Comparison Study on Polysaccharide Fractions from Laminaria

Oct 12, 2017 - Our previous study has suggested that the crude polysaccharide obtained from Laminaria japonica by acid assisted extraction (LP-A) have...
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Cite This: J. Agric. Food Chem. 2017, 65, 9790-9798

Comparison Study on Polysaccharide Fractions from Laminaria japonica: Structural Characterization and Bile Acid Binding Capacity Jie Gao,†,§ Lianzhu Lin,†,§ Baoguo Sun,‡ and Mouming Zhao*,†,‡,§ †

School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, P. R. China Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, P. R. China § Guangdong Food Green Processing and Nutrition Regulation Technologies Research Center, Guangzhou 510640, P. R. China ‡

ABSTRACT: Our previous study has suggested that the crude polysaccharide obtained from Laminaria japonica by acid assisted extraction (LP-A) have significant bile acid-binding capacity, which probably ascribed to its specific structure characterization. The relationship between structure characterization and bile acid-binding capacity of the purified LP-A fractions are still unknown. This paper conducted a comparison study on the structure characterization and bile acid-binding capacity of three LPA fractions (LP-A4, LP-A6, and LP-A8). The results indicated that LP-A4, LP-A6, and LP-A8, characterized as mannoglucan, fucomannoglucan, and fucogalactan, had significantly different structure characterization. Furthermore, the bile acid-binding capacity of LP-A8 was obviously higher than the other fractions, which may be attributed to its highly branched structure, abundant sulfate, fucose, and galactose in chemical composition and denser interconnected macromolecule network in molecular morphology. This study provides scientific evidence for the potential utilization of LP-A8 as an attractive functional food supplement candidate for the hyperlipidemia population. KEYWORDS: Laminaria japonica polysaccharides, fractions, structural characterization, atomic force microscopy, glycosidic linkage, bile acid-binding capacity

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INTRODUCTION Laminaria japonica is the most widely cultivated and consumed commercial edible brown seaweed around the world. Recently, the polysaccharides from L. japonica (LP) have attracted considerable attention due to their important biological properties and strongly diversified structure characterization. Alginic acid, fucoidan, and laminarin are believed to be the main water-soluble polysaccharides in L. japonica; their contents generally vary from 40% to 80% of dry weight of defatted algal biomass.1−3 Alginates are cell wall polysaccharides of L. japonica, which consist of alternating units of mannuronic and guluronic acids.4 Laminarans, a low molecular weight α-/β-glucans (5−10 kDa) with a degree of polymerization between 20 and 25, are the carbohydrate reserve of L. japonica.5 Fucoidans, containing substantial percentages of α-Lfucose and sulfate ester groups, are special sulfated polysaccharides with molecular weights ranging from 100 to 1600 kDa in the cell wall of L. japonica.6 Numerous published studies have demonstrated that alginic acid, fucoidan, and laminarin have significant biological properties, such as immunoregulation, hypolipidemic, antioxidant, anticancer, antiallergic, anticoagulant, and antithrombotic activities, which could be closely linked with their molecular weights distribution, sulfate content, and monosaccharide composition.7−13 However, there was limited information about the links between structural characteristics and bioactivity of LP. It is well-known that polysaccharide or dietary fiber can significantly reduce the blood lipid of hyperlipidemia patients because of its high bile acid-binding capacity. The explanation could be that the polysaccharides can bind bile acids and © 2017 American Chemical Society

enhance their intestinal elimination, consequently stimulate the conversion of cholesterol to bile acids in liver, leading to the decreased risk of cardiovascular diseases due to the reduced levels of total cholesterol and low-density lipoprotein (LDL) cholesterol.14,15 In earlier work, we found that the crude polysaccharide obtained from L. japonica by acid assisted extraction (LP-A) have significant bile acid-binding capacity, which probably ascribed to its specific structure characterization.16 The relationship between structure characterization and bile acidbinding capacity of the purified LP-A fractions are still unknown. This paper conducted a comparison study on the chemical composition, molecular weight distribution, molecular morphology, glycosidic linkage, repeating units, and bile acidbinding capacity of three highly purified LP-A fractions (LP-A4, LP-A6, and LP-A8). The results may be available as a scientific foundation for its application in functional foods.

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MATERIALS AND METHODS

Materials. L. japonica, harvested in July 2016 from Weihai (Shandong, China), was first washed with running tap water and deionized water, and then dried in an oven at 60 °C. The dried samples were further pulverized through a 50 mesh screen. Monosaccharide standards, cholic acid, taurocholic acid, and glycocholic acid were purchased from Sigma−Aldrich (St. Louis, MO, U.S.A.). Dextrans with different molecular weights were Received: Revised: Accepted: Published: 9790

August 29, 2017 October 11, 2017 October 12, 2017 October 12, 2017 DOI: 10.1021/acs.jafc.7b04033 J. Agric. Food Chem. 2017, 65, 9790−9798

Article

Journal of Agricultural and Food Chemistry

The methylated polysaccharide was hydrolyzed by 3 mL of formic acid (88%, v/v) at 100 °C for 6 h and further hydrolyzed in 3 mL of 2 mol/L trichloroacetic acid at 100 °C for 3 h. After being reduced with NaBH4 at 60 °C for 1 h, monosaccharides were converted into the alditol acetates by reacting with pyridine and acetic anhydride at 100 °C for 1 h. A TRACE DSQ GC-MS system (Thermo Fisher Scientific, U.S.A.) equipped with a TG-5SILMS capillary column (30 m × 0.25 mm, 0.25 μm, Thermo Fisher Scientific, U.S.A.) was used to analyze the glycosidic linkage. The initial temperature of column was 150 °C, increased to 180 °C at 10 °C/min, then to 220 °C at 2 °C/min, and increased to 240 °C at 5 °C/min, holding for 5 min; injection temperature: 230 °C. The ion source of mass spectrometer was set at 240 °C. The resulting peaks of alditol acetates were identified by their MS fragmentation patterns and the relative retention time in GC spectrum. Their relative amount was estimated as ratios of peak areas. Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR spectroscopy is a convenient and efficient method to obtain more valuable information about the structure and properties of polysaccharides. Polysaccharide was dissolved in 0.5 mL of D2O to a final concentration of 6% (w/v). The 1D and 2D NMR spectra, including 1H NMR, 13C NMR, H/H COSY, and HSQC were recorded on a Bruker DRX-500 spectrometer (Bruker, Rheinstetten, Germany) at 25 °C. Chemical shift was expressed in ppm.27 Molecular Morphology Observation. Atomic force microscopy (AFM) was applied to observe the molecular morphology of L. japonica polysaccharide. After the complete dissolution, 5 μL of 10 μg/ mL sample solution was deposited onto freshly cleaved mica surfaces. After air-drying for 4 h, the sample was examined using a Bruker Multimode 8 atomic force microscopy (Bruker Corporation, Germany). The AFM image was obtained with the resolution of 256 × 256 point.28 Bile Acid-Binding Capacity Assay. According to our previous study, 10 mg samples (cholestyramine as the positive control) were dissolved in 2 mL of 0.01 mol/L HCl to simulate gastric condition, and then incubated at 37 °C for 60 min with continuous shaking. After adjusting to pH 7.0 with 2 mL of 0.01 mol/L NaOH, the solution was mixed with 4 mL of 1% pancreatin and 4 mL of 500 μmol/L bile acid stock solution (prepared by 0.05 mol/L phosphates buffer, pH 7.0) so as to simulate the intestinal condition, subsequently incubated at 37 °C for 60 min with continuous shaking.16 The digested samples were transferred into dialysis membranes (Mw 12 000−14 000). These types of artificial intestines were placed in a flask containing 100 mL of deionized water and a plastic ball. To simulate human intestinal peristalsis, the flasks were shaken (90 rpm) at 37 °C for 3 h. After 3 h of digestion, an 800 μL aliquot of digested samples was taken and heated up to 95 °C for 5 min to inactivate the enzymes. To purify the bound bile acid, a solid-phase extraction was performed using RP-18 cartridges (Oasis HLB Plus LP Extraction Cartridges, Waters Corporation, U.S.A.). Bile acid was eluted from the cartridge by methanol and detected with high-performance liquid chromatography equipped with evaporative light-scattering mass detector (HPLC-ELSD). Chromatography was performed on a Nova-Park C18 steel column (5 μm × 250 mm × 4.6 mm, Waters) at room temperature. The mobile phase, methanol−water-glacial acetic acid (80:20:0.01, v/v/v), was delivered at a flow rate of 0.8 mL/min. The ELSD 2000ES (Alltech, U.S.A.) detector was performed at a nitrogen carrier gas flow of 2.0 L/min and drift tube temperature of 105 °C.29−31 Statistical Analysis. The data were presented as mean ± SD (n = 3) and evaluated by one-way analysis of variance (ANOVA) followed by the Duncan’s test. Differences were considered to be statistically significant difference if P < 0.05. All statistical analyses were carried out using SPSS for Windows, Version 17.0 (SPSS Inc., Chicago, IL, U.S.A.).

purchased from the National Institute for Food and Drug Control (Beijing, China). All the other reagents used in the present study were of analytical grade. Extraction and Purification of L. Japonica Polysaccharide. In this study, acid assisted extraction (AAE) of L. japonica polysaccharides was performed according to the previously reported methods with some modifications.17 The dried L. japonica powder (100 g) was immersed in ethanol (95%, v/v) reflux extraction for 3 h to defat. The residues were dried and subsequently extracted with 0.1 mol/L HCl (4000 mL) at 60 °C for 3 h. After extraction, the pH of the solution was adjusted to 7.0 with 1 mol/L NaOH. Then the extracting solution was filtered through a 200-mesh nylon filter cloth. The filtrate was centrifuged at 2000 × g for 15 min and the supernatant was concentrated to 1/5 of the initial volume, and then precipitated with 80% (v/v) ethanol at 4 °C for 24 h. After centrifugation, the precipitates were collected and washed with ethanol, acetone and ether by turns to remove lipids and pigments completely, followed by deproteinization with Sevage reagent (CHCl3/BuOH = 4:1, v/v) for 5 times.18 After dialyzing against distilled water for 72 h and freezedrying, the L. japonica polysaccharide (LP-A) was obtained. Ion-exchange chromatography and gel filtration column chromatography were used for the purification of the crude LP-A and this process was monitored by the phenol-sulfuric acid method.19 Each sample (160 mg) was dissolved in distilled water and then centrifuged. The supernatant was injected to a column of DEAE-Sepharose Fast Flow (2.6 × 40 cm2), eluting with a linear gradient solution of aqueous NaCl (0−1.0 mol/L, dissolved in 0.02 mol/L Tris-HCl buffer, pH = 7.6) at a flow rate of 0.8 mL/min. As a result, three LP-A fractions were obtained, and then dialyzed, concentrated, centrifuged, and loaded onto a Sephacryl S-400 column (1.6 × 100 cm2, GE Healthcare). The column was washed with distilled water at a rate of 0.2 mL/min. Each eluting peak was collected, concentrated, and lyophilized to give the highly purified polysaccharide named LP-A4, LP-A6, and LP-A8, which were used in the subsequent studies on its structure and bile acid-binding capacity.20,21 Determination of the Structural Characterization. The polysaccharide content was measured by phenole-sulfuric acid method.19 The uronic acid content was determined by sulfuric acidcarbazole method, and mannuronic acid (ManA) was used to make a standard curve.22 The sulfate content was determined turbidimetrically after hydrolysis of the samples with 1 mol/L HCl and addition of gelatin/BaCl2 solution.23 The monosaccharide composition was analyzed according to the method in our previous reports.16 The monosaccharide standards consisted of D-mannose, L-rhamnose, Dglucose, D-arabinose, L-fucose, D-galactose, D-Mannuronic acid, DGlucuronic acid and D-Galacturonic acid. The average molecular weight was identified by high-performance gel permeation chromatography.24 A Shimadzu LC-20A liquid chromatography instrument (Zhimadzu Corporation, Kyoto, Japan) equipped with a refractive index detector was used. Tandemly linked G6000PWXL and G3000PWXL columns (Tosoh Bioscience, Stuttgart, Germany) were used for separation of polysaccharides. Dextran standards with molecular weights of 5.22−2990 kDa were applied for calibration. The molecular weight was calculated by GPC software (Zhimadzu Corporation, Kyoto, Japan). FT-IR Spectra Analysis. The FT-IR spectra of the sample was recorded using a Bruker VERTEX 33 spectrometer (Bruker Corporation, Germany) with a scanning range from 4000 to 400 cm−1. Prior to measurement, the sample was ground with KBr powder and pressed into pellets according to KBr disk method.25 Glycosidic Linkage Analysis. Methylation analysis of polysaccharide was performed according to the method of Bradbury with minor modifications.26 The dried sample (10 mg) was dissolved in 5 mL of anhydrous dimethyl sulfoxide (DMSO). After immediately addition of NaOH powder (100 mg), CH3I (2 mL) was added slowly into the solution. The methylation was repeated until the polysaccharide was methylated completely. The completion of methylated polysaccharide was confirmed by the disappearance of the OH band (3200−3700 cm−1) in the IR spectrum.

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RESULTS AND DISCUSSION Purification and Structural Characterization. The results of the purification and structural characterizations of LP-A fractions are shown in Figure 1 and Table 1. After

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DOI: 10.1021/acs.jafc.7b04033 J. Agric. Food Chem. 2017, 65, 9790−9798

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Figure 1. Purification and molecular weight distribution of L. japonica polysaccharide. (A) Stepwise elution curve of crude LPs on a DEAE-52 column; (B) elution profile of three LP fractions on a Sephacryl S-400 column, and (C) HPGPC profile of three LP fractions on tandemly linked TSK-GEL G6000 and G3000 PWXL column.

Table 1. Structural Characterization of the Main Three Fractions from L. japonica Polysaccharidea

Data from three separate experiments are expressed as the mean ± SD (n = 3). Values with different superscripts are significantly different (ANOVA, Duncan’s test), p < 0.05. a

content of sulfate (2.31 ± 0.33%). On the contrary, LP-A8 had the lowest content of uronic acid (25.25 ± 0.55%) and the highest content of sulfate (29.06 ± 1.50%). The uronic acid and sulfate content of 59.26 ± 0.78% and 13.05 ± 0.11% was observed for LP-A6. Significant differences could be observed in the monosaccharides composition among these LP-A fractions. LP-A4 could be characterized as mannoglucan because it predominantly gave mannuronic, glucuronic, and mannose after hydrolysis (62.11%, 19.56% and 12.92% of total monosaccharides, respectively). LP-A6, characterized as fucomannoglucan, contained considerable amount of fucose, mannuronic and

purification with DEAE-Sepharose Fast Flow column and Sephacryl S-400 column chromatograph, the acid polysaccharide fractions (LP-A4, LP-A6, and LP-A8) were obtained. No significant absorption band was observed at 260 or 280 nm in the UV spectrum, indicating that these LP-A fractions contained no protein or nucleic acid (data not shown). The high purity of these fractions has been proved by HPLC analysis in Figure 1C. The molecular weight of LP-A4, LP-A6, and LP-A8 were calculated as 5.55 × 105 Da, 12.03 × 105 Da, and 9.78 × 105 Da, respectively. Compared with the other fractions, LP-A4 contained the highest content of uronic acid (89.64 ± 2.27%) and the lowest 9792

DOI: 10.1021/acs.jafc.7b04033 J. Agric. Food Chem. 2017, 65, 9790−9798

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Figure 2. FT-IR spectra analysis of three LP fractions (LP-A4, LP-A6, and LP-A8).

Table 2. Methylation Analysis and Mode of Linkage of LP Fractions relative molar ratio (%) retention time (min)

methylated sugar residues

linkages types

LP-A4

LP-A6

LP-A8

major mass fragment (m/z)

13.98 14.05 16.27 17.74 18.6 19.2 20.46 21.46

2,4-Me2-Fuc 2,3-Me2-Fuc 3,4-Me2-xyl 2,3-Me2-Man 2-Me1-Glc 2,3,4-Me3-Gal 3-Me1-Man 0-Me0-Man

→3)Fucp(1→ →4)Fucp(1→ →2)xylp(1→ →4)Manp(1→ →3,4)Glcp(1→ →6)Galp(1→ →2,4)Manp(1→ →2,3,4)Manp(1→

0 0 0 78.27 15.61 0 6.12 0

22.01 17.02 8.41 33.43 19.13 0 0 0

32.44 28.03 0 0 9.02 17.70 0 12.81

43,87,96,101,117,145,205 43,87,96,101,117,159,174,234 43,69,87,101,129,189,217,281 43,45,71,87,117,129,189,207,330 43,60,72,96,114,127,156,174,207,229,398 43,87,101,113,117,129,161,233,355 43,73,87,115,129,140,182,211 43,97,103,114,139,145,187,259,289,341

the bands around 820 and 850 cm−1 of LP-A4, LP-A6, and LPA8 was probably ascribed to their different sulfate group location in the structure of the polysaccharide chain. The band at 1035 cm−1 was assigned to C−O−C stretching vibration, which referred to the “finger print” region of polysaccharides and allowed the identification of specific chemical groups in every LP-A fractions. Glycosidic Linkage Analysis. Glycosidic linkage analysis of LP-A fractions was summarized in Table 2. The LP-A fractions were methylated and hydrolyzed to form alditol acetate derivatives, which were analyzed by GC-MS and identified by the retention time and characteristic iron fragments in MS spectra.37 It was possible that the uronic acid in LP-A fractions would be transformed into methyl esters and subsequently degraded by β-elimination under highly alkaline environments in the repeated methylation of Needs’ method. Hence the signal of uronic acid was not shown in GC−MS analysis.38 As summarized in Table 2, (1 → 4) linked mannopyranose (78.27%) was the largest residual amount of the backbone structure of LP-A4 with 15.61% of (1 → 3, 4) linked glucopyranose and 6.12% of (1 → 2, 4) linked mannopyranose as branched residues. The majority of the sugar residues in LPA6 were (1 → 4) linked mannopyranose (33.43%), (1 → 3) linked fucopyranose (22.01%) and (1 → 4) linked fucopyranose (17.02%); and the branched residue was (1 → 3, 4) linked glucopyranose (19.12%). LP-A6 showed the presence of 3 major residues, namely (1 → 3) linked fucopyranose (32.44%), (1 → 4) linked fucopyranose (28.03%) and (1 →

glucuronic (44.16%, 26.19% and 15.53% of total monosaccharides, respectively). LP-A8 was mainly composed of 57.88% of fucose and 21.79% of galactose, which could be identified as fucogalactan. Previous studies have suggested that polysaccharide from Laminaria japonica contained predominantly fucose, galactose, mannose, glucose, and sulfate group, which was very similar to the results of our present study.32,33 FT-IR Spectra Analysis. The FT-IR spectra of LP-A fractions were presented in Figure 2. The absorbance band at 3450 cm−1 represented the stretching vibration of O−H in the constituent sugar residues. The strong band around 2930 cm−1 was associated with stretching vibration of C−H in the sugar ring. The bands at 1635 and 1423 cm−1 were due to the stretching vibration of the deprotonated carboxylic group (COO−) and the bending vibration of C−H bond, respectively.34 A series of bands in the region 1300−500 cm−1 due to the C−O−S and SOS stretching vibrations (1258, 965, 850, 820, and 686 cm−1) for the sulfate group were observed, which is a characteristic component in fucoidans of brown seaweeds.35 Compared with LP-A4 and LP-A6, the broad asymmetric absorption bands in 1258, 965, 850, and 686 cm−1 indicated the presence of abundant sulfate groups in LP-A8, which was in accordance with their structure characterization in Table 1. The infrared spectrum showed the characteristic band of C− O−S stretching vibration at 820 and 850 cm−1 indicated that the equatorial sulfate groups located at position C-2 and the axial sulfate groups at position C-4 of fucopyranose residues.36 The infrared spectrum showed that the obvious difference in 9793

DOI: 10.1021/acs.jafc.7b04033 J. Agric. Food Chem. 2017, 65, 9790−9798

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Figure 3. 13C NMR spectrum (A, C, and E) and 1H NMR spectrum (B, D, and F) of LP-A4 (A and B), LP-A6 (C and D), and LP-A8 (E and F), respectively.

From the 1H NMR spectrum of LP-A4, the signals of anomeric protons at δ 4.74, 4.77, and 5.14 ppm were designated to H-1 of (1 → 4) linked β-D-ManpA, (1 → 2, 4) linked β-D-Manp, and (1 → 3, 4) linked α-D-GlcpA, respectively.32 The 1H NMR spectrum of LP-A6 presented five signals at δ 4.82, 5.05, 5.14, 5.21, and 5.34 ppm in the anomeric region, which originated from H-1 of (1 → 4) linked β-D-ManpA, (1 → 2) linked α-D-Xylp, (1 → 3, 4) linked α-DGlcpA, (1 → 3) linked α-L-Fucp, and (1 → 4) linked α-L-Fucp. In the 1H NMR spectra of LP-A8, signal at around δ 4.84, 5.12, 5.25, 5.29, and 5.43 ppm in the anomeric region was assigned to the H-1 of (1 → 2, 3, 4) linked β-D-ManpA, (1 → 3, 4)

6) linked galacopyranose (17.70%), with the branched residues of (1 → 2,3,4) linked mannopyranose (12.81%) and (1 → 3, 4) linked glucopyranose (9.02%). These molar ratios agree with the overall monosaccharide composition described above. The signal of the arabinose in LP-A4 and the galactose in LP-A6 was no indication owing to their low content (less than 5.5%). Generally, there were significant differences in the glycosidic linkage of the polysaccharide structure of three LP-A fractions. NMR Spectra Analysis. The spectra of 1H and 13C NMR of LP-A4, LP-A6, and LP-A8 in D2O at 20 °C are shown in Figure 3. The peak at around δ 4.70 ppm was attributed to the chemical shifts of HOD. 9794

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Journal of Agricultural and Food Chemistry Table 3. 1H and 13C NMR Chemical shift assignments of LP fractions in D2O

Figure 4. Molecular morphology of LP-A4, LP-A6, and LP-A8 was observed under atomic force microscope at the concentrations of 10 ug/mL.

linked α-D-GlcpA, (1 → 3) linked α-L-Fucp, (1 → 4) linked αL-Fucp, and (1 → 6) linked α-D-Galp. In the 13C NMR spectra, peaks in the range of δ 15−18 ppm belonged to the C-6 of fucose; and the strong peaks observed at around δ 173−176 ppm was a typical signal of uronic acid.32 On the basis of the cross peaks in 1H−1H correlation NMR spectra (COSY, not shown), the chemical shifts of H-1, H-2, H3, H-4, H-5, and H-6 of the sugar residues in LP-A fractions have been assigned and summarized in Table 3. The correlation peaks in 1H−13C correlation NMR spectra (HSQC, not shown) identified at δ 4.74/100.11, 4.77/101.42, and 5.14/ 100.24 ppm in the anomeric region of LP-A4 could be assigned to C-1 of (1 → 4) linked β-D-ManpA, (1 → 2, 4) linked β-DManp and (1 → 3, 4) linked α-D-GlcpA, respectively.39,40

Similarly, the chemical shifts of C-1, C-2, C-3, C-4, C-5, and C6 of the sugar residues in LP-A4, LP-A6, and LP-A8 were assigned completely using the correlation peaks in COSY and HSQC, as shown in Table 3. On the basis of the results of glycosidic linkage analysis and NMR spectra analysis, the possible repeating structural unit of LP-A4, LP-A6, and LP-A8 could be as shown in Figures 4 and 5. Molecular Morphology Observation. Atomic force microscopy (AFM) is a powerful technique for observing the three-dimensional structure and microcosmic surface shape of individual macromolecules, including polysaccharides.41,42 On the basis of the AFM images shown in Figure 4, the polysaccharide structure of LP-A fractions appeared with 9795

DOI: 10.1021/acs.jafc.7b04033 J. Agric. Food Chem. 2017, 65, 9790−9798

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Figure 5. (A) In vitro bile acid (cholic acid, taurocholic acid, and glycocholic acid) binding capacity of three LP fractions (LP-A4, LP-A6, and LP-A8) and cholestyramine (CT) based on dry matter (DM). (B) Predicted structure of the repeating units of three LP fractions and the schematic representation of the possible interaction between polysaccharide and bile acid during simulated digestion.

certain different, flexible, and worm-like chains with many branches. Compared with LP-A4, LP-A6, and LP-A8 appeared in an entangled and adherent macromolecule network with more branches and coiling or bending regions. Furthermore, the obvious difference observed in LP-A4 might be the result of the low content of sulfate and branches, which appeared as a flexible, curly, and thin linear structure with very few branches in the polysaccharide molecules. The mean thickness of polysaccharide chains in LP-A4, LPA6, and LP-A8 was 0.440 ± 0.04, 0.654 ± 0.01, and 0.825 ± 0.01 nm, respectively. The mean thickness of LP-A8 was significantly higher than the other fractions, suggesting the different structure of molecular aggregation of these fractions in aqueous solution. These results are similar to those obtained in carrageenan and our previous study.16,43 Bile Acid-Binding Capacity. Many studies have revealed that soluble dietary fibers or polysaccharides could reduce serum cholesterol and thus a decreased risk of cardiovascular disease (CVD).44 The presence of polysaccharide in the small intestine has been proved to prevent bile acid from being reabsorbed into the enterohepatic circulation, resulting in excess excretion in feces. This causes a depletion of bile acid in the liver and consequently cholesterol is rapidly catabolized in

the hepatocyte, thus lowering blood cholesterol concentration.44,45 Cholic acid (CA), glycocholic acid (GCA), and taurocholic acid (TCA) are the primary bile acids synthesized in the human body. As shown in Figure 5A, the bile acid-binding capacity of cholestyramine (CT) for CA and GCA was significantly higher than the LP-A fractions on an equal dry matter (DM) basis, however, the bile acid-binding capacity of LP-A8 for TCA was obviously higher than that of CT. Cholestyramine could bind 11.22 ± 0.59 μmol CA, 10.73 ± 0.56 μmol GCA, and 7.49 ± 0.56 μmol TCA per 100 mg dry matter, respectively. These values are similar to previously reported observations.46 The highest CA, GCA, and TCA binding capacity was observed in LP-A8 as 68.29%, 81.99%, and 161.72% with that of the cholestyramine resin on a same weight basis. On a dry matter basis, the bile acid-binding capacity of LP-A8 is very encouraging, which could be an indicator of its significantly higher healthful potential than that of the other fractions. The predicted structure of the repeating units of three LP-A fractions and the schematic representation of the possible interaction between polysaccharide and bile acid during simulated digestion are shown in Figure 5B. On the basis of the predicted structure of the repeating units and the AFM 9796

DOI: 10.1021/acs.jafc.7b04033 J. Agric. Food Chem. 2017, 65, 9790−9798

Article

Journal of Agricultural and Food Chemistry

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images, the schematic representation of LP-A fractions showed different molecular morphology in aqueous solution during simulated digestion. Compared with LP-A4 and LP-A6, LP-A8 appeared as a denser interconnected macromolecule network with more branches, which would easily trap the bile acid molecules. In comparasion with fucoidans from brown alga (sea cucumber or Laminaria japonica),6−10 the entangled and adherent macromolecule network in AFM images and a large amount of highly branched sugar residues were the unique structure characteristics of LP-A8. The mechanism of LP-A fraction binding bile acid might be similar to other plant polysaccharides, which might be related to its unique structural characteristics.45−47 In addition, LP-A8 with the highest bile acid-binding capacity showed the lowest content of uronic acid, highest content of sulfate, and the presence of highly branched sugar residue such as (1 → 2, 3, 4) linked β-D-ManpA, indicating the possible correlation between structural characterization and bile acidbinding capacity. Binding bile acids to polymers may eliminate the bile acid through the gastrointestinal tract, which may promote the metabolism of cholesterol in liver and consequently reduce serum cholesterol levels. The present study provides scientific evidence and advances in the potential utilization of LP-A8 as an attractive functional food supplement candidate for the hyperlipidemia population. However, further study is required to elucidate the precise molecular mechanism involved in the cholesterol-lowering capacity of LP-A using the experiment in human and animal models.

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AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: +86 20 87113914. E-mail: [email protected] (M.Z.). ORCID

Lianzhu Lin: 0000-0003-4657-1231 Mouming Zhao: 0000-0003-0221-3838 Present Address ∥

No. 381, Wushan Road, Tianhe District, Guangzhou City, Guangdong Province, China. Funding

This study was funded by the Guangzhou Science and Technology Plan Project (Project No. 20160402172). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.7b04033 J. Agric. Food Chem. 2017, 65, 9790−9798