Bioresource Technology 101 (2010) 6192–6199

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Structure of Agaricus spp. fucogalactans and their anti-inflammatoryand antinociceptive properties

Dirce L. Komura a, Elaine R. Carbonero b, Ana Helena P. Gracher a, Cristiane H. Baggio c, Cristina S. Freitas c,Rodrigo Marcon d, Adair R.S. Santos e, Philip A.J. Gorin a, Marcello Iacomini a,*

a Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba 81531-980, Brazilb Departamento de Química, Universidade Federal de Goiás, Campus Catalão, Catalão 75704-020, Brazilc Departamento de Farmacologia, Universidade Federal do Paraná, Curitiba 81531-980, Brazild Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Florianópolis 88049-900, Brazile Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Florianópolis 88040-900, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 June 2009Received in revised form 18 December 2009Accepted 29 January 2010Available online 2 April 2010

Keywords:Agaricus spp.FucogalactansNMR spectroscopyAntinociceptiveAnti-inflamatory

0960-8524/$ - see front matter � 2010 Published bydoi:10.1016/j.biortech.2010.01.142

* Corresponding author. Tel.: +55 41 3361 1655; faE-mail address: iacomini@ufpr.br (M. Iacomini).

Fucogalactans from Agaricus brasiliensis (EPF-Ab) and A. bisporus var. hortensis (EPF-Ah) were prepared viaby aqueous extraction and a purification procedure. EPF-Ab had Mw 19.4 � 103 g/mol and EPF-Ah Mw

31.1 � 103 g/mol. EPF-Ab had a (1 ? 6)-linked a-D-Galp main-chain partially substituted in O-2 bynon-reducing end-units of a-L-Fucp. EPF-Ah had a similar main-chain with O-2 substitution, but was par-tially methylated at HO-3, as well as having 2.5% non-reducing end-units of b-D-Gal. In mice, EPF-Ab gave39% antinociceptive inhibition (ID50 > 100 mg/kg) and no anti-inflammatory activity. EPF-Ah also gave aninhibition of 39% at ID50 0.33 mg/kg and also inhibited by 61% (ID50 5.0 mg/kg) total cell migration and by32% peritoneal capillary permeability, which is related to the anti-inflammatory effect. The small differ-ences in chemical structure in these polysaccharides thus modified their biological activities.

� 2010 Published by Elsevier Ltd.

1. Introduction

Among mushrooms that are widely known for their medicinalproperties or are appreciated in the culinary world, are Lentinulaedodes (= Lentinus edodes; shiitake), Agaricus brasiliensis (A. blazei),A. bisporus (champignon de Paris), A. bisporus var. hortensis (porto-bello), Pleurotus spp., and Ganoderma lucidum (Chang, 2005).

For centuries, mushrooms have been used as therapeutic agentsand in food, since they have high nutritional values, being suitablefor a balanced diet. This is due to their high protein and fiber con-tent with small amounts of fat (Manzi et al., 2001; Lakhanpal andRana, 2005). They also contain significant amounts of calcium, zinc,magnesium, iron and phosphorus (Mohacek-Grošev et al., 2001).

Hot-water extracts and decocts of mushrooms are used in or-iental medicine, where this practice began, especially in China. Thiswas due to their immunomodulatory and antitumoral properties,reduction of the blood pressure, among other attributes (Chang,1996; Wasser, 2002; Chen and Seviour, 2007; Zhang et al., 2007).

These practices are the basis of modern studies on the medici-nal properties of mushrooms, in which a great number of activemolecules have been identified in many species (Smith et al.,

Elsevier Ltd.

x: +55 41 3266 2042.

2002). Among these, prominent are polysaccharides which havebeen isolated from the fruiting bodies, mycelia, and culture media(Zhang et al., 2007).

Several polysaccharides have been isolated from basidiomyce-tes, such as linear or branched glucans and heterogalactans, whichcan contain O-methyl groups or a variety of side chains (Wasser,2002; Zhang et al., 2007).

Most heterogalactans have a (1 ? 6)-linked a-D-Galp main-chain with different substituents, mainly by fucose or mannose(Wasser, 2002; Rosado et al., 2003; Zhang et al., 2007; Carboneroet al., 2008a,b). However, there are few studies on their detailedstructure and biological activity.

Many of these polysaccharides have been evaluated as biologicalresponse modifiers, especially for their antitumor action (Wasserand Weis, 1999; Moradali et al., 2007; Zhang et al., 2007). Somestudies have shown that those extracted molecules of mushroomscan have other effects, such as anti-inflammatory and antinocicep-tive activity (Park et al., 2005; Carbonero et al., 2008b; Smiderleet al., 2008). These can occur due to a decrease in the concentrationof interleukins, interferon and tumor necrosis factor and inhibitionof inflammatory cells, such as lymphocytes and macrophages. How-ever, it is not possible to attribute a relation between structure andactivity because most of the investigations were carried out withcrude polysaccharide extracts (Lindequist et al., 2005; Poucheret

D.L. Komura et al. / Bioresource Technology 101 (2010) 6192–6199 6193

et al., 2006), although recent studies have described the anti-inflammatory and antinociceptive effects of fucomannogalactansand mannogalactans isolated of L. edodes (=Lentinus edodes) (Car-bonero et al., 2008b) and Pleurotus pulmonarius (Smiderle et al.,2008) respectively.

Among nociception models, the abdominal contortions test in-duced by acetic acid is described as typical for measuring visceralinflammatory pain. Although with a low specificity, the method issensitive, having been used as a screening tool for evaluating anal-gesic or anti-inflammatory properties of novel agents (Ikeda et al.,2001; Le Bars et al., 2001).

In order to compare chemical structure with biological proper-ties of mushroom polysaccharides, we now characterize structuresof fucogalactans isolated from A. brasiliensis and A. bisporus var.hortensis, as well as to determine their anti-inflammatory andantinociceptive properties.

2. Methods

2.1. Biological material

Dried fruiting bodies of A. brasiliensis and A. bisporus var. horten-sis were respectively provided by Cogumelo Sol de Minas Company(Wagner Passos), Baependi, State of Minas Gerais, and MakotoYamashita Company (Miriam Harumi Yamashita), São José dos Pin-hais, State of Paraná, Brazil.

2.2. Extraction and purification of fucogalactans

Extraction of crude polysaccharides and their purification wascarried out as in Fig. 1A. Wiley-milled powder fruiting bodies fromA. brasiliensis (500 g) and A. bisporus var. hortensis (236 g) wereeach extracted with 4:1 (v/v) chloroform–methanol (CHCl3–

B1

Volume (ml)

0 20 40 60 80

0.02

0.15

0.10

0.05

0.00

- 0.05

RI

(vol

ts)

-

RI

(vol

ts)

Mw 19.4 x 103 g/mol dn/dc 0.165 ml/g

FUCOGALACTANS

Agaricus brasiliensis (A. bisporus var. hortensi

CHCl3-M

Treatment with Fehling sol

Fehling supernatant(FS)

Residue I H2O at 4 ºC for 6 h (x 6)

Ethanol precipitate(PW)

Residue II

Insoluble fraction (IPW)

Ultrafiltration with 300 k

Eluted fraction(EFP)

Freeze-thawing, Centrifugation

A

Fig. 1. (A) Scheme of extraction and purification of fucogalactans from the Agaricus brasi(B1) and EPF-Ah (B2) determined by HPSEC.

MeOH) at �60 �C for 3 h (�3), to remove low molecular weightmaterial. In order to obtain polysaccharides, the defatted fruitingbodies were extracted with H2O at 4 �C for 6 h (�6; 2000 ml).The combined aq. extracts were evaporated to a small volumeand added to excess ethanol (EtOH, 3:1; v/v) to precipitate polysac-charide, which was collected by centrifugation at 8500 rpm at10 �C for 20 min. It was then dissolved in H2O, dialyzed against dis-tilled water for 20 h to remove low-molecular-weight carbohy-drates, giving rise to fractions PW-Ab and PW-Ah, respectively.These were frozen and then allowed to thaw slowly and resultinginsoluble material (fractions IPW-Ab and IPW-Ah) was removedunder the same centrifugation conditions. The supernatants (frac-tions SPW-Ab and SPW-Ah) were treated with Fehling solution(Jones and Stoodley, 1965) and precipitated Cu++ complexes (FP-Ab and FP-Ah respectively) were removed by centrifugation at9000 rpm for 15 min, at 25 �C. The precipitates were neutralizedwith acetic acid (HOAc), dialyzed against tap water (48 h), deion-ized with mixed ion exchange resins, and then freeze-dried.

Each fraction was further purified by ultrafiltration through amembrane with a 300 kDa Mr cut-off (Millipore� – polyethersulf-one) on filter holder (Sartorius – Model 16249), with compressedair at 10 psi as carrier gas.), giving rise to eluted (EFP) and retained(RFP) material (Fig. 1A).

2.3. Monosaccharide composition

Monosaccharide components of the polysaccharides (1 mg)were identified and their ratios were determined following hydro-lysis with 2 M trifluoroacetic acid (TFA, 1 ml) for 8 h at 100 �C, andconversion to alditol acetates (GC–MS) by successive sodium boro-hydride (NaBH4) and/or deuterated sodium borohydride (NaB2H4)reduction (pH 9–10), and acetylation with acetic anhydride–pyri-

B2

0 20 40 60 80

0.04

0.03

0.02

0.01

0.00

0.01

0.05

Volume (ml)

Mw 31.1 x 103 g/mol dn/dc 0.148 ml/g

Ab)s (Ah)

eOH (4:1)

ution, Centrifugation

Fehling precipitate(FP)

Low molecular weight material

Soluble fraction(SPW)

Ethanol supernatant

Aqueous extract (W)EtOH (3:1; v/v)

Da cut –off membrane

Retained fraction(RFP)

liensis (Ab) and A. bisporus var. hortensis (Ah). (B) Elution profiles of fractions EPF-Ab

6194 D.L. Komura et al. / Bioresource Technology 101 (2010) 6192–6199

dine (Ac2O–pyridine, 1:1; v/v) for 12 h at room temperature (Wol-from and Thompson, 1963a,b).

Alditol acetate mixtures formed from polysaccharides wereanalyzed by GC–MS using a Varian model 3300 gas chromatographlinked to a Finnigan Ion-Trap, Model 810-R12 mass spectrometer,incorporating a DB-225 capillary column (30 m � 0.25 mm i.d.)programmed from 50 to 220 �C at 40 �C/min, then hold.

2.4. Determination of homogeneity of polysaccharides and theirmolecular weight (Mw)

The homogeneity and molecular mass (Mw) of the crude puri-fied fractions (EFP-Ab and EFP-Ah) were determined by high per-formance steric exclusion chromatography (HPSEC), using arefractive index (RI) detector. The eluent was 0.1 M sodium nitrate(NaNO3), containing 0.5 g/l sodium azide (NaN3). The polysaccha-ride solutions were filtered through a membrane with 0.22 lmdiameter pores (Millipore). The specific refractive index increment(dn/dc) was determined using a Waters 2410 detector.

2.5. Methylation analysis of polysaccharides

Per-O-methylation of the polysaccharides (EPF-Ab and EPF-Ah;2 to 5 mg) was carried out using sodium hydroxide–dimethylsulphoxide–iodomethane (NaOH–Me2SO–MeI) (Ciucanu and Ker-ek, 1984). The per-O-methylated derivatives were hydrolyzed with45% aqueous formic acid (HCO2H, 1 ml) for 6 h at 100 �C, followedby NaB2H4 reduction and acetylation as above (Section 2.3), to givea mixture of partially O-methylated alditol acetates, which wasanalyzed by GC–MS (DB-225 column programmed at 40 �C min�1

to 210 �C).

2.6. Enantiomeric configuration of monosaccharides

The enantiomeric configuration of monosaccharides was deter-mined, following reductive amination with chiral 1-amino-2-pro-

Table 1Monosaccharide composition and yields of polysaccharide fractions obtained from Agaricu

Fraction Yield (%)a Monosac

Xyl

A. brasiliensis SPW-Ab 2.4 –FS-Ab 0.4 –PF-Ab 1.7 –EPF-Ab 1.6 –

A. bisporus var. hortensis SPW-Ah 2.1 5.2FS-Ah 1.5 6.4FP-Ah 0.3 –EFP-Ah 0.2 –

a Yields based on dry fungi (A. brasiliensis: 500 g and A. bisporus var. hortensis: 236 g)b Alditol acetates obtained on successive hydrolysis, NaBH4 and/or NaB2H4 reduction,c Confirmed by the presence of the fragments m/z 130 and 190, after hydrolysis, redu

Table 2Partially O-methylalditol acetates formed on methylation analysis of A. brasiliensis and A.

Partially O-methylated alditol acetatea Linkage typeb RT (min

2,3,4-Me3-Fuc Fucp-(1? 7.802,3,4,6-Me4-Gal Galp-(1? 9.802,3,4-Me3-Gal 6?)-Galp-(1? 14.913,4-Me2-Gal 2,6?)-Galp-(1? 21.50

a Analyzed by GC–MS after methylation, total acid hydrolysis, reduction (NaB2H4) andb Based on derived O-methylalditol acetates.c Retention time (min).

panol, followed by acetylation and GC analysis (Ultra-2 column,Hewlett–Packard) (Cases et al., 1995).

2.7. Nuclear magnetic resonance (NMR) spectroscopy

Mono- (13C, 1H and DEPT NMR) and bidimensional NMR spectra(HMQC, COSY, TOCSY, and coupled HMQC) were prepared using a400 MHz Bruker Avance spectrometer incorporating Fourier trans-form. This was carried out at 50 �C on samples dissolved in D2O.Chemical shifts are expressed in d relative to acetone at d 32.77(13C) and 2.21 1H), based on DSS (2,2-dimethyl-2-silapentane-3,3,4,4,5,5-d6-5-sulfonate sodium salt; d = 0.0 for 13C and 1H).

2.8. Experimental animals

Male Swiss mice (25–35 g) were kept in an automatically con-trolled temperature room (23 ± 2 �C) with 12 h light–dark cycles,water and food being freely available. They were acclimatized tothe laboratory for at least 2 h before testing and were used onlyonce for experiments. These were performed after approval ofthe protocol by the Institutional Ethics Committee of the Univer-sidade Federal de Santa Catarina and in accordance with currentguidelines for the care of laboratory animals and ethical guidelinesfor investigation of experimental pain in conscious animals (Zim-mermann, 1983). The numbers of animals and intensities of nox-ious stimuli used were the minimum necessary to demonstrateconsistent effects of the drug treatments.

2.9. Abdominal constriction, peritoneal capillary permeability andleukocytes infiltration caused by intraperitoneal injection of 0.6%acetic acid

Abdominal constrictions were induced according to proceduresdescribed previously (Lucena et al., 2007), which resulted in con-traction of the abdominal muscle together with stretching of thehind limbs in response to the intraperitoneal injection (i.p.) of0.6% acetic acid. At the beginning of each experiment, mice were

s brasiliensis and A. bisporus var. hortensis.

charide (%)b

Fuc Man 3-Me-Galc Gal Glc

9.7 14.0 – 59.3 17.0– 21.9 – 23.8 54.3

13.6 – – 83.3 3.113.9 – – 86.1 –

11.8 21.7 13.7 42.9 4.76.9 22.6 14.9 39.7 9.5

10.8 3.0 15.8 68.8 1.616.8 – 15.4 67.8 –

.and acetylation, and analyzed by GC–MS.ction with NaB2H4 and acetylation.

bisporus var. hortensis heteropolysaccharides (EPF-Ab and EPF-Ah, respectively).

)c Fraction (mol%) Mass fragmentation (m/z)

Ab Ah

14.2 14.8 89,101,115, 117,131,161,175– 2.5 87,101,117,129,145,161,205

71.8 64.8 87,101,117,129,161,173,189,23314.0 17.9 87,99,129,159,173,189,233

acetylation.

D.L. Komura et al. / Bioresource Technology 101 (2010) 6192–6199 6195

pre-treated intravenously with 2.5% Evans blue dye solution(10 ml/kg), used as a peritoneal capillary permeability marker.One hour later, they were treated with the samples (3–100 mg/kg) by the intraperitoneal route 30 min before acetic acid injection.Control animals received a similar volume of the saline solution(10 ml/kg, i.p.), used to dilute the EFP-Ab and EFP-Ah. After thechallenge, the mice were placed individually into glass cylindersof 20 cm diameter, and the abdominal constrictions were countedup to 20 min. Antinociceptive activity is expressed as the reductionin the number of abdominal constrictions [i.e., the difference be-tween control mice (mice pre-treated with saline) and animalspre-treated with EFP-Ab or EFP-Ah]. Immediately after the test,mice were sacrificed by cervical dislocation and the peritoneal cav-ity was washed with 1 ml of sterile saline plus heparin (25 IU/ml)and the volume collected with automatic pipettes. Total leukocytecounts were performed using a Neubauer chamber via opticalmicroscopy after diluting a sample of the peritoneal fluid withTürk solution (1:20). A sample of the collected fluid (700 ll) wascentrifuged at 1000 rpm for 10 min and the absorbance of thesupernatant was read at 610 nm with an ELISA analyzer. The peri-toneal capillary permeability induced by acetic acid is expressed interms of dye (lg/ml), which leaked into the peritoneal cavityaccording to the standard curve of Evans blue dye (Lucena et al.,2007).

74.5

69.7

103.

9

100.

7

80.3

71.7

71.1

69.4

A 72.4

72.2 18

.2

B

103.

9

100.

7

106.

3 80.3

81.6

74.5

72.4

72.2

71.7

71.1

69.4

70.0

68.2

18.2

58.9

63.7

69.7

A’

B’

ppm

ppm

ppm

ppm

Fig. 2. 13C NMR spectra, obtained at 50 �C, of fucogalactans from A. brasiliensis (A)and A. bisporus var. hortensis (B) in D2O. Inset of CH3-6 region from Fucp units (A0

and B0).

2.10. Statistical analysis

The results are expressed as means ± S.E.M., except that the ID50

values (i.e. the dose of EFP-Ab or EFP-Ah reducing the abdominalconstriction, peritoneal capillary permeability and leukocyte infil-tration responses by 50%, relative to the control value), which arepresented as geometric means accompanied by their respective95% confidence limits. The ID50 values were determined by linearregression from individual experiments using linear regressionGraphPad software (Graph Pad software, San Diego, CA, USA).The statistical significance of differences between groups was de-tected by ANOVA followed by the Newman–Keuls’ test. P valuesless than 0.05 were considered to be significant.

3. Results and discussion

Agaricus is an important fungal genus, because some of its spe-cies contain polysaccharides, which have several biological activi-ties, especially antitumor action. In order to obtain pureheteropolysaccharides from the fruiting bodies of A. brasiliensisand A. bisporus var. hortensis, each was defatted with hot CHCl3–MeOH, and submitted to aqueous extraction at 4 �C. The extractedpolysaccharides were recovered by ethanol precipitation, and weredialyzed against tap water, and the solution freeze-dried to givePW-Ab and PW-Ah respectively (Fig. 1A).

Fractionation and purification of PW-Ab and PW-Ah was carriedout by a freeze–thawing procedure, resulting in a respective cold-water soluble SPW-Ab (12 g) and SPW-Ah (4.96 g) and discardedinsoluble fractions.

Fraction SPW-Ab contained fucose, xylose, galactose and glu-cose, SPW-Ah had, besides these, xylose and 3-O-methyl-galactose(confirmed by the presence of the fragments m/z 130 and 190, afterhydrolysis, reduction with NaB2H4 and acetylation) (Table 1).

Fractions SPW-Ab and SPW-Ah gave heterogeneous HPSEC elu-tion profiles, so they were then treated with Fehling solution, therespective precipitates being FPW-Ab (8.5 g) and FPW-Ah(708 mg). Each was further fractionated by ultrafiltration througha 300 kDa Mr cut-off membrane, giving rise to eluted EFP-Ab andEFP-Ah and retained RFP-Ab and RFP-Ah fractions respectively(Fig. 1A). The EFP fractions, (EPF-Ab: 8.0 g and EPF-Ah: 472 mg)were homogeneous on HPSEC, and had 19.4 � 103 g/mol (dn/dc0.165 ml/g) for A. brasiliensis and 31.1 � 103 g/mol (dn/dc0.148 ml/g) for A. bisporus var. hortensis (Fig. 1B).

Fraction EFP-Ab contained only fucose and galactose comparedwith EFP-Ah, which also contained 3-O-methyl-galactose (Table 1),both being fucogalactans. Using the method of Cases et al. (1995),galactose and 3-O-methyl-galactose had the D- and the fucose res-idues the L-enantiomer.

EFP-Ab and EFP-Ah were submitted to methylation analysis(Ciucanu and Kerek, 1984) and GC–MS and resulting O-methylald-itol acetates showed the presence of branched structures, contain-ing mainly, non-reducing end-units of Fucp (2,3,4-Me3Fuc), besidesthe 6-O-(2,3,4-Me3Gal) and 2,6-di-O-substituted units (3,4-Me2Gal) of galactopyranose. Small amounts of the non-reducingend-units of Galp (2,3,4,6-Me4Gal) were only observed in theRFP-Ah isolated from A. bisporus var. hortensis (Table 2). Thisanalysis indicated that the ratio of units of Fucp: 2,6-di-O-Galp:6-O-Galp was approximately 1:1:5 and 1:1:4 for RFP-Ab from A.brasiliensis and RFP-Ah from A. bisporus var. hortensis respectively,these values being confirmed by the integration of H-1 signals(5.10:5.06:5.01) present in the 1H NMR spectrum.

Further NMR spectroscopy [1H, 13C (Fig. 2), HMQC (Fig. 3), DEPT(Fig. 4), TOCSY, COSY, coupled HMQC] was also helpful in elucidat-ing the fucogalactan structures (Table 3).13C NMR (Fig. 2A and B)and HMQC spectra (Fig. 3A and B), obtained using D2O as solvent,

δ 100.7/5.06

δ 103.9/5.10

δ 100.7/5.01

δ 80.3/3.84

δ 74.5/3.85

δ 71.1/3.89

δ 72.0/3.85

δ 69.9/4.20δ 71.1/4.08

δ 72.2/4.09δ 71.7/4.21

δ 69.4/69.7/3.72;3.93δ 70.0/3.65;4.01

δ 71.2/3.84

δ 72.4/3.89δ 72.4/4.03

A

δ 18.2/1.27

A’

δ 80.3/3.84δ 81.6/3.58

δ 106.3/4.64

δ 100.7/5.06δ 100.7/5.01

δ 100.6/5.01

δ 103.9/5.10

δ 69.4/69.7/3.72;3.93δ 70.0/3.65

δ 74.5/3.85δ 71.2/3.89

δ 71.2/3.84

δ 72.0/3.85

δ 72.2/4.09δ 71.7/4.21

δ 72.4/4.03

δ 68.2/4.30

δ 71.1/4.08

δ 70.1/3.88δ 58.9/3.43

δ 63.7/3.80B

δ 18.2/1.27

B’

Fig. 3. 1H (obs.), 13C HMQC spectra of fucogalactans EPF-Ab from A. brasiliensis (A) and EPF-Ah from A. bisporus var. hortensis (B) in D2O at 40 �C. Inset of CH3-6 region fromFucp units (A0 and B0).

6196 D.L. Komura et al. / Bioresource Technology 101 (2010) 6192–6199

had signals (C-1/H-1) at d 103.9/5.10, 100.7/5.06 and 100.7/5.01corresponding to Fucp units, 2,6-di-O- and 6-O-substituted Galpunits, respectively. Anomeric signals corresponding to non-reduc-ing end groups of b-Galp (d 106.3/4.64) and units of 3-Me-Galp(d 100.6/5.01) were present only in fraction EPF-Ah (Figs. 2B and3B), in agreement with the methylation data that indicated smallamounts of non-reducing end-units of b-Galp (Table 2). The glyco-sidic configurations were confirmed by the values of the couplingconstants JC-1,H-1 found in 1H/13C coupled HMQC spectra. Thenon-reducing end- of Fucp and the main-chain units (galactoseor 3-O-Me-galactose or both) had the a-configuration due torespective JC-1/H-1 167.8 and 172.6 Hz respectively, while those ofnon-reducing end-units of Galp had b-configuration, consistentwith JC-1,H-1 162.6 Hz. (Perlin and Casu, 1969).

The above methylation analysis indicated the presence of 6-O-and 2-O-substituted linkages (Table 2), these being confirmed byNMR spectroscopy. O-substituted C-2 signals were at d 80.3(Figs. 2–4), and substituted –CH2 groups of the 6-O-(Galp e 3-O-Me-Galp) and 2,6-di-O-substituted (Galp) units of the main-chainwere at d 69.4/69.7 and 70.0, respectively, giving rise to invertedsignals in the DEPT spectra (Fig. 4A and B).

The presence and position of O-methyl groups of the hetero-polysaccharides from A. brasiliensis and A. bisporus var. hortensiswere confirmed by d 58.9/3.43 and d 81.6/3.58 (C/H) signals corre-sponding to –OCH3 and O-substituted C-3 substituted/H-3, respec-tively (Figs. 2B and 3B; Table 3).

The signals of C-2/H-2 to C-6/H-6 at d 72.0/3.85, 71.2/3.84, 74.5/3.85, 69.9/4.20 and d 18.2/1.27 corresponded to Fucp units, while

100.

7

103.

9

80.3 74

.572

.4 .72

.271

.171

.769

.7

69.4

69.9

70.0

72.5

A

100.

7

103.

9

80.3

74.5

72.4

72.2

71.171.7

69.9

69.4

70.0

70.1

72.5

106.

3

81.6

68.2

58.9

63.7

68.5

69.7

78.3

76.1

73.6

B

100.

6

ppm

Fig. 4. 13C NMR DEPT spectra of fucogalactans EPF-Ab (A) and EPF-Ah (B) obtained respectively from A. brasiliensis and A. bisporus var. hortensis.

Table 31H and 13C NMR chemical shifts [expressed as d (ppm)] of the fucogalactans from A. brasiliensis (EPF-Ab) and A. bisporus var. hortensis (EPF-Ah).a

Units 1 2 3 4 5 6 –O-CH3

6a 6b

a-Fucp-(1? 13C 103.9 72.0 71.2 74.5 69.9 18.2 – –1H 5.10 3.85 3.89 3.85 4.20 1.27 – –

2,6?)-a-Galp-(1? 13C 100.7 80.3 71.1 72.2 71.7 69.7 69.7 –1H 5.06 3.84 4.08 4.09 4.21 3.70 4.01 –

?6)-a-Galp-(1? 13C 100.7 71.1 72.4 72.4 71.7 69.4 69.4 –1H 5.01 3.84 3.89 4.03 4.21 3.72 3.93 –

?6)-3-O-Me-a-Galp-(1? 13C 100.6 70.1 81.6 68.2 71.7 69.4 69.4 58.91H 5.01 3.88 3.58 4.30 4.21 3.72 3.93 3.43

a Assignments are based on 13C, 1H, DEPT, COSY, TOCSY and HMQC examination.

D.L. Komura et al. / Bioresource Technology 101 (2010) 6192–6199 6197

those at d 70.1/3.88, 81.6/3.58, 68.2/4.30, 71.7/4.21 and d 69.4/69.7/3.72/3.93 were from 3-O-Me-Galp units (Figs. 2–4; Table 3).Due to the small amount of b-Galp (2.5%) of the polymer isolatedfrom A. bisporus var. hortensis, the signals of C-2 (d 73.6), C-3 (d76.1), C-4 (d 71.1), C-5 (d 78.3) and C-6 (d 63.7) were enhancedin its DEPT spectrum (Fig. 4B).

Analysis of the fucogalactan EFP-Ab from A. brasiliensis showedit to consist of a (1 ? 6)-linked a-D-galactopyranosyl main-chain,

partially substituted at O-2 by non-reducing end-units of a-L-Fucp(Fig. 5A). That of the fucogalactan EFP-Ah from A. bisporus var. hort-ensis was similar, although the main-chain was partially O-methyl-ated and had a low proportion of non-reducing end-units of b-Galp(Fig. 5B).

Fucogalactans similar to those now described have been iso-lated from cultivated mycelium of Coprinus comatus (Fan et al.,2006) and fruiting bodies of Hericium erinaceus (Zhang et al.,

OH

OO

OHOH

OH

OO

OHOH

OH

OO

OOH

OH

OH

OO

OHOH

OH

OO

OOH

OH

O

OOHOH

OHO

OH

OH

H

n

A

OO

OHOH

O

OO

OOH

OH

OH

OO

OHOH

OH

OO

OOH

OH

O

OOHOH

OHO

OH OH

OHCH3

n

B

Fig. 5. Structure of the fucogalactans EPF-Ab and EPF-Ah, obtained respectively from Agaricus brasiliensis (A) and A. bisporus var. hortensis (B).

6198 D.L. Komura et al. / Bioresource Technology 101 (2010) 6192–6199

2006), which have side chains with low amounts of 3-O-methylr-hamnose. However, fucogalactans with a methylated main-chainhave not been previously described.

The interest in substances with analgesic activity has beenincreasing for treatment of several types of pain (neurogenic andinflammatory origins). Several animal models of nociception havebeen developed to test the activity of extracts and chemical com-pounds. The test now performed is a typical for visceral inflamma-tory nociception and allows an estimation of the antinociceptiveactivity of substances that act at a central and peripheric level. Itis able to evaluate anti-inflammatory action by the increase ofthe peritoneal capillary permeability (Evans Blue dye exudation)as well as for leukocyte migration to the peritoneal cavity (Lucenaet al., 2007).

In order to determine the biological action of the fucogalactansisolated from A. brasiliensis and A. bisporus var. hortensis, they weretested for probable antinociceptive and anti-inflammatory effects.This was at doses on mice of 10, 30 and 100 mg/kg for the fucoga-

0

10

20

30

40

EPF-Ah mg/kg, i.p.

0.6% Acetic Acid (450 μl, i.p.)

***

**

******

Inhibition: 72± 6%ID50 = 0.33 (0.14-0.80 mg/kg)

Num

ber o

f Writ

hes

#

***

Sal C Dexa Indo 1 3 10 Sal C Dexa In0.0

2.5

5.0

7.5

10.0

12.5

0.6% Acet

#

***Tota

l Leu

kocy

tes

(x 1

06)

0

20

40

60

EPF-Ab mg/kg, i.p.

0.6% Acetic Acid (450 μl, i.p.)

Inhibition: 39 ± 8%ID50 > 100 mg/kg

***

*

#

Num

ber o

f Writ

hes

Sal C Dexa Indo 10 30 100 Sal C Dexa In0

1

2

3

4

0.6% Aceti

***

#

Tota

l Leu

kocy

tes

(x 1

06 )A1 B1

A2 B2

Fig. 6. Effect of EFP-Ab and EFP-Ah (1–10 mg/kg, i.p.) on acetic acid-induced abdominal coleukocyte infiltration (C1 and C2, respectively) in mice. Data are expressed as means ± S#p < 0.001 vs. saline (sal), ANOVA followed by Newman–Keuls test.

lactan of A. brasiliensis (EFP-Ab) and 1, 3 and 10 mg/kg for that(EFP-Ah) of A. bisporus var. hortensis, thirty minutes before aceticacid administration. EPF-Ab inhibited 39 ± 8% at a dose of100 mg/kg (Fig. 6A1), while EPF-Ah had a better nociceptive re-sponse (Fig. 6A2). The dose of EPF-Ah calculated as necessary to in-hibit 50% of contortions (ID50) was 0.33 (0.14–0.80) mg/kg, and aninhibition of 39 ± 8% at the tested doses (Fig. 6A2) was observed.EPF-Ab did not give rise to anti-inflammatory activity, modifyingneither peritoneal capillary permeability nor leukocyte infiltrationcaused by acetic acid (Fig. 6B1 and C1).

EPF-Ah inhibited by 61 ± 13% leukocyte migration at doses of 3and 10 mg/kg, with an ID50 of 5 (2.7–9.2) mg/kg (Fig. 6B2). It alsoinhibited by 32 ± 6% the peritoneal capillary permeability at a doseof 3 mg/kg (Fig. 6C2).

Previous investigations have described the antinociception and/or anti-inflammatory action of heteropolysaccharides from basidi-omycetes. A fucomannogalactan of Lentinus edodes, which has a(1 ? 6)-linked a-Galp main-chain, partially substituted at O-2 by

do 1 3 10

EPF-Ah mg/kg, i.p.

ic Acid (450 μl, i.p.)

*****

Inhibition: 61± 13%ID50 = 5.0 (2.7-9.2 mg/kg)

***

0

5

10

15

EPF-Ah mg/kg, i.p.

0.6% Acetic Acid (450 μl, i.p.)

***

Inhibition: 32± 6%ID50 > 10 mg/kg

Evan

's B

lue

Dye

( μg/

ml)

#

* *

***

do 10 30 100

EPF-Ab mg/kg, i.p.

c Acid (450 μl, i.p.)

Sal C Dexa Indo 1 3 10

Sal C Dexa Indo 10 30 1000.0

2.5

5.0

7.5

EPF-Ab mg/kg, i.p.

0.6% Acetic Acid (450 μl, i.p.)

***

#

Evan

's B

lue

Dye

( μg/

ml) C1

C2

nstriction (A1 and A2, respectively), Evans blue leakage (B1 and B2, respectively), and.E.M.; n = 6–8 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control (C), and

D.L. Komura et al. / Bioresource Technology 101 (2010) 6192–6199 6199

b-Manp and a-Fucp units, totally inhibited nociception and leuko-cyte infiltration at a dose of 100 mg/kg and reduced peritoneal cap-illary permeability by 76% (Carbonero et al., 2008b). Anotherpolysaccharide already examined was a mannogalactan partiallysubstituted at O-2 by Manp isolated from P. pulmonarius, whichhas a similar main-chain and the same O-substitution position asthe fucogalactans from A. bisporus var. hortensis, inhibited nocicep-tion by 93% at a dose of 30 mg/kg (ID50 = 16.2 mg/kg) and did nothave anti-inflammatory activity (Smiderle et al., 2008).

These results show that the heterogalactans have antinocicep-tive and or anti-inflammatory action, which are related to theirstructures. The influence of the structure on the biological responsewas found with similar polymers with small differences in themain-chain, such as the presence of O-methyl groups, or in the sidechains. As a result, the determination of the fine structure of thepolysaccharides is important to understand the relation betweenstructure and biological activity. To determine the mechanism ofaction of these polymers other antinociceptive and/or anti-inflam-matory assays are being carried out.

Acknowledgements

The authors would like to thank the Brazilian funding agenciesCAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Supe-rior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tec-nológico), and Fundação Araucária for financial support, andWagner Passos (Cogumelo Sol de Minas Company) for donation ofthe fruiting bodies of Agaricus brasiliensis.

References

Carbonero, E.R., Gracher, A.H.P., Rosa, M.C.C., Torri, G., Sassaki, G.L., Gorin, P.A.J.,Iacomini, M., 2008a. Unusual partially 3-O-methylated a-galactan frommushrooms of the genus Pleurotus. Phytochemistry 69, 252–257.

Carbonero, E.R., Gracher, A.H.P., Komura, D.L., Marcon, R., Freitas, C.S., Baggio, C.H.,Santos, A.R.S., Torri, G., Gorin, P.A.J., Iacomini, M., 2008b. Lentinus edodesheterogalactan: antinociceptive and anti-inflammatory effects. Food Chem. 111(3), 531–537.

Cases, M.R., Cerezo, A.S., Stortz, C.A., 1995. Separation and quantitation ofenantiomeric galactoses and their mono-O-methylethers as theirdiastereomeric acetylated 1-deoxy-1-(2-hydroxypropylamino) alditols.Carbohydr. Res. 269 (2), 333–341.

Chang, R., 1996. Functional properties of edible mushrooms. Nutr. Rev. 54 (11), 91–93.Chang, S.T., 2005. The development of the mushroom industry in China, with a note

on possibilities for Africa. Acta Edulis Fungi 12, 3–19.Chen, J., Seviour, R., 2007. Medicinal importance of fungal b-(1 ? 3), (1 ? 6)-

glucans. Mycol. Res. 111 (6), 635–652.Ciucanu, I., Kerek, F., 1984. A simple and rapid method for the permethylation of

carbohydrates. Carbohydr. Res. 131, 209–217.Fan, J., Zhang, J., Tang, Q., Liu, Y., Zhang, A., Pan, Y., 2006. Structural elucidation of a

neutral fucogalactan from the mycelium of Coprinus comatus. Carbohydr. Res.341, 1130–1134.

Ikeda, Y., Ueno, A., Naraba, H., Oh-ishi, S., 2001. Involvement of vanilloid receptorVR1 and prostanoids in the acid-induced writhing responses of mice. Life Sci.69, 2911–2919.

Jones, J.K.N., Stoodley, R.J., 1965. Fractionation using copper complexes. MethodsCarbohydr. Chem. 5, 36–38.

Lakhanpal, T.N., Rana, M., 2005. Medicinal and nutraceutical genetic resources ofmushrooms. Plant Genet. Resour. 3 (2), 288–303.

Le Bars, D., Gozariu, M., Cadden, S.W., 2001. Animal models of nociception.Pharmacol. Rev. 53 (4), 597–652.

Lindequist, U., Niedermeyer, T.H.J., Jülich, W., 2005. The pharmacological potentialof mushrooms. Evid-Based Compl. Alt. 2 (3), 285–299.

Lucena, G.M.R.S., Gadotti, V.M., Maffi, L.C., Silva, G.S., Azevedo, M.S., Santos, A.R.S.,2007. Antinociceptive and anti-inflammatory properties from the bulbs ofCypura paludosa Aubl. J. Ethnopharmacol. 112, 19–25.

Manzi, P., Aguzzi, A., Pizzoferrato, L., 2001. Nutritional value of mushrooms widelyconsumed in Italy. Food Chem. 73, 321–325.

Mohacek-Grošev, V., Bozac, R., Pepples, G.J., 2001. Vibrational spectroscopiccharacterization of wild growing mushrooms and toadstools. Spectrochim.Acta A 57, 2815–2829.

Moradali, M.F., Mostafavi, H., Ghods, S., Hedjaroude, G.A., 2007. Immunomodulatingand anticancer agents in the realm of macromycetes fungi (macrofungi). Int.Immunopharmacol. 7, 701–724.

Park, Y.M., Won, J.H., Kim, Y.H., Choi, J.W., Park, H.J., Lee, K.T., 2005. In vivo andin vitro anti-inflammatory and anti-nociceptive effects of the methanol extractof Inonotus obliquus. J. Ethnopharmacol. 101, 120–128.

Perlin, A.S., Casu, B., 1969. Carbon-13 and proton magnetic resonance spectra of D-glucose-13C. Tetrahed. Lett. 34, 2919–2924.

Poucheret, P., Fons, F., Rapior, S., 2006. Biological and pharmacological activity ofhigher fungi: 20-year retrospective analysis. Cryptogamie Mycol. 27 (4), 311–333.

Rosado, F.R., Carbonero, E.R., Claudino, R.F., Tischer, C.A., Kemmelmeier, C., Iacomini,M., 2003. The presence of partially 3-O-methylated mannogalactan from thefruit bodies of edible basidiomycetes Pleurotus ostreatus ‘florida’ Berk. andPleurotus ostreatoroseus Sing. FEMS Microbiol. Lett. 221, 119–124.

Smiderle, F.R., Olsen, L.M., Carbonero, E.R., Marcon, R., Baggio, C.H., Freitas, C.S.,Santos, A.R., Torri, G., Gorin, P.A.J., Iacomini, M., 2008. A 3-O-methylatedmannogalactan from Pleurotus pulmonarius: structure and antinociceptiveeffect. Phytochemistry 69 (15), 2731–2736.

Smith, J.E., Rowan, N., Sullivan, R., 2002. Medicinal mushrooms: a rapidlydeveloping area of biotechnology for cancer therapy and other bioactivities.Biotechnol. Lett. 24, 1839–1845.

Wasser, S.P., 2002. Medicinal mushrooms as a source of antitumor andimmunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 60,258–274.

Wasser, S.P., Weis, A., 1999. Therapeutic effects of substances occurring in higherbasidiomycetes mushrooms: a modern perspective. Crit. Rev. Immunol. 19, 65–96.

Wolfrom, M.L., Thompson, A., 1963a. Reduction with sodium borohydride. MethodsCarbohydr. Chem. 2, 65–68.

Wolfrom, M.L., Thompson, A., 1963b. Acetylation. Methods Carbohydr. Chem. 2,211–215.

Zhang, A., Zhang, J., Tang, Q., Jia, W., Yang, Y., Liu, Y., Fan, J., Pan, Y., 2006. Structuralelucidation of a novel fucogalactan that contains 3-O-methyl rhamnose isolatedfrom the fruiting bodies of the fungus, Hericium erinaceus. Carbohydr. Res. 341,645–649.

Zhang, M., Cui, S.W., Cheung, P.C.K., Wang, Q., 2007. Antitumor polysaccharide frommushrooms: a review on their isolation process, structural characteristics andantitumor activity. Trends Food Sci. Technol. 18 (1), 4–19.

Zimmermann, M., 1983. Ethical guidelines for investigations of experimental painin conscious animals. Pain 16, 109–110.