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Fractionation, physicochemical properties and structural features of non-arabinoxylan polysaccharide from the seeds of Plantago asiatica L.
Food Hydrocolloids 55 (2016) 128e135
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Fractionation, physicochemical properties and structural features of non-arabinoxylan polysaccharide from the seeds of Plantago asiatica L. Jun-Yi Yin, Jun-Qiao Wang, Hui-Xia Lin, Ming-Yong Xie, Shao-Ping Nie* State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 June 2015 Received in revised form 10 October 2015 Accepted 6 November 2015 Available online 12 November 2015
Polysaccharide from seeds of Plantago asiatica L. is rich in Ara and Xyl. However, it contains small amounts of other monosaccharides, such as Rha, Gal and GalA. It is the first time to systematically elucidate structural features as well as fractionation, physicochemical properties of non-arabinoxylan polysaccharide from the seeds of Plantago asiatica L.. Non-arabinoxylan polysaccharide named as PLWE-NAX was successfully isolated and separated from the water extract of the seeds at a temperature of 50 C. PLWE-NAX was purified into five fractions by anion exchange chromatography and gel permeation chromatography. The non-arabinoxylan fractions included arabinogalactan (PLWE-1), xylan (PLWE-2) and pectin (PLWE-3, PLWE-4, PLWE-5). PLWE-2, PLWE-3, PLWE-4 and PLWE-5 were acidic polysaccharide. PLWE-4 was subjected for structural characterization by methylation analysis and 1D/2D NMR. It was mainly consisted of a-1,2-linked Rhap (25.01%), T-linked Araf (12.11%) and b-1,3,4-linked Xylp (20.01%) residues. Uronic acid in PLWE-4 was a-1,4-linked GalAp (31.55%). PLWE-4 had the back bone as following, /1)-a-GalAp-(4 / 1)-a-Rhap-(2 / 1)-a-GalAp-(4 / . © 2015 Elsevier Ltd. All rights reserved.
Keywords: Seeds of Plantago asiatica L. Non-arabinoxylan polysaccharide Structure Methylation NMR
1. Introduction Polysaccharide extracted from the seeds husk of Plantago family (psyllium) is one of functional biopolymers. The polysaccharide is considered to be arabinoxylan, for its high contents in Ara and Xyl. It has been widely used as laxatives because of its high viscosity and weak gelling properties. Moreover, the polysaccharide could improve gut health by facilitating propulsion of colon contents (Marlett, Kajs, & Fischer, 2000; Marteau et al., 1994), increasing short-chain fatty acid production (Hu, Nie, Li, & Xie, 2013; Hu, Nie, Min, & Xie, 2012) and being beneficial for lipid metabolism and colon microbiota (Hu et al., 2014). Psyllium polysaccharide usually has b-1,4-linked Xylp backbone which is highly branched, where most of C (O)-2 and/or C (O)-3 in b-1,4-linked Xylp residues in the backbone were substituted. Single arabinofuranose and xylopyranose, or the oligosaccharide residues could be found in the branch area of the polysaccharide (Guo, Cui, Wang, & Christopher Young, 2008; Kennedy, Sandhu, & Southgate, 1979; Samuelsen et al., 1999; Yin, Lin, Li, et al., 2012; Yin, Lin, Nie, Cui, & Xie, 2012). Galacturonic acid or glucuronic acid could be
* Corresponding author. E-mail address: [email protected] (S.-P. Nie). http://dx.doi.org/10.1016/j.foodhyd.2015.11.011 0268-005X/© 2015 Elsevier Ltd. All rights reserved.
detected in branch chain (Aisa et al., 2006; Kennedy et al., 1979; Samuelsen et al., 1999; Yin et al., 2012). Arabinoxylan is used to be considered to be the main component of psyllium polysaccharide, since the high content of Ara and Xyl have been found. Other non-arabinoxylan polysaccharide may be present. One of typical examples is monosaccharides, such as Rha, Man, Gal and Glc, have been detected in the psyllium polysaccharide (Aisa et al., 2006; Edwards, Chaplin, Blackwood, & Dettmar, 2003; Gong et al., 2015; Kennedy et al., 1979; Van Craeyveld, Delcour, & Courtin, 2009). It was even noted some fractions rich of Rha, Gal or GalA (Guo et al., 2008; Samuelsen et al., 1999). However, more detailed knowledge of structural features and functional properties of the non-arabinoxylan compounds from psyllium polysaccharide has not been clearly elucidated. Structural characteristics of polysaccharide from the seeds of Plantago asiatica L. have been investigated in our lab, and the results showed it was also arabinoxylan (Yin, Li, Nie, & Xie, 2013; Yin et al., 2012; Yin et al., 2012). However, there were probably some nonarabinoxylan fractions in the polysaccharide since it contained 0.92% of Gla, 2.28% of Rha and 3.98% of Gal (Yin et al., 2012). Recently, one kind of non-arabinoxylan polysaccharide (named as PLWE-NAX, non-arabinoxylan polysaccharide from the seeds of Planago asiatica L. water extract) was successfully isolated from the seeds of Planago asiatica L.. Therefore, this study was aimed at
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investigating physicochemical properties and structural features of fractions purified from PLWE-NAX, by monosaccharide compositions and methylation analysis, combined with FT-IR, GC/MS, 1D/2D NMR. It would be the first time to systematically explore the physicochemical properties and structural features of nonarabinoxylan polysaccharide from pysllium. 2. Material and methods 2.1. Material The seeds of Plantago asiatica L. were from Ji'an County (Jiangxi Province, China) and dried before use. Dextrans of different molecular weight (200 0000, 70,000, 40,000 and 10,000 Da), and Sephacryl™ S-400 HR were from Pharmacia Co. (Uppsala, Sweden). DEAE-A-52, mannose (Man), rhamnose (Rha), ribose (Rib), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc) were purchased from SigmaeAldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Hydroxylamine hydrochloride, pyridine, trifluoroacetic acid (TFA), methyl iodide and glucose were of analytical grade. Aqueous solutions were prepared with ultra-pure water from a Milli-Q water purification system (Millipore, Bedford, MA, USA). 2.2. Polysaccharide isolation and purification Polysaccharide was extracted and purified according to
procedure shown Figs. 1 and 2, respectively. Briefly, the seeds of Plantago asiatica L. were defatted with ethanol (80%, v/v) and dried at 50 C. Then, the defatted seeds (100 g) were extracted with deionized water (1000 mL) at 50 C for 3 h. The extraction procedure was taken twice, and the combined aqueous extracts were filtered and concentrated at 55 C. The solution was precipitated by ethanol at its final concentration of 80% for more than 12 h. After centrifugation, the precipitate was washed with anhydrous ethanol, acetone and diethyl ether, and finally lyophilized. The obtained fraction was named as PLWE-50. PLWE-50 was dissolved in water, and centrifugated at 4800 rpm for 10 min to get two layers. The supernatant was collected and defined as PLWE-NAX. PLWE-NAX was then subjected for purification by anion exchange chromatography and gel permeation chromatography (Fig. 2). 2.3. General analysis Neutral sugar content was determined by the phenol-sulfuric acid colorimetric method with Xyl as the standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956; Zhou, Xie, Wan, & Nie, 2008). Uronic acid content was determined by m-hydroxybiphenyl photometric procedure using D-glucuronic acid as the standard (Blumenkrantz & Asboe-Hansen, 1973). Protein content was determined by photometric assay, and bovine serum albumin was used as the standard (Bradford, 1976). Polysaccharide solutions were placed in liquid nitrogen for 5 min before subjected for freeze drying. The dried samples were
Seeds of of Plantago asiatica L. Extracted with 80% EtOH
Residue
Ethanol extract
Extracted with water at 50 for twice, 3 h/time, filtered Water extract Condensed under reduced pressure, 55 oC; precipitated with ethanol (80%), and centrifugated
Supernatant (discarded)
Precipitate Washed with ethanol, acetone and anhydrous diethyl ether, successively Crude polysaccharide (PLWE-50, black, yield of 1.58%) Centrifugation
Supernatant (PLWE-NAX, black, yields of 0.42%)
129
Underlayer (PLWE-AX, puce, yields of 0.83%)
Fig. 1. Extraction and fractionation of crude polysaccharide from the seeds of Plantago asiatica L.
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PLWE-NAX (860 mg) Fractionation by DEAE-A-52 column (2.6×40 cm) Eluted with water, dialyzed and lyophilized PLWE-1 (white, 22 mg)
Eluted with 0.5 M NaCl, dialyzed and lyophilized
Eluted with 1.0 M NaCl, dialyzed and lyophilized
PLWE-4 (dust color, 91.5 mg)
Eluted with 0.1 M NaCl, dialyzed and lyophilized
PLWE-5 (dust color, 17.9 mg)
Sub-fraction (white, 340 mg) Purified with Sephacryl TM S-400 HR 2.4×70 cm , dialyzed and lyophilized
PLWE-2 (white, 84.3 mg)
PLWE-3 (white, 142.5 mg)
Fig. 2. Fractionation and purification procedure of PLWE-NAX.
analyzed by a QUANTA 200F SEM (FEI Company, Hillsboro, USA) and viewed at an accelerating voltage of 20 kV. Thermogravimetric analysis was carried out with a simultaneous thermal analyzer TG/ DTA Pyris Diamond (PE Instruments, Waltham, USA), under nitrogen atmosphere at a flow rate of 100 mL/min and a heating rate of 10 C/min in the temperature range from ambient to 700 C, using platinum crucible. Polysaccharide was dissolved completely in ultra-pure water (1.0%, w/v) at 55 C. Rheological properties of samples were measured with cone plate (50 mm diameter with a gap of 0.047 mm) or parallel plate (50 mm diameter with a gap of 0.500 mm) geometry in an ARES G-2 Rheometer (TA instrument, New Castle, USA). Temperature was controlled by Thermo Cube (Solid State Cooling System, New York, USA). 2.4. Molecular weight and monosaccharide compositions Homogeneity and molecular weight of the fractions were analyzed by high performance gel permeation chromatography (HPGPC), on a HPLC system (UK6 injector and 515 HPLC pump, Waters, Milford, MA) equipped with a Waters Ultrahydrogel™ Linear column (7.8 300 mm), and a Waters 410 differential refractometer. Samples were dissolved at a concentration of 1.0 mg/ mL. Water was applied as mobile phase, and flow rate was 0.5 mL/ min. Dextran standards and glucose were used to calibrate the column and establish a standard curve. For monosaccharide compositions analysis, the polysaccharide was hydrolyzed by 2 M TFA at 100 C for 12 h, and analyzed by GC method (Chen, Xie, Wang, Nie, & Li, 2009).
acetateds by hydrolysis, reduction with NaBD4, and acetylation, followed by linkage analysis using an Agilent 7890-7000A GCeMS system with a SP-2330 column (30 m 0.25 mm, 0.2 mm film thickness; Supelco, Bellefonte, PA). Individual peaks of the PMAA and fragmentation patterns were identified by their mass spectra and relative retention time in GC (Carpita & Shea, 1989). Percentage of methylated sugar was estimated as ratio of the peak area (total ion current). 2.6. NMR Polysaccharide (about 25 mg) was exchanged with 99.9% of D2O for twice by freeze drying. Then, it was dissolved in 0.5 mL D2O completely, and filtered by membrane (0.45 mm) before NMR spectra collection. NMR spectra of the sample were recorded on a Bruker DRX-600 NMR spectrometer (Bruker, Rheinstetten, Germany) with a 5 mm probe. The 1H NMR spectrum was recorded by fixing HOD signal at d 4.70, with 128 summed transients and a spectral width of 9615.4 Hz. The 13C NMR spectrum was recorded by using acetone as an internal standard and fixing the methyl carbon signal at d 30.44. Acquisition and relaxation time were 0.91 and 2 s, respectively. A total of 42122 scans per spectrum were accumulated. Spectra of 1H, 13C, DQF-COSY (Double Quantum Filtered-Correlated Spectroscopy), TOCSY (Total Correlated Spectroscopy), NOESY (Nuclear Overhauser Effect Spectroscopy), HSQC (Heteronuclear Single Quantum Correlated Spectroscopy) and HMBC (Heteronuclear Multiple Quantum Correlated Spectroscopy) were collected by using the standard Bruker pulse sequence. 3. Results and discussion
2.5. Methylation and GCeMS analysis 3.1. Polysaccharide fractionation and molecular weight Methylation analysis of polysaccharide was conducted according to the method of previous reports (Ciucanu & Kerek, 1984; Yin et al., 2012) with some modifications. The methylated polysaccharide was converted into partially methylated alditol
There were two layers when PLWE-50 was centrifugated. The upper layer (collected and named as PLWE-NAX) was clear while it was viscous for the lower layer (collected and named as PLWE-AX,
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arabinoxylan from seeds of Planago asiatica L. water extract). Fig. 3 shows effect of shear rate on apparent viscosity of PLWE-50, PLWENAX and PLWE-AX, successively. The fractions showed shear thinning behavior as the apparent viscosity decreased with increasing shear rate, except PLWE-NAX. PLWE-NAX was almost Newtonian fluid because there was little difference in apparent viscosity at various shear rate. Among the three fractions, PLWE-AX had the highest viscosity. That both SEM microscope (S.Fig. 1) and thermogravimetric analysis results (S.Fig. 2) showed PLWE-NAX and PLWE-AX were obviously different. PLWE-NAX was flaky curly aggregates, while it was fibrous for PLWE-AX. From thermogravimetric analysis results, both of the two fractions had main decomposition stage at temperature between 150 C and 400 C. The temperature at the highest decomposition rate was 283.4 C for PLWE-NAX, while it was 297.5 C for PLWE-AX. The structure of PLWE-NAX was complexity as it was demonstrated to contain six different kinds of monosaccharides (Table 1). Therefore, PLWE-NAX was subjected for further purification. PLWENAX (860 mg) was separated by DEAE-A-52 column into PLWE-1 (water eluted fraction, 22 mg), PLWE-NAX-2 (0.1 M NaCl eluted fraction, 340 mg), PLWE-4 (0.5 M NaCl eluted fraction, 91.5 mg) and PLWE-5 (1.0 M NaCl eluted fraction, 17.9 mg). PLWE-NAX-2 was applied for purification by Sephacryl™ S-400 HR column to get another two sub-fractions, which were named as PLWE-2 (higher in molecular weight, 84.3 mg) and PLWE-3 (lower in molecular weight, 142.5 mg), respectively. HPGPC results of all the fractions are shown in Supplementary Data (S.Fig. 3). Because of the low yield of PLWE-1, it was not purified forward. PLWE-4 and PLWE-5 was homogeneous, and the average molecular weight was 594 and 953 kDa, respectively. The average molecular weight of PLWE-2 was much higher than that of PLWE-3.
3.2. Physicochemical properties and monosaccharide compositions Table 1 shows sugar, uronic acid and protein contents of the purified fractions. All of these fractions contained small amounts of protein. PLWE-2 had the highest sugar content (63.6%), while PLWE-5 had the lowest (28.0%). Fractions of PLWE-2, PLWE-3, PLWE-4 and PLWE-5 were detected to be rich in uronic acid contents. Among the five purified fractions, their monosaccharide compositions were different (Table 1). PLWE-1 was arabinogalactan because it was mainly composed of Ara (48.89%) and Gal (37.31%). The majority monosaccharide in PLWE-2 was Xyl (80.49%). PLWE-3, PLWE-4 and PLWE-5 mainly contained Rha, Ara, Xyl and Gal, but the molar ratios of monosaccharides were different. In conclusion,
Apparent viscosity (Pa.s)
100 10
PLWE-50
(1.0%)
PLWE-NAX (1.0%) PLWE-AX
(1.0%)
1 0.1 0.01 0.001 Shear rate (s-1)
Fig. 3. Effect of shear rate on apparent viscosity of different polysaccharide fractions, determined at 25 C.
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the water extracted fraction (PLWE-NAX) from the seeds of Plantago asiatica L. had four different types of polysaccharide, which were arabinogalactan, xylan, pectin, and arabinoxylan. 3.3. Structural features of PLWE-1 and PLWE-5 Only glycosidic linkages of PLWE-1 and PLWE-5 were characterized for their low yields. From methylation analysis results (Table 2), PLWE-1 was mainly composed of T-linked Araf (5.00%), 1,5-linked Araf (28.97%), T-linked Galp (8.00%), 1,3-linked Galp (24.34%), 1,3,6-linked Galp (15.92%) and 1,6-linked Glcp (5.33%). It indicated PLWE-1 probably had 1,3-linked Galp backbone. Other residues such as 1,3-linked Araf (2.41%), 1,2-linked Galp (3.88%), 1,4linked Galp (2.63%) and 1,4,6-linked Glcp (3.52%) were also detected in PLWE-1. Residue linkage type of PLWE-5 was different to PLWE-1. The majority residues of PLWE-5 were 1,2-linked Rhap (30.18%), 1,3-linked Xylp (9.98%) and 1,3,4-linked Xylp (20.90%). IR spectra of PLWE-1 and PLWE-5 are shown in S.Figs. 4 and 5, respectively. The band nearby 3400 cm 1 was assigned to the hydroxyl stretching vibration. Absorption in the region of 2930 cm 1 was because of CeH stretching vibration. There was a strong band between 900 and 1200 cm 1 both for PLWE-1 and PLWE-5, which was assigned to the stretching vibrations of the pyranose ring. Characteristic absorption of pyranose ring confirmed methylation analysis results for lots of pyranose residues determined in PLWE-1 and PLWE-5. Characteristic absorption at 892.9 cm 1 indicated bconfiguration of the sugar units existed in PLWE-5 (Barker, Bourne, Stacey, & Whiffen, 1954). 3.4. Structural features of PLWE-4 Main residues in PLWE-5 were similar to PLWE-4 as data shown in Table 2, and yield of PLWE-4 was much higher than that of PLWE5. Therefore, PLWE-4 was subjected for its fine structure characterization. Glycosidic linkages information of PLWE-4 is shown in Table 2. Main residues in PLWE-4 were 1,2-linked Rhap (25.01%), T-linked Araf (12.11%), 1,3-linked Araf (8.07%), 1,3-linked Xylp (9.37%), 1,2,4linked Xylp (5.79%), 1,3,4-linkd Xylp (20.01%), 1,3,6-linked Galp (5.95%) and 1,4,6-linked Galp (6.05%). Residues of 1,4-linked Galp increased remarkably, from “not determined” to be 29.36%, when uronic acid in PLWE-4 was reduced (Taylor & Conrad, 1972). It suggested that main uronic acid in PLWE-4 existed in 1,4-linked GalAp. Galacturonic acid content was determined to be 32.20% in PLWE-4 by anion exchange chromatography test, except a small amount of GlcA (3.42%). IR spectrum of PLWE-4 (S.Fig. 6) confirmed that it was acidic polysaccharide, containing pyranose ring and bconfiguration. The bands of 1735.3 cm 1 and 1256.0 cm 1 suggested some carboxyl groups were probably esterified (Chatjigakis et al., 1998; Cui & Chang, 2014). Spectra of 1H and 13C NMR of PLWE-4 are shown in Fig. 4. The 1H NMR determined at 323 K was aimed to obtain proton signals distributed in the area of d 4.6e4.9 (Fig. 4). More than four peaks were shown in the anomeric region (d 4.4e5.3) of the 1H NMR. Two peaks at d 1.16 and d 1.17 were from proton resonances of methyl groups in Rha. The corresponding resonance in the 13C spectrum was at d 16.74. According to 1H NMR spectrum, PLWE-4 mainly had four anomeric proton signals at d 5.14, 4.96, 4.93 and 4.47, which were labeled as A, B, C and D. The corresponding anomeric carbon signals in 13C NMR spectrum were labeled with the contribution of HSQC (Fig. 5). All 1H and 13C NMR of labeled residues were assigned from COSY (Fig. 6), TOCSY (not shown), HSQC and NOESY (Fig. 6) spectra. The conclusion and deduction of main residues' chemical shifts are shown in Table 3. Residue A was in a-configuration according to chemical shifts of
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Table 1 Physicochemical properties, molecular weight and monosaccharide compositions of different polysaccharide fractions from the seeds of Plantago asiatica L. Polysaccharide fractionsa
Sugar content (%)
Uronic acid content Protein content (%) (%)
Relative average molecular weight
Monosaccharide compositions (%)
PLWE-50 PLWE-NAX PLWE-AX PLWE-1 PLWE-2 PLWE-3 PLWE-4 PLWE-5
58.0 40.4 64.7 40.4 63.6 37.5 41.3 28.0
24.2 26.1 17.8 3.5 17.6 34.3 39.4 31.3
n.d. n.d. n.d. n.d. 1153028 463380 594047 953224
4.78 21.69 1.00 1.88 1.29 46.15 24.2 26.28
2.0 4.6 1.5 n.d. 0.1 0.1 2.3 4.8
Rhamnose Arabinose Xylose Mannose Glucose Galactose 18.58 19.10 20.71 48.89 13.44 16.84 19.32 23.14
68.00 43.83 75.05 1.99 80.49 17.39 46.33 36.99
1.09 1.19 n.d. 6.39 0.43 0.78 n.d. n.d.
1.98 2.13 1.05 3.54 0.57 n.d. n.d. 2.5
5.57 12.05 2.19 37.31 3.77 18.84 10.15 11.09
n.d., not determined. a PLWE-50 was the water extracted polysaccharide from the seeds of Plantago asiatica L. at a temperature of 50 C. It was separated into arabinoxylan (PLWE-AX) and nonarabinoxylan polysaccharide (PLWE-NAX). PLWE-1, PLWE-2, PLWE-3, PLWE-4 and PLWE-5 were different fractions purified from PLWE-AX by anion exchange chromatography and gel permeation chromatography as shown in Fig. 2.
Table 2 Methylation analysis results of PLWE-1, PLWE-4 and PLWE-5. Linkages
PLWE-1 (%)a
PLWE-4 (%)a
PLWE-5 (%)a
m/z (mass-to-charge ratio)
1,2-linked Rhap 1,2,4-linked Rhap 1,3,4-linked Rhap T-linked Araf 1,3-linked Araf 1,5-linked Araf 1,3-linked Xylp 1,4-linked Xylp 1,2,4-linked Xylp 1,3,4-linked Xylp T-linked Galp 1,2-linked Galp 1,3-linked Galp 1,4-linked Galp 1,6-linked Galp 1,3,6-linked Galp 1,4,6-linked Galp 1,4-linked Glcp 1,6-linked Glcp 1,4,6-linked Glcp
trace n.d. n.d. 5.00 2.41 28.97 n.d. n.d. n.d. n.d. 8.00 3.88 24.34 2.63 trace 15.92 trace trace 5.33 3.52
25.01 trace 2.44 12.11 8.07 n.d. 9.37 n.d. 5.79 20.01 trace n.d. 2.92 n.d. 2.27 5.95 6.05 n.d. n.d. n.d.
30.18 5.91 trace 2.72 6.90 n.d. 9.98 2.15 6.18 20.90 2.43 n.d. 3.66 trace trace 6.53 n.d. 2.45 n.d. n.d.
43,89,130,131,190 43,101,130,143,190,203 43,87,89,100,131,262 43,71,87,101,102,118,129,161 43,59,87,99,118,233 43,87,102,118,129,189 43,87,102,118,129,189 43,59,71,87,102,118,129,189 43,57,71,87,88,129,130,189,190 43,99,118 43,71,87,89,102,118,129,145,161,162,205 43,87,88,100,129,130,145,161,190,205 43,71,87,101,118,129,161,234 43,87,99,102,113,118,162,173,233 43,59,71,87,99,102,118,129,162,173,189,233 43,59,87,101,118,129,189,234 43,87,102,118,162 43,59,99,102,113,118,129,162,173,233 43,87,99,102,118,129,162,173,189,233 43,85,99,102,118,162,201,261
a
Peak percentage was calculated according to total ion current; n.d.: not determined.
anomeric carbon (d 99.12) and proton (d 5.14), with a relative high content in PLWE-4. Other proton of residue A (from H-2 to H-6) was assigned from COSY spectrum (Fig. 6). The corresponding chemical shifts of carbon were d 76.57, 69.50, 72.93, 71.30 and 16.52 for C-2, C-3, C-4, C-5 and C-6, successively, which was supported by HSQC spectrum. According to references (Neiwert, Holst, & Duda, 2014; Polle, Ovodova, Shashkov, & Ovodov, 2002; Qian, Cui, Nikiforuk, & Goff, 2012), residue A was attributed to be a-1,2-linked Rhap. Chemical shifts of anomeric proton of residue B and C were d 4.96 and d 4.93, respectively. The corresponding chemical shifts of anomeric carbon were d 99.12 for residue B and d 97.88 for residue C. The cross peaks of their other proton (H-2, H-3, H-4 and H-5) were assigned in the COSY spectrum (Table 3). For residue B, signals of C-2, C-3, C-4 and C-5 were at d 71.98, d 69.41, d 77.73 and d 70.83 as revealed by HSQC. Other carbon signals of residue C could also be assigned similarly. From methylation analysis results, the content of 1,4-linked GalAp was 29.36% among all the detected residues. Therefore, residue B and C were assigned to be 1,4-linked GalAp for their high intensity of anomeric carbon and proton. The residues of 1,4-linked GalAp in the polysaccharide were linked in a-configuration. Chemical shift of the C-6 was d 173.68. A full assignment of chemical shifts for 1,4-linked GalAp was in agreement with previous reports (Ding et al., 2015; Li, Cui, Nie, & Xie, 2014; Qian et al.,
2012). From 1H and 13C NMR, chemical shifts of proton (d 2.24) and carbon (d 19.82) were found which indicated part of carboxyl group in 1,4-linked GalAp was acetylated (Zhang, Nie, Yin, Wang, & Xie, 2014). It was consistent with IR result. Residues D whose chemical shift of anomeric proton was d 4.40 was assigned as 1,3,4-linked Xylp, as it had less intensive signal at d 102.77. It was in b-configuration. Other chemical shifts of protons and carbons were assigned by COSY and HSQC. The downfield chemical shift of C-3 (d 79.91) confirmed the residue was 1,3,4linked. The proton and carbon chemical shifts of residue D were supported by literature values (Apirattananusorn, Tongta, Cui, & Wang, 2008; Yin et al., 2012). HMBC and NOESY are powerful spectra in revealing glycosylic linkages between sugar residues. Intra-residue connectives could also be found. Intra-residues and inter-residues correlations in PLWE-4 are shown in Table 4. From NOESY spectrum, some interresidual cross peaks were observed, which were A H-1 to C H-1, A H-1 to C H-2, A H-1 to C H-4, A H-1 to C H-5; A H-1 to B H-4; B H-1 to C H-1, B H-1 to C H-2, B H-1 to C H-3, B H-1 to C H-4; B H-1 to A H2; C H-1 to A H-2, C H-1 to A H-3; C H-1 to B H-4, C H-1 to B H-5. According to above analysis results, main repeating unit of PLWE-4 was deduced as: /1)-a-GalAp-(4 / 1)-a-Rhap-(2 / 1)-a-GalAp(4 / .
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Fig. 4.
13
C and 1H NMR spectra of PLWE-4.
Fig. 5. Part of HSQC spectrum of PLWE-4.
4. Conclusion This is the first report about systematic exploration on physicochemical properties and structural characteristics of nonarabinoxylan polysaccharide, which was isolated and purified from the seeds of Plantago asiatica L.. There were three different kinds of polysaccharide. They were arabinogalactan (PLWE-1), xylan (PLWE-2) and pectin (PLWE-3, PLWE-4, PLWE-5). According to methylation analysis results, PLWE-1 mainly contained T-linked Araf, 1,5-linked Araf, T-linked Galp, 1,3-linked Galp and 1,3,6-linked Galp. PLWE-5 had residual linkages such as 1,2-linked Rhap, 1,3linked Araf, 1,3-linked Xylp, 1,2,4-linked Xylp, 1,3,4-linked Xylp and 1,3,6-linked Galp. Primary structure of PLWE-4 was elucidated by methylation and 1D/2D NMR spectra. However, more functional properties of the non-arabinoxylan fractions from the seeds of Plantago asiatica L. should be explored to elucidate the structurefunction relationship.
Fig. 6. Part of COSY (A) and NOESY (B) spectra of PLWE-4.
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Table 3 Chemical shifts of main residues in PLWE-4 from 1H/13C NMR. Residues
A B C D
Linkage information
a-1,2-linked Rhap a-1,4-linked GalAp a-1,4-linked GalAp b-1,3,4-linked Xylp
Chemical shifts of different proton/carbon (d) H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5/C-5
H-6/C-6
5.14/99.12 4.96/99.12 4.93/97.88 4.47/102.77
4.00/76.57 3.50/71.98 3.79/69.56 3.25/72.93
3.78/69.50 3.60/69.41 4.02/70.02 3.65/79.91
3.31/72.93 4.37/77.73 4.32/77.73 4.23/82.65
3.60/71.30 4.77/70.83 3.85/68.02 3.70/61.38
1.13/16.52 /173.68 /173.68 /
Table 4 Assignment of cross-peaks in the NOESY spectrum of PLWE-4. Residue (linkage information)
Proton
Chemical shift (d)
Connectivities Inter-connectivities
A (1,2-linked Rhap)
H-1
5.14
B (1,4-linked GalAp)
H-1
4.96
C (1,4-linked GalAp)
H-1
4.93
Intra-connectivities
Residue, proton
Chemical shift (d)
Residue, proton
Chemical shift (d)
C H-3 C H-2 B H-4 C H-4 C H-5 C H-1 C H-2 C H-3 A H-2 C H-4 A H-3 A H-2 B H-4 B H-5
3.60 3.78 4.32 4.37 4.75 4.93 3.78 3.99 4.00 4.32 3.78 4.00 4.37 4.77
A A A A
H-4 H-5 H-3 H-2
3.31 3.60 3.78 4.00
B H-2 B H-4
3.50 4.37
C H-2 C H-3 C H-4
3.79 3.99 4.32
Acknowledgments This study is financially supported by the National Natural Science Foundation of China (No: 31301434, 21564007), Research Fund for the Doctoral Program of Higher Education of China (No: 20133601120009), and Jiangxi Provincial Natural Science Foundation (20142BAB214004), which are gratefully acknowledged. The authors wish to thank Mr. Wei Zhou from Centre for Nutrition and Food Science, Queensland Alliance for Agricultural and Food Innovation, the University of Queensland, Australia for kind discussions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodhyd.2015.11.011. References Aisa, H., Abdurahim, M., Yili, A., Xui, L., Rakhmanberdyeva, R., & Sagdullaev, B. (2006). Polysaccharides of Plantago ovatax seeds. Chemistry of Natural Compounds, 42(3), 347e348. Apirattananusorn, S., Tongta, S., Cui, S. W., & Wang, Q. (2008). Chemical, molecular, and structural characterization of alkali extractable nonstarch polysaccharides from Job's tears. Journal of Agricultural and Food Chemistry, 56(18), 8549e8557. Barker, S. A., Bourne, E. J., Stacey, M., & Whiffen, D. H. (1954). Infra-red spectra of carbohydrates. Part I. Some derivatives of D-glucopyranose. Journal of the Chemical Society (Resumed), 171e176. Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54(2), 484e489. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1), 248e254. Carpita, N. C., & Shea, E. M. (1989). Linkage structure of carbohydrates by gas chromatography-mass spectrometry (GC-MS) of partially methylated alditol acetates. In C. J. Biermann, & G. D. McGinnis (Eds.), Analysis of carbohydrates by GLC and MS (pp. 157e216). Boca Raton: CRC Press. Chatjigakis, A., Pappas, C., Proxenia, N., Kalantzi, O., Rodis, P., & Polissiou, M. (1998). FT-IR spectroscopic determination of the degree of esterification of cell wall pectins from stored peaches and correlation to textural changes. Carbohydrate
Polymers, 37(4), 395e408. Chen, Y., Xie, M. Y., Wang, Y. X., Nie, S. P., & Li, C. (2009). Analysis of the monosaccharide composition of purified polysaccharides in Ganoderma atrum by capillary gas chromatography. Phytochemical Analysis, 20(6), 503e510. Ciucanu, I., & Kerek, F. (1984). A simple and rapid method for the permethylation of carbohydrates. Carbohydrate Research, 131(2), 209e217. Cui, S. W., & Chang, Y. H. (2014). Emulsifying and structural properties of pectin enzymatically extracted from pumpkin. LWT-Food Science and Technology, 58(2), 396e403. Ding, H. H., Cui, S. W., Goff, H. D., Chen, J., Wang, Q., & Han, N. F. (2015). Arabinanrich rhamnogalacturonan-I from flaxseed kernel cell wall. Food Hydrocolloids, 47(0), 158e167. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350e356. Edwards, S., Chaplin, M. F., Blackwood, A. D., & Dettmar, P. W. (2003). Primary structure of arabinoxylans of ispaghula husk and wheat bran. Proceedings of the Nutrition Society, 62(01), 217e222. Gong, L., Zhang, H., Niu, Y. G., Chen, L., Liu, J., Alaxi, S., et al. (2015). A novel alkali extractable polysaccharide from Plantago asiatic L. seeds and its radicalscavenging and bile acid-binding activities. Journal of Agricultural and Food Chemistry, 63(2), 569e577. Guo, Q., Cui, S. W., Wang, Q., & Christopher Young, J. (2008). Fractionation and physicochemical characterization of psyllium gum. Carbohydrate Polymers, 73(1), 35e43. Hu, J. L., Nie, S. P., Li, C., & Xie, M. Y. (2013). In vitro effects of a novel polysaccharide from the seeds of Plantago asiatica L. on intestinal function. International Journal of Biological Macromolecules, 54, 264e269. Hu, J. L., Nie, S. P., Min, F. F., & Xie, M. Y. (2012). Polysaccharide from seeds of Plantago asiatica L. increases short-chain fatty acid production and fecal moisture along with lowering pH in mouse colon. Journal of Agricultural and Food Chemistry, 60(46), 11525e11532. Hu, J. L., Nie, S. P., Wu, Q. M., Li, C., Fu, Z. H., Gong, J., et al. (2014). Polysaccharide from seeds of Plantago asiatica L. affects lipid metabolism and colon microbiota of mouse. Journal of Agricultural and Food Chemistry, 62(1), 229e234. Kennedy, J. F., Sandhu, J. S., & Southgate, D. A. (1979). Structural data for the carbohydrate of ispaghula husk ex Plantago ovata forsk. Carbohydrate Research, 75, 265e274. Li, J. E., Cui, S. W., Nie, S. P., & Xie, M. Y. (2014). Structure and biological activities of a pectic polysaccharide from Mosla chinensis Maxim. cv. Jiangxiangru. Carbohydrate Polymers, 105, 276e284. Marlett, J. A., Kajs, T. M., & Fischer, M. H. (2000). An unfermented gel component of psyllium seed husk promotes laxation as a lubricant in humans. The American Journal of Clinical Nutrition, 72(3), 784e789. Marteau, P., Flourie, B., Cherbut, C., Correze, J., Pellier, P., Seylaz, J., et al. (1994).
J.-Y. Yin et al. / Food Hydrocolloids 55 (2016) 128e135 Digestibility and bulking effect of ispaghula husks in healthy humans. Gut, 35(12), 1747e1752. Neiwert, O., Holst, O., & Duda, K. A. (2014). Structural investigation of rhamnoserich polysaccharides from Streptococcus dysgalactiae bovine mastitis isolate. Carbohydrate Research, 389, 192e195. Polle, A. Y., Ovodova, R., Shashkov, A., & Ovodov, Y. S. (2002). Some structural features of pectic polysaccharide from tansy, Tanacetum vulgare L. Carbohydrate Polymers, 49(3), 337e344. Qian, K. Y., Cui, S. W., Nikiforuk, J., & Goff, H. D. (2012). Structural elucidation of rhamnogalacturonans from flaxseed hulls. Carbohydrate Research, 362, 47e55. Samuelsen, A., Lund, I., Djahromi, J., Paulsen, B., Wold, J., & Knutsen, S. (1999). Structural features and anti-complementary activity of some heteroxylan polysaccharide fractions from the seeds o Plantago major L. Carbohydrate Polymers, 38(2), 133e143. Taylor, R. L., & Conrad, H. (1972). Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl group. Biochemistry, 11(8), 1383e1388. Van Craeyveld, V., Delcour, J. A., & Courtin, C. M. (2009). Extractability and chemical and enzymic degradation of psyllium (Plantago ovata Forsk) seed husk arabinoxylans. Food Chemistry, 112(4), 812e819.
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Yin, J. Y., Li, C., Nie, S. P., & Xie, M. Y. (2013). Structural characterization of purified polysaccharide fraction( PLP-1) from the seeds of Plantago asiatica L. Chemical Journal of Chinese Universities-Chinese, 34(12), 2728e2733. Yin, J. Y., Lin, H. X., Li, J., Wang, Y. X., Cui, S. W., Nie, S. P., et al. (2012). Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L. Carbohydrate Polymers, 87(4), 2416e2424. Yin, J. Y., Lin, H. X., Nie, S. P., Cui, S. W., & Xie, M. Y. (2012). Methylation and 2D NMR analysis of arabinoxylan from the seeds of Plantago asiatica L. Carbohydrate Polymers, 88(4), 1395e1401. Yin, J. Y., Nie, S. P., Li, J., Li, C., Cui, S. W., & Xie, M. Y. (2012). Mechanism of interactions between calcium and viscous polysaccharide from the seeds of Plantago asiatica L. Journal of Agricultural and Food Chemistry, 60(32), 7981e7987. Zhang, H., Nie, S. P., Yin, J. Y., Wang, Y. X., & Xie, M. Y. (2014). Structural characterization of a heterogalactan purified from fruiting bodies of Ganoderma atrum. Food Hydrocolloids, 36, 339e347. Zhou, C., Xie, M. Y., Wan, Y., & Nie, S. P. (2008). Study on determination of the contents of polysaccharides in Semen Plantaginis. Chinese Journal of Analysis Laboratory, 27(4), 10e13.
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Fractionation, physicochemical properties and structural features of non-arabinoxylan polysaccharide from the seeds of Plantago asiatica L. Jun-Yi Yin, Jun-Qiao Wang, Hui-Xia Lin, Ming-Yong Xie, Shao-Ping Nie* State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 June 2015 Received in revised form 10 October 2015 Accepted 6 November 2015 Available online 12 November 2015
Polysaccharide from seeds of Plantago asiatica L. is rich in Ara and Xyl. However, it contains small amounts of other monosaccharides, such as Rha, Gal and GalA. It is the first time to systematically elucidate structural features as well as fractionation, physicochemical properties of non-arabinoxylan polysaccharide from the seeds of Plantago asiatica L.. Non-arabinoxylan polysaccharide named as PLWE-NAX was successfully isolated and separated from the water extract of the seeds at a temperature of 50 C. PLWE-NAX was purified into five fractions by anion exchange chromatography and gel permeation chromatography. The non-arabinoxylan fractions included arabinogalactan (PLWE-1), xylan (PLWE-2) and pectin (PLWE-3, PLWE-4, PLWE-5). PLWE-2, PLWE-3, PLWE-4 and PLWE-5 were acidic polysaccharide. PLWE-4 was subjected for structural characterization by methylation analysis and 1D/2D NMR. It was mainly consisted of a-1,2-linked Rhap (25.01%), T-linked Araf (12.11%) and b-1,3,4-linked Xylp (20.01%) residues. Uronic acid in PLWE-4 was a-1,4-linked GalAp (31.55%). PLWE-4 had the back bone as following, /1)-a-GalAp-(4 / 1)-a-Rhap-(2 / 1)-a-GalAp-(4 / . © 2015 Elsevier Ltd. All rights reserved.
Keywords: Seeds of Plantago asiatica L. Non-arabinoxylan polysaccharide Structure Methylation NMR
1. Introduction Polysaccharide extracted from the seeds husk of Plantago family (psyllium) is one of functional biopolymers. The polysaccharide is considered to be arabinoxylan, for its high contents in Ara and Xyl. It has been widely used as laxatives because of its high viscosity and weak gelling properties. Moreover, the polysaccharide could improve gut health by facilitating propulsion of colon contents (Marlett, Kajs, & Fischer, 2000; Marteau et al., 1994), increasing short-chain fatty acid production (Hu, Nie, Li, & Xie, 2013; Hu, Nie, Min, & Xie, 2012) and being beneficial for lipid metabolism and colon microbiota (Hu et al., 2014). Psyllium polysaccharide usually has b-1,4-linked Xylp backbone which is highly branched, where most of C (O)-2 and/or C (O)-3 in b-1,4-linked Xylp residues in the backbone were substituted. Single arabinofuranose and xylopyranose, or the oligosaccharide residues could be found in the branch area of the polysaccharide (Guo, Cui, Wang, & Christopher Young, 2008; Kennedy, Sandhu, & Southgate, 1979; Samuelsen et al., 1999; Yin, Lin, Li, et al., 2012; Yin, Lin, Nie, Cui, & Xie, 2012). Galacturonic acid or glucuronic acid could be
* Corresponding author. E-mail address: [email protected] (S.-P. Nie). http://dx.doi.org/10.1016/j.foodhyd.2015.11.011 0268-005X/© 2015 Elsevier Ltd. All rights reserved.
detected in branch chain (Aisa et al., 2006; Kennedy et al., 1979; Samuelsen et al., 1999; Yin et al., 2012). Arabinoxylan is used to be considered to be the main component of psyllium polysaccharide, since the high content of Ara and Xyl have been found. Other non-arabinoxylan polysaccharide may be present. One of typical examples is monosaccharides, such as Rha, Man, Gal and Glc, have been detected in the psyllium polysaccharide (Aisa et al., 2006; Edwards, Chaplin, Blackwood, & Dettmar, 2003; Gong et al., 2015; Kennedy et al., 1979; Van Craeyveld, Delcour, & Courtin, 2009). It was even noted some fractions rich of Rha, Gal or GalA (Guo et al., 2008; Samuelsen et al., 1999). However, more detailed knowledge of structural features and functional properties of the non-arabinoxylan compounds from psyllium polysaccharide has not been clearly elucidated. Structural characteristics of polysaccharide from the seeds of Plantago asiatica L. have been investigated in our lab, and the results showed it was also arabinoxylan (Yin, Li, Nie, & Xie, 2013; Yin et al., 2012; Yin et al., 2012). However, there were probably some nonarabinoxylan fractions in the polysaccharide since it contained 0.92% of Gla, 2.28% of Rha and 3.98% of Gal (Yin et al., 2012). Recently, one kind of non-arabinoxylan polysaccharide (named as PLWE-NAX, non-arabinoxylan polysaccharide from the seeds of Planago asiatica L. water extract) was successfully isolated from the seeds of Planago asiatica L.. Therefore, this study was aimed at
J.-Y. Yin et al. / Food Hydrocolloids 55 (2016) 128e135
investigating physicochemical properties and structural features of fractions purified from PLWE-NAX, by monosaccharide compositions and methylation analysis, combined with FT-IR, GC/MS, 1D/2D NMR. It would be the first time to systematically explore the physicochemical properties and structural features of nonarabinoxylan polysaccharide from pysllium. 2. Material and methods 2.1. Material The seeds of Plantago asiatica L. were from Ji'an County (Jiangxi Province, China) and dried before use. Dextrans of different molecular weight (200 0000, 70,000, 40,000 and 10,000 Da), and Sephacryl™ S-400 HR were from Pharmacia Co. (Uppsala, Sweden). DEAE-A-52, mannose (Man), rhamnose (Rha), ribose (Rib), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc) were purchased from SigmaeAldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Hydroxylamine hydrochloride, pyridine, trifluoroacetic acid (TFA), methyl iodide and glucose were of analytical grade. Aqueous solutions were prepared with ultra-pure water from a Milli-Q water purification system (Millipore, Bedford, MA, USA). 2.2. Polysaccharide isolation and purification Polysaccharide was extracted and purified according to
procedure shown Figs. 1 and 2, respectively. Briefly, the seeds of Plantago asiatica L. were defatted with ethanol (80%, v/v) and dried at 50 C. Then, the defatted seeds (100 g) were extracted with deionized water (1000 mL) at 50 C for 3 h. The extraction procedure was taken twice, and the combined aqueous extracts were filtered and concentrated at 55 C. The solution was precipitated by ethanol at its final concentration of 80% for more than 12 h. After centrifugation, the precipitate was washed with anhydrous ethanol, acetone and diethyl ether, and finally lyophilized. The obtained fraction was named as PLWE-50. PLWE-50 was dissolved in water, and centrifugated at 4800 rpm for 10 min to get two layers. The supernatant was collected and defined as PLWE-NAX. PLWE-NAX was then subjected for purification by anion exchange chromatography and gel permeation chromatography (Fig. 2). 2.3. General analysis Neutral sugar content was determined by the phenol-sulfuric acid colorimetric method with Xyl as the standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956; Zhou, Xie, Wan, & Nie, 2008). Uronic acid content was determined by m-hydroxybiphenyl photometric procedure using D-glucuronic acid as the standard (Blumenkrantz & Asboe-Hansen, 1973). Protein content was determined by photometric assay, and bovine serum albumin was used as the standard (Bradford, 1976). Polysaccharide solutions were placed in liquid nitrogen for 5 min before subjected for freeze drying. The dried samples were
Seeds of of Plantago asiatica L. Extracted with 80% EtOH
Residue
Ethanol extract
Extracted with water at 50 for twice, 3 h/time, filtered Water extract Condensed under reduced pressure, 55 oC; precipitated with ethanol (80%), and centrifugated
Supernatant (discarded)
Precipitate Washed with ethanol, acetone and anhydrous diethyl ether, successively Crude polysaccharide (PLWE-50, black, yield of 1.58%) Centrifugation
Supernatant (PLWE-NAX, black, yields of 0.42%)
129
Underlayer (PLWE-AX, puce, yields of 0.83%)
Fig. 1. Extraction and fractionation of crude polysaccharide from the seeds of Plantago asiatica L.
130
J.-Y. Yin et al. / Food Hydrocolloids 55 (2016) 128e135
PLWE-NAX (860 mg) Fractionation by DEAE-A-52 column (2.6×40 cm) Eluted with water, dialyzed and lyophilized PLWE-1 (white, 22 mg)
Eluted with 0.5 M NaCl, dialyzed and lyophilized
Eluted with 1.0 M NaCl, dialyzed and lyophilized
PLWE-4 (dust color, 91.5 mg)
Eluted with 0.1 M NaCl, dialyzed and lyophilized
PLWE-5 (dust color, 17.9 mg)
Sub-fraction (white, 340 mg) Purified with Sephacryl TM S-400 HR 2.4×70 cm , dialyzed and lyophilized
PLWE-2 (white, 84.3 mg)
PLWE-3 (white, 142.5 mg)
Fig. 2. Fractionation and purification procedure of PLWE-NAX.
analyzed by a QUANTA 200F SEM (FEI Company, Hillsboro, USA) and viewed at an accelerating voltage of 20 kV. Thermogravimetric analysis was carried out with a simultaneous thermal analyzer TG/ DTA Pyris Diamond (PE Instruments, Waltham, USA), under nitrogen atmosphere at a flow rate of 100 mL/min and a heating rate of 10 C/min in the temperature range from ambient to 700 C, using platinum crucible. Polysaccharide was dissolved completely in ultra-pure water (1.0%, w/v) at 55 C. Rheological properties of samples were measured with cone plate (50 mm diameter with a gap of 0.047 mm) or parallel plate (50 mm diameter with a gap of 0.500 mm) geometry in an ARES G-2 Rheometer (TA instrument, New Castle, USA). Temperature was controlled by Thermo Cube (Solid State Cooling System, New York, USA). 2.4. Molecular weight and monosaccharide compositions Homogeneity and molecular weight of the fractions were analyzed by high performance gel permeation chromatography (HPGPC), on a HPLC system (UK6 injector and 515 HPLC pump, Waters, Milford, MA) equipped with a Waters Ultrahydrogel™ Linear column (7.8 300 mm), and a Waters 410 differential refractometer. Samples were dissolved at a concentration of 1.0 mg/ mL. Water was applied as mobile phase, and flow rate was 0.5 mL/ min. Dextran standards and glucose were used to calibrate the column and establish a standard curve. For monosaccharide compositions analysis, the polysaccharide was hydrolyzed by 2 M TFA at 100 C for 12 h, and analyzed by GC method (Chen, Xie, Wang, Nie, & Li, 2009).
acetateds by hydrolysis, reduction with NaBD4, and acetylation, followed by linkage analysis using an Agilent 7890-7000A GCeMS system with a SP-2330 column (30 m 0.25 mm, 0.2 mm film thickness; Supelco, Bellefonte, PA). Individual peaks of the PMAA and fragmentation patterns were identified by their mass spectra and relative retention time in GC (Carpita & Shea, 1989). Percentage of methylated sugar was estimated as ratio of the peak area (total ion current). 2.6. NMR Polysaccharide (about 25 mg) was exchanged with 99.9% of D2O for twice by freeze drying. Then, it was dissolved in 0.5 mL D2O completely, and filtered by membrane (0.45 mm) before NMR spectra collection. NMR spectra of the sample were recorded on a Bruker DRX-600 NMR spectrometer (Bruker, Rheinstetten, Germany) with a 5 mm probe. The 1H NMR spectrum was recorded by fixing HOD signal at d 4.70, with 128 summed transients and a spectral width of 9615.4 Hz. The 13C NMR spectrum was recorded by using acetone as an internal standard and fixing the methyl carbon signal at d 30.44. Acquisition and relaxation time were 0.91 and 2 s, respectively. A total of 42122 scans per spectrum were accumulated. Spectra of 1H, 13C, DQF-COSY (Double Quantum Filtered-Correlated Spectroscopy), TOCSY (Total Correlated Spectroscopy), NOESY (Nuclear Overhauser Effect Spectroscopy), HSQC (Heteronuclear Single Quantum Correlated Spectroscopy) and HMBC (Heteronuclear Multiple Quantum Correlated Spectroscopy) were collected by using the standard Bruker pulse sequence. 3. Results and discussion
2.5. Methylation and GCeMS analysis 3.1. Polysaccharide fractionation and molecular weight Methylation analysis of polysaccharide was conducted according to the method of previous reports (Ciucanu & Kerek, 1984; Yin et al., 2012) with some modifications. The methylated polysaccharide was converted into partially methylated alditol
There were two layers when PLWE-50 was centrifugated. The upper layer (collected and named as PLWE-NAX) was clear while it was viscous for the lower layer (collected and named as PLWE-AX,
J.-Y. Yin et al. / Food Hydrocolloids 55 (2016) 128e135
arabinoxylan from seeds of Planago asiatica L. water extract). Fig. 3 shows effect of shear rate on apparent viscosity of PLWE-50, PLWENAX and PLWE-AX, successively. The fractions showed shear thinning behavior as the apparent viscosity decreased with increasing shear rate, except PLWE-NAX. PLWE-NAX was almost Newtonian fluid because there was little difference in apparent viscosity at various shear rate. Among the three fractions, PLWE-AX had the highest viscosity. That both SEM microscope (S.Fig. 1) and thermogravimetric analysis results (S.Fig. 2) showed PLWE-NAX and PLWE-AX were obviously different. PLWE-NAX was flaky curly aggregates, while it was fibrous for PLWE-AX. From thermogravimetric analysis results, both of the two fractions had main decomposition stage at temperature between 150 C and 400 C. The temperature at the highest decomposition rate was 283.4 C for PLWE-NAX, while it was 297.5 C for PLWE-AX. The structure of PLWE-NAX was complexity as it was demonstrated to contain six different kinds of monosaccharides (Table 1). Therefore, PLWE-NAX was subjected for further purification. PLWENAX (860 mg) was separated by DEAE-A-52 column into PLWE-1 (water eluted fraction, 22 mg), PLWE-NAX-2 (0.1 M NaCl eluted fraction, 340 mg), PLWE-4 (0.5 M NaCl eluted fraction, 91.5 mg) and PLWE-5 (1.0 M NaCl eluted fraction, 17.9 mg). PLWE-NAX-2 was applied for purification by Sephacryl™ S-400 HR column to get another two sub-fractions, which were named as PLWE-2 (higher in molecular weight, 84.3 mg) and PLWE-3 (lower in molecular weight, 142.5 mg), respectively. HPGPC results of all the fractions are shown in Supplementary Data (S.Fig. 3). Because of the low yield of PLWE-1, it was not purified forward. PLWE-4 and PLWE-5 was homogeneous, and the average molecular weight was 594 and 953 kDa, respectively. The average molecular weight of PLWE-2 was much higher than that of PLWE-3.
3.2. Physicochemical properties and monosaccharide compositions Table 1 shows sugar, uronic acid and protein contents of the purified fractions. All of these fractions contained small amounts of protein. PLWE-2 had the highest sugar content (63.6%), while PLWE-5 had the lowest (28.0%). Fractions of PLWE-2, PLWE-3, PLWE-4 and PLWE-5 were detected to be rich in uronic acid contents. Among the five purified fractions, their monosaccharide compositions were different (Table 1). PLWE-1 was arabinogalactan because it was mainly composed of Ara (48.89%) and Gal (37.31%). The majority monosaccharide in PLWE-2 was Xyl (80.49%). PLWE-3, PLWE-4 and PLWE-5 mainly contained Rha, Ara, Xyl and Gal, but the molar ratios of monosaccharides were different. In conclusion,
Apparent viscosity (Pa.s)
100 10
PLWE-50
(1.0%)
PLWE-NAX (1.0%) PLWE-AX
(1.0%)
1 0.1 0.01 0.001 Shear rate (s-1)
Fig. 3. Effect of shear rate on apparent viscosity of different polysaccharide fractions, determined at 25 C.
131
the water extracted fraction (PLWE-NAX) from the seeds of Plantago asiatica L. had four different types of polysaccharide, which were arabinogalactan, xylan, pectin, and arabinoxylan. 3.3. Structural features of PLWE-1 and PLWE-5 Only glycosidic linkages of PLWE-1 and PLWE-5 were characterized for their low yields. From methylation analysis results (Table 2), PLWE-1 was mainly composed of T-linked Araf (5.00%), 1,5-linked Araf (28.97%), T-linked Galp (8.00%), 1,3-linked Galp (24.34%), 1,3,6-linked Galp (15.92%) and 1,6-linked Glcp (5.33%). It indicated PLWE-1 probably had 1,3-linked Galp backbone. Other residues such as 1,3-linked Araf (2.41%), 1,2-linked Galp (3.88%), 1,4linked Galp (2.63%) and 1,4,6-linked Glcp (3.52%) were also detected in PLWE-1. Residue linkage type of PLWE-5 was different to PLWE-1. The majority residues of PLWE-5 were 1,2-linked Rhap (30.18%), 1,3-linked Xylp (9.98%) and 1,3,4-linked Xylp (20.90%). IR spectra of PLWE-1 and PLWE-5 are shown in S.Figs. 4 and 5, respectively. The band nearby 3400 cm 1 was assigned to the hydroxyl stretching vibration. Absorption in the region of 2930 cm 1 was because of CeH stretching vibration. There was a strong band between 900 and 1200 cm 1 both for PLWE-1 and PLWE-5, which was assigned to the stretching vibrations of the pyranose ring. Characteristic absorption of pyranose ring confirmed methylation analysis results for lots of pyranose residues determined in PLWE-1 and PLWE-5. Characteristic absorption at 892.9 cm 1 indicated bconfiguration of the sugar units existed in PLWE-5 (Barker, Bourne, Stacey, & Whiffen, 1954). 3.4. Structural features of PLWE-4 Main residues in PLWE-5 were similar to PLWE-4 as data shown in Table 2, and yield of PLWE-4 was much higher than that of PLWE5. Therefore, PLWE-4 was subjected for its fine structure characterization. Glycosidic linkages information of PLWE-4 is shown in Table 2. Main residues in PLWE-4 were 1,2-linked Rhap (25.01%), T-linked Araf (12.11%), 1,3-linked Araf (8.07%), 1,3-linked Xylp (9.37%), 1,2,4linked Xylp (5.79%), 1,3,4-linkd Xylp (20.01%), 1,3,6-linked Galp (5.95%) and 1,4,6-linked Galp (6.05%). Residues of 1,4-linked Galp increased remarkably, from “not determined” to be 29.36%, when uronic acid in PLWE-4 was reduced (Taylor & Conrad, 1972). It suggested that main uronic acid in PLWE-4 existed in 1,4-linked GalAp. Galacturonic acid content was determined to be 32.20% in PLWE-4 by anion exchange chromatography test, except a small amount of GlcA (3.42%). IR spectrum of PLWE-4 (S.Fig. 6) confirmed that it was acidic polysaccharide, containing pyranose ring and bconfiguration. The bands of 1735.3 cm 1 and 1256.0 cm 1 suggested some carboxyl groups were probably esterified (Chatjigakis et al., 1998; Cui & Chang, 2014). Spectra of 1H and 13C NMR of PLWE-4 are shown in Fig. 4. The 1H NMR determined at 323 K was aimed to obtain proton signals distributed in the area of d 4.6e4.9 (Fig. 4). More than four peaks were shown in the anomeric region (d 4.4e5.3) of the 1H NMR. Two peaks at d 1.16 and d 1.17 were from proton resonances of methyl groups in Rha. The corresponding resonance in the 13C spectrum was at d 16.74. According to 1H NMR spectrum, PLWE-4 mainly had four anomeric proton signals at d 5.14, 4.96, 4.93 and 4.47, which were labeled as A, B, C and D. The corresponding anomeric carbon signals in 13C NMR spectrum were labeled with the contribution of HSQC (Fig. 5). All 1H and 13C NMR of labeled residues were assigned from COSY (Fig. 6), TOCSY (not shown), HSQC and NOESY (Fig. 6) spectra. The conclusion and deduction of main residues' chemical shifts are shown in Table 3. Residue A was in a-configuration according to chemical shifts of
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Table 1 Physicochemical properties, molecular weight and monosaccharide compositions of different polysaccharide fractions from the seeds of Plantago asiatica L. Polysaccharide fractionsa
Sugar content (%)
Uronic acid content Protein content (%) (%)
Relative average molecular weight
Monosaccharide compositions (%)
PLWE-50 PLWE-NAX PLWE-AX PLWE-1 PLWE-2 PLWE-3 PLWE-4 PLWE-5
58.0 40.4 64.7 40.4 63.6 37.5 41.3 28.0
24.2 26.1 17.8 3.5 17.6 34.3 39.4 31.3
n.d. n.d. n.d. n.d. 1153028 463380 594047 953224
4.78 21.69 1.00 1.88 1.29 46.15 24.2 26.28
2.0 4.6 1.5 n.d. 0.1 0.1 2.3 4.8
Rhamnose Arabinose Xylose Mannose Glucose Galactose 18.58 19.10 20.71 48.89 13.44 16.84 19.32 23.14
68.00 43.83 75.05 1.99 80.49 17.39 46.33 36.99
1.09 1.19 n.d. 6.39 0.43 0.78 n.d. n.d.
1.98 2.13 1.05 3.54 0.57 n.d. n.d. 2.5
5.57 12.05 2.19 37.31 3.77 18.84 10.15 11.09
n.d., not determined. a PLWE-50 was the water extracted polysaccharide from the seeds of Plantago asiatica L. at a temperature of 50 C. It was separated into arabinoxylan (PLWE-AX) and nonarabinoxylan polysaccharide (PLWE-NAX). PLWE-1, PLWE-2, PLWE-3, PLWE-4 and PLWE-5 were different fractions purified from PLWE-AX by anion exchange chromatography and gel permeation chromatography as shown in Fig. 2.
Table 2 Methylation analysis results of PLWE-1, PLWE-4 and PLWE-5. Linkages
PLWE-1 (%)a
PLWE-4 (%)a
PLWE-5 (%)a
m/z (mass-to-charge ratio)
1,2-linked Rhap 1,2,4-linked Rhap 1,3,4-linked Rhap T-linked Araf 1,3-linked Araf 1,5-linked Araf 1,3-linked Xylp 1,4-linked Xylp 1,2,4-linked Xylp 1,3,4-linked Xylp T-linked Galp 1,2-linked Galp 1,3-linked Galp 1,4-linked Galp 1,6-linked Galp 1,3,6-linked Galp 1,4,6-linked Galp 1,4-linked Glcp 1,6-linked Glcp 1,4,6-linked Glcp
trace n.d. n.d. 5.00 2.41 28.97 n.d. n.d. n.d. n.d. 8.00 3.88 24.34 2.63 trace 15.92 trace trace 5.33 3.52
25.01 trace 2.44 12.11 8.07 n.d. 9.37 n.d. 5.79 20.01 trace n.d. 2.92 n.d. 2.27 5.95 6.05 n.d. n.d. n.d.
30.18 5.91 trace 2.72 6.90 n.d. 9.98 2.15 6.18 20.90 2.43 n.d. 3.66 trace trace 6.53 n.d. 2.45 n.d. n.d.
43,89,130,131,190 43,101,130,143,190,203 43,87,89,100,131,262 43,71,87,101,102,118,129,161 43,59,87,99,118,233 43,87,102,118,129,189 43,87,102,118,129,189 43,59,71,87,102,118,129,189 43,57,71,87,88,129,130,189,190 43,99,118 43,71,87,89,102,118,129,145,161,162,205 43,87,88,100,129,130,145,161,190,205 43,71,87,101,118,129,161,234 43,87,99,102,113,118,162,173,233 43,59,71,87,99,102,118,129,162,173,189,233 43,59,87,101,118,129,189,234 43,87,102,118,162 43,59,99,102,113,118,129,162,173,233 43,87,99,102,118,129,162,173,189,233 43,85,99,102,118,162,201,261
a
Peak percentage was calculated according to total ion current; n.d.: not determined.
anomeric carbon (d 99.12) and proton (d 5.14), with a relative high content in PLWE-4. Other proton of residue A (from H-2 to H-6) was assigned from COSY spectrum (Fig. 6). The corresponding chemical shifts of carbon were d 76.57, 69.50, 72.93, 71.30 and 16.52 for C-2, C-3, C-4, C-5 and C-6, successively, which was supported by HSQC spectrum. According to references (Neiwert, Holst, & Duda, 2014; Polle, Ovodova, Shashkov, & Ovodov, 2002; Qian, Cui, Nikiforuk, & Goff, 2012), residue A was attributed to be a-1,2-linked Rhap. Chemical shifts of anomeric proton of residue B and C were d 4.96 and d 4.93, respectively. The corresponding chemical shifts of anomeric carbon were d 99.12 for residue B and d 97.88 for residue C. The cross peaks of their other proton (H-2, H-3, H-4 and H-5) were assigned in the COSY spectrum (Table 3). For residue B, signals of C-2, C-3, C-4 and C-5 were at d 71.98, d 69.41, d 77.73 and d 70.83 as revealed by HSQC. Other carbon signals of residue C could also be assigned similarly. From methylation analysis results, the content of 1,4-linked GalAp was 29.36% among all the detected residues. Therefore, residue B and C were assigned to be 1,4-linked GalAp for their high intensity of anomeric carbon and proton. The residues of 1,4-linked GalAp in the polysaccharide were linked in a-configuration. Chemical shift of the C-6 was d 173.68. A full assignment of chemical shifts for 1,4-linked GalAp was in agreement with previous reports (Ding et al., 2015; Li, Cui, Nie, & Xie, 2014; Qian et al.,
2012). From 1H and 13C NMR, chemical shifts of proton (d 2.24) and carbon (d 19.82) were found which indicated part of carboxyl group in 1,4-linked GalAp was acetylated (Zhang, Nie, Yin, Wang, & Xie, 2014). It was consistent with IR result. Residues D whose chemical shift of anomeric proton was d 4.40 was assigned as 1,3,4-linked Xylp, as it had less intensive signal at d 102.77. It was in b-configuration. Other chemical shifts of protons and carbons were assigned by COSY and HSQC. The downfield chemical shift of C-3 (d 79.91) confirmed the residue was 1,3,4linked. The proton and carbon chemical shifts of residue D were supported by literature values (Apirattananusorn, Tongta, Cui, & Wang, 2008; Yin et al., 2012). HMBC and NOESY are powerful spectra in revealing glycosylic linkages between sugar residues. Intra-residue connectives could also be found. Intra-residues and inter-residues correlations in PLWE-4 are shown in Table 4. From NOESY spectrum, some interresidual cross peaks were observed, which were A H-1 to C H-1, A H-1 to C H-2, A H-1 to C H-4, A H-1 to C H-5; A H-1 to B H-4; B H-1 to C H-1, B H-1 to C H-2, B H-1 to C H-3, B H-1 to C H-4; B H-1 to A H2; C H-1 to A H-2, C H-1 to A H-3; C H-1 to B H-4, C H-1 to B H-5. According to above analysis results, main repeating unit of PLWE-4 was deduced as: /1)-a-GalAp-(4 / 1)-a-Rhap-(2 / 1)-a-GalAp(4 / .
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Fig. 4.
13
C and 1H NMR spectra of PLWE-4.
Fig. 5. Part of HSQC spectrum of PLWE-4.
4. Conclusion This is the first report about systematic exploration on physicochemical properties and structural characteristics of nonarabinoxylan polysaccharide, which was isolated and purified from the seeds of Plantago asiatica L.. There were three different kinds of polysaccharide. They were arabinogalactan (PLWE-1), xylan (PLWE-2) and pectin (PLWE-3, PLWE-4, PLWE-5). According to methylation analysis results, PLWE-1 mainly contained T-linked Araf, 1,5-linked Araf, T-linked Galp, 1,3-linked Galp and 1,3,6-linked Galp. PLWE-5 had residual linkages such as 1,2-linked Rhap, 1,3linked Araf, 1,3-linked Xylp, 1,2,4-linked Xylp, 1,3,4-linked Xylp and 1,3,6-linked Galp. Primary structure of PLWE-4 was elucidated by methylation and 1D/2D NMR spectra. However, more functional properties of the non-arabinoxylan fractions from the seeds of Plantago asiatica L. should be explored to elucidate the structurefunction relationship.
Fig. 6. Part of COSY (A) and NOESY (B) spectra of PLWE-4.
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Table 3 Chemical shifts of main residues in PLWE-4 from 1H/13C NMR. Residues
A B C D
Linkage information
a-1,2-linked Rhap a-1,4-linked GalAp a-1,4-linked GalAp b-1,3,4-linked Xylp
Chemical shifts of different proton/carbon (d) H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5/C-5
H-6/C-6
5.14/99.12 4.96/99.12 4.93/97.88 4.47/102.77
4.00/76.57 3.50/71.98 3.79/69.56 3.25/72.93
3.78/69.50 3.60/69.41 4.02/70.02 3.65/79.91
3.31/72.93 4.37/77.73 4.32/77.73 4.23/82.65
3.60/71.30 4.77/70.83 3.85/68.02 3.70/61.38
1.13/16.52 /173.68 /173.68 /
Table 4 Assignment of cross-peaks in the NOESY spectrum of PLWE-4. Residue (linkage information)
Proton
Chemical shift (d)
Connectivities Inter-connectivities
A (1,2-linked Rhap)
H-1
5.14
B (1,4-linked GalAp)
H-1
4.96
C (1,4-linked GalAp)
H-1
4.93
Intra-connectivities
Residue, proton
Chemical shift (d)
Residue, proton
Chemical shift (d)
C H-3 C H-2 B H-4 C H-4 C H-5 C H-1 C H-2 C H-3 A H-2 C H-4 A H-3 A H-2 B H-4 B H-5
3.60 3.78 4.32 4.37 4.75 4.93 3.78 3.99 4.00 4.32 3.78 4.00 4.37 4.77
A A A A
H-4 H-5 H-3 H-2
3.31 3.60 3.78 4.00
B H-2 B H-4
3.50 4.37
C H-2 C H-3 C H-4
3.79 3.99 4.32
Acknowledgments This study is financially supported by the National Natural Science Foundation of China (No: 31301434, 21564007), Research Fund for the Doctoral Program of Higher Education of China (No: 20133601120009), and Jiangxi Provincial Natural Science Foundation (20142BAB214004), which are gratefully acknowledged. The authors wish to thank Mr. Wei Zhou from Centre for Nutrition and Food Science, Queensland Alliance for Agricultural and Food Innovation, the University of Queensland, Australia for kind discussions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodhyd.2015.11.011. References Aisa, H., Abdurahim, M., Yili, A., Xui, L., Rakhmanberdyeva, R., & Sagdullaev, B. (2006). Polysaccharides of Plantago ovatax seeds. Chemistry of Natural Compounds, 42(3), 347e348. Apirattananusorn, S., Tongta, S., Cui, S. W., & Wang, Q. (2008). Chemical, molecular, and structural characterization of alkali extractable nonstarch polysaccharides from Job's tears. Journal of Agricultural and Food Chemistry, 56(18), 8549e8557. Barker, S. A., Bourne, E. J., Stacey, M., & Whiffen, D. H. (1954). Infra-red spectra of carbohydrates. Part I. Some derivatives of D-glucopyranose. Journal of the Chemical Society (Resumed), 171e176. Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54(2), 484e489. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1), 248e254. Carpita, N. C., & Shea, E. M. (1989). Linkage structure of carbohydrates by gas chromatography-mass spectrometry (GC-MS) of partially methylated alditol acetates. In C. J. Biermann, & G. D. McGinnis (Eds.), Analysis of carbohydrates by GLC and MS (pp. 157e216). Boca Raton: CRC Press. Chatjigakis, A., Pappas, C., Proxenia, N., Kalantzi, O., Rodis, P., & Polissiou, M. (1998). FT-IR spectroscopic determination of the degree of esterification of cell wall pectins from stored peaches and correlation to textural changes. Carbohydrate
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