An aqueous extract containing polysaccharides was obtained from the giant mushroom Macrocybe titans, and it was purified by amylase treatment, freeze-thawing process and dialysis. The purified fraction (ESP) was analyzed by HPSEC and GC–MS which showed a homogenous polysaccharide with Mw 14.2 × 103 g/mol composed by galactose and fucose.
Trang 1Contents lists available atScienceDirect Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol
Fucogalactan from the giant mushroom Macrocybe titans inhibits melanoma
cells migration
Shayane da Silva Milhorinia, Fhernanda Ribeiro Smiderlea, Stellee Marcela Petris Biscaiab,
Fabio Rogerio Rosadoc, Edvaldo S Trindadeb, Marcello Iacominia,⁎
a Department of Biochemistry and Molecular Biology, Federal University of Parana, CEP 81531-980, Curitiba, PR, Brazil
b Department of Cellular Biology, Federal University of Parana, CEP 81531-980, Curitiba, PR, Brazil
c Department of Biosciences, Federal University of Parana, CEP 85950-000, Palotina, PR, Brazil
A R T I C L E I N F O
Keywords:
Macrocybe titans
Fucogalactan
Chemical characterization
Melanoma B16-F10
A B S T R A C T
An aqueous extract containing polysaccharides was obtained from the giant mushroom Macrocybe titans, and it was purified by amylase treatment, freeze-thawing process and dialysis The purified fraction (ESP) was ana-lyzed by HPSEC and GC–MS which showed a homogenous polysaccharide with Mw14.2 × 103g/mol composed
by galactose and fucose NMR and methylation analysis of ESP confirmed the presence of a fucogalactan with a (1→ 6)-linked α-D-Galp main chain partially substituted at O-2 by non reducing end units ofα-L-Fucp residues in the side chain Its biological activity was evaluated against murine melanoma cells B16-F10 The fucogalactan did not alter the viability, proliferative capacity and morphology of cells However, this polysaccharide was able
to reduce the cell migration in vitro at 40% (100μg/mL) and 33% (250 μg/mL) The results obtained showed that Macrocybe titans fucogalactan is a promising agent capable of altering melanoma cell migration without decrease the cell viability
1 Introduction
For millennia, mushrooms have been used in culinary and folk
medicine by mankind, not only for their unique taste, but also because
of their nutritional value and bioactive compounds (Patel & Goyal,
2012;Ruthes, Smiderle, & Iacomini, 2016) A great variety of bioactive
molecules has been isolated from mushrooms, such as proteins (Zhang
et al., 2014), glycoproteins (Cui et al., 2013), secondary metabolites
(Baby, Johnson, & Govindan, 2015), lipopolysaccharides (Wasser,
2011) and polysaccharides (Moreno et al., 2016;Meng, Cheng, Han,
Chen, & Wang, 2017) The latter molecules are potent antitumor and
immunomodulating agents isolated from mushrooms, according to
studies dated from 1976 until now (Patel & Goyal, 2012; Smiderle,
Ruthes, & Iacomini, 2014) These medicinal molecules have been
ob-tained from several mushrooms, such as Agaricus bisporus (Ruthes,
Rattmann, Carbonero, Gorin, & Iacomini, 2012), Agaricus brasiliensis
(Komura et al., 2010), Macrolepiota dolichaula (Samanta et al., 2015),
Lactarius rufus (Ruthes et al., 2012), Flammulina velutipes (Smiderle,
Carbonero, Sassaki, Gorin, & Iacomini, 2008), Lentinus edodes
(Carbonero et al., 2008), Pleurotus sajor-caju (Silveira et al., 2015),
among others However, there are some mushroom species that are
unexplored and may have a medicinal potential that is still unknown,
such as the giant edible mushroom Macrocybe titans (H.E Bigelow & Kimbr.) Pegler, Lodge & Nakasone This species is large and its fruit body is white andfirm and grows in large caespitose clusters which may exceed 30 kg of fresh weight (Pegler, Lodge, & Nakasone, 1998) De-spite of its peculiar morphology and popular consumption, M titans has never been studied about its polysaccharides and there are no reports about its biological properties Therefore, considering its large size, this mushroom can be a promising source to obtain different bioactive molecules in great yields, including the polysaccharides
The biologically active mushroom polysaccharides that were mostly isolated and studied from several Basidiomycetes are theβ-D-glucans (Smiderle, Sassaki, Griensven, & Iacomini, 2013) Besides that, there is another class of mushroom polysaccharides that presents more than one monosaccharide, and variable anomeric configuration, linkage and branching type, including the presence of methyl groups in its structure (Ruthes et al., 2016) Among these polysaccharides, there are the het-erogalactans, which are mostly composed by a main chain of (1→ 6)-linked α-D-Galp units, some of them partially methylated and sub-stituted mainly by fucose or mannose (Rosado et al., 2003; Komura
et al., 2010;Ruthes et al., 2013)
Heterogactans have been largely explored about their biological activities, such as immunomodulatory (Sun & Liu, 2009;Samanta et al.,
https://doi.org/10.1016/j.carbpol.2018.02.063
Received 21 November 2017; Accepted 21 February 2018
⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Federal University of Parana, CP 19046, Curitiba, PR, Brazil.
E-mail address: iacomini@ufpr.br (M Iacomini).
Available online 23 February 2018
0144-8617/ © 2018 Elsevier Ltd All rights reserved.
T
Trang 22015), anti-inflammatory and antinociceptive (Komura et al., 2010;
Silveira et al., 2015) anti-sepsis (Ruthes et al., 2012), anti-oxidative
(Ding, Hou, & Hou, 2012;Samanta et al., 2015) and also antitumor
activities (Jeff et al., 2013;Wang, Sun, Wu, Yang, & Tan, 2014) The
latter one has showed great interest by the scientific and medical
community because tumors are one of the main cause of morbidity and
death worldwide (Bae, Jang, Yim, & Jin, 2005; Liu, Zeng, Li, & Shi,
2016,Tian, Zhao, Zeng, Zhang, & Zheng, 2016) According to the World
Health Organization (WHO/Cancer, 2018), cancer related-deaths were
totalized 8.8 millions in 2015, which implies that approximately 1 in 6
deaths in the world is due to cancer Among the cancer types, the skin
cancer classified as melanoma is one of the most aggressive with the
highest rates of metastasis and mortality (Harries et al., 2016) It is the
eighth most common cancer that affects people from developed
coun-tries (Hamel et al., 2016) There are some studies that demonstrated
activity of mushroom polysaccharides against melanoma cells in vitro
(Bae et al., 2005; Han et al., 2006) and antitumor activity of such
molecules in vivo (Zhang, Yang, Chen, Hou, & Han, 2005)
Based on this, the aim of this study was to obtain polysaccharides
from Macrocybe titans fruiting bodies, that could exhibit some activity
against melanoma cells Furthermore, the active polysaccharide was
purified and its chemical structure was carefully analyzed until
com-plete elucidation
2 Material and methods
2.1 Biological material
The mushroom Macrocybe titans was kindly donated by Dr Fábio
Rogério Rosado from the Department of Bioscience, Federal University
of Paraná– Palotina-PR, Brazil
2.2 Extraction and purification procedures
The dried and powdered fruiting bodies (300 g) were submitted to
several purification steps as shown inFig 1 Firstly, the apolar
com-pounds were extracted with CHCl3:MeOH (2:1; v/v) at 60 °C during 3 h
(3×) Subsequently, the resulting residue was dried and extracted with
distilled water at room temperature under mechanical stirring during
6 h (3×) The aqueous extract was concentrated under reduced
pressure and precipitated with ethanol (3:1; v/v) The precipitated polysaccharides were obtained by centrifugation (10.000 rpm, at 4 °C, for 20 min) and dialyzed against distilled water (6–8 kDa) Dialysis was stopped when the eluted material contained no sugar, which was ver-ified by phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers,
& Smith, 1956) The polysaccharide fraction (P) was treated with α-amylase (Sigma-Aldrich) and precipitated with ethanol (3:1; v/v) The precipitate was dialyzed against tap water (6–8 kDa, for 24 h), con-centrated under reduced pressure and submitted to freezing and slow thawing (3×) until complete precipitation of cold-water insoluble polysaccharides (Gorin & Iacomini, 1984) After centrifugation (10.000 rpm, at 4 °C, for 20 min), the soluble material, which contained the cold-water soluble polysaccharides (SP), was purified by dialysis against distilled water through a membrane of 1000 kDa Mw cut-off, giving rise to retained (RSP) and eluted (ESP) fractions, that were concentrated under reduced pressure and freeze-dried The yield of each fraction was calculated in comparison to the initial weight of dried mushroom
2.3 Analysis of monosaccharide composition by GC–MS The polysaccharide fractions (1 mg) were hydrolyzed with 2 M TFA
at 100 °C for 8 h, followed by evaporation to dryness The dried car-bohydrate samples were dissolved in distilled water (100μL) and 1 mg NaBH4was added The solution was held at room temperature over-night to reduce aldoses into alditols (Sassaki et al., 2008) The product was dried and excess NaBH4was neutralized by the addition of acetic acid, and removed by the addition of methanol (×2) under a com-pressed air stream in a fume hood Acetylation of the alditols was performed in pyridine–Ac2O (200μL; 1:1, v/v), heated for 30 min at
100 °C The resulting alditol acetates were analyzed by GC–MS, and identified by their typical retention times and electron impact profiles The relative percentage of correspondent monosaccharides was calcu-lated by determination of each peak area with a Varian CP-3800 gas chromatograph coupled to an Ion-Trap 4000 mass spectrometer, using a VF5 column (30 m × 0.25 mm i.d.) programmed from 100 to 280 °C at
10 °C min−1, with He as carrier gas
2.4 Methylation analysis Per-O-methylation of the purified polysaccharide (ESP; 5 mg) was carried out using NaOH-Me2SO-MeI (Ciucanu & Kerek, 1984) After isolation of the products by neutralization (HOAc), dialysis, and eva-poration, the methylation process was repeated The per-O-methylated derivatives were hydrolyzed using 45% aqueous formic acid (1 mL) for
8 h at 100 °C followed by evaporation to dryness The residue was converted into partially O-methylated alditol acetates by reduction with NaBD4and acetylation with pyridine–Ac2O as describe above (Section 2.3), giving rise to a mixture of partially O-methylated alditol acetates, which was analyzed by GC–MS using a VF5 capillary column as de-scribed above (Section2.3) The derivatives were identified by their m/
z of positive ions and retention time, by comparison with standards, and the results were expressed as relative percentage of each component (Sassaki, Gorin, Souza, Czelusniak, & Iacomini, 2005)
2.5 Determination of homogeneity of polysaccharide fractions and molecular weight (Mw) of fucogalactan
The homogeneity of polysaccharide fractions (P, SP and ESP) was determined by high-performance size-exclusion chromatography (HPSEC) coupled to refractive index detector Four gel-permeation Ultrahydrogel columns in series with exclusion sizes of 7 × 106,
4 × 105, 8 × 104, and 5 × 103Da were used The eluent was 0.1 M aq NaNO2 containing 200 ppm aq NaN3 at 0.6 mL min−1 The sample, previously filtered through a membrane (0.22 μm), was injected (100μL loop) at a concentration of 1 mg mL−1
Fig 1 Scheme of extraction and purification of M titans fucogalactan.
Trang 32.6 Determination of relative molar mass
The retention time of fucogalactan (ESP) was compared with a
curve of dextran patterns of different molecular masses (Sigma-Aldrich)
(5.00 × 103; 9.40 × 103; 1.72 × 104; 4.02 × 104; 7.22 × 104;
1.24 × 105; 2.66 × 105; 4.87 × 105; 2.00 × 106), aiming to estimate its
relative molecular weight
2.7 Nuclear magnetic resonance (NMR) spectroscopy
NMR spectra (coupled-HSQC; HSQC-DEPT; COSY; TOCSY) were
obtained using a 400 MHz Bruker model Avance III spectrometer with a
5 mm inverse probe The analyses were performed at 70 °C and samples
(20 mg) were dissolved in D2O Chemical shifts are expressed in ppm
(δ) relative to resonance of acetone at δ 30.2 and 2.22 corresponding to
13C and1H signals, respectively
2.8 Cell culture
The cells used for cell culture were B16-F10 (BCRJ – Banco de
Células do Rio de Janeiro), maintained in Dulbecco’s Modified Eagle’s
Medium – DMEM (ThermoFisher, Waltham, MA, EUA, CAT
12800-017), supplemented with 10% (v/v) fetal bovine serum (FBS)
(ThermoFisher, 12657), 10 mM Hepes (Sigma-Aldrich, H-4034),
0,25μg/mL penicillin-streptomycin in 0,85% saline (ThermoFisher,
15140-148), 3.7 g/L sodium bicarbonate (ThermoFisher, 25080094) at
37 °C in 5% CO2in humidified atmosphere
2.9 Viability and proliferation assays
The B16-F10 cells were cultured in 96-well plates (600 cells/well)
After 24 h, the cells were exposed to treatment (100 or 250μg/mL) for
24, 48 or 72 h, with fucogalactan Afterwards, the cell viability was
determined using Neutral Red (Sigma-Aldrich, N6634) as described in
Borenfreund & Puerner (1985), and proliferation assay was performed
using protocol byGillies, Didier and Denton (1986), in the same plate
(Chiba, Kawakami, & Tohyama, 1998) with modifications All
experi-ments were compared to cells in the absence of treatment
2.10 Morphology and migration assays
The morphology and cell migration were determined using a
mi-croscope EVOS FL Auto (ThermoFisher) Therefore cells (2.5 × 104)
were cultured in 6-well plates, exposed or not (control) to fucogalactan
(100 and 250μg/mL), for 72 h After treatment, images were collected
using phase contrastfilter for analysis of morphology The scratch was
performed with a 10μL tip, cells were washed 2 times with medium,
andfinally added medium with 1% SFB The images were collected
every 30 min, for 10 h This assay was performed according to the
protocol describe byLiang, Park and Guan, (2007)with modifications
The images were analyzed using a T-scratch program (free software by
CSELab, ETH, Zurich, Switzerland)
2.11 Statistical analysis
Statistical analyses was performed using GraphPad Prism 5.0
soft-ware (GraphPad Softsoft-ware®, Inc.), being considered different p < 0.05
value, in different parametric tests, as indicated by each legend
3 Results and discussion
3.1 Isolation and structural characterization of fucogalactan from M titans
The dried and milled mushroom was used to prepare polysaccharide
extracts that were purified as described above (Section2.2) The
pur-ification process was accompanied by analyses of monosaccharide
composition (Table 1) and HPSEC (Fig 2) of the samples obtained after precipitation with ethanol (P), freeze-thawing treatment (SP), and elution through dialysis membrane (ESP) Crude polysaccharide extract (P) contained fucose, mannose, galactose and glucose This fraction (P) showed a heterogeneous elution profile on HPSEC (Fig 2) by the pre-sence of two blunt peaks (at∼43 min and ∼53 min), confirming the necessity of further purification steps
Fungi stock energy is glycogen and some species may contain high amounts of this polysaccharide, which is easily removed byα-amylase treatment (Synytsya & Novák, 2013) Therefore, this enzymatic hy-drolysis was performed followed by freeze and thawing process, giving rise to a soluble polysaccharide fraction (SP), after centrifugation Its elution profile on HPSEC still contained two peaks, although the one at
∼43 min was sharpened (Fig 2) and the glucose content reduced drastically (Table 1), indicating that a partial purification occurred Considering the difference of molecular weight observed on SP chro-matogram (Fig 2), a dialysis through a membrane of 1000 kDa Mw
cut-off was performed as purification step Only fucose and galactose were detected on the eluted fraction (ESP), and its HPSEC profile showed only one sharp peak at∼53 min (Fig 2) The molecular weight of the purified fraction ESP was estimated at 14.2 × 103g/mol
The methylation analysis (Table 2) was consistent with a poly-saccharide composed of (1→ 6)-linked Galp main chain, indicated by the presence of 2,3,4-Me3-Galp (56%) derivatives This fraction also presented 3,4-Me2-Galp (21.9%) and 2,3,4-Me3-Fucp (20.7%) derivates, indicating that the main chain is branched at O-2 position by non re-ducing end units of Fucp in the proportion of each 2–3 units of Galp
Table 1 Yields and monosaccharide composition of fractions obtained from M titans Fraction Yield (%) a Monosaccharides (%) b
Fuc Man Gal Glc
SP 2.0 13.7 7.3 60.2 18.8
a Yields relative to fungus dry weight.
b Alditol acetates obtained after hydrolysis, NaBH 4 reduction, and acetylation.
Fig 2 Elution profile of P, SP and ESP fraction determined by HPSEC (refractive index detector), eluted with 0.1 M NaNO 2
Table 2 Partially-O-methylalditol acetates formed on methylation analysis of fucogalactan iso-lated from M titans.
Partially O-methylated alditol acetates a Sample (mol%) b Linkage type c
2,3,4-Me 3 -Fucp 20.7 Fucp-(1→ 2,3,4,6-Me 4 -Galp 1.4 Galp-(1→ 2,3,4-Me 3 -Galp 56.0 6→)-Galp-(1→ 3,4-Me 2 -Galp 21.9 2,6→)-Galp-(1→
a Analyzed by GC–MS, after methylation, total acid hydrolysis, reduction with NABD 4
and acetylation.
b % of peak area relative to total peak area.
c Based on derived O-methylalditol acetates.
Trang 4Fig 3 HSQC-DEPT spectrum of fucogalactan (ESP), in D 2 O at 70 °C (chemical shifts are expressed in ppm) Correlations of CH 2 groups are flipped to negative phase, and are shown in grey color.
Fig 4 Viability of B16-F10 cells determined by Neutral Red Assay.
C = control (untreated cells); Treated = 100 or 250 μg/mL of Fucogalactan; Time 24, 48 and 72 h The results are representative of three independent experiments with technical quadruplicate Data are shown as mean ± SD, statistical analysis: One-way ANOVA, Tukey post-test, no difference compared with control group.
Fig 5 Proliferation of B16-F10 cells determined by Crystal Violet Assay.
C = control (untreated cells); Treated = 100 or 250 μg/mL of Fucogalactan; Time: 24, 48 and 72 h The results are representative of three independent experiments with technical quadruplicate Data are shown as mean ± SD, statistical analysis: One-way ANOVA, Tukey post-test, no difference compared with control group.
Trang 5The NMR analyses (coupled-HSQC; COSY; TOCSY, data not shown)
contributed to elucidate the chemical structure of the fucogalactan and
the signals were assigned according to the correlation among the NMR
spectra (coupled-HSQC; HSQC-DEPT; COSY; TOCSY) and to literature
values of similar fucogalactans isolated from other basidiomycetes
(Ruthes et al., 2012; Ruthes et al., 2013) The HSQC-DEPT (Fig 3)
showed signals in the anomeric region (C-1/H-1) atδ 101.5/5.07, 98.2/
4.98 and 98.1/5.03 corresponding to Fucp units, 6-O and
2,6-di-O-substituted Galp units, respectively The α-configuration of Galp and
Fucp units was confirmed by the values of the coupling constants J
C-1/H-1observed in1H/13C coupled-HSQC spectrum (data not shown), which
was 171 Hz for Galp and 170,6 Hz for Fucp units (Perlin and Casu,
1969) The substitution at O-2 of 2,6→ )-D-Galp-(1→ was confirmed by
signals atδ 77.8/3.84 and 77.8/3.82 Furthermore, the O-6 substitution
of all Galp units was identified by the negative phase resonances at δ
66.8/3.71 and 66.8/3.91 The signals atδ 15.6/1.25 and 15.5/1.23 are
attributed to CH3of Fucp units
The data showed that the M titans fucogalactan is composed of a
(1→ 6)-linked α-D-Galp main chain that is partially substituted at O-2
position by non reducing end units ofα-L-Fucp residues
3.2 Biological activity of fucogalactan from M titans
The literature data has shown that several polysaccharides present
antitumor activities (Ale, Maruyama, Tamauchi, Mikkelsen, & Meyer,
2011;Hung, Hsu, Chang, & Chen, 2012;Srinivasahan & Durairaj, 2015;
Tong et al., 2009; Zong, Cao, & Wang, 2012), and fucogalactans of
mushrooms have already been evaluated for their biological effect,
presenting a reduction in late mortality rate caused by polymicrobial
sepsis (Ruthes et al., 2012); antinoceptive and anti-inflammatory
ac-tivities in mice (Komura et al., 2010;Ruthes et al., 2013); and an
in-crease of the expression of TNF-α gene and NO by macrophages
(Mizuno et al., 2000)
In the present work, the effect of the fucogalactan isolated from M
titans on melanoma tumor cells was evaluated The cells were exposed
to different concentrations of fucogalactan, for different periods to
determine their viability after incubation with the polysaccharide No
difference was observed among the treatments and controls (Fig 4)
Proliferation of tumor cells is an important target when it is desired
to develop antitumor drugs (Ale et al., 2011; Hung et al., 2012)
Therefore, the fucogalactan was evaluated on a cell proliferation assay,
however no alteration was observed after incubation of B16-F10 cells
with different concentrations of fucogalactan (Fig 5) The next step was
to assess whether the treatment could affect the morphology of cells
Tumor cell morphology is quite characteristic, as seen in Fig 6
(control), showing adherent cells as well as rounded cells The treated
groups (100 or 250μg/mL) presented the same morphology of cancer
cells: as spindle-shaped and epithelial-like cells and stacked up on each
other (Fig 6)
Thus, without changes in cell viability, proliferation and
mor-phology, we then analyzed an important cell malignancy parameter,
which was cell migration
It is known that cell migration is one of the major cellular events and it is considered a hallmark of cancer, because it is through this mechanism that tumor cell can achieve metastasis and thus generate new secondary tumors (Hanahan & Weinberg, 2000, 2011)
After incubation of B16-F10 cells with fucogalactan, it was observed that this polysaccharide was able to change the migration behavior (Fig 7) As shown inFig 7, it can be seen that after 10 h the control cells migrated until closing the scratched area (0% open area) How-ever, the cells treated with both concentrations of fucogalactan (100 or
250μg/mL) still presented an average open area of 40% and 33%, re-spectively, after the same period The inhibition of cell migration was statistically different (**** p < 0.0001) when treated groups were compared with control group
By the results obtained, it was demonstrated that the fucogalactan extracted from Macrocybe titans does not alter the viability, prolifera-tion or morphology of melanoma cells However, it was able to reduce their migration, showing that this polysaccharide presented some an-titumor effect in vitro even with no toxicity against the tested cells Many authors correlate antitumor capacity of some drugs with cy-totoxicity or loss of cell viability (Ivanova, Krupodorova, Barshteyn, Artamonova, & Shlyakhovenko, 2014;Shang et al., 2011;Srinivasahan
& Durairaj, 2015) However, in this case, there is a great possibility of such drugs to damage or kill normal and healthy cells Thus, recently there is a search for compounds that do not alter the cellular viability, but rather cause cellular modifications that lead to antitumor effects, with minimum side effects (Biscaia et al., 2017;Meng, Liang, & Luo,
2016; Novaes, Valadares, Reis, Gonçalves, & Menezes, 2011; Tong
et al., 2009) Based on this, the results presented on this study showed a promising agent against melanoma cells, that is candidate of new stu-dies to evaluate its antitumor effect in vivo
4 Conclusion
A heteropolysaccharide was obtained from the giant edible mush-room Macrocybe titans by aqueous extraction It was chemically char-acterized and its biological activity on melanoma cells was evaluated GC–MS, NMR and HPSEC analyzes showed that this polysaccharide is a homogeneous fucogalactan with a (1→ 6)-linked α-D-Galp main chain partially substituted at O-2 by non reducing end units ofα-L-Fucp re-sidues in the side chain The activity of Macrocybe titans fucogalactan against B16-F10 cells wasfirstly reported in this study This polymer showed a promising antitumor effect because it did not affect the cell viability, proliferation and morphology; however it inhibited the mi-gration of melanoma cells Considering that M titans is a mushroom that reaches around 30 kg and therefore could provide high yields of polysaccharides; and that the purified fucogalactan showed potential effect against melanoma cells, the study of this mushroom showed to be interesting and should be continued in vivo
Fig 6 Morphology of B16-F10 cells after incubation with fucogalactan Cells were treated with fucogalactan (100 μg/mL or 250 μg/mL), for 72 h and the treatment did not alter the cell morphology Control cells received no treatment.
Trang 6The authors would like to thank the Brazilian funding agencies
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
and Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq)
References
Ale, M T., Maruyama, H., Tamauchi, H., Mikkelsen, J D., & Meyer, A S (2011)
Fucose-containing sulfated polysaccharides from brown seaweeds inhibit proliferation of
melanoma cells and induce apoptosis by activation of caspase-3 in vitro Marine
Drugs, 9(12), 2605–2621 http://dx.doi.org/10.3390/md9122605
Baby, S., Johnson, A J., & Govindan, B (2015) Secondary metabolites from Ganoderma.
Phytochemistry, 114, 66–101 http://dx.doi.org/10.1016/j.phytochem.2015.03.010 Bae, J S., Jang, K H., Yim, H., & Jin, H K (2005) Polysaccharides isolated from Phellinus gilvus inhibit melanoma growth in mice Cancer Letters, 218, 43–52 http://dx.doi org/10.1016/j.canlet.2004.08.002
Biscaia, S M P., Carbonero, E R., Bellan, D L., Borges, B S., Costa, C R., Rossi, G R., & Trindade, E S (2017) Safe therapeutics of murine melanoma model using a novel antineoplasic, the partially methylated mannogalactan from Pleurotus eryngii Carbohydrate Polymers, 178, 95–104 http://dx.doi.org/10.1016/j.carbpol.2017.08.
117 Borenfreund, E., & Puerner, J A (1985) A simple quantitative procedure using mono-layer cultures for cytotoxicity assays (HTD/NR-90) Journal of Tissue Culture Methods, 9(1), 7–9 http://dx.doi.org/10.1007/BF01666038
Carbonero, E R., Gracher, A H P., Freitas, C S., Komura, D L., Santos, A., Baggio, Iacomini, M (2008) Lentinus edodes heterogalactan: Antinociceptive and anti-in-flamatory efects Food Chemistry, 111, 531–537 http://dx.doi.org/10.1016/j phytochem.2007.06.018
Chiba, K., Kawakami, K., & Tohyama, K (1998) Simultaneous evaluation of cell viability
Fig 7 Cell migration Assay Cells were treated with fucogalactan (100 μg/mL or 250 μg/mL), for 72 h Control cells received no treatment On time 0 h, a scratch was produced on cells and the migration process was recorded during 10 h Fucogalactan altered cell migration in both concentrations Data are shown as mean ± SD, statistical analysis: One-way ANOVA, Dunnett post-test, **** p < 0.0001 compared with control group.
Trang 7by neutral red, MTT and crystal violet staining assays of the same cells Toxicology In
Vitro, 12(3), 251–258 http://dx.doi.org/10.1016/S0887-2333(97)00107-0
Ciucanu, I., & Kerek, F (1984) A simple and rapid method for the permethylation of
carbohydrates Carbohydrate Research, 131(2), 209–217 http://dx.doi.org/10.1016/
0008-6215(84)85242-8
Cui, F., Zan, X., Li, Y., Yang, Y., Sun, W., Zhou, Q., & Dong, Y (2013) Purification and
partial characterization of a novel anti-tumor glycoprotein from cultured mycelia of
Grifola frondosa International Journal of Biological Macromolecules, 62, 684–690.
http://dx.doi.org/10.1016/j.ijbiomac.2013.10.025
Ding, X., Hou, Y L., & Hou, W R (2012) Structure elucidation and antioxidant activity of
a novel polysaccharide isolated from Boletus speciosus Forst International Journal of
Biological Macromolecules, 50(3), 613–618 http://dx.doi.org/10.1016/j.ijbiomac.
2012.01.021
Dubois, M., Gilles, K A., Hamilton, J K., Rebers, P A., & Smith, F (1956) Colorimetric
method for determination of sugars and related substances Analytical Chemistry, 28,
350–356 http://dx.doi.org/10.1021/ac60111a017
Gillies, R J., Didier, N., & Denton, M (1986) Determination of cell number in monolayer
cultures Analytical Biochemistry, 159(1), 109–113
http://dx.doi.org/10.1016/0003-2697(86)90314-3
Gorin, P A J., & Iacomini, M (1984) Polysaccharides of the lichens Cetraria islandica and
Ramalina usnea Carbohydrate Research, 128(1), 119–132 http://dx.doi.org/10.1016/
0008-6215(84)85090-9
Hamel, J F., Pe, M., Coens, C., Martinelli, F., Eggermont, A M M., Brandberg, Y., &
Bottomley, A (2016) A systematic review examining factors influencing health
re-lated quality of life among melanoma cancer survivors European Journal of Cancer,
69, 189–198 http://dx.doi.org/10.1016/j.ejca.2016.10.008
Han, S B., Lee, C W., Kang, J S., Yoon, Y D., Lee, K H., Lee, K., & Kim, H M (2006).
Acidic polysaccharide from Phellinus linteus inhibits melanoma cell metastasis by
blocking cell adhesion and invasion International Immunopharmacology, 6, 697–702.
http://dx.doi.org/10.1016/j.intimp.2005.10.003
Hanahan, D., & Weinberg, R A (2000) The hallmarks of cancer Cell, 100, 57–70 http://
dx.doi.org/10.1007/s00262-010-0968-0
Hanahan, D., & Weinberg, R A (2011) Hallmarks of cancer: The next generation Cell,
144(5), 646–674 http://dx.doi.org/10.1016/j.cell.2011.02.013
Harries, M., Malvehy, J., Lebbe, C., Heron, L., Amelio, J., Szabo, Z., & Schadendorf, D.
(2016) Treatment patterns of advanced malignant melanoma (stage III-IV) – a review
of current standards in Europe European Journal of Cancer, 60, 179–189 http://dx.
doi.org/10.1016/j.ejca.2016.01.011
Hung, C., Hsu, B., Chang, S., & Chen, B (2012) Antiproliferation of melanoma cells by
polysaccharide isolated from Zizyphus jujuba Nutrition, 28(1), 98–105 http://dx.
doi.org/10.1016/j.nut.2011.05.009
Ivanova, T S., Krupodorova, T A., Barshteyn, V Y., Artamonova, A B., & Shlyakhovenko,
V A (2014) Anticancer substances of mushroom origin Experimental Oncology,
36(2), 58–66
Jeff, I B., Li, S., Peng, X., Kassim, R M R., Liu, B., & Zhou, Y (2013) Purification,
structural elucidation and antitumor activity of a novel mannogalactoglucan from the
fruiting bodies of Lentinus edodes Fitoterapia, 84, 338–346 http://dx.doi.org/10.
1016/j.fitote.2012.12.008
Komura, D L., Carbonero, E R., Gracher, A H., Baggio, C H., Freitas, C S., Marcon, R., &
Iacomini, M (2010) Structure of Agaricus spp fucogalactans and their
anti-in-flammatory and antinociceptive properties Bioresource Technology, 101, 6192–6199.
http://dx.doi.org/10.1016/j.biortech.2010.01.142
Liang, C., Park, A Y., & Guan, J (2007) In vitro scratch assay: A convenient and
in-expensive method for analysis of cell migration in vitro Nature Protocols, 2(2),
329–333 http://dx.doi.org/10.1038/nprot.2007.30
Liu, M M., Zeng, P., Li, X T., & Shi, L G (2016) Antitumor and immunomodulation
activities of polysaccharide from Phellinus baumii International Journal of Biological
Macromolecules, 91, 1199–1205 http://dx.doi.org/10.1016/j.ijbiomac.2016.06.086
Meng, X., Liang, H., & Luo, L (2016) Antitumor polysaccharides from mushrooms: A
review on the structural characteristics, antitumor mechanisms and
im-munomodulating activities Carbohydrate Research, 424, 30–41 http://dx.doi.org/10.
1016/j.carres.2016.02.008
Meng, M., Cheng, D., Han, L., Chen, Y., & Wang, C (2017) Isolation, purification,
structural analysis and immunostimulatory activity of water-soluble polysaccharides
from Grifola Frondosa fruiting body Carbohydrates Polymers, 157, 1134–1143 http://
dx.doi.org/10.1016/j.carbpol.2016.10.082
Mizuno, M., Shiomi, Y., Minato, K I., Kawakami, S., Ashida, H., & Tsuchida, H (2000).
Fucogalactan isolated from Sarcodon aspratus elicits release of tumor necrosis factor-a
and nitric oxide from murine macrophages Immunopharmacology, 46, 113–121.
http://dx.doi.org/10.1016/S0162-3109(99)00163-0
Moreno, R B., Ruthes, A C., Baggio, C H., Vilaplana, F., Komura, D L., & Iacomini, M.
(2016) Structure and antinociceptive effects of β-D-glucans from Cookeina
tricho-loma Carbohydrates Polymers, 141, 220–228 http://dx.doi.org/10.1016/j.carbpol.
2016.01.001
Novaes, M R C G., Valadares, F., Reis, M C., Gonçalves, D R., & Menezes, M C (2011).
The effects of dietary supplementation with Agaricales mushrooms and other
med-icinal fungi on breast cancer: Evidence-based medicine Clinics, 66(12), 2133–2139.
http://dx.doi.org/10.1590/S1807-59322011001200021
Patel, S., & Goyal, A (2012) Recent developments in mushrooms as anti-cancer
ther-apeutics: A review 3 Biotech, 2, 1–15
http://dx.doi.org/10.1007/s13205-011-0036-2
Pegler, D N., Lodge, D J., & Nakasone, K K (1998) The pantropical genus Macrocybe
gen nov Mycologia, 90(3), 494–504 http://dx.doi.org/10.2307/3761408
Perlin, A S., & Casu, B (1969) Carbon-13 and proton magnetic resonance spectra of
D-glucose- 13 C Tetrahedron Letters, 34, 2919–2924
http://dx.doi.org/10.1016/S0040-4039(01)88308-8
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 the fruit bodies of edible basidiomycetes Pleurotus ostreatus “florida” Berk and Pleurotus ostreatoroseus Sing FEMS Microbiology Letters, 221, 119–124 http://dx.doi org/10.1016/S0378-1097(03)00161-7
Ruthes, A C., Rattmann, Y D., Carbonero, E R., Gorin, P A J., & Iacomini, M (2012) Structural characterization and protective effect against murine sepsis of fucoga-lactans from Agaricus bisporus and Lactarius rufus Carbohydrate Polymers, 87, 1620–1627 http://dx.doi.org/10.1016/j.carbpol.2011.09.071
Ruthes, A C., Rattmann, Y D., Malquevicz-Paiva, S M., Carbonero, E R., Córdova, M M., Baggio, C H., & Iacomini, M (2013) Agaricus bisporus fucogalactan: Structural characterization and pharmacological approaches Carbohydrate Polymers, 92, 184–191 http://dx.doi.org/10.1016/j.carbpol.2012.08.071
Ruthes, A C., Smiderle, F R., & Iacomini, M (2016) Mushroom heteropolysaccharides: A review on their sources, structure and biological effects Carbohydrate Polymers, 136, 358–375 http://dx.doi.org/10.1016/j.carbpol.2015.08.061
Samanta, S., Nandi, A K., Sen, I K., Maity, P., Pattanayak, M., & Islam, S S (2015) Studies on antioxidative and immunostimulating fucogalactan of the edible mush-room Macrolepiota dolichaula Carbohydrate Research, 413, 22–29 http://dx.doi.org/ 10.1016/j.carres.2015.05.006
Sassaki, G L., Gorin, P A J., Souza, L M., Czelusniak, P A., & Iacomini, M (2005) Rapid synthesis of partially O-methylated alditol acetate standards for GC–MS: Some re-lative activities of hydroxyl groups of methyl glycopyranosides on Purdie methyla-tion Carbohydrate Research, 340, 731–739 http://dx.doi.org/10.1016/j.carres.2005 01.020
Sassaki, G L., Souza, L M., Serrato, R V., Cipriani, T R., Gorin, P A J., & Iacomini, M (2008) Application of acetate derivaties for gas chromatography mass spectrometry: Novel approaches on carbohydrates, lipids and amino acids analysis Journal of Chromatography A, 1208, 215–222 http://dx.doi.org/10.1016/j.chroma.2008.08.
083 Shang, D., Li, Y., Wang, C., Wang, X., Yu, Z., & Fu, X (2011) A novel polysaccharide from Se-enriched Ganoderma lucidum induces apoptosis of human breast cancer cells Oncology Reports, 25, 267–272 http://dx.doi.org/10.3892/or_00001070 Silveira, M L L., Smiderle, F R., Agostini, F., Pereira, E M., Bonatti-Chaves, M., Wisbeck, E., Iacomini, M (2015) Exopolysaccharide produced by Pleurotus sajor-caju: Its chemical structure and anti-inflammatory activity International Journal of Biological Macromolecules, 75, 90–96 http://dx.doi.org/10.1016/j.ijbiomac.2015.01.023 Smiderle, F R., Carbonero, E R., Sassaki, G L., Gorin, P A J., & Iacomini, M (2008) Characterization of a heterogalactan and some nutricional values present in the ed-ible mushroom Flammulina velutipes Food Chemistry, 108, 329–333 http://dx.doi org/10.1016/j.foodchem.2007.10.029
Smiderle, F R., Sassaki, G L., Griensven, L J L D V., & Iacomini, M (2013) Isolation and chemical characterization of a glucogalactomannan of the medicinal mushroom Cordyceps militaris Carbohydrate Polymers, 97, 74–80 http://dx.doi.org/10.1016/j carbpol.2013.04.049
Smiderle, F R., Ruthes, A C., & Iacomini, M (2014) Natural polysaccharides from mushrooms: Anti-nociceptive and anti-inflammatory properties In J.-M Merillon, &
K G Ramawat (Eds.) Polysaccharides – bioactivity and biotechnology (pp 1–25) Berlin Heidelberg: Springer-Verlag http://dx.doi.org/10.1007/SpringerReference_405358
Srinivasahan, V., & Durairaj, B (2015) In vitro and apoptotic activity of polysaccharide rich Morinda citrofolia fruit on MCF-7 cells Asian Journal of Pharamceutical and Clinical Research, 8(2), 190–193
Sun, Y., & Liu, J (2009) Purification, structure and immunobiological activity of a water-soluble polysaccharide from the fruiting body of Pleurotus ostreatus Bioresource Technology, 100, 983–986 http://dx.doi.org/10.1016/j.biortech.2008.06.036 Synytsya, A., & Novák, M (2013) Structural diversity of fungal glucans Carbohydrate Polymers, 92, 792–809 http://dx.doi.org/10.1016/j.carbpol.2012.09.077 Tian, Y., Zhao, Y., Zeng, H., Zhang, Y., & Zheng, B (2016) Structural characterization of
a novel neutral polysaccharide from Lentinus giganteus and its antitumor activity through inducing apoptosis Carbohydrate Polymers, 154, 231–240 http://dx.doi.org/ 10.1016/j.carbpol.2016.08.059
Tong, H., Xia, F., Feng, K., Sun, G., Gao, X., Sun, L., & Sun, X (2009) Structural char-acterization and in vitro antitumor activity of a novel polysaccharide isolated from the fruiting bodies of Pleurotus ostreatus Bioresource Technology, 100(4), 1682–1686.
http://dx.doi.org/10.1016/j.biortech.2008.09.004 Wang, D., Sun, S Q., Wu, W Z., Yang, S L., & Tan, J M (2014) Characterization of a water-soluble polysaccharide from Boletus edulis and its antitumor and im-munomodulatory activities on renal cancer in mice Carbohydrate Polymers, 105, 127–134 http://dx.doi.org/10.1016/j.carbpol.2013.12.085
Wasser, S P (2011) Current findings, future trends, and unsolved problems in studies of medicinal mushrooms Applied Microbiology and Biotechnology, 89, 1323–1332.
http://dx.doi.org/10.1007/s00253-010-3067-4
World Health Organization (2017) Cancer [Retrieved from http://www.who.int/med-iacentre/factsheets/fs297/en/]
Zhang, W., Yang, J., Chen, J., Hou, Y., & Han, X (2005) Immunomodulatory and anti-tumour effects of na exopolysaccharide fraction from cultivated Cordyceps sinensis (Chinese caterpillar fungus) on tumour-bearing mice Biotechnology and Applied Biochemistry, 42, 9–15 http://dx.doi.org/10.1042/BA20040183
Zhang, Y., Liu, Z., Ng, T B., Chen, Z., Qiao, W., & Liu, F (2014) Purification and characterization of a novel antitumor protein with antioxidant and deoxyr-ibonuclease activity from edible mushroom Pholiota nameko Biochimie, 99, 28–37.
http://dx.doi.org/10.1016/j.biochi.2013.10.016 Zong, A., Cao, H., & Wang, F (2012) Anticancer polysaccharides from natural resources:
A review of recent research Carbohydrate Polymers, 90(4), 1395–1410 http://dx.doi org/10.1016/j.carbpol.2012.07.026