Comparison of characterization, antioxidant and immunological activities of three polysaccharides from Sagittaria sagittifolia L.

Journal Pre-proof Comparison of characterization, antioxidant and immunological activities of three polysaccharides from Sagittaria sagittifolia L Jinyan Gu, Haihui Zhang, Hui Yao, Jie Zhou, Yuqing Duan, Haile Ma

PII:

S0144-8617(20)30113-2

DOI:

https://doi.org/10.1016/j.carbpol.2020.115939

Reference:

CARP 115939

To appear in:

Carbohydrate Polymers

Received Date:

14 November 2019

Revised Date:

21 December 2019

Accepted Date:

30 January 2020

Please cite this article as: Gu J, Zhang H, Yao H, Zhou J, Duan Y, Ma H, Comparison of characterization, antioxidant and immunological activities of three polysaccharides from Sagittaria sagittifolia L, Carbohydrate Polymers (2020), doi: https://doi.org/10.1016/j.carbpol.2020.115939

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Comparison of characterization, antioxidant and immunological activities of three polysaccharides from Sagittaria sagittifolia L. Jinyan Gu a, Haihui Zhang a, b, *, Hui Yao a, Jie Zhou a, Yuqing Duan a, b, *, Haile Ma a, b

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China.

b

Institute of Food Physical Processing, Jiangsu University, Zhenjiang 212013, China.

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a

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Corresponding author:

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*Haihui Zhang, Ph.D., Professor, School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

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Email: [email protected]

*Yuqing Duan, Ph.D., Professor, School of Food and Biological Engineering, Jiangsu

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University, Zhenjiang 212013, China

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Tel: +86 511 88780201

Fax: +86 511 88780201 Email:[email protected]

Highlights 1



Three processes were used to extract S. sagittifolia L. polysaccharides (SSs).



Extraction processes had significant effects on physicochemical properties of SSs.



Polysaccarides extracted by subcritical water had greater potential to be biologicalagents.

Abstract

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To investigate and compare the preliminary structural characteristics and biological activity in vitro of polysaccharides from Sagittaria sagittifolia L. (SSs) by different extration methods,

three polysaccharides (SSW, SSU, and SSP) were obtained with hot water, ultrasound-assisted,

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and subcritical water extraction. Their structural features were elucidated using High

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Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Scanning

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Electron Microscopy (SEM), Infrared Spectroscopy (IR), Atomic Force Microscopy (AFM), Zeta Potential and Congo red methods. Furthermore, the antioxidant activity and

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immunostimulatory effects were investigated in vitro. Molecular weight and monosaccharide composition analysis exhibited that SSW (2275.0 kDa), SSU (148.7 kDa), and SSP (1984.0

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kDa) were heteropolysaccharide with dramatically different monosaccharide species and mole ratios. In addition, SSP exhibited stronger antioxidant activity in vitro and more potent

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immunomodulatory activity than SSW and SSU. SSP has greater potential to be explored as biologicalagents for use in complementary medicine or functional foods.

Keywords:

Sagittaria sagittifolia L. polysaccharides 2

Ultrasound-assisted extraction Subcritical water extraction Preliminary structure Antioxidant and immunomodulatory activity

1. Introduction

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S. sagittifolia L. (Alismataceae) which is generally called Cigu or Yanweicao in

China, is a perennial aquatic plant belonging to the Alismatidae Sagittaria families. It is found in different countries around the world, predominately in North America,

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Europe and Asia (Ahmed Wani et al. 2015). It has long been utilized both as a healthy

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food source and as a restorative in traditional Chinese medicine for the adjuvant

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therapy of tuberculosis, night blindness, pancreatitis, diabetes, tracheitis and urinary tract infections. The health beneficial effects of S. sagittifolia L. are owing to its

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various bioactive ingredients, such as polysaccharides, proteins, minerals, vitamins and dietary fiber (Wani et al. 2015). Generally, polysaccharides are considered the

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main bioactive components in S. sagittifolia L., which possess various bioactivities, such as antioxidant (Zhang et al. 2019), immunological (Zhang et al. 2019), antitumor

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(Wang et al. 2016), hypoglycemic (You et al. 2018), and antimicrobial (Alboofetileh et al. 2019) activities. The extraction technique could play an important role in the yield and structural characteristic of the polysaccharides, as well as the biological activities (Chen et al. 2019). In general, hot water extraction has been broadly used as a traditional 3

technology for the extraction and preparation of polysaccharides since ancient time as a result of conveniently in operation and environmentfriendly characteristics (Yan et al. 2018). However, it is usually associated with a longer heating and extraction time, larger solvent amounts, which can damage the polysaccharide structures, and in turn, has lower extraction efficiency (Zhang et al. 2019). In recent decades, researches on the polysaccharides non-conventional extraction techniques, including the use of

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enzymes and non-conventional energy sources (microwave, ultrasound, subcritical

water and supercritical fluid) have been attracted more attention (Alboofetileh et al. 2019). Furthermore, these innovative extraction methods have the significantly effects

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on modification the chemical composition, molecular weight distribution and

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biological activity of the target polysaccharides (Huang et al. 2019). Chen et al. (2019) obtained the bamboo shoot polysaccharides with better digestibility from quadrangularis

through

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Chimonobambusa

ultrasound-

and

enzymeassisted

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extractions. Z. Y. Zhu et al. (2016) extracted polysaccharides from Cordyceps gunnii mycelia by using room-temperature water, hot-water extraction, microwave-assisted,

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ultrasound-assisted, and cellulase-assisted extraction methods. They found that microwave-assisted extraction was the simple and effective method for improving the

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antitumor activity by a distinct intrinsic viscosity and spatial conformation of C. gunnii polysaccharides. Alboofetileh et al. (2019) compared antibacterial, antiviral and cytotoxic activities of fucoidans obtained from Nizamuddinia zanardinii by using different extraction technologies (hot water, alcalase, flavourzyme, cellulase, viscozyme, ultrasound, microwaves, alcalase-ultrasound, microwave-ultrasound, and 4

subcritical water), and their results showed that fucoidans extracted by microwaves and subcritical water inhibited the growth of E. coli and those isolated by alcalase-ultrasound, microwave-ultrasound and subcritical water showed inhibitory effects against P. aeruginosa at 2 mg/mL, which may due to their various chemical compositions and molecular weights. Thus, extraction methods are closely related with physicochemical properties, structural characteristics, and biological activities.

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However, to the best of our knowledge, no study has been devoted to

comprehensively compare the effect of different extraction techniques on the yield, physicochemical characteristics and biological activities of the polysaccharides from

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S. sagittifolia L., furthermore, the relationship between chemical structure and

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antioxiant activity of S. sagittifolia L. polysaccharides has not been investigated. Therefore, in the present study, we aimed to extract water-soluble SSs from S.

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sagittifolia L. by using different extraction methods. The effects of extraction methods

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on the yield, chemical profile, as well as the monosaccharide composition, molecular weight, surface topography, and other preliminary structural features. The antioxidant

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activities of these SSs in vitro and the relationship between chemical structure and activity of polysaccharides from S. sagittifolia L. were also investigated

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comprehensively.

2. Materials and methods

2.1. Materials and chemicals

S. sagittifolia L. was purchased from a local market (Zhenjiang, China). The dry 5

S. sagittifolia L. (100 g) was crushed into powder, passed through a 60 mesh sieve. The powder was degreased with petroleum ether and subjected to soak in 80% ethanol at room temperature for 2 h to remove small molecule sugar and colored substances. The residue was air dried and sealed in airtight plastic bags before use. Monosaccharide standard samples were purchased from Aladdin Industrial Corporation (Shanghai, China). The 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′

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-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from

Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Medium Dulbecco’s modified Eagle’s (DMEM), 3 (4,5 dimethyltiazol 2 yl) 2,5 diphenyltetrazolium bromide (MTT)

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and lipopolysaccharide (LPS) were provided from Sigma Chemical Co. (St. Louis,

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USA). Other chemical reagents and solvents were analytical grade, provided by

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Sinopharm Chemical Reagents co., LTD (Shanghai, China).

2.2. Extraction of SSs by different methods

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2.2.1. Hot water extraction

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The grinded powder was mixed with distilled water with the solvent to raw

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material ratios (30 mL/g) in a big beaker, and then extracted at 100 ◦C for 2 h. The suspension was centrifuged (4500 rpm, 10 min) and concentrated to one-fifth of the initial volume using a rotary evaporator at 42 ◦C under reduced pressure.

2.2.2. Ultrasound-assisted extraction

Approximately 0.83 g of grinded powder was mixed with distilled water (25mL) 6

in a small beaker and placed in an ultrasonic cell disruptor (SL-400S, Nanjing Shunliu Instrument Co., Nanjing, China) working at a frequency of 25 kHz, input power of 120 W, equipped with digital time and temperature controller. The mixture was then extracted using extraction times (15 min), and extraction temperature (80 ◦C). The suspension was centrifuged (4500 rpm, 10 min) and concentrated to one-fifth of the

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initial volume using a rotary evaporator at 42 ◦C under reduced pressure.

2.2.3. Subcritical water extraction

Approximately 0.83 g of grinded powder was mixed with distilled water (25 mL)

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in a subcritical extraction tank, and then extracted at 170 ◦C for 16 min. The

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suspension was centrifuged (4500 rpm, 10 min). The supernatant was collected and

reduced pressure.

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concentrated to one-fifth of the initial volume using a rotary evaporator at 42 ◦C under

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2.2.4. Preparation for the polysaccharides

The above supernatant was precipitated with ethanol (80%) at 4 °C overnight.

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The precipitates were collected by centrifugation at 4500 r/min for 10 min. The

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proteins were removed by Sevage method (Liu et al. 2015), and then dialyzed with 3500 Da molecular weight cutoff membrane against distilled water for 48 h, and lyophilized to obtain the crude polysaccharides (SSW, SSU, and SSP).

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2.3. General physicochemical properties of SSs

2.3.1. Chemical analysis of SSs

The total carbohydrate, uronic acid, protein of SSs were determined by the phenol-sulfuric acid method (Dubois, Hamilton, Rebers, & Smith, 1956), via m-hydroxy biphenyl method (Blumenkrantz and Asboe-Hansen, 1973), and via the

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coomassie bright blue method (Bradford, 1976), respectively. The SSs yield (Y) was

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measured by the following equation:

Where C denotes the concentration of polysaccharides (mg/mL); V represents the

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volume of extraction solution; K is the dilution factor (mL); W denotes the weight of

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raw materials (g).

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2.3.2. Determination of zeta potential

The zeta potentials (Zp) measurements was accomplished in polysaccharide

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solutions (1 mg/mL) using Card Zeta potential/Particle sizer (Malvern, UK) at 25 °C.

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Each sample was repeated three times.

2.3.3. Determination of molecular weights of SSs

The determination of molecular weight was based on previous methods, with

minor modifications (Yan et al. 2018). Three kinds of polysaccharides were extracted by different processes prepared into 1 mg/mL solution with 0.1 mol/L NaCl and 8

filtered through 0.45μm membranes before injection. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and MWD (Mw/Mn) of SSs were mensurated by high performance size-exclusion chromatograph (HPSEC) combined with MultiAngle Laser Light Scattering detector (MALLS, DAWN HELEOS II, Wyatt Technology, USA), differential Refractive Index detector (Agilent G1362A, USA) and collocated with two columns (shodex OHpak SB-805 and shodex

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OHpak SB-806) in a series at 25℃. Sample elution was performed, 0.1M NaCl was

applied as the elution solvent, at a flow rate of 0.5 mL/min. The differential refractive index increment (dn/dc) of the SSW, SSU and SSP was 0.130, 0.125 and 0.138 mL/g.

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Astra software was used for data processing and analysis.

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2.3.4. Determination of monosaccharide compositions

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The common derivatization methods include: trimethylsilylation, saccharide nitrile acetate, as well as saccharide alcohol acetate and trifluoroacetate. In this

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experiment, the polysaccharide samples were derived by saccharide nitrile acetic acid

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esterification. Monosaccharides composition of SSs were determined by GC method on basis of the previous method with minor modification (Zhang, Chen, et al. 2019).

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SSs samples (10.0 mg) were hydrolyzed with sulfuric acid solution (2 mol/L, 5mL) at 100℃ for 8 h. After hydrolysis, the solution was neutralized with BaCO3, then the supernatant was concentrated and dried by rotary evaporator under low pressure. Subsequently, hydroxylamine hydrochloride (10.0 mg) and pyridine (1.0 mL) were added to hydrolysate at 90℃ water bath for 30min. After that, acetic anhydride (1.0 9

mL) were added, again reaction at 90℃ for 30 min, and the product obtained was sugar nitrile acetate derivatives. The standard monosaccharides were derivatived according to the above methods. The derivative was filtered through a 0.22 μm membrane and measured by a gas chromatograph (7890 A, Agilent Technologies, CA, USA) which was equipped with a HP-5 fused silica capillary column (30 m × 320 μm × 0.25 μm) and a flame ionization detector (FID). The detection conditions were as

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follows: N2 (30 mL/min), H2 (38 mL/min) and air (370 mL/min). The temperature of the detector and injector were 300 °C and 280 °C, and the oven temperature was

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maintained at 130℃ for 5 min, then rise to 240℃ with the rate of 4 ℃/min.

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2.3.5. FT-IR spectroscopic analysis

A Nicolet is 50 FT-IR spectrometer (Thermo Electron, Madison,WI, USA) was

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used to record Fourier transform–infrared (FT-IR) spectra of SSs at 4000-400 cm-1 by

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KBr tablet pressing method (He et al. 2018).

2.3.6. Congo-red assay

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The Triple helix structure of SSs were determine by Congored method (Xiong et

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al. 2019) with minor modifcations. Briefly, the solution of sample (2.0 mL, 1 mg/mL) was mixed with 2.0 mL of Congo-red solution (91 μmol/L) and then 1 M NaOH solution was gradually added to the mixtures to fnal NaOH concentration of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mol/L. After standing for 10 min, the UV absorption spectrum was scanned from 400 to 700 nm by a UV spectrophotometer (Implen Nanophotometer, Germany). 10

2.3.7. SEM observation

After the SSs powder was fixed on metal platform, and then coated with a thin gold layer, the freeze-dried samples were examined using a SEM (JSM-7800F, JEOL. Tokyo. Japan) (Wang et al. 2016) at an accelerating voltage of 15.0 kV and image magnification of 300× and 1000×.

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2.3.8. Determination of AFM

Polysaccharide samples (1mg) were dissolved in deionized water to 10 μg/mL.

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After stiring to dissolve it fully and filtered through 0.22 μm membranes, 10 μL of the

polysaccharide diluent droplets were placed on freshly cleaved mica surface and dried

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overnight at room temperature (J. Chen, Zhang, et al. 2019). Multimode 8 atomic

polysaccharides.

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force microscope (AFM) was used to directly observe the molecular morphology of

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2.4. Antioxidant activity assays in vitro

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2.4.1. DPPH radical scavenging activity

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The DPPH radical scavenging activity of SSs was performed on the basis of (Ji et al. 2019) with minor modification. Various concentrations of SSs (1.00-5.00mg/mL) were reacted with DPPH solution (0.1 mmol/L, previously dissolved in ethanol) for 30 min in the dark followed by absorbance determination at 517 nm. The reaction mixtures without DPPH were used as a control and vitamin C (VC) for positive control. The DPPH radical scavenging rate was calculated using the following 11

formulation: DPPH radical scavenging ability (%) = [1 - (A1 - A2)/A0] × 100 (1) Where A0: the absorbance without sample, A1: the absorbance of the sample and DPPH solution, and A2: the absorbance without DPPH.

2.4.2. Assay of ABTS scavenging activity

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The ABTS radical scavenging activities of SSs were fulfilled according to (Xiao et al. 2019) with appropriate modifications. Briefly, moderate amount of ABTS (7 mol/L) and persulfuric acid (2.45mol/L) were added into acquire the ABTS+· solution

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(7 mmol/L) in the dark for 12 h at room temperature. The mixture was diluted with

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deionized water to a working solution to get absorbance of 0.70 ± 0.02 at 734nm priors to use. Subsequently, 100 μL polysaccharide sample solution (1.00-5.00 mg/mL)

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was mixed with 100 μL ABTS working liquid, then placed in the dark for 10 min at room temperature after mixed thoroughly, followed with the absorbance was

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measured at 734 nm. Distilled water was used as a control and VC was used as

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positive control. The ABTS radical scavenging activity was counted as following equation:

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ABTS radical scavenging activity of SSs (%) = [1 - (A1 - A2)/A0] × 100 (2) Where A0: the absorbance without sample, A1: the absorbance of sample, A2: the absorbance without ABTS+· solution).

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2.4.3. Hydroxyl radical scavenging assay

Hydroxyl radical scavenging assay was executed by Fenton method in accordance with (L. Wang, Li, and Wang 2018). Briefly, 0.2 mL of different concentrations polysaccharide sample (1.00-5.00 mg/mL) was mixed with 1 mL of FeSO4 (0.15 mmol/L), 0.4mL of salicylic acid-ethanol (2 mmol/L), 1 mL of H2O2 (6

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mmol/L), and 0.4 mL distilled. Then the mixed solutions were incubated at 37℃ in water bath for 1h, measured at 510 nm. Distilled water was used as a control and VC

was taken as positive control. The hydroxyl radical scavenging rate was calculated as

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following formula:

Hydroxyl radical scavenging rate (%) = [1 - (A1 - A2)/A0] × 100 (3)

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Where A0: the control absorbance without sample, A1: the absorbance of sample; A2:

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the background absorbance without H2O2.

2.5. In vitro immunomodulatory activity assay

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2.5.1. Cell lines and culture

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Mouse macrophage line RAW264.7 was donated by Jiangsu university hospital (Zhenjiang, China). The cells were cultured in DMEM sugar medium containing 10%

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FBS, penicillin (100 IU/ml) and streptomycin (100 mg/L) in a humidified 5% CO2 atmosphere at 37℃ before use. Treatments included DMEM medium blank control group, LPS (lipopolysaccharide) positive control group (1 μg/mL), various concentrations (50, 100, 200 and 400 μg/mL) of SSW, SSU and SSP.

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2.5.2. Cell proliferation, phagocytosis and NO determination assay

RAW264.7 cells (2×105 cell/mL) was seeded onto 96-well culture plates for 12 h and then treated with control and different concentrations of polysaccharides samples for 24h. The effect of polysaccharides extracted by different method on the proliferation of macrophages was determined by the modified MTT method (Fan et al. 2016). The pinocytosis activity of RAW 264.7 cells was determined according to

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the neutral red staining method (Z. Wang, Cai, and He 2018). The level of nitric oxide (NO) in the supernatant was evaluated using Griess method (Yu et al. 2018).

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2.6. Statistical analysis

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All experiments were performed in triplicates at least. The data values were

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subjected to SPSS software version 16.0 and OriginPro Software 2017 to determine differences and obtain graphics and were expressed as means ± standard deviations

(ANOVA).

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(S.D.) with significance p < 0.05 after passing one-way analysis of variance

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3. Results and discussion

3.1. General physicochemical properties of SSs

3.1.1. Extraction yields and chemical composition

The extraction yields and chemical composition of the SSs from S. sagittifolia L. extracted with different extraction technologies are listed in Table 1. The yields of the 14

SSs were determined as SSW (12.99 ± 0.23%) < SSP (20.37 ± 0.54%) < SSU (24.46 ± 0.42%), which suggested that the sub-critical water extraction technology, especially ultrasonic assistance extraction technology could significantly increase the extraction yields of SSs. In general, subcritical water extraction method promotes solubilization applying high pressures and temperatures, and the high temperatures tend to weaken the hydrogen bond, followed with reducing the energy needed for

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division cell wall and increasing extraction efficiency (Muñoz-Almagro et al. 2019). While, ultrasound-assisted extraction can effectively accelerate the penetration of solvents into raw material through mechanical pulverization, thermal and cavitation

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effects of ultrasound, which would help increase the extraction rate (Zhao et al. 2018).

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Moreover, the lowest extraction yield of SSs was obtained during hot water extraction, which may due to high temperature and long time bring about polysaccharides

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degradation. This result was in agreement with those of (Guo, Shang, Zhou, Zhao, &

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Zhang, 2017; Zhang, Chen, Wen, et al., 2019), who reported higher yield with ultrasonic assistance and sub-critical water extraction than with hot water extractation.

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The highest total sugar content was obtained from SSU (63.57 ± 2.04%) via ultrasound-assisted extraction, and the lowest was obtained from SSW (50.04 ±

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1.74%) via hot water, while, uronic acid content was highest in SSP (8.62 ± 0.19%), followed by SSU (3.43 ± 0.15%), and SSW (2.75 ± 0.12%). In addition, all three polysaccharides contain proteins, and this result revealed that they may be glycoproteins containing polysaccharide transport chains and protein residues (Zhao et al. 2017) or residual free protein. 15

Table 1 The physicochemical properties of SSs extracted by different techniques. Samples

SSW

SSU

SSP

Polysaccharides yield (%)

12.99 ± 0.23

24.46 ± 0.42

20.37 ± 0.54

Total polysaccharides content

50.04 ± 1.74

63.57 ± 2.04

66.15 ± 2.13

Protein content (%)

7.78 ± 0.45

7.62 ± 0.21

3.13 ± 0.13

Uronic acid content (%)

2.75 ± 0.12

3.43 ± 0.15

8.62 ± 0.19

Mw

2275

149

Mn

2023

137

Mw/Mn

1.125

1.316

Zeta potential (mV)

-10.74

Average Molecular Weights

1984 1734

1.144

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(kDa)

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(%)

-11.23

-15.88

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Values are expressed as mean ± standard deviation (n = 3).

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3.1.2. Zeta potential of SSs

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2275 and 2023 kDa) and SSP (1984 and 1734 kDa

Characterization of polysaccharide charge can be indicative of the stability of a

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solution or colloid (G. Chen, Li, et al. 2019). Generally, the higher the absolute value of polysaccharide potential is, the more stable it is. Therefore, the zeta potentials of

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the three polysaccharides were determined. As can been shown from Table 1, SSP had the largest amount of negative charge. In general, the negative charge of the polysaccharide is mainly produced by the uronic acid, which is in agreement with the result in Section 3.1.1 that SSP owns the highest content of uronic acid. Thus, the larger negative charge reveals SSP had good stability, which was similar to the result 16

of polysaccharides from bamboo shoot (G. Chen, Li, et al. 2019).

3.1.3. Molecular weight of SSs

Molecular weight reflects the molecular chain of polysaccharide and is closely related to its biological activity (He et al. 2018). Therefore, the molecular weight of SSs needs to be evaluated. The molecular weight of the SSs are summarised in Table

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1. The weight average molecular weight (Mw) and number average molecular weight (Mn) of SSU were 149 and 137 kDa, respectively, were significantly smaller than

those of SSW (2275 and 2023 kDa) and SSP (1984 and 1734 kDa). Because it is

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difficult to dissolve macromolecular polysaccharide at low ultrasonic temperature,

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moreover, ultrasonic extraction would change the structure of polysaccharides and break the branch chain of polysaccharides (Zhong et al. 2015). Similar studies have

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displayed that the molecular weights of polysaccharides extracted by ultrasound assisted extraction are lower than that of conventional hot water extraction and

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subcritical water extraction (He et al., 2018; Zhang et al., 2019). Results also showed

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that molecular weights of SSP were lower than those of SSW, which may due to high temperature would destroy the molecular chains of polysaccharides and degrade

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polysaccharide molecules (You et al. 2018). Su, Lai, & Ng (2017) also confirmed a coincident situation when the extraction temperature rose from 70℃ to 121℃, the relative contents of the low molecular weight population of Grifola frondosa polysaccharides increased from 30.8 to 43.3%, which suggested that the high extraction temperature may have resulted in the degradation of the high molecular 17

weight population. In addition, polydispersity index of four polysaccharides was < 2, which mean that molecules dispersed less in aqueous solution and do not form larger aggregates (Jixian Zhang, Wen, Gu, et al. 2019).

3.1.4. Neutral monosaccharide composition of SSs

The monosaccharide composition of polysaccharides is often analyzed

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quantitatively and qualitatively by GC. The obtained GC results are shown in Table 2 and Fig.1. SSW, SSU and SSP all contained D-Glucose and D-Galactose in different molar percentages and SSU also included L-Rha and

D-Arabinose

although their

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amounts were very little. The results indicated that three polysaccharides have

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different monosaccharide constituent units; all classified as heteropolysaccharides. Yan et al. (2018) reported that four polysaccharides extracted from Hericium

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erinaceus via different solvents all comprised L-Rha, D-Ara, D-Man, D-Glc, and D-Gal with varying molar percentages. Zhang, Chen, Wen, et al. (2019) reported that the

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monosaccharide compositions of two fractions (SSP-W1 and SSP-S1) obtained from

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the S. sagittifolia L. difffer significantly, not only in proportion but also in type. Consequently, the monosaccharide composition and content may be decided by the

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extraction methods and separation processes. Table 2

Monosaccharide composition of SSs extracted by different methods (%). Monosaccharide composition

Molar Composition (mol %) SSW

SSU

(mol %) 18

SSP

L-Rhamnose

13.58

1.85

8.47

D-Arabinose

4.06

2.71

2.09

D-Xylose

N.D.

N.D.

N.D.

D-Mannose

N.D.

0.55

N.D.

D-Glucose

68.05

74.86

75.01

D-Galactose

14.31

20.03

14.43

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N.D.: Not detectable or lower than the limit of quantification.

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Fig. 1. GC analysis of monosaccharide constitute stemed from SSW, SSU, and SSP. (A) Standard monosaccharides; peaks: (1) L-Rhamnose, (2) D-Arabinose, (3) D-Xylose, (4) D-Mannose, (5) D-Glucose, (6) D-Galactose; (B) SSW, (C) SSU, (D) SSP.

3.1.5. FT-IR spectra characteristics

The absorption peak in the infrared spectrum can reflect the vibration of atoms or functional groups in the polysaccharide molecules. Because of its strong 19

characteristics, infrared spectrum is usually used to analyze the types and vibration of functional groups in the polysaccharides. Fig.2 displayed the FT-IR spectra of SSW, SSU and SSP among absorption bands in the 400–4000 cm-1 region. It is clear that the main absorption bands were similar in the three polysaccharides with a few differences in some absorption peaks. Similar parts included the broad and strong peak at approximately 3298 cm-1 and the small band at approximately 2976 cm-1 in

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the spectra of SSs attributed to the stretching vibrations of the hydroxyl groups and C-H stretching and bending vibrations, respectively (Nie et al. 2018). These two

absorption peaks belong to the characteristic groups of polysaccharides. The

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absorption peaks observed at approximately 1657 and 1400 cm-1 (1450 cm-1) were

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assigned to the asymmetric and symmetric stretching of the carboxylate anions groups (Yan et al. 2018). Occurrence of amide group vibrations at 1400 cm-1 (due to Amide

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III), 1537 cm-1 (due to Amide II) and 1657 cm-1 (due to Amide I) indicate that there is

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bound protein in these polysaccharides, which was consistent with the results of Thambiraj, Phillips, Koyyalamudi, & Reddy, (2018) and section 3.1.1 in this

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manuscript. There were also absorption peaks of sulfation groups (SO3-) at 1252 cm-1 on behalf of an asymmetrical S=O stretching vibration (Gu et al. 2020). In addition,

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the absorptions between 1000 and 1200 cm-1 (1070 and 1159 cm-1) can be in agreement with the ring vibrations overlapping with the stretching vibrations of the C-O-H side groups and the C-O-C glycosidic bond vibrations, which suggested that the three polysaccharides contained pyranose monomers in their structures (Nie et al. 2018). The peak at 857 cm-1 is a typical characteristic of α-D-glucopyranose ring 20

(Rong et al. 2019). Q. Yuan et al., (2019) reported that no obvious difference was observed among the characteristic organic groups of the polysaccharides from okra extracted by hot water, pressurized water and microwave assisted techniques. Alboofetileh et al., (2019) also found that three polysaccharides obtained with different extraction solvents from the brown alga Nizamuddinia zanardinii had similar FT-IR patterns. Our results were in agreement with above studies. However, a few

ro of

differences included the absorption of at 1023 cm-1 which was probably the stretching vibration of the C-N (Tang et al. 2016) where SSW and SSU had weak peak while

-p

SSP had hardly peak, which is consistent with their protein content.

3500

3000

2500

1410 1450 1252 1159 1070 857

1537

2000

1500

1000

500

-1

Wavenumber（ cm ）

Fig. 2. FT-IR spectra of SSs.

Jo

ur

4000

1023

1657

3298

na

2976

lP

re

SSW SSU SSP

3.1.6. Congo red test

The functional and biological activities of polysaccharides are related to their triple-helix conformation, especially in immunoregulation and antitumor activities, 21

which can be identified simply using the Congo red test (J. Chen, Zhang, et al. 2019). The polysaccharides containing triple helical structure could form a Congo red-polysaccharide complex, which will display a λmax red shift compared to the Congo red control (G. Chen, Chen, et al. 2019). When the biopolymers are under the action of strong alkaline, the triple helical conformations can be destroyed and a decline in red-shift effect of the Congo red-polysaccharide complex can be observed.

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The effects of NaOH concentration on the λmax of the Congo red-polysaccharide complexes are presented in Fig. 3. SSU and SSP displayed a large red shift in λmax

compared to the Congo red blank, indicating that these two polysaccharides possessed

-p

a triple-helix structure. However, SSW showed no signifcant red shift in λmax,

re

suggesting the polysaccharide did not have a triple-helical conformation in solution. These results might be due to variations in the hydrogen bond by different extration

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methods. The stability of the triple helical structure in polysaccharide solution

na

depends mainly on intramolecular and intermolecular hydrogen bonds and the rupture of the hydrogen bond will damage triple helical structure (Xiong et al. 2019). Long

ur

time and high temperature extraction, which may break the hydrogen bond, had a

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destructive effect on the triple helical structure of SSW.

22

510

SSW SSU SSP Blank

505

λmax/nm

500 495 490 485 480 0.0

0.1

0.2

0.3

0.4

0.5

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NaOH concentration (mol/L)

-p

Fig. 3. The maximum absorption wavelengths of Congo red-polysaccharide complex and Congo red solution at various concentrations of NaOH.

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3.1.7. SEM analysis of SSs

Previous studies indicated that extraction, purification, and preparation

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conditions could have influence on the structures, activities and surface morphology of polysaccharides (Ma et al. 2018). Fig.4 showed the surface morphology of SSs

na

determined by SEM at magnifications of 1,000 ×, and 3,000 ×. The SEM images of

ur

SSW in Fig. 4 presented some irregular lumpish particles and rough surface with a non-uniform size, indicating that high temperature and long time treatment could

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cause polysaccharide aggregation. However, SSU possessed relatively loose and flaky surfaces with some small pores and curly morphology, which may be caused by the substantial cavitation activity, turbulence shear, and instantaneous high pressure (Xu et al. 2018). Compared with the two processes above, subcritical water treatment mainly take advantage of high pressure to disaggregate polymer clusters by 23

destroying the noncovalent intra and inter-molecular bonds, but polysaccharide might reaggregat after this treatment (Huang et al. 2018) and this phenomenon leads to the

-p

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relatively even and big schistose surface of SSP, with some small particles.

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Fig. 4. Scanning electron micrographs (300×, 1000×) of SSW, SSU, and SSP.

lP

3.1.8. AFM

AFM is a useful tool to characterize morphology on the nanostructural, and the

na

random linear or spheres structures of polysaccharides (J. Chen, Zhang, et al. 2019). As shown in Fig. 5, the 2D and 3D image of SSW, SSU and SSP displayed many

ur

spherical and inhomogeneous lumps. The agglomerations suggest polysaccharide

Jo

molecules have gathered and that their structures were branched and entangled. The results were agreement with the Li Wang, Liu, & Qin, (2017). The spherical lumps of SSP in the diameter and height ranges of 1.49–7.65 nm and 54.08–116.95 nm were imaged. The SSU aggregated to form spherical particles with diameters ranging from 1.28 to 5.67 nm, heights ranging from 44.08 to 85.92 nm, which was a reduction in

24

diameter and height of the molecular chain of SSU compared with that of SSP. The reason may be that SSP contains more aldehyde acid rich in hydroxyl and carboxyl groups, which formed the hydrogen bonds and provided strong intermolecular and intramolecular interactions with each other (Gao et al. 2017). SSW molecules were aggregated with diameter from 1.16 to 9.68 nm and height of 60.62 to 163.74 nm. The aggregates of SSW are bigger than these of SSP and SSU, while the content of uronic

ro of

acid is the lowest. It manifested that the significant different in the molecular

conformation conformation of SSs may be related to monosaccharide composition and ratio (Wang et al. 2019). The results of SEM and AFM were in accordance with

Jo

ur

na

lP

re

-p

each other.

25

ro of -p re lP

na

Fig. 5. Comparison of AFM images of SSW (A), SSU (B), and SSP (C) at 10 μg /mL.

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3.2. In vitro antioxidant activity

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3.2.1. DPPH radical scavenging ability

DPPH radical is a stable at room temperature and was extensively applied to

estimate the free radical scavenging activity of natural compounds (Y. Wang et al. 2018). The principle of this method is that the original fuchsia-red DPPH radical is reduced to yellow (non-free radical form, DPPH-H) by providing hydrogen and 26

electrons, and the maximum absorption value of the original fuchsia-red at 517 nm is reduced (Shen et al. 2018). Fig. 6A showed the scavenging activities of SSW, SSU, SSP and Vc against the DPPH radical. The three polysaccharides demonstrated a concentration-dependent scavenging activity against DPPH within the concentration from 0 mg/mL to 5.0 mg/mL, however the scavenging ability of each polysaccharide is distinct. At the concentration of 5.0 mg/mL, the scavenging effects of SSW, SSU,

ro of

and SSP on DPPH were 61.49%, 54.20%, and 66.54%, respectively, which were much lower than that of Vc (94.36%, 5.0 mg/mL). The scavenging ability of SSP was

higher than the other two polysaccharides (SSW and SSP), which might be due to SSP

re

3.2.2. ABTS+ radical scavenging ability

-p

diverted more hydrogen atoms to neutralize DPPH free radicals (Xiao et al. 2019).

lP

ABTS radical assay is a simplified method to determine the antioxidant capacities of polysaccharides (Yuan et al. 2018). The ABTS radical scavenging

na

experiment is based on the transfer of electrons from antioxidants to ABTS radicals

ur

(Liu et al. 2019). As shown in Fig. 6B, all these SSs exhibited significant scavenging ability on ABTS in a concentration dependent manner. As the concentration increased,

Jo

the scavenging effect of SSP increased significantly from 33.41% to 94.59%, and the SSW and SSP reached 91.19% and 86.24% at 5.0 mg/mL, respectively, which were stronger than that of the result reported by Zhang, Chen, et al. (2019), but less than that of Vc (99.92%).

27

3.2.3. Hydroxyl radical scavenging ability

Hydroxyl radicals are the most active radicals in biological systems. They could readily pass through cell membranes, forming breaking DNA strands, and have the ability to give rise to cancer, mutagenesis, and cytotoxicity (Cui et al. 2018). Hydroxyl radical is one of the reactive oxygen radicals, which can cause serious

ro of

damage to the functional biomolecular in living cells (Liu et al. 2017), therefore it is significant to scavenge the hydroxyl radicals. The hydroxyl free radical scavenging

activity of three polysaccharides was measured with Vc as a positive control and the

-p

results were shown in Fig. 6C. Obviously, their scavenging effect against hydroxyl radical correlated positively well with increasing concentrations. Interestingly, when

re

the concentration increased from 0 to 1.0 mg/mL, the hydroxyl radical scavenging

lP

activities of SSW, SSU and SSP were almost same. But, the scavenging effects of SSW and SSP were higher than that of SSU when the concentration was higher than

na

1.0 mg/mL. For three kinds of polysaccharide components and Vc, the scavenging hydroxyl radical effects decreased in the order of SSP > SSW > SSU > Vc. These

ur

results showed that these polysaccharides of S. sagittifolia L. demonstrated great

Jo

capacity for scavenging hydroxyl radicals, especially SSP and SSW.

28

ro of -p

lP

re

Fig. 6. In vitro antioxidant activity of SSs. (A) DPPH radical scavenging assay, (B) ABTS radical scavenging assay, (C) Hydroxyl radical scavenging assay.

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3.2.4 Correlation of structure and antioxidant activity

It is well known that the antioxidant activities were influenced by several factors,

ur

such as content of uronic acid, molecular weight, monosaccharide composition and

Jo

ratios, as well as types of sugar (Lo et al. 2011). Although the role of the structure in antioxidant activity of these polysaccharides remained indistinct, some relationships can be extrapolated from current study as follows. The results showed that SSP has the higher antioxidant activities than SSW and SSU, which could be associated with the high molecular weight (2241kDa), uronic acid content (8.62%) and the amount of rhamnose (8.47%), big schistose and clumpy structures, triple helix configuration and 29

larger Zeta potential of SSP. Shen et al. (2018) investigated polysaccharides (WL-CPS-1, NaCl-CPS-1 and Glu-CPS-1) of different Mw (1.02 × 103 kDa, 1.12 × 103 kDa and 1.33 × 103 kDa), extracted from the Nostoc flagelliforme and found higher antioxidant activity values for high Mw polysaccharides. In other investigation by Fimbres-Olivarria et al. (2018), it was found that the rhamnose content in the polysaccharide from the Navicula sp. was the main determinant factor associated with

ro of

the antioxidant properties. It has been widely established that existence of uronic acid

appeared to be imperative in testifying the antioxidant ability of polysaccharides (Zhu et al. 2019); the higher the content of uronic acid, the greater the antioxidant

-p

capability (Liu et al. 2019). Z. Y. Zhu et al. (2016) extracted polysaccharides from

re

Cordyceps gunnii mycelia by different extraction methods. They found that the polysaccharides that contained clumpy structures, indicating the extension of the

lP

chain, exhibited better biological activity than the polysaccharides that contained a

na

flaky appearance and helix structure. In Our results were good consistent with the above reports. The potential connection between structure and biological activity of

ur

polysaccharides can be very intricate. Studies in relevance between structure and

Jo

function of polysaccharides remained to be further investigated.

3.5. Immunomodulatory activities of the polysaccharide fractions

3.5.1. Effects on macrophage viability

To investigate the effect of SSW, SSU and SSP on the RAW264.7 viability, MTT assays were used to examine cell proliferation. As shown in Fig. 7A, compared with 30

the blank control, the three polysaccharides (50, 100, 200, 400 μg/mL) remarkably promoted the proliferation of RAW264.7 macrophages in a dose-dependent manner (P < 0.05). The appreciation rate of SSU was higher than SSW and SSP at the concentration of 50 and 100 μg/mL, but at the concentration of 200 and 400 μg/mL, the appreciation rate of SSP was higher than SSW and SSU. The three polysaccharides all have no inhibition on macrophage proliferation in the

ro of

concentration range of 50–400 μg/mL.

3.5.2. Effects on phagocytic ability

-p

Macrophages are an important part of the body’s immune system, activated

re

macrophages and recruiting other immune cells to activate specific defense mechanisms (Yu et al. 2017). Therefore, phagocytic ability was examined by neutral

lP

red phagocytosis assay in the present study. As shown in Fig. 7B, phagocytosis indices for SSW, SSU and SSP in treated concentrations were all higher than 1.0,

na

suggesting that all of them had the ability to activate RAW 264.7 cells. Compared to the blank control, the three polysaccharides all significantly enhanced the

ur

phagocytosis of neutral red (p<0.01), and as SSW, SSP (50, 100, 200 μg/mL) and SSU

Jo

(50, 100 μg/mL) concentration increased, the phagocytosis activity enhanced. When the concentration of polysaccharides was further increased, the phagocytosis index decreased, but the value is still higher than the blank control. There were obvious differences between the three fractions, and SSP showed a stronger phagocytic ability than SSW and SSU. The phagocytosis index of SSP at a concentration of 200 μg/mL

31

was even close to that of the positive control (LPS, 1 μg/mL).

3.5.3. Effects on NO production

Studies have shown that NO has a wide range of immunological significance and plays a key role in the immune system as an important signal transduction medium (Nie et al. 2018). As shown in Fig. 7C, untreated RAW264.7 cells produced a small

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amount of NO, and the three polysaccharides increased the production of NO in a trend consistented with that of phagocytic activity at the concentration between 50 and 400 μg/mL. The amount of NO production stimulated by each fraction followed

Jo

ur

na

lP

re

-p

the order SSP > SSU > SSW.

Fig. 7. Effect of SSs on the (A) proliferation, (B) phagocytic activity, (C) NO production of mouse macrophages. *p<0.05, **p<0.01, contract with the control group.

32

4. Conclusions

Influences of different extract methods on the extraction yield, physicochemical properties, and biological activity in vitro of the S. sagittifolia L. polysaccharide were investigated in this study. Experimental results indicated that SSU had the highest yield. SSP had the higher molecular weight (2275kDa), uronic acid content (8.62%)

ro of

and the amount of rhamnose (8.47%), and larger Zeta potential than SSW and SSU, induced stronger capabilities of DPPH, ABTS and hydroxyl radical scavenging activity, higher levels of macrophage proliferation and phagocytosis, as well as

-p

stimulated higher NO production. These results indicate subcritical water extraction is

an effective technique for improving the bioactivities potential of S. sagittifolia L.

re

polysaccharide and SSP may be developed as natural biological agents for the

na

Acknowledgements

lP

pharmaceutical and functional food industry.

This study was supported by National Key R & D Program, China

ur

(2016YFD0400303), Key Research and Development Plan of Jiangsu Province

Jo

(BE2016350, BE2017353) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Key Technology R & D Program of Zhenjiang (No. SH2019011).

33

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