Structural characteristics of Medicago Sativa L. Polysaccharides and Se-modified polysaccharides as well as their antioxidant and neuroprotective activities

Journal Pre-proofs Structural characteristics of Medicago Sativa L. polysaccharides and Se-modified polysaccharides as well as their antioxidant and neuroprotective activities Xuegui Liu, Shuangshuang Xu, Xiaodan Ding, Dandan Yue, Jun Bian, Xue Zhang, Gonglin Zhang, Pinyi Gao PII: DOI: Reference:

S0141-8130(19)35107-4 https://doi.org/10.1016/j.ijbiomac.2019.10.078 BIOMAC 13572

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

5 July 2019 9 September 2019 8 October 2019

Please cite this article as: X. Liu, S. Xu, X. Ding, D. Yue, J. Bian, X. Zhang, G. Zhang, P. Gao, Structural characteristics of Medicago Sativa L. polysaccharides and Se-modified polysaccharides as well as their antioxidant and neuroprotective activities, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.078

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Structural characteristics of Medicago Sativa L. polysaccharides and Se-modified polysaccharides as well as their antioxidant and neuroprotective activities. Xuegui Liua,b, Shuangshuang Xua, Xiaodan Dinga, Dandan Yueb, Jun Biana, Xue Zhanga, Gonglin Zhangc, Pinyi Gaoa*

a

College of Pharmaceutical and Biological Engineering, Shenyang University of Chemical

Technology, Shenyang, Liaoning 110142, PR China b

Institute of Functional Molecules, Shenyang University of Chemical Technology, Shenyang,

Liaoning 110142, PR China c

College of Environment and Safety Engineering, Shenyang University of Chemical Technology,

Shenyang 110142, PR China

Corresponding author College of Pharmaceutical and Biological Engineering Shenyang University of Chemical Technology 11 Street, Shenyang Liaoning P.R. China Tel.: +86-13840113203 E-mail: [email protected]

Abstract Medicago Sativa L., a nutrient-rich plant used as feed for cattle and sheep, is widely planted globally. This study investigated the structural characteristics and activities of three kinds of novel polysaccharides (APS1, APS2 and APS3) isolated from the stems of M. sativa as well as its two selenium modified products (Se-APS2 and Se-APS3). APS1 (Mw = 13.4 KDa) and APS2 (Mw = 11.2 KDa) were composed of rhamnose, arabinose, mannose and galactose with different molar ratio, APS3 (Mw = 18.6 KDa) was composed of rhamnose, arabinose, fructose, mannose and galactose. All APS1-3 contained 1→3/1→6/1→4/1→2 glycosidic bonds in a ratio of 0.74:0.09:0.05:0.12, 0.34:0.20:0.36:0.10 and 0.63:0.17:0.06:0.14, respectively. The selenium content of Se-APS2 (Mw = 9.0 KDa) and Se-APS3 (Mw = 10.2 KDa) were 1.05 and 2.57 µg/mg, respectively. Their surface morphology and thermal stability were determined by scanning electron microscope (SEM) and thermal analysis (TGA), respectively. Further, the antioxidant and neuroprotective activities of the three natural polysaccharides and two Se-polysaccharides were studied. Interestingly, Se-polysaccharides not only exhibited higher antioxidant activity, but also higher neuroprotective activity compared to natural polysaccharides. Key words: Selenium polysaccharide; Antioxidant activity; Neuroprotective activity. Abbreviations: ABTS, 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate acid); 13CNMR, Carbon 13 Nuclear Magnetic Resonance; DPPH, 2.2-Diphenyl-1-picrylhydrazyl; FT-IR, Fourier transform infrared;

1H-1H-COSY, 1H-1H-correlated

spectroscopy;

1HNMR,

Proton Nuclear Magnetic

Resonance; HPGPC, High-performance gel permeation chromatography; HPLC, High performance liquid chromatography; HMBC, 1H detected heteronuclear multiple-boned correlation; HSQC, 1H detected heteronuclear single-quantum coherence. NMR, Nuclear magnetic resonance; SEM, 1

Scanning electron microscope; TFA, Trifluoroacetic acid; TGA, Thermogravimetric analysis; UV, Ultraviolet; Vc, Vitamin C. 1. Introduction Polysaccharides, regarded as one of the four basic substances of life, are found in plants, animals and microorganisms. In recent years, plant polysaccharides have received much attention from researchers due to their complex chemical structures and physiological activities. Previous studies indicate that polysaccharides possess various biological activities such as anti-tumor effects [1, 2], antioxidant [3, 4], anti-diabetic and immunity regulation bioactivities [5, 6]. Medicago sativa L., a Leguminosae perennial herb, which is widely distributed in many parts of the world, is not only used as a forage crop and human food, but also contributes towards soil nutrients and water conservation [7, 8]. It is reported that M. sativa contains bioactive compounds, including polysaccharides, flavonoids, saponins, proteins, amino acids and vitamins [9, 10]. Besides, some previous works reported that M. sativa is rich in polysaccharides and possess a variety of biological activities including immunoregulatory [11, 12], antioxidant [13], cytoprotective [14], antiinflammatory functions [15]. Therefore, there is a need to explore the structural characteristics and other physical properties of polysaccharides from M. sativa for further application. To improve the biological activities of polysaccharides or confer them with new bioactivities, many scholars have explored various methods of structural modification of polysaccharides. Among them, selenide modification of polysaccharides has received extensive attention. It is well-known that Selenium (Se) regulates human physiological functions by modifying the expression of selenoproteins [16]. Selenoenzymes, an essential trace element, influences the physiological functions of the human body, including antioxidants, immune system function and cancer 2

prevention [17, 18]. According to previous reports, selenium and polysaccharides can directly influence the physiological functions in human body, while selenide modification can significantly increase the physiological activities of polysaccharides, such as antioxidant, antihyperlipidemic [19], antidiabetic [20] and anti-inflammatory activity [21]. Many previous studies have reported that M. sativa polysaccharides possess antioxidant activity [13], but there are few literatures comparing the physiological activities of M. sativa natural polysaccharides and their Se-modified products, especially in terms of their neuroprotective functions. Therefore, this study compared the antioxidant and neuroprotective activities of selenized polysaccharides with natural polysaccharides, to explore the effects of Se-modification on the functions of M. sativa polysaccharide, which laid the foundation for further research on the derivatization of M. sativa polysaccharides. 2. Materials and methods 2.1 Materials and Reagents Medicago sativa L. hay was purchased from Inner Mongolia, China. Monosaccharide standards (Dglucose, D-fructose, D-xylose, D-mannose, D-arabinose, D-galactose, L-rhamnose and Dgalacturonic acid) and glucan standards (T4, T10, T20, T40, T200, T500) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). AB-8 resin, DEAE-52 cellulose and Sephadex G-200 were purchased from Ruida Henghui Co., Ltd. (Beijing, China). All other reagents and chemicals were of analytical and chromatographic grade. 2.2 Extraction and Isolation. The dried stems of Medicago Sativa L (2.0 kg), pulverized to ~1 cm in length by grinder, were refluxed with petroleum ether (2 h) and ethanol (3 h) to remove small molecular compounds, and 3

were then extracted three times with distilled water by reflux condensation. The crude extract was then mixed with neutral protease and cultured at 50 oC for 1 h, then inactivated at 90 oC, and centrifuged to obtain the supernatant. A Sevage reagent containing N-butyl alcohol and chloroform (1:4, v/v), was added to the supernatant and was sonicated vigorously for half an hour to remove the protein, then the mixture was centrifuged and the supernatant was collected. The supernatant was decolored in an AB-8 resin column and eluted with distilled water. The solution was then concentrated using a rotary evaporator (R-3, BUCHI, Switzerland) to produce the crude polysaccharide. Then the crude polysaccharide was separated in DEAE-52 Cellulose column, by eluting with distilled water and a NaCl gradient system (0.1 mol/L、0.2 mol/L、0.3 mol/L、0.5 mol/L). These fractions were sampled at 7 min intervals by an automatic sampler (BSZ-100; Shanghai Huxi, China) and were determined through phenol-sulfuric acid method [22]. The obtained five fractions were purified by Sephadex G-200 column (distilled water) to get high purity fractions. Three major fractions, eluted with 0.1 mol/L 、 0.2 mol/L 、 0.3 mol/L NaCl, were concentrated and dried to yield the polysaccharide powder. 2.3 Analysis of monosaccharide composition The acid hydrolysis of each polysaccharide was carried out according previously reported methods. [23]. Each polysaccharide sample (5 mg) was hydrolyzed by 10 mL trifluoroacetic acid (TFA) (2 M) at 110 oC for 6 h. The HPLC analysis of hydrolyzed polysaccharide was performed using a LC3000 high performance liquid chromatograph system equipped with a sugar column and refractive index detector. The acid hydrolysate was eluted with a mixture of acetonitrile and purified water in a ratio of 88:12 (v/v) with a flow rate of 0.4 mL/min. The monosaccharide composition was determined by comparing with the retention time of standard sugars (rhamnose, fructose, arabinose, 4

mannose, and galactose) diluted to 5 mg/mL. 2.4 Periodate oxidation-Smith degradation reaction Each polysaccharide sample (20 mg) was oxidized with 15 mM of 25 mL sodium periodate (NaIO4) in dark. The absorbance of the 0.1 mL reaction solution (diluted to 25 mL with distilled water) was monitored at 223 nm every 12 h using a UV spectrophotometer. The reaction was terminated by 2 mL glycol when the absorbance was stable. The spectrophotometric method was used to confirm the consumption of NaIO4 and the sodium hydroxide (NaOH) solution (0.005 M) was used to determine the yield of formic acid [24]. The standard consumption curve of NaIO4 was obtained by measuring the absorbance of NaIO4 solutions at different concentrations (0, 3, 6, 9, 12, 15 mmol/L). The remaining solution was dialyzed with distilled water for 72 h, using molecular weight cut-off 3500 Da dialysis membrane. 70 mg of NaBH4 was then added to reduce the reaction solution for 24 h at 22 oC. The excess NaBH4 was neutralized using acetic acid. The solution was then dialyzed again using distilled water for 72 h. The reaction liquid was concentrated by rotary evaporation, followed by hydrolysis with 2 M TFA at 110 oC (10 mL) for 6 h in oil bath. The redundant TFA was removed 12 times by methanol via rotary evaporation. The final solution was analyzed using HPLC with an amino column and refractive index detector, eluted with a mixture of acetonitrile and purified water in a ratio of 88:12 (v/v) at a flow rate of 0.3 mL/min. 2.5 Synthesis of selenium polysaccharides Two of polysaccharides (APS2, APS3) were used to obtain Se-polysaccharides via nitric acidsodium selenite method [19]. Briefly, polysaccharide was added to the mixture (70 mg sodium selenite (Na2SeO3) and 10 mL of 0.6% HNO3 solution) with stirring. The mixture was reacted for 10 h at 70 oC, then cooled to room temperature. NaOH (0.1 M) was then added to adjust the PH to 5

7 followed by dialysis (molecular weight cut-off: 1000 Da) to remove Na2SeO3. Finally, the solution was concentrated and dried to obtain Se-polysaccharides (Se-APS2 and Se-APS3). 2.6 Analysis of selenium content The selenium polysaccharides (5 mg) were added to a solution of 10 mL HNO3 and 4 mL HClO4 and the mixture was reacted for 18 h in the dark. After digestion at 180 oC, 10 mL HCl (1.0 mg/mL) was added and the mixture was concentrated to 1 mL. The solution was reacted for 1 h at 90 oC after the addition of 10 mL HCl (6 mol/L), then diluted ten times with 10% hydrochloric acid, and the final product was analyzed using atomic fluorescence spectrometer (SK-2003A, JINSUOKUN Co., Ltd., China) to determine the selenium content. Standard selenium solutions with different concentrations (0, 5, 10, 20, 40 ng/mL) were used to develop a standard curve. 2.7 Molecular weight determination The weight average molecular weight (Mw) of samples was determined by high-performance gel permeation chromatography (HPGPC). First, 10 mg standard solutions of glucose (T4, T10, T20, T40, T200, T500) were dissolved with pure water and mixed fully to yield 10 mg/mL standard solution. Then, APS1 (10 mg), APS2 (10 mg), APS3 (10 mg) Se-APS2 (10 mg) and Se-APS3 (10 mg) were dissolved with pure water to obtain sample solutions (10 mg/mL) and measured by HPGPC system (Shimadzu Co., Japan). The above samples (30 μL) were loaded on an A TSK gel G5000PW column (I.D = 7.5 mm, L = 300 mm), eluted with Potassium dihydrogen phosphate solution (0.02 mol/L). The detector was 20A Refractive index detector. 2.8 FT-IR spectroscopy analysis Each polysaccharide and Se-polysaccharide fraction were mixed with a moderate amount of dried potassium bromide (KBr) to prepare a disc for IR Spectrometer (NEXUS 470, Thermo Nicolet Co) 6

characterization. 2.9 NMR spectroscopy The dried APS2 (25 mg), APS3 (25 mg), APS4 (25mg), Se-APS3 (25 mg) and Se-APS4 (25 mg) were dissolved with 99% deuteroxide (0.55 mL) for 1H NMR and

13C

NMR analysis ，

respectively. 2D-NMR including HSQC, 1H-1H-COSY and HMBC, were performed using 2DNMR spectrometer (ARX-500, Bruker Inc., Germany) at room temperature. 2.10 Scanning Electron Microscope (SEM) The powders of the three dried polysaccharide and two Se-products, were coated with gold, and scanned using a scanning electron microscope (SEM, SU8010, Hitachi, Japan). 2.11 Thermal analysis (TGA) Thermogravimetric analysis (TG) and Differential Thermal analysis (DTA) of this samples were tested using a thermal analyzer (STA 449C, NETZSCH, Germany) with a heating cycle from 25 oC to 900 oC at a rate of 10 °C/min under nitrogen (65 mL/min). 2.12 Antioxidant activity of polysaccharide 2.12.1 DPPH free-radical scavenging assay Each sample (10 mg) was dissolved in 50% ethanol at various concentrations (0.005, 0.02, 0.08, 0.3, 0.8, 2, 5 mg/mL). The freshly prepared DPPH ethanol solution (100 µL, 0.2 mmol/L) and 100 µL samples solution of the different concentrations were seeded into 96 well plates, respectively. The mixture was incubated in the dark for 30 min, and the absorbance was measured at 517 nm using microplate reader (Synergy-2, Gene Co., Ltd., China). At the same time, 100 µL of 0.2 mmol/L DPPH ethanol solution was mixed with 100 µL of 50% ethanol as blank [25], Vitamin C was taken as positive standard. and the scavenging percentage was calculated by the following 7

equation: Scavenging effect (%) = (1-(A1-A2)/A0 ) × 100 % Where A1 is the absorbance of a mixture of 100 µL sample and 100 µL DPPH solution; A2 is the absorbance of a mixture of 100 µL sample and 100 µL of 50% ethanol solution, and A0 is the absorbance of a mixture of 100 µL DPPH and 100 µL of 50% ethanol solution. 2.12.2 ATBS free-radical scavenging assay A stock solution consisting of 2 mL ATBS (7 mmol/L) and 2 mL Potassium persulfate (K2S2O8) was incubated in the dark at 20 - 25 oC for 12 - 16 h. The stock solution was diluted with absolute ethanol until the absorbance at 734 nm was 0.70  0.02. The samples solution (10 µL) at different concentrations (0.005, 0.02, 0.08, 0.3, 0.8, 2, 5 mg/mL) were mixed with 190 µL stock solution in 96 well plates, respectively. The mixture was then kept in the dark for 10 min. Subsequently, the absorbance after the reaction was measured at 734 nm via the enzyme marker detector. ATBS stock solution (190 µL) mixed with 10 µL of 50% ethanol was used as a blank [26]. Vitamin C was taken as positive standard. The scavenging percentage was calculated using the following equation: Scavenging effect (%) = (1 –(A1-A2)/A0) × 100% Where A1 is the absorbance of a mixture of 10 µL sample and 190 µL ABTS stock solution; A2 is the absorbance of a mixture of 10 µL sample and 190 µL of 50% ethanol, and A0 is the absorbance of a mixture of 190 µL ABTS stock solution and 10 µL of 50% ethanol. 2.13 The neuroprotective activity of SH-SY5Y cells induced by H2O The model of SH-SY5Y cell line (Stem Cell Bank, Chinese Academy of Sciences, China) injury induced by H2O2 was established, and the neuroprotective activities of the compounds (APS1, APS2, APS3, Se-APS2, Se-APS3) were tested using the Cell CountingKit-8(CCK8, Dojindo Laboratories, 8

Japan) method as previously reported [27, 28]. SH-SY5Y cells were seeded at 96 well plates (1×104/well) and incubated at 37 oC for 24 h. Each compound at various concentrations (25, 50, 100 μmol/L) was add to the wells and cultured in incubator for 1 h, then, 350 μmol/L H2O2 was added to the 96 well plates and incubated for 4 h. Finally, CCK-8 (10μL) was added into the 96-well microplates and measured at 450 nm by the microplate reader. Trolox was used as positive control and all experiments were performed in triplicate. 3, Results and discussion 3.1 Extraction, isolation and purification of ASP1-3 Crude polysaccharides were obtained by hot water extraction from M. sativa stem, after separation using DEAE-52 Cellulose and purification via Sephadex G-200 column, to obtain three major polysaccharide fractions (APS1, APS2 and APS3) were obtained. 3.2 Analysis of monosaccharide composition and their Molecular weight After completed acid hydrolysis and HPLC analysis, the monosaccharide composition of the polysaccharide was determined through comparison with the standard retention time. The results showed that APS1 was mainly composed of rhamnose, arabinose, mannose and galactose, in a mole ratio of 0.44:0.13:0.11:0.32, respectively. On the other hand, APS2 was mainly composed of rhamnose, arabinose, mannose and galactose, in a mole ratio of 0.50:0.22:0.07:0.21, respectively. While APS3 was composed of rhamnose, arabinose, fructose, mannose and galactose, in a mole ratio of 0.56:0.19:0.18:0.05:0.02, respectively. Based on the standards calibration curve of glucan (lgMw=-0.4632RT+11.37639 (R2=0.97105)), the molecular weights (Mw) of APS1, APS2, APS3, Se-APS2 and Se-APS3 were found to be 13.4 KDa, 11.2 KDa, 18.6 KDa, 9.0 KDa and 10.2 KDa, respectively. The reduction of the molecular weight of Se-polysaccharides could have been caused 9

by hydrolysis of the polysaccharides under acidic reaction conditions. 3.3. Periodate oxidation-Smith degradation reaction The results for the periodate oxidation method of the three samples (APS1, APS2 and APS3) are shown in Table. 1. According to the standard curve of periodate acid consumption (Y=0.03884X0.01429 (R2=0.99485)), One mole of sugar residues consumed 0.35 (APS1), 0.86 (APS2) and 0.54 (APS3) moles of sodium periodate, and generated 0.085, 0.200 and 0.170 moles of formic acid, respectively. It was revealed that APS1, APS2 and APS3 were all composed of monosaccharide which were (1→6)-linked, (1→3)-linked, (1→2)-linked or (1→4)-linked. The HPLC spectrum results after smith degradation revealed that APS1 contained ethylene glycol, glycerol, erythritol, Rha and Ara, indicating the presence of →3)-L-Rhap-(1→ and →3)-D-Arap-(1→ residue linkage. APS2 contained ethylene glycol, glycerol, erythritol, Rha, Ara, and Gal, indicating the presence of →3)-L-Rhap-(1→, →3)-D-Araf-(1→ and →3)-D-Galp-(1→ residue linkage. While APS3 contained ethylene glycol, glycerol, erythritol, Rha, Ara, and Fru, indicating the presence of→3)L-Rhap-(1→, →3)-D-Araf-(1→ and →3)-D-Fruf-(1→ residue linkage. The ratio of ethylene glycol, glycerol and erythritol in APS1, APS2 and APS3 were 0.28:0.64:0.08, 0.24:0.49:0.20, and 0.24:0.65:0.11, respectively. Combined with the results of periodate oxidation, it was revealed that APS1 contained (1→3)-linked (74.0%), (1→6)-linked (8.5%), (1→4)-linked (4.5%) and (1→2)-linked (12.5%) monosaccharide. On the other hand, APS2 contained (1→3)linked (34.0%), (1→6)-linked (20.0%), (1→4)-linked (36.0%) and (1→2)-linked (10.0%) monosaccharide. while APS3 contained (1→3)-linked (63.0%), (1→6)-linked (17.0%), (1→4)linked (6.0%) and (1→2)-linked (14.0%) monosaccharide. 3.4 Selenium content analysis 10

According to the standard curve (Y=149.7725+58.5725X (R=0.9977)), the selenium content in APS2 and APS3 were 1.05 and 2.57 µg/mg, respectively. 3.5. Analysis of FT- IR spectrum Fig. 1 shows the FI-IR spectra of the samples (APS1, APS2, APS3, Se-APS2 and Se-APS3). The strong peaks at 3422 cm−1, 3425 cm−1, 3428 cm−1, 3428 cm−1, and 3426 cm−1 were ascribed to O−H stretching vibrations of APS1, APS2, APS3, Se-APS2 and Se-APS3, respectively. The peaks at 2926 cm−1 (APS1), 2932 cm−1 (APS2) and 2924 cm−1 (APS3, Se-APS2, Se-APS3) were assigned to the C−H stretching vibrations of methyl groups [29, 30]. Peaks at 1652 cm−1 of APS1, 1616 cm−1 of APS2, 1632 cm−1 of APS3, 1626 cm−1 of Se-APS2 and 1625 cm−1 of Se-APS3 were caused by bound water [31]. The peaks at 1384 cm−1 were the variable angle vibrations of C-H [24]. The two strong peaks ranging from 1156 - 1075 cm−1 indicates the presence of furan glycosides. The peaks at 858 cm−1 (APS2), and 860 cm−1 (APS1, APS3, Se-APS2, Se-APS3) confirmed the presence of α-type glycosidic linkage [32]. Compared with the polysaccharide fractions, the new absorption peaks of Se-polysaccharides appeared at 894 cm-1 (Se-APS2), 898 cm-1 (Se-APS3) and 824 cm-1 (Se-APS3), due to the vibration of Se-O-C or Se-OH, respectively [19], indicating that selenium was successfully bound to the polysaccharides. 3.6.NMR analysis spectroscopy The structural characteristics of the samples were confirmed via NMR spectroscopy. The 1H NMR spectrum and 13C-NMR of APS1-3 are shown in Fig. 2. The results show that the 1H NMR and 13C NMR spectrum of the three polysaccharides were highly similar. From the 1H NMR spectrum of APS1-3, the singles at δ 1.16 - 1.19 ppm were assigned to the proton of α-L-rhamnose methyl [33], indicating that the three polysaccharides contained rhamnose. The signals between δ 3.31 - 4.30 11

ppm were assigned to H-2 to H-5 (or H-6) of the glycosidic ring [34]. From previous studies, the 1H singles at δ 2.00 - 2.17 ppm, 5.59 ppm and 5.75 ppm were assigned to the protons in O-acetylgroups, H-2 and H-1 of 2-O-acetyl-β-D-Manp-(1→, respectively [35, 36]. While the peak at 4.71 ppm of APS1, 4.69 ppm of APS2 and 4.70 of APS3 were designated as the chemical shifts of HOD [37]. From the 13C NMR spectrum of APS1-3, the peaks at 16.4-16.6 ppm were assigned to the carbon of α-L-rhamnose methyl [38]. The presence of C-2 to C-6 in the residues were revealed through the signals between 62.4 - 78.8 ppm [39]. The signals at 173.9 - 174.7 ppm from 13C NMR spectrum of APS1-APS3 confirmed the presence of 2-O-acetyl-β-D-Manp-(1→ [40]. Based on the 1H NMR spectrum of APS1, the peaks at 5.19, 5.02, 4.95, and 4.34 ppm were assigned to the anomeric protons of the α-L-Araf, α-L-Rhap, α-D-Galp and β-D-Manp, respectively. The cross peaks at 5.19/109.5, 5.02/107.5, 4.95/97.7, 4.34/107.4 ppm from the HSQC spectrum of APS1 verified those residues, respectively [41, 42, 43]. In 1H-1H-COSY spectrum of APS1, the single at 1.18/2.31 ppm was assigned to the H-4 proton of methyl in α-L-rhamnose. Such correlations could have occurred as follows: H-2/H-3 (5.58/3.69 ppm) of 2-O-acetyl-β-D-Manp-(1→, H-1/H-2 (4.95/3.50 ppm) of α-D-Galp, H-1/H-2 (5.18/3.70 ppm) of α-L-Araf, H-1/H-2 (5.02/4.00 ppm) of α-L-Rhap, and H-1/H-2 (4.35/3.64 ppm) of β-D-Manp. Combined with the results of periodate oxidation-Smith degradation reaction, the structural elements of APS1 were elucidated as follow: 2-O-acetyl-β-D-Manp-(1→3)-α-L-Araf-1(1→4)- α-L-Rhap-(1→ →3)- α-L-Rhap-(1→4)- α-L-Rhap-(1→ According to 1H NMR of APS2, the singles at δ 5.18, 5.03, 4.94, 4.35 ppm were attributed to the anomeric protons of the four residues. The singles at δ 107.2, 108.9, 101.3, 103.2 ppm from APS2 12

were assigned to anomeric carbon of the four residues [39]. The cross peak at δ 1.18/16.5 ppm from HSQC spectrum confirmed the presence of α-L-rhamnose. While the cross peaks at δ 5.18/107.2, 4.94/101.3, 5.03/108.9, 4.35/103.2 of APS2 from HSQC were assigned to α-L-Araf [32], α-L-Rhap, α-D-Galp and β-D-Manp, respectively [41, 43]. In the HMBC spectrum, the cross peak at 5.03/71.5 was assigned to the H-1 of →3)-α-D-Galp-(1→ to its C-4. The cross peaks at 4.41/100.1 was assigned to the H-1 of β-D-Manp and C-1 of α-L-Rhap. The signals at 3.36/103.5 were assigned to H-2 of O-acetyl-β-D-Manp and C-1 of β-D-Manp while 1.18/79.1 was the methyl proton and C-4 of α-L-Rhap. The signal at 5.18/79.1 was assigned to the H-1 and C-4 of α-L-Rhap. Combined with the results of periodate oxidation-Smith degradation reaction, the structural elements of APS2 were elucidated as follow: →3)-α-L-Rhap-(1→4)-α-L-Rhap-(1→ →3)-α-D-Galp-(1→4)-α-D-Galp-(1→ β-D-Manp-(1→2)-O-acetyl-β-D-Manp-(1→ β-D-Manp-(1→3)-α-L-Rhap-(1→ Compared with the 1H-NMR spectrum of APS2, the single at 4.94 ppm disappeared in Se-APS2 indicating that α-L-Rha was hydrolyzed under acidic reaction conditions and then removed by dialysis. The peaks at 5.75 and 5.58 ppm disappeared indicating that O-acetyl-β-D-Manp was hydrolyzed into β-D-Manp. Comparing 13C-NMR of Se-APS2 with that of APS2, the unlinked C-6 peak of β-D-Man at 62.1 ppm was disappeared, indicating that C-6 of the glycosyl group was substituted by selenium [44]. From the HSQC spectrum of APS3, the cross peaks at 4.52/103.8, 5.13/109.8, 4.98/107.6, 5.15/98.8, 4.39/103.3 ppm were identified as β-D-Manp [33], α-D-Galp, α-L-Rhap [42], α-L-Araf, β-D-Fruf 13

[24], respectively. The single 3.90/84.5 ppm was loaded at H-3/C-3 of → 3)- α-D-Galp-(1 → . According to the 1H-1H-COSY spectrum of APS3, the correlations were established as follows: H1/H-2 (5.13/2.23 ppm) of α-D-Galp, H-1/H-2 (5.15/3.08 ppm) of α-L-Araf, H-1/H-2 (4.98/2.14 ppm) of α-L-Rhap, H-1/H-2 (4.35/3.74 ppm) of β-D-Manp, and H-1/H-2 (4.39/1.35 ppm) of β-D-Fruf. According to the cross peaks at 2.23/4.39, 5.13/2.14, 3.08/5.15 alongside the results of periodate oxidation-Smith degradation reaction, the structural elements of APS3 were elucidated as follow: →3)-β-D-Fruf-(1→2)-α-D-Galp-(1→ α-D-Galp-(1→2)- α-L-Rhap-(1→ →3)-α-L-Araf-(1→3)- α-L-Rhap-(1→ The 1H-NMR and 13C-NMR spectrum of Se-APS3 were similar to those of Se-APS2, the peak at 62.3 ppm disappeared due to the substitution of C-6 [44]. 3.7 Scanning Electron Microscope The surface morphology of APS1, APS2 and APS3 was studied by SEM observation as shown in Fig. 4A, B, C. The results revealed that the structure of APS1 was flaky with a smooth surface, APS2 was an irregular sheet with loose porosity. A comparison with APS1 and APS2, APS3 showed lump and fragment with a rough surface structure. Combined with adsorption dynamic analysis, it could be inferred that the larger the molecular weight, the more structural fragments, which is more beneficial for the combination of Se and polysaccharide, thus the selenium content of Se-APS3 was higher than that of Se-APS2. As shown in Fig. 4 D, E, both Se-APS2 and Se-APS3 exhibited fragmented structure with a few spherical structures attached to the surface. This could have been caused by the acidic and high temperature reaction conditions, which broke down the slatted structure of polysaccharide into 14

fragments. The SEM results showed that the structure and morphology of APS were changed by selenide modification. 3.8 Thermal analysis In Fig. 5, the TG curve of APS1 shows that the degradation procedure was divided into three steps. The first mass loss of approximately 21.8% occurred below 230 oC due to the vaporization of absorbed and bonded water. The second mass loss of 37.9% (230 - 489 oC) and the third mass loss of 8.8% (489 - 900 oC) were due to the pyrolysis of polysaccharide skeletons. Similar to APS1, the TG curves of APS2-3 and Se-APS2-3 revealed three-step of degradation process with the same degradation principle, the first mass loss: for APS2, APS3, Se-APS2 and Se-APS3 were 14.3%, 15.43%, 18.8% and 14.1%, respectively and occurred below 230 oC. The second mass loss: for APS2 and APS3 were 48.3% and 45.14%, respectively and occurred in the range of 230 - 500 oC, respectively. While for Se-APS2 and Se-APS3 were 49.9% and 48.6%, respectively and occurred in the range of 230 - 560 oC. The third mass loss: for APS2 and APS3 were 6.7% and 7.7%, and occurred in the range of 500 oC to 900 oC, respectively. However, Se-APS2 and Se-APS3 were 7.9% and 6.5%, respectively, and occurred in the range of 560 - 900 oC. From the DTA curve of the samples, the exothermic peak of APS1-3 and Se-APS2-3 were at 658.2, 639.5, 632.6, 639.9, 645.1 oC, respectively. The TG and DTA curves revealed that the first and second mass loss of the samples were exothermic reactions, while the third weightlessness was as a result of endothermic reactions. These results showed that the selenium modification of the polysaccharide had no distinct effects on their thermal stability. 3.9. In vitro antioxidant activities of polysaccharide fractions In this study, the scavenging effects on DPPH radical and ABTS radical was used to estimate the in 15

vitro antioxidant activity of APS1-3 and Se-APS2-3. The samples ability to scavenge free radicals was closely related to the antioxidant ability. The DPPH radical had a characteristic absorbance at 517 nm. As shown in Fig. 6a, all polysaccharides and Se-polysaccharide displayed indistinct scavenging ability compared to ascorbic acid at the same concentration. The scavenging activities of APS1-3 and Se-APS2-3 increased with the concentration of polysaccharides ranging from 0.005 to 5.000 mg/mL. This figure shows that the DPPH free radical scavenging activity of Se-polysaccharides was stronger than that of natural polysaccharides. At high concentration (5 mg/mL), the scavenging effects of Se-APS2 and Se-APS3 were 88.1% and 92.0%, respectively. These results indicate that the selenium modification of polysaccharides can significantly increase their antioxidant effects in vitro. As shown in Fig. 6b, the ABTS radical scavenging effects of APS1-3 and Se-APS2-3 exhibited a dose-dependent pattern and were lower than those of the positive control Vc at various tested concentrations. These results indicate that the scavenging activity of selenium modified polysaccharides was significantly higher than that of natural polysaccharide. In summary, the results of DPPH and ABTS radical scavenging rate indicated that both natural polysaccharides and selenium modified polysaccharides had specific antioxidant activities and the Se-polysaccharide had more significant antioxidant effect than natural polysaccharide. According to the results, the scavenging effect of Se-APS3 on DPPH free radicals at 5 mg/mL was close to that of Vc which was a very fascinating revelation. It has been reported in the literature that both the Semodified polysaccharides of Sargassum pallidum and tea have stronger scavenging effect on DPPH radicals than natural polysaccharides [45, 46] and the Se-modified polysaccharides of Pleurotus ostreatus have a stronger scavenging effect on ABTS radicals [47]. These studies indicated that 16

polysaccharides modified by selenization exhibited stronger scavenging abilities on DPPH and ABTS radicals than their ordinary polysaccharides, indicating better antioxidant activities. 3.10. The neuroprotective activity of SH-SY5Y cells induced by H2O2 The neuroprotective effects of polysaccharides were evaluated in H2O2-induced SH-SY5Y cell line damage, and the cell viability were calculated as shown in Fig. 7. Compared to the cells of the control group in which cell viability was 100%, the cell viability of SH-SY5Y cells treated with H2O2 was reduced to around 61%. From Fig. 7, it was found that all the samples had dose-dependent neuroprotective activity. Compared with the three natural polysaccharides, APS1 showed remarkable neuroprotective activity at different concentrations, while APS2 and APS3 had no neuroprotective activities. However, compared with APS2 and APS3, the neuroprotective activity of Se-APS2 and Se-APS3 were obviously enhanced. At a concentration of 100 µM, the neuroprotective activity of Se-APS2 reached 79% and that of Se-APS3 reached 86%. These results indicate that selenide modification of M. sativa polysaccharide enhanced its neuroprotective activity significantly. 4. Conclusion In conclusion, three polysaccharides (APS1, APS2, APS3) were isolated from the stem of M. sativa. The structural analysis of the three polysaccharides showed that APS1 was mainly linked by 1→3 glycosidic bonds. APS2 was mainly linked by 1→4 glycosidic bonds while APS3 was linked by 1→3 glycosidic bonds. APS2 and APS3 were then modified by Se and their antioxidant and neuroprotective activities were compared to that of natural polysaccharides. The results showed that both polysaccharides and Se-polysaccharides displayed significant free radical scavenging ability and neuroprotective activity. At high concentrations, the DPPH free radical scavenging ability of 17

the polysaccharides increased in the order of APS1 < APS2 < APS3 < Se-APS3 < Se-APS2. The ABTS free radical scavenging ability increased in the order of APS3 < APS1 < APS2
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24

Table 1 Periodate oxidation and glycosidic linkages position of APS1, APS2, and APS3.

Samples

NaIO4

HCOOH

consumption/mol

production/mol

hexose

hexose

APS1

0.35

APS2 APS3

1→6

1→3

1→2

1→4

0.085

8.5%

74%

12.5%

4.5%

0.86

0.200

20%

34%

10%

36%

0.54

0.170

17%

63%

14%

6%

Figure legends Fig. 1. FT-IR spectrum. (a)APS2 and Se-PAP2, (b) APS3 and Se-PAP3. Fig. 2. 1H-NMR and 13C-NMR of APS1-3 and Se-APS2-3. (a) 1H-NMR of APS1-3 and Se-APS23, (b) 13C-NMR of APS1-3 and Se-APS2-3. Fig. 3. The scanning electron micrographs of APS1-3 and Se-APS2-3 at magnification (×5.00 k). The lyophilized samples were coated with a thin layer of gold and photographed by a scanning electron microscope. (a) APS1(×5.00 k); (b) APS2 (×5.00 k); (c) APS3(×5.00 k); (d) Se-APS2 (5.00 k); (e) Se-APS3 (5.00 k). Fig. 4. TG and DTA curves of APS1-3 and Se-APS2-3 under N2 at a heating rate of 10 oC/min. (a) TG and DTA curves of APS1; (b) TG and DTA curves of APS2; (c) TG and DTA curves of APS3; (d) TG and DTA curves of Se-APS2;(e) TG and DTA curves of Se-APS3; Fig. 5. Antioxidant activities of APS1-3 and APS2-3 with different methods. (a) Scavenging activity on DPPH radicals; (b) Scavenging activity on ABTS radicals. Fig. 6. Neuroprotective activity of SH-SY5Y cells induced by H2O2 of APS1-3 and Se-APS2-3.

25

a

2 Se-APS3 APS3 2 100

858.77

90

Transmittance %

80

894.27

70

532.92

2932.58 60

1616.42 3425.53

50

1384.51

2924.84

1075.18

1631.28

40

3428.87 30 4000

3000

2000

1000

Wavennumbers cm

b

0

-1

Se-APS3 APS3

105

824.22

100

898.16

Transmittance %

95 90 85

860.44

80

2924.18

534.99

1632.59

75

1384.36

70

1076.41

3428.81 65

1156.63

3426.07 4000

3000

2000

Wavenumbers cm

1000

0

-1

Fig 1. FT-IR spectrum. (a) APS2 and Se-PAP2, (b) APS3 and Se-PAP3.

a

b

Fig. 2. 1H-NMR and 13C-NMR of APS1-3 and Se-APS2-3. (a) 1H-NMR of APS1-3 and Se-APS23, (b) 13C-NMR of APS1-3 and Se-APS2-3.

26

Fig. 3. The scanning electron micrographs of APS1-3 and Se-APS2-3 at magnification (×5.00 k). The lyophilized samples were coated with a thin layer of gold and photographed by a scanning electron microscope. (a) APS1(×5.00 k); (b) APS2 (×5.00 k); (c) APS3(×5.00 k); d) Se-APS2 (5.00 k); (e) Se-APS3 (5.00 k). b

TG DTA

110

100 10 90

90

50 40

weight (%)

80

70

5

60 50 40

0

DTA(uV/mg)

60

DTA (uV/mg)

5

0

200

400

600

800

200

400

600

800

0

1000

200

temperature (oC)

temperature (oC)

d

0

40 30

0

1000

60 50

0

30

30

5 70

TG DTA 10

110

DTA (uV/mg)

80

80 70

TG DTA 10

110 100

10 90

weight (%)

c

TG DTA

110

100

weight (%)

a

e

400

800

1000

TG DTA

110

100

600

temperature (oC)

100 10

90

90

50 40

weight (%)

60

80

5

60 50

0

30

70

DTA (uV/mg)

5

70

DTA (uV/mg)

weight (%)

80

40

0

30

20 0

200

400

600

800

1000

0

temperature (oC)

200

400

600

800

1000

temperature (oC)

Fig. 4. TG and DTA curves of APS1-3 and Se-APS2-3 under N2 at a heating rate of 10 oC/min. (a) TG and DTA curves of APS1; (b) TG and DTA curves of APS2; (c) TG and DTA curves of APS3; (d) TG and DTA curves of Se-APS2;(e) TG and DTA curves of Se-APS3.

27

a

Vc APS1 APS2 APS3 Se-APS2 Se-APS3

100 90

scavenging effect (%)

80 70 60 50 40 30 20 10 0 0

1

2

3

4

5

concentration (mg/mL)

b

Vc APS1 APS2 APS3 Se-APS2 Se-APS3

scavenging effect (%)

100

50

0 0

1

2

3

4

5

concentration (mg/mL)

Fig. 5. Antioxidant activities of APS1-3 and APS2-3 with different methods. (a) Scavenging activity on DPPH radicals; (b) Scavenging activity on ABTS radicals.

Fig. 6. Neuroprotective activity of SH-SY5Y cells induced by H2O2 of APS1-3 and Se-APS2-3. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. the H2O2-treated cells group.

28

Highlights

1, Three natural polysaccharides (APS1-3) isolated from Medicago Sativa L. stem and their Semodified products (Se-APS2-3) were synthesized. 2, The monosaccharide composition and glycoside bond binding sites of APS1-3 were determined. 3, The molecular weight, surface morphology and thermal stability were compared between polysaccharides and Se- polysaccharide. 4, The antioxidant activities and neuroprotective activity of Se- polysaccharides in vitro were higher than that of natural polysaccharides.

29