalsystems does not occur enzymatically and requires thepresence of iron or transition metals [24,25]. Table 2 shows

Table 1Chemical composition of tissue and extract from G. saccatummushroom (g/100 g dry weight)

Components %

Carbohydrates 42.30±2.52Proteins 37.05±1.03Moisture 9.01±1.07Lipids 1.44±0.32Ashes 10.20±1.33

1163C.M.P. Guerra Dore et al. / International Immunopharmacology 7 (2007) 1160–1169

the rate of inhibition of hydroxyl radicals, expressed asinhibition rate. Also, the table shows that the inhibition is dosedependent and the inhibition rate obtained at 0.07 mg/mL and0.27 mg/mL was 30.0± 0.32% and 77.0±1.76%, respectively.The glucan extract showed efficient lipid peroxidationinhibition of 15.9±0.61 to 59.1±0.31. Our results confirmthe antioxidant properties of the extract of this mushroom atthe concentration tested (Table 2).

3.4. Action of extract in carrageenan-induced pleurisy

The results obtained showed a high number of polymorpho-nuclear cells (3230 cells/mm3) in the pleural infiltrate ofpositive controls (carrageenan-treated animals). When theanimals were submitted to the action of carrageenan andpreviously treated with glucan extract (30 mg/kg), 1375 cells/mm3 were obtained (Fig. 4), corresponding to a reduction of

Fig. 2. 13C NMR spectroscopy of glucan extract from G. saccatum.

Fig. 3. 1H NMR spectroscopy of glucan extract from G. saccatum.

1164 C.M.P. Guerra Dore et al. / International Immunopharmacology 7 (2007) 1160–1169

57.60% in the number of cells. On the other hand, at a 50 mg/kg concentration, a reduction of 38.93% occurred in thenumber of cells in the infiltrate (pb0.001), as measured byANOVA. The Tukey–Kramer test was highly significant inrelation to the control group (pb0.001).

3.5. Nitric oxide (NO)

Fig. 5 shows the level of nitrate/nitrite produced aftercarrageenan administration and previous treatment withglucan extract at concentrations of 30 (GS 30) and 50 mg/kg(GS 50). The positive control (animals with carrageen) showed23.92±1.79 nmol of nitrate/nitrite. The animals that receivedglucan (30mg/kg) had a value of 20.36±1.02 c of nitrate/nitrite(pb0.001). At 50 mg/kg, however, the nitrate/nitrite level was14.53± 1.8 nmol (pb0.001).

3.6. Croton oil-induced ear edema test

The animals in this experiment received a subcutaneousinjection of croton oil (0.1 mL of 1%) solution in saline withacetone. Ear volume was measured in all the groups: positive

control rats that received croton oil, saline (negative control)and glucan extracts (10, 30 and 50 mg/kg). The glucan extractreduced the edema by 60.4% at 10 mg/kg. At 30 mg/kg thereduction was 76.2%. With 50 mg/kg, however, the reductionwas 21.3%, demonstrating that treatment was inefficient athigh extract concentrations of this mushroom (Fig. 6). Anumber of experiments were performed to demonstrate itsaction mechanisms on ear edema. Thus, L-NAME (60 mg/kg)and diclofenac (5 mg/kg) inhibitors of NOS and cycloox-igenase were used, respectively. The results showed thatpretreatment with these inhibitors and administration ofglucan extract reduced the edema, for L-NAME+GS(10 mg/kg) and diclofenac+GS (10 mg/kg) by 86.23% and89.2% (pb0.001), respectively (Fig. 7).

Table 2Effect of mushroom polysaccharide extracts from G. saccatum onsuperoxide and hydroxyl radical generation and microsomal lipidperoxidation

Concentration(mg/mL)

Inhibitionsuperoxideradical (%)

Inhibitionhydroxylradical (%)

Inhibition ofmicrosomal lipidperoxidation (%)

0.07 51.2±0.73 30.0±0.32 15.9±0.720.11 72.5±0.24 50.0±1.79 29.1±1.320.20 71.0±1.44 64.0±2.92 46.6±0.620.27 88.4±0.48 77.0±1.76 59.1±0.31

Each value represents mean±SEM (n=6).

Fig. 4. Antiinflammatory effect of glucan extract from G. saccatum oncarrageenan induced pleurisy. The experimental animals were treatedwith glucan extract from G. saccatum at 30 mg/kg and 50 mg/kg. Dataobtained from animal experiments were expressed as mean±SD. Thedifferences between treatment and control were tested using byANOVA. A value of pb0.001 was considered statistically significant.

Fig. 5. Effect of glucan extract from G. saccatum in the NO productionfrom pleural exudate of Wistar rats with carrageenan induced pleurisy.The animals were treated with glucan extract from G. saccatum at30 mg/kg and 50 mg/kg. Control: Wistar rats with carrageenan inducedpleurisy. Data obtained from animal experiments (n=7) were ex-pressed as mean±SD. The differences between treatment and controlwere tested using by ANOVA. A value of ⁎⁎⁎pb0.001 and ⁎⁎pb0.5was considered statistically significant.

Fig. 6. Effect of glucan extract G. saccatum on croton-oil-induced earedema assay in mice Balb/c. The animals were treated with 10 mg/kg,30 mg/kg or 50 mg/kg of glucan extract from G. saccatum. Dataobtained from animal experiments were expressed as mean±SD. Thedifferences between treatment and control were tested using byANOVA. A value of pb0.001 was considered statistically significant.

1165C.M.P. Guerra Dore et al. / International Immunopharmacology 7 (2007) 1160–1169

3.7. Histological analysis

Histological examination of ear edema sections revealedsignificant tissue damage to animals treated with croton oil.Thus, when ear sections were compared, the animals withcroton oil (Fig. 8A) and the animals treated with extracts (Fig.8C, D, E) were similar to those that received only saline (Fig.8B), demonstrating that the extract of this fungus dramaticallyreduced leukocyte infiltration.

3.8. Cytotoxicity test

Cytotoxicity tests are based on the capacity of cells toconvert tetrazolium (MTT), which is made up of bluecoloration called formazan. However, the live cells have thiscapacity [20]. The evaluation of the transformation of MTTinto formazan in the mononuclear cells of the peripheralblood is performed using ELISA at 540 and 620 nm. Theincubation of the cells with extracts (0.5–1.5 mg/mL)resulted in moderate cytotoxic action for the tested extractswhen compared with the control (pb0.001). However, at0.25 mg/mL the viability of cells was 100%. Althoughcytotoxic, these extracts did not reach IC50 nor are theycapable, at these concentrations, of killing half of the cellsinserted into the wells (Fig. 9).

4. Discussion

In this study, we investigated the chemical composi-tion of extracts from the native fungi G. saccatum. Theirantioxidant, cytotoxicity and antiinflammatory effectswere also analyzed. Chemical analyses and 13C and 1HNMR showed that extracts from the fungi G. saccatum

had high polysaccharide content that is composed of β-glucose units (β-glucan). According to Yalin et al. [26],the presence of β-glucose is observed by a signal at103.5 ppm. These spectra also showed that parts of thesepolysaccharides are linked to proteins. The 1H NMRconfirms this possibility by the results of chemicalsignals at 0.5–2.7 ppm [21]. Carbonero et al. [27]reported that a peculiar characteristic of the â(1?3)-glucan spectra is the presence of at least six signals ofsimilar magnitude (110–60 ppm). Our results are inaccordance with those obtained by these authors.

Studies of antioxidant activity with G. saccatumshowed that the glucan extract of this mushroom has aninhibitory action on the formation of hydroxyl radicalsin a dose-dependent way. These radicals are highlydeleterious. Polysaccharide extracts from fungi ofdifferent classes have showed high scavenger activityon free radicals [28]. This activity is dependent onprotein portion linked to polysaccharide chain [14]. Inaddition, we found the glucan extract G. saccatuminhibited the generation of superoxide radicals, as wellas the lipid peroxidation. Although the antioxidantmechanism of polysaccharides extracts is still not fullyunderstood, factors related to polysaccharide such asmonosaccharide residues (mainly glucose), molecularweight and water solubility are very important.

The intrapleural administrationof carrageenan impliesthe induction of an inflammatory process with increasedneutrophils and mononuclear leukocytes number [29].The inflammation in the respiratory pathway induceshistamine, thromboxane A2, leukotrienes, cytokines and

Fig. 7. Effect of compounds on croton-oil-induced ear edema assay in mice Balb/c. The Data obtained from animal experiments were expressed asmean±SD. The animals were treated as described in methods. The differences between treatment and control were tested using by ANOVA. Avalueof pb0.001 was considered statistically significant.

1166 C.M.P. Guerra Dore et al. / International Immunopharmacology 7 (2007) 1160–1169

nitric oxide release [30]. Pleural fluid volume and theamount of cells increased in the initial 12 h followed bya decline. The antiinflammatory effect of glucan G.saccatum extract on carrageenan-induced pleurisy wasstudied. We observed that the glucan extract decreasedthe number of cells from pleural fluid of rats. There isevidence that inhibitors of NOS reduce the productionof prostaglandins by COX through reduced synthesis ofNO, these decreased several inflammatory symptomssuch as vessel dilation [31]. Thus, it could be related tothe inhibition of diapedesis of cells as mononuclear

leukocytes to the inflammation site when we used theglucan extract.

In the last few years, NO has been recognized as animportant messenger in diverse pathophysiologicalfunctions, including neuronal transmission, vascularrelaxation, immune modulation and cytotoxicity againsttumor cells [32]. The value of nitrate/nitrite produced inthe pleural cavity of rats by carrageenan administrationand previous treatment with glucan G. saccatum extractat a concentration of 50 mg/kg (GS 50), showed that theanimals receiving 50 mg/kg of glucan extract decreased

Fig. 8. Histological analysis (HEX200) of the ear from rats Balb/c with Croton oil-induced ear edema test and treated with glucan extract from G.saccatum. (A) Positive control (croton oil), the arrow shows the leukocytes cells; (B) negative control (saline); (C) animals treated with glucan extractfrom G. saccatum at 10 mg/kg; (D) animals treated with glucan extract at 30 mg/kg; (E) animals treated with glucan extract at 50 mg/kg.

1167C.M.P. Guerra Dore et al. / International Immunopharmacology 7 (2007) 1160–1169

NO levels by 41.5% (pb0.001) in compare to thecontrol group (carrageenan). These effects suggest anantiinflammatory effect of glucan G. saccatum extract.Oxide nitric synthase is classified into two isozymes;constitutive NOS (cNOS) and inducible (iNOS) [33]. L-NAME is an inhibitor of NOS and diclofenac hasinhibitory effect on cyclooxygenase. Interestingly, ourdata have showed that simultaneous administration of L-NAME or diclofenac and glucan extract produces supra-additive antiinflammatory effect in ear edema inducedby croton oil. These results have revealed synergisticinteraction between iNOS or COX inhibitors and glucanextract, which have led us to suggest that theantiinflammatory effect of glucan extract from G.saccatum is mediated by inhibition of both NOS andCOX.

Acknowledgments

The authors are grateful for the financial support pro-vided by CAPES-PROCAD and Conselho Nacional deTecnologia e Pesquisa (CNPq), Process No. 475867/03-3.

References

[1] Chang ST. Global impact of edible and medicinal mushrooms onhuman welfare in the 21st century: non green revolution. Int JMed Mushrooms 1999;1:1–8.

[2] Marino RH, Eira AF, Kuramae EE, Queiroz EC. Morphomole-cular characterization of Pleurotus ostreatus (Jacq. Fr.) Kummer

strains in relation to luminosity and temperature of frutification.Sci Agric (Piracicaba, Braz) 2003;60:531–5.

[3] Brum IS, Spritzer PM, Paris F, Maturana MA, Audran F, SultanC. Association between androgen receptor gene CAG repeatpolymorphism and plasma testosterone level in post menopausalwomen. J Soc Gynecol Invest 2005;12:135–41.

[4] Soto MK, Wighrit JE. Taxonomia del genero Geastrum(basidiomycetes, Lycoperdales) em la Provincia de BuenosAires, Argentina. Bol Soc Argent Bot 2000;134:185–201.

[5] Ponce De León P. A revisión of the family Geastraceae.Fieldiana, Bot 1968;31:303–49.

[6] Baseia IG, Galvão TC. Some interesting Gasteromycetes(Basidiomycota) in dry areas from Northeastern Brazil. ActaBot Bras 2002;16:1–8.

[7] Tellez BMC. Importancia ecológica y econômica de los hongosem la reserva de la biosfera de la Sierra de Manantlán, Jalisco.Tesis Professional, Facultad de Ciências, UDG, 1987.

[8] Tomo INI. Atlas de las plantas medicinales tradicionalesmexicanas. México: Instituto Nacional Indigenista; 1994.

[9] Spector J. Refinement of the Coomassie blue method of proteinquantification: a simple and linear spectrophotometric assay of0.5 to 50 mg of protein. Anal Biochem 1978;86:142–6.

[10] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F.Colorimetric method for determination of sugars, and relatedsubstances. Anal Biochem 1956;28:350–6.

[11] AAOC. Association Of Official Analitical Chemists. Officialmethods of analysis. 14 ed. Washington: AAOC-Association OfOfficial Analitical Chemists; 1984. p. 988–1141.

[12] Ponti V, Dianzani MU, Cheeseman K, Slater TF. Studies on thereduction of nitroblue tetrazolium chloride mediated through theaction of NADH and phenazine methosulphate. Chem BiolInteract 1978;23:281–91.

[13] Smirnoff N, Cumbes QJ. Hydroxyl radical scavenging activity ofcompatible solutes. Phytochemistry 1989;28:1057–60.

[14] Liu F, Ooi VE, Chang ST. Free radical scavenging activities ofmushroom polysaccharide extracts. Life Sci 1997;60:763–71.

[15] Zhang Q, Yu P, Li Z, Zhang H, Xu Z, Li P. Antioxidant activitiesof sulfated polysaccharide fractions from Porphyra haitanensis.J App Phycol 2003;15:305–10.

[16] Tubaro A, Decorti G, Zilli C, Bartolli KF, Della Loggia R,Mallardi F, et al. Impairment of motor coordination induced bydoxorubicin in mice. Pharmacol Res Commun 1985;17:535–45.

[17] Zitterl-Eglseer K, Sosa S, Jurenitsch J, Schubert-Zsilavecz M,Della Loggia R, Tubaro A, et al. Anti-oedematous activitiesof the main triterpendiol esters of marigold (Calendula offi-cinalis L.). J Ethnopharmacol 1997;57:139–44.

[18] Segura L, Vila R, Gupta MP, Esposito-Avella M, Adzet T,Canigueral S. Antiinflammatory activity of Anthurium cerro-campanense Croat in rats and mice. J Ethnopharmacol 2002;61:243–8.

[19] Cuzzocrea S, Costantino G, Zingarelli B, Caputi AP. Beneficialeffects of Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTbap),a superoxide dismutase mimetic, in carrageenan-induced pleu-risy. Free Radical Biol Med 1998;26:25–33.

[20] Mosmann T. Rapid colorimetric assay for cellular growth andsurvival: application to proliferation and cytotoxicity assays. JImmunol Methods 1983;65:55–63.

[21] Gonzaga MLC, Ricardo NMPS, Heatley F, Soares SA. Isolationand characterization of polysaccharides from Agaricus blazeiMurril. Carbohydr Polym 2005;60:43–9.

[22] Kawagishi H, Kanao T, Inagaki R, Mizuno T, Shimura K, Ito H,et al. Formolysis of a potent antitumor (1?6)-â-D-glucan–protein

Fig. 9. Effects of glucan extract from G. saccatum on the viability ofPMBC cells. The cells were incubated for 4 h without (control) or withglucan extract from G. saccatum (from 0.25 to 1.5 μg). Cell viabilitywas evaluated by MTT assay. Results are expressed as % of controland are the mean±standard error of 6 different experiments. Thedifferences between treatment and control were tested using byANOVA. A value of pb0.001(⁎⁎) and pb0.5 (⁎) was consideredstatistically significant.

1168 C.M.P. Guerra Dore et al. / International Immunopharmacology 7 (2007) 1160–1169

complex from Agaricus blazei fruiting bodies and antitumoractivity of the resulting products. Carbohydr Polym 1990;12:393–403.

[23] Zhou YC, Zheng RL. Phenolic compounds and analog as super-oxide anion scavengers and antioxidants. Biochem Pharm 1991;42:1177–9.

[24] Bulkley GB. The role of oxygen free radicals in human diseaseprocesses. Surgery 1983;94:407–11.

[25] Conner EM, Grisham MB. Inflammation, free radicals andantioxidants. Nutrition 1996;12:274–7.

[26] Yalin W, Yavanjiang P, Cuirong S. Isolation, purification andstructural investigation of a water-soluble polysaccharide fromSolanum lyratum Thunb. Int J Biol Macromol 2005;15:241–5.

[27] Carbonero ER, Montai AV, Woranovicz-Barreira SM, Gorin PAJ,Iacomini M. Polysaccharides of lichenized fungi of three Cladinaspp significance as chemotypes. Phytochemistry 2002;61:681–6.

[28] Wasser SP, Weis AL. Medicinal properties of substancesoccurring in higher Basidiomycetes mushrooms: current per-spective (review). Int J Med Mushrooms 1999;1:31–62.

[29] Fujisawa H, Nakagawa S, Ohkubo Y, Matsui M, Yamaguchi S,Wamura M, et al. Local and systemic expression of induciblenitric oxide synthase in comparison with that of cyclooxygenase-2 in rat carrageenin-induced pleurisy. Nitric Oxide 2005;12:80–8.

[30] Eum SY, Maghni K, Hamid Q, Eidelman DH, Campbell H,Isogai S, et al. Inhibition of allergic airways inflammation andairway hyperresponsiveness in mice by dexamethasone: role ofeosinophils, IL-5, eotaxin, and IL-13. J Allergy Clin Immunol2003;111:1049–61.

[31] Salvemini D. Regulation of cyclooxygenase enzymes by nitricoxide. Cell Mol Life Sci 1997;53:576–82.

[32] Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physio-logic messenger. Ann Intern Med 1994;120:227–37.

[33] Moncada S, Higgs A. The L-arginine-nitric oxide pathway. NEng J Med 1993;329:2002–12.

1169C.M.P. Guerra Dore et al. / International Immunopharmacology 7 (2007) 1160–1169