In vitro antioxidant and immunostimulating activities of polysaccharides from Ginkgo biloba leaves

Accepted Manuscript In vitro antioxidant and immunostimulating activities of polysaccharides from Ginkgo biloba leaves

Qi Ren, Jing Chen, Yu Ding, Jianghua Chen, Song Yang, Zhenhua Ding, Qianying Dai, Zhien Ding PII: DOI: Reference:

S0141-8130(18)34447-7 https://doi.org/10.1016/j.ijbiomac.2018.11.276 BIOMAC 11153

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

25 August 2018 18 November 2018 29 November 2018

Please cite this article as: Qi Ren, Jing Chen, Yu Ding, Jianghua Chen, Song Yang, Zhenhua Ding, Qianying Dai, Zhien Ding , In vitro antioxidant and immunostimulating activities of polysaccharides from Ginkgo biloba leaves. Biomac (2018), https://doi.org/ 10.1016/j.ijbiomac.2018.11.276

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ACCEPTED MANUSCRIPT In vitro antioxidant and immunostimulating activities of polysaccharides from Ginkgo biloba leaves Qi Ren1#, Jing Chen2#, Yu Ding 3, Jianghua Chen4, Song Yang4, Zhenhua Ding2, Qianying Dai1*, Zhien Ding1*

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1 School of Tea and Food Science & Technology, Anhui Agricultural University, Hefei 230036, China

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2 Anhui Institute of Product Qaulity Supervison and Inspection, Hefei 230051, China

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3 First Affiliated Hospital of Anhui Medical University, Hefei 230022, China

4 Agro-products Processing Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031,

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 Corresponding author; Tel/fax: +86 551 65786982; E-mail address:[email protected] (Q-Y. Dai); [email protected] (Z-E.Ding) # These authors contributed equally to this study and share first authorship. 1

ACCEPTED MANUSCRIPT Abstract: Ginkgo biloba leaves (GBLs) are used as herbal dietary supplements and medicine worldwide. In this study, crude GBL polysaccharides (GBPSs) were extracted and further purified on a DEAE (diethylaminoethanol) Sepharose Fast Flow column to yieldobtain GBPS-2 and GBPS-3. The molecular weights of GBPS-2 and

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GBPS-3 werehad molecular weights of 672 and 723 kDa, respectively. GBPS-2 and

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GBPS-3 were typical acidic heteropolysaccharides, composed of mannose (Man),

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rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), and arabinose (Ara) (molar ratio: 0.08:0.12:0.16:0.06:0.11:1.00:0.32)

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and Man, Rha, GlcA, GalA, Gal, and Ara (molar ratio: 0.92:1.00:0.83:0.11:0.42:0.23),

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respectively. GBPS-2 and GBPS-3 exhibited limited scavenging abilities for the hydroxyl and 2,2-diphenyl-1-picrylhydrazyl radicals as well as noticeable scavenging

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effects on superoxide radicals and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic

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acid) radicals. Furthermore, GBPS-2 and GBPS-3 significantly increased the phagocytosis of macrophages and promoted the production of NO, tumor necrosis

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factor (TNF)-α, interleukin (IL)-1β, and IL-6. Thus, GBPS-2 and GBPS-3 exhibit

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potential for useapplication as functional food supplements. Keywords: Ginkgo biloba; Polysaccharides; Antioxidant activity; Immunostimulating activity

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ACCEPTED MANUSCRIPT 1. Introduction Ginkgo biloba L., regarded as a “living fossil,” has existed on Earth for 200 million years [1]. G. biloba leaf (GBL), one of the most well-known plant-derived medicinal materials, has been used for treating asthma in China for several thousand

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years [2]. Fifty million G. biloba trees are distributed worldwide. They are

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particularly common in China, the United States, and France. These trees produce

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approximately 8000 tons of dried leaves to meet the annual commercial demand for GBL products [3]. GBL has been widely accepted for use as an herbal dietary

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supplement or medicine in many European countries as well as in the United States.

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In 2001, 4.5 million–5.1 million pounds of dried GBLs were consumed [3,4]. The GBL extract contain 22%–27% flavone glycosides, 2.8%–3.4% terpene lactones

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(including ginkgolides A, B, and C), 2.6%–3.2% bilobalide, and <5 ppm of ginkgolic

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acids [5]. Some studies and clinical trials have demonstrated that GBL has various health-promoting effects, including antioxidant, anti-inflammatory, antidiabetic,

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hepatoprotective, antimicrobial, antihypertensive, and anticancer effects [4,6,7].

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Polysaccharides, composed of numerous monosaccharides joined by glycosidic linkages, exhibit many biological functions in organisms, such as structural support, antigenic determinants, and energy storage [8,9]. Numerous studies have revealed that natural plant-derived polysaccharides exhibit many bioactivities, such as antioxidant, anti-inflammatory, immunomodulatory, antitumor, antidiabetic, and antiobesity activities [10–12]. Some natural polysaccharides have been widely used as nutritional dietary additives in functional foods [10,13]. Antioxidant and immunomodulatory 3

ACCEPTED MANUSCRIPT activities are the primary bioactivities of polysaccharides and have been extensively investigated in recent years [14]. For instance, studies on polysaccharides from Lupinus luteus L. [15], swollen culms of Zizania latifolia [14], and Paxillus involutus [16] have demonstrated that these polysaccharides exhibit superior antioxidant and

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immunomodulatory activities.

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GBL polysaccharides (GBPSs), critical bioactive components in GBLs, have

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received considerable attention due to their numerous health-promoting bioactivities [17]. Studies have mainly focused on the antioxidant, antitumor, hepatoprotective, and

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anti-inflammatory activities of GBPSs. Chen et al. obtained the crude polysaccharides

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from Ginkgo biloba exocarp and found the crude polysaccharides exhibited noticeable scavenging activity for hydroxyl radical, which was greater than that for the positive

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control [3]. However, the antioxidant activities of purified GBPSs should be further

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comparatively investigated. HoweverFurthermore, reports on the immunomodulatory activity evaluated using RAW264.7 cells have been limited. Here, we extracted crude

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GBPS samples from GBLs and purified them on a diethylaminoethanol (DEAE)

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Sepharose Fast Flow column to obtain GBPS-2 and GBPS-3. The chemical properties, molecular weights, and monosaccharide compositions of GBPS-2 and GBPS-3 were investigated. In addition, in vitro antioxidant and immunomodulatory activities were evaluated. 2. Materials and methods 2.1.Materials GBLs were obtained from Bozhou Hongyu Co., Ltd. (Bozhou, China). DEAE– 4

ACCEPTED MANUSCRIPT Sepharose Fast Flow columns were obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). Monosaccharide standards, including ribose (Rib), glucose (Glc), rhamnose (Rha), fucose (Fuc), galacturonic acid (GalA), mannose (Man), galactose (Gal), xylose (Xyl), glucuronic acid (GlcA) and arabinose (Ara), were

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obtained from Sigma Chemical Co. (St. Louis, MO, USA), along with (DPPH),

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2,2-diphenyl-1-picrylhydrazyl

(LPS)

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2,2ʹ-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), lipopolysaccharide and

3-methyl-1-phenyl-2-pyrazolin-5-one

(PMP).

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased

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from Solarbio Life Sciences (Beijing, China). Dulbecco’s modified Eagle medium (DMEM), streptomycin–penicillin, trypsin–ethylenediaminetetraacetic acid (EDTA),

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and fetal calf serum were obtained from Gibco/Invitrogen (Carlsbad, CA, USA).

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Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were obtained from Nanjing Jiancheng Bioengineering

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Institute (Nanjing, China). All other chemical reagents used here were analytical

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grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2.GBPS-2 and GBPS-3 preparation GBPSs were extracted as reported previously [13]. In brief, GBLs was milled into a powder. After the removal of the small molecules, such as polyphenols, pigments, monosaccharides, and oligosaccharides, by using ethanol (70%, v/v) three times for 8 h at 70°C, the sample was dried at 50°C until it reached a stable weight. Hot water (1:10, w/v) was used to extract GBPSs from samples three times at 70°C over 3 h. 5

ACCEPTED MANUSCRIPT The obtained extract solutions were combined, precipitated using three volumes of dehydrated ethanol, and maintained overnight at 4°C. The precipitates were then centrifuged (4000 rpm, 15 min) and washed using absolute ethanol and acetone. Subsequently, proteins was removed from the precipitates by using the Sevag method

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(CHCl3/nBuOH = 5:1, the ratio of GBPS to Sevag reagent was 10:1). Finally, the

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solution was dialyzed and lyophilized to obtain crude GBPS for further purification.

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GBPS solution (10 mg/mL, 20 mL) was loaded onto a DEAE Sepharose Fast Flow column (2.6 × 50 cm2) and eluted stepwise by using various concentrations of

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NaCl solutions (0, 0.1, 0.3, and 0.5 M) at a flow rate of 1.0 mL/min. The eluates were

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then collected and assessed using the phenol–sulfuric acid method [18]. Two main fractions were finally obtained, GBPS-2 and GBPS-3.

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2.3. Chemical composition analysis

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The carbohydrate, uronic acid, protein, and total polyphenol contents of GBPS-2 and GBPS-3 were determined using the phenol–sulfuric acid method with glucose as

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the standard [18], the m-hydroxydiphenyl–sulfuric acid method with galacturonic acid

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as the standard [19], the Bradford method with bovine serum albumin as the standard [20], and the Folin–Ciocalteu method with gallic acid as the standard [21], respectively. The monosaccharide compositions of GBPS-2 and GBPS-3 were evaluated using the Agilent 1100 high-performance liquid chromatography (HPLC) system (Agilent, USA) with a Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm, Agilent Technologies, USA) according to the PMP derivation method [22] under the following chromatographic conditions: acetonitrile and phosphate-buffered 6

ACCEPTED MANUSCRIPT saline (PBS, 0.1 M, pH 6.7) in a ratio of 17:83 (v/v), flow rate of 1.0 mL/min at 35°C, and detector wavelength of 245 nm. 2.4.Determination of homogeneity and molecular weight The homogeneity and molecular weights of GBPS-2 and GBPS-3 were

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determined using Waters 1525 HPLC system equipped with a refractive index detector,

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which in turn was equipped with a TSK G4000PWXL column (7.8 × 300 mm2; Tosoh

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Corp., Tokyo, Japan). The mobile phase was deionized water at a flow rate of 0.5 mL/min. Molecular weight was calibrated on the basis of D-series dextrans standards

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with known molecular weights.

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2.5. Fourier-transform infrared spectroscopic analysis A Nicolet iS50 Fourier-transform infrared (FT-IR) spectrometer (Thermo Fish

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Scientific Inc., Waltham, MA, USA) was used to evaluate the FT-IR spectrum of

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GBPS-2 and GBPS-3 over 400–4000 cm-1. 2.6.In vitro antioxidant activity assay

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2.6.1. Hydroxyl radical scavenging activity assay

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The hydroxyl radical scavenging activities of GBPS-2 and GBPS-3 were evaluated according to a slightly modified version of the method by Wang et al. [14]. In brief, the test mixture solution comprised 50 μL of GBPS solutions (0, 0.125, 0.25, 0.5, 1.0, 1.5, 2.0, and 4.0 mg/mL), 50 μL of H2O2 (0.3%), 50 μL of FeSO4 (9 mmol/L), and 25 μL of salicylic acid–ethanol solutions (9 mmol/L), which were pipetted into a 96-well plate. The plate was incubated at 37°C for 30 min, and the absorbance (Abs) was measured at 532 nm. The hydroxyl radical scavenging activities of GBPS-2 and 7

ACCEPTED MANUSCRIPT GBPS-3 were calculated using the following equation: Hydroxyl radical scavenging activity (%) =

𝐴𝑏𝑠0 −(𝐴𝑏𝑠1 −𝐴𝑏𝑠2 ) 𝐴𝑏𝑠0

× 100,

where Abs0 is the Abs of the blank control (water instead of sample), Abs1 the Abs of GBPS-2 and GBPS-3 samples, and Abs2 the Abs of the GBPS-2 and GBPS-3 samples

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under same conditions as Abs1, but with salicylic acid–ethanol solution rather than

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FeSO4–H2O2 solution.

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2.6.2. DPPH radical scavenging activity assay

The DPPH radical scavenging activities of GBPS-2 and GBPS-3 were evaluated

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according to a modified version of a previously reported method [23]. In brief, the

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reaction mixture comprised 50 μL of GBPS solutions (0, 0.125, 0.25, 0.5, 1.0, 1.5, 2.0, and 4.0 mg/mL), 100 μL of deionized water, and 25 μL of methanolic DPPH solution

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(0.4 mM), and the mixture was pipetted into a 96-well plate. After the mixture

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underwent reaction in the dark for 30 min, Abs was measured at 517 nm. The DPPH radical scavenging activities of GBPS-2 and GBPS-3 were calculated using the

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following formula:

𝐴𝑏𝑠1 −𝐴𝑏𝑠2 𝐴𝑏𝑠0

) × 100,

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DPPH radical scavenging activity (%) = (1 −

where Abs0 is the Abs of the blank control (water instead of sample), Abs1 the Abs of GBPS-2 and GBPS-3 samples, and Abs2 the Abs of the GBPS-2 and GBPS-3 samples under same conditions as those of Abs1, but with methanol rather than DPPH solution. 2.6.3. Superoxide anion radical scavenging activity assay The superoxide anion radical scavenging activities of GBPS-2 and GBPS-3 were assessed using a modified version of a previously described method [24]. In brief, the 8

ACCEPTED MANUSCRIPT reaction mixture comprised 50 μL of GBPS-2 or GBPS-3 solutions (0, 0.125, 0.25, 0.5, 1.0, 1.5, 2.0, and 4.0 mg/mL), 75 μL of phosphate buffer (0.15 M, pH 7.4), 50 μL of FeSO4 (0.75 mM), 50μL of H2O2 (0.01%, w/v), and 50 μL of ferroin solution (0.75 mM). After the mixture underwent reaction at room temperature for 5 min, the Abs of

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the reaction solution was measured at 560 nm. Superoxide radical scavenging

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activities of GBPS-2 and GBPS-3 were calculated using the following formula:

Superoxide radical scavenging activity (%) = (1 −

𝐴𝑏𝑠1 −𝐴𝑏𝑠2 𝐴𝑏𝑠0

) × 100,

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where Abs0 is the Abs of the blank control (water instead of sample), Abs1 the Abs of

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GBPS-2 and GBPS-3 samples, and Abs2 the Abs of the GBPS-2 and GBPS-3 samples under same conditions as Abs1, but with 0.1 M phosphate buffer rather than 4-nitro

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blue tetrazolium chloride solution.

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2.6.4. ABTS radical scavenging activity assay The ABTS radical scavenging activities of GBPSs were evaluated according to a

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modified version of a previously described method [25]. Firstly, ABTS (7.0 mM) was

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mixed with potassium persulfate (K2S2O8, 4.95 mM) at room temperature in the dark for 12 h to generate an ABTS radical solution. PBS (0.2 M, pH 7.4) was used to dilute the ABTS solution and obtain a working solution (Abs at 734 nm = 0.7 ± 0.02). Subsequently, 20 μL of GBPS-2 or GBPS-3 solutions (0, 0.125, 0.25, 0.5, 1.0, 1.5, 2.0, and 4.0 mg/mL) was mixed with 200 μL of the working solution. After reaction for 6 min at 25°C, the Abs at 734 nm of the mixture was evaluated. ABTS radical scavenging activities of GBPS-2 and GBPS-3 were calculated using the following 9

ACCEPTED MANUSCRIPT formula: ABTS free radical scavenging activity (%) = (1 −

𝐴𝑏𝑠1 −𝐴𝑏𝑠2 𝐴𝑏𝑠0

) × 100,

where Abs0 is the Abs of the blank control (water instead of sample), Abs1 the Abs of GBPS-2 and GBPS-3 samples, and Abs2 the Abs of the GBPS-2 and GBPS-3 samples

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(PBS instead of ABTS solution).

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2.7. In vitro immunomodulatory activity assay.

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2.7.1. RAW264.7 cell culture

RAW264.7 cells were incubated in DMEM supplemented with 10% (v/v) fetal

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calf serum and 1% (v/v) penicillin–streptomycin in a humidified incubator with a 5%

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CO2 atmosphere at 37ºC. 2.7.2. Cell viability assay

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The effects of GBPS-2 and GBPS-3 on RAW264.7 cell viability were evaluated

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using MTT assays. In brief, 200 μL of suspended RAW264.7 cells were plated per well in a 96-well culture plate and incubated for 12 h. The culture medium was

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replaced by 200 μL of new medium with various concentrations of GBPS-2 or

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GBPS-3 (0, 50, 100, and 200 μg/mL). After incubation for 24 h, the old culture medium was discarded; the MTT solution (50 μL, 2 mg/mL) was added to each well; and the plate was further incubated for 4 h at 37°C. After the MTT solution was discarded, 200 μL of dimethyl sulfoxide was added to dissolve the formazan crystals. Subsequently, a microplate reader was used to the measure the Abs at 570 nm. Cell viability was calculated using the following formula: Cell viability (%) = Abs𝑠𝑎𝑚𝑝𝑙𝑒 /𝐴𝑏𝑠𝐵𝑙𝑎𝑛𝑘 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 . 10

ACCEPTED MANUSCRIPT 2.7.3. Phagocytosis assay The effects of GBPS-2 and GBPS-3 on phagocytic activity were evaluated using a neutral red phagocytosis assay according to a reported method with some modifications [14]. In brief, 200 μL of suspended RAW264.7 cells were plated in

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96-well plates and incubated for 24 h. The culture medium was replaced by 200 μL of

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GBPS-2 or GBPS-3 solutions at various concentrations (0, 50, 100, and 200 μg/mL)

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and positive control (LPS, 10.0 μg/mL) in new medium and incubated for 24 h. After removal of the old suspension, 100 μL of the neutral red solution at a concentration of

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0.075% was added to each well, and the cells were further incubated for 1 h. The

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neutral red solution was decanted, and the cells were washed two times by using PBS to remove the residual neutral red solution. Finally, 100 μL of cell lysis buffer (1.0 M

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acetic acid/ethanol = 1:1, v/v) was added to each well. The Abs of the mixture at 540

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nm was measured after the mixture had been incubated at room temperature overnight. The macrophage phagocytosis index of devouring neutral red was evaluated using the

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following equation:

Phagocytosis index = 𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒 /𝐴𝑏𝑠𝑏𝑙𝑎𝑛𝑘 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 . 2.7.4. NO, TNF-α, IL-1β, and IL-6 assays In total, 1000 μL of suspended RAW264.7 cells was plated in a 12-well plate and incubated for 24 h. The culture medium was replaced by 1000 μL of GBPS-2 or GBPS-3 solutions with various concentrations (0, 50, 100, and 200 μg/mL) and positive control (LPS, 10.0 μg/mL) in new medium and incubated for 24 h. NO 11

ACCEPTED MANUSCRIPT concentrations were measured using commercially available kits. TNF-α, IL-1β, and IL-6 levels in supernatants were determined using ELISA kits according to the manufacturer protocol. 2.7.5. RNA preparation and quantitative real-time reverse-transcription polymerase

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chain reaction analysis

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In total, 1000 μL of suspended RAW264.7 cells was plated in a 12-well plate and

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incubated for 24 h. The culture medium was replaced by 1000 μL of GBPS-2 or GBPS-3 solution at different concentrations (0, 50, 100, and 200 μg/mL) and positive

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control (LPS, 10.0 μg/mL) in new medium and incubated for 12 h. RNA preparation

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and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) analysis were performed according to an approach described previously [26].

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MiniBEST Universal RNA Extraction Kit (TaKaRa Bio. Inc., Dalian, China) was used

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to extract RNA from the cells. cDNA was synthesized from total RNA using PrimeScript RT Master Mix (TaKaRa Bio. Inc.), according to manufacturer protocol.

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An RNA qRT-PCR was performed using SYBR Green Master Mix (TaKaRa Bio. Inc.)

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in an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primer sequences, including those for inducible nitric oxide synthase (iNOS), TNF-α, IL-1β, and IL-6, are listed in Table 1. 3. Results and discussion 3.1. Preparation and preliminary characterization of GBPS-2 and GBPS-3 Crude GBPS was prepared from the GBLs through hot-water extraction, ethanol precipitation, deproteinization procedures, dialysis, and freeze-drying. Crude GBPS 12

ACCEPTED MANUSCRIPT yield was 9.73% ± 1.34%. Three purified fractions, namely GBPS-1, GBPS-2, and GBPS-3, were obtained after separation on a DEAE Sepharose Fast Flow column (Fig. 1a). The yields of GBPS-1, GBPS-2, and GBPS-3 yields were 1.62% ± 0.63%, 9.45% ± 1.67%, and 32.87% ± 3.11%, respectively. GBPS-1 was difficult to collect;

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therefore, GBPS-2 and GBPS-3 were used for further experiments.

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Natural carbohydrate, uronic acid, protein, and total polyphenol contents of

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GBPS-2 and GBPS-3 are presented in Table 2. The carbohydrate contents in GBPS-2 and GBPS-3 were 71.46 ± 1.93 and 44.32 ± 1.18, respectively. GBPS-2 and GBPS-3

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were typical acidic polysaccharides with high uronic acid contents of 12.49% ± 0.98%

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and 38.37% ± 1.62% respectively. Other researchers have also reported high uronic acid content in GBPSs; this characteristicwhich may contribute to GBPS-2 and

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GBPS-3 bioactivities of GBPS-2 and GBPS-3 [3]. In this study, the measured protein

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and total polyphenol contents were <0.3%. In addition, the ultraviolet spectra of GBPS-2 and GBPS-3 (Fig. 1b) revealed a minor peak between 260 and 280 nm,

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indicating the presence of few proteins or polyphenols in the samples.

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The results of the high-performance gel permeation chromatography (GPC) analysis (Fig. 1c and 1d) reflected the homogeneity and molecular weights of GBPS-2 and GBPS-3. Two clear, single, narrow, and symmetrical peaks were observed, suggesting the homogeneity of GBPS-2 and GBPS-3. Calibration with standard dextrants indicated that the molecular weights of GBPS-2 and GBPS-3 were 672 and 723 kDa, respectively. The monosaccharide compositions of GBPS-2 and GBPS-3 were evaluated through HPLC (Table 2 and Fig. 1e). Both GBPS-2 and GBPS-3 were 13

ACCEPTED MANUSCRIPT typical heteropolysaccharides, but their monosaccharide compositions and molar ratios differed considerably. SpecificallyThereinto, GBPS-2 was composed of Man, Rha, GlcA, GalA, Glc, Gal, and Ara (molar ratio: 0.08:0.12:0.16:0.06:0.11:1.00:0.32), whereas GBPS-3 was composed of Man, Rha, GlcA, GalA, Gal and Ara (molar ratio:

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0.92:1.00:0.83:0.11:0.42:0.23).

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The FT-IR spectra of GBPS-2 and GBPS-3 are illustrated in Fig. 1f; these results

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reflect the characteristic absorption peaks of acidic polysaccharides. In the spectra of both GBPS-2 and GBPS-3 spectra, the broad bands at approximately 3400 cm-1 and

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2920 cm-1 were assigned to O-H and C-H stretching vibrations, respectively [27,28].

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Two strong absorption peaks at approximately 1611 cm-1 and 1400 cm-1 were attributable to the strong characteristic asymmetric and symmetric stretching

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vibrations of COO- and C=O, confirming the presence of carboxyl group in GBPS-2

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and GBPS-3 [29,30]. The FT-IR spectra analysis results indicated that GBPS-2 and GBPS-3 were acidic polysaccharides, consistent with the observed chemical

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properties. The complex bands ranging from 1000–1200 cm-1 were assigned to C-OH

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bending vibrations and coupled C-O,C-C stretching [24,31]. 3.2. Antioxidant activities of GBPS-2 and GBPS-3 3.2.1. Hydroxyl radical scavenging activity The hydroxyl radical, one of the strongest free radicals, can induce oxidative damage

to

adjacent

biomolecules,

resulting

in

cytotoxicity,

mutagenesis,

carcinogenesis, and other diseases [9,14]. In this study, dose-dependent decrease in the hydroxyl radical scavenging activities of GBPS-2 and GBPS-3 were clearly 14

ACCEPTED MANUSCRIPT observed with the increase in sample concentrations from 125 to 2000 μg/mL (Fig. 2a). No increase in scavenging activity was observed when the concentrations of GBPS-2 and GBPS-3 were higher than 2000 μg/mL. The hydroxyl radical scavenging activities of GBPS-2 and GBPS-3 at the concentration of 4000 μg/mL were 34.43% ±

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2.49% and 35.62% ± 1.56%, respectively, which were much lower than those of the

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scavenging abilities compared with the positive control.

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positive control. Both GBPS-2 and GBPS-3 exhibited weaker hydroxyl radical

3.2.2. DPPH radical scavenging activity

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DPPH radical scavenging activity is commonly used for evaluating the antiradical

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effects of natural polysaccharides considering polysaccharides’ hydrogen-donating abilities and stability after acceptance of an electron or hydrogen radical [30]. DPPH

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radical scavenging activities of GBPS-2 and GBPS-3 are plottedshowed in Fig. 2b.

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Both GBPS-2 and GBPS-3 demonstrateddisplayed a significant dose-dependent increase at concentrations of 125–4000 μg/mL. The DPPH radical scavenging

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activities of GBPS-2 and GBPS-3 were 38.35% ± 2.87% and 44.59% ± 3.65%,

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respectively, at the concentration of 4000 μg/mL. These results indicated that compared with the positive control, both GBPS-2 and GBPS-3 exhibited limited scavenging abilities for the DPPH radical compared with the positive control. 3.2.3. Superoxide anion radical scavenging activity The superoxide anion radical is generated during numerous biological and photochemical reactions and is considered a weak oxidant. However, the superoxide anion radical may form stronger reactive oxidative species, including hydrogen 15

ACCEPTED MANUSCRIPT peroxide, hydroxyl radical, and singlet oxygen, which can cause oxidative damage [3,27]. In this study, GBPS-2 and GBPS-3 exhibited similar levels of antioxidant activity for the superoxide anion radical, as denoted in Fig. 2c. At concentrations from 125 to 1000 μg/mL, the superoxide anion radical scavenging activities of GBPS-2 and

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GBPS-3 increased with polysaccharide concentrations. This correspondent increase

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slowed when the GBPS-2 and GBPS-3 concentrations exceeded 1000 μg/mL. The

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superoxide anion radical scavenging activities of GBPS-2 and GBPS-3 were more than 60% at the concentration of 1000 μg/mL, suggesting that compared with the

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compared with the positive control.

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positive control, GBPS-2 and GBPS-3 affected the superoxide radicals considerably

3.2.4. ABTS radical scavenging activity

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ABTS radical scavenging activity has been widely used to evaluate the total

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antioxidant activities of polysaccharides [32]. Therefore, the ABTS radical scavenging activities of GBPS-2 and GBPS-3 were investigated in this study. GBPS-2

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and GBPS-3 exhibited significant concentration-dependent increase in antioxidant

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activities from 125 to 4000 μg/mL. At the concentration of 4000 μg/mL, the ABTS radical scavenging activities of GBPS-2 and GBPS-3 were 74.34% ± 2.10% and 82.01% ± 1.26%, respectively, close to the corresponding positive control values. Thus, GBPS-2 and GBPS-3 exhibited antioxidant activities in experiments with the ABTS radical. The result showed both GBPS-2 and GBPS-3 exhibited limited scavenging abilities for the hydroxyl and DPPH radicals and noticeable scavenging effects on 16

ACCEPTED MANUSCRIPT superoxide and ABTS radicals. Thereinto, the antioxidant activity of GBPS-3 was superior to that of GBPS-2 for the DPPH, superoxide anion, and ABTS radicals, potentially because of the higher content of uronic acid in GBPS-3 [24]. Yuan et al. purified two kinds of polysaccharides from mulberry leaves (MLP-3a and MLP-3b)

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and found MLP-3b showed higher antioxidant activity than MLP-3a, which might be

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due to the higher content of uronic acid of MLP-3b [21]. Hu et al. reported that

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carboxyl groups are critical in the antioxidant activities of polysaccharides for

more easily than hydroxyl groups can [9].

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scavenging radicals because carboxyl groups can donate electron or hydrogen radicals

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3.3. In vitro immunomodulatory activities of GBPS-2 and GBPS-3 Accumulating evidence suggests that immunity-enhancing activity, one of the

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most critical bioactivities of polysaccharides, is indispensable to their role as

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antitumor agents [33,34]. Numerous studies have reported that plant-derived polysaccharides exhibit immunomodulatory activities, which are superior to from

other

sources

[35,36].

However,

research

on

the

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polysaccharides

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immunomodulatory activity of GBPS is limitedlacking. Therefore, by using RAW264.7 cells, some parameters of immune activation were measured to evaluate the immunostimulatory activities of GBPS-2 and GBPS-3.

3.3.1. Effects of GBPS-2 and GBPS-3 on macrophage viability Natural polysaccharides must be subjected to toxicological evaluation to determine the safety of their use prior to investigationevaluation of their 17

ACCEPTED MANUSCRIPT immunostimulating activities. Macrophage viability is a critical indicator of immunoactivation [22]. Therefore, here, the effects of GBPS-2 and GBPS-3 on the viability of RAW264.7 cells were evaluated using MTT assay. As indicated in Fig. 3a, neitherboth GBPS-2 norand GBPS-3 exhibited no cytotoxicity toward RAW264.7

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cells at the tested concentrations of 50–200 μg/mL. For GBPS-2, a significant

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promotion of macrophage proliferation was observed at a concentration of 200 μg/mL

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compared with the blank control (p < 0.05). However, GBPS-3 did not affect RAW264.7 cell viability.

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3.3.2. Effects of GBPS-2 and GBPS-3 on phagocytosis

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Macrophage phagocytosis is a crucial part of the first and imperative step in an immune response; this activity is considered one of the most distinct characteristics of

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activated macrophages [37,38]. Therefore, the effects of GBPS-2 and GBPS-3 on

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macrophage phagocytosis were evaluated using the neutral red uptake method in this study. As indicated in Fig. 3b, GBPS-2 and GBPS-3 significantly increased

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macrophage phagocytosis dose-dependently compared with the blank control (p <

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0.05). The results suggested that both GBPS-2 and GBPS-3 critically contributed to immune activation by eliciting an increase in macrophage phagocytosis. Other natural polysaccharides have also been reported to enhance macrophage phagocytosis [14,22]. 3.3.3. Effects of GBPS-2 and GBPS-3 on NO and cytokine production When macrophages are activated by external conditions, they release numerous cell factors, including NO, TNF-α, IL-1β, and IL-6; these are directly involved in 18

ACCEPTED MANUSCRIPT inhibiting cancer cells and removing microbial infections [22,37]. NO, a critical cytotoxic mediator released from macrophages, plays a pivotal role in inflammatory reactions, immune responses, and antiviral and antitumor activities [14,33]. TNF-α, a major proinflammatory cytokine produced chiefly by activated macrophages, can

PT

induce the secretion of other immunoregulatory and inflammatory mediators, such as

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IL-6 and IL-1β, to regulate immune response [39]. IL-6 and IL-1β, two types of

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proinflammatory cytokines, also play vital roles in immune responses and host defense [40,41]. Accordingly, inciting increases in the levels of these cytokines by

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using natural polysaccharides may be an effective strategy for improving immune

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response. Therefore, the effects of GBPS-2 and GBPS-3 on the production of NO, TNF-α, IL-1β, and IL-6 were investigated in further assessment of the

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immunostimulatory activities of these polysaccharides. As showedindicated in Fig. 4,

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the lowest production of NO, TNF-α, IL-1β, and IL-6 was observed in the blank control. Compared with the blank control, treatments with various concentrations of

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GBPS-2 or GBPS-3 (50, 100, or 200 μg/mL) for 24 h significantly stimulated the

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production of NO, TNF-α, IL-1β, and IL-6 in a concentration-dependent manner (p < 0.05). Thus, both GBPS-2 and GBPS-3 exhibited significant immunostimulating effects on murine macrophage cells RAW264.7 by promoting cytokine secretion. The effects of GBPS-2 and GBPS-3 on mRNA expression of iNOS, TNF-α, IL-6, and IL-1β were also examined using qPCR to confirm whether GBPS-2 and GBPS-3 enhance NO and cytokine production by inducing gene expression. The mRNA expressions of iNOS, TNF-α, IL-6, and IL-1β were significantly greater than those in 19

ACCEPTED MANUSCRIPT the blank control (Fig. 4b, 4d, 4f, and 4h). Therefore, GBPS-2 and GBPS-3 may have stimulated NO and cytokine secretion in RAW264.7 cells through the upregulation of iNOS, TNF-α, IL-6, and IL-1β expression. Accumulating evidence suggests that various natural polysaccharides can stimulate NO, TNF-α, IL-6, and IL-1β production

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in macrophages, in turn stimulating the immune responses. Ma et al. reported that

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polysaccharides from Strongylocentrotus nudus eggs significantly increase the levels

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of NO and secretion of cytokines, including TNF-α, IL-6, and IL-1β, in RAW264.7 cells [40]. Similarly, Li et al. reported that nonstarch polysaccharides from Chinese

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yam can promote NO, IL-6, and TNF-α release in RAW264.7 cells [41]. Interestingly,

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the results showed that GBPS-2 displayed higher immunostimulating activity than GBPS-3, which was contrary to the result of antioxidant activity. Accumulating

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evidence suggests that the biological activities of polysaccharides are closely related

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to the chemical properties, molecular weights, monosaccharide compositions, glycosidic linkages, and branch degrees of polysaccharides [42,43]. In the current

CE

study, however, the structural characteristics of GBPS-2 and GBPS-3 were not

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reported. Moreover, descriptions of the molecular mechanism underlying the relevant biological activities are limited. Accumulating evidence suggests that the biological activities of polysaccharides are closely related to the chemical properties, molecular weights, monosaccharide compositions, glycosidic linkages, and branch degrees of polysaccharides [42,43]. Therefore, the relationship between the biological activities of GBPS-2 and GBPS-3 and their structures should be explored in future. 4. Conclusions 20

ACCEPTED MANUSCRIPT In the present study, two main polysaccharide fractions, namely GBPS-2 and GBPS-3, were extracted from GBLs; their average molecular weights were 672 and 723

kDa,

respectively.

GBPS-2

and

GBPS-3

were

typical

acidic

heteropolysaccharides, composed of Man, Rha, GlcA, GalA, Glc, Gal, and Ara (molar

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ratio: 0.08:0.12:0.16:0.06:0.11:1.00:0.32) and Man, Rha, GlcA, GalA, Gal, and Ara

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(molar ratio: 0.92:1.00:0.83:0.11:0.42:0.23), respectively. GBPS-2 and GBPS-3

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exhibited limited scavenging abilities for the hydroxyl and DPPH radicals and exhibited noticeable effects on the superoxide and ABTS radicals. In addition,

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GBPS-2 and GBPS-3 significantly increased phagocytosis and promoted NO and

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cytokine production in RAW264.7 cells. Therefore, GBPS-2 and GBPS-3 have potential for use in functional food supplements.

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Acknowledgments

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The study was supported by the National Natural Science Foundation of China

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(31772057).

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Polym. 132 (2015) 378-396.

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ACCEPTED MANUSCRIPT Figure Captions Fig. 1. (a) Stepwise elution curve of crude GBPS on a DEAE Sepharose Fast Flow column, (b) ultraviolet spectra of GBPS-2 and GBPS-3, (c) GPC profile of GBPS-2, (d)

GPC

profile

of

GBPS-3,

(e)

chromatograms

of

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3-methyl-1-phenyl-2-pyrazolin-5-one derivatives of mixed monosaccharide standards,

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GBPS-2-1, and GBPS-3-1, and (f) FT-IR spectra of GBPS-2-1 and GBPS-3-1.

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Fig. 2. Scavenging effects of GBPS-2 and GBPS-3 on hydroxyl (a), DPPH (b), superoxide (c), and ABTS (d) radicals.

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Fig. 3. In vitro effects of GBPS-2 and GBPS-3 on the proliferation (a) and

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phagocytosis (b) indexes of RAW264.7 cells.

Fig. 4. In vitro effects of GBPS-2 and GBPS-3 on NO (a), TNF-α (c), IL-1β (e), and

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IL-6 (g) production and iNOS (b), TNF-α (d), IL-1β (f), and IL-6 (h) mRNA

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expression of RAW264.7 cells.

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Annealing temperature (oC) 60 60 60

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60

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60

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Table 1 Primer sequences used in qPCR Gene Sequence (5’-3’) iNOS F: GAAGCTGAGGCCCAGGAGGA R: GAACAAGGTGGCCAGGTCCC TNF-α F: CCCTCACACTCAGATCATCTTCT R: GCTACGACGTGGGCTACAG IL-1β F: GGG CTG CTT CCA AAC CTT TG R: GCT TGG GAT CCA CAC TCT CC IL-6 F: TAGTCCTTCCTACCCCAATTTCC R: TTGGTCCTTAGCCACTCCTTC GAPDH F: AAGGCTGTGGGCAAGGTCAT R: CGTCAGATCCACGACGGACA

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GBPS-3 44.32 ± 1.18 38.37 ± 1.62 0.23 ± 0.03 723

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0.92 1.00 0.83 0.11 0.42 0.23

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Table 2 Preliminary characterization of GBPS-2 and GBPS-3 Item GBPS-2 Carbohydrate (%) 71.46 ± 1.93 Protein (%) 0.26 ± 0.02 Uronic acid (%) 12.49 ± 0.98 Total polyphenols (mg GAE/100 mg) Molecular weight (kDa) 672 Monosaccharide composition (mol) Man 0.08 Rha 0.12 GlcA 0.16 GalA 0.06 Glc 0.11 Gal 1.00 Ara 0.32

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Fig. 1. (a) Stepwise elution curve of crude GBPS on a DEAE Sepharose Fast Flow column, (b) ultraviolet spectra of GBPS-2 and GBPS-3, (c) GPC profile of GBPS-2, (d) GPC profile of GBPS-3, (e) chromatograms of PMP derivatives of mixed monosaccharide standards, GBPS-2-1, and GBPS-3-1, and (f) FT-IR spectra of GBPS-2-1 and GBPS-3-1.

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Fig. 2. Scavenging effects of GBPS-2 and GBPS-3 on hydroxyl (a), DPPH (b),

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superoxide (c), and ABTS (d) radicals.

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phagocytosis (b) indexes of RAW264.7 cells.

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Fig. 3. In vitro effects of GBPS-2 and GBPS-3 on the proliferation (a) and

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Fig. 4. In vitro effects of GBPS-2 and GBPS-3 on NO (a), TNF-α (c), IL-1β (e), and IL-6 (g) production and iNOS (b), TNF-α (d), IL-1β (f), and IL-6 (h) mRNA expression of RAW264.7 cells. 35

ACCEPTED MANUSCRIPT HIGHLIGHTS 

Three kinds of polysaccharides were obtained from Ginkgo biloba leaves.



Physicochemical characterization and functional property of GBPS-2 and GBPS-3 were preliminarily investigated. GBPS-2 and GBPS-3 were typical acidic heteropolysaccharides.



GBPS-2 and GBPS-3 exhibited noticeable antioxidant and immunomodulatory

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activities.

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