aLaboratório de Glicobiologia, Departamento de Bioquímica, Universidade Federal do Rio Grande do Norte-UFRN, Natal-RN, Brazil bLaboratório de Biologia, Universidade Estadual do Ceará, UECE, Iguatú-CE, Brazil
cLaboratório 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 Mata da Estrela-RN. This species has curative properties for eye infections and diseases such as asthma. The tissues of this mushroom contain carbohydrates, proteins, lipids, moisture and ashes in amounts of 42.3%, 37.05%, 9.01, 1.4% and 10.2%, respectively. An extract from this mushroom was characterized by chemical analyses and13C and1H NMR spectroscopy. It contains high amount of glucose and traces of galactose. The signal appearing at 103.5 ppm was assigned to C1 of β-glucose. The signals observed between 20 and 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 at 0.27 mg/mL. The glucan (30 mg/kg) reduces the polymorphonuclear cell migration (57.6%). The ear edema induced by croton oil was inhibited by glucan (60.4% at 10 mg/kg) and by its association with diclofenac (5 mg/kg) (89.2%) orL-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 degree of the polymorphonuclear cell migration. We concluded that the glucan has antioxidant, and antiinflammatory properties as well as its antiinflammatory 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 medicinal products and their by-products amounted to US$ 6 billion. In Brazil, the largest barriers to commercializing
mushrooms are related to the widespread belief in their poisonous nature, eating habits and low productivity. However, the consumption of edible fungi is growing significantly because of their high nutritional value and increased availability. The main commercial mush- rooms are the Agaricus bisporus Lange (champignon), Lentinula edodes Berk (shiitake) and species of the gender Pleurotus[2]. The interest in these organisms is ⁎Corresponding author.
antiviral, antithrombotic, hypocholesterolemic, hypoli- pidemic and antioxidant properties. These activities are related to a wide range of substances such as esters, linoleic and oleic acids, proteins, enzymes, vitamins and polysaccharides[3].
The Basidiomycete Geastrum saccatum is a saprobic fungus 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 of Geastrum have been catalogued in Brazil[6]. This mushroom, popularly known as star of the land, is used in popular medicine by obstetricians and healers and has curative properties for eye infections and diseases, such as asthma[7,8].
It has long been recognized that many naturally occurring substances in plants and fungi have antioxi- dant activities. Several species of mushroom contain a great variety of molecules, scavenger-free radicals or reactive oxygen species such as polysaccharides and phenolic compounds [9,10]. This explains why mush- rooms have recently become attractive as nutritionally beneficial foods and as a source material for the deve- lopment of drugs. Several types of inflammatory tissue injury are mediated by reactive oxygen metabolites. The most likely sources of these oxidizing agents are the phagocytic leukocytes (e.g., neutrophils, mono- cytes, macrophages and eosinophils) that invade the tissue. It is becoming increasingly apparent that in addition to promoting cytotoxicity, reactive oxygen metabolites 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 were analyzed.
2. Materials and methods 2.1. Mushroom
Fresh mushroom fruiting bodies of G. saccatum, Filo Basidiomycota, class Basydiomicete, subclass Agaricomyce- tidae, order Phallales, family Geastraceae were obtained from a private Atlantic Forest reserve in the state of Rio Grande do Norte, Brazil. The mushroom was identified by Prof. Iuri Goulart Baseia, Department of Botany, Ecology and Zoology of UFRN.
2.2. Chemicals
(St. Louis, MO, USA). The RPMI 1640 medium was purchased 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 were used in croton oil-induced ear edema tests and the rats in pleurisy. 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 and powdered. The powder was delipidated with acetone and placed in solution in a proportion of 10 g of powder to 100 mL of distilled water at 100 °C. We have used water because it is the most 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 and proteins, 3 volumes of ethanol was added to the supernatant (see Fig. 1). After centrifugation (10000×g, 4 °C), the precipitate was evaporated in vacuum to obtain a solid glucan extract.
2.5. Chemical analysis
Total protein of the extract was determined by Spector's[9]
method. Total sugar content was determined by phenol sulfuric acid method[10]. The chemical composition was determined by AOAC[11]. The glucan extract was hydrolyzed with 2 M HCl. The resulting monosaccharides were converted to their alditol acetate derivates and analyzed by gas–liquid chromatography (GLC).
2.6.13C and1H nuclear magnetic resonance spectroscopy (NMR)
13
C and 1H NMR spectroscopy experiments were performed using a 200-MHz Varian Mercury 200 Magneto Oxford spectrometer at 60 °C, with the sample dissolved in D2O.
2.7. In vitro antioxidant tests
Superoxide radicals[12]were generated in 3 mL Tris–HCl buffer (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-
Hydroxyl radicals were generated by [13] in sodium phosphate buffer (150 mM, pH 7.4), which contained 0.15 mM FeSO4–EDTA, 2 mM sodium salicylate, 6 mM H2O2and varying concentrations of polysaccharide extract. In
the control, sodium phosphate buffer replaced H2O2. The
solutions were incubated at 37 °C for 1 h and detected by monitoring the absorbance at 510 nm.
Lipid peroxidation was performed by preparing liver microsomes from male Wistar rats according to Liu et al.
[14]. The microsomes (200–300 μg mL− 1) were incubated at
37 °C for 1 h with varying concentrations of polysaccharide extract, 10 μM FeSO4and 0.1 mM ascorbic acid in 1.0 mL
potassium phosphate buffer solution (0.2 M, pH 7.4). The reaction was stopped by 20% trichloroacetic acid (1.0 mL) and 0.67% 2-thiobarbituric acid (1.5 mL) in succession, and the solution was then heated at 100 °C for 15 min. The color reaction of malondialdehyde–thiobarbituric acid complex was detected by monitoring the absorbance at 532 nm. The control was performed without FeSO4 and ascorbic acid. The percentage of antioxidant activity of the samples was evaluated according to the following formula: Inhibition rate (%) = (A0− A) / (A0− Ae) × 100%, where A0 is the absor-
bance of the free radical generation system, A is the absorbance of the test sample and Ae is the absorbance of
the control[15].
2.8. Croton oil-induced ear edema test
described [16–18]. Mice were anesthetized with ketamine hydrochloride and xylazine hydrochloride in a proportion of 1:1 (v/v) at a concentration of 1 mL/kg of animal weight. A total of 20 μL 0.4 μg of croton oil in acetone was applied to the inner surface of the right ear of each mouse. After 24 h the glucan extract was injected intravenously in concentrations of 10, 20 and 50 mg/kg. The left ear remained untreated. Control animals received only the irritant. The oil-induced edema of the other groups of animals was also pretreated withL-NAME at 60 mg/kg or diclofenac at 5 mg/kg (i.p.). After 30 min glucan G. saccatum extract (10 mg/kg) was injected intravenously. The animals were sacrificed by cervical dislocation 24 h after. The difference in thickness between the two plugs before and after treatment was taken as a measure of edematous response.
2.9. Action of extract in carrageenan-induced pleurisy The rats were anesthetized with ketamine hydrochloride and xylazine hydrochloride in the proportion of 1:1 (v/v) at a concentration of 1 mL/kg. The animals had been previously treated with 30 or 50 mg/kg i.p. of the glucan extract with the exception of the control group. After 30 min the animals were submitted to a skin incision at the level of the sixth intercostal space. The muscles were dissected and 0.2 mL saline solution containing 1% (w/v) carrageenan was injected into the pleural cavity. The skin incision was closed with a suture and the animals were allowed to recover. The animals were sacrificed Fig. 1. Extraction of the glucans from G. saccatum.
counted by optical microscope after fixation with Türk's solution.
2.10. Measurement of nitrite-nitrate concentration in pleural exudates
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 by incubation with nitrate reductase (670 mU/mL) and NADPH (160 μM) at room temperature for 3 h. The total nitrite concentration in the samples was then measured using the Griess reaction, by adding 100 μL of Griess reagent to the 100- μL sample. Optical density was measured at 550 nm. Nitrite concentration was calculated by comparing OD550 with
standard solutions of sodium nitrite prepared in H2O.
2.11. Colorimetric MTT (tetrazolium) assay
Extract cytotoxicity was measured as previously described by Mosmann[20]. The leukocytes were obtained from human peripheral blood, incremented with heparin and centrifuged with Phycoll isopaque (Histopaque-1077). The cells were washed successively with RPMI 1640 medium and supple- mented with 50 μM 2-mercaptoethanol and 5–10% fetal bovine serum, in a 5% CO2atmosphere. A total of 100 μL of
the solution was added to the cells in a ratio of 1 × 106cells/ well. These cells had been incubated for 4 h with different concentrations (0.5, 1.0 and 1.5 μg/mL) of glucan G. saccatum extract and stock MTT solution (10 μL/ 100 μL medium RPMI) was added to all assay wells, and the plates were incubated at 37 °C for 4 h. Acid-isopropanol (100 μL of 0.04 N HCl) was added to all the wells and mixed thoroughly to dissolve the dark blue crystals. The plates were read at 570 nm. 2.12. Histological examination
The mice were sacrificed for histological examination and ear biopsies were taken 48 h after croton oil-induced edema. The tissue slices were fixed in 10% neutral-buffered formaldehyde, embedded in paraffin and sectioned. The sections 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 data evaluation with p b 0.05 accepted as statistically significant. 3. Results
carbohydrates, 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 the presence mainly carbohydrates (84%) and proteins (7.7%). High amounts of glucose and traces of galactose were found by nmol Thus, the extract of G. saccatum contains high levels of polysaccharides, composed mainly of a glucan.
3.2.13C and1H NMR spectroscopy of the extract from G. saccatum
The 13C NMR spectroscopy analysis of extracts of this mushroom revealed a relative simplicity. This is due to the homopolysaccharide type present in these organisms. Fig. 2
shows that the13C NMR spectrum of the G. saccatum glucan
extract, the absence of signals at 180–120 ppm in this figure shows that this spectrum was not contaminated by phenolic compounds. The presence of glucose was observed through a signal at 103.5 ppm, characteristic of the β-configuration. Other important signals in the present spectra are those in the area of 60–80 ppm, where they are related to C2, C3, C4, C5 and C6 of that carbohydrate. This spectrum displays signals in the area between 28.5 and 33.0 ppm. According to Gonzaga et al. [21], the presence of signals between 20 and 40 ppm suggests characteristic glucan–protein compound structure.
The1H spectra (Fig. 3) showed a chemical shift in the anomeric region at 4–6 ppm. Signals of 4.1 and 4.6 were obtained 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 extract concentrations (0.07, 0.11, 0.20 and 0.27 mg/mL). The results showed 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 of extract. Thus, it can be observed (seeTable 2) that the inhibition rate of the superoxide radicals of the sample tested is proportional to the increased concentration in a dose-dependent way.
The formation of radical hydroxyls (HO·) in biological systems does not occur enzymatically and requires the presence of iron or transition metals[24,25].Table 2shows Table 1
Chemical composition of tissue and extract from G. saccatum mushroom (g/100 g dry weight)
Components %
the rate of inhibition of hydroxyl radicals, expressed as inhibition rate. Also, the table shows that the inhibition is dose dependent and the inhibition rate obtained at 0.07 mg/mL and 0.27 mg/mL was 30.0 ± 0.32% and 77.0 ± 1.76%, respectively. The glucan extract showed efficient lipid peroxidation inhibition of 15.9 ± 0.61 to 59.1 ± 0.31. Our results confirm the antioxidant properties of the extract of this mushroom at the 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 of positive controls (carrageenan-treated animals). When the animals were submitted to the action of carrageenan and previously treated with glucan extract (30 mg/kg), 1375 cells/ mm3were obtained (Fig. 4), corresponding to a reduction of Fig. 2.13C NMR spectroscopy of glucan extract from G. saccatum.
57.60% in the number of cells. On the other hand, at a 50 mg/ kg concentration, a reduction of 38.93% occurred in the number of cells in the infiltrate (p b 0.001), as measured by ANOVA. The Tukey–Kramer test was highly significant in relation to the control group (p b 0.001).
3.5. Nitric oxide (NO)
Fig. 5 shows the level of nitrate/nitrite produced after carrageenan administration and previous treatment with glucan extract at concentrations of 30 (GS 30) and 50 mg/kg (GS 50). The positive control (animals with carrageen) showed 23.92 ± 1.79 nmol of nitrate/nitrite. The animals that received glucan (30 mg/kg) had a value of 20.36 ± 1.02 c of nitrate/nitrite (p b 0.001). At 50 mg/kg, however, the nitrate/nitrite level was 14.53 ± 1.8 nmol (p b 0.001).
3.6. Croton oil-induced ear edema test
The animals in this experiment received a subcutaneous injection of croton oil (0.1 mL of 1%) solution in saline with acetone. 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 extract reduced the edema by 60.4% at 10 mg/kg. At 30 mg/kg the reduction was 76.2%. With 50 mg/kg, however, the reduction was 21.3%, demonstrating that treatment was inefficient at high extract concentrations of this mushroom (Fig. 6). A number of experiments were performed to demonstrate its action 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 that pretreatment with these inhibitors and administration of glucan extract reduced the edema, for L-NAME+GS
(10 mg/kg) and diclofenac + GS (10 mg/kg) by 86.23% and 89.2% (p b 0.001), respectively (Fig. 7).
Effect of mushroom polysaccharide extracts from G. saccatum on superoxide and hydroxyl radical generation and microsomal lipid peroxidation Concentration (mg/mL) Inhibition superoxide radical (%) Inhibition hydroxyl radical (%) Inhibition of microsomal lipid peroxidation (%) 0.07 51.2 ± 0.73 30.0 ± 0.32 15.9 ± 0.72 0.11 72.5 ± 0.24 50.0 ± 1.79 29.1 ± 1.32 0.20 71.0 ± 1.44 64.0 ± 2.92 46.6 ± 0.62 0.27 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 on carrageenan induced pleurisy. The experimental animals were treated
Fig. 5. Effect of glucan extract from G. saccatum in the NO production from pleural exudate of Wistar rats with carrageenan induced pleurisy. The animals were treated with glucan extract from G. saccatum at 30 mg/kg and 50 mg/kg. Control: Wistar rats with carrageenan induced pleurisy. Data obtained from animal experiments (n = 7) were ex- pressed as mean ± SD. The differences between treatment and control were tested using by ANOVA. A value of ⁎⁎⁎p b 0.001 and ⁎⁎p b 0.5 was considered statistically significant.
Fig. 6. Effect of glucan extract G. saccatum on croton-oil-induced ear edema assay in mice Balb/c. The animals were treated with 10 mg/kg,
3.7. Histological analysis
Histological examination of ear edema sections revealed significant tissue damage to animals treated with croton oil. Thus, when ear sections were compared, the animals with croton 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 dramatically reduced leukocyte infiltration.
3.8. Cytotoxicity test
Cytotoxicity tests are based on the capacity of cells to convert tetrazolium (MTT), which is made up of blue coloration called formazan. However, the live cells have this capacity[20]. The evaluation of the transformation of MTT into formazan in the mononuclear cells of the peripheral blood is performed using ELISA at 540 and 620 nm. The incubation of the cells with extracts (0.5–1.5 mg/mL) resulted in moderate cytotoxic action for the tested extracts when compared with the control (p b 0.001). However, at 0.25 mg/mL the viability of cells was 100%. Although cytotoxic, these extracts did not reach IC50 nor are they
capable, at these concentrations, of killing half of the cells inserted into the wells (Fig. 9).
4. Discussion
In this study, we investigated the chemical composi- tion of extracts from the native fungi G. saccatum. Their antioxidant, cytotoxicity and antiinflammatory effects
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 at 103.5 ppm. These spectra also showed that parts of these polysaccharides are linked to proteins. The 1H NMR confirms this possibility by the results of chemical signals 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 of similar magnitude (110–60 ppm). Our results are in accordance with those obtained by these authors.
Studies of antioxidant activity with G. saccatum showed that the glucan extract of this mushroom has an inhibitory action on the formation of hydroxyl radicals in a dose-dependent way. These radicals are highly deleterious. Polysaccharide extracts from fungi of different classes have showed high scavenger activity on free radicals [28]. This activity is dependent on protein portion linked to polysaccharide chain[14]. In addition, we found the glucan extract G. saccatum inhibited the generation of superoxide radicals, as well as the lipid peroxidation. Although the antioxidant mechanism of polysaccharides extracts is still not fully understood, factors related to polysaccharide such as monosaccharide residues (mainly glucose), molecular weight and water solubility are very important.
The intrapleural administration of carrageenan implies the induction of an inflammatory process with increased neutrophils and mononuclear leukocytes number [29]. Fig. 7. Effect of compounds on croton-oil-induced ear edema assay in mice Balb/c. The Data obtained from animal experiments were expressed as mean ± SD. The animals were treated as described in methods. The differences between treatment and control were tested using by ANOVA. A value of p b 0.001 was considered statistically significant.
nitric oxide release [30]. Pleural fluid volume and the amount of cells increased in the initial 12 h followed by a decline. The antiinflammatory effect of glucan G. saccatum extract on carrageenan-induced pleurisy was studied. We observed that the glucan extract decreased the number of cells from pleural fluid of rats. There is evidence that inhibitors of NOS reduce the production of prostaglandins by COX through reduced synthesis of
leukocytes to the inflammation site when we used the glucan extract.
In the last few years, NO has been recognized as an important messenger in diverse pathophysiological functions, including neuronal transmission, vascular relaxation, immune modulation and cytotoxicity against tumor cells[32]. The value of nitrate/nitrite produced in the pleural cavity of rats by carrageenan administration 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 extract from 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.
NO levels by 41.5% (p b 0.001) in compare to the control group (carrageenan). These effects suggest an antiinflammatory 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 has