Chemical characterization, antioxidant properties and anticancer activity of exopolysaccharides from Floccularia luteovirens

Journal Pre-proof Chemical characterization, antioxidant properties and anticancer activity of exopolysaccharides from Floccularia luteovirens Zhengjie Liu, Yingchun Jiao, Hongyun Lu, Xiaoli Shu, Qihe Chen

PII:

S0144-8617(19)31099-9

DOI:

https://doi.org/10.1016/j.carbpol.2019.115432

Reference:

CARP 115432

To appear in:

Carbohydrate Polymers

Received Date:

25 August 2019

Revised Date:

19 September 2019

Accepted Date:

3 October 2019

Please cite this article as: Liu Z, Jiao Y, Lu H, Shu X, Chen Q, Chemical characterization, antioxidant properties and anticancer activity of exopolysaccharides from Floccularia luteovirens, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115432

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Chemical characterization, antioxidant properties and anticancer activity of exopolysaccharides from Floccularia luteovirens

Zhengjie Liu1, Yingchun Jiao3, Hongyun Lu1, Xiaoli Shu2, Qihe Chen1,∗

1Department

of Food Science and Nutrition, Zhejiang University, Hangzhou, P. R. China;

of Agriculture and Biotechnology, Zhejiang University, Hangzhou, P. R. China;

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2College

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[email protected] [email protected]

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Agriculture and Animal Husbandry College, Qinghai University, Xining 810016, P. R. China

Corresponding author: Qihe Chen, Department of Food Science and Nutrition,

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*

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Running title: Characterization, structure and antioxidant activity of EPS

Zhejiang University, Yuhangtang Rd.866, Hangzhou 310058, P. R. China

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Highlights 



EPSs were purified from Floccularia luteovirens in the submerged culture. The fractions had good antioxidant activities and different thermal properties.



ALF1 was proven to inhibit tumor cells without affecting the normal 1

cells. 

ALF1 could suppress oxidant stress by improving the activities of SOD, GSH-Px and CAT.



ALF1 could decrease ROS level, and stabilized mitochondrial membrane

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potential.

Abstract

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Two polysaccharides, ALF1 and ALF2 were obtained from the fermentation liquid of

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Floccularia luteovirens. These fractions had good performance in scavenging radicals and ALF1 exhibited obvious antioxidant activities. Further, linkage analysis and NMR

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were used to characterize the structures of ALF1. Linkage and NMR data

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→4)-β-D-Manp→,

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comprehensively showed that ALF1 mainly contained six kinds of linkage type units 1,3-α-Fucp→,

α-L-Araf-C1→,

→6)-β-D-Galp-C1→,

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→4)-α-D-GlcAp-(1→ and →3)-β-D-Glcp(1→. In addition, ALF1 had good bioactivities such as anticancer and antioxidant activities. ALF1 was proven to be able to inhibit tumor cells without affecting the normal cells. Besides, ALF1 improved the

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activities of SOD, GSH-Px and CAT, and decreased the production of MDA which result in protecting PC12 cells against H2O2-induced oxidative stress. ALF1 decreased ROS production, and stabilize mitochondrial membrane potential. The findings indicated that the fermentation liquid of Floccularia luteovirens could be used as a potential natural source of antioxidant.

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Keywords: Floccularia luteovirens; exopolysaccharides; antioxidant activity; structural characterization; anticancer activity

1. Introduction

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Oxidation is an essential process for the production of energy to many organisms.

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Under pathological conditions, however, the concentrations of reactive oxygen

species (ROS) are usually over physiological limits leading to oxidative stress (Gülçin,

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Oktay, Küfrevioǧlu, & Aslan, 2002; Liu & Jiang, 2012). A multitude of evidence

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indicated that ROS was closely related to coronary heart disease, carcinogenesis, atherosclerosis and other diseases associated with the aging (Wei, Cheng, Wei, &

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Zhang, 2012). Currently, synthetic antioxidants had unwanted side effects mostly (Qi et al., 2005). Therefore, it is essential to develop and utilize effective natural

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antioxidants to replace the synthetic antioxidants (Singh & Rajini, 2004). Several exogenous antioxidants isolated from natural resources had been proved to have antioxidant capability, such as polysaccharides, tocopherol, ascorbic acid, flavonoids,

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β-carotene, anthocyanins, and tannins (Soong & Barlow, 2004). Among these antioxidants, polysaccharides are considered the most representative components and proven oxidation resistance (Ferreira et al., 2015; Wang, Hu, Nie, Yu, & Xie, 2016). Previously, people would like to focus on polysaccharides from plants (Ramarathnam, Osawa, Ochi, & Kawakishi, 1995). However, polysaccharides from edible 3

mushrooms have provoked great interest as sources of novel potential antioxidants in recent time (Datta et al., 2019; Liang et al., 2019; Zhang, T. et al., 2018). In general, fungus polysaccharides have strong antioxidant activities which was indicated in vast literatures and could be explored for anti-tumor, immunomodulatory and antioxidant

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uses (Datta et al., 2019; Lee et al., 2003; Wang et al., 2012). Floccularia luteovirens (known as Armillaria luteo-virens Sacc.), as a

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well-known unique Chinese medicinal and edible basidiomycete, is mainly distributed

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in the meadows and grasslands of Qinghai-Tibet plateau (Chen, Shao, Tao, & Wen, 2015). As a traditional Tibetan medicine, F. luteovirens is frequently used for the

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treatment of neurasthenia, dizziness, insomnia, headaches, infantile convulsions and

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numbness in limbs. To the best of our knowledge, F. luteovirens was found to show significantly biological activity against radiation, anti-hypoxia, anticancer in the

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previous report (Feng et al., 2006). However, there are rare literatures describing the antioxidant activity of polysaccharides from F. luteovirens. Fruiting bodies of F. luteovirens could be harvested only once per year in the wild, thus the production was limited. As a result, it has actual application to obtain exopolysaccharides by

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submerged culture.

In this study, we extracted and purified EPS fractions from the cultured broth of

F. luteovirens under the submerged cultivation. Our research team had well optimized the submerged culture conditions in the past few years (Jiao, Chen, Zhou, Zhang, & Chen, 2008; Xu, Fu, Chen, & Liu, 2011). Here, we investigated the chemical 4

characterization, thermal properties and in vitro antioxidant activities of the fractions. The structural characterization, anticancer activity and the most effective fraction (ALF1) were further characterized. Besides, the antioxidant capacity of ALF1 for cellular oxidative stress was evaluated. This study has practical significance for novel

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potential antioxidants used in food industry.

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2. Materials and Methods 2.1. Materials and reagents

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The mycelia of F. luteovirens were isolated and screened from the F. luteovirens

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fruiting body growing in the steppe of Qilian County, Qinghai, China (Fig. S1). This culture collection which had been originally isolated in our laboratory, was originally

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named as A. luteo-virens Sacc QH (CGMCC No.1884). It was maintained on potato dextrose agar (PDA) slant at 25 °C and monthly subcultured. Commercial kits for

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SOD (superoxide dismutase), GSH-Px (glutathione peroxidase) CAT (catalase) and MDA (methylene dioxyamphetamine) were purchased from Beyotime (Shanghai, China). All other chemicals and reagents were purchased from Aladdin and were of

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analytical grade. The HepG2 (human hepatoma), LO2 (normal human hepatocyte cell line) and PC12 cells (rat pheochromocytoma line 12) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 2.2. Cultivation of the submerged mycelia from F. luteovirens

In this work, an airlift fermenter was used to conduct the fermentation pocess. 5

Derived from the previous study (Jiao et al., 2008), the optimized medium compositions composed of 31.26 g/L glucose, 0.45 g/L MgSO4·7H2O, 0.50 g/L KH2PO4, 1.00 g/L K2HPO4, and 1.06 g/L yeast extract powder, pH unadjusted. Prior to 5 L bioreactor experiments, the seed culture was prepared with slant culture

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medium and cultivated for 10 days at 25 °C and 100 rpm in 250-mL flasks (100 mL liquid culture medium). Under 5 L bioreactor experiments, seed culture was used to

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inoculate a 5 L bioreactor (with 4.0 L working volume) at 5% (v/v) inoculum’s level.

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During the cultivation process, a control unit was used to monitor the temperature, pH, and dissolved oxygen level. The cultivation process was conducted at 25 °C with an

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aeration of 1.5 L/min.

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2.3. Extraction and purification of EPSs

After incubating for 15 d at 25 °C, the airlift fermenter was unloaded and the

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supernatant was collected by filtration. The supernatant was mixed with four volumes of ethanol and stored for 12 h-24 h at 4 °C. After centrifugation at 8000×g for 20 min, the precipitate containing EPS was collected. The precipitate was re-dissolved in

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deionized water and subsequently deproteinized, decolored, dialyzed and lyophilized to obtain the crude EPS. The crude EPS solution was further purified and fractionated by a cellulose

DEAE-52 column (2.6 cm×40 cm) (Wang, Liu, & Qin, 2017) with a step gradient of NaCl (0, 0.1, 0.2, 0.3 M) at 1 mL/min. An automatic collector was used to collect the 6

dominating EPS fractions (5 mL/tube) according to the phenol-sulfuric acid method (DuBois, Gilles, Hamilton, Rebers, & Smith, 1956). Two purified EPS fractions, named ALF1 and ALF2 respectively, were obtained. After lyophilization, the EPS fractions was weighed to evaluate the respective yield.

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The EPS fractions were finally purified on a Sephacryl S-200 gel filtration column (2.0 cm × 60 cm). The column was eluted with ultrapure water at a flow rate of 1

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mL/min and the EPS fractions were collected with an automatic fraction collector (5

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mL/tube). After lyophilization, total carbohydrate content was estimated by the phenol–sulfuric acid method (DuBois et al., 1956) using glucose as the standard.

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Protein content was assayed by the method of Bradford (Bradford, 1976).

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2.4. Structural characterization and thermal analysis

2.4.1. Molecular weight detection

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The molecular weights of ALF1 and ALF2 were determined with ASEC-MALLS-RI system. This system and the samples processing method was based on our previous work (Gu, Jiao, Wu, Liu, & Chen, 2017) and proved to be reliable and precise. The

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SEC column (Ultrahydrogel 250, 7.8 mm × 300 mm, Waters, Kitashinagawa, Japan) was eluted with 0.15 M aqueous NaCl solution at a flow rate of 0.5 mL/min at 25 °C and the value of refractive index increment (dn/dc) was 0.138 mL/g. After that, samples were detected by a Waters 2414 refractive index detector and a DAWN HELEOS II MALLS detector. A calibration curve was established according to the 7

dextran standards with different molecular weights (2500, 4600, 7100, 10000, 21400, 41100, 84400, 133800, 200000 Da). 2.4.2. Analysis of monosaccharide compositions The 1-phenyl-3-methyl-5-pyrazolone high performance liquid chromatography

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(PMP-HPLC) method (Li et al., 2016) was used to determine the monosaccharide compositions of ALF1 and ALF2. The following standard sugars used in this study

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were fucose (Fuc), galactose (Gal), arabinose (Ara), xylose (Xyl), glucuronic acid

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(GlcA), galacturonic acid (GalA), glucose (Glu), rhamnose (Rha), mannose (Man), ribose (Rib) (Gu et al., 2017). The HPLC equipped with a ZORBAX Eclipse

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XDB-C18 column (5 μm, 4.6 mm × 250 mm) was used to analyze the investigated

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samples. 2.4.3. FT-IR spectroscopy analysis

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A Nicolet AVA TAR370 Fourier-transform infrared spectrophotometer (FTIR, Nicolet, Madison) was used to record the IR spectra of ALF1 and ALF2. The samples were mixed with spectroscopic grade potassium bromide (KBr) powder at a ratio of 1:50 (m/m) and then measured in the frequency range of 4000-400 cm-1 at room

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temperature (Lin et al., 2017).

2.4.4. Glycosidic linkage analysis The glycosidic linkage analysis was conducted by permethylation of the EPS (ALF1) followed by hydrolysis, reduction and acetylation as described by the literature (Kim, Reuhs, Michon, Kaiser, & Arumugham, 2006). GC–MS (Agilent Technologies, 8

6890A/5975C), as mentioned above, was then employed to analyze the mixtures. The oven temperature was initially set as 140 °C and hold for 2 min, risen at 3 °C/min to 230 °C and hold for 3 min. One microliter of sample was injected and carried by helium gas at 1.0 mL/min with a split ratio at 10:1. The mass spectrum of partially

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methylated alditol acetates (PMAAs) was determined by EI ion source detector with the same conditions as the monosaccharide composition analysis. Finally, the

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glycosidic linkages were determined that referred to the online spectral database

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(CCRC: http://www.ccrc.uga.edu/specdb/ms/pmaa/pframe.html) and the reported literatures (Kim et al., 2006; Sassaki, Gorin, Souza, Czelusniak, & Iacomini, 2005).

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2.4.5. Thermal analysis of EPS fractions

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The thermal properties of ALF1 and ALF2 were measured with differential scanning calorimetry (DSC, ZCEC-130263F, Mettler Toledo). With a scan rate of 10 °C/min,

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10 mg of each sample were heated from 20 °C to 500 °C in a covered aluminum pan under nitrogen purge (Lee, Li, Chen, & Park, 2017). 2.4.6. Nuclear magnetic resonance (NMR) spectroscopy For nuclear magnetic resonance (NMR) spectroscopic analysis, the lyophilized EPS

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fractions (60 mg) were dissolved in 0.6 mL D2O and lyophilized three times (Wei et al., 2016). 1D and 2D nuclear magnetic resonance (NMR) spectra experiments (1H NMR,

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C NMR,

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C NMR-DEPT 135, 1H-1H COSY, HSQC, HMBC, TCOSY and

NOESY) were performed on a 600 MHz NMR spectrometer (DD2, Agilent) at 25 °C with acetone as internal standard. 9

2.5. Evaluation for in vitro antioxidant activities

2.5.1. DPPH radical scavenging activity A previously described method was used to conduct the 1,1-Diphenyl-2-picrylhydrazy (DPPH) radical scavenging effect assay of EPS fractions with slight modifications

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(Xu et al., 2009). The results are expressed in scavenging ability (%). Synthetic

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antioxidants butylated hydroxyanisole (BHA) was used as the positive control. 2.5.2. Ferrous ion chelating ability

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The ferrous ion chelating ability of EPS fractions was conducted according to previously described method with slight modifications (Kalın, Gülçin, & Gören,

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2015). The data are demonstrated in scavenging ability (%). Ethylenediaminetetra

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acetic acid disodium salt (EDTA-2Na) was used as the positive control, which had remarkable ferrous ion chelating ability.

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2.5.3. ABTS radical cation scavenging assay

The 2,2’-azino-bis-(3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt (ABTS) assay was carried out using the T-AOC Assay Kit (Beyotime Institute of

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Biotechnology, China). Before use, ABTS stock solution was mixed with 2.45 mM potassium persulfate in the dark and stored for 12-16 h at room temperature to prepare the ABTS radical cation (ABTS·+) solution. The resulting ABTS·+ solution was diluted in order to adjust the absorbance value to 0.70 ± 0.05 at 734 nm. Samples or Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylicacid) standard were 10

mixed with diluted ABTS·+ solution in a ratio of 1:20 (v/v). After storing for 2-6 min in the dark at room temperature, the absorbance of the mixture was measured at the absorbance of 734 nm. Results were expressed as mmol/g Trolox equivalent antioxidant capacity (TEAC).

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2.5.4. Ferric reducing antioxidant power (FRAP) assay The FRAP assay was carried out using the FRAP method kit (Beyotime Institute of

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Biotechnology, China). 2,4,6-tripyridyl-s-triazine (TPTZ) dilution, detective buffer

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and TPTZ solution were mixed respectively in a ratio of 10:1:1 (v/v) after thawing to form the working solution, which should be freshly prepared. Before use, the working

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solution was warmed to 37 °C. For the assay operation, the sample (5 μL) with the

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concentration of 5 mg/mL and the positive control (Trolox) were mixed with 180 μL of FRAP working solution. After preserved for 5 min at 37 °C, the resulting solution

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was measured at 593 nm. FeSO4 values were used to express the activity, which were calculated using the standard curves. 2.5.5. Oxygen radical antioxidant capacity (ORAC) assay The ORAC assay of EPS fractions ALF1 and ALF2 was performed according to

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Prior’s method with minor modification (Prior et al., 2003). This procedure was carried out on a Fluoroskan Ascent FL plate-reader (Thermo Fisher Scientific, USA) with filters selecting an emission wavelength of 538 nm and an excitation wavelength of 485 nm. The final ORAC values were calculated according to the net area under the decay curves and expressed as mmol/g TEAC. 11

2.6. Antiproliferation activity assay The antiproliferation activities of ALF1 in HepG2 (human hepatoma) and LO2 (human normal hepatocytes) cells were evaluated by MTT assays. Cells were seeded in 96-well plates at the concentration of 5 × 103 cells in DMEM medium containing

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10% fetal bovine serum, respectively, and incubated at 37 °C under 5% CO2. The cells subsequently were treated with different concentrations of ALF1 (0, 200, 400,

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800, 1000 μg/mL) for 24 h. Then 20 μL MTT (5 mg/mL) was added to each well and

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incubated for 4 h at 37 °C. After removing the culture medium, 150 μL dimethyl sulphoxide (DMSO) was added to each well to solubilize formazan. Absorbance was

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measured at 570 nm.

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2.7. Protection effect of ALF1 on H2O2-induced oxidative damage in PC12 cells PC12 cells (rat pheochromocytoma line 12) were used in this assay. In order to obtain

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stable experimental results, we used different concentrations (100, 200, 300, 400 and 500 μM) of H2O2 to treat the cultured PC12 cells for 4 h to verify an appropriate concentration in the cell injury model. Before this assay, PC12 cells were seeded in

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6-well plates (concentration of 1 × 105 cells) and 96-well plates (concentration of 5 × 103 cells) and then incubated for 24 h at 37 °C under 5% CO2. Subsequently, the cells treated with different methods were divided into four groups: (1) Sample group (treated with ALF1 of 200, 400, 800, 1000 μg/mL); (2) Positive control group (treated with ascorbic acid); (3) Blank control group (treated with DMEM medium); (4) 12

Damage group (treated with DMEM medium). After incubated for 12 h, sample group, positive control group and damage group were treated with 200 μM H2O2 for 4 h while blank control group was treated with DMEM medium again. After different treatments above, the cell viability was evaluated by MTT assays.

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2.8. Determination of reactive oxygen species (ROS) level

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ROS level of cells was evaluated by 2’,7’-dichlorofluorescin diacetate (DCFH-DA)

fluorescent staining. In brief, the cells treated with various methods above were

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washed in PBS three times and incubated with 10 μM DCFH-DA for 30 min at 37 °C

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in dark. Subsequently, a fluorescence microplate reader (Thermo Fisher Scientific, USA) was employed to measure the fluorescence at 488nm excitation and 525 nm

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emission wavelength. The level of intracellular ROS was expressed with the fluorescence intensity. The final statistical results were expressed as percentage of the

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control group.

2.9. Measurement of lactate dehydrogenase (LDH) release The release of LDH into the culture medium was quantified with a commercial LDH

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assay kit. In brief, after cells were treated with various methods, the medium was collected. The LDH activity was measured at 450 nm to detect LDH activity according to the manufacturer’s instructions and expressed as a percentage of the control. 2.10. Determination of SOD, GSH-Px, CAT and MDA levels 13

After the various treatments, the cells in 6-well plates were lysed by lysis buffer and centrifuged to collected the supernatant for the next analysis. The measurements of protein concentrations, MDA levels and activities of SOD, GSH-Px, CAT were conducted with commercial assay kits according to the manufacturer’s instructions.

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2.11. Mitochondrial membrane potential (MMP) determination

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The changes in mitochondrial membrane potential were evaluated by Rhodamine 123 (RH123) staining. In briefly, the cells treated with various methods were washed in

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PBS three times and incubated with 2 μM Rhodamine 123 in serum-free DMEM for

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30 min at 37 °C. Subsequently, the cells were washed twice in PBS and observed by fluorescence microscope (Nikon, Japan). The fluorescence intensity was analyzed by

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2.12. Statistical analysis

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Image-Pro Plus 6.0 software and were expressed as percentage of the control.

Results were presented as means ± standard deviations for three replicates for each sample. The data were statistically analyzed with the SPSS statistical software (SPSS

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Inc, USA). A probability of P< 0.05 was considered as statistical significance.

3. Results and Discussion 3.1. Extraction, purification and monosaccharide compositions of EPS The crude EPS was collected from culture broth and then fractionated by DEAE-25 14

column. The water-eluted EPS (ALF2) and salt-eluted EPS (ALF1) were collected (Fig. 1A), and the yields were 68.52% for ALF1 and 29.23% for ALF2. The two EPS fractions were further purified with Sephacryl S-200 column and both of them appeared as only a single symmetrical peak indicating the two EPS fractions were

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homogenous polysaccharides (Fig. 1B-C). The two fractions contained over 95% of

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carbohydrates and no protein was detected (Table 1).

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The molecular weights of ALF1 and ALF2 were estimated by the SEC-MALLS-RI system. The average molecular mass of ALF1 was estimated to be 2.836×104 Da,

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while ALF2 was estimated to be 1.866 ×104 Da. A PMP-HPLC method was used to

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analyze the monosaccharide compositions of EPS fractions. According to the monosaccharide standards, the major monosaccharides of ALF1 were mannose and

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glucose (Table 1). The molar ratio of monosaccharide compositions in ALF1 was described as follows: Man:Glc:Fuc:Gal:GlcA:Ara = 1.00:0.65:0.39:0.47:0.29:0.01. However, in ALF2, the major monosaccharides were glucose and galactose with the

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molar ratio of Glc:Man:Rha:Gal = 1.00:0.40:0.34:0.54. 3.2. FT-IR spectroscopy, thermal analysis of two EPS fractions Fig. S2 indicated the FT-IR spectra of the purified EPS fractions, which performed for the determination of the structural features. The characteristic strong broad band near 3384 cm-1 corresponded to the presence of hydroxyl stretching in hydrogen bonds of 15

the polysaccharide. The C-H stretching vibration was demonstrated by the peak around 2930 cm-1 (Manrique & Lajolo, 2002). As we can see in the figure, ALF1 had a stronger absorption peak at 2930 cm-1 than ALF2. An absorbance at 1609 cm-1 of ALF1 and one at 1651 cm-1 of ALF2 indicated the bound water. The peak at 1413

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cm−1 of ALF1 was due to the characteristics of -CH2 deformation mode (Buriti et al., 2014), and O-H deformation vibrations and C-O stretching vibrations were indicated

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by the peaks in the region of 1240 and 1420 cm-1. It has been reported that the bands

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around 1200-1000 cm-1 were defined as the fingerprint area for polysaccharides, because they were assigned to C-O-C stretching of glycosidic bonds and to C-OH

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bending (Cai et al., 2016), as a result the absorption peaks at 1031 cm-1 (ALF1) and

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1116 (ALF2) cm-1 indicated that the sugar rings of two polysaccharide fractions were pyranose rings. In addition, the peaks at 807 cm-1 of ALF1 and 891 cm-1 of ALF2

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proved the characteristic absorption of mannose (Kato, Nitta, & Mizuno, 1973). In this work, we also conducted DSC measurement in order for the thermal behavior examination of the purified EPS fractions. As data presented in Fig. S3, the DSC curve of ALF1 showed one exothermic sharp peak at 51.5 °C and two endothermic

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sharp peaks at 43 °C and 160.5 °C. The exothermic peak at 51.5 °C stood for the crystallization. The endothermic sharp peaks at 160.5 °C indicated a melting point. In view of ALF2, only one endothermic sharp peak at 163.3 °C was observed, which means there was just a melting point. Herein, no exothermic peak is observed. These data implied during the heating procedure no oxidization reaction or cross-linking 16

reaction occurred.

3.3. Evaluation for in vitro antioxidant activities of two EPS fractions DPPH is a stable free radical that could be scavenged once encounters a

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proton-donating substance. As presented in Fig. 2A, ALF1 and ALF2 could scavenge

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DPPH radicals effectively and the scavenging activities were improved with the increase of concentration (0.1-5 mg/mL). The highest scavenging rates of ALF1 and

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ALF2 were 76.37 ± 3.02 and 74.71 ± 2.37% at 5.0 mg/mL, respectively, which were

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close to that of BHA (78.20 ± 2.31%), indicating a wide-used antioxidant with good radical-scavenging activity. The 50% inhibitory concentration (IC50) values of the two

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EPS fractions were summarized in Table 2. The DPPH radical scavenging effect of ALF1 (703.73 μg/mL) was slightly better than ALF2 (763.70 μg/mL). The IC50 values

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of ALF1 and ALF2 were close to the result reported by Liu (Liu et al., 2017), in which the IC50 value of EPS from Grifola frondosa fermentation broth was 0.60 mg/L. Previously reported, the polysaccharides isolated from fruiting bodies of edible

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mushrooms had certain DPPH radical activity (Chen et al., 2017; Liang et al., 2019). Herein, the results mentioned above indicated that the EPS isolated from the submerged fermentation of F. luteovirens was a useful antioxidant.

Considering the previous study, ferrous ions are considered as effective 17

pro-oxidants in the food system (Yamauchi, Tatsumi, Asano, Kato, & Ueno, 1988). Therefore, the high ferrous-ion chelating abilities of EPS from F. luteovirens would be beneficial. As presented in Fig. 2B, the chelating ability of ALF1 and ALF2 on ferrous ion was improved with the increase of concentration from 0.1 to 5 mg/mL. The

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highest ferrous ion chelating ratio of ALF1 and ALF2 were 78.03 ± 2.31 and 68.77 ± 2.77% at 5.0 mg/mL, respectively. In comparison with EDTA-2Na, which represented

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a chelating ability of 98.70% at 5 mg/mL, ALF1 and ALF2 could chelate ferrous ion

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with good performance.

ABTS radical cation scavenging assay is also used to evaluate the antioxidant

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activity of two fractions. Compared with Trolox, ALF1 and ALF2 showed significant

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antioxidant capacity (Fig. 2C). Results were expressed in Trolox equivalent antioxidant capacity (TEAC). In ABTS assay, two EPS fractions achieved different

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TEAC values. The TEAC value of ALF1 was 0.094 mmol Trolox/g and that of ALF2 was 0.089 mmol Trolox/g. Chen et al. (Chen et al., 2017) found that the polysaccharides from Tricholoma lobayense fruiting bodies had the scavenging activities towards ABTS radicals with IC50 value of 500 µg/mL. In this assay, the EPS

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presented strong antioxidant activity of ABTS radicals as well. FRAP assay was based on a reaction between antioxidant and TPTZ-Fe3+ complex. According to the standard curves and regression equations, the corresponding FeSO4 values of ALF1 (0.113

mmol/g) and ALF2 (0.096 mmol/g) were calculated and listed in Fig. 2C, indicating that ALF1 and ALF2 showed remarkable reducing power. Polysaccharides isolated 18

from wild Armillaria ostoyae mushrooms presented the antioxidant activities at 24.8 and 29.0 μmol/g in FRAP assay (Siu, Xu, Chen, & Wu, 2016). Compared with the results above, ALF1 and ALF2 presented outstanding reducing powers. As compared with assaying data in Fig. 2C, ALF1 showed higher TEAC value

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(0.341 mmol/g), while the TEAC value of ALF2 was 0.248 mmol/g. In contrast with ABTS assay, the TEAC value of ALF1 from ORAC assay was 3.6 times higher, and

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that of ALF2 was 2.8 times higher. That is due to that the two methods are based on

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different reaction mechanisms (Wang et al., 2016). Previously reported, if compared with ORAC assay, ABTS assay gives an underestimate of the antioxidant capacity of

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samples of a more complex nature (Zulueta, Esteve, & Frigola, 2009). Therefore,

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ORAC assay was more standard, which meant the data of ORAC from different laboratories were comparable (Zulueta et al., 2009). Herein, the EPS fractions of F.

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luteovirens exhibited remarkable antioxidant capacity in contrast with other edible mushrooms, such as Tricholoma matsutake, Lentinus polychrous and Ganoderma (Ferreira et al., 2015; Thetsrimuang, Khammuang, Chiablaem, Srisomsap, & Sarnthima, 2011; You et al., 2013). It was reported that the monosaccharide

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compositions, types of glycosidic linkage, content of uronic acid and molecular weights are the determining factors that influenced antioxidant activities of EPS (Jiang, Wang, Liu, Gan, & Zeng, 2011; Sun, Zhang, Zhang, & Niu, 2010; Wang et al., 2016; Yang et al., 2009; You et al., 2011). Thus, we have good reason to believe that chemical composition and structural characteristics had a great influence on the 19

antioxidant activities of EPS from F. luteovirens. In view of the ALF1’s better performance in the in vitro antioxidant activities and the high purification yield, the elaborate structure and antioxidant activities evaluation of ALF1 were further investigated in the following study.

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3.4. Glycosidic linkages of the purified ALF1 polysaccharide

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As presented in Table 2 and Figure S4, ALF1, containing 4 terminal residues (t-Araf,

t-Fucp and t-Manp, t-Galp), 8 linear glycosidic residues (2-Fucp, 3-Glcp, 2-Manp,

-p

2-Galp, 6-Manp, 4-Galp, 4-GlcAp, and 6-Galp) and six branching glycosidic residues

re

(2,3-Manp, 2,4-Galp, 2,6-Manp, 3,6-Glcp, 2,6-Glcp, and 2,6-Galp). ALF1 was mainly composed of t-linked Manp (14.23 mol%), 2-linked Manp (15.29 mol%) and

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2,6-linked Glcp (11.44 mol%) (Table 3). It implied that ALF1 fraction possessed the

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complex structure with polysaccharides compositions and linkage types.

3.5. Structural characterization by NMR spectroscopy for ALF1 NMR spectroscopy is a convenient technique for elucidation of the structural

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properties in polysaccharide. The literature values were used as references to compared with the NMR chemical shifts in this work (Chen et al., 2017; Wei et al., 2016). By means of chemical analyses and NMR spectroscopy, the structural information of ALF1 isolated from F. luteovirens was studied. 1H and

13

C NMR

spectra of ALF1 in D2O were shown in Fig. 3. In 1H NMR spectrum of ALF1, weak 20

signals appeared at 5.08-5.62 ppm indicated that ALF1 was composed of a small amount of α-glycoside bonds, and strong peaks around 4.49 ppm showed it mainly contained of β-glycoside bonds. The chemical shifts variation from δ3.2 ppm to δ4.2 ppm was considered as the signals correlating with the protons existing on C2-C6. 13

C NMR spectra of ALF1 were crowded in a narrow region within

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The 1H and

3.0-5.3 ppm (1H NMR) and 60-110 ppm (13C NMR), which are typical for

ro

polysaccharides. In the anomeric region of the 1H NMR spectrum of ALF1, five

In the anomeric region of the

13

-p

signals were observed at δ 5.62-5.08, δ 4.94-4.85, δ 4.40, δ3.96-3.40, δ 1.39-0.95 ppm. C NMR spectrum, carbon resonances appeared at δ

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109.45-102.37, δ 98.39, δ 78.58-70.53, δ 69.55, δ 68.25, δ 63.71-63.11, δ 32.88-32.39,

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δ 17.99 ppm (Fig. 3B). Predominant signals between δC60.0 to δC82.0 ppm represented oxygen substituted carbon in monosaccharide residues. Besides, (1→3/4)

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glycosidic linkages and (1→6) glycosidic linkages were confirmed by the signals at δC80.0–81.8, and 67.4–70.0 (Yu, X. et al., 2017). Moreover, acetyl groups and the CH3 of mannitol residues were confirmed by the signal of δC22.2 - 29.4 ppm and 13.0

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-18.0 ppm (Liang et al., 2019).

1

H-1H COSY, 1H-13C HSQC, and

13

C HMBC spectral analysis was detected for

structural characterization of ALF1 (Fig. 4A, B, C). Combining the data of linkage analysis, it revealed that ALF1 has mainly contained six kinds of sugar residues: residue A as →4)-β-D-Manp→, B as 1,3-α-Fucp→, C as α-L-Araf-C1→, and D as 21

→6)-β-D-Galp-C1→, E as →4)-α-D-GlcAp-(1→, F as →3)-β-D-Glcp(1→. 1H and 13

C NMR spectral assignments of ALF1 were summarized by spectral analysis of

COSY, HSQC and HMBC (Table S1) (Chen et al., 2019; Datta et al., 2019; Liang et al., 2019; Wang et al., 2019). Previous studies indicated that the specific

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monosaccharide composition, glycosidic linkage, and the degree of branching were closely related to the antioxidant capabilities (He et al., 2017; Yu, Li, Du, Mou, &

ro

Wang, 2017). The structure-activity relationship of antioxidant polysaccharides has

-p

not been fully explained. Its advanced structure will be further elucidated in the

re

further work.

lP

3.6. Antiproliferative activities on LO2 and HepG2 cells

Many studies have reported that natural polysaccharides from basidiomycetes have

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antiproliferative effects on cancer cells (Cui et al., 2014; Datta et al., 2019; Liu et al., 2015; Peng, Zhang, Zeng, & Kennedy, 2005; Tsai, Song, Shih, & Yen, 2007; You et al., 2013; Zhang, Y. et al., 2018). Thus, it’s obligated to investigate the

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pharmacological activity of ALF1. However, some drugs that inhibit cancer cells also affect normal cells (Lu et al., 2019). Accordingly, we used LO2 cells as the control. Fig. 5A demonstrated that ALF1 did not exhibit statistically significant effects on the proliferation of LO2 cells, which indicated that ALF1 had no cytotoxicity in normal cells. On the other hand, unlike LO2 cells, ALF1 inhibited the proliferation of HepG2 22

cells in a dose-dependent manner (Fig. 5B) and the inhibition rate reached 43.60% at the concentration of 1000 μg/mL. The result implied that ALF1 could inhibit tumor cells without affecting the normal cells.

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3.7. Effects of H2O2 and ALF1 on the viability of PC12 cells

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The PC12 cells were treated with various concentrations of H2O2 to verify an appropriate concentration in the cell injury model. As shown in Fig. 6A, the viability

-p

of PC12 cells decreased significantly after the treatment of H2O2 in a dose-dependent

re

manner. The viability of PC12 cells which incubated with 200 μM H2O2 reached

in the further analysis.

lP

49.07 ± 4.81%. Thus, 200 μM H2O2 was used for the induction of oxidative damage

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As shown in Fig. 6B, comparing to the damage group, the viability of PC12 cells

pre-treated with ALF1 increased significantly in a dose-dependent manner. The viability of cells pre-treated with 1000 μg/mL ALF1 reached 85.23% of the control

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value (the blank control). The result indicated that the pre-treatment of ALF1 could promote proliferation of PC12 cells to resist H2O2-induced oxidative damage

effectively.

3.8. Effects of ALF1 on the change of ROS and LDH release in treated PC12 cells 23

Excessive ROS levels resulted in cell damage. Antioxidants could be used to reduce ROS content to protect cells (Poljsak, Suput, & Milisav, 2013). As presented in Fig. 6C, the level of ROS in PC12 cells increased significantly after the treatment of 200 μM H2O2 (p < 0.01). The ROS levels in PC12 cells pre-treated with ALF1 decreased

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in a dose-dependent manner. 800 and 1000 μg/mL ALF1 represented better inhibitory effect than the positive controls (VC). Moreover, there was no significant difference

ro

between the ROS levels in cells pre-treated with of 1000 μg/mL ALF1 and the blank

-p

control group. These data indicated that ALF1 had positive effect of eliminating intracellular ROS.

re

Lactate dehydrogenase (LDH) was a stable cytoplasmic enzyme. The LDH

lP

leakage was a vital indicator to evaluate cell damage (Jin et al., 2019). As shown in Fig. 6D, LDH release increased significantly after the treatment of 200 μM H2O2 (P <

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0.01). However, ALF1 exhibited consistent protective activity in a dose-dependent manner. 800 and 1000 μg/mL ALF1 represented better protective effect than positive controls (VC). Overall, these results indicated that ALF1 could contribute to the

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protection of PC12 cells against H2O2-induced oxidative stress. 3.9. Effects of ALF1 on activities of SOD, GSH-Px, CAT and MDA content Previous reports demonstrated that the excessive accumulation of ROS resulted in increase of MDA content and decrease of the activity of enzymes such as SOD, GSH-Px and CAT (Zhang et al., 2019). In addition, polysaccharides from Flammulina 24

velutipes, Morchella importuna, corn silk and Sphallerocarpus gracilis were proved to protect cells against oxidative stress via decrease MDA content and increase SOD, GSH-Px and CAT activities (Guo, Liu, Wang, Shi, & Zhang, 2018; Guo et al., 2019; Hu et al., 2017; Xiong, Li, Chen, Chen, & Huang, 2016). Consequently, we evaluated

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the antioxidant effects of ALF1 by measuring MDA content and activities of SOD, GSH-Px and CAT enzymes.

ro

As demonstrated in Table 3, comparing with the blank controls, the activities of

-p

SOD, GSH-Px and CAT of the damage group decreased sharply after the treatment, and meanwhile, the MDA level of damage group increased obviously. However, the

re

cells which pre-treated with ALF1 had significantly increased activities of SOD,

lP

GSH-Px and CAT comparing to the damage group. With the treatment of ALF1, the MDA content in PC12 cells also decreased significantly, which was the product of

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lipid peroxidation (Xiong et al., 2016). In this study, ALF1 increased activities of SOD, GSH-Px and CAT and decreased MDA levels in a dose-dependent manner. Therefore, the findings suggested that enzymatic mechanism might be the way to

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protect cells against oxidative stress.

3.10. Analysis of mitochondrial membrane potential in PC12 cells The dysfunction of mitochondria caused by oxidative stress was due to the ROS accumulation which lead to changes in mitochondrial membrane potential (Ahmad, 25

Ijaz, Shabbiri, Ahmed, & Rehman, 2017; Lemasters & Nieminen, 1997). Consequently, we evaluated the antioxidant effects of ALF1 by mitochondrial membrane potential investigation. In this study, the changes in mitochondrial membrane potential were evaluated by Rhodamine 123 staining. As shown in Fig. 7A,

of

in contrast with the blank controls, the fluorescence intensity of the damage group decreased sharply after the treatment. In contrast, the fluorescence intensity of

ro

ALF1-treated PC12 cells increased in a dose-dependent manner and the fluorescence

-p

intensity of cells treated with 800 and 1000 μg/mL ALF1 reached 79.25% and 83.76% respectively, which were higher than the positive control (75.32%) (Fig. 7B). This

re

result showed that pretreatment of ALF1 had an effective protection on cells against

lP

mitochondrial dysfunction. Thus, the findings indicated that ALF1 could effectively reverse the decrease of mitochondrial membrane potential in H2O2-induced PC12

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cells.

4. Conclusion

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This is the first report of the exopolysaccharides from the fermentation mycelia of Floccularia luteovirens and two EPS fractions (ALF1 and ALF2) were obtained. The chemical characterizations, thermal properties and in vitro antioxidant activities of ALF1 and ALF2 were investigated. Results demonstrated that ALF1 had better antioxidant activity than ALF2 in five different in vitro antioxidant capacity assays. 26

The elaborate structure of ALF1 was elucidated using linkage analysis and 2D NMR. ALF1 mainly contained six kinds of linkage type units as →4)-β-D-Manp→, 1,3-α-Fucp→, α-L-Araf-C1→, →6)-β-D-Galp-C1→, →4)-α-D-GlcAp-(1→ and →3)-β-D-Glcp(1→. In addition, ALF1 had good bioactivities such as anticancer and

of

antioxidant activities. ALF1 was proven to be able to inhibit tumor cells without affecting normal cells. Besides, ALF1 improved the activities of SOD, GSH-Px and

ro

CAT, and decreased the production of MDA which result in protect PC12 cells against

-p

H2O2-induced oxidative stress. ALF1 could decrease ROS production, and stabilize mitochondrial membrane potential. In this study, we evaluated the antioxidant

re

activities of EPS from F. luteovirens in vitro and in cells, and investigated the

lP

mechanism of ALF1 to protect cells against oxidative stress. Thus, we suggested the

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EPS derived from this fungus might be developed as a functional food component.

Notes: The authors declare no competing financial interest. Acknowledgements

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This study was financially supported by the National Nature Science Foundation of China (31960471, 31871904) and Nature Science Foundation of Zhejiang Province (LR13C200002).

27

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33

Fig. 1. Stepwise elution curve of the EPSs fractionated using DEAE-52 column (A) and elution curves subsequently obtained using Sephacryl S-200 column (B-C).

Fig. 2. Antioxidant capacities of EPS in five different assays. (A) Scavenging ability

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of two EPS fractions of F. luteovirens on DPPH radicals. (B) Chelating ability of EPS fractions from F. luteovirens on ferrous ions. Each value is expressed as the mean ±

ro

standard deviation. The different letters represent the statistical differences at P < 0.05

-p

among the investigated groups.

lP

re

Fig. 3. 1H-NMR (A) and 13C-NMR (B) spectra of ALF1.

Fig. 4. 1H-1H COSY (A), 1H-13C HSQC (B), 1H-13C HMBC (C) spectral analysis of

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ALF1 polysaccharide.

Fig. 5. Effects of ALF1 on the viability of LO2 and HepG2 cells. (A) the human normal hepatocytes LO2, (B) human hepatoma HepG2. All data are expressed as

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mean ± S.D. The different letters represent the statistical differences at P < 0.05 among the investigated groups.

34

Fig. 6. Effects of ALF1 on the viability, ROS generation and LDH leakage of PC12 cells treated with H2O2. (A) The cells were incubated with H2O2 of various concentrations. (B) Protection effect of ALF1 on the viability of PC12 cells. (C) ROS levels in PC12 cells. (D) LDH leakage of PC12 cells. All data are expressed as mean

of

± S.D. The different letters represent the statistical differences at P < 0.05 among the

ro

investigated groups.

-p

Fig. 7. Effects of ALF1 on the mitochondrial membrane potential of PC12 cells. (A) the fluorescence in PC12 cells with the exposure of H2O2. Each image was

re

representative of 20 randomly observed fields; (B) fluorescence intensity analysis. All

lP

data are expressed as mean ± S.D. The different letters represent the statistical

Jo

ur na

differences at P < 0.05 among the investigated groups.

35

Table 1. Yields and chemical compositions of EPS fractions from culture broth of F. luteovirens. ALF1

ALF2

Yield (%)

68.52 ± 1.13

29.23 ± 0.45

Total carbohydrate (%)

96.62 ± 4.74

95.62 ± 3.13

Protein (%)

NDa

ND

Man

35.58

17.41

Glc

23.19

Fuc

13.86

Gal

16.73

GlcA

10.31

of

Sample

0.0

ND, not detected.

Jo

ur na

a

43.90

36

0.0

-p

re 0.33

lP

Ara Rha

ro

Monosaccharide Compositions (%)

23.82

0.0

0.0 14.86

Table 2. Glycosidic linkages of ALF1 by methylation analysis. Linkage pattern

MW

PMAA

(Da)

Molar ratio (%)

1,4-di-O-acetyl-2,3,5-tri-O-methyl arabinitol

279

0.35

t-Fuc(p)

1,5-di-O-acetyl-6-deoxy-2,3,4-tri-O-methyl fucitol

293

4.27

t-Man(p)

1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol

323

14.23

t-Gal(p)

1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol

323

5.32

2-Fuc(p)

1,2,5-tri-O-acetyl-6-deoxy-3,4-di-O-methyl fucitol

321

8.69

3-Glc(p)

1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl glucitol

351

5.97

2-Man(p)

1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl mannitol

351

15.29

2-Gal(p)

1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl galactitol

351

3.49

6-Man(p)

1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl mannitol

4-Gal(p)

1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol

4-GlcA(p)

1,4,5-tri-O-acetyl-1,6,6-tri-deuterio-2,3,6-tri-O-methyl glucitol

2,3-Man(p)

ro

of

t-Ara(f)

5.51

351

2.35

353

10.42

1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl mannitol

379

1.03

6-Gal(p)

1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol

351

2.23

2,4-Gal(p)

1,2,4,5-tetra-O-acetyl-1,6,6-tri-deuterio-3,6-di-O-methyl galactitol

381

0.84

3,6-Glc(p)

1,3,5,6-tetra-O-acetyl-1,6,6-tri-deuterio-2,4-di-O-methyl glucitol

381

5.67

2,6-Man(p)

1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl mannitol

379

0.67

2,6-Glc(p)

1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl glucitol

379

11.44

2,6-Gal(p)

1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl galactitol

379

2.23

Jo

ur na

lP

re

-p

351

37

Table 3. Effects of ALF1 on SOD, GSH-Px, CAT and MDA in H2O2-induced PC12 cells. GSH-Px (U/mg protein)

CAT (U/mg protein)

MDA (nmol/mg protein)

Blank control

105.47 ± 5.56a

184.32 ± 2.76a

42.37 ± 2.25a

1.13 ± 0.08a

Damage group

53.29 ± 1.37b

83.65 ± 2.17b

9.65 ± 0.51b

3.23 ± 0.15b

ALF1 (200 μg/mL)

68.52 ± 1.76c

132.65 ± 1.99c

32.83 ± 1.38c

2.59 ± 0.12c

ALF1 (400 μg/mL)

78.72 ± 3.27d

143.17 ± 7.59d

35.25 ± 1.87d

ALF1 (800 μg/mL)

84.37 ± 3.12e

166.40 ± 6.99e

38.83 ± 0.58e

ALF1 (1000 μg/mL)

93.61 ± 3.44f

172.02 ± 7.22f

40.26 ± 2.13f

1.33 ± 0.07f

Positive control

78.74 ± 2.03d

167.62 ± 8.88e

37.89 ± 2.01g

1.83 ± 0.05g

of

SOD (U/mg protein)

2.16 ± 0.08d 1.65 ± 0.12e

ro

-p

Group

re

All data are expressed as mean ± S.D. The different letters represent the

Jo

ur na

lP

statistical differences at P < 0.05 among the groups.

38