A Novel Alkali Extractable Polysaccharide from Plantago asiatic L

Article pubs.acs.org/JAFC

A Novel Alkali Extractable Polysaccharide from Plantago asiatic L. Seeds and Its Radical-Scavenging and Bile Acid-Binding Activities Lu Gong,∥,† Hua Zhang,∥,§ Yuge Niu,*,† Lei Chen,† Jie Liu,† Sierkemideke Alaxi,† Pingping Shang,† Wenjuan Yu,# and Liangli (Lucy) Yu*,†,⊥ †

Institute of Food and Nutraceutical Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China § School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China # Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai 200240, China ⊥ Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, United States S Supporting Information *

ABSTRACT: A new acidic polysaccharide (PLP) was isolated and characterized from Plantago asiatic L. seeds by hot alkali extraction and chromatographic purification using DEAE cellulose and Sephacryl S-400 columns. PLP has a molecular weight of 1.15 × 106 Da, and a monosaccharide composition of xylose (Xyl), arabinose (Ara), glucuronic acid (GlcA), and galactose (Gal) in a molar ratio of 18.8:7.2:6.1:1. The results of methylation analysis, FT-IR, and 1D and 2D NMR indicated that PLP was a highly branched heteroxylan of β-1,4-linked Xylp backbone with three α-GlcAp-(1→3)-Araf attached to the O-3 position and one α-T-linked-GlcAp and one α-Araf-(1→5)-Araf attached to the O-2 position every eight monosaccharide residues. PLP exhibited scavenging abilities against hydroxyl, peroxyl anion, and DPPH radicals in vitro and showed significant binding capacities against cholic and chenodeoxycholic acids, suggesting its possible cholesterol-lowering activity. The results demonstrated the potential use of PLP in functional foods and nutraceuticals. KEYWORDS: Plantago asiatic L. polysaccharides, monosaccharide composition, structure analysis, bile acid-binding capacity, antioxidant

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INTRODUCTION Plantago L. (Plantaginaceae) is a perennial herb that has more than 270 species all over the world. Polysaccharides from Plantago L., such as psyllium, are important health components.1−4 Psyllium is an arabinoxylan derived from the seed husk of Plantago ovata and is a commercial dietary fiber with a bile-acid binding activity, which might be important for its cholesterol-lowering activity.5 Psyllium is mostly produced in India. In China, Plantago major, Plantago asiatic, and Plantago depressa have been used in functional foods, and P. asiatic is most widely distributed.6 The hot water extractable polysaccharides from P.asiatica L. seeds exhibited antioxidant activity in vitro7 and were shown to promote defecation.8 Polysaccharides may bind bile acids and enhance their intestinal elimination and consequently stimulate the conversion of cholesterol to bile acids in liver.9 As a result, the levels of total plasma and low-density lipoprotein (LDL) cholesterol may be reduced, which may reduce the risk of cardiovascular diseases. Additionally, polysaccharides with antioxidant capacities could suppress LDL oxidation and consequently reduce the risk of cardiovascular disease. Our previous studies showed that the physicochemical properties and bile acid-binding capacities of psyllium were closely related to its chemical and molecular structures.9 Alkaliextractable polysaccharides may have bioactivities different from the water-extractable ones because the alkali-sensitive chemical bonds between polysaccharides and cell wall of plants could be broken during extraction, leading to the release of © XXXX American Chemical Society

polysaccharide(s) with different chemical and molecular structures.10,11 To date, little is known about the alkaliextractable polysaccharides of P. asiatic L. seeds. The present study aimed to investigate the alkali-extractable P. asiatic L. seed polysaccharides. The ground seeds of P. asiatic L. were extracted with alkali and purified by column chromtographies. The chemical structures of individual polysaccharides were characterized by methylation, GC, HPLC, GC-MS, and 1D and 2D NMR spectroscopy analyses. In addition, their hydroxyl, peroxyl, and DPPH radicalscavenging capacities and bile acid-binding activity were examined. The results may serve as a scientific foundation for developing their possible application in functional foods and nutraceuticals.

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MATERIALS AND METHODS

Materials. The seeds of P. asiatic L. were collected and identified by Dr. Haibo Yin (Liaoning University of Traditional Chinese Medicine) in 2012. Diethylaminoethyl (DEAE) cellulose was purchased from Whatman International Ltd. (Kent, UK). Sephacryl S-400 was purchased from GE Healthcare (Uppsala, Sweden). Dextrans with different molecular weights and standard monosaccharaides including arabinose, glucose, mannose, glucuronic acid, galactose, rhamnose, fucose, and xylose were purchased from the National Institute for Food and Drug Control (Beijing, China). Deuterated Received: October 28, 2014 Accepted: December 23, 2014

A

DOI: 10.1021/jf505909k J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

monosaccharide standard or the hydrolyzed polysaccharide sample solution was mixed with 50 μL of 0.5 M PMP in methanol. The reaction was carried out at 70 °C for 30 min and neutralized with 100 μL of 0.3 M hydrochloric acid, followed by extraction with chloroform (1.0 mL) three times. The aqueous layer was filtered through a 0.45 μm pore membrane filter for HPLC analysis (Agilent Technologies Co. Ltd., Santa Clara, CA, USA) at 250 nm with a Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm, Agilent, Palo Alto, CA, USA). The mobile phase was composed of ammonium acetate solution (pH 5.5) and acetonitrile at a ratio of 78:22 (v/v). Infrared Spectral Analysis. The Fourier transform infrared (FTIR) spectrum was determined on a FT-IR spectrophotometer with an ATR accessory (Nicolet 6700, Thermo Fisher, Waltham, MA, USA) in the range of 4000−400 cm−1. Methylation and GC-MS Analysis. Methylation analysis of polysaccharide was performed according to the method of Needs and Selvendran with minor modifications.16 The dried PLP (10 mg) was dissolved in 5 mL of anhydrous DMSO with added NaOH powder (100 mg). CH3I (2 mL) was added slowly into the solution. The methylated polysaccharide was extracted with chloroform. The methylation was repeated until the polysaccharide was methylated completely. The completion of methylation of PLP was confirmed by the disappearance of the OH band (3200−3700 cm−1) in the IR spectrum. The methylated polysaccharide was hydrolyzed in 3 mL of formic acid (88%, v/v) at 100 °C for 6 h and further hydrolyzed in 3 mL of 2 M 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 and subjected to linkage analysis using an Agilent 7890A5975C GC-MS system (Agilent Technologies Co. Ltd., Santa Clara, CA, USA) equipped with a DB-5MS column (30 m × 0.25 mm, 0.25 μm, Agilent, Palo Alto, CA, USA). The oven temperature was from 100 to 190 °C at 20 °C/min, increased to 260 °C at 3 °C/min, and increased to 300 °C at 10 °C/min. 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. PLP (50 mg) was dissolved in 0.5 mL of D2O. The 1D and 2D NMR spectra, including 1H NMR, 13C NMR, HH COSY, HSQC, and HMBC, were recorded on a Bruker AV-500 MHz NMR spectrometer (Bruker, Rheinstetten, Germany) at 30 °C. Relative DPPH•-Scavenging Capacity (RDSC). DPPH•-scavenging capacity was determined following a previously reported laboratory protocol17 using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). Trolox was employed as an antioxidant standard. Briefly, 100 μL of PLP, Trolox standard solution, or blank was added into each well, then 100 μL of 0.092 mM DPPH• solution was added to initiate the antioxidant radical reaction. The absorbance of the reaction mixture at 515 nm was recorded every minute for 1.5 h, and each sample was tested three times in parallel. The relative scavenging capacity (RDSC) was calculated as micromoles of Trolox equivalents (TE) per gram of PLP using the area under the curve (AUC) calculation. Hydroxyl Radical-Scavenging Capacity (HOSC). A previously reported laboratory protocol18 was applied to evaluate the HOSC value of PLP using a Synergy 2 Multi-Mode Microplate Reader (BioTek). Fluorescein (FL) prepared in 75 mM sodium phosphate buffer (pH 7.4) was used as the molecular probe and Trolox as the antioxidant standard. One hundred and seventy microliters of FL solution (9.28 × 10−8 M), 30 μL of PLP, standard, or blank, 40 μL of 0.199 M hydrogen peroxide, and 60 μL of 3.43 M FeCl3 were added into each well successively to initiate the reaction. The fluorescence was recorded every minute for 6 h. The excitation wavelength was 485 nm, and emission wavelength was 528 nm. The experiment was conducted three times in parallel for each sample. The HOSC value was expressed as micromoles of TE per gram of PLP based on the AUC.

water (D2O) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). Fluorescein (FL), iron(III) chloride, 6hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), dimethyl sulfoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), cholic and chenodexycholic acids, diphorase, β-nicotinamide adenine dinucleotide, and 3-α-hydroxysterol dehydrogenase were purchased from Sigma-Aldrich (St. Louis, MO, USA). myo-Inositol, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), nitro blue tetrazolium (NBT), and 3-methyl-1-phenyl-2-pyrazolin-5-one (PMP) were purchased from J&K Scientific (Beijing, China). Acetic anhydride, pyridine, and sodium borohydride were purchased from Sinopharm (Beijing, China). Other reagents for isolation were of analytical grade without further purification. Extraction, Isolation, and Purification of Polysaccharide (PLP). The dried seeds of P. asiatica L. (100 g) were ground into powder and then defatted by ethanol (95%, v/v) extraction for 9 h. The residues were extracted by hot water (1:30, m/v) three times at 80 °C for 2 h each time. After filtering, the residues were extracted by 0.5 M NaOH (1:25, m/v) twice at 80 °C for 2 h each time. The combined supernatants were neutralized by 37.5% hydrochloric acid and dialyzed with 3500 Da molecular weight cutoff membrane against distilled water for 72 h. The solution was deproteinized according to a Sevag method.12 Fifteen milliliters of 30% hydrogen peroxide was added to 100 mL of solution to remove the pigment at 60 °C for 6 h. The resulting aqueous solution was collected after centrifugation at 4500 rpm for 10 min and then precipitated using 4-fold volumes of ethanol (95%, v/v). The precipitates were collected by centrifugation (4500 rpm × 10 min), rinsed with anhydrous ethanol, acetone, and ether, and dried with nitrogen (2 L/min, room temperature) to yield crude polysaccharides. Three milliliters of crude polysaccharide solution (100 mg/mL) was applied to a DEAE column (2.5 cm × 30 cm, Whatman International Ltd.) and eluted first with distilled water at a rate of 0.2 mL/min for 24 h, followed by 0.62 M NaCl solution at the same rate. Fractions were combined according to the result of phenol−sulfuric acid determination. The eluent was concentrated, centrifuged, and loaded onto a Sephacryl S-400 column (1.6 cm × 100 cm, 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 pure polysaccharide named PLP. Purity and Molecular Weight Determination. The purity and molecular weight of PLP were determined using a high-performance size exclusion chromatograph (Agilent, Santa Clara, CA, USA) equipped with a gel filtration column (Shodex SUGAR KS-805, 8 mm i.d. × 300 mm, Showa Denko, Japan) and a refractive index detector. Deionized water as the mobile phase eluted at a flow rate of 1.0 mL/min. The molecular weight of PLP was calculated by the standard curve prepared with a series of dextran standards with different molecular weights (180, 2700, 5250, 9750, 13050, 36800, 64650, 135,350, 300,600, and 2,000,000 Da, respectively). Monosaccharide Composition Analysis. PLP (5 mg) was dissolved in 1 mL of 2 M trifluoroacetic acid (TFA) and kept at 100 °C for 8 h. After being completely hydrolyzed, the excess TFA was removed by codistillation with the added 0.1% HCl−methanol. The residual was dissolved in 1 mL of distilled water for further derivatization.13 The hydrolysate was applied to monosaccharide composition analysis using a GC method.14 Briefly, monosaccharide standards including arabinose, glucose, mannose, glucuronic acid, galactose, rhamnose, fucose, and xylose and the hydrolyzed polysaccharide samples were reduced by NaBH4 at 65 °C for 30 min, followed by incubation with acetic anhydride and pyridine at 100 °C for 2 h. The resulting alditol acetates were applied to a GC (Agilent) equipped with a flame ionization detector and a HP-5 capillary column (30 m × 0.32 mm, 0.25 μm). The oven temperature was raised from 110 to 220 °C at 5 °C/min, held for 2 min, increased to 240 °C at 2 °C/min, held for 2 min, and finally increased to 280 °C at 10 °C/min. Precolumn derivatization with PMP was used for the identification and quantification of the monosaccharides including uronic acid.15 After mixing with 50 μL of 0.6 M sodium hydroxide, 50 μL of B


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Figure 1. GC chromatograms: standard monosaccharides (A) and monosaccharide composition of PLP (B). Peaks: 1, rhamnose; 2, arabinose; 3, fucose; 4, xylose; 5, inositol; 6, mannose; 7, glucose; 8, galactose. HPLC chromatograms of PMP derivatives: standard monosaccharaides (C) and monosaccharide composition of PLP (D). Peaks: 1, mannose; 2, rhamnose; 3, glucuronic acid; 4, glucose; 5, galactose; 6, xylose and arabinose; 7, fucose. protocol.19 Fluorescein (FL) was used as the fluorescent probe with Trolox as the antioxidant standard. In brief, 30 μL of PLP, standard, or

Oxygen Radical Absorbance Capacity (ORAC). The ORAC assay was conducted according to a previously described laboratory C


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Figure 2. FT-IR spectrum of PLP.

Table 1. Methylation Analysis of PLP retention time (min)

methylated sugar

11.827 12.203 12.291 13.181 13.306 13.344 24.027

2,5-Me2-Araf 2,3 -Me2-Araf 2,3,5-Me3-Araf 3-Me-Xylp 2,3-Me2-Xylp 2-Me-Xylp 2,3,4,6-Me4-GluAp

mass fragments(m/z) 43, 43, 43, 43, 43, 43, 43,

58, 87, 45, 87, 87, 85, 71,

87, 99, 113, 117, 129, 159, 233 101, 117, 129, 189 71, 87, 101, 117, 129, 145, 161 99, 126, 129, 189 101, 117, 129, 161, 189, 233 101, 117, 127, 159, 201, 233 101, 117, 129, 161, 203

blank was added into 225 μL of 81.63 nM FL solution. After 20 min of preheating at 37 °C in a Synergy 2 Multi-Mode Microplate Reader (BioTek), 25 μL of 0.36 M AAPH was successively added into each well to initiate the reaction. The fluorescence was measured every minute for 2 h at 37 °C. The excitation wavelength was 485 nm, and emission wavelength was 535 nm. The ORAC value was calculated on the basis of AUC and reported as micromoles of TE per gram of PLP. Bile Acid-Binding Capacity Assay. According to a previous laboratory protocol,20 25 mg of each sample was dissolved in 0.25 mL of 0.01 mol/L HCl to simulate gastric condition and then incubated at 37 °C for 60 min with continuous shaking. The solution was adjusted to pH 7.0 by adding 25 μL of 0.1 mol/L NaOH and mixed with 1.25 mL of bile acid stock solution (400 μM in 0.01 M phosphates buffer, pH 7.0) to simulate the intestinal condition. The mixture was incubated for another 60 min at 37 °C, followed by centrifugation at 4500 rpm for 15 min. The supernatant was collected to quantify the unbound bile acid. One hundred microliters of each sample or bile acid standard was mixed with 125 μL of 1.22 mmol/L nicotinamide adenine dinucleotide, 5 mmol/L NBT, 100 μL of diphorase (625 units/L), and 100 μL of 3-α-hydroxysterol dehydrogenase solution (625 units/L). After incubation at ambient temperature for 1 h, 100 μL of 1.33 mmol/L phosphoric acid was added to stop the reaction. The absorbance of each mixture was determined at 530 nm with cholestyramine resin as a positive control and the phosphate buffer without bile acid as the reagent blank. The bile acid-binding capacity (mg/g sample) was calculated according to the standard curves of

molar ratio

linkage type

0.62 0.25 0.22 0.44 1.85 0.66 1

1,3-linked Araf 1,5-linked Araf 1-linked Araf 1,2,4-linked Xylp 1,4-linked Xylp 1,3,4-linked Xylp 1-linked GluAp

cholic and chenodeoxycholic acids, respectively. Triplicate tests were conducted. Statistical Analysis. Data were expressed as the mean ± standard deviation (SD) for each assay. Differences between means were determined by the analysis of one-way ANOVA and Tukey’s test, using SPSS (SPSS for Windows, version rel. 16.0, SPSS Inc., Chicago, IL, USA). Statistical significance was declared at P < 0.05.

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RESULTS AND DISCUSSION Isolation, Purification, and Molecular Weight of PLP. The crude polysaccharides were obtained from the dry seeds of P. asiatic L. by hot alkaline extraction and ethanol precipitation with a yield of approximately 5.45% (w/w). After purification with an ion exchange column (DEAE-32), the alkali-extractable polysaccharide PLP was further purified using a Sephacryl S400 column chromatograph. No significant absorption band was observed at 260 or 280 nm in the UV spectrum, indicating that PLP contained no protein or nucleic acid (data not shown). PLP showed a single, sharp, and symmetric peak by HPLC analysis, and its purity was calculated as 99.2% (data not shown). The molecular weight of PLP was calculated 1.15 × 106 Da using a dextran standard curve. Monosaccharide Composition. The result of GC analysis showed that the monosaccharide composition of PLP was D


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Figure 3. continued

E


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Figure 3. 13C (A) and 1H (B) NMR spectra of PLP; (C) 1H/1H COSY spectrum; (D) HSQC spectrum (anomeric region); (E) HMBC spectrum (anomeric region).

Table 2. 13C and 1H Chemical Shifts of PLP chemical shifts, δ sugar residue

C1/H1

C2/H2

C3/H3

C4/H4

C5/H5

H5′

(A) α-1,3-linked Araf (B) α-1,5-linked Araf (C) α-T-linked Araf (D) α-T-linked GlcAp (E) β-1,3,4-linked Xylp (F) β-1,2,4-linked Xylp (G) β-1,4-linked Xylp

107.90/5.32 104.75/5.18 105.87/5.16 98.47/5.04 102.50/4.79 101.41/4.55 100.85/4.46

79.41/4.41 80.12/4.11 79.41/4.31 70.82/3.62 75.26/3.46 78.91/3.56 72.56/3.28

83.44/3.99 76.66/3.92 83.51/3.98 72.22/3.74 72.22/3.79 78.92/3.73 75.26/3.44

81.88/4.46 84.54/3.64 81.95/4.39 84.57/4.22 72.43/3.62 80.12/4.22 70.42/3.62

60.80/3.83 64.63/4.00 60.94/3.76 76.63/3.93 68.90/3.44 61.04/3.83 64.74/3.99

3.79 3.31

C6

176 3.28 3.77 3.33

measure the possible uronic acid and glucose contents of PLP. The peaks of PMP-labeled monosaccharides in Figure 1D showed the presence of xylose, arabinose, glucuronic acid, and galactose without glucose. Taken together, these data indicated no presence of glucose in the alkali extractable polysaccharide

xylose, arabinose, glucose, and galactose at a molar ratio of 18.8:7.2:6.1:1 according to peak areas and response factors (Figure 1A,B). Considering that glucuronic acid could be reduced to glucose by NaBH4 during acetylation and measured as glucose, a PMP derivatization analysis was further used to F


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Journal of Agricultural and Food Chemistry Table 3. Significant Connectivities Observed in the HMBC Spectrum for the Anomeric Protons/Carbons of the Sugar Residues of PLP observed connectivities residue A

sugar linkage 1,3-linked Araf

H 1/C 1 δH/δC

δH/δC

residue

atom

107.90

72.22 83.44 81.88

E A A

C3 C3 C4

5.32

B

1,5-linked Araf

5.18 104.75

78.91 84.54

F B

C2 C4

C

T-linked Araf

5.16 105.87

60.94 81.95

B C

C5 C4

D

T-linked GlcAp

5.04 98.47

78.91 83.44

F A

C2 C3

E

1,3,4-linked Xylp

4.79 102.50

3.62

E

H4

F

1,2,4-linked Xylp

4.55 101.41

4.22

F

H4

G

1,4-linked Xylp

4.46

72.43 80.12 72.56

E F G

C4 C4 C2

100.85

Figure 5. Free radical-scavenging activities of PLP. Data were expressed as Trolox equivalent (TE) in μmol TE/g. Free radicalscavenging activities were calculated using the area under the curve (AUC) against the Trolox standard. Vertical bars represent SD.

from P. asiatic L. seeds. The monosaccharide composition of PLP was xylose, arabinose, glucuronic acid, and galactose at a molar ratio of 18.8:7.2:6.1:1. It contained no protein or nucleic acid. The content of uronic acid was about 18.4%. Structural Characterization of PLP. The FT-IR spectrum of PLP is shown in Figure 2. The assignments of functional groups were conducted according to previous literature.14,21,22 The band centered at 3312 cm−1 arose from OH stretching vibrations of hydroxyl groups. The strong band at 2923 cm−1 was characteristic of C−H stretching vibrations of a CH2 group. Moderate IR bands at 1600 and 1416 cm−1 arose from asymmetric and symmetric stretching vibrations of carboxylate generated by the reaction of KBr and carboxylic acid. They implied the presence of carboxylic acid, supporting the presence of glucuronic acid suggested by the result of PMP derivatization analysis. Strong overlapped IR bands at 900−1200 cm−1 corresponded to coupled C−O, C−C stretching, and C−OH bending vibrations. An IR band at 899 cm−1 was characteristic of β-anomeric configuration. Methylation followed by GC-MS analysis was employed to obtain more structural information on PLP. Demonstrated by the FT-IR spectrum, PLP was methylated completely and then hydrolyzed under an acidic condition to form partially methylated alditol acetates followed by GC-MS analysis.

Figure 6. Bile acid-binding capacity of PLP. CA and CDCA represent cholic and chenodeoxycholic acids, respectively. Resin means cholestyramine resin, which is the positive control. Vertical bars represent the SD. ∗ represents significant differences (P < 0.05).

According to the results in Table 1, seven homogeneous peaks were observed by GC analysis. According to their retention time, the peaks were identified as 2,5-di-Me-arabitol, 2,3-di-Me-arabitol, 2,3,5-tri-Me-arabitol, 3-Me-xylitol, 2,3-diMe-xylitol, 2-Me-xylitol, and 2,3,4,6-tetra-Me-gluctiol with a molar ratio of 0.62:0.25:0.22:0.44:1.85:0.66:1, which was in good agreement with the monosaccharide composition. Due to its low content (3%) and possible structural damage during the methylation reaction, the galactose residues were not detected in the GC-MS analysis. The results implied that the possible structure of PLP was a highly branched araboxylan mainly composed of 1,4-linked Xylp as the backbone with side chains

Figure 4. Proposed repeating unit of PLP. G


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grains (18.6−24.5 μmol TE/g)33 but lower than those of tobacco seed flour (44−74 μmol TE/g).32 Previous studies19,34 have shown that polysaccharides possess a radical-scavenging effect due to their specific structure features. Their antioxidant effects are related to the number of available active hydroxyl groups in the polysaccharide structure. The results from this study suggested that PLP could effectively scavenge the three free radicals in vitro by either a hydrogen atom or an electron transfer mechanism. It was possibly dependent on the available active hydroxyl groups in its structure. Bile Acid-Binding Capacity of PLP. Cholic acid (CA) and chenodeoxycholic acid (CDCA) are two primary bile acids synthesized in the liver from cholesterol. Binding bile acids to polymers may enhance their elimination through the gastrointestinal tract, which may promote the metabolism of cholesterol in liver and consequently lower the total plasma and LDL cholesterol levels.9 As shown in Figure 6, the CA binding capacity for PLP was 65% of that for cholestyramine resin, whereas its CDCA binding capacity was 60% of that for cholestyramine resin on a per same weight basis. These data suggested the potential of PLP in reducing total plasma and LDL cholesterol levels because cholestyramine resin was a commercially used cholesterol-lowering agent. The mechanism of PLP binding bile acid might be similar to that for other psyllium-related and derived polysaccharides and might be related to its highly branched structure.5 These results warrant additional research in investigating the cholesterol-lowering capacity of PLP from P. asiatic L. for its potential application in cholesterol-lowering functional foods.

attached to the O-2 or O-3 position.23,24 The side chains consisted of 1,3-linked Araf, 1, 5-linked Araf, T-linked Araf, and T-linked GlcA. According to the 1H NMR of PLP (Figure 3A), seven signals were displayed in the anomeric region between δ 4.4 and 5.5. Generally, the chemical shifts of α-linked Araf and β-linked Xylp were between δ 5.1 and 5.4 and between δ 4.4−4.8, respectively.25−27 By comparison with the results of methylation analysis, the anomeric proton signals at δ 4.79, 4.55, and 4.46 and the anomeric carbon signals at δ 102.50, 101.41, and 100.85 corresponded with H-1 and C-1 of β-1,3,4-linked Xylp (residue E), β-1,2,4-linked Xylp (residue F), and β-1,4-linked Xylp (residue G), respectively. The β-anomeric configuration was in a good agreement with the appearance of an FT-IR band at 899 cm−1 (Figure 2). The anomeric proton signals at δ 5.32, 5.18, and 5.16 and the anomeric carbon signals at δ 107.9, 104.75, and 105.87 (Figure 3B) could be attributed to H-1 and C-1 of α-1,3-linked Araf (residue A), α-1,5-linked Araf (residue B), and α-T-linked Araf (residue C). The typical signal at δ 176 was ascribed to C-6 of the α-T-linked GlcAp (residue D).28 From the H−H−COSY spectrum (Figure 3C), the H-1 signal for α-1,3-linked Araf at δ 5.32 might correlate with H-2 at δ 4.41. Similarly, H-3 (δ 3.99), H-4 (δ 4.46), H-5 (δ 3.83), and H-5′ (δ 3.79) were assigned successively. According to these results and the HSQC spectrum (Figure 3D), C-2, C-3, C-4, and C-5 of α-1,3-linked Araf were assigned to δ 79.41, 83.44, 81.88, and 60.80, respectively. Signals of other residues were assigned by the same procedure. All carbon and proton signals are summarized in Table 2.29 The signals of some terminal residues such as T-linked Xyl and T-linked Gal were not detected by NMR spectroscopy, possibly due to their low molecular ratio in PLP. For polysaccharides, the HMBC spectra are considered an important tool to give information about the sequence of glycoside residues.30 From the HMBC spectrum of PLP (Figure 3E), a cross peak was observed between H-1 (δ 5.32) of residue A and C-3 (δ 72.22) of residue E, which implied residue A was linked to O-3 of residue E. The cross peak D/F (H1/C2) was also found, indicating residue D was linked to O2 of residue F. Other cross peaks were assigned by a similar procedure, and they are summarized in Table 3. Taking all information together, PLP was a highly branched heteroxylan of β-1,4-linked Xylp backbone possibly with three α-GlcAp-(1→3)-Araf attached to the O-3 position and one αT-linked-GlcAp and one α-Araf-(1→5)-Araf attached to the O2 position every eight monosaccharide residues (Figure 4). Furthermore, the glucuronic acid content was greater in PLP compared to the hot water extractable P. asiatic L. polysaccharide.31 The side chains of PLP were also different from that of water-extractable P. asiatic L. polysaccharides. Antioxidant Activity of PLP in Vitro. The DPPH•scavenging capacity was a widely accepted assay for evaluating the radical-scavenging capacity of natural compounds based on a single electron transfer (SET) mechanism.14 PLP exhibited a strong scavenging capacity against DPPH• with a relative DPPH•-scavenging capacity (RDSC) value of 60.5 μmol TE/g (Figure 5). It was much stronger than that of the tobacco seed flour and wheat grains at 2.65−4.37 μmol TE/g32 and 0.91− 1.53 μmol TE/g,33 respectively. PLP had ORAC and HOSC values of 32.65 and 21.6 μmol TE/g, respectively. The ORAC value of PLP was comparable to that of the tobacco seed flour (25−53 μmol TE/g)32 and wheat grains (15.7−35.8 μmol TE/ g).33 The HOSC value of PLP was comparable to that of wheat

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Y.N.) Phone: (86)-21-34204538. Fax: (86)-21-34204107. Email: [email protected]. *(L.Y.) Phone: (301) 405-0761. Fax: (301) 314-3313. E-mail: [email protected]. Author Contributions ∥

L.G. and H.Z. contributed equally to this work.

Funding

This work was partly supported by a grant from the National Natural Science Funds of China (Grant 31401490), grants from the National High Technology Research and Development Program of China (Grants 2013AA102202 and 2013AA102207), a special fund for Agro-scientific Research in the Public Interest (Grant 201203069), and a grant from Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd. Notes

The authors declare no competing financial interest.

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A Novel Alkali Extractable Polysaccharide from Plantago asiatic L

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