Accepted Manuscript

Title: Physicochemical properties and structural

characterization of a galactomannan from Sophora
alopecuroides L. seeds

Author: Rui Guo Lianzhong Ai Nannan Cao Juan Ma Yan Wu

Jinhong Wu Xiangjun Sun

PII: S0144-8617(15)01236-9
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2015.12.058
Reference: CARP 10656

To appear in:

Received date: 31-10-2015

Revised date: 21-12-2015
Accepted date: 22-12-2015

Please cite this article as: Guo, R., Ai, L., Cao, N., Ma, J., Wu, Y., Wu,
J., and Sun, X.,Physicochemical properties and structural characterization of a
galactomannan from Sophora alopecuroides L. seeds, Carbohydrate Polymers (2015),
http://dx.doi.org/10.1016/j.carbpol.2015.12.058

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Highlights

►Neutral F25 from Sophora alopecuroides L. seeds was characterized for the first

time.

►The macromolecular solution behavior of F25 was revealed in different manners.

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►The proposed structure of F25 was clarified and confirmed as a galactomannan.

ip
►3D molecular modeling of F25 was preliminarily established.

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an
Physicochemical properties and structural characterization of a galactomannan
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from Sophora alopecuroides L. seeds
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Rui Guo a, 1, Lianzhong Ai b, 1, Nannan Cao a, Juan Ma a, Yan Wu a, *, Jinhong Wu a,

Xiangjun Sun a
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a
Key Laboratory of Urban Agriculture (South), School of Agriculture and Biology,
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Shanghai Jiao Tong University, Shanghai 200240, China

b
School of Medical Instrument and Food Engineering, University of Shanghai for

Science and Technology, Shanghai 200093, China

Page 1 of 40
1
These authors contributed equally to this work and should be considered co-first

authors.

* Corresponding author: Yan Wu

Tel.: +86-21-34205715

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Fax: +86-21-34206918

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E-mail address: wuy@sjtu.edu.cn

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p te
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Page 2 of 40
1 ABSTRACT

2 A water-soluble neutral polysaccharide (F25) was fractionated from crude

3 polysaccharides of Sophora alopecuroides L. seeds. Its physicochemical properties

4 and structural characterization were investigated. The I2-KI and Congo red reactions

t
5 showed F25 possessed many branches without triple-helical conformation, which

ip
6 coincided with methylation and NMR analyses, i.e., F25 was a galactomannan with

cr
7 1,4-linked β-D-Manp backbone distributed randomly (M/G 1.48-1.84) by major

8 T-α-D-Galp (86.82%) or minor 1,4-linked α-D-Galp (13.18%) at O-6. Compared with

us
9 [η] of deionized water (2.98 dL/g), it was indicated that NaCl (2.89-2.90 dL/g) and

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10 NaOH (2.27-2.28 dL/g) could affect intrinsic interactions and conformation of F25 in

11 solution, leading to molecular non-entanglements and intramolecular associations.

M
12 This was demonstrated by 3D molecular modeling. Furthermore, the amorphous

13 nature of F25 was revealed with higher crystallinity (25.7%) and crystallite size (7.6
d

14 nm) compared to other polysaccharides. In this study, the possible relationships

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15 between molecular structure and physicochemical properties were obtained for F25,

16 which could be potentially applied in food and pharmaceutical industries.

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17
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18 Keywords: Sophora alopecuroides L. seeds; Galactomannan; Physicochemical

19 properties; Structural characterization; 3D molecular modeling

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20

21

22

Page 3 of 40
23 1. Introduction

24 In recent years, polysaccharides (including gums, mucilages and hydrocolloids)

25 from plants are enjoying accelerating development in food and pharmaceutical

26 industries. They could serve as therapeutic agents, excipients, thickeners, stabilizers,

t
27 emulsifiers, encapsulants, coating agents and texture modifiers due to their

ip
28 considerable availability, diverse functionality, non-cytotoxicity and ease of

cr
29 modification (Archana et al., 2013; Timilsena, Adhikari, Kasapis & Adhikari, 2016;

30 Wei et al., 2015; Zhao et al., 2014). In addition, there are increasing researches related

us
31 to the structural elucidation, molecular modification and diverse bioactivities of

an
32 polysaccharides. For example, Xie et al. (2015) reported that the sulfated modification

33 of polysaccharide from Cyclocarya paliurus was more favorable for antioxidant

M
34 behavior. With respect to bioactive arabinogalactan from Lycium ruthenicum, it was

35 found that (1→3)-linked galactan backbone was essential for complement fixating
d

36 activity, while the macrophage stimulation could be triggered by arabinosyl side

te

37 groups and galactan backbone (Peng, Liu, Lei & Wang, 2016). It is evident, therefore,

38 that the relationship between molecular structure and bioactivity for polysaccharides
p

39 was probably ascribed to monosaccharide composition, glycosidic linkage, molecular

ce

40 conformation, functional groups and branching features. Furthermore, they will lay

41 molecular basis for precise interpretation on structure-function/bioactivity and

Ac

42 conformation-function/bioactivity relationships of polysaccharides (Ferreira, Passos,

43 Madureira, Vilanova & Coimbra, 2015).

44 As popular materials or ingredients being frequently and widely applied, the

45 physicochemical properties of polysaccharides are becoming more and more

46 important in order to develop property-based applications. Among them, solution

47 properties are in increasing demand for industrial processing in food and

Page 4 of 40
48 pharmaceutical fields. The solution properties could shed light upon hydrodynamic

49 behaviors of polysaccharides in the construction of food and pharmaceutical products

50 during the process of real production (Shao, Zhu, Qin, Fang & Sun, 2015).

51 Specifically, the intrinsic viscosity [η] will provide insight on the macromolecular

t
52 properties of polysaccharide in solution, which has been demonstrated by a number of

ip
53 studies related to the effects of salts, sugars and alkali on [η] and conformation of

cr
54 polysaccharides from plants (Behrouzian, Razavi & Karazhiyan, 2014; Guo et al.,

55 2013; Shao, Zhu, Qin, Fang & Sun, 2015; Yin, Nie, Guo, Wang, Cui & Xie, 2015)

us
56 From the viewpoint of polysaccharide-based production and processing, it is

an
57 necessary to require not only identification of chemical structure but also relationships

58 between structure and physicochemical properties of polysaccharides.

M
59 In our previous study (Guo, Cao, Wu & Wu, 2015), F25, as a water-soluble

60 neutral fraction, was obtained from the seeds of traditional Chinese herb Sophora
d

61 alopecuroides L.. It was mainly consisted of β-mannose and α-galactose (97.52 mol%
te

62 in total) and the M/G ratio was 1.48. Besides, F25 had a relatively low weight-average

63 molecular weight Mw (288.81 kDa) and [η] (1.69-1.81 dL/g in deionized water). In
p

64 order to explore various applications of F25 in food and pharmaceutical realms, the
ce

65 accurate chemical structure and physicochemical properties are required to be figured

66 out. As of now, however, little information of polysaccharides from S. alopecuroides

Ac

67 L. seeds is available, particularly in aspects of solution properties, crystallinity and

68 fine structure. Hence, in this study, the physicochemical properties and structural

69 elucidation/modeling of F25 were extensively investigated in order to better

70 understand and predict potential applications in food and pharmaceutical industries.

71 2. Materials and methods

72 2.1. Fractionation and separation of F25

Page 5 of 40
73 Briefly, the crude polysaccharides of S. alopecuroides L. seeds (CSAP) were

74 firstly obtained under optimal procedure of water extraction (50.99:1 v/w, 70.68 oC,

75 4.15 h), and then 2% (w/v) CTAB (Cetyltrimethylammonium bromide) was added at

76 room temperature (~ 25 oC) to precipitate the acidic fraction overnight. Afterwards,

t
77 the neutral supernatant of CSAP was fractionated by stepwise ethanol precipitation at

ip
78 4 oC overnight, during which the precipitate obtained at a final concentration of 25%

cr
79 (v/v) ethanol was collected and denoted as F25 (Guo, Cao, Wu & Wu, 2015).

80 2.2. I2-KI analysis of F25

us
81 1 mg/ml solution of F25 (2 mL) was mixed with iodine reagent (1.2 mL,

an
82 containing 0.2% KI and 0.02% I2, w/v) for 10 min. After that, a UV-Vis

83 spectrophotometer (TU-1810PC, Beijing Purkinje General Instrument Co., Ltd.,

M
84 Beijing, China) was used to measure the absorbance in the range of 300-700 nm at

85 room temperature using a quartz cuvette with 1 cm optical length.

d

86 2.3. Congo red analysis of F25

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87 2.5 mg/mL solution of F25 (2 mL) reacted with 80 µM Congo red (2 mL) in a

88 concentration gradient of NaOH solutions (0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30,
p

89 0.35, 0.40, 0.45 and 0.50 M) for 10 min (Palacios, García-Lafuente, Guillamón &
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90 Villares, 2012; Wang, Xu, Zhang, Liu, Sun & Zha, 2015). Afterwards, the maximum

91 absorption wavelengths ranging from 400-800 nm were obtained for F25-Congo red
Ac

92 mixture at various concentrations of NaOH by the TU-1810PC UV-Vis

93 spectrophotometer. Besides, deionized water added with Congo red was considered as

94 the control.

95 2.4. Intrinsic viscosity of F25

96 The [η] of F25 in different solvents (deionized water, 0.1 M NaCl and 0.5 M

97 NaOH) were determined by an Ubbelohde capillary viscometer (Model 1836, I.D.

Page 6 of 40
98 0.38 mm, viscometer constant 2.947×10-3 mm2/s2, Jiaojiang Glass Instrument Factory,

99 Taizhou, China) in a water bath at 25±0.1 oC. Each relative viscosity ηr was gauged

100 within the range of 1.2-2.2 after equilibration for 10 min, while different solvents

101 were used as control accordingly. Based on the isoionic dilution procedure, dilution of

t
102 F25 samples in situ was achieved in triplicate with respective solvent (Goh,

ip
103 Matia-Merino, Pinder, Saavedra & Singh, 2011). Afterwards, Huggins (Eq. (1)),

cr
104 Kraemer (Eq. (2)) and Fedors (Eq. (3)) equations were applied for the calculation of

105 [η].

us
106 (1)

an
107 (2)

108 (3)
M
109 where ηsp, c and cm are the specific viscosity (dimensionless), concentration and its
d

110 upper limit (g/dL), respectively. K′ is the Huggins coefficient (dimensionless), and K″
te

111 is the Kraemer coefficient (dimensionless). In the Huggins-Kraemer plot, [η] (dL/g)

112 was obtained as the average of intercepts by extrapolating to zero concentration. In

p
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113 the Fedors plot, however, [η] was derived from the slope of linear function with

114 higher determination coefficient R2 (> 0.9997).

Ac

115 2.5. X-ray diffraction analysis of F25

116 The X-ray diffraction measurement of F25 was implemented on an X-ray

117 Polycrystaline Diffractometer (D8 Advance, Bruker Co., Karlsruhe,

118 Baden-Württemberg, Germany) operated at 40 kV and 40 mA with a Cu-Kα radiation

119 at room temperature. The diffraction intensities of F25 were counted at 4o/min in the

120 range of 2θ = 5-70o with a scan step of 0.02o. The X-ray diffraction analysis was

121 carried out using DIFFRAC EVA V2.0 software.

7

Page 7 of 40
122 The crystalline index (CI), i.e., the degree of crystallinity, and the apparent

123 crystallite size (ACS) were calculated automatically according to the following

124 equations (Popescu, Popescu, Lisa & Sakata, 2011):

125 (4)

t
ip
126 (5)

cr
127 where Acr is the sum of crystalline integral areas, Atl is the total integral area of F25

128 diffractogram, λ is the wavelength of X-ray radiation (0.154056 nm), β is the

us
129 half-height width (rad) of the maximum diffraction band and θ is the Bragg angle (o)

130 corresponding to the band.

131

132
2.6. Methylation analysis of F25

an
The methylation procedure of F25 was performed in compliance with Ciucanu et
M
133 al. with minor modifications (Ciucanu & Kerek, 1984; Guo et al., 2011; Xing, Cui,

134 Nie, Phillips, Goff & Wang, 2014). 2-3 mg powders of F25 were vacuum-dried
d

135 (DZF-6050 Vacuum Drying Oven, Shanghai Yiheng Scientific Instruments Co., Ltd.,
te

136 Shanghai, China) at 80 oC for 5 h and desiccated with P2O5 overnight. Afterwards, the
p

137 dried samples were dissolved in 0.5 mL anhydrous DMSO and sonicated (THC-20B
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138 Digital Control Ultrasonic Extractor, Jining Tianhua Ultrasonic Electronic Instrument

139 Co., Ltd., Shandong, China) at 45 oC for 4 h prior to 85 oC water bath under constant
Ac

140 stirring for 1 h, in order to reach a complete dissolution of F25. 20 mg NaOH

141 (vacuum-dried at 80 oC for 5 h and desiccated with P2O5 overnight) were added to the

142 solution with constant agitation at room temperature for 3 h. Then, the methylation

143 reaction was initiated by the addition of 0.3 mL CH3I and kept for 2.5 h under

144 constant stirring at room temperature. The mixture was extracted with 2 mL CH2Cl2

145 and partitioned against deionized water (3-5 mL) for three times. The organic phase

Page 8 of 40
146 obtained after partitions was filtered through an anhydrous Na2SO4 column with

147 CH2Cl2 eluting twice, and the methylated F25 was dried and recovered by evaporation

148 under a stream of N2 at room temperature. Further hydrolysis in 0.5 mL 4 M

149 trifluoroacetic acid occurred at 100 oC for 6 h with constant stirring under N2

t
150 environment. After that, the hydrolysates were cooled at room temperature and dried

ip
151 with continuous N2 flow. 0.3 mL deionized water, one drop of 1% (v/v) NH4OH and

cr
152 1-5 mg NaBD4 were then added, in sequence, and kept stirring overnight to reduce

153 hydrolyzed samples. Afterwards, the reduced products were acetylated with 0.5 mL

us
154 acetic anhydride at 100 oC under constant stirring for 2 h before N2 evaporation. As a

an
155 result, the partially methylated alditol acetates (PMAAs) were collected and loaded (1

156 µL) into a gas chromatography-mass spectroscopy (GC-MS) system (TRACE

M
157 GC2000/TRACE MS, ThermoQuest Finnigan, San Diego, California, USA) equipped

158 with a SP™-2330 column (30 m × 0.25 mm × 0.20 µm; Supelco Inc., Bellefonte,
d

159 Pennsylvania, USA) and an ion trap MS detector. The GC program was set as: 1.0
te

160 mL/min flow rate of helium carrier, 250 oC injection temperature, 160-210 oC at 2
o
161 C/min (held for 8 min at 210 oC) and then 210-240 oC at 5 oC/min (held for 6 min at
p

162 240 oC) for column temperature.

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163 2.7. NMR analysis of F25

164 30 mg F25 in 0.5 ml 99.9% (v/v) D2O (Cambridge Isotope Laboratories, Inc.,
Ac

165 Cambridge, Massachusetts, USA) were swirled for 2 h at room temperature before

166 complete dissolution at 4 oC overnight, and then lyophilized for two days (FreeZone®

167 Triad™ Freeze Dry System, Model 7400030, Labconco Co., Kansas City, Missouri,

168 USA). The procedure was repeated thrice to ensure the complete substitution of -OH

169 with -OD in F25 (de Souza, Lucyszyn, Ferraz & Sierakowski, 2010; Xing, Cui, Nie,

170 Phillips, Goff & Wang, 2014). Prior to NMR testing, the samples were dissolved in

Page 9 of 40
171 0.5 mL D2O with trimethylsilyl propionate (TSP) as an internal standard (-0.017 ppm

172 and -1.8 ppm for 1H and 13C, respectively) (Cheng & Neiss, 2012). The 1D 1H and 13C

173 NMR experiments were conducted on a Bruker Avance III 600 MHz NMR

174 spectrometer (Bruker Co., Karlsruhe, Baden-Württemberg, Germany) at 50 oC. The

t
175 2D NMR measurements, including 1H-1H correlation spectroscopy (COSY), 1H-1H

ip
1
176 total correlation spectroscopy (TOCSY), H-13C heteronuclear single quantum

cr
177 coherence spectroscopy (HSQC) and 1H-13C heteronuclear multiple bond correlation

178 spectroscopy (HMBC), were also carried out. Chemical shifts were expressed in ppm.

us
179 2.8. Molecular modeling of F25

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180 According to Miao and his co-workers (Miao, Huang, Jia, Cui, Jiang & Zhang,

181 2015; Miao, Ma, Huang, Jiang, Cui & Zhang, 2015), twenty-four β-D-Manp
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182 backbone units attached by sixteen α-D-Galp pendant groups were employed to

183 produce schematic structures of F25 using ChemBioOffice® Ultra 13.0 (PerkinElmer,
d

184 Inc., Waltham, Massachusetts, USA), of which the 3D molecular structure was further
te

185 optimized by energy minimization based on the calculation of molecular mechanics

186 (MM2). Then, 3D molecular modeling of F25 with the inter-/intra-hydrogen bonds
p

187 was visualized via Discovery Studio 2.5 (Accelrys Software Inc., San Diego,
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188 California, USA).

189 3. Results and discussion

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190 3.1. I2-KI analysis of F25

191 According to Yamada, Yanahira, Kiyohara, Cyong and Otsuka (1985), the

192 relatively longer and more branched chains of polysaccharide could be determined

193 spectrophotometrically by the absence of the maximum absorption peak at 565 nm,

194 otherwise, the shorter and less branched chains of polysaccharide will be implied. As

195 shown in Fig. 1A, the maximum absorption peak of F25 was centered at ~ 351 nm

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196 while no peak was observed around 565 nm, which demonstrated that many branched

197 chains were attached to the backbone of F25. It is in agreement with the analysis

198 based on the monosaccharide composition and M/G ratio (Guo, Cao, Wu & Wu,

199 2015). Besides, the negative reaction between F25 and iodine reagent manifested no

t
200 starches were present in F25.

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201 3.2. Congo red analysis of F25

cr
202 It is well known that Congo red tends to form complexes with polysaccharides in

203 a triple helical conformation, which, as a result, leads to a bathochromic shift of the

us
204 maximum absorption wavelength compared to the positive (e.g., laminarin) and

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205 negative (e.g., dextran) controls (Palacios, García-Lafuente, Guillamón & Villares,

206 2012; Wang et al., 2014). Meanwhile, descending behavior of the maximum
M
207 absorption wavelength for polysaccharide-Congo red complexes has been extensively

208 reported for bioactive glucans from, e.g., Huangshan floral mushroom (Wang, Xu,
d

209 Zhang, Liu, Sun & Zha, 2015), Lentinus edodes (Wang et al., 2014), Jinqian
te

210 mushroom (Liu, Du, Wang, Zha & Zhang, 2014) and Calocybe gambosa (Villares,

211 2013), which is possibly due to the breakdown of hydrogen bonding or hydrophobic
p

212 interactions between glucan and Congo red at high concentrations of NaOH, giving
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213 rise to molecular helix-coil transition thereafter (Wang, Xu, Zhang, Liu, Sun & Zha,

214 2015; Xu, Yan & Zhang, 2012). In turn, it also signified that the triple-helical
Ac

215 conformation is a crucial attribute of polysaccharide embodying specific biological

216 response and activity-enhancing effect, which has been demonstrated by the

217 relationship between triple helical conformation of lentinan, a β-(1→3)-D-glucan, and

218 its antitumor activity (Surenjav, Zhang, Xu, Zhang & Zeng, 2006; Xu, Yan, Tang,

219 Chen & Zhang, 2014). As shown in Fig. 1B, however, the maximum absorption

220 wavelengths both decreased progressively with NaOH concentration increment,

11

Page 11 of 40
221 showing no specific bathochromic shift for F25-Congo red mixture, which indicated

222 that there are no triple helical conformations of F25 in solution. Similarly, this has

223 been revealed for other polysaccharides from Umbilicaria esculenta (Du, Liu & Wang,

224 2015), Prunella vulgaris Linn (Li, Huang, Fu, Yue, Liu & You, 2015), Allium

t
225 macrostemon Bunge (neutral fraction) (Zhang, Wang, Wang, Ma, Ye & Zeng, 2015)

ip
226 and Dimocarpus longan Lour. (LPII-IV) (Yi et al., 2013). According to Guo, Cao,

cr
227 Wu and Wu (2015), the conformation of F25 was considered as a random coil. It

us
228 should be noteworthy that, in addition to the influence of monosaccharides, glycosidic

229 linkages, molecular weight, functional groups and branching properties, the random

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230 coil conformation could contribute to high immunostimulatory activity for

231 polysaccharides, such as galactoglucomannan and pectic arabinogalactan (Ferreira,

M
232 Passos, Madureira, Vilanova & Coimbra, 2015).

233 3.3. Intrinsic viscosity of F25

d

234 As a criterion of hydrodynamic volume of single polysaccharide chain in certain

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235 solvent, [η] is capable of providing conformational information due to its relevance to
p

236 molecular structure, molecular weight, solvent property, hydrodynamic parameters

ce

237 and even macromolecular dimensions (Guo, Wu, Cao, Guo, Sun & Wu, 2015; Yin,

238 Nie, Guo, Wang, Cui & Xie, 2015). Therefore, both intercept- and slope-dependent
Ac

239 methods of [η] determination were employed to investigate the effect of different

240 solvents commonly used on molecular structure of F25 in solution, which could

241 establish a valuable foundation for more advanced analysis of molecular

242 characterization, e.g., static and dynamic light scattering. As shown in Table 1, it is

243 evident that the largest [η] (2.98 dL/g) was obtained in deionized water, while the

244 smallest [η] (2.27-2.28 dL/g) in 0.5 M NaOH. This is consistent with that reported for

245 PLCP from Plantago asiatica L., whereas much less reduction of [η] (2.89-2.90 dL/g)
12

Page 12 of 40
246 in 0.1 M NaCl was observed for F25, which is probably ascribed to much less content

247 of uronic acid in F25 (0.73%, w/v) as compared with that in PLCP (16.62%, w/v)

248 (Guo, Cao, Wu & Wu, 2015; Yin, Nie, Guo, Wang, Cui & Xie, 2015; Yin et al.,

249 2012). It is believed that charges in the polysaccharide chains could induce expansion

t
250 and cooperative aggregation of molecules resulting in high [η], which are partially

ip
251 screened by salts to reduce electrostatic repulsions among adjacent polysaccharide

cr
252 chains and, thus, decrease hydrodynamic volume to a larger extent (Shao, Zhu, Qin,

253 Fang & Sun, 2015). Moreover, the ions with different sizes, valences and charges

us
254 have been demonstrated to exert pronounced effects on [η] and polyelectrolytic

an
255 parameters (such as salt tolerance and Smidsrød-Haug stiffness), which could indicate

256 interactions and conformations of polysaccharide chains under given conditions (Shao,
M
257 Zhu, Qin, Fang & Sun, 2015). However, the screen effect of NaCl on the neutral

258 chains of F25 was mainly focused on the hydroxyl groups, giving rise to slight change
d

259 of F25 conformation. For guar gum, on the contrary, Ma and Pawlik (2007) and Wang,
te

260 He, Guo, Zhao and Tang (2015) revealed that [η] was higher in salt solutions, which

261 was due to the chaotropic (destruction of water structure to promote polymeric
p

262 dissolution) and kosmotropic (competitive hydration of free water molecules for
ce

263 polymer) characteristics of ions at relative high concentrations. In terms of the

264 addition of NaOH, many internal forces, such as electrostatic interactions,

Ac

265 intermolecular hydrogen bonding, hydrophobic interactions and covalent interactions,

266 in polysaccharide could be vastly disrupted regardless of polyelectrolytic nature (Guo

267 et al., 2013; Yin, Nie, Guo, Wang, Cui & Xie, 2015). In addition, a little discrepancy

268 of [η] was observed when compared to the previous one (1.69-1.81 dL/g in deionized

269 water, 25±0.1 oC), which is probably attributed to some local aggregations and

270 self-associations of F25 dissolved incompletely or interfered by hydrophobic

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Page 13 of 40
271 interactions under normal conditions (Ma & Pawlik, 2007; Sébastien, Christophe,

272 Mario, Pascal, Michel & Aurore, 2014).

273 On the other hand, the values of K′ were determined to estimate solvent-solute

274 interaction and self-aggregation of polysaccharide. In general, higher K′ will be

t
275 obtained if solute-solute interaction, i.e., self-aggregation, is superior to solvent-solute

ip
276 interaction (Wang, He, Guo, Zhao & Tang, 2015). Specifically, K′ varies from 0.3 to

cr
277 0.4 for polysaccharide in a good solvent and 0.5-0.8 in a θ solvent (an unperturbed

278 state), while it could be higher than 1 in case of aggregations. Meanwhile, K′+K″ =

us
279 0.4-0.6 should be reached for the absence of molecular associations (Guo, Wu, Cao,

an
280 Guo, Sun & Wu, 2015). The results from Table 1 indicated that F25 was dispersed

281 unperturbedly in deionized water (0.66), while 0.1 M NaCl (0.47) was possibly served
M
282 as a good solvent. Besides, no intra-/inter-molecular entanglements were observed in

283 0.1 M NaCl (0.54), although some local associations of F25 chains occurred in
d

284 deionized water (0.61). As for K′ (1.14) obtained for F25, however, there was the
te

285 presence of self-aggregation of polymeric molecules in 0.5 M NaOH, which was

286 demonstrated by the existence of intramolecular associations (0.73). According to

p

287 Guo et al. (2013), the elimination of cooperative aggregates and even molecular
ce

288 degradation in a strong alkali system were likely to be accountable. Furthermore, F25

289 in those solvents were all exhibited as random coils via the exponent analysis of
Ac

290 power-law correlation (ηsp = acb) (Guo, Cao, Wu & Wu, 2015).

291 As compared with [η] of other galactomannans, such as Caesalpinia pulcherrima

292 (12.02 dL/g, deionized water, 25±0.5 oC) (Thombre & Gide, 2013), Dimorphandra

293 gardneriana (8.67 dL/g, deionized water, 25±0.01 oC) (de Almeida et al., 2015) and

294 Prosopis ruscifolia (7.65 dL/g, bideionized water, 25±0.1 oC) (Busch, Kolender,

295 Santagapita & Buera, 2015), the [η] of F25 were much lower, which were comparable

14

Page 14 of 40
296 to that from Peltophorum pterocarpum (3.14 dL/g, deionized water, 25±0.1 oC)

297 (Nwokocha & Williams, 2014). Therefore, it implied a relatively high critical

298 concentration (not obtained in this study) of F25. Besides, only comparatively high

299 concentrations of F25 could play a dramatic role in solution properties, e.g.,

t
300 rheological behavior (Nwokocha & Williams, 2014).

ip
301 Table 1

cr
Deionized water 0.1 M NaCl 0.5 M NaOH
[η] (dL/g)

us
Huggins-Kraemer 2.98 2.89 2.27
Fedors 2.98 2.90 2.28
Parameters

an
K′ 0.66 0.47 1.14
K″ -0.05 0.07 -0.41
K′+K″ 0.61 0.54 0.73
M
b 1.11 1.08 1.14
302
d
303 3.4. X-ray diffraction analysis of F25
te

304 It is well known that X-ray diffraction is widely used for the evaluation of

305 crystalline characteristics, such as crystallinity, crystallite size and orientation, in

p

306 different substances, especially the metal material. With respect to polysaccharides,
ce

307 however, the abundant hydroxyl groups situated on the polymeric molecules could

308 contribute to the formation of intra-/inter-molecular hydrogen bonds, which is likely

Ac

309 to result in various degrees of crystalline arrangements and amorphous-crystalline

310 transitions (Liyanage, Abidi, Auld & Moussa, 2015; Popescu, Popescu, Lisa & Sakata,

311 2011). As shown in the diffractogram of F25 (Fig. 1C), broad peaks centered at ~ 2θ

312 = 20o and 40o were apparently observed, which is in agreement with various

313 galactomannans and their derivatives (Albuquerque et al., 2014; Gong, Liu, Chen,

314 Han, Gao & Zhang, 2012; Jiang, Jian, Cristhian, Zhang & Sun, 2011; Vendruscolo et

15

Page 15 of 40
315 al., 2009), indicating that there was a relatively large amount of amorphous structures

316 in F25 with a crystallinity of 25.7%, which was close to those of Gleditsia sinensis

317 (29.7%), Gleditsia melanacantha (27.8%) and Gleditsia microphylla (28.5%) (Jiang,

318 Jian, Cristhian, Zhang & Sun, 2011). Noteworthily, although higher value was

t
319 obtained for Cassia grandis (33.4%) (Albuquerque et al., 2014), the crystallinity of

ip
320 F25 is still larger than those of guar gum (3.86-13.2%) (Mudgil, Barak & Khatkar,

cr
321 2012) and its derivatives (0.67-3.01%) (Wang, Li, Du, Li & Li, 2013), suggesting the

322 existence of more organized structures in F25 due to its chemical composition,

us
323 structural properties and preparation procedure. In fact, other physicochemical

an
324 properties, such as rheology, thermodynamics, flexibility, swelling, solubility and

325 opaqueness of polysaccharide, are directly susceptible to the crystalline arrangement

M
326 (Ghribi et al., 2015). In addition, it has been reported that molecular weight (Jiang,

327 Jian, Cristhian, Zhang & Sun, 2011), drying processes (Albuquerque et al., 2014;
d

328 Vendruscolo et al., 2009), lateral substitutions (e.g., carboxymethyl and fluorinated
te

329 groups) (Gong, Liu, Chen, Han, Gao & Zhang, 2012; Wang, Li, Du, Li & Li, 2013),

330 enzymatic depolymerization (Mudgil, Barak & Khatkar, 2012), addition of co-solute
p

331 (Almrhag, George, Bannikova, Katopo & Kasapis, 2012) and other physicochemical
ce

332 treatments (i.e., ultrasound and alkali) (Ballesteros, Cerqueira, Teixeira & Mussatto,

333 2015; Mohammad Amini, Razavi & Mortazavi, 2015) could exert pronounced effects
Ac

334 on the crystallinity of polysaccharides. In terms of the apparent crystallite size, F25

335 (7.6 nm) was larger than guar galactomannans (4.7 and 6.6 nm) (Liyanage, Abidi,

336 Auld & Moussa, 2015), Lentinus edodes polysaccharides (1.7-2.1 nm) (Wang et al.,

337 2014) and celluloses (5.00-6.52 nm) under different enzymatic treatments (Cao & Tan,

338 2005).

16

Page 16 of 40
 i
cr
us
an
M
339
340 ed
(A) (B)
pt
ce
Ac

341
342 (C)
343 Fig. 1

344

17

Page 17 of 40
345 3.5. Methylation analysis of F25

346 The results of methylation analysis for F25 were presented in Table 2 and Fig.

347 S1. It is apparent that three main peaks of PMAAs were observed (Fig. S1A), i.e.,

348 1,5-di-O-acetyl-(1-deuterio)-2,3,4,6-tera-O-methyl hexitol (C, 22.72 mol%),

t
349 1,4,5-tri-O-acetyl-(1-deuterio)-2,3,6-tri-O-methyl hexitol (D, 36.25 mol%) and

ip
350 1,4,5,6-tera-O-acetyl-(1-deuterio)-2,3-di-O-methyl hexitol (F, 33.20 mol%), which

cr
351 were identified as T-D-Galp, 1,4-linked D-Manp and 1,4,6-linked D-Manp,

352 respectively, according to the monosaccharide composition of F25 (Guo, Cao, Wu &

us
353 Wu, 2015) and comparison with mass spectra of PMAAs (Carpita & Shea, 1989).

an
354 Besides, small amounts (> 1.4 mol%) of T-D-Glcp (1.49 mol%) and 1,4-linked

355 D-Galp (3.45 mol%) were also assigned by combination with NMR analysis (referred
M
356 to section 3.6). Therefore, the preliminary structure of F25 was proposed as a

357 galactomannan with 1,4-linked D-Manp backbone glycosylated with T-D-Galp or

d

358 1,4-linked D-Galp at O-6, which is consistent with the results from GC-MS/NMR
te

359 analysis for fenugreek gum (Muschin & Yoshida, 2012) and Cassia grandis seed

360 galactomannan (Albuquerque et al., 2014). However, different substitution patterns,

p

361 namely single T-D-Galp side chains with/without T-D-Araf branches attached at O-6
ce

362 of mannopyranosyl main chain, were found for other galactomannans from, e.g.,

363 Caesalpinia ferrea var. ferrea (de Souza, Lucyszyn, Ferraz & Sierakowski, 2010),
Ac

364 Senna tora (Pawar & Lalitha, 2014), Ceratonia siliqua L. (Sébastien, Christophe,

365 Mario, Pascal, Michel & Aurore, 2014; Simões, Nunes, Domingues & Coimbra, 2011)

366 and Prosopis ruscifolia (Busch, Kolender, Santagapita & Buera, 2015). This might be

367 due to uncompleted methylation of galactomannan (Albuquerque et al., 2014),

368 additional analyses by other techniques (e.g., 2D-NMR, esp. HMBC) (Muschin &

369 Yoshida, 2012), minor structures determined by novel experimental designs (e.g.,

18

Page 18 of 40
370 combined methods of specific enzymolysis, ESI-MS/MS and GC-qMS) (Simões,

371 Nunes, Domingues & Coimbra, 2011), as well as various structural characterizations

372 related to distribution of galactosyl residues (Sébastien, Christophe, Mario, Pascal,

373 Michel & Aurore, 2014). Moreover, the M/G ratio (2.28-2.65) of F25 could be

t
374 obtained directly from the GC of PMAAs (Table 3), which is slightly greater than that

ip
375 (1.48) via high performance anion-exchange chromatography coupled with pulsed

cr
376 amperometric detection (HPAEC-PAD) (Guo, Cao, Wu & Wu, 2015). It is possible

377 that higher quantitative determination of M/G ratio by GC is induced by

us
378 undermethylation, incomplete hydrolysis, degradation and demethylation during

an
379 hydrolysis, atelic reduction and acetylation, as well as selective losses of volatile

380 components (related to D-Galp) of F25 during solvent evaporation (Blakeney, Harris,
M
381 Henry & Stone, 1983; Harris, Henry, Blakeney & Stone, 1984).

382
d

383 Table 2
te

Residues Retention times PMAAs Fragments Linkage Molar ratiosb

(min) (diagnostic ions, m/z) patterns (mol%)
B 11.32 1,5-di-O-acetyl-(1-deuterio)-2,3,4,6 43, 59, 71, 87, 101, 102, 118, T-D-Glcp 1.49
p

a
(1.00) -tera-O-methyl hexitol 129, 145, 161, 162, 205
C 12.28 1,5-di-O-acetyl-(1-deuterio)-2,3,4,6 43, 59, 71, 87, 101, 102, 118, T-D-Galp 22.72
ce

(1.08) -tera-O-methyl hexitol 129, 145, 161, 162, 205

D 16.35 1,4,5-tri-O-acetyl-(1-deuterio)-2,3,6 43, 59, 71, 87, 99, 102, 113, 1,4-linked 36.25
(1.44) -tri-O-methyl hexitol 118, 129, 162, 173, 233 D-Manp
Ac

E 16.65 1,4,5-tri-O-acetyl-(1-deuterio)-2,3,6 43, 59, 71, 87, 99, 102, 113, 1,4-linked 3.45
(1.47) -tri-O-methyl hexitol 118, 129, 162, 173, 233 D-Galp
F 21.90 1,4,5,6-tera-O-acetyl-(1-deuterio)-2, 43, 85, 87, 99, 102, 118, 127, 1,4,6-linked 33.20
(1.93) 3-di-O-methyl hexitol 162, 201, 261 D-Manp
384 a
Values between parentheses indicated retention times relative to T-D-Glcp.
385 b
Calculated from the ratio of peak areas.
386
387 Table 3

M/G ratios
a 1
Monosaccharides HPAEC-PAD GC (PMAAs) H NMRd 13
C NMR

19

Page 19 of 40
 Peak areasb Peak heightsc Anomeric signalsd C-4 (Man) Split signalse
D-Manp (M) 1.48 2.65 2.28 1.51 1.84 1.77
D-Galp (G) 1.00 1.00 1.00 1.00 1.00 1.00
388 a
Referred to Guo, Cao, Wu and Wu (2015).
389 b
Referred to Simões, Nunes, Domingues and Coimbra (2011).
390 c
Referred to Sébastien, Christophe, Mario, Pascal, Michel and Aurore (2014).
391 d
Referred to Chaubey and Kapoor (2001) and Vieira, Mendes, Gallão and de Brito (2007).

t
392 e
Referred to Cunha, Vieira, Arriaga, de Paula and Feitosa (2009).

ip
393

cr
394 3.6. NMR analysis of F25

395 According to preliminary structural investigation (Guo, Cao, Wu & Wu, 2015),

us
396 F25 was considered initially as a galactomannan. In this study, 1D and 2D NMR

397 spectra were employed to further identify the fine structure based on the assignment

an
398 of resonance signals of protons and carbons in F25. For this, the 1D 13C and 1H NMR

399 spectra (150.92 MHz and 600.13 MHz, respectively) were obtained (Fig. 2A-B) and
M
400 compared with the NMR data of other seed galactomannans (Table 4). It is clear that

401 the chemical shifts of proton and carbon signals are mainly influenced by the
d

402 resonance frequency of instrument as well as temperature and concentration of sample,

te

403 which was, particularly, embodied by the appearance of H-1 signals (M1/M1′) and
p

404 well resolved peaks (1H: M5′ and M6′; 13

C: M2′, M3 and G4) for F25 recorded at
ce

405 higher resonance frequency and temperature (Fig. 2A vs. Fig. S3A, Fig. 2B vs. Fig.

406 S3B). As shown in Fig. 2B, there were three proton signals (5.01, 4.75 and 4.74 ppm)
Ac

407 in the anomeric region, which were assigned typically as H-1 of T-α-D-Galp,

408 1,4,6-β-D-Manp and 1,4-β-D-Manp, respectively. Meanwhile, the M/G ratio of F25

409 could also be obtained directly from the relative peak areas of those signals, which is

410 in concert with the value reported previously (Table 3). It is noteworthy that various

411 physicochemical properties and health benefits of galactomannan could be affected by

412 M/G ratio, such as flexibility of chain conformation, synergistic interaction,

413 polymeric solubility, rheological and emulsifying characteristics, and relief of chronic
20

Page 20 of 40
414 diseases (Busch, Kolender, Santagapita & Buera, 2015; Cui, Ikeda & Eskin, 2007).

415 With regard to other proton assignments (from H-2 to H-6), however, it was difficult

416 to indentify them because their 1D NMR peaks were seriously overlapped (Fig. 2B),

417 and 2D signals in 1H-1H COSY and 1H-1H TOCSY (Fig. S2) were too ambiguous to

t
13
418 be distinguished. On the contrary, C NMR signals (Fig. 2A) were readily revealed

ip
419 and, consequently, the corresponding proton signals were unveiled by the

cr
420 identification of cross peaks in 1H-13C HSQC spectrum (Fig. 2C). Similarly, three
13
421 anomeric signals of C NMR were observed, which were assigned to C-1 of

us
422 1,4,6-β-D-Manp (101.22 ppm), 1,4-β-D-Manp (101.17 ppm) and T-α-D-Galp (99.84

an
423 ppm). Besides, the M/G ratio is slightly higher than that from 1H NMR anomeric

424 region (Table 3), although the nuclear Overhauser enhancements have little impact on
M
13
425 the relative areas of C NMR anomeric peaks (Vieira, Mendes, Gallão & de Brito,

426 2007).
d

427 In terms of distribution of α-D-Galp on the mannopyranosyl main chain of F25,

te

428 the analysis of C-4 split peaks (β-D-Manp) was carried out in relation to other seed

429 galactomannans (Table S1). In the splitting region, Three peaks, which were centered
p

430 at 77.76, 77.54 and 77.45 ppm, were attributed to consecutive di-substituted
ce

431 β-D-Manp (peak I), diad mono-substituted β-D-Manp (peak II) and non-substituted

432 β-D-Manp (peak III), respectively (Buriti et al., 2014; Cunha, Vieira, Arriaga, de
Ac

433 Paula & Feitosa, 2009; de Souza, Lucyszyn, Ferraz & Sierakowski, 2010). Meanwhile,

434 the proportion of peak II (46.45%) is larger than peak I (33.41%) and peak III

435 (20.14%), which is in accordance with Caesalpinia ferrea var. ferrea seed

436 galactomannan (de Souza, Lucyszyn, Ferraz & Sierakowski, 2010), indicating a large

437 number of α-D-Galp (M/G 1.77) distributed randomly on the mannopyranosyl

438 backbone of F25.

21

Page 21 of 40
439 In order to determine the glycosidic sequences of monosaccharide residues in

440 F25, the 1H-13C HMBC spectrum (Fig. 2D) was used where the correlation signals

441 (M/M′ H1-M/M′ C4 and M/M′ C1-M/M′ H4) between two β-D-Manp were marked,

442 suggesting that the mannopyranosyl backbone of F25 was connected by (1→4)

t
443 linkages. In addition, the appearance of G H1-M′ C6 cross peak demonstrated the

ip
444 existence of 1,4,6-β-D-Manp, i.e., α-D-Galp were attached to β-D-Manp at O-6 via

cr
445 (1→6) linkages. Nevertheless, it is worth noting that G H1-G C3, G H1-G C4 and G

446 H1-G C5 correlation signals were also detected, similar to the results reported by

us
447 Muschin and Yoshida (2012), which could be explained by the fact that more than

an
448 two α-D-Galp were glycosylated as substituents on the mannopyranosyl main chain of

449 F25, in addition to T-α-D-Galp lateral groups. According to the results from Table 2,
M
450 the (1→4) linkages were probably accountable. Therefore, the proposed structure of

451 F25 was substantiated as a galactomannan with 1,4-linked β-D-Manp backbone

d

452 branched randomly (M/G 1.48-1.84) with major T-α-D-Galp (86.82%) or minor
te

453 1,4-linked α-D-Galp (13.18%) at O-6 (shown in Table 4).

454
p

455
ce

456

457
Ac

22

Page 22 of 40
 i
cr
us
an
M
458 ed
pt
459 (A) (B)
ce
Ac

460
461 (C) (D)
462 Fig. 2

23

Page 23 of 40
 i
cr
463 Table 4
Origins Resonance frequencies Temperatures Concentrations Residues Chemical shifts (ppm)
(oC)

us
(MHz) (mg/ml) H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6/C-6
S. alopecuroides L. 400 25 60 T-α-D-Galp 5.01/99.88 3.81/69.55 3.94/69.32 3.98/69.32 3.90/71.39 3.75/61.31
(F25) 600 50 60 5.01/99.84 3.82/69.47 3.92/70.43 3.99/70.32 3.89/72.28 3.75/62.23
b a
Prosopis ruscifolia 500 25 10-20 - /99.48 -/69.20 -/70.04 -/70.04 -/72.08 -/62.03

an
Cassia grandisc 600 45 10 5.01/99.61 3.81/69.24 3.93/70.19 3.99/70.12 3.89/72.10 3.74/61.97
Caesalpinia 500 70 10 5.02/99.46 3.84/69.07 4.11/70.60 4.00/69.93 3.89/71.82 3.75/61.77
pulcherrimad

M
Caesalpinia ferrea 400 70 25 5.51/99.4 4.34/69.1 4.40/70.1 4.47/69.9 4.38/71.8 4.24/61.7
e
var. ferrea
Dimorphandra 500 70 1 5.01/99.59 3.83/69.21 3.91/70.07 4.00/70.04 3.88/71.90 3.76/61.87
f
gardneriana Tul.
Prosopis juliflorag 500

ed 85 10
1,4-β-D-Manp
-/99.63
4.74/101.25
4.74/101.17
-/69.25
4.12/71.00
4.11/70.94
-/70.29
3.80/72.51
3.81/72.48
-/70.12
3.81/77.63
3.82/77.54
-/71.96
3.55/76.09
3.55/76.07
-/61.94
3.90/61.62
3.89/61.58
pt
-/100.77 -/70.61 -/72.08 -/77.81 -/75.82 -/61.48
4.73/100.96 4.11/70.74 3.80/72.23 3.81/77.37 3.55/75.86 3.90/61.34
4.73/100.75 4.11/70.60 3.79/72.07 3.85/77.30 3.55/75.70 3.89/61.19
ce

5.21/100.7 4.60/70.5 4.29/72.1 4.37/77.0 4.05/75.6 4.29/61.2

4.71/100.72 4.13/70.68 3.79/71.95 3.85/77.20 3.54/75.80 3.89/61.32
-/100.77 -/70.75 -/72.21 -/77.47 -/75.84 -/61.40
Ac

1,4,6-β-D-Manp 4.74/101.25 4.12/71.00 3.80/72.51 3.82/77.94 3.74/74.39 3.97/67.62

4.75/101.22 4.12/71.03 3.81/72.48 3.83/77.88 3.74/74.41 3.94/67.59
-/100.77 -/70.61 -/72.08 -/77.81 -/74.06 -/67.27
a
Not mentioned.
4.74/101.15 4.11/70.74 3.80/72.23 3.83/77.64 3.75/74.20 3.96/67.37
b-g
Adapted from Busch, Kolender, Santagapita & Buera (2015), Albuquerque et al. 4.74/100.57 - - 3.82/77.34 3.74/74.04 3.81/67.24
(2014), Buriti et al. (2014), de Souza, Lucyszyn, Ferraz & Sierakowski (2010), Cunha, 5.26/100.5 4.60/70.5 4.29/72.1 4.37/77.5 4.22/74.1 4.39/67.2
Vieira, Arriaga, de Paula & Feitosa (2009) and Vieira, Mendes, Gallão & de Brito (2007), 4.37/77.3
respectively. 4.73/100.70 - - 3.82/77.40 3.74/74.11 3.96/67.27
-/100.77 -/70.75 -/72.21 -/77.69 -/75.84 -/67.39

464
24

Page 24 of 40
465 3.7. Molecular modeling of F25

466 In order to provide a stereochemical structure of F25, its 2D structural model

467 (Fig. 3A) was converted to 3D patterns (Fig. 3B-C), which showed random coil

468 conformations for F25. This is consistent with the results of molecular

t
469 characterization of F25 reported previously (Guo, Cao, Wu & Wu, 2015). In addition,

ip
470 less intra-hydrogen bonds could be observed at the junction zones of F25

cr
471 entanglements due to energy minimization and equilibrium during molecular

472 dynamics simulation (Fig. 3C). It has been recognized that β-(1→4) linkages could

us
473 result in stiffer chains compared to β-(1→2) and β-(1→3) linkages, while α-linkages

an
474 lead to a more flexible conformation (Laws & Marshall, 2001). Therefore, much more

475 intra-hydrogen bonding interactions could be obtained for (1→3)(1→6)-α-D-glucan

M
476 (Miao, Huang, Jia, Cui, Jiang & Zhang, 2015) and (1→4)(1→6)-α-D-glucan (Miao,

477 Ma, Huang, Jiang, Cui & Zhang, 2015) from the visualization of 3D modeling, giving
d

478 rise to low viscosities inferior to that of F25 (preliminary data). It has been well
te

479 known that interactions, induced by electrostatic forces, Van der Waals forces or

480 hydrogen bonds, among chains could contribute to 3D networks of polysaccharides in

p

481 solution (De Vuyst & Degeest, 1999). Hence, there were probably more
ce

482 intra-hydrogen bonding interactions over inter-hydrogen bonding interactions in F25

483 owing to comparatively small macromolecular parameters (such as radius of gyration,

Ac

484 hydrodynamic radius and persistence length) and [η], which is in good agreement

485 with analyses of other 3D molecular models (Lambo-Fodje, Leeman, Wahlund,

486 Nyman, Öste & Larsson, 2007; Miao, Huang, Jia, Cui, Jiang & Zhang, 2015; Miao,

487 Ma, Huang, Jiang, Cui & Zhang, 2015). On the other hand, based on the effect of

488 M/G ratio via molecular modeling, the simultaneous existence of substituted (hairy

489 regions) and unsubstituted (smooth regions) mannosyl residues for galactomannans

25

Page 25 of 40
490 with low M/G ratios, e.g., 1.48-1.84 for F25, has been proved to be inclined to

491 establish intra-molecular interactions between hairy and smooth regions, thereafter

492 forming intra-molecular junction zones via hydrogen bonding (as shown in Fig. 3C)

493 (Wu, Li, Cui, Eskin & Goff, 2012). According to the results of NMR analysis, F25

t
494 was determined as a glactomannan with 1,4-linked β-D-Manp backbone branched

ip
495 mainly with T-α-D-Galp at O-6. Through MM3 force field method, the average

cr
496 values of molecular partition function for α-(1→3), α-(1→4), α-(1→6) and β-(1→4)

497 linkages were 4.95, 4.65, 10.00 and 4.10 deg2, respectively, which inferred the

us
498 greatest stiffness of β-(1→4)-linked structure (Dowd, French & Reilly, 1992; Dowd,

an
499 Reilly & French, 1994; Dowd, Zeng, French & Reilly, 1992). Consequently, it is

500 likely to indicate that under same conditions of polysaccharide concentration and
M
501 molecular weight, F25 would be more rigid than other 3D modeled glucans, resulting

502 in a more extended conformation with relatively higher viscosity (Miao, Huang, Jia,
d

503 Cui, Jiang & Zhang, 2015; Miao, Ma, Huang, Jiang, Cui & Zhang, 2015). Moreover,
te

504 with regard to the aforementioned fragments (I, II and III) of F25, smaller values of

505 molecular partition function under computer simulation (MM3) were obtained for I
p

506 (17 deg2) and II (51 deg2) than for III (66 deg2), which signifies the relatively stiff
ce

507 nature of F25 conformation possibly ascribed to steric interactions of bulky α-D-Galp

508 groups and stabilization by hydrogen bonds between α-D-Galp and β-D-Manp
Ac

509 (Petkowicz, Reicher & Mazeau, 1998).

510

511

512

513

26

Page 26 of 40
 i
cr
us
514

an
M
ed
pt
ce
Ac

515

516 (A) (B) (C)

517 Fig. 3

518

27

Page 27 of 40
519 4. Conclusions

520 In this study, a water-soluble neutral fraction (F25) was obtained from S.

521 alopecuroides L. seeds, and its fine structure was substantiated as a galactomannan

522 with 1,4-linked β-D-Manp backbone attached optionally by major T-α-D-Galp

t
523 (86.82%) or minor 1,4-linked α-D-Galp (13.18%) at O-6. Solution properties showed

ip
524 no triple-helical conformation of F25, which possessed many side chains (M/G

cr
525 1.48-1.84). Besides, different solvents, i.e., deionized water, 0.1 M NaCl and 0.5 M

526 NaOH, could exert pronounced influences on inter-/intra-molecular entanglements

us
527 and associations of F25, resulting in its conformational change and rearrangement of

an
528 molecular cross-linkings between F25 and solvent, such as electrostatic interactions,

529 hydrogen bonding, hydrophobic interactions and covalent interactions. These

M
530 signified the relationships between molecular structure and physicochemical

531 properties for F25, which showed certain advantages in food and pharmaceutical
d

532 applications. Nevertheless, further precise studies are required to understand the
te

533 conformational behaviors of F25 in solution by other advanced techniques, e.g.,

534 atomic force microscopy (AFM), static (SLS) and dynamic (DLS) light scatterings as
p

535 well as RIS Metropolis Monte Carlo (RMMC)-based computer simulation (Guo,
ce

536 Kang, Wu, Cui, Hu & Yada, 2015). On the other hand, it should gain more insight

537 into the molecular mechanisms of various bioactivities for F25.

Ac

538 Acknowledgements

539 This study was supported financially by the National Natural Science Foundation

540 of China (Grant Nos. 31271822 and 31471623).

541 References

542 Albuquerque, P. B. S., Barros, W., Jr., Santos, G. R. C., Correia, M. T. S., Mourão, P.

543 A. S., Teixeira, J. A., & Carneiro-da-Cunha, M. G. (2014). Characterization and

28

Page 28 of 40
544 rheological study of the galactomannan extracted from seeds of Cassia grandis.

545 Carbohydrate Polymers, 104, 127-134.

546 Almrhag, O., George, P., Bannikova, A., Katopo, L., & Kasapis, S. (2012). Networks

547 of polysaccharides with hydrophilic and hydrophobic characteristics in the

t
548 presence of co-solute. International Journal of Biological Macromolecules,

ip
549 51(1-2), 138-145.

cr
550 Archana, G., Sabina, K., Babuskin, S., Radhakrishnan, K., Fayidh, M. A., Babu, P. A.

551 S., Sivarajan, M., & Sukumar, M. (2013). Preparation and characterization of

us
552 mucilage polysaccharide for biomedical applications. Carbohydrate Polymers,

an
553 98(1), 89-94.

554 Ballesteros, L. F., Cerqueira, M. A., Teixeira, J. A., & Mussatto, S. I. (2015).
M
555 Characterization of polysaccharides extracted from spent coffee grounds by

556 alkali pretreatment. Carbohydrate Polymers, 127, 347-354.

d

557 Behrouzian, F., Razavi, S. M. A., & Karazhiyan, H. (2014). Intrinsic viscosity of cress
te

558 (Lepidium sativum) seed gum: Effect of salts and sugars. Food Hydrocolloids, 35,

559 100-105.
p

560 Blakeney, A. B., Harris, P. J., Henry, R. J., & Stone, B. A. (1983). A simple and rapid
ce

561 preparation of alditol acetates for monosaccharide analysis. Carbohydrate

562 Research, 113(2), 291-299.

Ac

563 Buriti, F. C. A., dos Santos, K. M. O., Sombra, V. G., Maciel, J. S., Teixeira Sá, D. M.

564 A., Salles, H. O., Oliveira, G., de Paula, R. C. M., Feitosa, J. P. A., Monteiro

565 Moreira, A. C. O., Moreira, R. A., & Egito, A. S. (2014). Characterisation of

566 partially hydrolysed galactomannan from Caesalpinia pulcherrima seeds as a

567 potential dietary fibre. Food Hydrocolloids, 35, 512-521.

29

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568 Busch, V. M., Kolender, A. A., Santagapita, P. R., & Buera, M. P. (2015). Vinal gum,

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786 Figure captions

787 Fig. 1 Preliminary characterization of physicochemical properties of F25: (A) UV

788 visible spectrum in the presence of I2-KI; (B) Maximum absorption

789 wavelengths of F25-Congo red mixture at various NaOH concentrations; (C)

t
790 X-ray diffractogram.

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791 Fig. 2 13C (A), 1H (B), HSQC (C) and HMBC (D) spectra of F25. G: T-α-D-Galp; M:

cr
792 1,4-β-D-Manp; M′: 1,4,6-β-D-Manp.

793 Fig. 3 Molecular modeling of schematic structures of F25: (A) 2D model; (B) 3D

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794 model without energy minimization; (C) 3D model with energy minimization.

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795

796
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797
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798 Table captions

799 Table 1 Comparison of viscometric parameters of F25 in different solvents

800 Table 2 Results of methylation analysis for F25

801 Table 3 Comparison of M/G ratios of F25

t
802 Table 4 1H and 13
C NMR chemical shifts for F25 in D2O compared with other seed

ip
803 galactomannans

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804

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805

806

807

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