Immunomodulatory Effects of Enzymatic Synthesized α

Nutrition and Food Branch of China Association of Gerontology and Geriatrics,. Beijing 100050, People's Republic of China. ¶. Department of Food Scie...
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Immunomodulatory Effects of Enzymatic Synthesized #Galactooligosaccharides and Evaluation of the Structure-activity Relationship Zhuqing Dai, Wanyong Lyu, Xiaoli Xiang, Yuhong Tang, Bing Hu, Shiyi Ou, and Xiaoxiong Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01939 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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Journal of Agricultural and Food Chemistry

Immunomodulatory Effects of Enzymatic Synthesized α-Galactooligosaccharides and Evaluation of the Structure-activity Relationship

Zhuqing Dai,†,‡,ǁ Wanyong Lyu,§,ǁ Xiaoli Xiang,† Yuhong Tang,† Bing Hu,† Shiyi Ou, ¶



and Xiaoxiong Zeng†,*

College of Food Science and Technology, Nanjing Agricultural University, Nanjing

210095, People’s Republic of China ‡

Institute of Farm Product Processing, Jiangsu Academy of Agricultural Sciences,

Nanjing 210014, People’s Republic of China §

Nutrition and Food Branch of China Association of Gerontology and Geriatrics,

Beijing 100050, People’s Republic of China ¶

Department of Food Science and Engineering, Jinan University, Guangzhou

510632, Guangdong, People’s Republic of China

*To whom correspondence should be addressed. Tel & Fax: +86 25 84396791; E-mail: [email protected] (X. Zeng). ǁ These authors contributed equally to this study and share first authorship. 1

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ABSTRACT

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In this study, α-galactooligosaccharides (α-GOSs) were synthesized by using

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galactose as the substrate and α-galactosidase from Aspergillus niger as the catalyst.

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In the reaction, synthesized products of U1, U2, U3 and U4 were detected by HPLC.

5

By mass spectrometry, NMR and 1-phenyl-3-methyl-5-pyrazolone derivatization, U1

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was

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α-D-Galp-(1→2)-α-D-Gal, α-D-Galp-(1→3)-α-D-Gal, α-D-Galp-(1→4)-α-D-Gal;

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U2 was identified to be α-D-Galp-(1→6)-α-D-Gal; U3 was the mixture of

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galacto-trisaccharides linked by one α-(1→6)-glycosidic linkage and one other

the

mixture

of

disaccharides

linkage;

of

was

α-D-Galp-(1→1)-α-D-Gal,

10

α-glycosidic

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α-D-Galp-(1→6)-α-D-Galp-(1→6)-α-D-Gal. Afterwards, the synthesized α-GOSs

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(U1, U2, U3, U4 and their mixture) as well as α-GOSs (manninotriose, stachyose,

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ciceritol and verbascose) obtained from natural materials were used as subjects to

14

evaluate their immunomodulatory effects in vitro by culturing mouse macrophage

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RAW264.7. The results showed that α-GOS with higher degree of polymerization

16

had better immunomodulatory activity, while to a certain extent, α-GOS linked with

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α-(1→6)-galactosidic linkage showed better immunomodulatory effect.

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KEYWORDS: α-Galactooligosaccharide; Enzymatic synthesis; Immunomodulatory

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effect; Structure-activity relationship

U4

2

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identified

as

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INTRODUCTION

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α-Galactooligosaccharides (α-GOSs) are typical functional oligosaccharides that

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comprised of 2-10 monosaccharide units with α-galactosidic linkages. A variety of

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beneficial health effects has been reported for this group of compounds, such as

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regulating gut microflora, reducing the risk of cancer, enhancing cardiovascular

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function, improving immune activities, maintaining urinary tract health, modulating

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inflammatory response, lowering blood pressure and blood lipids, resisting bacterial

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and virus and preventing osteoporosis.1-5 Currently, most of the well documented

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α-GOSs are present in natural legumes or Chinese herbal medicines.2,6 Thus, most of

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the reported α-GOSs are extracted from raw materials, and there is relatively little

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research on their enzymatic synthesis.

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GOSs can be divided into β-GOSs and α-GOSs, both considered to be prebiotics.

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Meanwhile, it has become a mature technology for commercial production of

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β-GOSs by enzymatic synthesis. Related reports have been widely published, for

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example, using lactose as the substrate and β-galactosidase as the catalyst have

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become a typical method to synthesize β-GOSs.7,8 It has been reported that different

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sources of β-galactosidase could mediate the formation of different types of

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β-glycosidic bond. For example, β-galactosidase from Aspergillus oryzae catalyzed

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the systhesis of β-GOSs with β-(1→6)-glycosidic linkge; β-galactosidase from

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Bifidobacterium

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β-(1→3)-glycosidic linkge; β-galactosidase from Bacillus circulans mediated the

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synthesis of β-GOSs with β-(1→4)-glycosidic linkge.9-11 Compared to this, the study

bifidum

catalyzed

the

production

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with

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of enzymatic synthesis of α-GOSs is much less than that of β-GOSs.12-16 For

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example, raffinose was synthesized with sucrose and galactose as the substrates with

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α-galactosidase from Absidia corymbifera as the catalyst.13 It has been reported that

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α-galactosidase from A. niger APC-9319 could effectively catalyze a reverse

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reaction of galactose to produce α-GOSs.14 The α-GOSs contained 58%

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α-galactobiose, 28% α-galactotriose, and 14% oligosaccharides larger than

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α-galactotriose. However, the specific structures of α-GOSs were unknown in the

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study. Therefore, α-GOSs were synthesized and characterized in this study to further

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understand their biological function.

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The human immune system is an important defense system that maintains the

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integrity of the body and prevents the body from being invaded by microorganisms.

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Macrophages are important immune cells of the innate immune system of the body,

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and have various immune effects such as phagocytosis of foreign substances,

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secretion of immune factors and processing of antigens. A series of immune

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functions of macrophages are mainly achieved by the release of proinflammatory or

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chemical agents such as nitric oxide (NO), prostaglandin E2 (PGE2), interferon (IFN)

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and interleukin (IL).17-19 The release of these proinflammatory factors is also an

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important way to maintain self-stabilization when the body is injured.20 Functional

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oligosaccharides have been reported to promote the proliferation of bifidobacterium

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and other probiotics while inhibit the growth of pathogen.2,3,21 Their prebiotic

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function may play an important role in regulating the immune response.3,22-25

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However, most of the existing studies of α-GOSs used mixture of α-GOSs extracted 4

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from natural materials, which lacked the inquiry of structure-activity relationship.

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Therefore, both synthesized α-GOSs and natural α-GOSs (manninotriose, stachyose,

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ciceritol and verbascose) from raw materials were used as subjects to evaluate their

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immunomodulatory activities in the present study. This study was aimed to provide a

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theoretical basis for further study of the immunomodulatory activity of α-GOSs and

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the structure-activity relationship.

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MATERIALS AND METHODS

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Materials and Chemicals. α-D-galactosidase from A. niger DS was got from

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Amano Enzyme Co., Ltd. (Nagoya, Japan). p-Nitrophenyl-α-D-galactopyranoside

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(Galα-pNP), 1-phenyl-3-methyl-5-pyrazalone (PMP), galactose and activated

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charcoal (Darco G-60, 100 mesh) were purchased from Sigma-Aldrich Chemical Co.

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(St. Louis, MO, USA). Celite 535 was purchased from Fluka Co. (Buchs,

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Swiltzerland). Acetonitrile (grade of high performance liquid chromatography

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(HPLC)) was purchased from Hanbon Science and Technology Co., Ltd. (Jiangsu,

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China). Membrane filters (0.45 µm) were obtained from Millipore Co. (Bedford,

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MA, USA). All other chemicals were analytical grade.

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Assay of α-D-Galactosidase Activity. The α-D-galactosidase activity was

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measured according to previous report,26 and one unit of enzyme activity was

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defined as the amount of enzyme (product weight) hydrolyzing 1 µmol of pNP from

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Galα-pNP per minute. In brief, the reaction was carried out in 2.0 mL solution of 1.0

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mM Galα-pNP (dissolved in pH 5.5 sodium acetate buffer) with an appropriate

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amount of enzyme. After incubated at 37 ℃ for 10 min, the reaction was stopped 5

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by adding 8.0 mL 0.5 M Na2CO3 solution and the absorbance (Abs) at 405 nm of

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reaction mixture was measured spectrophotometrically. As a result, the activity of

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α-D-galactosidase was 9.48 U/mg.

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Synthesis of α-GOS. Galactose was dissolved in distill water and treated under

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125 ℃ and 110 kPa conditions for 20 min, affording a 96% supersaturated

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galactose solution. The galactose solution was then transferred to a 60 ℃ water

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bath and cooled to the reaction temperature. A certain amount of α-D-galactosidase

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(35U/g galactose) dissolved in pH 5.5 sodium acetate buffer was added and the

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galactose solution was adjust to a concentration of 90%. The reaction was then

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activated at 60 ℃ once the enzyme was added, and the reaction was monitored by

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interval sampling and analysis with HPLC. For termination the reaction, the reaction

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mixture was heated for 10 min under 100 ℃. All assays were run in triplicate.

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Chromatographic Determination of Reaction Products. An Agilent 1100 series

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HPLC system with a refraction index detector (RID) was used to analyze the

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reaction mixture. The separation of sugars was completed on a Sugar-D column (4.6

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mm × 250 mm, Nacalai Tesque Inc., Tokyo, Japan). The mobile phase was

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acetonitrile-water (75:25, v/v) at a flow rate of 1.0 mL/min. Injection volume was 20

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µL. Standard sugars were used to identify the peaks by comparing the retention

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times and to quantify the reaction products by an external calibration curve using its

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corresponding standard solutions. The synthetic yield was defined as the percentage

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of the amount of produced products to the amount of galactose added. The standard

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sugars were isolated and purified from reaction mixture as described below. 6

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Purification and Characterization of Synthesized α-GOSs. The reaction solution

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was centrifuged at 5000 g for 10 min, the resulting supernatant was filtered through

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a 0.45 µm filter and then loaded onto an activated charcoal-Celite preparative

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column (3.0 × 50 cm, Yamazen Co., Osaka, Japan). The column was eluted with a

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linear gradient ethanol-water solution (from 0 to 20%) at a flow rate of 4 mL/min.

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The eluent was collected (10 mL/tube) and monitored by phenol-sulfuric acid

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method. Those with sugars were further analyzed by HPLC, collected separately and

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concentrated by rotate evaporation. Briefly, galactose was eluted at 4-5% (v/v)

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aqueous ethanol solution, U1 was eluted with 9-10% aqueous ethanol solution, U2

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was eluted with 8% aqueous ethanol solution, U3 was eluted with 10-11% aqueous

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ethanol solution and U4 was eluted with 13% aqueous ethanol solution. As some

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fractions could not be completely separated by activated charcoal-Celite preparative

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column, they were further separated by polyacrylamide Biogel P-2 column (1.5 × 90

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cm, BioRad, Richmond, USA). The column was eluted by ultrapure water at a flow

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rate of 0.25 mL/min, and the eluates were treated as mentioned above. As results,

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four fractions were collected, concentrated and frzzed-dried, affording U1, U2, U3

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and U4 as white powder.

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The structures of synthesized products were identified by mass spectrometry

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with an electrospray ionization source (ESI-MS) and nuclear magnetic resonance

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spectroscopy (NMR). The Mariner system 5304 mass spectrometer (Applied

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Biosystems, Foster City, CA, USA) was used to obtain MS spectra by direct

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injection. The Bruker Avance DRX-500 spectrometer (Bruker, Karlsruhe, Germany) 7

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was used to record 1H and

13

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D2O. The residual solvent signal was used as internal standard. The chemical shifts

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(δ) are given in ppm, and coupling constants (J) are given in Hz.

C NMR spectra at 300 K. Samples were dissolved in

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Analysis of PMP Derivatives. The four synthetic products (U1, U2, U3 and U4)

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were further analyzed by PMP derivatization according to reported method.27 Briefly,

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100 µL of 5 mg/mL product solution was mixed with 100 µL of 0.6 M NaOH

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solution, half of the mixture was then derivatized with 100 µL of 0.5 M PMP

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solution (dissolved in methanol) for 2 h at 70 ℃. After derivatization, 0.3 M HCl

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solution was used to neutralize the reaction solution. The resulting solution was

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concentrated by rotate evaporation to dryness, and the residue was dissolved with

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distilled water and chloroform was added to extract the water phase. After filtered

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through a 0.45 µm membrane, the water phase was analyzed by an Agilent HPLC

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system equiped with Eclipse Plus C18 column (4.6 × 250 mm, 5 µm, Agilent) and

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diode array detector at a flow rate of 1.0 mL/min at 30 ℃. The mobile phase was

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consisted of 83% phosphate-buffered saline (PBS, 0.1 M, pH 6.7) and 17%

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acetonitrile (v/v).

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Cell Culture. The murine macrophage RAW264.7 cells were cultured in complete

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DMEM (supplemented with 10% newborn calf serum, 100 IU/mL penicillin, and

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100 IU/mL streptomycin) and incubated in a humidified 5% CO2 incubator at 37 ℃.

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The adherent cells were kept for cell passage and used for further culture when the

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cells were spread over 80% of the bottom of the culture dishes.

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Assay of RAW264.7 Cell Viability. The RAW264.7 cell viability was measured 8

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according to the MTT-based colorimetric method.28 Briefly, the RAW264.7 cells

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were seeded into a 96-well flat-bottom plate (100 µL/well) when suspended at a

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density of 1 × 106 cells/mL in DMEM. They were incubated under 5% CO2 at 37 ℃

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for 12 h. The nonadherent cells were washed with PBS twice. After that, 100 µL of

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different samples dissolved in DMEM with a series of concentrations (final

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concentrations of 25.0, 50.0, 100.0, 200.0 and 400.0 µg/mL) were added to each well,

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respectivrly. Positive control (10.0 µg/mL LPS) and blank control (complete DMEM

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alone) were also added. The cells were incubated as mentioned above for 12, 24, 36

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and 48 h, respectively. After each incubation, each well was added 200 µL of MTT

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solution (0.5 mg/mL) and then incubated further at 37 ℃ for 4 h. Finally, 150 µL of

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DMSO was added to each well to dissolve formazan crystals. A Synergy 2

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Multimode Microplate Reader (BioTeK Instruments, Inc., Winooski, VT, USA) was

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used to measure the Abs at 570 nm. The cell viability rate was calculated according

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to the formula below:

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Cell viability = Abssample/Absblank control

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Assay of Phagocytosis of RAW264.7 Cells. The cell phagocytosis was measured

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according to the reported method29 with modification. The prior stage culture of

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RAW264.7 cells was done as described above. The samples at concentrations of 25.0,

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50.0, 100.0, 200.0 and 400.0 µg/mL were added to a 96-well plate (100 µL/well).

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Positive control (10.0 µg/mL LPS) and blank control (complete DMEM alone) were

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also added. The plate was incubated at 37 ℃ in a 5% CO2 incubator for 24 h.

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Subsequently, each well (100 µL/well) was added with 0.075% of neutral red 9

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solution and further incubated for 1 h. Then, the wells were washed with PBS twice

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to remove excess neutral red. After that, each well was added with 100 µL of cell

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lysate (0.1 M glacial acetic acid:ethanol=1:1, v/v), and the plate was incubated

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overnight at 37 ℃. Finally, the Abs at 540 nm of each well was determined and the

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phagocytosis index was calculated by using the following equation:

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Phagocytosis index = Abssample/Absblank control

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Assay of NO and Cytokines. The NO amount released by peritoneal macrophages

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was measured by an NO assay kit from Nanjing Jiancheng Bioengineering Institute

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(Nanjing, China). The levels of cytokines (IL-6, IL-1β and IFN-γ) were measured by

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using ELISA kits (Nanjing Jiancheng Bioengineering Institute) according to the

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manufacturer’s protocol.

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Statistical Analysis. The data are expressed as means ± SD and subjected to

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one-way analysis of variance (ANOVA). Duncan’s new multiple range test was

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performed to determine the significant difference using SPSS 18.0 software (SPSS

188

Inc., Chicago, IL, USA).

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RESULTS AND DISCUSSION

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Synthesis and Characterization of α-GOS. As shown in Figure 1, 4 peaks for

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synthesized products were observed in the HPLC chromatogram by using galactose

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as the substrate and α-galactosidase as the catalyst. The products were isolated from

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the reaction mixture by using activated charcoal-Celite column and Biogel P-2

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column to afford fractions of U1, U2, U3 and U4, respectively. Purified U1, U2, U3

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and U4 were used to draw the standard curves by HPLC according to the peak area 10

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and sample concentration. The concentration of each synthetic product was

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calculated from the standard curve. As a result, the synthetic yield was 25%. Among

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the synthesized products, U1 accounted for 32%, U2 accounted for 40%, U3

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accounted for 15% and U4 accounted for 13%. The yield of α-GOSs synthesis by

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enzymatic method is usually less than 30%. For example, in a enzymatic reaction

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using α-galactosidase from Lactobacillus reuteri as the catalyst, a maximum yield of

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26% (w/v) trisaccharide was achieved using melibiose as the substrate.16 Improved

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enzymatic synthesis for higher yields needs to be further investgated.

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The MS analysis of synthetic products is shown in Figure 2. The mass spectra of

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U1 and U2 showed significant peak at m/z 365 for [M+Na]+, indicating that their

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molecular weights were 342 with disaccharide structure. The mass spectra of U3 and

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U4 gave peak at m/z 538.9 for [M+Cl]-, implying their molecular weights were 502.9

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with trisaccharide structure. In general, the shift of α-pyranoside bond on the C-1 is

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more than δ 5.0 ppm. Meanwhile, the coupling constant J of α-pyranoside bond is

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usually between 2-4 Hz, while the β-pyranoside bond is usually between 7-9 Hz,

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which will be able to distinguish between the two different types of linkage. The

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shift of the C displacement in 13C NMR is also different. If substitution occurs, the

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C-1 anomeric carbon shifts its chemical shifts from δ 90-95 ppm to δ 97-103 ppm,

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the C-6 anomeric carbon shifts its chemical shifts from δ 60-65 ppm to δ 67-70

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ppm.10 As shown in Figure 3, the characteristic signals of U2 at δ 5.27 ppm (J = 3.8

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Hz) and 5.02-4.97 ppm (J = 3.48 Hz) in the 1H NMR spectrum indicated that the

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galactosyl residues were connected through α-linkage. Furthermore, in the 13C NMR 11

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spectrum of U2, there was only one signal peak of C-6 at δ 61.18 ppm. Therefor, U2

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was identified to be α-D-Galp-(1→6)-α-D-Gal. Similar to U2, the 1H NMR

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spectrum of U4 (Figure 4) showed the characteristic chemical shift signals of

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galactose residues at δ 5.27 ppm (J = 3.8 Hz), 5.02 ppm (J = 3.3 Hz) and 4.98 ppm

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(J = 3.7 Hz), indicating that the glycosidic bonds of U4 were also α-linkages.

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Meanwhile, according to its

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61.19 ppm, U4 was identified as α-D-Galp-(1→6)-α-D-Galp-(1→6)-α-D-Gal. The

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results indicated that the synthesized products were mainly linked with

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α-(1→6)-glycosidic linkage, which is consistant with previous report.16 However,

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the 1H and

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(shown in supplementary material, Figures S1 and S2), suggesting that they were

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not single compound. Therefor, the PMP derivatization was carried out to further

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determine their compositions.

13

13

C NMR spectrum, only one signal peak of C-6 at δ

C NMR spectra of U1 and U3 showed miscellaneous information

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PMP can react with reducing sugars under mild conditions and the products have

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no stereoisomer with a strong UV absorption at 245 nm. After derivatized by PMP,

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the hydrophobicity of reduced sugar improved while the derivative is charged and

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can be separated well afterwards. As shown in Figure 5, U2 and U4 showed only

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one peak after derivatization (the retention time at 15.6 min for PMP, the retention

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time at 43 min for galactose), while U1 had four peaks (except for the overlapped

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peak with U2). Since α(1→5)-glycosidic linkage can not be formed, it is therefore

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assumed

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α-D-Galp-(1→2)-α-D-Gal, α-D-Galp-(1→3)-α-D-Gal, α-D-Galp-(1→4)-α-D-Gal.

that

U1

was

the

mixture

of

12

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U3 showed two peaks after PMP derivatization (except for the PMP peak), as the

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α-(1→6)-glycosidic bond occupied the highest proportion in the products, U3 was

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speculated as the mixture of galacto-trisaccharides linked by a α-(1→6)-glycosidic

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bond and an other type α-glycosidic linkage.

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Effect of α-GOS on Macrophage Viability. The cell viability of macrophage is

245

an indicator of immune activation and is the basis for subsequent experiments. The

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effects of different treatment groups on cell viability are shown in supplementary

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material (Table S1), and the cell proliferation index reached the maximum when

248

treated for 24 h in almost all treatment groups. After 24 h, the cell proliferation index

249

decreased with the increase of culture time. Therefor, the treatment time was set at

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24 h for the following experiments. When compared with different concentrations,

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the cell proliferation index reached the maximum at concentration of 100 or 200

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µg/mL. By comparing the effect of different samples, it was found that all α-GOSs

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treatments promoted the proliferation of RAW264.7 cells, while no significant

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differences was observed between differnet α-GOS treatments.

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Effect of α-GOS on Macrophage Phagocytosis. Activated macrophages not only

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involve in specific and non-specific immune regulation, but also play an important

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role in the connection between the two immune responses. The macrophage

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phagocytosis is a significant sign of macrophage activation.30 As shown in Table 1,

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when the concentrations of different α-GOS treatments were less than 200 µg/mL,

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the phagocytosis index increased with the increase of the sample concentrations,

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showing a dose-dependent relationship. Comparing different α-GOS treatments on 13

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cell phagocytosis at the concentration of 200 µg/mL, all the α-GOS treatments

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showed higher phagocytosis index than positive control, while ciceritol and

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verbascose exhibited significant higher phagocytosis than other α-GOSs (p < 0.05).

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The results indicated that ciceritol and verbascose had better effects in promoting

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macrophage phagocytosis than other structure of α-GOS.

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Effects of α-GOS on Production of NO and Cytokines. Once activated, the

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macrophages will release NO and a large number of cytokines, such as IFN and

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PGE2, which play an important role in against exogenous pathogens and

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anti-cancer.31 NO is a neurotransmitter modulator that acts as a vasodilator as well as

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pathogens resistant. NO is produced by L-arginine under the action of inducible

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nitric oxide synthase (iNOS).32 It has been reported that the immune reponses of

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anti-tumor, antiviral and inflammatory modulation are closely linked to the release of

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NO.33 As shown in Table 2, the treatment of α-GOSs at concentration of 200 or 400

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µg/mL showed better effect on reseasing NO. The effect of α-GOSs on promotion of

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NO also exhibited a dose-dependent manner. When compared the treatment groups,

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the levels of NO in α-GOSs groups were lower than those of positive control at low

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concentrations. However, when the treatment concentration reached 200 µg/mL,

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they showed similar effect as positive control. Among the α-GOS treatments, U4,

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stachyose and verbascose significantly protomted the release of NO than other

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α-GOS groups (p < 0.05). For the four synthesized products, U2 showed

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significantly higher level of NO than U1 at 100 and 400 µg/mL concentrations; U4

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had significantly higher level of NO than U3 at 200 and 400 µg/mL concentrations 14

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(p < 0.05).

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Cytokines are small molecule proteins that are immunologically regulated by

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immune cells. TNF-α and IL-1β are considered as "early response cytokines".

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Upregulation of these cytokine on the inner cell wall may contribute to the migration

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of phagocytic cells to the injury site.34,35 In addition, IFN-γ may promote antigen

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presentation, cytokine production, the lethal effect of monocyte-macrophages and

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natural killer cells.36 The effects of different treatments on TNF-α, IL-1β and IFN-γ

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secretion are shown in Table 3-5. The levels of TNF-α, IFN-γ and IL-1β in α-GOS

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treatment groups were significantly higher than those of blank control. Besides, all

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treatment groups showed the highest levels of cytokines when treated at

294

concentration of 200 µg/mL. When the concentration was less than 200 µg/mL, the

295

levels of TNF-α, IFN-γ and IL-1β increased with the increase of treatment

296

concentratios, showing a dose-dependent relationship. The results showed that the

297

production of TNF-α in U3, U4, stachyose and verbascose groups was significantly

298

higher than that of other treatment groups (p < 0.05). Stachyose, ciceritol and

299

verbascose treatments significantly promoted the secretion of IL-1β compared with

300

other groups (p < 0.05). Meanwhile, U4, stachyose and verbascose exhibited better

301

effect in promoting the release of IFN-γ. Comparing the effects of four synthesized

302

products on production of TNF-α, IFN-γ and IL-1β, it revealed that U2 showed

303

better effects than U1, and U4 showed better effects than U3. However, the

304

differences were not statically significant in some cases.

305

In this study, it was found that α-GOSs significantly promoted macrophages 15

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phagocytosis and secretion of NO, TNF-α, IFN-γ and IL-1β when treating

307

macrophages RAW264.7 in vitro. It also showed a dose-dependent manner when the

308

concentration less than 200 µg/mL. The results indicated that α-GOSs had positive

309

immune regulation functions, which is consistant with our previous report.37 Related

310

reports also indicated that oligosaccharides promoted the secretion of these cytokines

311

in macrophages, and this immune regulation function was mainly through the

312

activation of a series of signaling pathways like toll-like receptor, nuclear factor and

313

the mitogen-activated protein kinase pathway.25,38-40 The prebiotic effect of GOSs

314

has been well studied and it has been reported that the degree of polymerization (DP),

315

the type of linkage, and the monosaccharide composition are the three factors

316

affecting their probiotic activity.41 In this study, we demnonstread that DP and the

317

type of linkage also affected greatly the immunomodulatory effects of α-GOSs.

318

When comparing the immunoregulatory function of α-GOSs with different

319

structures, it was found that verbascose showed the best effects, followed by

320

stachyose. In structure, verbascose and stachyose were both raffinose family

321

oligosaccharides, while verbascose has five monosaccharide units and stachyose has

322

four monosaccharide units. These results indicated that α-GOSs with higher DP

323

could exhit better immunomodulatory effect. It has also been reported that α-GOSs

324

with higher DP released more TNF-α.42 While comparing the effects of four

325

synthesized products on production of TNF-α, IFN-γ and IL-1β, U2 had better

326

effects than U1, U4 had better effects than U3. It is shown to a certain extent that

327

α-GOS with α-(1→6)-glycosidic linkage is more effective. However, this 16

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phenomenon needs further investigation. Among these α-GOSs, U4, manninotriose

329

and ciceritol had similar structure that they all comprised of three monosaccharide

330

units with two galactose and one different monosaccharide end. Comparing the

331

effects of these three α-GOSs, U4 showed the best effects on NO, TNF-α and

332

IFN-γ promotion, indicating that oligosaccharides with galacose unit exibited better

333

immunomodulatory effect. However, manninotriose and ceritol secreted more IL-1β

334

than U4, which is contrary with previous results. Thus, the effect of monosaccharide

335

composition on immunomodulatoty activity needs to be further investigated.

336

In conclusion, α-GOSs were synthesized by using galactose as the substrate

337

under supersaturated concentration and A. niger α-galactosidase as the catalyst.

338

Among them, U1 was the mixture of disaccharides of α-D-Galp-(1→1)-α-D-Gal,

339

α-D-Galp-(1→2)-α-D-Gal, α-D-Galp-(1→3)-α-D-Gal, α-D-Galp-(1→4)-α-D-Gal;

340

U2 was α-D-Galp-(1→6)-α-D-Gal; U3 was the mixture of galacto-trisaccharides

341

linked by a α-(1→6) glycosidic bond and a other type of α-glycosidic linkage; U4

342

was α-D-Galp-(1→6)-α-D-Galp-(1→6)-α-D-Gal. Both the newly synthesized and

343

the naturally occurring α-GOSs exhibited immunomodulatory effects by promoting

344

the secreation of NO and immune cytokines. Furthermore, α-GOSs with higher DP

345

exhibited better immune regulation activity.

346

ASSOCIATED CONTENT

347

Supporting information

348

Figures for 1H and

349

treatments on RAW 264.7 cell proliferation at different treatment times.

13

C NMR spectra of U1 and U3. Table for effects of different

17

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350

AUTHOR INFORMATION

351

Corresponding Author

352

*Phone: +86-25-84396791. E-mail: [email protected] (X Zeng).

353

ORCID

354

Xiaoxiong Zeng: 0000-0003-2954-3896

355

Funding

356

This work was partly supported by Grants-in-Aid for scientific research from the

357

National Natural Science Foundation of China (31171750), a project funded by the

358

Priority Academic Program Development of Jiangsu Higher Education Institutions

359

(PAPD) and a grant funded by Jiangsu Key Laboratory of Quality Control and

360

Further Processing of Cereals & Oils.

361

Notes

362

The authors declare no competing financial interest.

363

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Figure Captions Figure 1. Determination of enzymatic synthesis reaction products by HPLC Figure 2. ESI-MS spectra of synthetic products of U1 (A), U2 (B), U3 (C) and U4 (D) Figure 3. 1H (A) and 13C NMR (B) spectra of product U2 Figure 4. 1H (A) and 13C NMR (B) spectra of product U4 Figure 5. HPLC chromatograms of PMP derivatives of U1, U2, U3 and U4

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Table 1. Effects of Different Treatments on RAW264.7 Cell Phagocytosis Index Treatments

Cell phagocytosis index 25 µg/mL

50 µg/mL

100 µg/mL

NC

1.00 ± 0.11

LPS

1.19 ± 0.02

α-GOS mixture

1.26 ± 0.09 cd, A

1.26 ± 0.03 bc, A

200 µg/mL

400 µg/mL

1.27 ± 0.07 ab, A

1.33 ± 0.11 a, A

1.26 ± 0.12 abc, A

U1

1.13 ± 0.03 a, A

1.22 ± 0.06 ab, B

1.21 ± 0.07 a, B

1.24 ± 0.05 a, B

1.19 ± 0.04 a, AB

U2

1.16 ± 0.03 abc, A

1.21 ± 0.04 ab, AB

1.23 ± 0.04 ab, AB

1.25 ± 0.08 a, B

1.22 ± 0.05 ab, AB

U3

1.15 ± 0.03 ab, A

1.21 ± 0.02 ab, B

1.24 ± 0.02 ab, BC

1.30 ± 0.06 a, D

1.27 ± 0.03 bc, CD

U4 Manninotriose

1.24 ± 0.06 bc, A

1.29 ± 0.06 c, A

1.28 ± 0.03 b, A

1.30 ± 0.03 a, A

1.27 ± 0.03 abc, A

1.19 ± 0.05 abc, A

1.21 ± 0.04 ab, AB

1.24 ± 0.06 ab, AB

1.28 ± 0.07 a, B

1.22 ± 0.03 ab, AB

Stachyose

1.16 ± 0.1 abc, A

1.17 ± 0.02 a, A

1.23 ± 0.05 ab, AB

1.28 ± 0.06 a, B

1.22 ± 0.08 ab, AB

Ciceritol

1.49 ± 0.15 e, ABC

1.54 ± 0.03 e, BC

1.60 ± 0.07 d, C

1.43 ± 0.05 b, AB

1.40 ± 0.03 d, A

1.36 ± 0.09 d, A

1.38 ± 0.04 d, A

1.46 ± 0.09 c, A

1.61 ± 0.12 c, B

1.33 ± 0.08 cd, A

Verbascose

a-d represents significant differences between different α-GOS treatments of same concentration (P < 0.05). A-D represents significant differences between different concentrations of same α-GOS treatment (P < 0.05).

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Table 2. Effects of Different Treatments on NO Level of RAW264.7 Cell Treatments

NO Production(µmol/L) 25 µg/mL

50 µg/mL

100 µg/mL

NC

10.4 ± 1.8

LPS

36.4 ± 2.1

α-GOS mixture

200 µg/mL

15.1 ± 0.7 ab, A

26.8 ± 0.9 bc, B

30.5 ± 2.5 bcd, C

32.4 ± 1.8 a, C

400 µg/mL

33.0 ± 1.8 ab, C

U1

12.1 ± 2.5 a, A

22.0 ± 1.2 abc, B

24.0 ± 3.7 a, B

31.1 ± 1.4 a, C

32.0 ± 2.0 a, C

U2

15.0 ± 1.7 ab, A

24.1 ± 2.5 abc, B

30.5 ± 2.5 bcd, C

34.8 ± 1.2 ab, D

37.9 ± 0.4 bc, D

U3

15.6 ± 1.5 ab, A

23.0 ± 1.7 abc, B

27.0 ± 1.1 abc, C

32.6 ± 1.3 a, D

30.9 ± 0.4 a, D

U4 Manninotriose

18.2 ± 1.6 bc, A

26.0 ± 2.1 abc, B

33.3 ± 1.2 cde, C

38.9 ± 2.4 bcd, D

40.3 ± 3.0 c, D

15.9 ± 2.2 ab, A

21.3 ± 1.9 ab, B

26.7 ± 1.0 ab, C

31.2 ± 1.0 a, D

33.5 ± 2.8 ab, D

Stachyose

17.7 ± 2.5 bc, A

25.8 ± 1.7 abc, B

34.4 ± 3.0 de, C

40.2 ± 0.3 cd, D

38.3 ± 1.5 bc, D

Ciceritol

16.7 ± 1.6 ab, A

20.8 ± 2.4 a, B

28.6 ± 2.1 abcd, C

35.9 ± 2.1 abc, D

34.3 ± 1.2 ab, D

Verbascose

22.2 ± 1.2 c, A

27.5 ± 2.6 c, B

36.9 ± 1.2 e, C

43.7 ± 3.5 d, D

37.6 ± 2.0 bc, C

a-d represents significant differences between different α-GOS treatments of same concentration (P < 0.05). A-D represents significant differences between different concentrations of same α-GOS treatment (P < 0.05).

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Table 3. Effects of Different Treatments on TNF-α Levels of RAW264.7 Cell Treatments

TNF-α production (ng/L) 25 µg/mL

50 µg/mL

100 µg/mL

200 µg/mL

400 µg/mL

62.6 ± 7.0 ab, AB

66.6 ± 3.8 abc, B

61.5 ± 3.1 bc, AB

NC

48.2 ± 4.5

LPS

61.0 ± 0.7

α-GOS mixture

58.8 ± 3.1 ab, A

61.3 ± 1.9 a, AB

U1

56.8 ± 2.2 a, AB

60.4 ± 2.3 a, BC

61.0 ± 1.3 a, BC

64.5 ± 3.3 ab, C

54.7 ± 3.9 a, A

U2

62.2 ± 2.2 cd, AB

63.3 ± 1.6 ab, AB

63.2 ± 2.4 ab, AB

64.7 ± 4.4 ab, B

59.1 ± 3.3 ab, A

U3

59.2 ± 1.4 abc, A

60.5 ± 2.2 a, A

63.4 ± 1.0 ab, A

69.8 ± 3.7 cd, B

63.0 ± 2.0 bc, A

U4 Manninotriose

63.6 ± 1.7 d, A

63.6 ± 2.7 ab, A

64.4 ± 4.0 ab, A

70.7 ± 1.5 cd, B

64.1 ± 8.9 bc, A

59.0 ± 2.4 abc, A

63.2 ± 2.8 ab, A

64.9 ± 1.6 ab, A

61.5 ± 2.9 a, A

61.3 ± 4.6 abc, A

Stachyose

65.1 ± 1.6 d, A

65.7 ± 3.5 b, A

68.1 ± 3.1 b, A

69.2 ± 0.8 bcd, A

65.2 ± 3.7 bc, A

Ciceritol

62.0 ± 2.9 bcd, A

62.2 ± 0.8 ab, A

63.1 ± 1.8 ab, A

62.4 ± 3.2 a, A

59.8 ± 1.3 abc, A

64.0 ± 2.8 d, A

67.6 ± 2.1 b, AB

69.3 ± 6.5 b, AB

72.8 ± 3.2 d, B

66.6 ± 1.2 c, AB

Verbascose

a-d represents significant differences between different α-GOS treatments of same concentration (P < 0.05). A-D represents significant differences between different concentrations of same α-GOS treatment (P < 0.05).

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Table 4. Effects of Different Treatments on IL-1β Levels of RAW264.7 Cell Treatments

IL-1β production (ng/L) 25 µg/mL

50 µg/mL

100 µg/mL

NC

5.40 ± 0.27

LPS

7.75 ± 0.47

α-GOS mixture

7.22 ± 0.27 ab, A

7.31 ± 0.35 ab, A

200 µg/mL

400 µg/mL

7.33 ± 0.12 ab, A

7.54 ± 0.37 ab, A

7.28 ± 0.2 ab, A

U1

6.87 ± 0.18 a, A

6.92 ± 0.11 a, A

7.09 ± 0.21 a, A

7.35 ± 0.53 a, A

7.10 ± 0.43 a, A

U2

7.03 ± 0.3 a, A

7.28 ± 0.33 ab, AB

7.42 ± 0.25 ab, AB

7.56 ± 0.28 ab, B

7.18 ± 0.18 ab, AB

U3

7.19 ± 0.26 ab, A

7.24 ± 0.3 ab, A

7.49 ± 0.16 ab, AB

7.73 ± 0.15 abc, B

7.49 ± 0.09 bc, AB

U4 Manninotriose

7.44 ± 0.08 bc, A

7.47 ± 0.17 bc, A

7.71 ± 0.3 bcd, AB

7.85 ± 0.08 abc, B

7.68 ± 0.22 cd, AB

7.57 ± 0.47 bcd, A

7.86 ± 0.39 cd, A

8.03 ± 0.1 cd, A

8.10 ± 0.45 bcd, A

7.90 ± 0.11 de, A

Stachyose

7.93 ± 0.03 de, A

7.99 ± 0.09 d, A

8.12 ± 0.42 de, A

8.35 ± 0.7 cd, A

7.95 ± 0.02 de, A

Ciceritol

7.69 ± 0.14 cde, A

7.82 ± 0.23 cd, AB

7.93 ± 0.2 cd, AB

8.24 ± 0.41 cd, B

7.78 ± 0.08 cd, A

8.08 ± 0.18 e, A

8.27 ± 0.39 d, A

8.53 ± 0.47 e, A

8.70 ± 0.42 d, A

8.23 ± 0.32 e, A

Verbascose

a-e represents significant differences between different α-GOS treatments of same concentration (P < 0.05). A-D represents significant differences between different concentrations of same α-GOS treatment (P < 0.05).

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Table 5. Effects of Different Treatments on IFN-γ Levels of RAW264.7 Cell Treatments

IFN-γ production (ng/L) 25 µg/mL

50 µg/mL

100 µg/mL

NC

81.4 ± 6.7

LPS

113.4 ± 5.5

α-GOS mixture

107.9 ± 4.2 cd, A

111.3 ± 2.7 c, AB

112.2 ± 3.9 cd, AB

200 µg/mL

400 µg/mL

119.2 ± 4.7 cde, C

114.2 ± 1.3 c, BC

U1

92.4 ± 2.1 a, A

93 ± 3.2 a, A

95.3 ± 0.3 a, A

97.1 ± 5.9 a, A

92.7 ± 0.6 a, A

U2

92.0 ± 4.7 a, A

96.4 ± 2.2 a, AB

101.5 ± 4.6 ab, B

112.2 ± 3.5 b, C

101.2 ± 1.7 b, B

U3

112.3 ± 1.7 de, A

113.3 ± 2.9 cd, A

117.3 ± 3.5 de, AB

120.2 ± 4.4 cde, B

114.6 ± 6.1 c, AB

U4 Manninotriose

115.2 ± 2.5 e, A

117.0 ± 2.6 d, AB

119.2 ± 2.1 e, AB

122.3 ± 5.9 de, B

116.6 ± 3.2 c, AB

102.8 ± 1.7 b, A

105.4 ± 2 b, AB

107.3 ± 4.6 bc, AB

115.7 ± 6.3 bcd, C

111.7 ± 4.2 c, BC

Stachyose

114.3 ± 1.1 e, A

115.8 ± 5.5 cd, A

122.4 ± 4.0 e, B

123.7 ± 0.7 e, B

114.0 ± 2.4 c, A

Ciceritol

106.3 ± 3.3 bc, A

103.6 ± 2.6 b, A

107.9 ± 2.7 bc, A

114.1 ± 2.4 bc, B

105.1 ± 4.3 b, A

Verbascose

112.9 ± 3.3 e, A

114.6 ± 4 cd, A

121.3 ± 7.9 e, AB

125.5 ± 0.2 e, B

115.5 ± 5.5 c, A

a-e represents significant differences between different α-GOS treatments of same concentration (P < 0.05). A-D represents significant differences between different concentrations of same α-GOS treatment (P < 0.05).

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