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Analysis of pectin from Panax ginseng flower buds and their binding activities to galectin-3
International Journal of Biological Macromolecules 128 (2019) 459–467
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Analysis of pectin from Panax ginseng flower buds and their binding activities to galectin-3 Liangnan Cui, Jiayi Wang, Rui Huang, Ya Tan, Fan Zhang, Yifa Zhou, Lin Sun ⁎ Jilin Province Key Laboratory on Chemistry and Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University, Changchun 130024, PR China
a r t i c l e
i n f o
Article history: Received 20 November 2018 Received in revised form 23 January 2019 Accepted 23 January 2019 Available online 28 January 2019 Keywords: Ginseng flower buds Polysaccharides Pectin Galectin-3
a b s t r a c t Water-soluble pectic polysaccharides isolated from Panax ginseng flower buds (WGFPA) were completely fractionated into six homogeneous fractions (WGFPA-1a, WGFPA-2a, WGFPA-3a, WGFPA-1b, WGFPA-2b and WGFPA-3b) by a combination of ion-exchange and size exclusion chromatographies. Monosaccharide composition, enzymatic hydrolysis and 13C nuclear magnetic resonance (NMR) spectra analysis were combined to characterize their structural features. Furthermore, the interactions between these polysaccharides and galectin-3 were evaluated by biolayer interferometry assay. The results showed that WGFPA-1a, WGFPA-2a and WGFPA-3a were rhamnogalacturonan I (RG-I) type pectin with abundant side chains, including α-L-1,5arabinan, β-D-1,4-galactan, arabinogalactan I (AG-I) and arabinogalactan II (AG-II), exhibiting strong binding activities to galectin-3 with apparent KD values 4.9 μM, 0.71 μM and 0.24 μM, respectively. WGFPA-1b, WGFPA-2b and WGFPA-3b were homogalacturonan (HG) type pectin covalently linked with different ratios of rhamnogalacturonan II (RG-II) domains, showing weaker or no interactions with galectin-3. This study provides useful structural information for further investigation on the structure-activity relationship of ginseng flower buds pectin. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Pectin is a group of acidic heteropolysaccharide, which is considered to be the most complicated plant cell wall polysaccharide [1]. Galacturonic acid (GalA), galactose (Gal), arabinose (Ara), and rhamnose (Rha) are the main monosaccharides of pectin, organized into distinct structural domains. Pectin is known to consist of linear homogalacturonan (HG) domain as smooth region and branched rhamnogalacturonan I and II (RG-I and RG-II) domains as hairy regions [2]. HG is a linear α-(1,4)-linked D-GalpA homopolymer, in which GalA residues can be partly methyl-esterified at C-6 and partly acetyl-esterified at O-2 and/or O-3 [3]. RG-I is composed of [→2)-αL-Rhap-(1 → 4)-α-D-GalpA-(1→] repeating units as backbone, decorated with Ara- and Gal- containing side chains at O-4 of Rha residues. The side chains of RG-I consist of arabinogalactan I and II (AG-I and AG-II), arabinan and galactan [4]. RG-II is a low molecular weight (5–10 kDa) pectic polysaccharide, which consists of a HG
⁎ Corresponding author. E-mail addresses: [email protected] (L. Cui), [email protected] (J. Wang), [email protected] (R. Huang), [email protected] (Y. Tan), [email protected] (F. Zhang), [email protected] (Y. Zhou), [email protected] (L. Sun).
https://doi.org/10.1016/j.ijbiomac.2019.01.129 0141-8130/© 2019 Elsevier B.V. All rights reserved.
backbone containing unusual sugars such as 3-deoxy-D-manno-2octulosonic acid (Kdo), 3-deoxy-D-lyxo-2-heptulosaric acid (Dha), apiose (Api) and aceric acid (AceA) [5]. Panax ginseng C. A. Meyer (ginseng) is a well-known traditional Chinese medicine, and has been used for thousands of years as a panacea in the Orient. The whole plant of ginseng, including roots, leaves, flower buds and fruits, can be used in medicine, among which the roots are considered to be the major medicinal part [6]. Saponins are the principal active components of all parts of ginseng, which have been extensively studied [7,8]. Polysaccharides are the most abundant component of ginseng. Up to now, the structures and pharmaceutical activities of polysaccharides from roots, leaves and fruits of ginseng have been investigated [9–12]. Our lab has performed a series of studies on the structures and biological activities of polysaccharides from ginseng roots, especially on pectin-derived polysaccharides [13–19]. Ginseng flower buds are usually used as healthy tea due to their medicinal potential such as anti-fatigue and immunity-enhancement. To our knowledge, the structures and biological activities of polysaccharides from ginseng flower buds have not been reported. In this study, water-soluble pectic polysaccharides were extracted from ginseng flower buds, systematically fractionated and structurally characterized. The binding activities of different polysaccharide fractions to galectin-3, which is a molecular target in the development of
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anti-cancer therapeutics, were evaluated. This study provides structural information of water-soluble pectic polysaccharides from ginseng flower buds, which will promote the applications of polysaccharides from ginseng flower buds in functional food or medicine. 2. Materials and methods 2.1. Materials Panax ginseng flower buds were collected from Changbai Mountain, Jinlin, China. DEAE –Cellulose was purchased from Shanghai Chemical Reagent Research Institute (Shanghai, China). Sepharose CL-6B and Sephacryl S-200 were purchased from GE Healthcare (Pittsburgh, USA). endo-1,5-α-Arabinanase (E.C. 3.2.1.99), α-Arabinofuranosidase (E.C. 3.2.1.55), endo-1,4-β-Galactanase (E.C. 3.2.1.89), β-Galactosidase (E.C. 3.2.1.23) from Aspergillus niger and pectate lyase (E.C. 4.2.2.2) from Aspergillus sp were purchased from Megazyme (Bray, Ireland). All other reagents were of analytical or HPLC grade made in China. 2.2. General methods Total carbohydrate and uronic acid contents were determined by phenol‑sulfuric acid and m-hydroxyl diphenyl methods, using glucose (Glc) and galacturonic acid (GlcA) as standards independently [20,21]. Kdo and Dha were colorimetrically determined using the modified thiobarbituric acid (TBA) method [22]. Protein content was determined by the Bradford method, with bovine serum albumin (BSA) as standard [23]. Monosaccharide composition was analyzed by PMP pre-column derivatization and HPLC method as previously described [18]. To determine the ash content, polysaccharide (100 mg) was heated in a muffle furnace at 550 °C for 5 h until the sample reached a constant weight [24]. Homogeneity and molecular weight (Mw) were determined by high performance size exclusion chromatography (HPSEC) on a TSK-gel G-3000PWXL column or TSK-gel G-4000PWXL column (7.8 × 300 mm, TOSOH, Tokyo, Japan) coupled to a Shimadzu HPLC system (Tokyo, Japan). 2.3. Extraction and fractionation of polysaccharides from ginseng flower buds Dried ginseng flower buds (500 g) were washed to remove the dust and immersed in 8 L distilled water for 12 h, then extracted with distilled water at 100 °C for 3 h. The extraction was repeated 3 times. After filtration, the filtrates were concentrated under 60 °C and centrifuged. The supernatant was precipitated by the addition of 95% ethanol (3 volumes). After centrifugation, the precipitate was re-dissolved in water, then dialyzed using dialysis membrane (MWCO 3500 Da) and lyophilized to get water-soluble ginseng flower polysaccharides (WGFP). WGFP (30 g) was dissolved in 300 mL distilled water and applied to a DEAE-Cellulose column (Cl¯), eluting with water to give the un-bound fraction (WGFPN) and then with 0.5 M NaCl to give the bound fraction (WGFPA). WGFPA was applied to DEAE-Cellulose column again, and eluted with stepwise 0, 0.1, 0.2, 0.3 and 0.5 M NaCl to produce five fractions WGFPA-N, WGFPA-1, WGFPA-2, WGFPA-3 and WGFPA-4, respectively. Major fractions WGFPA-1, WGFPA-2 and WGFPA-3 were further applied to Sephacryl S-200 or Sepharose CL-6B column to give pectic fractions a (WGFPA-1a, WGFPA-2a and WGFPA-3a) and pectic fractions b (WGFPA-1b, WGFPA-2b and WGFPA-3b). All of the collected fractions were concentrated, dialyzed using dialysis membrane (MWCO 3500 Da) and lyophilized. 2.4. Enzymatic hydrolysis WGFPA-1a, WGFPA-2a and WGFPA-3a (15 mg) were separately dissolved in 1.5 mL sodium acetate buffer (100 mM, pH 4.0). Digestions were carried out with a combination of endo-1,4-β-Galactanase, β-
Galactosidase, endo-1,5-α-Arabinanase and α-Arabinofuranosidase (2 U/mL of each enzyme) at 40 °C for 24 h. The enzyme was inactivated by heating at 100 °C for 10 min and removed by centrifugation. The supernatant was dialyzed (molecular weight cut-off of 3500 Da) against distilled water. Polymeric fraction inside of the dialysis membrane was collected. WGFPA-1b, WGFPA-2b and WGFPA-3b (5 mg) were separately dissolved in 500 μL Tris HCl buffer (50 mM, pH 8.0) and incubated with 3 U of pectate lyase at 40 °C for 24 h. After enzyme inactivation by boiling for 10 min and centrifugation at 10000 rpm for 5 min, solutions were passed through a 0.22 μm filter prior to further HPSEC analysis. 2.5. NMR spectroscopy 13 C NMR spectrum was recorded at 25 °C with a Bruker Avance 600 MHz spectrometer (Bruker, Karlsruhe, Germany), operating at 150 MHz. Samples (20 mg) were dissolved in D2O (99.8%, 0.5 mL), freeze-dried, and re-dissolved in D2O (0.5 mL). Chemical shifts were given in ppm, with acetone as an internal chemical shift reference.
2.6. Biolayer interferometry The affinities of WGFP-related pectic fractions for galectin-3 were measured by biolayer interferometry using a ForteBio Octet RED 96 instrument (Fortebio, Fremont, CA, USA). Ni-NTA biosensors (Fortebio) were hydrated with PBS for 10 min prior to performing the experiment. The concentration of His-tagged Gal-3 was 10 μg/mL, and monitoring was as follows: initial baseline for 120 s, loading for 120 s, baseline for 120 s, association for 100 s, and dissociation for 100 s. For Ni-NTA biosensors, the regeneration buffer was 10 mM glycine (pH 2.0), and recharging (10 mM NiCl2 in H2O) was done for 60 s. The kinetics buffer was PBS (pH 7.2) with 0.1% Tween 20 and 1% BSA. To determine binding kinetics, five concentrations of each sample were dissolved in kinetics buffer (3.9, 7.8, 15.6, 31.2 and 62.4 μM for WGFPA-1a; 0.7, 1.4, 2.8, 5.6 and 11.2 μM for WGFPA-2a; 0.2, 0.4, 0.8, 1.6 and 3.2 μM for WGFPA3a; 78.1, 156.2, 312.4, 624.8 and 1249.6 μM for WGFPA-1b; 28.7, 57.4, 114.8, 229.6 and 459.2 μM for WGFPA-2b; 0.3, 0.6, 1.2, 2.4 and 4.8 μM for WGFPA-3b). Data were analyzed using a 1: 1 binding stoichiometry, and the equilibrium dissociation constant, KD, was calculated using Octet® RED96 Data Analysis Software version 7.0 (Fortebio). 3. Results 3.1. Preparation of pectin from ginseng flower buds Water-soluble ginseng flower buds polysaccharides (WGFP, yield 5.2%) were obtained following hot water extraction and ethanol precipitation. WGFP contained 82% of carbohydrate, 10% of ash and 3% of protein. Monosaccharide composition analysis (Table 1) showed that WGFP
Table 1 Yields, Mws and monosaccharide compositions of fractions from WGFP. Fraction
Yeild Mw Monosaccharide composition (mol%) (%W) (KDa) GalA Rha Gal Ara Glc GlcA Man Xyl Fuc
WGFP WGFPA WGFPA-1a WGFPA-1b WGFPA-2a WGFPA-2b WGFPA-3a WGFPA-3b
5.2a 55.2b 14.3b 47.1b 22.9b 43.3b 18.2b 51.3b
a b
15.8 3.2 86.5 8.7 302.5 27.2
30.1 49.9 3.9 56.9 14.3 70.3 19.0 82.2
4.2 5.0 3.1 6.0 9.0 10.2 13.4 3.6
32.4 22.2 47.2 11.9 35.3 7.4 27.5 6.3
18.5 17.4 39.4 17.2 39.1 9.4 35.9 5.3
Yield in relation to ginseng flower buds. Yield in relation to fraction applied onto column.
5.3 1.7 2.1 1.7 0.8 0.3 0.9 1.2
2.6 1.6 4.0 2.1 1.3 0.9 0.7 1.8
4.4 1.1 0.3 1.7 0.3 – 0.9 –
2.1 0.8 – 1.7 – 1.6 1.6 0.8
0.4 0.2 – 0.8 – – 0.1 –
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mainly consisted of Gal (32.4%), GalA (30.1%) and Ara (18.5%), with minor Glc (5.3%), mannose (Man) (4.4%), Rha (4.2%), GlcA (2.6%), xylose (Xyl) (2.1%) and fucose (Fuc) (0.4%). The negative result of I 2 -KI assay indicated that WGFP did not contain starch-like polysaccharide. WGFP was then applied to a DEAE-cellulose column (Cl¯), eluting with H2O and 0.5 M NaCl to give one neutral polysaccharide WGFPN (yield 10.9%) and one acidic polysaccharide WGFPA (yield 55.2%) (Fig. 1). The structure of WGFPN was not discussed in this paper. WGFPA contained around 50% of GalA, suggesting it was pectic polysaccharide. It was further fractionated by DEAE-cellulose column (Cl¯), eluting sequentially with water, 0.1 M, 0.2 M, 0.3 M and 0.5 M NaCl, respectively. Five fractions named WGFPA-H (yield 2.0%), WGFPA-1 (yield 15.3%), WGFPA-2 (yield 33.6%), WGFPA-3 (yield 23%) and WGFPA-4 (yield 3.0%) were obtained. Due to the low yield, WGFPA-H and WGFPA-4 were not further analyzed. The other three fractions were then fractionated by size exclusion chromatography. Each fraction was separated into two sub-fractions (Fig. 2). At last, six homogenous polysaccharide fractions WGFPA-1a (yield 14.3%), WGFPA-1b (yield 47.1%), WGFPA-2a (yield 22.9%), WGFPA-2b (yield 43.3%), WGFPA-3a (yield 18.2%) and WGFPA-3b (yield 51.3%) were got. 3.2. Structural feature analysis of pectin from ginseng flower buds The obtained homogeneous polysaccharide fractions were then structurally analyzed. Among these polysaccharides, fractions a (WGFPA-1a, WGFPA-2a and WGFPA-3a) had higher molecular weight, lower yield and lower GalA content than their corresponding fractions b (WGFPA1b, WGFPA-2b and WGFPA-3b) (Table 1). Fractions a mainly contained Gal, Ara, GalA and Rha residues. The ratio of Rha/GalA in WGFPA-1a, WGFPA-2a and WGFPA-3a were 0.8, 0.6 and 0.7, respectively, typical for RG-I type pectin [25]. WGFPA-1b, WGFPA-2b and WGFPA-3b were mainly composed of GalA, suggesting HG was the major pectin type in these fractions. Meanwhile, RG-II was also detected in fractions b by TBA assay. According to their structural similarity and difference, we will analyze the structures of fractions a and fractions b separately. 3.2.1. Structural feature analysis of WGFPA-1a, WGFPA-2a and WGFPA-3a WGFPA-1a, WGFPA-2a and WGFPA-3a were all composed of high contents of Gal and Ara (Table 1). Since Gal and Ara are the major sugars in neutral side chains and Rha is the branching point, the ratio of Ara + Gal/Rha could roughly reflect the amount/length of
Fig. 2. Elution profiles of (A) WGFPA-1 and (B) WGFPA-2 on a Sephacryl S-200 column; (C) WGFPA-3 on a Sepharose CL-6B column (-●- total sugar; -○- uronic acid). Vo is void volume and Vt is total volume.
Fig. 1. Fractionation scheme for WGFP by anion-exchange and size-exclusion chromatographies.
side chain in RG-I [15,26]. For WGFPA-1a, WGFPA-2a and WGFPA3a, Ara + Gal/Rha ratios were 27.9, 8.3 and 4.7 (Table 2), respectively, suggesting the amount/length of side chains in these fractions were in the same order.
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Table 2 Mw and compositional features for fractions a and fraction a-ara-gal. Fraction
WGFPA-1a WGFPA-1a-ara-gal WGFPA-2a WGFPA-2a-ara-gal WGFPA-3a WGFPA-3a-ara-gal
Monosaccharide composition (mol%) GalA Rha
Gal
Ara
3.9 4.9 14.3 30.0 19.0 33.8
47.2 61.4 35.3 34.1 27.5 27.2
39.4 19.1 39.1 10.8 35.9 7.2
3.1 3.8 9.0 18.1 13.4 21.9
Mw Rha/ Ara + (KDa) GalA Gal/ Rha
Gal/ Rha
Ara/ Rha
15.8 15.7 86.5 62.0 302.0 174.0
15.2 16.2 3.9 1.9 2.0 1.2
12.7 5.0 4.3 0.6 2.7 0.3
0.8 0.8 0.6 0.6 0.7 0.7
27.9 21.2 8.3 2.5 4.7 1.6
Their structures were then analyzed by 13C NMR spectrum (Fig. 3), and the chemical shift assignments were listed in Table 3. The signals at δ 100.61 and δ 173.08 were attributed to C-1 and C-6 of α-D-1,4-
GalpA, and the resonance at δ 16.79 and δ 16.35 were assigned to C-6 of α-L-1,2,4-Rhap and α-L-1,2-Rhap [15], respectively, which indicated they had RG-I backbone of alternating α-L-Rhap and α-D-GalpA residues. The low intensity signals for Rha and GalA in the spectrum of WGFPA-1a was consistent with its monosaccharide composition. The anomeric carbon resonances at δ 109.10, δ 108.13, δ 107.31 and δ 106.96 could be assigned to C-1 of α-L-t-Araf, α-L-1,3-Araf, α-L-1,5Araf and α-L-1,3,5-Araf, respectively. Signals at δ 104.22, δ 103.57, δ 103.25, δ 103.01 and δ 102.5 were assigned to the anomeric carbon of β-D-1,4-Galp, β-D-1,3,6-Galp, β-D-t-Galp, β-D-1,6-Galp and β-D-1,3Galp. The complex and overlapping signals at δ 60-δ 85 were attributed to C-2 to C-6 of different linkages of Gal and Ara [27–30]. Through comparison of these NMR spectra, we found WGFPA-1a was mainly composed of β-D-1,6-Galp, β-D-1,3-Galp, β-D-1,3,6-Galp, α-L-1,3-Araf, αL-1,5-Araf and α-L-t-Araf residues, which was consistent with the
Fig. 3. 13C NMR spectra of (A) WGFPA-1a; (B) WGFPA-2a; (C) WGFPA-3a.
L. Cui et al. / International Journal of Biological Macromolecules 128 (2019) 459–467 Table 3 13 C NMR spectral assignments of WGFPA-1a, WGFPA-2a and WGFPA-3a. Fraction
Residues
Chemical shift, δ(ppm) C-1
WGFPA-1a
WGFPA-2a
WGFPA-3a
→4)-α-GalpA-(1→ 100.61 →2)-α-Rhap-(1→ \ \ →5)-α-Araf-(1→ 107.31 →3)-α-Araf-(1→ 108.13 T-Araf 109.1 →6)-β-Galp-(1→ 103.01 →3)-β-Galp-(1→ 102.5 →3,6)-β-Galp-(1→ 103.57 T-Galp 103.25 →4)-α-GalpA-(1→ 100.62 →2)-α-Rhap-(1→ \ \ →3,5)-α-Araf-(1→ 106.96 →5)-α-Araf-(1→ 107.37 T-Araf 109.08 →4)-β-Galp-(1→ 104.22 →6)-β-Galp-(1→ 103.01 T-Galp 103.25 →4)-α-GalpA-(1→ 100.62 →2)-α-Rhap-(1→ \ \ →3,5)-α-Araf-(1→ 106.96 →5)-α-Araf-(1→ 107.32 T-Araf 109.08 →4)-β-Galp-(1→ 104.21 →6)-β-Galp-(1→ 103.01 →3)-β-Galp-(1→ 102.51 →3,6)-β-Galp-(1→ 103.25 T-Galp 103.25
C-2
C-3
C-4
C-5
C-6
\ \ \ \ \ \ 78.98 81.19 70.61 70.14 71.68 \ \ \ \ \ \ \ \ 80.67 81.12 71.68 70.58 \ \ \ \ \ \ \ \ 80.67 80.67 71.68 70.59 \ \ \ \ \ \
\ \ \ \ 76.44 83.49 76.44 72.46 79.97 79.97 \ \ \ \ \ \ 83.76 76.59 76.41 73.17 72.46 \ \ \ \ \ \ 83.75 78.96 76.42 73.17 72.46 80.05 80.05 \ \
\ \ \ \ \ \ \ \ 83.71 68.5 70.6 68.32 \ \ \ \ \ \ 81.12 82.19 83.73 77.52 68.5 \ \ \ \ \ \ 81.12 82.19 83.71 77.52 68.8 70.59 68.31 \ \
\ \ \ \ \ \ 61.13 60.94 74.14 73.25 73.25 \ \ \ \ \ \ 66.35 66.74 60.95 74.37 \ \ \ \ \ \ \ \ 66.35 66.74 60.95 74.37 \ \ \ \ \ \ \ \
\ \ 16.35
69.73 61.13 69.73 \ \ 173.08 16.79
60.61 69.73 \ \ 173.08 16.37
60.61 69.73 61.13 69.73 \ \
structural feature of AG-II side chains [4]. WGFPA-2a contained α-L1,5-Araf, α-L-1,3,5-Araf and β-D-1,4-Galp glycosyl linkages, indicating it might possess β-D-1,4-galactan and α-L-1,5-arabinan or AG-I side chains. WGFPA-3a had more complex side chains than the other two fractions. It contained almost all kinds of glycosidic linkages mentioned-above. Thus, WGFPA-3a might contain a mixture of α-L-1,5-arabinan, β-D-1,4-galactan, AG-I and AG-II as side chains. To further analyze the side chain structures of WGFPA-1a, WGFPA-2a and WGFPA-3a, they were degraded by endo-1,4-β-D-galactanase, β-Dgalactosidase, endo-1,5-α-L-arabinanase and α-L-arabinofuranosidase in combination. After removing released oligosaccharides by dialysis, the left polysaccharide fractions (WGFPA-1a-ara-gal, WGFPA-2a-ara-gal and WGFPA-3a-ara-gal) were obtained. Their major monosaccharide compositions and molecular weights were listed in Table 2. WGFPA-1aara-gal had the same molecular weight compared with WGFPA-1a, indicating WGFPA-1a was not greatly degraded by these enzymes. The ratio of Ara/Rha or Gal/Rha was calculated and used to compare the change of Ara and Gal before and after enzymatic degradation. The result showed that Gal residues in WGFPA-1a were hardly degraded by the enzymes, and Ara residues were degraded by 60%, suggesting that WGFPA-1a did not contain β-D-(1,4)-Galp residues and contain α-L-1,5-Araf or α-L-1,3-Araf, agreed with AG-II side chain structure analyzed by NMR. WGFPA-2a-ara-gal (62.0 KDa) had lower molecular weight than WGFPA-2a (86.5 KDa), indicating an effective enzymatic hydrolysis. The ratio of Gal/Rha and Ara/Rha decreased significantly by 51% and 86%, respectively. Therefore, side chains such as β-D-1,4-galactan, α-L-1,5-arabinan or AG-I side chains existed in WGFPA-2a, which was consistent with the NMR result. The incomplete removal of Gal residues from WGFPA-2a might be due to the branching of Ara on them. These enzyme treatments also reduced the Gal and Ara level of WGFPA-3a by 40% and 89%, producing WGFPA-3a-ara-gal with a lower molar weight. This result proved that WGFPA-3a also contained β-D-1,4-galactan, α-L-1,5arabinan or AG-I side chains. However, less Gal residues in WGFPA-
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3a were hydrolyzed than WGFPA-2a, which suggested that other linkages of Gal residues such as β-D-1,3-Galp, β-D-1,6-Galp or β-D-1,3,6-Galp might also exist. Thus, WGFPA-3a had a mixture of α-L-1,5-arabinan, β-D-1,4-galactan, AG-I and AG-II side chains. 3.2.2. Structural feature analysis of WGFPA-1b, WGFPA-2b and WGFPA-3b The content of GalA in WGFPA-1b, WGFPA-2b and WGFPA-3b was 56.9%, 70.3% and 82.2% (Table 1), respectively, suggesting they contained HG domain. TBA assay is a very sensitive reaction used to identify Kdo and Dha, which are characteristic monosaccharides in RG-II [22]. All fractions b contained TBA-positive constituents, indicating RG-II domain existed in these fractions. Other monosaccharides such as Rha, Gal, Ara, GlcA and Fuc might belong to RG-II domain in fractions b. Their structures were further analyzed by 13C NMR spectrum (Fig. 4). The major resonance signals at δ 99.03, δ 67.94, δ 68.53, δ 77.85, δ 70.94 and δ 174.64 were attributed to C-1 to C-6 of α-D-1,4-GalpA, respectively. The signal at δ 170.77 was assigned to C-6 of methyl-esterified α-D1,4-GalpA, and δ 52.74 were assigned to the methyl group, suggesting HG segments in these fractions were methyl-esterified [15]. The degree of methyl-esterification (DM) can be estimated by the integral peak area of the signals at δ 174.64 and δ 170.77, which were assigned to un-esterified carboxyl (U) and esterified carboxyl (E), respectively. Using formula Area (E)/Area (E + U), DM was estimated to be ~40%, ~30% and ~20% for WGFPA-1b, WGFPA-2b and WGFPA-3b, respectively [18]. WGFPA-1b showed more complex spectrum than WGFPA-2b and WGFPA-3b. Besides major signals for α-D-1,4-GalpA residues, some characteristic signals representing for RG-II domain were also found. Signals at δ 107.45, δ 103.25 and δ 96.6 were assigned to anomeric carbons of α-Araf, α-Arap and 2-O-Me-α-Xylp, respectively. Signals at δ 96.03 and δ 92.06 were assigned to C-2 of α-Kdop and α-AcefA [31,32]. The spectrum of WGFPA-2b was similar to that of WGFPA-1b, but characteristic signals for RG-II domain were in lower intensity, indicating the ratio of RG-II was lower in WGFPA-2b. The NMR spectrum of WGFPA-3b demonstrated that HG was the dominant structure in this fraction. To further characterize the structure of WGFPA-1b, WGFPA-2b and WGFPA-3b, pectate lyase which is able to cleavage of α-D-(1,4)galacturonan was used to degrade these fractions. The molecular weight change of enzymatic degradation product was detected by HPSEC (Fig. 5). As can be seen, the three fractions were all degraded by pectate lyase, producing two oligosaccharide pools (OP I and OP II) with molecular weight of 1.8 KDa (OP I, retention time ~14.7 min) and 1.1 KDa (OP II, retention time ~15.3 min). These results suggested that WGFPA-1b, WGFPA-2b and WGFPA-3b all contained HG domain, consistent with the NMR analysis. After enzymatic degradation, one polysaccharide pool (PP) which was resistant to pectate lyase hydrolsyis was also found in WGFPA-1b and WGFPA-2b. The molecular weight of this pool from WGFPA-1b and WGFPA-2b was 3.3 KDa and 4.6 KDa, respectively, in accordance with that of monomeric RG-II [5]. The polysaccharide pool standing for RG-II domain in degradation product of WGFPA-3b was in extremely low abundance, indicating WGFPA-3b was mainly composed of HG and very less RG-II. Based on these analyses, WGFPA-1b, WGFPA2b and WGFPA-3b all contained covalently-linked HG and RG-II domains, where RG-II was predominant in WGFPA-1b, HG was predominant in WGFPA-3b, and nearly equivalent HG and RG-II was present in WGFPA-2b. 3.3. Detection the binding of pectin from ginseng flower buds to galectin-3 The interaction between ginseng flower buds pectin and galectin-3 was measured by biolayer interferometry (BLI) using the Ni-NTA sensor [33]. The association and dissociation curves of pectic fractions from ginseng flower buds at various concentrations were shown in Fig. 6. Nonspecific binding has been eliminated as in the absence of galectin-3, polysaccharides hardly bound to sensors even at the highest concentrations
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Fig. 4. 13C NMR spectra of (A) WGFPA-1b; (B) WGFPA-2b; (C) WGFPA-3b.
used in the study. Analysis of the BLI data suggested that three RG-I type pectin WGFPA-1a, WGFPA-2a and WGFPA-3a exhibited strong binding avidity to galectin-3, with apparent KD values of 4.90 μM for WGFPA1a, 0.71 μM for WGFPA-2a and 0.24 μM for WGFPA-3a. For covalentlylinked HG and RG-II fractions, only WGFPA-2b could bind to galectin-3 (KD 48.41 μM), while WGFPA-1b and WGFPA-3b did not exhibit binding to galectin-3. Different binding avidities of pectic fractions from ginseng flower buds to galectin-3 depend on their various structures. WGFPA1a, WGFPA-2a and WGFPA-3a possessed strong binding activity to galectin-3 due to their RG-I structures. Both the backbone and abundant side chains in these RG-I fractions might contribute to the binding [13]. WGFPA-3a had a higher molecular weight than that of WGFPA-2a, indicating the existence of more binding sites, which resulted in higher binding avidity. The lower binding activity of WGFPA-1a to galectin-3 than WGFPA-2a and WGFPA-3a was probably caused by less RG-I backbone and lower molecular weight. WGFPA-1b, WGFPA-2b and WGFPA-3b all contained covalently-linked HG and RG-II structures, but the ratios of RG-II domain to HG domain in these fractions were different. WGFPA-
1b was mainly composed of RG-II domain. The high methylesterification, high degree of substitution on the backbone, low content of Gal residues and low molecular weight of this fraction might make it not binding to galectin-3. WGFPA-3b was composed of methylesterified HG domain. Our lab previously found that HG domain could also bind to galectin-3, and un-esterified and non-substituted ‘smooth’ backbone is crucial for the binding [34]. Here, the methyl-esterification occurring on the HG backbone in WGFPA-3b might cause the absence of binding activity. Interestingly, WGFPA-2b containing around equivalent ratio of HG and RG-II possessed the binding ability to galectin-3. It was speculated that a synergistic effect might occur when RG-II was covalently-linked with HG in some specific molar ratio, like the synergistic effect between RG-I with HG [34]. 4. Discussion Water-soluble polysaccharides from ginseng roots have been studied for many years, and their structures were well characterized
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Fig. 5. HPSEC of fractions b and their products after enzymatic degradation (A) WGFPA-1b and its degradation products; (B) WGFPA-2b and its degradation products; (C) WGFPA-3b and its degradation products. Vo is void volume and Vt is total volume.
[13–15,18]. In our previous study, water-soluble ginseng polysaccharides (WGP, yield 10.7%) were extracted from ginseng roots with hot water and precipitated with ethanol [18]. WGP was then applied to DEAE-Cellulose column, eluting with H2O and 0.5 M NaCl to give neutral polysaccharide (WGPN) and acidic polysaccharide (WGPA). WGPA was applied to DEAE-Cellulose column again, and eluted with stepwise H2O, 0.1, 0.2, 0.3 and 0.5 M NaCl to produce five fractions WGPA-N, WGPA-1,
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WGPA-2, WGPA-3 and WGPA-4, respectively. WGPA-1, WGPA-2, WGPA-3 and WGPA-4 were further purified by Sepharose CL-6B column to give six pectic fractions (WGPA-1-RG, WGPA-2-RG, WGPA-1HG, WGFPA-2-HG, WGPA-3-HG and WGPA-4-HG). However, there were few studies about polysaccharides from ginseng flower buds. Here, we extracted and systematically fractionated water-soluble polysaccharides from ginseng flower buds and characterized the structures of pectin fractions. The yield for water-soluble polysaccharides of ginseng flower buds was 5.2%, which was lower than the yield of polysaccharides from roots (10.7%). The mass ratio of neutral polysaccharide to acidic polysaccharide is 78:22 for ginseng roots and 16:84 for ginseng flower buds, indicating higher proportion of pectin in ginseng flower buds polysaccharides. The structural features of pectin fractions from both roots and flower buds of ginseng were listed in Table 4. Among pectin fractions, RG-I domains containing high amounts of AG side chains and HG domains alone or covalently linked to RG-II domains were major structures. The ratio for RG-I to HG (+RG-II) was 1:4.7 in ginseng root pectin and 1:2.4 for ginseng flower pectin. As can be seen, RG-I or AG fractions in ginseng root pectin (WGPA-1-RG and WGPA-2-RG) mainly contained type II AG, with molecular weight of 100 and 110 KDa, while RG-I fractions in ginseng flower buds (WGFPA-1a, WGFPA-2a and WGFPA-3a) contained diverse neutral side chains including galactan (G), arabinan (A), AG-I and/or AG-II, and their molecular weight were from 15.8 KDa to 302.5 KDa. The content of GalA residues in HG fractions from ginseng roots was between 62.4% and 92.1%, and DM in these fractions was 5.0% to 30.0%. Due to the covalently linkage of RG-II, the content of GalA in HG fractions of ginseng flower buds was lower than that in ginseng roots, and the DM for these fractions was from 20.0% to 40.0%. Although pectin from ginseng leaves has not been characterized comprehensively like that from roots and flower buds, it also comprises of HG, RG-I, and RG-II domains with RG-II more abundant in leaves [35–37]. Different compositions and structures of polysaccharides in distinct organs of ginseng might be related to their various physiological functions in the plant. Galactin-3 has become a molecular target in the development of anti-cancer therapeutics because of its crucial role in the development and metastasis of various cancers [38]. Natural plant polysaccharides have great potential to be developed as galactin-3 antagonists. Up to now, besides galactomannans, pectin-derived polysaccharides have been identified as main galactin-3 inhibitors. It has been found that pectin-derived polysaccharides with different structures, such as galactan (M-galactan from modified citrus pectin [39]; potato galactan [40]), AG (MCP-N from modified citrus pectin [39]; RN1 from the flowers of Panax notoginseng [41]), RG-I (RG-I-4 and RG-I-3A from ginseng roots [13,19]; RG-2a from modified citrus pectin [42]; PPc from pumpkin [43]) and HG (HG-2b and HG-3p from modified citrus pectin [34]) could bind galactin-3 in different levels. Among these polysaccharides, RG-I type pectin usually showed better binding activities to galactin-3 duo to its complex structures. Our results also revealed that RG-I fractions (WGFPA-1a, WGFPA-2a and WGFPA-3a) purified from water-soluble ginseng flower buds polysaccharides had better binding activities to galactin-3 than other covalently linked HG and RG-II fractions (WGFPA-1b, WGFPA-2b and WGFPA-3b). Among RG-I fractions, WGFPA-2a and WGFPA-3a had better binding avidity than WGFPA-1a, which might be due to their higher molecular weights and higher ratios of RG-I backbone. In addition, some synergistic effect might exist between RG-II and HG domain for binding to galactin-3 when they were in some specific molar ratio. 5. Conclusions In summary, water-soluble pectic polysaccharides were extracted from ginseng flower buds and separated into homogeneous RG-I-type and HG-type pectin. RG-I fractions with
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Fig. 6. Biolayer interferometry analysis of pectin from ginseng flowers (A) WGFPA-1a; (B) WGFPA-2a; (C) WGFPA-3a; (D) WGFPA-1b; (E) WGFPA-2b; (F) WGFPA-3b.
abundant side chains had strong binding avidity to galectin-3. HG fractions exhibited different binding avidities to galectin-3 depending on the ratio of RG-II domain linked. These results provide valuable information for comprehending the structural features of pectin from ginseng flower buds and screening new active galectin-3 inhibitors.
Funding This work was supported by the National Natural Science Foundation of China (grant numbers 31770852), the Scientific and Technologic Foundation of Jilin Province (20180101004JC) and the Natural Science Foundation of Changchun City of China (17YJ004).
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Table 4 Structural features of pectin fractions purified from ginseng roots and flower buds. Parts of ginseng
Roots
Flower Buds
Fractions
WGPA-1-RG WGPA-2-RG WGPA-1-HG WGPA-2-HG WGPA-3-HG WGPA-4-HG WGFPA-1a WGFPA-2a WGFPA-3a WGFPA-1b WGFPA-2b WGFPA-3b
Ratio (%)
DM (%)
Mw (KDa)
Monosaccharide composition (%mol) GalA
Rha
Gal
Ara
4.7 12.7 9.6 33.8 29.8 9.4 4.4 16.2 8.8 15.2 30.5 24.8
– – 30.0 20.0 10.0 5.0 – – – 40.0 30.0 20.0
100.0 110.0 3.5 6.5 16.0 45.0 15.8 86.5 302.5 3.2 8.7 27.2
1.8 5.3 62.4 83.6 90.9 92.1 3.9 14.3 19.0 56.9 70.3 82.2
0.2 4.1 1.6 3.0 1.5 – 3.1 9.0 13.4 6.0 10.2 3.6
56.2 44.4 15.2 5.1 3.5 5.9 47.2 35.3 27.5 11.9 7.4 6.3
34.0 40.9 7.1 4.6 2.2 – 39.4 39.1 35.9 17.2 9.4 5.3
Conflicts of interest The authors declare no conflict of interest.
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Analysis of pectin from Panax ginseng flower buds and their binding activities to galectin-3 Liangnan Cui, Jiayi Wang, Rui Huang, Ya Tan, Fan Zhang, Yifa Zhou, Lin Sun ⁎ Jilin Province Key Laboratory on Chemistry and Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University, Changchun 130024, PR China
a r t i c l e
i n f o
Article history: Received 20 November 2018 Received in revised form 23 January 2019 Accepted 23 January 2019 Available online 28 January 2019 Keywords: Ginseng flower buds Polysaccharides Pectin Galectin-3
a b s t r a c t Water-soluble pectic polysaccharides isolated from Panax ginseng flower buds (WGFPA) were completely fractionated into six homogeneous fractions (WGFPA-1a, WGFPA-2a, WGFPA-3a, WGFPA-1b, WGFPA-2b and WGFPA-3b) by a combination of ion-exchange and size exclusion chromatographies. Monosaccharide composition, enzymatic hydrolysis and 13C nuclear magnetic resonance (NMR) spectra analysis were combined to characterize their structural features. Furthermore, the interactions between these polysaccharides and galectin-3 were evaluated by biolayer interferometry assay. The results showed that WGFPA-1a, WGFPA-2a and WGFPA-3a were rhamnogalacturonan I (RG-I) type pectin with abundant side chains, including α-L-1,5arabinan, β-D-1,4-galactan, arabinogalactan I (AG-I) and arabinogalactan II (AG-II), exhibiting strong binding activities to galectin-3 with apparent KD values 4.9 μM, 0.71 μM and 0.24 μM, respectively. WGFPA-1b, WGFPA-2b and WGFPA-3b were homogalacturonan (HG) type pectin covalently linked with different ratios of rhamnogalacturonan II (RG-II) domains, showing weaker or no interactions with galectin-3. This study provides useful structural information for further investigation on the structure-activity relationship of ginseng flower buds pectin. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Pectin is a group of acidic heteropolysaccharide, which is considered to be the most complicated plant cell wall polysaccharide [1]. Galacturonic acid (GalA), galactose (Gal), arabinose (Ara), and rhamnose (Rha) are the main monosaccharides of pectin, organized into distinct structural domains. Pectin is known to consist of linear homogalacturonan (HG) domain as smooth region and branched rhamnogalacturonan I and II (RG-I and RG-II) domains as hairy regions [2]. HG is a linear α-(1,4)-linked D-GalpA homopolymer, in which GalA residues can be partly methyl-esterified at C-6 and partly acetyl-esterified at O-2 and/or O-3 [3]. RG-I is composed of [→2)-αL-Rhap-(1 → 4)-α-D-GalpA-(1→] repeating units as backbone, decorated with Ara- and Gal- containing side chains at O-4 of Rha residues. The side chains of RG-I consist of arabinogalactan I and II (AG-I and AG-II), arabinan and galactan [4]. RG-II is a low molecular weight (5–10 kDa) pectic polysaccharide, which consists of a HG
⁎ Corresponding author. E-mail addresses: [email protected] (L. Cui), [email protected] (J. Wang), [email protected] (R. Huang), [email protected] (Y. Tan), [email protected] (F. Zhang), [email protected] (Y. Zhou), [email protected] (L. Sun).
https://doi.org/10.1016/j.ijbiomac.2019.01.129 0141-8130/© 2019 Elsevier B.V. All rights reserved.
backbone containing unusual sugars such as 3-deoxy-D-manno-2octulosonic acid (Kdo), 3-deoxy-D-lyxo-2-heptulosaric acid (Dha), apiose (Api) and aceric acid (AceA) [5]. Panax ginseng C. A. Meyer (ginseng) is a well-known traditional Chinese medicine, and has been used for thousands of years as a panacea in the Orient. The whole plant of ginseng, including roots, leaves, flower buds and fruits, can be used in medicine, among which the roots are considered to be the major medicinal part [6]. Saponins are the principal active components of all parts of ginseng, which have been extensively studied [7,8]. Polysaccharides are the most abundant component of ginseng. Up to now, the structures and pharmaceutical activities of polysaccharides from roots, leaves and fruits of ginseng have been investigated [9–12]. Our lab has performed a series of studies on the structures and biological activities of polysaccharides from ginseng roots, especially on pectin-derived polysaccharides [13–19]. Ginseng flower buds are usually used as healthy tea due to their medicinal potential such as anti-fatigue and immunity-enhancement. To our knowledge, the structures and biological activities of polysaccharides from ginseng flower buds have not been reported. In this study, water-soluble pectic polysaccharides were extracted from ginseng flower buds, systematically fractionated and structurally characterized. The binding activities of different polysaccharide fractions to galectin-3, which is a molecular target in the development of
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anti-cancer therapeutics, were evaluated. This study provides structural information of water-soluble pectic polysaccharides from ginseng flower buds, which will promote the applications of polysaccharides from ginseng flower buds in functional food or medicine. 2. Materials and methods 2.1. Materials Panax ginseng flower buds were collected from Changbai Mountain, Jinlin, China. DEAE –Cellulose was purchased from Shanghai Chemical Reagent Research Institute (Shanghai, China). Sepharose CL-6B and Sephacryl S-200 were purchased from GE Healthcare (Pittsburgh, USA). endo-1,5-α-Arabinanase (E.C. 3.2.1.99), α-Arabinofuranosidase (E.C. 3.2.1.55), endo-1,4-β-Galactanase (E.C. 3.2.1.89), β-Galactosidase (E.C. 3.2.1.23) from Aspergillus niger and pectate lyase (E.C. 4.2.2.2) from Aspergillus sp were purchased from Megazyme (Bray, Ireland). All other reagents were of analytical or HPLC grade made in China. 2.2. General methods Total carbohydrate and uronic acid contents were determined by phenol‑sulfuric acid and m-hydroxyl diphenyl methods, using glucose (Glc) and galacturonic acid (GlcA) as standards independently [20,21]. Kdo and Dha were colorimetrically determined using the modified thiobarbituric acid (TBA) method [22]. Protein content was determined by the Bradford method, with bovine serum albumin (BSA) as standard [23]. Monosaccharide composition was analyzed by PMP pre-column derivatization and HPLC method as previously described [18]. To determine the ash content, polysaccharide (100 mg) was heated in a muffle furnace at 550 °C for 5 h until the sample reached a constant weight [24]. Homogeneity and molecular weight (Mw) were determined by high performance size exclusion chromatography (HPSEC) on a TSK-gel G-3000PWXL column or TSK-gel G-4000PWXL column (7.8 × 300 mm, TOSOH, Tokyo, Japan) coupled to a Shimadzu HPLC system (Tokyo, Japan). 2.3. Extraction and fractionation of polysaccharides from ginseng flower buds Dried ginseng flower buds (500 g) were washed to remove the dust and immersed in 8 L distilled water for 12 h, then extracted with distilled water at 100 °C for 3 h. The extraction was repeated 3 times. After filtration, the filtrates were concentrated under 60 °C and centrifuged. The supernatant was precipitated by the addition of 95% ethanol (3 volumes). After centrifugation, the precipitate was re-dissolved in water, then dialyzed using dialysis membrane (MWCO 3500 Da) and lyophilized to get water-soluble ginseng flower polysaccharides (WGFP). WGFP (30 g) was dissolved in 300 mL distilled water and applied to a DEAE-Cellulose column (Cl¯), eluting with water to give the un-bound fraction (WGFPN) and then with 0.5 M NaCl to give the bound fraction (WGFPA). WGFPA was applied to DEAE-Cellulose column again, and eluted with stepwise 0, 0.1, 0.2, 0.3 and 0.5 M NaCl to produce five fractions WGFPA-N, WGFPA-1, WGFPA-2, WGFPA-3 and WGFPA-4, respectively. Major fractions WGFPA-1, WGFPA-2 and WGFPA-3 were further applied to Sephacryl S-200 or Sepharose CL-6B column to give pectic fractions a (WGFPA-1a, WGFPA-2a and WGFPA-3a) and pectic fractions b (WGFPA-1b, WGFPA-2b and WGFPA-3b). All of the collected fractions were concentrated, dialyzed using dialysis membrane (MWCO 3500 Da) and lyophilized. 2.4. Enzymatic hydrolysis WGFPA-1a, WGFPA-2a and WGFPA-3a (15 mg) were separately dissolved in 1.5 mL sodium acetate buffer (100 mM, pH 4.0). Digestions were carried out with a combination of endo-1,4-β-Galactanase, β-
Galactosidase, endo-1,5-α-Arabinanase and α-Arabinofuranosidase (2 U/mL of each enzyme) at 40 °C for 24 h. The enzyme was inactivated by heating at 100 °C for 10 min and removed by centrifugation. The supernatant was dialyzed (molecular weight cut-off of 3500 Da) against distilled water. Polymeric fraction inside of the dialysis membrane was collected. WGFPA-1b, WGFPA-2b and WGFPA-3b (5 mg) were separately dissolved in 500 μL Tris HCl buffer (50 mM, pH 8.0) and incubated with 3 U of pectate lyase at 40 °C for 24 h. After enzyme inactivation by boiling for 10 min and centrifugation at 10000 rpm for 5 min, solutions were passed through a 0.22 μm filter prior to further HPSEC analysis. 2.5. NMR spectroscopy 13 C NMR spectrum was recorded at 25 °C with a Bruker Avance 600 MHz spectrometer (Bruker, Karlsruhe, Germany), operating at 150 MHz. Samples (20 mg) were dissolved in D2O (99.8%, 0.5 mL), freeze-dried, and re-dissolved in D2O (0.5 mL). Chemical shifts were given in ppm, with acetone as an internal chemical shift reference.
2.6. Biolayer interferometry The affinities of WGFP-related pectic fractions for galectin-3 were measured by biolayer interferometry using a ForteBio Octet RED 96 instrument (Fortebio, Fremont, CA, USA). Ni-NTA biosensors (Fortebio) were hydrated with PBS for 10 min prior to performing the experiment. The concentration of His-tagged Gal-3 was 10 μg/mL, and monitoring was as follows: initial baseline for 120 s, loading for 120 s, baseline for 120 s, association for 100 s, and dissociation for 100 s. For Ni-NTA biosensors, the regeneration buffer was 10 mM glycine (pH 2.0), and recharging (10 mM NiCl2 in H2O) was done for 60 s. The kinetics buffer was PBS (pH 7.2) with 0.1% Tween 20 and 1% BSA. To determine binding kinetics, five concentrations of each sample were dissolved in kinetics buffer (3.9, 7.8, 15.6, 31.2 and 62.4 μM for WGFPA-1a; 0.7, 1.4, 2.8, 5.6 and 11.2 μM for WGFPA-2a; 0.2, 0.4, 0.8, 1.6 and 3.2 μM for WGFPA3a; 78.1, 156.2, 312.4, 624.8 and 1249.6 μM for WGFPA-1b; 28.7, 57.4, 114.8, 229.6 and 459.2 μM for WGFPA-2b; 0.3, 0.6, 1.2, 2.4 and 4.8 μM for WGFPA-3b). Data were analyzed using a 1: 1 binding stoichiometry, and the equilibrium dissociation constant, KD, was calculated using Octet® RED96 Data Analysis Software version 7.0 (Fortebio). 3. Results 3.1. Preparation of pectin from ginseng flower buds Water-soluble ginseng flower buds polysaccharides (WGFP, yield 5.2%) were obtained following hot water extraction and ethanol precipitation. WGFP contained 82% of carbohydrate, 10% of ash and 3% of protein. Monosaccharide composition analysis (Table 1) showed that WGFP
Table 1 Yields, Mws and monosaccharide compositions of fractions from WGFP. Fraction
Yeild Mw Monosaccharide composition (mol%) (%W) (KDa) GalA Rha Gal Ara Glc GlcA Man Xyl Fuc
WGFP WGFPA WGFPA-1a WGFPA-1b WGFPA-2a WGFPA-2b WGFPA-3a WGFPA-3b
5.2a 55.2b 14.3b 47.1b 22.9b 43.3b 18.2b 51.3b
a b
15.8 3.2 86.5 8.7 302.5 27.2
30.1 49.9 3.9 56.9 14.3 70.3 19.0 82.2
4.2 5.0 3.1 6.0 9.0 10.2 13.4 3.6
32.4 22.2 47.2 11.9 35.3 7.4 27.5 6.3
18.5 17.4 39.4 17.2 39.1 9.4 35.9 5.3
Yield in relation to ginseng flower buds. Yield in relation to fraction applied onto column.
5.3 1.7 2.1 1.7 0.8 0.3 0.9 1.2
2.6 1.6 4.0 2.1 1.3 0.9 0.7 1.8
4.4 1.1 0.3 1.7 0.3 – 0.9 –
2.1 0.8 – 1.7 – 1.6 1.6 0.8
0.4 0.2 – 0.8 – – 0.1 –
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mainly consisted of Gal (32.4%), GalA (30.1%) and Ara (18.5%), with minor Glc (5.3%), mannose (Man) (4.4%), Rha (4.2%), GlcA (2.6%), xylose (Xyl) (2.1%) and fucose (Fuc) (0.4%). The negative result of I 2 -KI assay indicated that WGFP did not contain starch-like polysaccharide. WGFP was then applied to a DEAE-cellulose column (Cl¯), eluting with H2O and 0.5 M NaCl to give one neutral polysaccharide WGFPN (yield 10.9%) and one acidic polysaccharide WGFPA (yield 55.2%) (Fig. 1). The structure of WGFPN was not discussed in this paper. WGFPA contained around 50% of GalA, suggesting it was pectic polysaccharide. It was further fractionated by DEAE-cellulose column (Cl¯), eluting sequentially with water, 0.1 M, 0.2 M, 0.3 M and 0.5 M NaCl, respectively. Five fractions named WGFPA-H (yield 2.0%), WGFPA-1 (yield 15.3%), WGFPA-2 (yield 33.6%), WGFPA-3 (yield 23%) and WGFPA-4 (yield 3.0%) were obtained. Due to the low yield, WGFPA-H and WGFPA-4 were not further analyzed. The other three fractions were then fractionated by size exclusion chromatography. Each fraction was separated into two sub-fractions (Fig. 2). At last, six homogenous polysaccharide fractions WGFPA-1a (yield 14.3%), WGFPA-1b (yield 47.1%), WGFPA-2a (yield 22.9%), WGFPA-2b (yield 43.3%), WGFPA-3a (yield 18.2%) and WGFPA-3b (yield 51.3%) were got. 3.2. Structural feature analysis of pectin from ginseng flower buds The obtained homogeneous polysaccharide fractions were then structurally analyzed. Among these polysaccharides, fractions a (WGFPA-1a, WGFPA-2a and WGFPA-3a) had higher molecular weight, lower yield and lower GalA content than their corresponding fractions b (WGFPA1b, WGFPA-2b and WGFPA-3b) (Table 1). Fractions a mainly contained Gal, Ara, GalA and Rha residues. The ratio of Rha/GalA in WGFPA-1a, WGFPA-2a and WGFPA-3a were 0.8, 0.6 and 0.7, respectively, typical for RG-I type pectin [25]. WGFPA-1b, WGFPA-2b and WGFPA-3b were mainly composed of GalA, suggesting HG was the major pectin type in these fractions. Meanwhile, RG-II was also detected in fractions b by TBA assay. According to their structural similarity and difference, we will analyze the structures of fractions a and fractions b separately. 3.2.1. Structural feature analysis of WGFPA-1a, WGFPA-2a and WGFPA-3a WGFPA-1a, WGFPA-2a and WGFPA-3a were all composed of high contents of Gal and Ara (Table 1). Since Gal and Ara are the major sugars in neutral side chains and Rha is the branching point, the ratio of Ara + Gal/Rha could roughly reflect the amount/length of
Fig. 2. Elution profiles of (A) WGFPA-1 and (B) WGFPA-2 on a Sephacryl S-200 column; (C) WGFPA-3 on a Sepharose CL-6B column (-●- total sugar; -○- uronic acid). Vo is void volume and Vt is total volume.
Fig. 1. Fractionation scheme for WGFP by anion-exchange and size-exclusion chromatographies.
side chain in RG-I [15,26]. For WGFPA-1a, WGFPA-2a and WGFPA3a, Ara + Gal/Rha ratios were 27.9, 8.3 and 4.7 (Table 2), respectively, suggesting the amount/length of side chains in these fractions were in the same order.
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Table 2 Mw and compositional features for fractions a and fraction a-ara-gal. Fraction
WGFPA-1a WGFPA-1a-ara-gal WGFPA-2a WGFPA-2a-ara-gal WGFPA-3a WGFPA-3a-ara-gal
Monosaccharide composition (mol%) GalA Rha
Gal
Ara
3.9 4.9 14.3 30.0 19.0 33.8
47.2 61.4 35.3 34.1 27.5 27.2
39.4 19.1 39.1 10.8 35.9 7.2
3.1 3.8 9.0 18.1 13.4 21.9
Mw Rha/ Ara + (KDa) GalA Gal/ Rha
Gal/ Rha
Ara/ Rha
15.8 15.7 86.5 62.0 302.0 174.0
15.2 16.2 3.9 1.9 2.0 1.2
12.7 5.0 4.3 0.6 2.7 0.3
0.8 0.8 0.6 0.6 0.7 0.7
27.9 21.2 8.3 2.5 4.7 1.6
Their structures were then analyzed by 13C NMR spectrum (Fig. 3), and the chemical shift assignments were listed in Table 3. The signals at δ 100.61 and δ 173.08 were attributed to C-1 and C-6 of α-D-1,4-
GalpA, and the resonance at δ 16.79 and δ 16.35 were assigned to C-6 of α-L-1,2,4-Rhap and α-L-1,2-Rhap [15], respectively, which indicated they had RG-I backbone of alternating α-L-Rhap and α-D-GalpA residues. The low intensity signals for Rha and GalA in the spectrum of WGFPA-1a was consistent with its monosaccharide composition. The anomeric carbon resonances at δ 109.10, δ 108.13, δ 107.31 and δ 106.96 could be assigned to C-1 of α-L-t-Araf, α-L-1,3-Araf, α-L-1,5Araf and α-L-1,3,5-Araf, respectively. Signals at δ 104.22, δ 103.57, δ 103.25, δ 103.01 and δ 102.5 were assigned to the anomeric carbon of β-D-1,4-Galp, β-D-1,3,6-Galp, β-D-t-Galp, β-D-1,6-Galp and β-D-1,3Galp. The complex and overlapping signals at δ 60-δ 85 were attributed to C-2 to C-6 of different linkages of Gal and Ara [27–30]. Through comparison of these NMR spectra, we found WGFPA-1a was mainly composed of β-D-1,6-Galp, β-D-1,3-Galp, β-D-1,3,6-Galp, α-L-1,3-Araf, αL-1,5-Araf and α-L-t-Araf residues, which was consistent with the
Fig. 3. 13C NMR spectra of (A) WGFPA-1a; (B) WGFPA-2a; (C) WGFPA-3a.
L. Cui et al. / International Journal of Biological Macromolecules 128 (2019) 459–467 Table 3 13 C NMR spectral assignments of WGFPA-1a, WGFPA-2a and WGFPA-3a. Fraction
Residues
Chemical shift, δ(ppm) C-1
WGFPA-1a
WGFPA-2a
WGFPA-3a
→4)-α-GalpA-(1→ 100.61 →2)-α-Rhap-(1→ \ \ →5)-α-Araf-(1→ 107.31 →3)-α-Araf-(1→ 108.13 T-Araf 109.1 →6)-β-Galp-(1→ 103.01 →3)-β-Galp-(1→ 102.5 →3,6)-β-Galp-(1→ 103.57 T-Galp 103.25 →4)-α-GalpA-(1→ 100.62 →2)-α-Rhap-(1→ \ \ →3,5)-α-Araf-(1→ 106.96 →5)-α-Araf-(1→ 107.37 T-Araf 109.08 →4)-β-Galp-(1→ 104.22 →6)-β-Galp-(1→ 103.01 T-Galp 103.25 →4)-α-GalpA-(1→ 100.62 →2)-α-Rhap-(1→ \ \ →3,5)-α-Araf-(1→ 106.96 →5)-α-Araf-(1→ 107.32 T-Araf 109.08 →4)-β-Galp-(1→ 104.21 →6)-β-Galp-(1→ 103.01 →3)-β-Galp-(1→ 102.51 →3,6)-β-Galp-(1→ 103.25 T-Galp 103.25
C-2
C-3
C-4
C-5
C-6
\ \ \ \ \ \ 78.98 81.19 70.61 70.14 71.68 \ \ \ \ \ \ \ \ 80.67 81.12 71.68 70.58 \ \ \ \ \ \ \ \ 80.67 80.67 71.68 70.59 \ \ \ \ \ \
\ \ \ \ 76.44 83.49 76.44 72.46 79.97 79.97 \ \ \ \ \ \ 83.76 76.59 76.41 73.17 72.46 \ \ \ \ \ \ 83.75 78.96 76.42 73.17 72.46 80.05 80.05 \ \
\ \ \ \ \ \ \ \ 83.71 68.5 70.6 68.32 \ \ \ \ \ \ 81.12 82.19 83.73 77.52 68.5 \ \ \ \ \ \ 81.12 82.19 83.71 77.52 68.8 70.59 68.31 \ \
\ \ \ \ \ \ 61.13 60.94 74.14 73.25 73.25 \ \ \ \ \ \ 66.35 66.74 60.95 74.37 \ \ \ \ \ \ \ \ 66.35 66.74 60.95 74.37 \ \ \ \ \ \ \ \
\ \ 16.35
69.73 61.13 69.73 \ \ 173.08 16.79
60.61 69.73 \ \ 173.08 16.37
60.61 69.73 61.13 69.73 \ \
structural feature of AG-II side chains [4]. WGFPA-2a contained α-L1,5-Araf, α-L-1,3,5-Araf and β-D-1,4-Galp glycosyl linkages, indicating it might possess β-D-1,4-galactan and α-L-1,5-arabinan or AG-I side chains. WGFPA-3a had more complex side chains than the other two fractions. It contained almost all kinds of glycosidic linkages mentioned-above. Thus, WGFPA-3a might contain a mixture of α-L-1,5-arabinan, β-D-1,4-galactan, AG-I and AG-II as side chains. To further analyze the side chain structures of WGFPA-1a, WGFPA-2a and WGFPA-3a, they were degraded by endo-1,4-β-D-galactanase, β-Dgalactosidase, endo-1,5-α-L-arabinanase and α-L-arabinofuranosidase in combination. After removing released oligosaccharides by dialysis, the left polysaccharide fractions (WGFPA-1a-ara-gal, WGFPA-2a-ara-gal and WGFPA-3a-ara-gal) were obtained. Their major monosaccharide compositions and molecular weights were listed in Table 2. WGFPA-1aara-gal had the same molecular weight compared with WGFPA-1a, indicating WGFPA-1a was not greatly degraded by these enzymes. The ratio of Ara/Rha or Gal/Rha was calculated and used to compare the change of Ara and Gal before and after enzymatic degradation. The result showed that Gal residues in WGFPA-1a were hardly degraded by the enzymes, and Ara residues were degraded by 60%, suggesting that WGFPA-1a did not contain β-D-(1,4)-Galp residues and contain α-L-1,5-Araf or α-L-1,3-Araf, agreed with AG-II side chain structure analyzed by NMR. WGFPA-2a-ara-gal (62.0 KDa) had lower molecular weight than WGFPA-2a (86.5 KDa), indicating an effective enzymatic hydrolysis. The ratio of Gal/Rha and Ara/Rha decreased significantly by 51% and 86%, respectively. Therefore, side chains such as β-D-1,4-galactan, α-L-1,5-arabinan or AG-I side chains existed in WGFPA-2a, which was consistent with the NMR result. The incomplete removal of Gal residues from WGFPA-2a might be due to the branching of Ara on them. These enzyme treatments also reduced the Gal and Ara level of WGFPA-3a by 40% and 89%, producing WGFPA-3a-ara-gal with a lower molar weight. This result proved that WGFPA-3a also contained β-D-1,4-galactan, α-L-1,5arabinan or AG-I side chains. However, less Gal residues in WGFPA-
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3a were hydrolyzed than WGFPA-2a, which suggested that other linkages of Gal residues such as β-D-1,3-Galp, β-D-1,6-Galp or β-D-1,3,6-Galp might also exist. Thus, WGFPA-3a had a mixture of α-L-1,5-arabinan, β-D-1,4-galactan, AG-I and AG-II side chains. 3.2.2. Structural feature analysis of WGFPA-1b, WGFPA-2b and WGFPA-3b The content of GalA in WGFPA-1b, WGFPA-2b and WGFPA-3b was 56.9%, 70.3% and 82.2% (Table 1), respectively, suggesting they contained HG domain. TBA assay is a very sensitive reaction used to identify Kdo and Dha, which are characteristic monosaccharides in RG-II [22]. All fractions b contained TBA-positive constituents, indicating RG-II domain existed in these fractions. Other monosaccharides such as Rha, Gal, Ara, GlcA and Fuc might belong to RG-II domain in fractions b. Their structures were further analyzed by 13C NMR spectrum (Fig. 4). The major resonance signals at δ 99.03, δ 67.94, δ 68.53, δ 77.85, δ 70.94 and δ 174.64 were attributed to C-1 to C-6 of α-D-1,4-GalpA, respectively. The signal at δ 170.77 was assigned to C-6 of methyl-esterified α-D1,4-GalpA, and δ 52.74 were assigned to the methyl group, suggesting HG segments in these fractions were methyl-esterified [15]. The degree of methyl-esterification (DM) can be estimated by the integral peak area of the signals at δ 174.64 and δ 170.77, which were assigned to un-esterified carboxyl (U) and esterified carboxyl (E), respectively. Using formula Area (E)/Area (E + U), DM was estimated to be ~40%, ~30% and ~20% for WGFPA-1b, WGFPA-2b and WGFPA-3b, respectively [18]. WGFPA-1b showed more complex spectrum than WGFPA-2b and WGFPA-3b. Besides major signals for α-D-1,4-GalpA residues, some characteristic signals representing for RG-II domain were also found. Signals at δ 107.45, δ 103.25 and δ 96.6 were assigned to anomeric carbons of α-Araf, α-Arap and 2-O-Me-α-Xylp, respectively. Signals at δ 96.03 and δ 92.06 were assigned to C-2 of α-Kdop and α-AcefA [31,32]. The spectrum of WGFPA-2b was similar to that of WGFPA-1b, but characteristic signals for RG-II domain were in lower intensity, indicating the ratio of RG-II was lower in WGFPA-2b. The NMR spectrum of WGFPA-3b demonstrated that HG was the dominant structure in this fraction. To further characterize the structure of WGFPA-1b, WGFPA-2b and WGFPA-3b, pectate lyase which is able to cleavage of α-D-(1,4)galacturonan was used to degrade these fractions. The molecular weight change of enzymatic degradation product was detected by HPSEC (Fig. 5). As can be seen, the three fractions were all degraded by pectate lyase, producing two oligosaccharide pools (OP I and OP II) with molecular weight of 1.8 KDa (OP I, retention time ~14.7 min) and 1.1 KDa (OP II, retention time ~15.3 min). These results suggested that WGFPA-1b, WGFPA-2b and WGFPA-3b all contained HG domain, consistent with the NMR analysis. After enzymatic degradation, one polysaccharide pool (PP) which was resistant to pectate lyase hydrolsyis was also found in WGFPA-1b and WGFPA-2b. The molecular weight of this pool from WGFPA-1b and WGFPA-2b was 3.3 KDa and 4.6 KDa, respectively, in accordance with that of monomeric RG-II [5]. The polysaccharide pool standing for RG-II domain in degradation product of WGFPA-3b was in extremely low abundance, indicating WGFPA-3b was mainly composed of HG and very less RG-II. Based on these analyses, WGFPA-1b, WGFPA2b and WGFPA-3b all contained covalently-linked HG and RG-II domains, where RG-II was predominant in WGFPA-1b, HG was predominant in WGFPA-3b, and nearly equivalent HG and RG-II was present in WGFPA-2b. 3.3. Detection the binding of pectin from ginseng flower buds to galectin-3 The interaction between ginseng flower buds pectin and galectin-3 was measured by biolayer interferometry (BLI) using the Ni-NTA sensor [33]. The association and dissociation curves of pectic fractions from ginseng flower buds at various concentrations were shown in Fig. 6. Nonspecific binding has been eliminated as in the absence of galectin-3, polysaccharides hardly bound to sensors even at the highest concentrations
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Fig. 4. 13C NMR spectra of (A) WGFPA-1b; (B) WGFPA-2b; (C) WGFPA-3b.
used in the study. Analysis of the BLI data suggested that three RG-I type pectin WGFPA-1a, WGFPA-2a and WGFPA-3a exhibited strong binding avidity to galectin-3, with apparent KD values of 4.90 μM for WGFPA1a, 0.71 μM for WGFPA-2a and 0.24 μM for WGFPA-3a. For covalentlylinked HG and RG-II fractions, only WGFPA-2b could bind to galectin-3 (KD 48.41 μM), while WGFPA-1b and WGFPA-3b did not exhibit binding to galectin-3. Different binding avidities of pectic fractions from ginseng flower buds to galectin-3 depend on their various structures. WGFPA1a, WGFPA-2a and WGFPA-3a possessed strong binding activity to galectin-3 due to their RG-I structures. Both the backbone and abundant side chains in these RG-I fractions might contribute to the binding [13]. WGFPA-3a had a higher molecular weight than that of WGFPA-2a, indicating the existence of more binding sites, which resulted in higher binding avidity. The lower binding activity of WGFPA-1a to galectin-3 than WGFPA-2a and WGFPA-3a was probably caused by less RG-I backbone and lower molecular weight. WGFPA-1b, WGFPA-2b and WGFPA-3b all contained covalently-linked HG and RG-II structures, but the ratios of RG-II domain to HG domain in these fractions were different. WGFPA-
1b was mainly composed of RG-II domain. The high methylesterification, high degree of substitution on the backbone, low content of Gal residues and low molecular weight of this fraction might make it not binding to galectin-3. WGFPA-3b was composed of methylesterified HG domain. Our lab previously found that HG domain could also bind to galectin-3, and un-esterified and non-substituted ‘smooth’ backbone is crucial for the binding [34]. Here, the methyl-esterification occurring on the HG backbone in WGFPA-3b might cause the absence of binding activity. Interestingly, WGFPA-2b containing around equivalent ratio of HG and RG-II possessed the binding ability to galectin-3. It was speculated that a synergistic effect might occur when RG-II was covalently-linked with HG in some specific molar ratio, like the synergistic effect between RG-I with HG [34]. 4. Discussion Water-soluble polysaccharides from ginseng roots have been studied for many years, and their structures were well characterized
L. Cui et al. / International Journal of Biological Macromolecules 128 (2019) 459–467
Fig. 5. HPSEC of fractions b and their products after enzymatic degradation (A) WGFPA-1b and its degradation products; (B) WGFPA-2b and its degradation products; (C) WGFPA-3b and its degradation products. Vo is void volume and Vt is total volume.
[13–15,18]. In our previous study, water-soluble ginseng polysaccharides (WGP, yield 10.7%) were extracted from ginseng roots with hot water and precipitated with ethanol [18]. WGP was then applied to DEAE-Cellulose column, eluting with H2O and 0.5 M NaCl to give neutral polysaccharide (WGPN) and acidic polysaccharide (WGPA). WGPA was applied to DEAE-Cellulose column again, and eluted with stepwise H2O, 0.1, 0.2, 0.3 and 0.5 M NaCl to produce five fractions WGPA-N, WGPA-1,
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WGPA-2, WGPA-3 and WGPA-4, respectively. WGPA-1, WGPA-2, WGPA-3 and WGPA-4 were further purified by Sepharose CL-6B column to give six pectic fractions (WGPA-1-RG, WGPA-2-RG, WGPA-1HG, WGFPA-2-HG, WGPA-3-HG and WGPA-4-HG). However, there were few studies about polysaccharides from ginseng flower buds. Here, we extracted and systematically fractionated water-soluble polysaccharides from ginseng flower buds and characterized the structures of pectin fractions. The yield for water-soluble polysaccharides of ginseng flower buds was 5.2%, which was lower than the yield of polysaccharides from roots (10.7%). The mass ratio of neutral polysaccharide to acidic polysaccharide is 78:22 for ginseng roots and 16:84 for ginseng flower buds, indicating higher proportion of pectin in ginseng flower buds polysaccharides. The structural features of pectin fractions from both roots and flower buds of ginseng were listed in Table 4. Among pectin fractions, RG-I domains containing high amounts of AG side chains and HG domains alone or covalently linked to RG-II domains were major structures. The ratio for RG-I to HG (+RG-II) was 1:4.7 in ginseng root pectin and 1:2.4 for ginseng flower pectin. As can be seen, RG-I or AG fractions in ginseng root pectin (WGPA-1-RG and WGPA-2-RG) mainly contained type II AG, with molecular weight of 100 and 110 KDa, while RG-I fractions in ginseng flower buds (WGFPA-1a, WGFPA-2a and WGFPA-3a) contained diverse neutral side chains including galactan (G), arabinan (A), AG-I and/or AG-II, and their molecular weight were from 15.8 KDa to 302.5 KDa. The content of GalA residues in HG fractions from ginseng roots was between 62.4% and 92.1%, and DM in these fractions was 5.0% to 30.0%. Due to the covalently linkage of RG-II, the content of GalA in HG fractions of ginseng flower buds was lower than that in ginseng roots, and the DM for these fractions was from 20.0% to 40.0%. Although pectin from ginseng leaves has not been characterized comprehensively like that from roots and flower buds, it also comprises of HG, RG-I, and RG-II domains with RG-II more abundant in leaves [35–37]. Different compositions and structures of polysaccharides in distinct organs of ginseng might be related to their various physiological functions in the plant. Galactin-3 has become a molecular target in the development of anti-cancer therapeutics because of its crucial role in the development and metastasis of various cancers [38]. Natural plant polysaccharides have great potential to be developed as galactin-3 antagonists. Up to now, besides galactomannans, pectin-derived polysaccharides have been identified as main galactin-3 inhibitors. It has been found that pectin-derived polysaccharides with different structures, such as galactan (M-galactan from modified citrus pectin [39]; potato galactan [40]), AG (MCP-N from modified citrus pectin [39]; RN1 from the flowers of Panax notoginseng [41]), RG-I (RG-I-4 and RG-I-3A from ginseng roots [13,19]; RG-2a from modified citrus pectin [42]; PPc from pumpkin [43]) and HG (HG-2b and HG-3p from modified citrus pectin [34]) could bind galactin-3 in different levels. Among these polysaccharides, RG-I type pectin usually showed better binding activities to galactin-3 duo to its complex structures. Our results also revealed that RG-I fractions (WGFPA-1a, WGFPA-2a and WGFPA-3a) purified from water-soluble ginseng flower buds polysaccharides had better binding activities to galactin-3 than other covalently linked HG and RG-II fractions (WGFPA-1b, WGFPA-2b and WGFPA-3b). Among RG-I fractions, WGFPA-2a and WGFPA-3a had better binding avidity than WGFPA-1a, which might be due to their higher molecular weights and higher ratios of RG-I backbone. In addition, some synergistic effect might exist between RG-II and HG domain for binding to galactin-3 when they were in some specific molar ratio. 5. Conclusions In summary, water-soluble pectic polysaccharides were extracted from ginseng flower buds and separated into homogeneous RG-I-type and HG-type pectin. RG-I fractions with
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Fig. 6. Biolayer interferometry analysis of pectin from ginseng flowers (A) WGFPA-1a; (B) WGFPA-2a; (C) WGFPA-3a; (D) WGFPA-1b; (E) WGFPA-2b; (F) WGFPA-3b.
abundant side chains had strong binding avidity to galectin-3. HG fractions exhibited different binding avidities to galectin-3 depending on the ratio of RG-II domain linked. These results provide valuable information for comprehending the structural features of pectin from ginseng flower buds and screening new active galectin-3 inhibitors.
Funding This work was supported by the National Natural Science Foundation of China (grant numbers 31770852), the Scientific and Technologic Foundation of Jilin Province (20180101004JC) and the Natural Science Foundation of Changchun City of China (17YJ004).
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Table 4 Structural features of pectin fractions purified from ginseng roots and flower buds. Parts of ginseng
Roots
Flower Buds
Fractions
WGPA-1-RG WGPA-2-RG WGPA-1-HG WGPA-2-HG WGPA-3-HG WGPA-4-HG WGFPA-1a WGFPA-2a WGFPA-3a WGFPA-1b WGFPA-2b WGFPA-3b
Ratio (%)
DM (%)
Mw (KDa)
Monosaccharide composition (%mol) GalA
Rha
Gal
Ara
4.7 12.7 9.6 33.8 29.8 9.4 4.4 16.2 8.8 15.2 30.5 24.8
– – 30.0 20.0 10.0 5.0 – – – 40.0 30.0 20.0
100.0 110.0 3.5 6.5 16.0 45.0 15.8 86.5 302.5 3.2 8.7 27.2
1.8 5.3 62.4 83.6 90.9 92.1 3.9 14.3 19.0 56.9 70.3 82.2
0.2 4.1 1.6 3.0 1.5 – 3.1 9.0 13.4 6.0 10.2 3.6
56.2 44.4 15.2 5.1 3.5 5.9 47.2 35.3 27.5 11.9 7.4 6.3
34.0 40.9 7.1 4.6 2.2 – 39.4 39.1 35.9 17.2 9.4 5.3
Conflicts of interest The authors declare no conflict of interest.
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