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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Biomass Engineering Research Center, Ministry of Education, Nanchang University, Nanchang, Jiangxi 330047, PR China
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, PR China
c
Center for Biorening and Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN 55108, USA
b
a r t i c l e
i n f o
Article history:
Received 28 December 2012
Received in revised form 22 January 2013
Accepted 3 March 2013
Available online 13 March 2013
Keywords:
Bamboo leaves
Hemicellulosic polysaccharides
Chemical structure
a b s t r a c t
Bamboo leaves are considered as an important source of bioactive molecules. In this work, leaves from
young bamboo (Phyllostachys pubescens Mazel) aged 3 months were subjected to aqueous extraction and
2% NaOH solution extraction followed by precipitation in ethanolwater medium with different ethanol
concentrations. The dissolved hemicellulosic polysaccharides presented a total recovery of 67.83% based
on the total hemicellulose content in bamboo leaves. Chemical analysis of the fractions was performed
by sugar composition analysis, Fourier-transform infrared spectrometry, and 1D nuclear magnetic resonance imaging. The results revealed that all polysaccharide fractions contained xylose, arabinose, glucose,
galactose, ribose, and uronic acid. The polysaccharides from young bamboo leaves mainly consisted of
arabinoxylans, arabinogalactans, and non-cellulosic -d-glucans having (13)- and (14)-glucosidic
linkages. The content of these polysaccharides was found to vary among the fractions depending on the
separation method. Finally, the thermal behavior was also discussed.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
In the last decade, increasing attention has been paid to biorenery processes for improving the utilization of lignocellulosic
raw materials to obtain new sources of chemicals and energy that
can replace oil-derived ones. Among the main components of lignocellulosic biomass, hemicellulosic polysaccharides are some of
the most interesting biorenery products. The term hemicellulose
denotes a wide variety of polysaccharides isolated from plant cell
wall.
As an important biomass, bamboo plants are commonly huge.
Their stem, unlike those of other grasses, is distinctly wood-like and
perennial, and their leaves survive more than one year. As the most
economically valuable bamboo species, Phyllostachys pubescens
Mazel is widely planted in China, with an annual production of
about seven billion roots. Nowadays, large amounts of bamboo
leaves are produced as a sub-product during the utilization process
of bamboo. However, these bamboo leaves are unused and generally discarded, which is a waste of forestry resource and biomass
because these leaves can be used to extract active polysaccharides (Wilkie & Woo, 1976, 1977) and generate high-value chemical
compounds, such as avonoids (Xie et al., 2013) and avone-Cglycosides (Zhang, Jiao, Liu, Wu, & Zhang, 2008).
The chemical structure and physical property of hemicellulosic polysaccharides from bamboo stems have been extensively
characterized (Luo, Peng, et al., 2012; Peng, Wang, et al., 2012;
Peng, Zhang, et al., 2012; Peng, Peng, Bian, Xu, & Sun, 2011; Wen
et al., 2011; Yang, Zhong, Yuan, Peng, & Sun, 2013). Wen et al.
(2011) found that alkali-soluble hemicelluloses prepared from the
bamboo Bambusa rigida consist a backbone of -(14)-linked
d-xylopyranosyl units with branches of arabinose and 4-O-methyld-glucuronic acid. The hemicelluloses fractionated by ionic liquid
followed by alkaline extraction from the bamboo Phyllostachys
sulphurea (P. sulphurea) were conrmed to consist mainly of 4-Omethyl--d-glucurono--l-arabino--d-xylans (Yang et al., 2013).
In our previous research, arabinoxylans have been isolated from
stems of the bamboo P. pubescens Mazel with different ages (Luo,
Peng, et al., 2012; Peng, Wang, et al., 2012; Peng, Zhang, et al.,
2012). The results demonstrate that the chemical properties of this
bamboos hemicelluloses slightly vary at different growing stages.
Meanwhile, studies on bamboo leaf polysaccharides are limited
(Wilkie & Woo, 1976, 1977). Wilkie and Woo (1976) found that
the ratios of (13)- to (14)-glucosidic linkages in total hemicelluloses were as follows: 1:2.5 for leaves and 1:8.0 for stems of
the bamboo Arundinaria japonica (A. japonica); 1:2.6 for young and
1:2.4 for old leaves of the bamboo Arundinaria anceps (A. anceps);
and 1:19.5 for young and 1:15.6 for old nodes of the bamboo A.
anceps. They also found that the structural properties of the hemicellulosic polysaccharides in bamboo leaves change with increased
maturity (Wilkie & Woo, 1976), and that the composition and structural features of hemicellulosic polysaccharides in bamboo leaves
generally differ from those in bamboo stems. However, the composition and structure of polysaccharides in leaves of the bamboo P.
pubescens Mazel are far from being completely understood. Accordingly, our group was prompted to investigate the physicochemical
characteristics of hemicellulosic polysaccharides in leaves of young
P. pubescens Mazel bamboo. Given that physicochemical features
are considered to be the basis of further utilization of hemicellulosic polysaccharides from young bamboo leaves, the chemical
structure and thermal properties of young bamboo leaves must be
elucidated.
In this study, water-soluble hemicellulosic polysaccharides and
alkali-soluble fractions precipitated from ethanolwater media
with different ethanol concentrations were isolated from delignied leaves of young P. pubescens Mazel bamboo aged 3 months. The
composition and structural features of the various hemicelluloses
were characterized by monosaccharide analysis, Fourier-transform
infrared (FT-IR) spectrometry, and 1D nuclear magnetic resonance
(1D NMR) imaging. The thermal properties were also evaluated by the thermogravimetry (TG)/differential thermogravimetry
(DTG) technique. This research aimed to fractionate hemicellulosic
polysaccharides, obtain structural information, and discuss thermal
performance.
2. Materials and methods
2.1. Materials
The leaves of P. pubescens Mazel aged 3 months were collected at
a local farm (Guanxi Village, Meiling Town, Nanchang City, China).
The starting time of bamboo age was deemed as the point at which
the bamboo shoots were above the ground. The young bamboo
leaves were dried under sunlight and then ground to pass through
a 40 mesh screen followed by a 100 mesh screen. The raw material
was chemically characterized using standard methods of China (GB
2677.9-94, GB/T 2677.8-94, GB/T 2677.3-93, GB/T 2677.6-94, GB/T
2677.5-93, and GB/T 2677.5-93). The composition (%, w/w) of the
young bamboo leaves was analyzed as cellulose 18.59%, hemicellulose 16.54%, Klason lignin 12.32%, ash 5.93%, benzeneethanol
extractives 10.27%, hot water extractives 18.99%, and 1% NaOH
extractives 13.25% based on the weight of oven dried bamboo leaf
powder. Distilled deionized water (Milli-Q) was used as the solvent
for the preparation of all reagent solutions. Trimethylchlorosilane (TMS) (>99.9%) and hexamethyldisilane (HMDS) (>98.0%) were
obtained from Sigma. All other reagents used were analytical grade.
2.2. Extraction of hemicellulosic polysaccharides from bamboo
leaves
Hemicellulosic polysaccharides were extracted from bamboo
leaves according to previous methods with slight modications
(Bian, Peng, Peng, Xu, & Sun, 2010; Peng et al., 2011). Fig. 1 shows
the scheme for extracting hemicellulosic fractions with water and
2% NaOH. Briey, wax-free bamboo leaf powder was delignied
with 0.6% NaClO2 at pH 4.24.7 for 2 h at 75 C. Then, the delignied leaf holocellulose was extracted with distilled water at 80 C
for 3 h under a solid-to-liquid ratio of 1:20 (g/mL). After ltration,
the ltrate was concentrated and then precipitated in three volumes of 95% ethanol, and the water-soluble hemicellulosic fraction
was obtained after freeze drying. The water-insoluble residue was
263
further treated with 2% NaOH at 55 C for 2.5 h and ltered thereafter. The alkaline ltrate was neutralized with acetic acid to pH
5.5 and concentrated at reduced pressure. The concentrated solution containing alkali-soluble polysaccharides was precipitated
in ethanolwater medium with different ethanol mass concentrations. Finally, four alkali-soluble hemicellulosic polysaccharide
fractions were precipitated. Hereafter, BLHW denotes the watersoluble hemicellulosic fraction extracted with distilled water at
80 C for 3 h with a solid-to-liquid ratio of 1:20 (g/mL). BLH20 ,
BLH40 , BLH60 , and BLH80 denote the alkali-soluble hemicellulosic
fractions isolated with 2% NaOH and sequentially precipitated in
graded ethanol solutions with increased concentrations of 20%,
40%, 60%, and 80% ethanol, respectively.
264
Pellet
Freeze-dried
Water-soluble hemicellulosic fraction
(BLHW )
Water-insoluble residue
Treated with 2% NaOH at 55 C for 2.5 h under
a solid to liquid ratio of 1:20 (g/mL)
Residue
(Cellulose)
Filtrate
(Discard)
Filtrate
(1) Neutralized with acetic acid to pH 5.5
(2) Concentrated at reduced pressure to 30 mL, and then
sequentially precipitated in 20%, 40%, 60%, and 80% ethanol
Pellet
Freeze-dried
Hemicellulosic fractions
(BLH20, BLH40, BLH60, BLH80 )
Fig. 1. Scheme of the extraction of hemicellulosic fractions from bamboo leaves.
Table 1
Yield of hemicellulosic polysaccharides isolated, and content of neutral sugars (relative percentage) and uronic acids (percentage dry sample, w/w) in the corresponding
hemicellulosic fractions.
Hemicellulosic fractions
Yielda (%)
Yieldb (%)
Arabinose
Xylose
Glucose
Galactose
Ribose
Uronic acid
Uro/Xyl
Ara/Xyl
a
b
Total
BLHW
BLH20
BLH40
BLH60
BLH80
3.08
0.51
26.37
16.79
20.24
33.80
2.80
3.24
0.19
1.57
5.38
0.89
45.21
30.38
14.45
8.24
1.71
1.28
0.04
1.49
25.82
4.27
7.22
85.33
4.23
2.09
1.14
2.09
0.02
0.08
27.69
4.58
43.14
43.97
1.62
7.99
3.28
1.43
0.03
0.98
5.86
0.97
36.35
40.47
1.42
16.61
5.14
2.03
0.02
0.90
67.83
11.22
150
989
100
% Transmittance
265
BLH80
797
1047
1403
1454
BLH60
1081
50
BLH40
BLH20
1081
1565 1415
-50
1732
1240
1645
-100
2000
1161
BLHW
896 797
1565
1373 1324
1500
1047
1000
500
Wavenumbers (cm-1 )
Fig. 2. FT-IR spectra of isolated polysaccharides.
266
Fig. 3.
13
C NMR spectra of polysaccharide fractions BLH40 (a) and BLH60 (b) in DMSO-d6 solvent.
267
Table 2
Assignment of signals in the 13 C NMR and 1 H NMR spectra of the fractions.
C (ppm)
175.20
107.13
103.57
101.78
101.67
101.49
99.95
85.93
82.23
80.33
78.78
77.78
77.00
76.59
75.21
74.77
74.14
73.92
73.24
73.12
72.91
72.49
69.47
69.19
65.70
63.25
61.78
61.37
61.23
60.62
H (ppm)
4.27
4.95
3.97
3.82
3.69
3.63
4.76
3.26
4.49
3.05
3.87/3.16
3.46
5.45
5.33
4.99
3.51
3.36
2.55
2.51
1.67
a
b
Assignment
Structural units
C-6
C-1
C-1
C-1
C-1/H-1
C-1/H-1
C-1
C-4/H-4
C-4
C-2/H-2
C-4
C-3/H-3
C-4
C-5
C-4/H-4
C-3/H-3
C-3
C-3/H-3
C-2/H-2
C-3
C-2
C-2/H-2
C-4
C-3
C-5
C-5/H-5eq a /H-5ax b
C-5/H-5
C -6
C -6
C-6
H-1
H-1
H-1
H-2
OCH3
CH2
CD3
CH3
4-O-Methyl--d-glucuronic acid
-l-Arabinofuranosyl
-d-Xylopyranosyl
(14)-Linked--d-xylopyranosyl at non-reducing end
3-O-Acetyl-(14)-linked--d-xylopyranosyl
-d-Glucopyranosyl
-l-Galactopyranosyl
-l-Arabinofuranosyl
4-O-Methyl--d-glucuronic acid
-l-Arabinofuranosyl
-d-Glucopyranosyl
-l-Arabinofuranosyl
Internal (14)-linked--d-xylopyranosyl
4-O-Methyl--d-glucuronic acid
(14)-Linked--d-xylopyranosyl
3-O-Acetyl-(14)-linked--d-xylopyranosyl
-d-Glucopyranosyl
(14)-Linked--d-xylopyranosyl
2-O-Acetyl-(14)-linked--d-xylopyranosyl
4-O-Methyl--d-glucuronic acid
4-O-Methyl--d-glucuronic acid
(14)-Linked--d-xylopyranosyl
(14)-Linked--d-xylopyranosyl at non-reducing end
-l-Galactopyranosyl
(14)-Linked--d-xylopyranosyl at non-reducing end
(14)-Linked--d-xylopyranosyl
-l-Arabinofuranosyl
-l-Galactopyranosyl
-l-Galactopyranosyl
-d-Glucopyranosyl
-l-Arabinofuranosyl linked to O-3 of xylan
-l-Arabinofuranosyl linked to O-2 of xylan
4-O-Methyl--d-glucuronic acid
-d-Glucopyranosyl
4-O-Methyl--d-glucuronic acid
Residual ethanol
DMSO-d6
Residual ethanol
268
Fig. 4.
H NMR spectra of the polysaccharide fraction BLH40 (a) and hemicellulosic fraction BLH60 (b) in DMSO-d6 solvent.
Table 3
Thermogravimetric characteristics.
Sample
Temperature range ( C)
Tpeak ( C)
BLHW
199383
483516
557637
286
493
615
48.78
2.29
3.48
4.00
1.07
0.72
25.03
74.97
BLH20
BLH40
197354
194359
297
288
60.71
54.60
8.14
7.66
19.83
25.45
80.17
74.55
BLH60
199370
510555
286
534
43.98
2.14
4.56
0.70
31.81
68.19
BLH80
200380
501544
298
527
34.39
3.64
3.07
1.32
35.33
64.67
70
144 C
60
50
-3
40
30
20
-4
BLHW
100
286 C
340 C
300 400 500
200
-5
600
100
90
80
70
60
50
40
30
20
10
BLH20
100
700
TG (%)
80
70
60
50
40
30
20
BLH40
100
288 C
200
300
400
500
600
700
600
510 C
555 C
370 C
-1
534 C
80
70
-2
60
-3
50
40
30
700
-4
BLH60
100
200
286 C
300 400
-5
500
600
700
Temperature ( C)
0
501 C 544oC
200 C
90
-1
380 C
80
527 C
70
-2
60
50
-3
40
DTG (%min)
100
TG (%)
500
199 C
90
Temperature ( C)
30
200
100
TG (%)
0
-1
-2
-3
-4
-5
-6
-7
-8
-9
359 C
90
DTG (%min)
194 C
297 C
300 400
Temperature ( C)
Temperature ( C)
100
0
-1
-2
-3
-4
-5
-6
-7
-8
-9
197 C 354oC
DTG (%min)
TG (%)
80
TG (%)
199 C
90
DTG (%min)
557 C 637oC
o
0
516 C
o
o
383 C
615 C -1
o
o 493 C
483 C
-2
100
269
DTG (%min)
298 C
BLH80
-4
100
200
300
400
500
600
700
Temperature ( C)
Fig. 5. TG-DTG curves of the polysaccharide fractions.
and 4.0, and 4.0 and 3.0 ppm, respectively. The anomeric protons
of terminal arabinofuranosyl units linked to O-3 and O-2 of xylans
were indicated by two weak resonances at 5.45 and 5.33 (5.34 in
Fig. 4b) ppm (Sun et al., 2005). The weak signals at 4.99 (H-1) and
3.36 (OCH3 ) ppm were attributed to 4-O-methyl--d-glucuronic
acid residues (Bian et al., 2010). In Fig. 4b, the signal at 3.36 ppm
was overlapped by other signals. The -anomeric proton signal
of (14)-linked--d-xylopyranosyl units acetylated at C-3 was
observed at 4.27 ppm (Peng, Zhang, et al., 2012; Yuan et al., 2011),
and the -anomeric proton signal at 4.20 ppm corresponding to
-d-xylopyranosyl units was not found (Yuan et al., 2011). The
signals at 4.49 and 4.76 ppm in 1 H NMR spectra of BLH40 and
BLH60 were assigned to H-2 from 2-O-acetyl--d-xylopyranosyl
units and H-3 from 3-O-acetyl--d-xylopyranosyl units,
respectively (Yuan et al., 2011). The absence of the signal at
2.0 ppm, originating from aliphatic acetates (acetyl groups in
hemicelluloses) in both the BLH40 and BLH60 spectra, was good
evidence of ester bond cleavage during hemicellulosic polysaccharide extraction with 2% NaOH (Kim et al., 2011). The signals
corresponding to H-2, H-3, H-4, and H-5 of -d-xylopyranosyl
units appeared at 3.05, 3.26 (3.25 in Fig. 4b), 3.63, and 3.87/3.16
(3.17 in Fig. 4b) ppm, respectively (Bian et al., 2010; Peng, Zhang,
et al., 2012; Sun, Jing, et al., 2011; Sun et al., 2005; Yuan et al.,
2011). The chemical shifts of 3.87 and 3.16 (3.17 in Fig. 4b) ppm
originated from the equatorial and axial protons linked at C-5 of
270
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