An α-D-galactan and a β-D-glucan from the mushroom Amanita muscaria: Structural characterization and antitumor activity against melanoma

Polysaccharides α-D-galactan (GAL-Am) and β-D-glucan (GLC-Am) were obtained from Amanita muscaria fruiting bodies. They were purified using different methodologies, such as Fehling precipitation (for both fractions), freeze-thawing process and ultrafiltration (for GLC-Am).

Carbohydrate Polymers 274 (2021) 118647 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol An α-D-galactan and a β-D-glucan from the mushroom Amanita muscaria: Structural characterization and antitumor activity against melanoma Matheus Zavadinack a, Daniel de Lima Bellan b, Jessica Loren da Rocha Bertage a, Shayane da Silva Milhorini a, Edvaldo da Silva Trindade b, Fernanda Fogagnoli Simas b, Guilherme Lanzi Sassaki a, Lucimara M.C Cordeiro a, Marcello Iacomini a, * a b Department of Biochemistry and Molecular Biology, Federal University of Paran´ a, Curitiba, PR CEP 81531-980, Brazil Department of Cell Biology, Federal University of Paran´ a, Curitiba, PR CEP 81531-980, Brazil A R T I C L E I N F O A B S T R A C T Keywords: A muscaria Polysaccharides α-Galactan β-Glucan Chemical structure Antimelanoma properties Polysaccharides α-D-galactan (GAL-Am) and β-D-glucan (GLC-Am) were obtained from Amanita muscaria fruiting bodies They were purified using different methodologies, such as Fehling precipitation (for both fractions), freeze-thawing process and ultrafiltration (for GLC-Am) Results showed that the GAL-Am has (1 → 6)-linked Galp main chain branched at O-2 by terminal Galp units and has not been previously reported Besides, GLC-Am has (1 → 3)-linked Glcp in the main chain, substituted at O-6 by (1 → 6)-linked β-Glcp units Both are watersoluble, with 9.0 × 103 g/moL and 1.3 × 105 g/moL, respectively GAL-Am and GLC-Am presented a selec­ tive proliferation reduction against B16-F10 melanoma cell line, not affecting non tumoral BALB/3T3 fibroblast cell line Furthermore, both fractions reduced clonogenic capacity of melanoma cell line over an extended period of time These results were obtained without modulations in B16-F10 cell adhesion, reinforcing the biological activities towards cell proliferation impairment and eliciting these polysaccharides as promising compounds to further exploration of their antimelanoma properties Introduction The fungal fruiting body, popularly known as mushroom, is an important source of nutrients and fibers, such as β-glucans (Abreu et al., 2019), being considered a delicacy food in many cultures and countries (White et al., 2019) Commonly known as fly agaric mushroom, A muscaria (L.:Fr) has toxic and hallucinogenic molecules in its composition such as muscimol, muscarina, ibotenic acid and muscazone, besides heterocyclic alkaloids in small amounts (Kondeva-Burdina et al., 2019; Ruthes et al., 2013a,b) Despite crude mushroom show toxicity, it is consumed in regions of Europe, North America and Japan after boiling in salt water and steeping in vinegar (Coville, 1898; Kiho et al., 1992) In addition, edible mushrooms present a diverse range of biological properties, including immunomodulatory, hypolipidemic, antibacterial, anti-inflammatory, hepatoprotective and antitumor effects (Morales et al., 2020) Amongst mushrooms biological activities, their antitumor properties are especially relevant and explored Cancer stands out as the second leading cause of death worldwide (Bray et al., 2018) One of the few cancer types with a rising incidence rate is melanoma, the most lethal form of skin cancer given its fast progression to the metastatic stage (Ward & Farma, 2017) Although recent advances in targeted and im­ munotherapies, cancer and particularly melanoma treatment are mostly palliative and accompanied of several debilitating adverse effects (Ramos-Casals et al., 2020), thus engendering the search for new ther­ apeutic approaches Polysaccharides obtained from mushrooms have already demonstrated its potential to modulate malignancy related traits in cancer models, reducing cell proliferation, inducing selective cyto­ toxicity, impairing migration and metastasis and decreasing tumor growth (Kothari et al., 2018; Ren et al., 2012) Their pharmacological relevance already reached cancer clinical trials in China and Japan, as illustrated by PSP - a heteropolysaccharide mainly constituted of Dglucose with a main chain α-(1 → 4) and β-(1 → 3)-linked units - and PSK – mainly constituted of glucan β-(1 → 4)-linked with (1 → 6)-β-gluco­ pyranosidic side chains – both obtained from the mushroom Coriolus versicolor (Habtemariam, 2020; Kobayashi et al., 1995) It is known that polysaccharides biological properties are * Corresponding author E-mail address: iacomini@ufpr.br (M Iacomini) https://doi.org/10.1016/j.carbpol.2021.118647 Received 31 March 2021; Received in revised form September 2021; Accepted September 2021 Available online September 2021 0144-8617/© 2021 Elsevier Ltd This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/) M Zavadinack et al Carbohydrate Polymers 274 (2021) 118647 Fig Extraction and purification scheme of the β-D-glucan (GLC-Am) and the α-D-galactan (GAL-Am) from A muscaria (L.:Fr) Fig HSQC spectra of the α-galactan (GAL-Am) The sample was analyzed in D2O at 70 ◦ C in a Bruker Avance III 600 MHz (chemical shifts are expressed in δ ppm) intrinsically linked to their composition and chemical structure (Ruthes et al., 2013a,b; Vetvicka & Yvin, 2004) and there is limited information in the literature regarding polysaccharides from A muscaria Ruthes et al (2013a,b) isolated a fucomannogalactan formed by a (1 → 6)linked α-D-galactopyranosyl main chain partially substituted for nonreducing end units at O-2 mainly by α-L-Fucp and β-D-Manp and a (1 → 3),(1 → 6) β-D-glucan Regarding biological effects of these polysaccharides, the (1 → 3),(1 → 6) β-D-glucan showed potent inhibi­ tion of inflammatory pain (Ruthes et al., 2013a,b) and significant anti­ tumor activity against Sarcoma 180 in mice (Kiho et al., 1992) Therefore, in the present study we advance in the knowledge of A muscaria polysaccharides, describing for the first time the chemical structure of an unmethylated α-D-galactan, not yet reported for mush­ rooms, as well as the structure of a β-D-glucan Moreover, their possible M Zavadinack et al Carbohydrate Polymers 274 (2021) 118647 Fig 2D NMR analysis of the GAL-Am fraction COSY (A), TOCSY (B), HMBC and 1H (C), and HSQC-TOCSY - in blue and green - with superimposed HSQC - in red - (D) corre­ lation maps of the α-D-galactan (GAL-Am) from A muscaria (L.:Fr) The sample was analyzed in D2O at 70 ◦ C in a Bruker Avance III 600 MHz (chemical shifts are expressed in δ ppm) n = carbon number from the sugar ring The symbols ' and " indicate the units 2,6→)-α-D-Galp-(1 → and α-D-Galp(1→, respectively, while the absence of them represent the 6→)-α-D-Galp-(1 → units M Zavadinack et al Carbohydrate Polymers 274 (2021) 118647 Fig (continued) Fig 2D NOESY analysis of the GAL-Am fraction The sample was analyzed in D2O at 70 ◦ C in a Bruker Avance III 600 MHz (chemical shifts are expressed in δ ppm) antimelanoma activities were further explored against the murine mel­ anoma B16-F10 cell line 2.2 General extraction and purification processes General extraction and purification processes performed on the A muscaria are represented in the flowchart (Fig 1) The extraction processes were carried out using 270.86 g dehydrated fruiting bodies The material was defatted with chloroform-methanol (2:1; v/v) at 65 ◦ C for h using a Soxhlet apparatus Then, the residue was submitted to aqueous extraction at room temperature (25 ◦ C) for h (5×, L each) under stirring The aqueous extract was concentrated, and the poly­ saccharides precipitated by ethanol addition (3:1, v/v), collected by centrifugation (20 min, 8000 rpm, 25 ◦ C) and dialyzed (6–8 kDa membrane) against tap water for 24 h Insoluble materials that formed during the dialysis process were removed by centrifugation (20 min, Material and methods 2.1 Biological material A muscaria fruiting bodies were collected at the Biological Sciences Sector gardens (25◦ 26′ 48.4′′ S 49◦ 14′ 16.5′′ W) of the Federal University of Paran´ a in Curitiba, Paran´ a - Brazil The respective mushrooms were cleaned, freeze-dried and then grounded to a powder M Zavadinack et al Carbohydrate Polymers 274 (2021) 118647 Table Chemical shiftsa of the correlation between the 1H/1H and 1H/13C of the adjacent carbons in monosaccharides units of the GAL-Am 6→)-α-Galp-(1→ 1 H-1/ H-2 H-2/13C-3 H-3/1H-4 H-4/1H-5 H-5/13C-6a H-5/13C-6b a b c d Correlations 2,6→)-α-Galp-(1→ b 4.98/3.88 3.88/68.4c 3.85/4.03b 4.03/4.17b 3.91/66.7c 3.71/66.7c Correlations d b H-1/ H-2 C-2/1H-3 H-3/13C-4 H-4/1H-5 13 C-5/1H-6a 13 C-5/1H-6b 5.02/3.97 75.1/3.98c 3.98/66.2c 4.25/4.17b 68.8/3.91c 68.8/3.71c 13 α-Galp-(1→ 1 5.15/3.88b 3.88/69.5b 3.94/4.03d 4.03/4.18d 4.18/3.76c – H-1/ H-2 H-2/13C-3 H-3/1H-4 H-4/1H-5 H-5/1H-6c – H/13C Assignments are based on 1H and HSQC analysis and are expressed as ppm Signals in COSY spectrum Signals in HSQC-TOCSY/HSQC spectrum Signals in TOCSY spectrum 10,000 rpm, 25 ◦ C) The soluble polysaccharides were named S-Am and submitted to Fehling treatment (Brito et al., 2018) Cu2+-complexed polysaccharides recovered by centrifugation (20 min, 10,000 rpm, 25 ◦ C) gave rise to the fraction (I-1) while the Cu2+-uncomplexed polysaccharides were named as S-1 fraction Both were neutralized with HOAc, dialyzed (2 kDa cut-off membrane) and deionized with mixed ion exchange resins and then freeze dried The latter one (S-1) was subjected to freeze-thawing (Gorin & Iacomini, 1984) cycles until no more coldwater insoluble polysaccharides appear After each cycle, the precipi­ tated fraction was recovered by centrifugation (10,000 rpm, 20 min, ◦ C) and the soluble portion was named as S-2 This was again treated with Fehling solution and Cu2+-uncomplexed polymers (fraction S-3) were further submitted to ultrafiltration (Millipore®; polyethersulfone; kDa membrane) on a filter holder (Sartorius – Model 16,249) with compressed air at 10 psi carrier gas, generating the retained (GLC-Am) and the eluted polysaccharide Sample I-1, due to the presence of contaminating glucans, suffered a second precipitation with Fehling reagent, originating a purified gal­ actan polysaccharide (GAL-Am) 2.4 Methylation analysis by GC–MS GAL-Am and GLC-Am were subjected to methylation process adapted from Ciucanu and Kerek (1984) by dissolving the sample in DMSO followed by addition of NaOH powder and methyl iodide, kept under stirring for 30 at 25 ◦ C Samples were maintained overnight; the reaction was neutralized with acetic acid and lyophilized The methylation process was repeated thrice for each sample The per-Omethylated derivatives were hydrolyzed with 45% formic acid (1 mL) at 100 ◦ C for 15 h (Carbonero et al., 2012) Acid excess was removed by lyophilization followed by reduction with NaBD4 and acetylation generating a mixture of partially O-methylated alditol acetates de­ rivatives which were analyzed by GC–MS using a Varian (model 4000) gas chromatograph equipped with VF5-MS capillary columns The injector temperature was maintained at 210 ◦ C and the oven tempera­ ture increased from 50 ◦ C (maintained min) to 90 ◦ C (20 ◦ C/min, then maintained for min), 180 ◦ C (5 ◦ C/min, then maintained for min) and to 210 ◦ C (3 ◦ C/min, then maintained for min) Helium was used as the carrier gas at a flow rate of 1.0 mL/min Partially O-methylated alditol acetates were identified by the ion m/z by comparing their pos­ itive ions with standards The results are expressed as a relative per­ centage of each component (Sassaki et al., 2005) 2.3 Monosaccharide composition Polysaccharides (5 mg) were hydrolyzed with 2M TFA at 100 ◦ C for h and analyzed by GC–MS as alditol acetate derivatives using a Varian (model CP-3800) gas chromatograph coupled to an Ion-Trap 4000 mass spectrometer using a VF5-MS column programmed from 100 to 280 ◦ C at 10 ◦ C/min, with He as the carrier gas The monosaccharides obtained were identified by their typical retention times and electron impact profiles in comparison with standards (Sassaki et al., 2008) 2.5 Controlled Smith degradation of the β-glucan (GLC-Am) Fraction GLC-Am (50 mg) was solubilized in 25 mL of distilled water and oxidized with 25 mL of 0.1 M sodium periodate at room temperature in the dark under stirring for 72 h (Delgobo et al., 1998) The material was then dialyzed (2 kDa, cut-off membrane) for 24 h against tap water Subsequently it was reduced with NaBH4 to pH ~8 and kept at room Table H and 13C NMR chemical shiftsa of fractions GAL-Amb, GLC-Amc and GLC-Smd from A muscaria (L.:Fr) Fractions Units GAL-Am 6→)-α-D-Galp-(1→ α-D-Galp-(1→ 2,6→)-α-D-Galp-(1→ GLC-Am β-D-Glcp-(1→ 3→)-β-D-Glcp-(1→ 3,6→)-β-D-Glcp-(1→ GLC-Sm a b c d 3→)-β-D-Glcp-(1→ 13 C H 13 C H 13 C H 13 C H 13 C H 13 C H 13 C H 98.1 4.98 95.7 5.15 98.0 5.02 103.2 4.27 103.2 4.55 103.2 4.55 103.0 4.53 69.6 3.88 69.6 3.88 75.1 3.97 73.7 3.09 72.8 3.35 72.8 3.35 72.9 3.31 68.4 3.85 69.5 3.94 66.9 3.98 76.7 3.14 86.3/86.7 3.50 86.1 3.51 86.2 3.49 69.5 4.03 69.5 4.03 66.2 4.25 70.2 3.14 68.7 3.29 68.7 3.29 68.4 3.25 68.8 4.17 71.0 4.18 68.8 4.17 76.4 3.29 76.4 3.29 74.8 3.54 76.4 3.27 Assignments are based on 13C NMR, 1H and HSQC analysis and are expressed as ppm GAL-Am: α-D-galactan isolated from A muscaria GLC-Am: β-D-glucan isolated from A muscaria GLC-Sm: GLC-Am that was submitted to a controlled Smith degradation, according to material and methods, Section 2.5 a b 66.7 3.91 61.2 3.76 66.7 3.91 60.9 3.72 60.9 3.72 68.5 4.11 60.9 3.71 66.7 3.71 61.2 3.76 66.7 3.71 60.9 3.50 60.9 3.50 68.5 3.58 60.9 3.48 M Zavadinack et al Carbohydrate Polymers 274 (2021) 118647 Table Partially O-methylated alditol acetates of the α-D-galactan (GAL-Am) and β-D-glucan (GLC-Am) polysaccharides purified from A muscaria Partially O-methylated alditol acetatesa Rtb Relative peak area %c d 2,3,4,6-Me4-Gal 2,3,4,6-Me4-Glc 2,3,4-Me3-Gal 2,4,6-Me3-Glc 2,3,4-Me3-Glc 2,4-Me2-Glc 3,4-Me2-Gal a b c d e 14.678 14.414 16.214 15.423 15.802 17.057 17.418 Linkage types GAL-Am GLC-Am 15 – 71 – – – 14 – 19 – 45 13 23 – e Galp-(1→ Glcp-(1→ 6→)-Galp-(1→ 3→)-Glcp-(1→ 6→)-Glcp-(1→ 3,6→)-Glcp-(1→ 2,6→)-Galp-(1→ GC–MS analysis on a Varian 4000 capillary column; Retention time (min) Based on derived O-methylalditol acetates GAL-Am: α-D-galactan isolated from A muscaria GLC-Am: β-D-glucan isolated from A muscaria temperature for 20 h, neutralized with acetic acid and dialyzed (2 kDa) for 24 h Then, it was hydrolyzed with TFA pH 2.0 at 100 ◦ C for 30 min, dialyzed (2 kDa) (Ruthes et al., 2015), subsequently precipitated by ethanol (3:1, v/v) and lyophilized The residual polysaccharide was named GLC-Sm and analyzed by NMR spectroscopy during 24 h and transferred to a mm NMR tube The analyses were carried out at temperature of 70 ◦ C The chemical shifts are expressed in δ (ppm) relative to signals from solvent Me2SO-d6 at δ 39.7 (13C) and 2.5 (1H) or external reference of acetone (at δ 30.2 and 2.22 for 13C and 1H, respectively) 2.6 Determination of homogeneity and relative molecular weight 2.8 Total sugar and protein content determination The homogeneity and relative Mw were determined by high perfor­ mance steric exclusion chromatography (HPSEC) using a refractive index (RI) detector and Waters Ultrahydrogel columns – 120, 250, 500 and 2000 were coupled in series The eluent was 0.1 M NaNO3, con­ taining 0.5 g/L NaN3 Each sample was dissolved in the solvent (1 mg/ mL) and filtered through a membrane (0.22 μm, Millipore) The relative Mw was determined using a calibration curve of dextran standards (9.4 kDa, 17.2 kDa, 40.2 kDa, 72.2 kDa, 124 kDa, 266 kDa and 487 kDa, from Sigma) The data obtained were analyzed by the Wyatt Technology ASTRA program Total sugar content present in GAL-Am and GLC-Am fractions was determined spectrophotometrically following the method of Dubois (Dubois et al., 1956) Galactose and glucose were used to construct the calibration curve for determination of the total sugar content in GAL-Am and GLC-Am, respectively The protein content present in the samples was determined using a 96-well plate by the Bradford method (Bio-Rad, Hercules, CA, USA) (Bradford, 1976) with bovine serum albumin (BSA) as the standard and performed according to the manufacturer's instructions 2.9 In vitro antitumor activity of polysaccharides 2.7 Nuclear magnetic resonance (NMR) spectroscopy 2.9.1 Cell culture and polysaccharide preparation The B16-F10 murine melanoma (BCRJ, Code 0046) and the nontumorigenic fibroblast BALB/3T3 clone A31 (ATCC, Code CCL-163) cell lines were used Cells were cultivated in Dulbecco's Modified Ea­ gle's Medium (DMEM; Gibco, Cat 12800-017, Waltham, Massachusetts, USA), supplemented with 10% fetal bovine serum (FBS; Gibco, Cat NMR analyses were performed on a Bruker Avance III HD spec­ trometer (Bruker), at base frequencies of 400 MHz (1H), and 100 MHz (13C), and on a Bruker Avance III 600 MHz spectrometer (Bruker Ger­ many) An aliquot of each sample (20 mg) was dissolved in 500 μL of solvent (D2O or Me2SO-d6) and left under stirring in a magnetic stirrer Fig HSQC spectra of the β-glucan (GLC-Am) The sample was analyzed in Me2SO-d6 at 70 ◦ C, at base frequencies of 400 MHz (1H) (chemical shifts are expressed in δ ppm) M Zavadinack et al Carbohydrate Polymers 274 (2021) 118647 Fig GAL-Am and GLC-Am cell cytotoxicity and proliferation (A) Neutral red and (B) crystal violet B16-F10 melanoma cell line (C) Neutral red and (D) crystal violet – Balb/3T3 non-tumor cell line These results represent the set of at least three biologically independent experiments Control represented as a dotted line 12657029, Waltham, Massachusetts, USA), 0.25 μg/mL penicillin/ streptomycin (Thermo Fisher, Cat 15140122, Waltham, Massachusetts, USA), 1.57 g/L sodium bicarbonate (Merck, Cat 36486, Kenilworth, New Jersey, USA), and maintained in a humidified incubator at 37 ◦ C and 5% CO2 For subculture and seeding, cells were always used with flask confluence not higher than 80%, detached with trypsin/EDTA (Gibco, Cat 15400054, Waltham, Massachusetts, USA) and counted in hemocytometer GAL-Am and GLC-Am were dissolved at a final con­ centration of mg/mL in DMEM without FBS and sterilized in 0.22 μm membranes (Millipore, Cat SLGV033RS, Kenilworth, New Jersey, USA) following the preparation of serial dilutions in the desired concentra­ tions DMEM without FBS was used as control in the same volume as the polysaccharides were fixed, stained with CV and imaged, following area measurement and counting using ImageJ Fiji Software (Schindelin et al., 2012) 2.9.4 Cell adhesion assay Cell adhesion ability was analyzed on plastic and on pre-coated Matrigel® (BD Biosciences, Cat 356234, San Jose, California, USA) plates For plate coating, 50 μL of 20 μg/mL Matrigel® diluted in cold PBS was pipetted per well on 96 well plates and kept overnight at ◦ C Wells were washed once with PBS before cell seeding For cell treatment, B16-F10 cells (1.2 × 104 cells/well) were seeded in well plates and then exposed to 10 or 100 μg/mL GAL-Am or GLC-Am for 72 h, following cell detachment with mM EDTA and counting with a hemocytometer × 104 cells were seeded per well and kept for 2h30m in a cell incu­ bator After the adhesion period, wells were washed with PBS (37 ◦ C) for removal of non-adherent cells Remaining adherent cells were fixed and stained with CV, following dye elution and absorbance reading in a microplate reader at 570 nm 2.9.2 Cytotoxicity and proliferation analysis B16-F10 cells (0.5 × 103 cells/well) or BALB/3T3 cells (0.2 × 104 cells/well) were seeded in 96 well plates and exposed or not (control group) to 10, 100 and 1000 μg/mL of GAL-Am or GLC-Am for 72 h Cytotoxicity was assessed by the Neutral Red (NR) assay (Borenfreund & Puerner, 1985), while cell proliferation was analyzed using Crystal Vi­ olet (CV) dye (Gillies et al., 1986) 2.9.5 Statistical analysis Significant differences between experimental groups were deter­ mined by unpaired t-test with Welch's correction, using GraphPad Prism software Data presented as median ± interquartile range unless stated otherwise 2.9.3 Clonogenic capacity assay B16-F10 clonogenic capacity was analyzed in two different ap­ proaches: in the first model, B16-F10 cells (1.2 × 104 cells/well) were seeded in well plates and then exposed to 10 or 100 μg/mL GAL-Am or GLC-Am for 72 h, following cell detachment, subsequent seeding in low density (5.5 × 102 cells/well, well plate) and maintained in culture without any treatments for more 96 h In the second approach, non-pretreated B16-F10 cells were seeded in the same low density conditions in the presence of 10 or 100 μg/mL GAL-Am or GLC-Am for 96 h Colonies Results and discussion 3.1 Chemical structure of the α-D-galactan (GAL-Am) from A muscaria The purification of the galactan (GAL-Am fraction) was developed sequentially and are depicted in Fig The crude soluble fraction S-Am (Suppl Fig 1A), obtained after ethanolic precipitation and dialysis, was M Zavadinack et al Carbohydrate Polymers 274 (2021) 118647 Fig GAL-Am and GLC-Am reduce B16-F10 colony formation capacity (A) Representative images of pre-treated colony formation assay (a- Control, b- GAL-Am 100 μg/mL, c- GLC-AM 100 μg/mL) (B) Colony counting – pre-treatment (C) Colony area – pre-treatment (D) Colony counting – simultaneous treatment (D) Colony area – simultaneous treatment These results represent the set of at least three biologically independent experiments Data shown as median ± interquartile range for B and D, and as mean ± SD for C and E treated with Fehling solution, generating the Cu2+-complexed fraction I1 (Suppl Fig 1B), which after a new treatment with Fehling solution, gave rise to fraction GAL-Am, containing the purified galactan (Fig 2) Results showed that the total sugar content was 95% and protein was absent in GAL-Am GAL-Am showed a homogeneous elution profile in HPSEC analysis, with Mw 9.08 × 103 g/mol (Suppl Fig 2A) and composed of 99% of galactose (Suppl Fig 3A) NMR analyses (1H, 13C, Suppl Figs and 5, HSQC, COSY, TOCSY, HSQC-TOCSY, HMBC and NOESY spectroscopy, Figs 2, and 4) contributed to elucidate the α-D-galactan structure through the signals and intermolecular correlations observed The α-configuration was confirmed by the coupling constant JC-1,H-1 = 173 Hz observed in coupled HSQC spectrum (Perlin & Casu, 1969) It is possible to observe in COSY (Fig 3A) and TOCSY (Fig 3B) experiments signals at δ 4.98/3.85, 5.02/3.97 and 5.15/3.88 indicating the corre­ lation between H-1 and H-2 of the three different monosaccharide groups present in the HSQC spectra (Fig 2) HMBC with superimposed H (Fig 3C) and NOESY (Fig 4) were performed to confirm the linkage between the different monosaccharide units In HMBC experiment, it is possible to observe signals that correlate the C-2 of the 2,6→)-α-D-Galp(1→ units with the H-1 of the α-D-Galp-(1→ units and between the C-1 and the H-6 of the 6→)-α-D-Galp-(1→ units, which indicates the pres­ ence of the linkage types → and → 6, respectively, which can also be observed in the result of the Nuclear Overhauser enhancement M Zavadinack et al Carbohydrate Polymers 274 (2021) 118647 Fig B16-F10 cell adhesion (A) Cell adhesion on plastic (B) Cell adhesion on Matrigel® These results represent the set of four biologically independent ex­ periments Control represented as a dotted line experiment, indicating a spatial molecular proximity relationship be­ tween the cited molecules In HSQC-TOCSY spectrum with super­ imposed HSQC (Fig 3D) the correlation between H-6 and C-5 (δ 3.91/ 68.8, 3.71/68.8, 3.91/68.8, 3.71/68.8, 3.76/71.0 for 5/6a, 5/6b, 5/6a, 5/6b, 5/6c, respectively) is indicated for the three monosaccharides units Through the results obtained, we conclude that the anomeric signals at δ 98.1/4.98 and 98.0/5.02 in the HSQC spectra correspond to 6-O-linked and 2,6-O-linked α-D-Galp units, respectively, and at δ 95.7/ 5.15 corresponds to C-1 of terminal α-D-Galp units linked to C-2 of Galp units in the main chain Signals at δ 66.7/3.91 and 66.7/3.71 were assigned to substituted C-6, while those at δ 61.2/3.76 corresponding to unsubstituted C-6 from terminal units (Brito et al., 2018; Carbonero et al., 2008) The signals at δ 75.1/3.97 and at δ 69.6/3.88 correspond to substituted and non-substituted C-2 from Galp units, respectively The other observed correlations are indicated in the Table ac­ cording to signals already reported by Brito et al (2018), Zhang et al (2013), Carbonero et al (2008) and Rosado et al (2003) Thus, the assignments of all carbons and hydrogen from the GAL-Am are sum­ marized in Table The analysis of methylated derivatives of GAL-Am fraction (Table and Suppl Fig 6) agrees with NMR analyses The mainly derivative observed was the 1,5,6-O-Ac3-2,3,4-Me3-galactitol (71%) from (1 → 6)linked Galp units from main chain Approximately 14% of the main chain units were 2-O-substituted by terminal Galp units (15%), which could be confirmed by the presence of 1,2,5,6-O-Ac4-3,4-Me2-galactitol and 1,5-O-A2-2,3,4,6-Me4-galactitol derivatives, respectively Polysaccharides having (1 → 6)-linked Galp units partially substituted at O-2 by different side chains, constituting hetero­ polysaccharides, have already been reported for A muscaria (Ruthes et al., 2013a,b) and other mushrooms, such as Agaricus bisporus (Komura et al., 2010), Flammulina velutipes (Zhang et al., 2012), Fomitella fraxinea (Cho, Yun, et al., 2011) and Ganoderma atrum (Zhang et al., 2014) It is worth to note that all of them had distinct chemical characteristics when compared with the α-D-galactan reported in the present work As far as we know, α-D-galactan homopolymer has not been described from the mushroom A muscaria until now Besides, α-D-galactans have previ­ ously been observed in mushrooms of the genus Pleurotus, having (1 → 6)-linked and partially 3-O-methylated α-D-Galp in the main chain (Brito et al., 2018; Carbonero et al., 2008; Rosado et al., 2003) Conversely, none of them showed 2-O-substitution by α-D-Galp non-reducing end units Finally, (1 → 6)-linked α-D-Galp units 2-O-substituted and without the presence of methyl groups at C-3 have not yet been reported in the literature 3.2 Chemical structure of the (1 → 3),(1 → 6)-β-D-glucan (GLC-Am) from A muscaria For the GLC-Am purification, the Cu2+-uncomplexed fraction S-1 (Suppl Fig 7A) obtained after the treatment with Fehling solution of SAm fraction has undergone to freezing and thawing process (2×) and a new treatment with Fehling solution (Fig 1), generating the Cu2+uncomplexed fraction S-3 (Suppl Fig 7B) In order to separate the mixture of polysaccharides present in the S-3 fraction, an ultrafiltration (3 kDa membrane) was performed, which gave rise the retained fraction GLC-Am, containing the purified glucan (Fig 5) Results showed that the total sugar content was 96% and the protein was absent in this fraction GLC-Am showed a Mw of 1.3 × 105 g/mol with a homogeneous elution profile in the HPSEC analysis (Suppl Fig 2B) It was composed of 98.8% of glucose (Suppl Fig 3B), confirming the presence of a glucan In the methylation data (Table and Suppl Fig 8) it is possible to identify and characterize a (1 → 3),(1 → 6)-linked D-glucan repre­ sented by the presence of 1,5-O-Ac2-2,3,4,6-Me4-glucitol (19%), 1,3,5O-Ac3-2,4,6-Me3-glucitol (45%), 1,5,6-O-Ac3-2,3,4-Me3-glucitol (13%) and 1,3,5,6-O-Ac4-2,4-Me2-glucitol (23%) derivatives, suggesting a β-Dglucan with (1 → 3)-linked main chain, according to the analysis of residual polysaccharide (GLC-Sm fraction) obtained after controlled Smith degradation (Suppl Fig 9) The six signals observed at δ 103.0/ 4.53, 86.2/3.49, 76.4/3.27, 72.9/3.31, 68.4/3.25 and 60.9/3.71;3.48 are relatives to C1/H1, C3/H3, C5/H5, C2/H2, C4/H4, C6/H6a;b, respectively, also referenced by de Jesus et al (2018); Morales et al (2020) and characteristic of a linear (1 → 3)-β-D-glucan (Table 2) The HSQC spectra (Fig 5) of GLC-Am presented signals relative to the (1 → 3),(1 → 6)-linked D-glucan, with anomeric signals at δ 103.2/ 4.55 and 103.2/4.27 (C1/H1), substituted C3/H3 signals at δ 86.3/3.50, and substituted C6/H6 signals at δ 68.5/4.11 and 68.5/3.58 The H6/C6 signals from terminal β-D-Glcp units can be observed at δ 60.9/3.72 and 60.5/3.50 The NMR chemical shifts are present in Table and were based on 1H, 13C (Suppl Figs 10 and 11), HSQC (Fig 5) and comparison with literature data (Bhanja et al., 2014; Kono et al., 2017; Morales et al., 2020; Ruthes et al., 2015; Zhu et al., 2015) (1 → 3),(1 → 6) β-D-glucans had already been isolated from the mushroom A muscaria and presented different Mw and branching degree when compared to GLC-Am fraction Kiho et al (1992) obtained a glucan with estimated Mw of 9.5 × 103 g/mol and (1 → 3)-linked main chain substituted by two (1 → 6)-linked D-Glcp units at every seven monosaccharide units in the main chain Besides, Ruthes et al (2013b) isolated a (1 → 3),(1 → 6) β-D-glucan with Mw 1.6 × 104 g/mol substituted at O-6 mostly by terminal β-D-Glcp units In addition, GLCAm appears to have a Mw similar to the (1 → 3),(1 → 6) β-D-glucan Carbohydrate Polymers 274 (2021) 118647 M Zavadinack et al isolated from Lactarius rufus (Ruthes et al., 2013a), but differs in the degree of branching and in the composition of the side chains characteristics already described as important traits eliciting mushroom polysaccharides as anticancer compounds (Lemieszek & Rzeski, 2012) Mushrooms heterogalactans have already shown antitumor activities (Ruthes et al., 2016) Some of these polysaccharides present a degree of similarity to GAL-Am, with α-D-galactose (1 → 6)-linked in the backbone and inducing in vitro cytotoxicity and proliferation reduction against cancer cells lines (Pires et al., 2017) A fucogalactan obtained from Macrocybe titans mushroom, which presented a (1 → 6)-linked α-Dgalactose main chain partially substituted at O-2 by α-L-Fucp units with a Mw of 14.2 kDa did not induce cytotoxicity nor proliferation changes in B16-F10 cell line, however it was able to reduce cell migration in a similar concentration to GAL-Am colony formation reduction effects (100 μg/mL) (Milhorini et al., 2018) A fucomannogalactan with a Mw of 17.1 kDa obtained from the mushroom Hypsizygus marmoreus with (1 → 6)-linked α-D-galactose main chain partially substituted at O-2 by α-LFucp and β-D-Manp was non-cytotoxic to B16-F10 cell line at concen­ trations from to 100 μg/mL, but was able to reduce cell colony area – similar to GAL-Am - at the highest concentration (Oliveira et al., 2019) Both previously cited polysaccharides containing a (1 → 6)-linked α-Dgalactose main chain present a relative low Mw close to GAL-Am (9.05 kDa), which could contribute to its biological effects, as already described for other polysaccharides as a relevant structural feature to­ wards higher biological activity (Cho, Lee, & You, 2011; Choi & Kim, 2013) Acute and long-term adverse effects related to cancer treatment are still one of the major concerns and barriers in drug development Most of the induced adverse effects revolve around the consequences of target­ ing cell molecules and mechanics common to cancer and normal cells (Nurgali et al., 2018) Hence, GAL-Am and GLC-Am selective reduction of B16-F10 proliferation indicates a promising feature of these poly­ saccharides that could potentially be translated to direct antitumor ac­ tivity without compromising non-tumor cells 3.3 GAL-Am and GLC-Am selectively reduce melanoma cells viability and proliferation GAL-Am and GLC-Am significantly reduced murine melanoma B16F10 neutral red uptake in all tested concentrations, as showed in Fig 6A (26.2%, 27.9% and 26.8% for GAL-Am; 22.5%, 23.5% and 32.7% for GLC-Am; concentrations of 10, 100 and 1000 μg/mL, respectively) Similarly, both polysaccharides reduced B16-F10 prolif­ eration at all tested concentrations (Fig 6B; 21%, 26.3% and 34.4% for GAL-Am; 27.4%, 34.2% and 37.5% for GLC-Am; concentrations of 10, 100 and 1000 μg/mL, respectively) Strikingly, although GAL-Am and GLC-Am induced significant reductions in B16-F10 proliferation (and consequently decrease the neutral red color measure), they did not impaired murine fibroblast 3T3 cell line in the same manner, with only GAL-Am at 1000 μg/mL decreasing cell viability The smaller concen­ trations (10 and 100 μg/mL) that did not induce any effects on 3T3 cell line were chosen to be used in the next assays 3.4 GAL-Am and GLC-Am cause B16-F10 colony formation impairment in extended cultivation periods that persists with treatment withdraw GAL-Am and GLC-Am treatment were able to affect B16-F10 colony formation capacity, both when cells were pre-treated and seeded to form colonies for 96 h without the polysaccharides (Fig 7A–B; 24.8% and 26.2% reduction in comparison to control group for GAL-Am and GLCAm, respectively, at 100 μg/mL for both compounds) and when cells were seeded to form colonies in the polysaccharide presence (Fig 7D; 32.0% and 24.9% reduction in comparison to control group for GAL-Am and GLC-Am, respectively, at 100 μg/mL for both compounds) In both conditions and concentrations tested (10 and 100 μg/mL), GAL-Am and GLC-Am were also able to significantly reduce colonies mean area when compared to control group (Fig 7C and E) CRediT authorship contribution statement Matheus Zavadinack, Jessica Loren da Rocha Bertage, Shayane da Silva Milhorini and Guilherme Lanzi Sassaki: Carbohydrate Chemistry Experiments, Writing - Original Draft; Daniel de Lima Bellan, Edvaldo da Silva Trindade and Fernanda Fogagnoli Simas: Biological Experiments, Writing - Original Draft, Lucimara M C Cordeiro and Marcello Iaco­ mini: Writing - Review & Editing, Supervision, Funding acquisition 3.5 B16-F10 cell adhesion is not modulated by GAL-Am nor GLC-Am B16-F10 treatment with GAL-Am or GLC-AM at 10 or 100 μg/mL did not alter its capacity to adhere neither in a plastic substrate nor in Matrigel® coated wells, as showed in Fig The absence of adhesion modulation reinforces the previous results, as it shows that both treat­ ments did not reduce the number of adherent cells, but rather reduced their capacity to proliferate both in normal culture conditions and in low confluence Although not fully understood, it is clearly that polysaccharide structural features, such as molecular weight, monosaccharide compo­ sition and type of glycosidic linkage, play an important role in poly­ saccharide biological activity (Xu et al., 2017) Several β-glucans obtained from mushrooms with similar structure to GLC-Am are described to present direct and indirect cytotoxic and anti-proliferative activity against tumor cells (Pandya et al., 2019; Ren et al., 2012) One example is lentinan, a β-glucan (1 → 3)-linked with β-(1 → 6)-glucose branches, from the mushroom Lentinus edodes with a Mw ranging from 400 to 800 kDa (Pandya et al., 2019) In a similar range of concentra­ tions as used in the present paper, a purified lentinan (605.4 kDa) was able to significantly reduce the proliferation of murine breast cancer cell line MCF-7 and murine sarcoma cell line S180 (Zhang et al., 2015) Another similar β-glucan from L edodes presented a very similar selec­ tive cytotoxicity as demonstrated by GLC-Am against liver cancer cell line H22 but not affecting human liver normal cell line HL7702 (Wang et al., 2017) Interestingly, as GLC-Am presents similar structure to these two polysaccharides but significantly smaller molecular weight, we suggest that the β-(1 → 3)-glucose backbone and (1 → 6)-β-glucose branches play an important role in the biological activity of β-glucans Moreover, GLC-Am glucose linkage and its water solubility are chemical Acknowledgements The authors would like to thank the Brazilian funding agencies: CAPES (Funding Code 001) and CNPq (404717/2016-0, 301719/20160, 307314/2018-9) for financial support, the Chemical Technicians Thiago J dos Santos and Arquimedes Paix˜ ao de Santana Filho for GC–MS and NMR analyses, 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of Food Composition and Analysis https://doi.org/10.1016/j.jfca.2015.01.019 12 ... 3.27 Assignments are based on 13C NMR, 1H and HSQC analysis and are expressed as ppm GAL-Am: α-D-galactan isolated from A muscaria GLC-Am: β-D-glucan isolated from A muscaria GLC-Sm: GLC-Am that... Based on derived O-methylalditol acetates GAL-Am: α-D-galactan isolated from A muscaria GLC-Am: β-D-glucan isolated from A muscaria temperature for 20 h, neutralized with acetic acid and dialyzed... M Zavadinack et al Carbohydrate Polymers 274 (2021) 118647 Table Partially O-methylated alditol acetates of the α-D-galactan (GAL-Am) and β-D-glucan (GLC-Am) polysaccharides purified from A muscaria