Optimation for preparation of oligosaccharides from flaxseed gum and evaluation of antioxidant and antitumor activities in vitro

Journal Pre-proofs Optimation for preparation of oligosaccharides from flaxseed gum and evaluation of antioxidant and antitumor activities in vitro Chen Yang, Chao Hu, Hao Zhang, Wenchao Chen, Qianchun Deng, Hu Tang, Fenghong Huang PII: DOI: Reference:

S0141-8130(19)33846-2 https://doi.org/10.1016/j.ijbiomac.2019.10.241 BIOMAC 13742

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International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

24 May 2019 11 October 2019 26 October 2019

Please cite this article as: C. Yang, C. Hu, H. Zhang, W. Chen, Q. Deng, H. Tang, F. Huang, Optimation for preparation of oligosaccharides from flaxseed gum and evaluation of antioxidant and antitumor activities in vitro, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.241

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Optimation for preparation of oligosaccharides from flaxseed gum and evaluation of antioxidant and antitumor activities in vitro

Chen Yang1*, Chao Hu1, Hao Zhang1, Wenchao Chen1, Qianchun Deng1, Hu Tang1*, Fenghong Huang1

1 Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Oil Crops and Lipids Process Technology National & Local Joint Engineering Laboratory, Hubei Key Laboratory of Lipid Chemistry and Nutrition, Key Laboratory of Oilseeds Processing, Ministry of Agriculture and Rural affairs, No. 2 Xudong 2nd Road, Wuhan 430062, China. * These two authors are both corresponding authors. Correspondence: [email protected] and [email protected]

Abstract Flaxseed oligosaccharides (FGOS) were prepared by degradation of flaxseed gum (FG) using enzymatic method. Factors affecting the enzymatic hydrolysis of FG were investigated by single factor and orthogonal tests. In the optimum hydrolysis conditions (reaction time 12 h, temperature 50 °C, pH 4.5, cellulase concentration 100 U/mL), the reducing sugar ratio and extraction yield of FGOS were 33.6 ± 0.35% and 56.8 ± 0.41%, respectively. The average molecular weight of FGOS was about 1.6 kDa, which consists of mannose, galactose, glucose, arabinose, glucuronic acid, xylose, rhamnose, ribose, galacturonic acid. Fourier-transform infrared spectra and NMR indicated that FG was successfully degraded to FGOS. FGOS exhibited better antioxidant activities than FG on scavenging hydroxyl, ABTS and DPPH radicals. In vitro cytotoxicities experiments reveal FGOS acquire the ability of antiproliferation against HepG2 and Hela cells in a dose-dependent manner.

Key words: oligosaccharides preparation; flaxseed polysaccharides antioxidant activity; antitumor activity.

1

1. Introduction Flaxseed (Linum usitatissimum L.), one of the most important oilseed crops, is a functional food source due to its high content of dietary fiber and oil rich in α-linolenic acid [1]. The flaxseed mucilage, known as flaxseed gum (FG), represents about 3.5% to 10.2% of the total seed weight [2] and is a heterogeneous polysaccharide consisting of neutral arabinoxylan and highly acidic rhamnose-containing polysaccharide [3]. FG is easy to incorporate into beverages and dairy products [4], and its possible synergistic effects with proteins could also be valuable in foods. However, further use of FG is limited because their high and heterogeneous molecular weight (1.7×101–5.7×103 kDa) [5]. Study also indicated that the high viscosity of flaxseed polysaccharides restricts their pharmaceutical application [6]. Oligosaccharides are short-chain polymers (containing 2–10 monosaccharide units) with low molecular weight, which have drawn great attention for their applications in various fields [7]. They have specific biological activities such as aiding proliferation of bifidobacteria, regulation of gastrointestinal function, liver protection, antitumor and antiaging properties, reduction of cardiovascular risk, et al. [8, 9]. Exploring the preparation methods of flaxseed oligosaccharides from FG will promote the use of FG, and produce healthy oligosaccharides with higher economic value. Previous study has shown that it was hard to depolymerize FG using only acid degradation or enzymatic method because of the complicated branched structure of flaxseed polysaccharide [10]. Arabinoxylan-oligosaccharides could be prepared from flaxseed mucilage by Guilloux et al. using enzymatic hydrolysis method [11]. Liang et al. also obtained flaxseed oligosaccharides from FG using H2O2 oxidation method [12]. However, these methods needed long extraction times at high temperature or complicated purification processes, and result in low product yield. Till now, there is almost no research has attempted to combine various methods on degrading FG to prepare oligosaccharides. There are also few reports about the biological activity of the oligosaccharides derived from FG. Thus, this study aimed to optimize enzymatic hydrolysis conditions for FG to gain higher yield of FGOS with less energy cost. This work also evaluated the anti-tumor activity of FGOS for the first 2

time, which shields the light on developing the flaxseed oligosaccharides as functional foods or candidate medicine.

2. Materials and methods 2.1. Materials Flaxseed gum (90% purity) was obtained from Xinjiang Linseed Biological Technology Co. Ltd. (Yili, Xinjing, China). Cellulase (50,000 U/g), dextran standards and analytical monosaccharide standards: rhamnose （Rha）, arabinose （Ara）, xylose（Xyl）, mannose（Man）, glucose（Glc）,

galactose（Gal）, ribose

（Rib）, glucuronic acid （GluA）, galacturonic acid（GalA）, fucose (Fuc), glucosamine (GluN) and galactosamine (GalN) were purchased from Sigma-Aldrich (Novozymes, Bagsvaerd, Denmark). H2O2 (30%, v/v) was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) and 2, 2-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) were purchased from MOLBASE (Shanghai, China). 2.2. Methods 2.2.1. Preparation of flaxseed oligosaccharide (FGOS) from FG FG powder was dispersed in 0.2 M H2O2 solution to obtain a 2% (w/v) suspension. It was pretreated in an automatic high-pressure steam sterilizer at 120 °C for 1.0 h. Cellulase was then added into the mixture to enzymatic hydrolysis was carried out under the optimum conditions. The reaction was stopped by boiling for 15 min and then the product was filtered. FGOS powder was finally obtained by lyophilized for analysis of physical and chemical properties and biological activities. The viscosity of degradation products was measured by SNB-1 digital viscometer from Shanghai Pingxuan Scientific Instrument Co.Ltd (Shanghai, China). The FOS yield (%) = w/M ×100, where w is the weight of obtained FGOS powder and M is the weight of FG powder used. 2.2.2. Single factor test Single-factor tests of enzymatic degradation were designed to optimize controllable variables including cellulase concentration (from 5 to 200 U/mL), pH (from 3.5 to 5.5), reaction time (from 2 to 16 h) and reaction temperature (from 40 to 60 °C). Reducing sugar (RS) was determined by the dinitrosalicylic acid colorimetric method with 3

glucose as standard [13] while total sugar (TS) was detected by the sulfuric acid-phenol method with glucose as standard [14]. The reducing sugar ratio, RSR= RS/TS. 2.2.3. Orthogonal test The best condition for preparing FGOS was further investigated by orthogonal tests. Four abovementioned factors (A: reaction time, B: temperature, C: pH, and D: enzyme concentration) were inspected and three levels of each factor were selected to carry out L9 (34) orthogonal tests (Table 1). Table 1. The variables investigated and their levels. Variable Levels Variables Investigated 1 2 50 100 A: Enzymatic concentration (U/mL） B: pH 4.5 5.0 C: Reaction time (h) 8 12 40 45 D:Reaction temperature (℃)

3 150 5.5 16 50

2.2.4. FT-IR (Fourier Transform Infrared) spectra and NMR (Nuclear Magnetic Resonance) analysis FT-IR analysis was conducted using Thermo Nicolet 5700 (Madison, WI, USA). Samples were mixed with KBr and made pellets. Samples were also analyzed as the contrast FTIR spectra were recorded in mid-IR region 4000–400 cm−1. The 30 mg of samples were dissolved in 2 mL of D2O three times and lyophilized. 1

H and 13C NMR spectra were recorded at 25 °C by a Bruker DRX-500 spectrometer

(Bruker, Rheinstetten, Germany) and operated at 400 MHz. 2.2.5. Evaluation of molecular weight The molecular weight distribution of FG and FGOS was determined by highperformance liquid chromatography–size exclusion chromatography (HPLC-SEC) analysis (LC-20AD; Shimadzu, Kyoto, Japan), equipped with an evaporative light scattering detector (ELSD-LT II Shimadzu). Sample (2 mg/mL) was filtered through a 0.45 μm polyether sulfone filter (Millipore) before inject into the system. The SEC columns in series were a TSKgel G5000PWXL and a TSKgel G3000PWXL (7.8 mm × 30 cm, 7 μm; Tosoh Bioscience, Tokyo, Japan). Ultrapure water was used as the mobile phase with 0.5 mL/min for 30 min. Columns were operated at 40 °C and ELSD was carried out at 110 °C in a drift tube. During determination, the air flow rate was 3.0 L/min. To calculate the molecular weight of FGOS, dextran standards with number 4

average Mw of 800, 2000, 5000, 10000, 126000, 289000, and 500000 Da were used to establish a standard curve with elution time and log Mw as horizontal and vertical coordinates respectively. The molecular weight of FGOS was also determined by the matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF-MS) analysis according to Huiqing Sun et al [15] with modification. Briefly, 20 mg/mL 2, 5Dihydroxybenzoic acid (DHB) dissolved 0.1%TFA: CAN (70:30, V:V) with 1mM NaCl was used as a matrix. 2.0 μL of matrix and 2.0 μL of sample (10mg/mL) were mixed and then 1μL of the obtained mixture was applied on the sample plate and allowed to dry at room temperature. Then the Mw of sample was detected with the Mass spectrometer (Ultraflextreme, Bruker Daltonik GmbH, Leipzig, Germany). The instrument was operated at an accelerating voltage of 25.10kV with an extra voltage of 13.41 kV. 2.2.6. Monosaccharide composition analysis FG and FGOS (20 mg) were treated with 2 M trifluoroacetic acid (TFA) at 110 °C for 2 h. Methanol (200 μL) was added into the hydrolyzed solution after cooling, and then dried with N2. The hydrolyzates were then derived with 1- phenyl -3- methyl -5pyrazolone (PMP) according to a previously described method [16], as well as the monosaccharides standards. They were dissolved in 0.6 M NaOH (0.18 g/L for each sample), 50 μL solution were mixed with 50 μL0.5 M PMP solution in methanol with a stopper. The reaction was conducted at 70 °C for 100 min, then the mixture was cooled and neutralized with 0.3 M HCl (2.0 mL). Extraction using chloroform was repeated three times. The sample for HPLC analysis was obtained by filtering through 0.45 μm nylon membrane. Analysis was performed on an Agilent 1100 series LC (Agilent Technologies, Waldbronn, Germany). Samples were isolated with a Zorbax Extend-C18 column (250 × 4.6 mm, 5 μm; Agilent Corporation, CA, USA). The injection volume was 20 μL with flow rate 1.0 mL/min and peaks were detected at 250 nm. The mobile phase consisted of 10 mM ammonium acetate (plus 1 mL glacial acetic acid per 100 mL)–acetonitrile (83:17, v/v). Columns were operated at 30 °C. 2.2.7. Antioxidant activity of FGOS The hydroxyl radical (OH·) scavenging capacity of samples was determined 5

according to the method of Garrido et al. [17] with minor modifications. Ascorbic acid and glucose solutions were used as positive and negative controls respectively. Test samples (2.0 mL) were mixed with 0.5 ml of salicylic acid-ethanol solution (9 mM), 0.5 ml FeSO4 (9 mM) and 0.5 ml H2O2 (8.8 mM). The mixtures were incubated for 60 min at 37 °C, after which the absorbance at 510 nm was recorded as Ax. The absorbance of the blank control was A0. The absorbance of each sample with H2O2 replaced by an equivalent volume of pure water was noted as Ax0. The OH· scavenging rate (HRSR) was calculated according to the equation, HRSR [%] = A0 − (Ax − Ax0)/A0×100. The DPPH scavenging activities of samples were measured using the modified method of Souza et al. [18]. Varying concentrations of FGOS (0.5 mL) were mixed with 2.5 mL DPPH (75 μM solution in ethanol). The reaction mixture was shaken and incubated for 30 min in the dark at room temperature. The absorbance of the resulting solution was determined (using distilled water as a reference) at 517 nm. The absorbance of samples of different concentration was recorded as Ax, and that of 0.5 mL H2O with equivalent DPPH was A0. The DPPH scavenging rate (DRSR) was calculated according to the equation, DRSR [%] = (A0−Ax)/A0×100. ABTS radical scavenging was measured according to the method described by Re et al. [19] with slight modifications. ABTS radical stock solutions were prepared by combining equal volumes of 7 mM ABTS and 2.45 mM K2S2O8 in deionized water, followed by incubation in the dark for 24 h at room temperature. The ABTS solution was diluted with 10 mM phosphate buffer (pH 7.4) to give a final absorbance of 0.700 ± 0.020 at 734 nm to obtain the ABTS working solution. Varied concentrations of FGOS (30 μL) as well as control samples were mixed with 3.0 mL ABTS working solution. The absorbance of ABTS solution before and after adding scavenger was recorded as A0 and Ax respectively. The ABTS radical scavenging rate (ARSR) was calculated according to the equation, ARSR [%] = (A0 − Ax)/A0×100. 2.2.8. Cytotoxicity against cancer cells HeLa, HepG2 and MRC-5 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and 1% penicillin–streptomycin (Invitrogen; Carlsbad, CA, USA). Cells were maintained in a humidified atmosphere at 37 °C, 5% CO2, and cells in the logarithmic growth stage 6

were used for experiments. FGOS was dissolved in dimethylsulfoxide (Sigma Chemical, St. Louis, MO, USA) at a stock concentration of 10 mg/mL and diluted with water. Cells were seeded into 96-well flat-bottom plates at a density of 1×104 cells per well and cultured overnight, then treated with FGOS, FG or inulin for 24 h. Cells were incubated with 10 μL of MTT solution for 1 h at 37 °C and the absorbance was then determined at 450 nm using an ELx800 spectrophotometer (BioTek, Winooski, VT, USA). The effect of FGOS on inducing apoptosis in two cancer cell lines was examined by flow cytometry analysis. After 24 h treatment, the cells were collected and washed with a cold phosphate buffer saline (PBS) solution twice. The cells were then stained with FITC-annexin V and PI by following the manufacturer’s instructions (FITC Annexin V Apoptosis Detection Kit I (BD, CA, USA)).

3. Results and discussion 3.1 Preparation of FGOS from FG FG was pre-treated by H2O2 alone under high-pressure steam sterilization and the RSR would not increase significantly even the H2O2 concentration rise up from 0.2 M to 0.6 M. Thus, efforts were made to further depolymerize the FG by using cellulase, which raised the RSR of reaction filtrate. The final FGOS was obtained as faint yellow powder with as much as yield of 56.8 ± 0.41% under the optimal enzymatic condition. In vitro antioxidant and anti-tumor activity of FGOS was also evaluated to provide a theoretical reference and practical basis for its product development. 3.2 Single-factor test results 3.2.1 Effect of enzyme concentration on FGOS hydrolysis The RSR of the degradation products was measured under the four controllable variables, which was an important characteristic property reflected the degradation level of products [20, 21]. Ideally, the TS and RS content of the reaction filtrate should be determined using a mixture of all the monosaccharides within the same composition in the heteropolysaccharide. However, the TS content of the reaction filtrate among each experimental group had almost no change whether using Glc, Man, Gal or their mixture as standard, while the detected RS changed with the reaction condition. 7

Considering that glucose is one of the main components of FG and its convenience as most literature reported, we chose to use glucose as a standard to determine RSR, whose variation could help us determine the optimal preparation conditions. Figure 1(A) shows the results of FG hydrolysis at different enzyme concentrations (0, 5, 10, 25, 50, 100 and 200 U/mL), while the other variables were set as reaction time 12 h, pH 4.5, and reaction temperature 50 °C. The RSR of the pretreated reaction filtrate without further enzymatic hydrolysis was 19.43 ± 0.59%. As the enzyme concentration was increased, increasing RSR of the products was observed. These results suggested that cellulase treatment resulted in substantial depolymerization of the FG in a dosedependent manner. Considering the commercial cost of enzyme preparation, 100 U/mL was deemed to be the optimum concentration for reaction. 3.2.2 Effect of reaction time on FGOS hydrolysis The results of FG hydrolysis over different reaction times are shown in Fig. 1(B). The reaction time was set as 2, 4, 8, 12, 16, or 24 h, while the other reaction conditions were set as enzyme concentration 100 U/mL, pH 4.5, and reaction temperature 50 °C. When the reaction time was in the range 2 – 8 h, there was little change in RSR of the products. Decreased RSR was observed at 16 h compared with 12 h and the RSR at 16 h was no change compared to 24 h. This might because the RS reacted with the trace impurities protein or cellulase itself, and formed malanodin by maillard reaction, for the OD420 value (associated with maillard reaction product) at 16 h was greater than 12 h, while protein content was less than that of 12 h. At this time, the consuming RS was more than the newly producing RS by enzymatic hydrolysis [22]. Thus, to avoid side reaction products, the optimum time for the enzymatic hydrolysis was set at 12 h. 3.2.3 Effect of pH on FGOS hydrolysis Fig. 1(C) shows the results of FG hydrolysis at different pH (3.5, 4.0, 4.5, 5.0 and 5.5). Other reaction conditions were enzyme concentration 100 U/mL, reaction temperature 50 °C, and reaction time 16 h. There was a marked increase in the RSR of the products at pH 4.5 compared with 4.0. At higher pH, the RSR decreased sharply. Thus, the optimum pH for further hydrolysis of FG was 4.5. The cellulase from Aspergillus niger is active in the pH range 4.0–6.0 [23]. Here, the optimal pH for this enzyme was in a narrow range around 4.5, which might be due to the specific reaction 8

buffer system. 3.2.4 Effect of temperature on FGOS further hydrolysis The results of FG hydrolysis at different temperatures (40, 45, 50, 55 and 60 °C) are shown in Fig. 1(D). The other reaction conditions were enzyme concentration 100 U/mL, pH 4.5, and reaction time 12 h. The RSR of the products increased obviously on going from 40 to 50 °C, and then decreased at 55 °C. The optimum reaction temperature was thus 50 °C, above which the enzyme was presumably inactive. 3.3 Orthogonal experiments results Orthogonal experiments were designed to investigate the best combination of conditions for preparing FGOS. Four controllable variables enzyme concentration (A), pH (B), reaction time (C), and reaction temperature (D) were chosen as factors to carry out L9(34) orthogonal tests (Table 2) . By comparing R values in Table 2, the order of influence of each variable on the degradation of FGOS was enzyme concentration (A) > pH (B) ≈ reaction time (C) > reaction temperature (D). It is obvious that the contribution of factors A, B and C was significant to FG degradation. The optimum reaction conditions were: cellulase concentration 100 U/mL, reaction time 12 h, pH 4.5, reaction temperature 50 °C Table 2. Orthogonal experiments arrangements and results Experimental no.

Concentration(U/mL) pH Level (A) Level (B)

Time(h) Level(C)

Temperature(℃)

Level(D)

Reducing sugar ratio/RSR (%)

1 1 1 1 1 2 1 2 2 2 3 1 3 3 3 4 2 1 2 3 5 2 2 3 1 6 2 3 1 2 7 3 1 3 2 8 3 2 1 3 9 3 3 2 1 *K1 93.64 97.90 96.58 95.97 *K2 97.94 94.21 98.35 97.53 *K3 98.04 97.51 94.68 96.11 R 1.47 1.23 1.22 0.52 *Kn is the sum RSR values of the corresponding level n under each factor; R = Kmax/3 − Kmin/3, it represents the influence degree for each factor.

31.49 31.37 30.77 33.56 31.06 33.31 32.85 31.78 33.42

9

Flaxseed gum under H2O2 oxidation is subsequently hydrolyzed by cellulase and got 33.6 ± 0.35% RSR in the optimal conditions. The RSR which reflects the level of degradation of FG, was quit close to the previous data (37.81%) [12] but consumed shorter reaction time. Our method also improved the average yield of oligosaccharides (56.8±0.41%) compared that of Guilloux et al. (35%) [11]. Excluding the protein content, the purity of FGOS was about 95.2%. It suggested that the enzyme-assisted H2O2 oxidative method was effective for preparation of FGOS by degrading FG with high yield and shorter reaction time. 3.4 FTIR and NMR analysis The FTIR spectrum is an important tool for studying the molecular structure of polysaccharides. Fig. 2A showed the FTIR spectra of the purified FGOS and FG. Both of them displayed broad OH stretching vibrations of hydroxyl groups at around 3400 cm−1 [24]. FGOS showed a weaker C–H stretching band of CH2 groups around 2924 cm−1 than FG. The strong absorption band at 1645 cm−1 was contributed to the stretching vibration of the deprotonated carboxylic group from amide I [25], indicating FG have glucosamine, which were consistent with the result of monosaccharide composition. And 1610 cm−1 in FGOS were assigned to the C=O asymmetric stretching vibrations of carboxyl groups [24]. The weak band at 1252 cm−1 in two samples corresponded to the C–H deformation vibration. The absorbance at around 1414 cm−1 in FG and 1410 cm−1 in FGOS corresponded to the C−H bending vibration [26]. The absorption peak at around 1060 cm−1 or 1067 cm−1 could be assigned to C-O-C stretching vibration, which suggested that the monosaccharides (glucose or mannose) of FGOS and FG had pyranose rings [27, 28]. The band at 874 cm−1 indicated the β-pyranoside linkage of the glycosylic residue in both FG and FGOS [29, 30]. The spectra of 1H and

13

C NMR were shown in Fig. 2B and C. The 1H-NMR of

FGOS showed that there are 9 main signal peaks in the range of 4.8~5.5 ppm, which corresponding to 9 the peaks in the 100~104 ppm range in

13

C NMR. This result

indicated that the FGOS was mainly composed of 9 types of monosaccharides. Combined with chemical analysis and literature report, the signals of anomeric proton at 4.742 /4.872 ppm and 4.925/4.945 ppm were designated as β-D-Gal and β-D-Man, respectively. The signals of 5.033/5.041 ppm and 5.131/5.154 ppm were assigned to α10

D-Gal and α-D-Glc [31]. The signals at and 5.285/5.255 ppm 5.425/5.411 ppm were assigned to α-L-Ara and α-D-GlcA, respectively [32]. The weak peak around 1.2 ppm also indicated the presence of rhamnose [33], which was consistent with the analysis result of monosaccharide composition. Both FG and FGOS revealed the typical carbohydrate absorption peaks by the signals of 60–80 ppm where they were related to C2, C3, C4, C5, and C6 from the glycosidic ring [34]. Resonances of FGOS at about 175.33 ppm and 175.48ppm are probably originated from the carboxyl group of 1–4-linked galacturonic acid units[35]. The absorption band assigned to carboxyl groups in FGOS also looked larger than that in FG, suggested that FGOS might have more carboxyl groups. Moreover, Based on chemical shifts reported in the literature, signals at 70.91, 74.02, 70.59, 71.23, 80.21 and 83.99 ppm were correspond to C-2 of different glycosidic linkage [36, 37]. Peaks at 19.43 ppm and 65.57 ppm were attributed to C-6 of α-L-Rha [38] and β-D-Gal [39]. The FGOS exhibited more peaks associated to C-2 and C-6 glycosidic linkage, suggested degradation of FG exposed more C-2 or C-6 positions groups. Our results indicated that FG and FGOS had the characteristics of typical acid pyranoses and the structure of FG was changed by H2O2 oxidation combined with cellulose hydrolysis. 3.5 Molecular weight of FGOS The molecular weights of FG and FGOS were determined by HPLC-SEC analysis (Fig. 3). Based on the average molecular weights and elution times of a series of dextran standards, a standard curve equation was calculated as logMw = −0.4539t + 13.7630 (R2 = 0.9863). The elution time of the FGOS peak was 23.28 min and the weight average molecular weight (Mw) of FGOS was calculated to be 1568 Da, similar to the value reported by Liang et al. [12]. There was a dramatic decrease in the molecular weight of FGOS compared with FG (about 635 kDa). The theoretical degree of polymerization of FGOS was about 8.71 based on the molecular weight of glucose (180 Da). The MALDI-TOF-MS was used to detect the molecular weights of degraded product. As shown in Fig. 3C, FGOS is a mixture of containing both oligosaccharides with different molecular weights, from 461.3 to 1630.8 m/z according to literature [40], the degree of polymerizations of oligosaccharide were 4, 5, 6, 8, 9, for 660 Da, 886 Da, 1091 Da, 1404 Da and 1630 Da respectively. Considering the deviation in HPLC-GPC analysis of small molecule weight polysaccharides, this result was consistent with the 11

previous data. It turned out that FG has been degraded to oligosaccharides. We also monitored the viscosity of the hydrolysates during the preparation of FGOS. The original 2% FG solution demonstrated high viscosity, up to 5886.33 mPa.s−1 at 25 °C. The viscosity of final solution was reduced to ~0.5 mPa.s−1 at 25 °C. Taken together, our results suggest that enzymatically-assisted H2O2 oxidation has good performance in the degradation of flaxseed polysaccharides into flaxseed oligosaccharides with lower molecular weight. 3.6 Monosaccharide composition of FGOS Results in Fig. 4 and Table 3 show the monosaccharide composition of FGOS. We found that FGOS consists of nine monosaccharides. As shown in Table 3, FGOS is mainly composed of mannose, galactose and glucose (34.91%, 27.30% and 22.86% mole percentages respectively). The proportions of arabinose, glucuronic acid and xylose were quite low (5.21%, 5.43% and 1.06% respectively). FG and FGOS consist of the same nine monosaccharides but in different molar ratios. Glucuronic acid, galacturonic acid and ribose were detected in our FGOS, unlike the product reported by Liang et al. [12]. There was no fructose in FG or FGOS in our study, while 7.54% fructose was found in the oligosaccharides prepared by Liang et al. The differences depend on the original plant source, as polysaccharide composition in flaxseed varies according to the flax variety, growth period and growth environment [41]. In this study, the mole percentage of main sugar units including mannose, galactose and glucose were decreased whereas the other sugar units were increased after flaxseed polysaccharides degraded. This was assisted with several reports demonstrated that the contents of monosaccharide would be changed when compared the different Mw fractions after polysaccharides degradation [42, 43]. Flaxseed polysaccharide consists of acid polysaccharide and neutral polysaccharide. Generally, acid polysaccharides are more susceptible to degradation than neutral polysaccharides [44]. Our results suggest that this method effectively degraded the FG and preferentially degraded the acidic parts in FG, lead to the minor increase of glucuronic acid, galacturonic acid and ribose in FGOS.

Table 3 Monosaccharide compositions of FG and FGOS 12

Mixed standards

FG

FGOS

Monosaccharide

Retention time (min)

Peak area percent/ %

Relative correctio n factor

Peak area percent/%

Mole percent/%

Peak area percent/%

Mole percent/%

Man Glc Gal Ara GlcA Xyl Rha GalA Rib GlcN

16.17 33.96 38.75 42.79 24.59 41.28 22.5 27.86 21.15 20.42

14.98 9.59 8.76 11.87 5.83 11.02 8.83 5.48 8.67 8.59

0.56 0.87 0.95 0.7 1.43 0.76 0.94 1.52 0.96 0.97

51.77 20.49 22.01 1.66 2.59 0.39 0.6 0.3 0.1 0.08

39.00 24.12 28.37 1.58 5.02 0.4 0.77 0.62 0.13 0.08

47.15 19.76 21.55 5.58 2.85 1.05 0.96 0.82 0.29 /

34.91 22.86 27.30 5.21 5.43 1.06 1.21 1.66 0.37 /

3.7 Antiradical activity of FGOS 3.7.1 Hydroxyl radical scavenging The hydroxyl radical is one the most active reactive oxygen species, and can induce serious damage to biomolecules [45]. The hydroxyl scavenging ability of FGOS and FG is shown in Fig. 5(A). The hydroxyl radical scavenging effect of ascorbic acid increased rapidly in the range 0.5 to 2.0 mg/mL, and its efficiency was nearly 100% when the ascorbic acid concentration was >2.0 mg/mL. The negative control glucose showed very low OH· scavenging capacity even at the highest concentration tested. The hydroxyl radical scavenging activity of FGOS displayed clear concentration dependence, reaching 78.3% at 5.0 mg/mL. FG showed some effect in scavenging hydroxyl radicals, but significantly lower than that of FGOS at the same concentration. The hydroxyl radical induces hydrogen abstraction from a carbon-bonded hydrogen or oxygen-bonded hydrogen on a carbohydrate, forming a carbon-centered radical and H2O. The later one was unexpected due to the higher bond dissociation energy need from the oxygen–hydrogen bond than from the carbon-bonded hydrogen [46, 47]. In this study, carbon-bonded hydrogen of FGOS is more likely abstracted by the hydroxyl radical to scavenge the radicals, as previous report [12]. 3.7.2 DPPH radical scavenging DPPH radical scavenging ability is commonly used as an indicator of antioxidant 13

activity of medicines and food [48]. Fig. 5(B) shows the DPPH scavenging ability of samples tested in this study. The DPPH scavenging ability of FGOS was concentration dependent, and it reached 76.9% at 5 mg/m. The positive control (ascorbic acid) exhibited over >98.1% DPPH radical scavenging ability even at a very low concentration. FG showed an extremely low scavenging ability, as did the negative control, glucose. Thus, the DPPH scavenging ability of FGOS was much better than that of FG, but not as strong as that of ascorbic acid. DPPH is susceptible to proton radical scavengers. In general, C-2 and C-6 hydroxyls of carbohydrate are mainly involved in proton transfer reactions [49]. From the 13C NMR spectrum, the degradation of FG (FGOS) exposed more C-2 and C-6 positions groups, thus greatly improved DPPH capture at these positions to decrease the level of proton as previous report [50]. 3.7.3 ABTS radical scavenging Results in Fig. 5(C) show the ABTS radical scavenging ability of FGOS, ascorbic acid, FG and glucose. ABTS scavenging capacity is frequently used by agricultural and food scientists to measure antioxidant capacity. The maximum observed ABTS scavenging effect of FGOS was 75.6% when the concentration reached 25 mg/mL, and the scavenging capacity increased markedly with concentration up to 5.0 mg/mL. Ascorbic acid exhibited strong ABTS radical scavenging ability even when its concentration was <5.0 mg/mL. As a result, FGOS exhibited good ABTS radical scavenging ability when compared with FG, though not better than ascorbic acid. FG showed little obvious ABTS radical scavenging ability at ≤5 mg/mL. Data were not recorded at >5 mg/mL FG as its viscosity made it impossible to prepare a liquid solution. The antioxidant of biological activities of polysaccharides could be affected by many factors including the monosaccharide composition [51], the molecular weight, the triple helical chain structure [21] and the solubility [21]. It was concluded that the higher viscosity of flaxseed gum extracted under different temperature, the weaker antioxidant activity was observed [52]. The high viscosity of flaxseed gum may be detrimental to the reaction between hydroxide groups and free radicals [53]. Moreover, poor water solubility could hurt the absorption bioactive compounds in vivo, resulting in low bioavailability and limited clinical application [54]. In this study, FG and FGOS showed 14

similar glycosidic bond characteristics and monosaccharide composition, but FGOS has superior water solubility with antioxidant activity to FG. We suggested the degradation of flaxseed gum caused the glycosidic bond to break and improved solubility, so that more functional groups exposed which could capture more free radicals. However, more experimental data needed to verify the relationship between polysaccharide structure and antioxidant activity. 3.8 Cytotoxicity against cancer cells To throw light on the possible use of flaxseed oligosaccharide against two high mortality worldwide cancers [55], we examined the cytotoxicity effects of FGOS on human cervical cancer cell HeLa and liver cancer cell HepG2. Cell viability of HeLa and HepG2 cells exposed to different samples after 24 h is shown in Figure 6. Inulin is a natural fructan (average DP 10~30), as a prebiotic, it has excellent antioxidant and could induce dendritic cell apoptosis [56, 57]. Thus, we used inulin as positive control, and the untreated FG as negative control. As shown in Fig. 6A, FGOS treatment remarkably decreased the viability of HeLa and HepG2 cells in a dose-dependent manner. The concentration of FGOS that inhibited the growth of Hela cells and HepG2 cells for 50% viability (IC50) were approximately 0.37 and 0.51 mg/mL, respectively. Lower viability of HeLa cells than HepG2 cells was detected at the same concentration of FGOS, suggested HeLa cells were more sensitive to FGOS than HepG2 cells. It is obvious that 0.4 mg/mL FG failed to affect the proliferation of HeLa or HepG2 cells. Furthermore, both FG and FGOS displayed no toxicity to MRC-5 cells (human lung fibroblasts) when the concentration is lower than 0.4 mg/ml [58]. But the cell viability would decrease 24.8% when the FGOS concentration raised to 0.8mg/mL and the IC50 of FGOS against MRC-5 was 3.19 mg/mL, which was much higher than against the cancer cells. Considering the high viscosity of cell culture solution containing FG, it was difficult and meaningless to evaluate the cytotoxicity of FG at higher concentration. Thus, we suggest that FG had no effect on the proliferation of these two types of cancer cell. Recently studies demonstrated that oligosaccharides like chitosan oligosaccharides [59], stachyose [60] and apple oligosaccharides [61] caused inhibitory effects on the proliferation of different carcinoma cells inducing cell cycle arrest and cell apoptosis 15

via the caspase-dependent mitochondrial pathway. To further explore the mechanisms of the inhibitory effects of FGOS, cell apoptosis was assessed by flow cytometric analysis using Annexin V-PI dual staining assay. As shown in Fig. 6B, the percentage of later apoptotic cells in the upper right quadrant for Hela treated with FGOS at 0.4 mg/mL (near the IC50 values for these two cells) was increased to 21.4±1.3% in comparison with the untreated cells (1.66 ± 0.2%, P < 0.05). On the other hand, the proportion of later apoptotic HepG2 cells after FGOS treated was also increased to 12.2 ±0 .95% compared with the untreated cells (4.69 ± 0.7%, P < 0.05). Our results showed that FGOS induced later apoptosis in these two cancer cell lines, which directly caused the inhibition of cell viability. Several studies suggested that the low molecular weight oligosaccharides had a better bioactivity [62] and is a critical factor in determining the cell apoptosis potential of polysaccharides [63]. Comparing the effects of FG and FGOS on these two cancer cells, the toxicity of FGOS to cancer cells might attribute to the revealed sugar structure and small molecular size by depolymerization of FG. However, the mechanism of FGOS inhibited the proliferation of cancer cells and the effect of FGOS against tumor in vivo still need to be further verified.

4. Conclusion FGOS was successfully prepared by degradation of FG using H2O2 oxidation, followed by cellulose hydrolysis. The average molecular weight of FGOS was about 1.6 kDa, which consists of mannose, galactose, glucose, arabinose, glucuronic acid, xylose, rhamnose, ribose, galacturonic acid. FGOS exhibited better antioxidant activities than FG on scavenging hydroxyl, ABTS and DPPH radicals. Furthermore, FGOS displayed significant, dose-dependent activities against HepG2 and HeLa cancer cells. Our study suggests that FGOS could potentially be used as an antioxidant and anti-tumor drug candidate in food and biomedical industry. However, the structure of FGOS and its functional mechanism against tumor in vivo need to be further explored.

Acknowledgments 16

This work was supported by the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Agricultural Sciences (1610172019009)，the Earmarked Fund for China Agriculture Research System (CARS-14), the Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2013-OCRI) and the National Natural Science Foundation of China (31701560). References [1] K. Prasad, A. Dhar, Flaxseed and Diabetes, Curr Pharm Design 22(2) (2016) 141-144. [2] S. Nybroe, A. Astrup, C.R. Bjornvad, Dietary supplementation with flaxseed mucilage alone or in combination with calcium in dogs: effects on apparent digestibility of fat and energy and fecal characteristics, Int J Obesity 40(12) (2016) 1884-1890. [3] J. Warrand, P. Michaud, L. Picton, G. Muller, B. Courtois, R. Ralainirina, J. Courtois, Structural investigations of the neutral polysaccharide of Linum usitatissimum L. seeds mucilage, Int J Biol Macromol 35(3-4) (2005) 121-125. [4] L. Qin, S.Y. Xu, W.B. Zhang, Effect of enzymatic hydrolysis on the yield of cloudy carrot juice and the effects of hydrocolloids on color and cloud stability during ambient storage, J Sci Food Agr 85(3) (2005) 505-512. [5] J. Warrand, P. Michaud, L. Picton, G. Muller, B. Courtois, R. Ralainirina, J. Courtois, Large-scale purification of water-soluble polysaccharides from flaxseed mucilage, and isolation of a new anionic polymer, Chromatographia 58(5-6) (2003) 331-335. [6] Z.S. Zhang, X.M. Wang, X.F. Mo, H.M. Qi, Degradation and the antioxidant activity of polysaccharide from Enteromorpha linza, Carbohyd Polym 92(2) (2013) 2084-2087. [7] S.I. Mussatto, I.M. Mancilha, Non-digestible oligosaccharides: A review, Carbohyd Polym 68(3) (2007) 587-597. [8] J. Courtois, Oligosaccharides from land plants and algae: production and applications in therapeutics and biotechnology, Curr Opin Microbiol 12(3) (2009) 261-73. [9] V. Bali, P.S. Panesar, M.B. Bera, R. Panesar, Fructo-oligosaccharides: Production, Purification and Potential Applications, Crit Rev Food Sci Nutr 55(11) (2015) 1475-90. [10] L.E. Rasmussen, A.S. Meyer, Endogeneous beta-D-xylosidase and alpha-L-arabinofuranosidase activity in flax seed mucilage, Biotechnol Lett 32(12) (2010) 1883-1891. [11] K. Guilloux, I. Gaillard, J. Courtois, B. Courtois, E. Petit, Production of Arabinoxylan-oligosaccharides from Flaxseed (Linum usitatissimum), J Agr Food Chem 57(23) (2009) 11308-11313. [12] S. Liang, W.Z. Liao, X. Ma, X.F. Li, Y. Wang, H2O2 oxidative preparation, characterization and antiradical activity of a novel oligosaccharide derived from flaxseed gum, Food Chem 230 (2017) 135144. [13] S. Dygert, L.H. Li, D. Florida, J.A. Thoma, Determination of reducing sugar with improved precision, Anal Biochem 13(3) (1965) 367-74. [14] G.K. Dubois M, Hamilton J, Rebers P, Smith F, Colorimetric method for determination of sugars and related substances, Anal Chem (28) (1956) 350-356. [15] H.Q. Sun, W.W. Song, L.J. Zhang, X.Y. Yang, Z.Y. Zhu, R.C. Ma, D.Y. Wang, Structural characterization 17

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Tables and Figures 20

Table 1. The variables investigated and their levels. Table 2. Orthogonal experiments arrangements and results. *Kn is the sum RSR values of the corresponding level n under each factor; R = Kmax/3 − Kmin/3, it represents the influence degree for each factor. Table 3. Monosaccharide compositions of FGOS. Figure 1. Effect of enzyme concentration (A), Reaction time (B), pH (C) and temperature

(D) on FG degradation. Figure 2. FTIR spectrum (A), 1H NMR spectrum (B) and 13C NMR spectrum (C) of FG

and FGOS Figure 3.The molecular weight for FG (A), FGOS (B) and the MALDI-TOF-MS

spectrum of FGOS (C). Figure 4. HPLC chromatogram of monosaccharide compositions for monosaccharide

standards (A) and FGOS (B). Figure 5．Antiradical activity results (A) hydroxyl radical scavenging, (B) DPPH

radical scavenging, (C) ABTS radical scavenging. Figure 6. Cytotoxicity of FG, inulin and FGOS against three cells（A）, Flow

cytometric analysis of FGOS on HepG2 and Hela cells (B).

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