- Email: [email protected]
Isolation, structure, and surfactant properties of polysaccharides from Ulva lactuca L. from South China Sea
Accepted Manuscript Title: Isolation, structure, and surfactant properties of polysaccharides from Ulva lactuca L. from South China Sea Author: Hua Tian Xueqiong Yin Qinghuan Zeng Li Zhu Junhua Chen PII: DOI: Reference:
S0141-8130(15)00372-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.05.031 BIOMAC 5115
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
17-4-2015 19-5-2015 21-5-2015
Please cite this article as: H. Tian, X. Yin, Q. Zeng, L. Zhu, J. Chen, Isolation, structure, and surfactant properties of polysaccharides from Ulva lactuca L. from South China Sea, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.05.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Isolation, structure, and surfactant properties of polysaccharides
2
from Ulva lactuca L. from South China Sea
3
Hua Tiana,b, Xueqiong Yina,b *, Qinghuan Zengb, Li Zhub, Junhua Chenb
ip t
1
4 5
a
6
University, Haikou, Hainan 570228, P.R. China
7
b
8
Haikou, Hainan, 570228, P.R. China
cr
Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan
us
Hainan Provincial Fine Chemical Engineering Research Center, Hainan University,
*Corresponding author at:
11
Address: School of Materials and Chemical Engineering, Hainan University, 58th
12
Renmin Road, Haikou, Hainan, 570228, PR China.
13
Tel.: +86 898 66279161
14
Fax: +86 898 66291383;
15
19 20
China Sea.
21
2. The extraction conditions were optimized, getting the highest yield of 17.57%.
22
3. The structure of the polysaccharides was characterized.
23
4. The polysaccharides expressed good surfactant property.
16 17 18
te
d
M
10
Ac ce p
an
9
Mobile: +86 13138907588
E-mail addresses: [email protected]
Highlights:
1. Two sulfated polysaccharides were isolated from Ulva lactuca L. from the South
1
Page 1 of 25
24
5. The polysaccharides are potential as natural additives in food.
25 26
ABSTRACT: Two polysaccharides (ULP1 and ULP2) were isolated through
28
ultrasonic-assisted extraction from green seaweed Ulva lactuca L. which was
29
collected from the South China Sea. The highest yield of 17.57% was obtained under
30
the conditions of 2%NaOH, 90 °C, material/water mass ratio 1:80, liquid extraction
31
5h and subsequent ultrasound-assisted extraction 1h. The structure of ULPs were
32
characterized with periodate oxidation followed by Smith degradation, 1H-NMR, and
33
13
34
189kDa, 230kDa, respectively. The structural characteristics of ULP1 and ULP2 were
35
quite similar. They were composed of rhamnose, xylose, glucose, and glucuronic acid.
36
The content of rhamnose, xylose, glucose, and glucuronic acid, sulfate was 51.2%,
37
12.3%, 20.1%, 16.4%, 12.0% for ULP1, respectively, and 60.8%, 14.2%, 8.2%,
39 40 41 42 43
cr
us
an
te
d
M
C-NMR spectroscopy, FTIR, and GPC. The molecular weights of ULP1, ULP2 were
Ac ce p
38
ip t
27
16.8%, 26.8%, respectively, for ULP2. Both ULP1 and ULP2 showed good surface activity. 5mg/mL ULP1（2.62×10-2mmol/L）decreased the water surface tension to 51.63mN/m. The critical micellular concentration of ULP1, ULP2 was 1.01 mg/mL (5.3×10-3mmol/L) and 1.14mg/mL (5.0×10-3mmol/L), respectively.
Key words: Ulva lactuca L. polysaccharide; structure; surface property
44 45 46
1. Introduction In recent years, marine resources have gained a lot of attention owing to the 2
Page 2 of 25
shortage of land resources. Marine bioresources, including fish, sea animals,
48
seaweeds, bacteria, fungi are abundant and important sources of nutrients, chemicals,
49
and materials. Natural chemicals from marine bioresources are widely investigated
50
and used due to their good biological activities. Polysaccharides are one of the natural
51
chemicals that can be isolated from marine bioresources, especially from seaweeds.
52
Polysaccharides possess various pharmacological activities, including immune
53
regulation, anti-oxidation, antiviral activities, anti-oncological activity, anti-
54
coagulation, and anti-aging effects [1]. As a result of the latter activities, they can be
55
used in a wide gamut of industrial sectors such as food, pharmaceuticals, cosmetic,
56
nutrition, and biomaterials [2].
M
an
us
cr
ip t
47
Surfactants are an important class of compounds to perform emulsifying,
58
solubilizing, wetting, dispersing and foaming in many products, such as detergents,
59
fabric softeners, emulsions, paints, adhesives, inks, anti-fogs, laxatives, biocides,
60
cosmetic, etc. Surfactants are amphiphilic compounds, which means they contain both
62 63 64
te
Ac ce p
61
d
57
hydrophobic groups (alkyl or acyl) and hydrophilic groups (hydroxyl, carboxyl group, sulfate, sulfonate, phosphate, amines, etc.). Owing to pollution and health issues caused by synthesized surfactants, natural surfactants, biomass-based surfactants, and biosurfactants with good biodegradability, low toxicity, and ecological acceptability
65
are increasingly in demand, especially in the fields of food, cosmetics and
66
pharmaceuticals [3]. Sugar-based surfactants are greener surfactants, due to their
67
biomass origin and good surface properties. The reported sugar-based surfactants are
68
usually prepared through biofermentation (such as rhamnolipids, sophorolipids, and
3
Page 3 of 25
mannosylerythritol lipid [4] or chemical modification (such as alkyl glucosides,
70
glucosyl alkane, alkyl cellulose, carboxymethyl cellulose, etc [5,6]. Surface activity of
71
natural polysaccharides has not been reported up until now. On the other hand, in the
72
fields of cosmetics, food, and pharmaceutical, other ingredients with antioxidant
73
activity are also needed to maintain a long shelf life [7]. Natural polysaccharides with
74
multifunctionality are of high potential in these fields.
75
Ulva lactuca L. is a type of common green algae in the division Chlorophyta found
76
in Europe, North and South America, the Caribbean Islands, Africa, Indian Ocean
77
Islands, South-west Asia, China, Pacific Islands, Australia, and New Zealand. A large
78
quantity of Ulva lactuca L. is washed up on beaches every year. Decomposition of
79
Ulva lactuca L produces methane, hydrogen sulphide, and other gases that are not
80
environmentally friendly. Polysaccharides are the main structural chemicals of alga
81
and Ulva lactuca L. is no exception as a potentially renewable source of useful
82
polysaccharides. In 1963, Pervical [8] reported the isolation, purification and chemical
84 85 86
cr us
an
M
d
te
Ac ce p
83
ip t
69
structural study of sulphated polysaccharide from Ulva lactuca L. They isolated 3 fractions and found that the polysaccharides are mainly composed of rhamnose, xylose, glucose, and glucuronic acid, with a sulfate group attached to rhamnose. Brading [9] and his coworker (1954) extracted a water-soluble polysaccharide
87
through boiling with Na2CO3 solution and subsequent ethanol precipitation. They
88
were able to identify the following composition: 31% rhamnose, 9.4% xylose, 19.2%
89
glucuronic acid, 7.7% glucose, and 15.9% sulfate. The chemical composition is
90
complex, and may be affected by species, growth conditions, origin, seasons, etc
4
Page 4 of 25
[10,11]. Although the chemical structure of polysaccharides from different Ulva
92
lactuca L. species are different [12], it is widely accepted that they display an unusual
93
chemical composition and a regular structure of uronic acids, sulfate groups [13,14],
94
and rare sugars, such as rhamnose and iduronic acid [15,16]. Methyl group makes
95
Ulva lactuca L. polysaccharide hydrophobic, while carboxyl, sulfate, hydroxyl make
96
it hydrophilic, which might result in an amphiphlic property and good surface activity
97
of Ulva lactuca L. polysaccharide. However, the surface activity of Ulva lactuca L.
98
polysaccharide has not been reported until now. Natural systems generally have more
99
than one polysaccharide fraction whose structural differences are almost certain.
an
us
cr
ip t
91
Therefore, it is incumbent to ascertain those differences for any long-term chemical
101
use.
M
100
In this paper, two polysaccharide fractions (ULPs) were separated from a green
103
seaweed Ulva lactuca L. collected in April, on the seashore of South China Sea in
104
Hainan province, China. The polysaccharide extraction conditions were optimized
106 107 108
te
Ac ce p
105
d
102
through orthogonal experiments. The structure of the polysaccharides was studied by Fourier transformation infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR),
and
gel-permeation
chromatography
(GPC).
The
monosaccharide
composition of ULP was measured by chemical analysis, gas chromatography (GC)
109
and high-pressure liquid chromatography (HPLC) after hydrolysis. The surface
110
property of the polysaccharides were determined to explore their potential application
111
in food, cosmetics and pharmaceuticals.
112
2. Experimental
5
Page 5 of 25
2.1. Materials
114
Ulva lactuca L. was collected in April from Baishamen Beach, Hainan province,
115
China. The seaweed was identified by Prof. Meihua Liu. Monosaccharide standards
116
(D-galactose (Gal), L-rhamnose (Rha), L-arabinose (Ara), L-fucose (Fuc), D-glucose
117
(Glc), D-xylose (Xyl), D-mannose (Man), and D-glucuronic acid (GlcA)) and
118
dextrans (MW: 196 kDa, 43.5 kDa, 9.9 kDa, 1.46 kDa, and 106 Da) were purchased
119
from Sigma Chemical Co. (St. Louis, MO, USA).
120
2.2. Extraction of crude polysaccharide
121
A fresh sample of green seaweed Ulva lactuca L. was cleaned with tapped water
122
followed by distilled water, air-dried, and then pulverized. Polysaccharides were
123
isolated through ultrasonic-assisted extraction in basic water. A typical procedure was
124
as follows. 2 g of the seaweed was soaked in 80mL diluted NaOH solution, boiled 4h
125
at 70°C in a water bath, and then extracted under ultrasonication for 1h at 70°C. The
126
supernatant was filtered and concentrated to 30 mL, followed by deproteination
128 129 130
cr
us
an
M
d
te
Ac ce p
127
ip t
113
applying the Sevage method [17]. The resultant liquid was dialyzed for 48h against deionized water in a dialysis bag having a cut-off 3500 Da. Two polysaccharide fractions (ULP1 and ULP2) were obtained through stepwise ethanol precipitation that had a final concentration of 30%, 40%, 50%, 60%, 70% and 80% [18], followed by
131
lyophilization. The polysaccharide content was determined by measuring the total
132
sugars using the standard phenol-sulfuric acid method [19].
133
2.3. Measurement of molecular weights
134
The molecular weights of ULP1, ULP2 were measured on a GPC using a Waters 515
6
Page 6 of 25
instrument fitted with two connecting columns (Ultrahydrogel 120 and Ultrahydrogel
136
500, Waters, USA). The eluent was 0.05% NaN3 with a flow rate of 0.6 mL/min at
137
40°C. The measurements were monitored with a Waters 2414 refractive index
138
detector. The GPC system was calibrated before sample analysis with dextran
139
standards (MW: 196 kDa, 43.5 kDa, 9.9 kDa, 1.46 kDa, and 106 Da).
140
2.4. FTIR and NMR analysis
141
The FTIR spectra were measured on a Bruker TENSOR27 spectrometer with a
142
scanning width ranging over 4000–500 cm−1 after immobilizing the samples in KBr.
143
The 1H-NMR and
144
spectrometer at 25°C. The polysaccharide samples were dissolved in D2O. The
145
concentration of the samples was 20 mg/mL for 1H-NMR and 40 mg/mL for
146
NMR.
147
2.5. Analysis of monosaccharide composition
148
2.5.1. Modification with 1-phenyl-3-methyl-5-pyrazolone (PMP)
150 151 152
cr us
an
C-NMR spectra were recorded on a Bruker AV 400 NMR
13
C-
te
d
M
13
Ac ce p
149
ip t
135
The ULP fraction was hydrolyzed according to the method of Lv [20]. Approximately 50 mg of the ULP fraction was dissolved in 5.0 mL of 3mol/L trifluoroacetic acid (TFA) in a 10 mL ampoule. The ampoule was kept in an oven at 120°C for 6 h to hydrolyze the polysaccharides to monosaccharides. The acid was removed through
153
co-distillation with methanol. The hydrolyzed products were modified with PMP.
154
100μl monosaccharide aqueous solution was mixed with 100μl of 0.3 mol/L NaOH,
155
and then 120μl of 0.5 mol/L PMP methanol solution was added. The reaction was
156
allowed to run for 1h at 70°C and then cooled to room temperature and neutralized
7
Page 7 of 25
with 100μl 0.3 mol/L HCl solution. The resultant solution was evaporated to dryness
158
and redissolved in distilled water. Finally, the sample solution was extracted with 15
159
mL chloroform; the process was repeated three times, and finally the aqueous layer
160
was filtered through a 0.22μm membrane [21]. Analysis was carried out using HPLC
161
with a Waters xbridge-C18 column and a UV detector. The HPLC was run using the
162
following conditions: mobile phase, 0.1M potassium phosphate buffer (pH10)-
163
acetonitrile (83:17) at a flow rate 1.0mL/min; column temperature, 30°C; injection
164
volume, 20μl; UV detector, WL245nm. The standard sugars (L-rhamnose, L-fucose,
165
L-arabinose, D-xylose, D-mannose, D-galactose, D-glucose, D-glucuronic acid) were
166
processed likewise.
167
2.5.2. Gas chromatography
168
The polysaccharide was hydrolyzed and dried as described in 2.5.1. The dried
169
samples were dissolved in 0.5mL pyridine, containing 10 mg hydroxylammonium
170
chloride and 1 mg inositol (as internal reference). The modification was run for 30
172 173 174
cr
us
an
M d
te
Ac ce p
171
ip t
157
min at 90°C under nitrogen. Once at room temperature, 0.5mL acetic anhydride was added to further react at 90°C for another 30 min. The resulted solution was directly analyzed with gas chromatography, using the following conditions: column: DB-1701 (30m×0.32mm×1μm) (Agilent Technologies Co., Ltd., USA); detector: flame-
175
ionization detector; gas flow: H2 40mL/min, air 400mL/min, N2 25ml/min; injection
176
temperature: 250°C; detector temperature: 250°C; column temperature: gradient 170-
177
250°C（2°C /min）. The standard sugars were measured according to the same
178
procedure [22].
8
Page 8 of 25
2.5.3. Smith degradation
180
ULP (50 mg) was dissolved in 50 mL 15 mmol/L NaIO4 and stirred at 5°C in the
181
dark. Approximately 20 uL solution was sampled every 24 h, diluted to 250 mL, and
182
followed by spectroscopy at 250nm. Excess ethylene glycol was added to the solution
183
when the absorbance was stable. The content of formic acid was determined by
184
titration using a NaOH solution. The oxidized polysaccharide solution was dialyzed
185
against distilled water and then reduced with NaBH4 at 25°C for 24 h. The excess
186
NaBH4 was decomposed by addition of 25% acetic acid until pH 5.5. The
187
polysaccharide was dialyzed against distilled water and then lyophilized. The
188
oxidized-reduced polysaccharide was hydrolyzed with 2 mol/L TFA at 105°C for 4 h.
189
The excess CF3CO2H was removed by evaporation in the presence of methanol. The
190
resultant materials were subjected to aceytlation and GC analysis [23].
193 194 195 196
cr
us
an
M
d
te
192
2.5.4. Determination of the amounts of sulfate and uronic acid
Ac ce p
191
ip t
179
The sulfate group content in polysaccharides was determined by the barium chloridegelatin method after hydrolysis of corresponding fractions with 1 mol/L HCl [24]. The content of uronic acid was determined by the carbazole-sulfuric acid method, using glucuronic acid as the standard [25].
197
2.6. Surface activity measurement
198
Steady-state fluorescence measurement was used to measure the critical micellar
199
concentration (CMC) of the polysaccharides. Pyrene was used as the probe, which
200
was dissolved in acetone to obtain a solution of 1 × 10-4 mol•L-1 Pyrene/acetone.
9
Page 9 of 25
201
0.1mL pyrene/acetone solution was added to a 10mL volumetric flask. Acetone was
202
blow-dried with N2. Then a 10 mL polysaccharide solution of different concentrations
203
was added to the volumetric flask, where the final concentration of pyrene was 1 × 10-
204
6
205
immediately placed in a temperature-controlled water bath at 37°C in total darkness
206
for 48 h. The fluorescence was measured by a FL-700 fluorescence spectrophotometer
207
(Hitachi, Tokyo, Japan) at 298K, with a slit opening for both excitation and emission
208
at 2.5 nm. Pyrene was excited at 335 nm, and the emission spectrum was collected in
209
over 360–500 nm at a scanning rate of 300 nm/min [26].
ip t
cr
us
an
The surface tensions of polysaccharides were measured at 25 °C and ambient
M
210
mol• L-1. The samples were homogenized using ultrasonication for 1h and
pressure, using a JK99B tensiometer (Shanghai Zhongchen Digital Instruments
212
Company). The tensiometer was calibrated with ultra pure water before use. The
213
platinum ring was cleaned by flame, while the glassware was cleaned with strong
214
basic solution and rinsed with tap water and ultra-pure water. The solution with
216 217 218
te
Ac ce p
215
d
211
concentration 5mg/mL was prepared for the measurement. Each sample was measured in triplicate[5].
3. Results and discussion
3.1. Extraction and purification of ULPs
219
Sulfated polysaccharides were isolated from a green seaweeds Ulva lactuca L. which
220
was collected from the South China Sea. Owing to the low yield of polysaccharide
221
obtained by extraction in water, ultrasonic-assisted extraction was used to optimize
222
the extraction conditions. On the basis of the single factor experiments, orthogonal
10
Page 10 of 25
experiments were carried out to identify an optimal extraction process. Table 1 listed
224
the results of the orthogonal experiments [L9(34)]. Each factor had three levels to be
225
optimized, in which NaOH concentration (1.0%, 1.5%, 2%), solid/liquid ratio (1:60,
226
1:70, 1:80), extraction time (4+1, 5+1, 6+1 h), and temperatures (70°C, 80°C, 90°C)
227
were evaluated. The yields of deproteinated polysaccharides were used to evaluate the
228
process. As shown in Table 1, the results indicated that the magnitude of the effect of
229
the order of the factors on the yield was: extraction temperature > the extraction time
230
> NaOH concentration > the solid/liquid ratio. The highest polysaccharide yield
231
(17.57%) was obtained under the conditions of 2% NaOH, 90°C, material/water ratio
232
= 1:80, liquid extraction time of 5 h, and subsequent ultrasound-assisted extraction 1
233
h.
M
an
us
cr
ip t
223
d
234
te
235
237 238 239 240 241 242
Ac ce p
236
Table 1. Results of the orthogonal experiments L9(34)
NO.
NaOH (A, %)
solid/liquid ratio (B, g/g)
extraction time (C, h)
extraction temperatur e (D, °C)
Yield (%)
1
1(1)
1(1:60)
1(4+1) *
1(70)
9.33
2
1(1)
2(1:70)
2(5+1)
2(80)
17.44
3
1(1)
3(1:80)
3(6+1)
3(90)
14.48 11
Page 11 of 25
2(1.5)
1(1:60)
2(5+1)
3(90)
15.88
5
2(1.5)
2(1:70)
3(6+1)
1(70)
9.83
6
2(1.5)
3(1:80)
1(4+1)
2(80)
13.58
7
3(2)
1(1:60)
3(6+1)
2(80)
13.30
8
3(2)
2(1:70)
1(4+1)
3(90)
15.99
9
3(2)
3(1:80)
2(5+1)
1(70)
k1
0.1375
0.1283
0.1296
0.1160
k2
0.1309
0.1442
0.1632
0.1477
k3
0.1498
0.1457
0.1253
0.1545
R
0.0189
0.0174
0.0379
0.0385
Best conditions
cr
an
A3
B3
C2
D3
A3 B3 C2 D3
M
Best levels
D> C >A > B
15.65
us
The sequence of the factors
ip t
4
*4+1 refers to alkali extraction for 4 h and subsequent ultrasonic-assisted extraction
244
for 1 h; the remainders are similar.
245
247 248 249 250 251
3.2. GPC and FTIR
Ac ce p
246
te
d
243
The ULP was separated through a stepwise precipitation with ethanol solution. Two
fractions with highest content (ULP1, ULP2) in the crude polysaccharide were obtained with 70% and 80% ethanol, respectively. The percentage of ULP1 and ULP2 was 33.44% and 15.05%, respectively. The GPC curves revealed that each fraction was represented by a broad and symmetrical peak (Fig.1). According to the
252
calibration with dextran standards, the number-average molecular weights of ULP1
253
and ULP2 were 189kDa and 230kDa, respectively. The molecular weights of ULPs
254
were in the range of reported molecular weights (5.3×105 to 3.6×106 g/mol) of
255
polysaccharides from genera UlVa seaweeds [27]. The polydispersity index was 1.31 12
Page 12 of 25
and 1.69 for ULP1 and ULP2, respectively, indicating that the polysaccharides were
257
relatively homogeneous in molecular weight distribution.
258 259 260
(Figure 1)
an
us
cr
ip t
256
M
261
d
a: ULP1 b: ULP2
266
Ac ce p
te
c: acidized ULP1
267
IR spectra showed no significant difference between ULP1 and ULP2. The strong
268
absorption band at 3445cm-1 was the O-H stretching vibration of sugar, resulting from
269
intermolecular and intramolecular hydrogen bonding. The peak near 2930 cm-1 was
270
due to C-H stretching vibration, while that near 1418cm-1 was due to the shear
262 263 264 265
(Figure 2)
The FTIR spectra of ULP1, ULP2, and acidized ULP1 are shown in Fig. 2, where
acidized ULP was obtained after acidifying ULP1 with diluted hydrochloric acid. The
13
Page 13 of 25
vibration of CH2. The peak at 1070cm-1 was attributed to the stretching vibration of C-
272
O-C. The absorption at 1635cm-1 was due to the asymmetric stretch vibration of COO-
273
. A new peak showed up at 1720cm-1 after acidification, which further verified that
274
the ULP contained carboxylic groups. The absorption peaks at 1260 cm-1 and 850 cm-
275
1
276
3.3. Monosaccharide analysis
277
The sugar content of the polysaccharides was determined using a PMP-derivation
278
method. ULP1 and ULP2 were hydrolyzed with 2mol/L TFA, subsequently modified
279
with PMP, and then monitored by HPLC. The HPLC spectra are shown in Fig. 3A.
280
Compared to the standard sugars, both ULP1 and ULP2 were composed of rhamnose,
281
xylose, glucose, and glucuronic acid. The contents of the sugars were listed in Table
282
2. It is observed that rhamnose was the main composition of the both fractions. The
283
content of rhamnose, xylose, glucose, glucuronic acid was 51%, 12%, 20%, 17%,
284
respectively, for ULP1, and 61%, 14%, 8%, 17%, respectively, for ULP2. ULP1 has a
286 287 288
cr us
an
M
d
te
Ac ce p
285
corresponded to the –SO3H and C-O-S groups, respectively [28].
ip t
271
lower content of rhamnose and sulfate than ULP2, while a higher content of glucose than ULP2. The contents of uronic acid were determined by chemical analysis by the sulfuric-carbazole reaction[18], which were consistent with those from HPLC. The sulfate content of ULP1 and ULP2 was 12.0% and 26.8%, respectively.
289
14
Page 14 of 25
ip t cr
290
(Figure 3)
us
291 292
Furthermore, the hydrolyzed products were derived by acetic anhydride and
294
detected by GC. The GC spectra of ULPs and the standard sugars are shown in Fig.
295
3B. It was verified that ULP1 and ULP2 were composed of rhamnose, glucose, and
296
xylose. Glucuronic acid was absent in Fig. 3B owing to its high boiling point. The
297
compositions of ULPs are similar with those of Ulva polysaccharides from other
298
species, with rhamnose, xylose, and glucuronic acid being the main components.
299
However, in the reported Ulva polysaccharides, the content of the sugars are in the
301 302 303
M
d
te
Ac ce p
300
an
293
range of rhamnose (16.8-45.0%), xylose (2.1-12.0%), glucose (0.5-6.4%), uronic acid (6.5-19.0%), and sulfate (16.0-23.2%) [29]. ULPs have a higher amount of rhamnose than the reported Ulva polysaccharides, which might give ULPs special properties.
304 305 306 307
15
Page 15 of 25
Table 2. The monosaccharide compositions of ULPs (%) fractions
Rha
Xyl
Glu
GAa
GAb
sulfate
ULP1
51.2
12.3
20.1
16.4
16.8
12.0
ULP2
60.8
14.2
8.2
16.8
16.8
26.8
ip t
308
a: obtained through PMP-HPLC method; b: obtained through barium chloride-gelatin
310
method.
us
cr
309
311
Periodate oxidation followed by Smith degradation with 2mol/L CF3CO2H at
313
105°C for 4 h was carried out on ULP1 and ULP2. The by-products from the
314
degradation were measured by GC using inositol as the internal standard. The spectra
315
are shown in Fig. 3C. As observed, only glycerol and rahmnose were detected after
316
the degradation, while erythritol is absent. The results indicated that there were no
317
1→4 and 1→4,6 linked residues in the polysaccharides. The structure is different
318
from those reported polysaccharides from Ulva, which contain 1→4 gycosidic bonds
322
Ac ce p
te
d
M
an
312
323
connected by oxidative glycosidic bonds 1→, 1→6, 1→2, or 1→2, 6, and unoxidized
324
glycosidic bonds [30].
319 320 321
for most sugars. In ULPs, rahmnose was present with a 1→4 linkage, which contained a sulfate at the 3 position[22]. There was formic acid formed after periodate oxidation, which revealed that there were 1→ and 1→6 glycosidic bonds. According to the amount of NaIO4 consumed and formic acid generated, the other residues were
325 326 327 16
Page 16 of 25
328
3.5. NMR analysis
us
cr
ip t
329
331
(Figure 4)
332
1
H-NMR and
13
an
330
C-NMR spectra of ULP1 and ULP2 were recorded in D2O at
room temperature. The spectra of ULP 1 and ULP2 were similar. The spectra of ULP2
334
are shown in Fig. 4. According to the resonance signals of anomeric H-1 at 4.5~5.5
335
ppm in the 1H-NMR spectra of ULP2 (Fig.4A), ULP2 contained both α-type and β-
336
type glycosidic bonds. The chemical shift at 4.92 ppm was due to the anomeric H-1 of
337
α-L-Rha [31]. The peak at 4.64 ppm was due to the H-1 signal of β-D-xylose, and that
339 340 341
d
te
Ac ce p
338
M
333
at 4.44 ppm due to the H-1 of β-D-xylose. The amount of glucose was low which resulted in invisible signal of H-1 proton NMR. The strong resonance signal owing to –CH3 at 1.31 ppm confirmed the presence of L-Rhamnose [32,33]. The
13
C-NMR spectrum of ULP2 is shown in Fig. 4B. The signal at 102.0 ppm
342
was attributed to the C-1 of D-GlcA, and that at 98.7 ppm attributed to C-1 of α-
343
(1→4)-L-Rha. The peaks at 77.6, 72.6 wee owing to C-4 and C-2,3 of glucouronic
344
acid. Those peaks at 76.9, 67.7, 66.9 ppm were the signals from C-4, C-2, and C-5 of
345
rhamnose. The peak at 61.2 ppm was due to C-5 of xylose. The peak at 56.2 ppm was
17
Page 17 of 25
346
due to the methyl of rhamonse. The signal at 174.17 ppm was assigned to –C=O of D-
347
glucuronic acid. The peak at 15.62 ppm was the characteristic signal of C6 of Rha
348
[34]. Those small signals not assigned are derived from xylose and glucose.
ip t
349
3.6. Surface properties
351
The surface tension of ULP1 and ULP2 in water were measured at room temperature,
352
with the sample concentration being 5mg/mL. The data was measured in triplicate at
353
the same conditions. ULP1 and ULP2 showed reduced water surface tensions, being
354
51.63mN/m and 57.37mN/m, respectively. ULP1 showed better ability than ULP2 as
355
evidenced by the fact that the water surface tension decreased. It was also observed
356
that equilibrium was not easy to achieve during the measurement due to large
357
molecular size and high viscosity. Therefore, steady-state fluorescence was used to
358
measure the critical micellular concentration (CMC) of the polysaccharides.
360 361 362 363
us
an
M
d
te
Pyrene was used as the fluorescence probe to provide indirect measurement of the
Ac ce p
359
cr
350
surface properties of the polysaccharides has been amply demonstrated previously [35]. The ratio of intensities of the first and third peaks I373/I383 reveals the polarity level of the microenvironment where pyrene is located [28]. The plots of fluorescence I373/I383 ratio versus logarithm of ULPs concentration in aqueous solution are shown
364
in Fig. 4. In Fig. 4, a continuous decrease in the fluorescence intensity I373/I383 with an
365
increase in ULPs concentration occurred. The decrease of fluorescence intensity
366
I373/I383 indicated that pyrene was sensing a more hydrophobic environment, which
367
resulted from the aggregation of ULPs and incorporation of pyrene into the
18
Page 18 of 25
hydrophobic area of the ULPs micelles. It is widely accepted that pyrene I373/I383 for
369
surfactants is well fitted by a Boltzmann function [36]. Function 1 and function 2
370
were the Boltzmann functions of ULP1 and ULP2, respectively. According to the
371
Boltzmann functions, the CMC of ULP1 and ULP2 was about 1.01 mg/mL (5.3×10-
372
3
373
good surface activities.
374
Y1
375
Y2
ip t
368
1.23
(1)
0.84 1.05 1 e ( x0.48) / 0.46
(2)
an
1 e
( x 0.44 ) / 0.41
us
0.66
cr
mmol/L) and 1.14mg/mL (5.0×10-3mmol/L), respectively, indicating ULPs have
376
M
377 378
d
2.0
—■—ULP1
1.6
Ac ce p
I1/I3
—◆—ULP2 1.4
te
1.8
1.2
1.0
-2.0
379 380 381
-1.5
-1.0
-0.5
0.0
0.5
1.0
logC
(Figure 5)
382
The self-aggregation of ULPs in aqueous solution was due to their amphiphilic
383
structures, in which they contain hydrophilic –OHs, -SO3Na and hydrophobic methyls
384
of rhamnose, in accordance with the structural results. ULP1 and ULP2 have high
385
contents of rhamnose (51.2% and 60.8%), which gives them good surface properties.
19
Page 19 of 25
Surface activity is affected by the chemical structure of the samples, especially the
387
balance of hydrophilic and hydrophobic groups. For ULPs, methyl of rhamnose is the
388
hydrophobic group, while sulfate plays important role on the hydrophilicity. ULP1
389
has lower rhamnose content and much lower sulfate content (shown in Table 2, 51.2%
390
rha, 12.0% sulfate) than ULP2 (60.8% rha, 26.8% sulfate), resulting in ULP1
391
expressing better surface activity than ULP2. There are no reports on the surface
392
activities of algae polysaccharides until now. The natural origin and good surface
393
properties of Ulva polysaccharides makes them potentially useful as surfactants in
394
food pharmaceutical and cosmetic applications.
395
4. Conclusions
M
an
us
cr
ip t
386
Two sulfated polysaccharides were extracted from Ulva lactuca L. collected
397
from the South China Sea through ultrasonic-assistant extraction, and followed by
398
stepwise ethanol precipitation. The polysaccharides consisted of rhamnose, xylose,
399
glucose, glucuronic acid and sulfate. The polysaccharides contain 1→, 1→6, 1→2,
401 402 403
te
Ac ce p
400
d
396
1→2,6, or unoxidized glycosidic bonds, different from the reported polysaccharides from other Ulva species. The present polysaccharides have good surface properties. Surface properties of natural polysaccharides were first reported, which might inspire more concerns on polysaccharides and make them potential feedstocks as food,
404
cosmetic, and pharmaceutical components. The specific information of sugar
405
sequence could not be obtained due to the unsuccessful methylation analysis of ULPs.
406
Further work is required to specify the structures. And the relationship between the
407
amphiphilic structure and the surface properties should be more detailed.
20
Page 20 of 25
408
Acknowledgments
410
The authors acknowledge the National Science Foundation of China (Project No.
411
21264007, 21466011), Key Scientific and Technological Project of Haikou city
412
(2010-084), and the Young Researcher Project of Hainan University (qnjj1221) for
413
financial support. We also thank Prof L.A. Lucia for helpful discussions regarding the
414
preparation of the manuscript.
us
cr
ip t
409
an
415
Figure Captions
417
Fig. 1 The GPC distributions of ULPs
418
Fig. 2 The FTIR spectra of ULPs
419
Fig. 3 HPLC spectra of PMP modified sugars (A), GC chromatograms of sugar after
420
acetylation (B), and GC chromatograms of ULPs products from Smith
421
degradation (C)
422
Fig. 4 The 1H-NMR (A) and 13C-NMR (B) spectra of ULP2
423
Ac ce p
te
d
M
416
424 425 426 427 428
Fig. 5 The plot fluorescence I373/I383 ratio versus logarithm ULPs concentration in aqueous solution
References[1] Nikapitiya C, De Zoysa M, Jeon Y J, et al. Isolation of sulfated
429
anticoagulant compound from fermented red seaweed Grateloupia filicina, Journal
430
of the World Aquaculture Society. 38 (2007) 407-417.
431 432
[2] Pujol C A, Errea M I, Matulewicz M C, et al, Antiherpetic activity of S1, an algal derived sulphated galactan, Phytotherapy Research. 10 (1996) 410-413.
433
[3] Rebello S., Asok A. K., Mundayoor S., Jisha M. S., Surfactants: toxicity,
434
remediation and green surfactants, Environmental Chemistry Letter. 12 (2014) 21
Page 21 of 25
435 436 437
275–287. [4] Lourith, N., Kanlayavattanakul, M., Natural surfactants used in cosmetics: glycolipids, International Journal of Cosmetic Science. 31 (2009) 255–261. [5] Zhang, T., Marchan, R. E., Novel Polysaccharide Surfactants: The Effect of
439
Hydrophobic and Hydrophilic Chain Length on Surface Active Properties, J.
440
Colloid Interface Sci. 177 (1996) 419–426.
ip t
438
[6] Melo, M. R. S., Feitosa, J. P. A., Freitas, A. L. P., De Paula, R. C. M., Isolation and
442
characterization of soluble sulfated polysaccharide from the red seaweed
443
Gracilaria cornea. Carbohydr. Polym. 49 (2002) 491-498.
us
445
[7] Yanishlieva, N. V, Marinova, E., Pokorny, J., Natural antioxidants from herbs and spices, Eur. J. Lipid Sci. Technol. 108 (2006) 776–793.
an
444
cr
441
[8] Percival, E., Wold, J. K., The acid polysaccharide from the green seaweed Ulva
447
lactuca. Part II. The site of the ester sulphate, J. Chem. Soc. 1040 (1963) 5459-
448
5468.
M
446
[9] Brading, J. W., Georg-Plant, M. M. T., Hardy, D. M. The polysaccharide from the
450
alga Ulva lactuca. Purification, hydrolysis, and methylation of the polysaccharide,
451
J. Chem. Soc. (1954) 319-324.
452
[10] Nagaoka, M., Shibata, H., Kimura-Takagi, I., Hashimoto, S., Aiyama, R.,
453
Ac ce p
te
d
449
454 455 456 457 458
Ueyama, S., Yokokura, T. Anti-ulcer effects and biological activities of polysaccharides from marine algae, Biofactors. 12 (2000) 267-274.
[11] Costa C., Alves A., Pinto P. R., Sousa R. A., Silva E. A. B., Reis R. L., Rodrigues A. E, Characterization of ulvan extracts to assess the effect of different steps in the extraction procedure, Carbohydr. Polym. 88 (2012) 537- 546.
[12] Hernández-Garibay, E., Zertuche-González, J. A., Pacheco-Ruíz, I, Isolation and
459
chemical characterization of algal polysaccharides from the green seaweed Ulva
460
clathrata (Roth) C. Agardh, J. Appl. Phycol. 23 (2011) 537-542.
461
[13] Qi, H., Huang, L., Liu, X., Liu, D., Zhang, Q., Liu, S. Antihyperlipidemic
462
activity of high sulfate content derivative of polysaccharide extracted from Ulva
463
pertusa(Chlorophyta), Carbohydr. Polym. 87 (2012) 1637-1640.
464
[14] Qi, H., Zhang, Q., Zhao, T., Chen, R., Zhang, H., Niu, X., Li, Z. Antioxidant 22
Page 22 of 25
activity of different sulfate content derivatives of polysaccharide extracted from
466
Ulva pertusa(Chlorophyta) in vitro, Int. J. Biol. Macromol. 37 (2005) 195-199.
467
[15] Jaulneau, V., Lafitte, C., Corio-Costet, M. F., Stadnik, M. J., Salamagne, S.,
468
Briand, X., Dumas, B., An Ulva armoricana extract protects plants against three
469
powdery mildew pathogens, European journal of plant pathology. 131 (2011) 393-
470
401.
ip t
465
[16] Shao, P., Chen, M., Pei, Y., & Sun, P., In intro antioxidant activities of different
472
sulfated polysaccharides from chlorophytan seaweeds Ulva fasciata, Int. J. Biol.
473
Macromol. 59 (2013) 295-300.
us
cr
471
[17] Navarini, L., Gilli, R., Gombac, V., Abatangelo, A., Bosco, M., Toffanin, R.,
475
Polysaccharides from hot water extracts of roasted Coffea arabica beans: isolation
476
and characterization, Carbohydr. Polym. 40 (1999) 71-81.
an
474
[18] Huang, X., Wang, D., Hu, Y., Lu, Y., Guo, Z., Kong, X., Sun, J., Effect of
478
sulfated astragalus polysaccharide on cellular infectivity of infectious bursal
479
disease virus, Int. J. Biol. Macromol. 42 (2008) 166-171.
d
M
477
[19] Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P., & Smith, F., Colorimetric
481
method for determination of sugars and related substances, Anal. Chem. 28 (1956)
482
350-356.
484 485 486 487 488 489 490
Ac ce p
483
te
480
[20] Lv, Y., Yang, X., Zhao, Y., Ruan, Y., Yang, Y., Wang, Z., Separation and quantification of component monosaccharides of the tea polysaccharides from Gynostemma pentaphyllum by HPLC with indirect UV detection, Food Chem. 112 (2009) 742-746.
[21] Hsu, W. K., Hsu, T. H., Lin, F. Y., Cheng, Y. K., Yang, J. P. W., Separation, purification, and α-glucosidase inhibition of polysaccharides from Coriolus versicolor LH1 mycelia, Carbohydr. Polym. 92 (2013) 297-306. [22] Sun, H. H., Mao, W. J., Chen, Y., Guo, S. D., Li, H. Y., Qi, X. H., Xu, J.,
491
Isolation,
492
polysaccharides from marine fungus Penicillium sp. F23-2, Carbohydr. Polym. 78
493
(2009) 117-124.
494
chemical
characteristics
and
antioxidant
properties
of
the
[23] Gutiérrez, A., Prieto, A., Martínez, A. T., Structural characterization of 23
Page 23 of 25
495
extracellular polysaccharides produced by fungi from the genus Pleurotus,
496
Carbohydr. Res. 281 (1996) 143-154. [24] Souza, Bartolomeu W.S., et al, Chemical characterization and antioxidant activity
498
of sulfated polysaccharide from the red seaweed Gracilaria birdiae, Food
499
Hydrocolloids. 27 (2012) 287–292.
503 504 505
cr
502
Biochem. 4 (1962) 330-334.
[26] Mathias, J. H., Rosen, M. J., Davenport, L., Fluorescence study of premicellar aggregation in cationic gemini surfactants, Langmuir. 17 (2001) 6148-6154.
us
501
[25] Bitter, T., Muir, H. M., A modified uronic acid carbazole reaction, Anal.
[27] Alves, A., Sousa, R., Reis, R., A practical perspective on ulvan extracted from green algae, Journal of Applied Phycology. 25 (2013) 407-424.
an
500
ip t
497
[28] Yang, B., Jiang, Y., Zhao, M., Chen, F., Wang, R., Chen, Y., & Zhang, D.,
507
Structural characterisation of polysaccharides purified from longan (Dimocarpus
508
longan Lour.) fruit pericarp, Food Chem. 115 (2009) 609-614.
M
506
[29] Lahaye, M., Robic, A., Structure and functional properties of ulvan, a
510
polysaccharide from green seaweeds, Biomacromolecules. 8 (2007) 1765-1774.
511
[30] Baird, J. K., Holroyde, M. J., Ellwood, D. C., Analysis of the products of Smith
512
degradation of polysaccharides by glc of the acetylated, derived aldononitriles and
513
alditols, Carbohydr. Res. 27 (1973) 464-467.
515 516 517 518 519 520 521
te
Ac ce p
514
d
509
[31] Lahaye, M., Inizan, F., Vigoureux, J., NMR analysis of the chemical structure of ulvan and of ulvan-boron complex formation. Carbohydr Polym. 36 (1998) 239249.
[32] Guentas, L., Pheulpin, P., Michaud, P., Heyraud, A., Gey, C., Courtois, B., Courtois, J., Structure of a polysaccharide from a Rhizobium species containing 2deoxy-β-d- arabino-hexuronic acid, Carbohydr. Res. 332 (2001) 167-173. [33] I, Uhliariková, et al, Positional specifity of acetylxylan esterases on natural polysaccharide: an NMR study, Biochim Biophys Acta. 1830 (2013) 3365–3372.
522
[34] D, Zhang, Three sulphated polysaccharides isolated from the mucilage of mud
523
snail, Bullacta exarata philippi: Characterization and antitumour activity, Food
524
Chem. 138 (2013) 306–314. 24
Page 24 of 25
525
[35] G, Basu Ray, Pyrene absorption can be a convenient method for probing critical
526
micellar concentration (cmc) and indexing micellar polarity, J Colloid Interface
527
Sci. 294 (2006) 248–254. [36] Aguiar, J., et al, On the determination of the critical micelle concentration by the
529
pyrene 1:3 ratio method, Journal of Colloid & Interface Science. 258 (2003) 116–
530
122.
ip t
528
Ac ce p
te
d
M
an
us
cr
531
25
Page 25 of 25
S0141-8130(15)00372-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.05.031 BIOMAC 5115
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
17-4-2015 19-5-2015 21-5-2015
Please cite this article as: H. Tian, X. Yin, Q. Zeng, L. Zhu, J. Chen, Isolation, structure, and surfactant properties of polysaccharides from Ulva lactuca L. from South China Sea, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.05.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Isolation, structure, and surfactant properties of polysaccharides
2
from Ulva lactuca L. from South China Sea
3
Hua Tiana,b, Xueqiong Yina,b *, Qinghuan Zengb, Li Zhub, Junhua Chenb
ip t
1
4 5
a
6
University, Haikou, Hainan 570228, P.R. China
7
b
8
Haikou, Hainan, 570228, P.R. China
cr
Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan
us
Hainan Provincial Fine Chemical Engineering Research Center, Hainan University,
*Corresponding author at:
11
Address: School of Materials and Chemical Engineering, Hainan University, 58th
12
Renmin Road, Haikou, Hainan, 570228, PR China.
13
Tel.: +86 898 66279161
14
Fax: +86 898 66291383;
15
19 20
China Sea.
21
2. The extraction conditions were optimized, getting the highest yield of 17.57%.
22
3. The structure of the polysaccharides was characterized.
23
4. The polysaccharides expressed good surfactant property.
16 17 18
te
d
M
10
Ac ce p
an
9
Mobile: +86 13138907588
E-mail addresses: [email protected]
Highlights:
1. Two sulfated polysaccharides were isolated from Ulva lactuca L. from the South
1
Page 1 of 25
24
5. The polysaccharides are potential as natural additives in food.
25 26
ABSTRACT: Two polysaccharides (ULP1 and ULP2) were isolated through
28
ultrasonic-assisted extraction from green seaweed Ulva lactuca L. which was
29
collected from the South China Sea. The highest yield of 17.57% was obtained under
30
the conditions of 2%NaOH, 90 °C, material/water mass ratio 1:80, liquid extraction
31
5h and subsequent ultrasound-assisted extraction 1h. The structure of ULPs were
32
characterized with periodate oxidation followed by Smith degradation, 1H-NMR, and
33
13
34
189kDa, 230kDa, respectively. The structural characteristics of ULP1 and ULP2 were
35
quite similar. They were composed of rhamnose, xylose, glucose, and glucuronic acid.
36
The content of rhamnose, xylose, glucose, and glucuronic acid, sulfate was 51.2%,
37
12.3%, 20.1%, 16.4%, 12.0% for ULP1, respectively, and 60.8%, 14.2%, 8.2%,
39 40 41 42 43
cr
us
an
te
d
M
C-NMR spectroscopy, FTIR, and GPC. The molecular weights of ULP1, ULP2 were
Ac ce p
38
ip t
27
16.8%, 26.8%, respectively, for ULP2. Both ULP1 and ULP2 showed good surface activity. 5mg/mL ULP1（2.62×10-2mmol/L）decreased the water surface tension to 51.63mN/m. The critical micellular concentration of ULP1, ULP2 was 1.01 mg/mL (5.3×10-3mmol/L) and 1.14mg/mL (5.0×10-3mmol/L), respectively.
Key words: Ulva lactuca L. polysaccharide; structure; surface property
44 45 46
1. Introduction In recent years, marine resources have gained a lot of attention owing to the 2
Page 2 of 25
shortage of land resources. Marine bioresources, including fish, sea animals,
48
seaweeds, bacteria, fungi are abundant and important sources of nutrients, chemicals,
49
and materials. Natural chemicals from marine bioresources are widely investigated
50
and used due to their good biological activities. Polysaccharides are one of the natural
51
chemicals that can be isolated from marine bioresources, especially from seaweeds.
52
Polysaccharides possess various pharmacological activities, including immune
53
regulation, anti-oxidation, antiviral activities, anti-oncological activity, anti-
54
coagulation, and anti-aging effects [1]. As a result of the latter activities, they can be
55
used in a wide gamut of industrial sectors such as food, pharmaceuticals, cosmetic,
56
nutrition, and biomaterials [2].
M
an
us
cr
ip t
47
Surfactants are an important class of compounds to perform emulsifying,
58
solubilizing, wetting, dispersing and foaming in many products, such as detergents,
59
fabric softeners, emulsions, paints, adhesives, inks, anti-fogs, laxatives, biocides,
60
cosmetic, etc. Surfactants are amphiphilic compounds, which means they contain both
62 63 64
te
Ac ce p
61
d
57
hydrophobic groups (alkyl or acyl) and hydrophilic groups (hydroxyl, carboxyl group, sulfate, sulfonate, phosphate, amines, etc.). Owing to pollution and health issues caused by synthesized surfactants, natural surfactants, biomass-based surfactants, and biosurfactants with good biodegradability, low toxicity, and ecological acceptability
65
are increasingly in demand, especially in the fields of food, cosmetics and
66
pharmaceuticals [3]. Sugar-based surfactants are greener surfactants, due to their
67
biomass origin and good surface properties. The reported sugar-based surfactants are
68
usually prepared through biofermentation (such as rhamnolipids, sophorolipids, and
3
Page 3 of 25
mannosylerythritol lipid [4] or chemical modification (such as alkyl glucosides,
70
glucosyl alkane, alkyl cellulose, carboxymethyl cellulose, etc [5,6]. Surface activity of
71
natural polysaccharides has not been reported up until now. On the other hand, in the
72
fields of cosmetics, food, and pharmaceutical, other ingredients with antioxidant
73
activity are also needed to maintain a long shelf life [7]. Natural polysaccharides with
74
multifunctionality are of high potential in these fields.
75
Ulva lactuca L. is a type of common green algae in the division Chlorophyta found
76
in Europe, North and South America, the Caribbean Islands, Africa, Indian Ocean
77
Islands, South-west Asia, China, Pacific Islands, Australia, and New Zealand. A large
78
quantity of Ulva lactuca L. is washed up on beaches every year. Decomposition of
79
Ulva lactuca L produces methane, hydrogen sulphide, and other gases that are not
80
environmentally friendly. Polysaccharides are the main structural chemicals of alga
81
and Ulva lactuca L. is no exception as a potentially renewable source of useful
82
polysaccharides. In 1963, Pervical [8] reported the isolation, purification and chemical
84 85 86
cr us
an
M
d
te
Ac ce p
83
ip t
69
structural study of sulphated polysaccharide from Ulva lactuca L. They isolated 3 fractions and found that the polysaccharides are mainly composed of rhamnose, xylose, glucose, and glucuronic acid, with a sulfate group attached to rhamnose. Brading [9] and his coworker (1954) extracted a water-soluble polysaccharide
87
through boiling with Na2CO3 solution and subsequent ethanol precipitation. They
88
were able to identify the following composition: 31% rhamnose, 9.4% xylose, 19.2%
89
glucuronic acid, 7.7% glucose, and 15.9% sulfate. The chemical composition is
90
complex, and may be affected by species, growth conditions, origin, seasons, etc
4
Page 4 of 25
[10,11]. Although the chemical structure of polysaccharides from different Ulva
92
lactuca L. species are different [12], it is widely accepted that they display an unusual
93
chemical composition and a regular structure of uronic acids, sulfate groups [13,14],
94
and rare sugars, such as rhamnose and iduronic acid [15,16]. Methyl group makes
95
Ulva lactuca L. polysaccharide hydrophobic, while carboxyl, sulfate, hydroxyl make
96
it hydrophilic, which might result in an amphiphlic property and good surface activity
97
of Ulva lactuca L. polysaccharide. However, the surface activity of Ulva lactuca L.
98
polysaccharide has not been reported until now. Natural systems generally have more
99
than one polysaccharide fraction whose structural differences are almost certain.
an
us
cr
ip t
91
Therefore, it is incumbent to ascertain those differences for any long-term chemical
101
use.
M
100
In this paper, two polysaccharide fractions (ULPs) were separated from a green
103
seaweed Ulva lactuca L. collected in April, on the seashore of South China Sea in
104
Hainan province, China. The polysaccharide extraction conditions were optimized
106 107 108
te
Ac ce p
105
d
102
through orthogonal experiments. The structure of the polysaccharides was studied by Fourier transformation infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR),
and
gel-permeation
chromatography
(GPC).
The
monosaccharide
composition of ULP was measured by chemical analysis, gas chromatography (GC)
109
and high-pressure liquid chromatography (HPLC) after hydrolysis. The surface
110
property of the polysaccharides were determined to explore their potential application
111
in food, cosmetics and pharmaceuticals.
112
2. Experimental
5
Page 5 of 25
2.1. Materials
114
Ulva lactuca L. was collected in April from Baishamen Beach, Hainan province,
115
China. The seaweed was identified by Prof. Meihua Liu. Monosaccharide standards
116
(D-galactose (Gal), L-rhamnose (Rha), L-arabinose (Ara), L-fucose (Fuc), D-glucose
117
(Glc), D-xylose (Xyl), D-mannose (Man), and D-glucuronic acid (GlcA)) and
118
dextrans (MW: 196 kDa, 43.5 kDa, 9.9 kDa, 1.46 kDa, and 106 Da) were purchased
119
from Sigma Chemical Co. (St. Louis, MO, USA).
120
2.2. Extraction of crude polysaccharide
121
A fresh sample of green seaweed Ulva lactuca L. was cleaned with tapped water
122
followed by distilled water, air-dried, and then pulverized. Polysaccharides were
123
isolated through ultrasonic-assisted extraction in basic water. A typical procedure was
124
as follows. 2 g of the seaweed was soaked in 80mL diluted NaOH solution, boiled 4h
125
at 70°C in a water bath, and then extracted under ultrasonication for 1h at 70°C. The
126
supernatant was filtered and concentrated to 30 mL, followed by deproteination
128 129 130
cr
us
an
M
d
te
Ac ce p
127
ip t
113
applying the Sevage method [17]. The resultant liquid was dialyzed for 48h against deionized water in a dialysis bag having a cut-off 3500 Da. Two polysaccharide fractions (ULP1 and ULP2) were obtained through stepwise ethanol precipitation that had a final concentration of 30%, 40%, 50%, 60%, 70% and 80% [18], followed by
131
lyophilization. The polysaccharide content was determined by measuring the total
132
sugars using the standard phenol-sulfuric acid method [19].
133
2.3. Measurement of molecular weights
134
The molecular weights of ULP1, ULP2 were measured on a GPC using a Waters 515
6
Page 6 of 25
instrument fitted with two connecting columns (Ultrahydrogel 120 and Ultrahydrogel
136
500, Waters, USA). The eluent was 0.05% NaN3 with a flow rate of 0.6 mL/min at
137
40°C. The measurements were monitored with a Waters 2414 refractive index
138
detector. The GPC system was calibrated before sample analysis with dextran
139
standards (MW: 196 kDa, 43.5 kDa, 9.9 kDa, 1.46 kDa, and 106 Da).
140
2.4. FTIR and NMR analysis
141
The FTIR spectra were measured on a Bruker TENSOR27 spectrometer with a
142
scanning width ranging over 4000–500 cm−1 after immobilizing the samples in KBr.
143
The 1H-NMR and
144
spectrometer at 25°C. The polysaccharide samples were dissolved in D2O. The
145
concentration of the samples was 20 mg/mL for 1H-NMR and 40 mg/mL for
146
NMR.
147
2.5. Analysis of monosaccharide composition
148
2.5.1. Modification with 1-phenyl-3-methyl-5-pyrazolone (PMP)
150 151 152
cr us
an
C-NMR spectra were recorded on a Bruker AV 400 NMR
13
C-
te
d
M
13
Ac ce p
149
ip t
135
The ULP fraction was hydrolyzed according to the method of Lv [20]. Approximately 50 mg of the ULP fraction was dissolved in 5.0 mL of 3mol/L trifluoroacetic acid (TFA) in a 10 mL ampoule. The ampoule was kept in an oven at 120°C for 6 h to hydrolyze the polysaccharides to monosaccharides. The acid was removed through
153
co-distillation with methanol. The hydrolyzed products were modified with PMP.
154
100μl monosaccharide aqueous solution was mixed with 100μl of 0.3 mol/L NaOH,
155
and then 120μl of 0.5 mol/L PMP methanol solution was added. The reaction was
156
allowed to run for 1h at 70°C and then cooled to room temperature and neutralized
7
Page 7 of 25
with 100μl 0.3 mol/L HCl solution. The resultant solution was evaporated to dryness
158
and redissolved in distilled water. Finally, the sample solution was extracted with 15
159
mL chloroform; the process was repeated three times, and finally the aqueous layer
160
was filtered through a 0.22μm membrane [21]. Analysis was carried out using HPLC
161
with a Waters xbridge-C18 column and a UV detector. The HPLC was run using the
162
following conditions: mobile phase, 0.1M potassium phosphate buffer (pH10)-
163
acetonitrile (83:17) at a flow rate 1.0mL/min; column temperature, 30°C; injection
164
volume, 20μl; UV detector, WL245nm. The standard sugars (L-rhamnose, L-fucose,
165
L-arabinose, D-xylose, D-mannose, D-galactose, D-glucose, D-glucuronic acid) were
166
processed likewise.
167
2.5.2. Gas chromatography
168
The polysaccharide was hydrolyzed and dried as described in 2.5.1. The dried
169
samples were dissolved in 0.5mL pyridine, containing 10 mg hydroxylammonium
170
chloride and 1 mg inositol (as internal reference). The modification was run for 30
172 173 174
cr
us
an
M d
te
Ac ce p
171
ip t
157
min at 90°C under nitrogen. Once at room temperature, 0.5mL acetic anhydride was added to further react at 90°C for another 30 min. The resulted solution was directly analyzed with gas chromatography, using the following conditions: column: DB-1701 (30m×0.32mm×1μm) (Agilent Technologies Co., Ltd., USA); detector: flame-
175
ionization detector; gas flow: H2 40mL/min, air 400mL/min, N2 25ml/min; injection
176
temperature: 250°C; detector temperature: 250°C; column temperature: gradient 170-
177
250°C（2°C /min）. The standard sugars were measured according to the same
178
procedure [22].
8
Page 8 of 25
2.5.3. Smith degradation
180
ULP (50 mg) was dissolved in 50 mL 15 mmol/L NaIO4 and stirred at 5°C in the
181
dark. Approximately 20 uL solution was sampled every 24 h, diluted to 250 mL, and
182
followed by spectroscopy at 250nm. Excess ethylene glycol was added to the solution
183
when the absorbance was stable. The content of formic acid was determined by
184
titration using a NaOH solution. The oxidized polysaccharide solution was dialyzed
185
against distilled water and then reduced with NaBH4 at 25°C for 24 h. The excess
186
NaBH4 was decomposed by addition of 25% acetic acid until pH 5.5. The
187
polysaccharide was dialyzed against distilled water and then lyophilized. The
188
oxidized-reduced polysaccharide was hydrolyzed with 2 mol/L TFA at 105°C for 4 h.
189
The excess CF3CO2H was removed by evaporation in the presence of methanol. The
190
resultant materials were subjected to aceytlation and GC analysis [23].
193 194 195 196
cr
us
an
M
d
te
192
2.5.4. Determination of the amounts of sulfate and uronic acid
Ac ce p
191
ip t
179
The sulfate group content in polysaccharides was determined by the barium chloridegelatin method after hydrolysis of corresponding fractions with 1 mol/L HCl [24]. The content of uronic acid was determined by the carbazole-sulfuric acid method, using glucuronic acid as the standard [25].
197
2.6. Surface activity measurement
198
Steady-state fluorescence measurement was used to measure the critical micellar
199
concentration (CMC) of the polysaccharides. Pyrene was used as the probe, which
200
was dissolved in acetone to obtain a solution of 1 × 10-4 mol•L-1 Pyrene/acetone.
9
Page 9 of 25
201
0.1mL pyrene/acetone solution was added to a 10mL volumetric flask. Acetone was
202
blow-dried with N2. Then a 10 mL polysaccharide solution of different concentrations
203
was added to the volumetric flask, where the final concentration of pyrene was 1 × 10-
204
6
205
immediately placed in a temperature-controlled water bath at 37°C in total darkness
206
for 48 h. The fluorescence was measured by a FL-700 fluorescence spectrophotometer
207
(Hitachi, Tokyo, Japan) at 298K, with a slit opening for both excitation and emission
208
at 2.5 nm. Pyrene was excited at 335 nm, and the emission spectrum was collected in
209
over 360–500 nm at a scanning rate of 300 nm/min [26].
ip t
cr
us
an
The surface tensions of polysaccharides were measured at 25 °C and ambient
M
210
mol• L-1. The samples were homogenized using ultrasonication for 1h and
pressure, using a JK99B tensiometer (Shanghai Zhongchen Digital Instruments
212
Company). The tensiometer was calibrated with ultra pure water before use. The
213
platinum ring was cleaned by flame, while the glassware was cleaned with strong
214
basic solution and rinsed with tap water and ultra-pure water. The solution with
216 217 218
te
Ac ce p
215
d
211
concentration 5mg/mL was prepared for the measurement. Each sample was measured in triplicate[5].
3. Results and discussion
3.1. Extraction and purification of ULPs
219
Sulfated polysaccharides were isolated from a green seaweeds Ulva lactuca L. which
220
was collected from the South China Sea. Owing to the low yield of polysaccharide
221
obtained by extraction in water, ultrasonic-assisted extraction was used to optimize
222
the extraction conditions. On the basis of the single factor experiments, orthogonal
10
Page 10 of 25
experiments were carried out to identify an optimal extraction process. Table 1 listed
224
the results of the orthogonal experiments [L9(34)]. Each factor had three levels to be
225
optimized, in which NaOH concentration (1.0%, 1.5%, 2%), solid/liquid ratio (1:60,
226
1:70, 1:80), extraction time (4+1, 5+1, 6+1 h), and temperatures (70°C, 80°C, 90°C)
227
were evaluated. The yields of deproteinated polysaccharides were used to evaluate the
228
process. As shown in Table 1, the results indicated that the magnitude of the effect of
229
the order of the factors on the yield was: extraction temperature > the extraction time
230
> NaOH concentration > the solid/liquid ratio. The highest polysaccharide yield
231
(17.57%) was obtained under the conditions of 2% NaOH, 90°C, material/water ratio
232
= 1:80, liquid extraction time of 5 h, and subsequent ultrasound-assisted extraction 1
233
h.
M
an
us
cr
ip t
223
d
234
te
235
237 238 239 240 241 242
Ac ce p
236
Table 1. Results of the orthogonal experiments L9(34)
NO.
NaOH (A, %)
solid/liquid ratio (B, g/g)
extraction time (C, h)
extraction temperatur e (D, °C)
Yield (%)
1
1(1)
1(1:60)
1(4+1) *
1(70)
9.33
2
1(1)
2(1:70)
2(5+1)
2(80)
17.44
3
1(1)
3(1:80)
3(6+1)
3(90)
14.48 11
Page 11 of 25
2(1.5)
1(1:60)
2(5+1)
3(90)
15.88
5
2(1.5)
2(1:70)
3(6+1)
1(70)
9.83
6
2(1.5)
3(1:80)
1(4+1)
2(80)
13.58
7
3(2)
1(1:60)
3(6+1)
2(80)
13.30
8
3(2)
2(1:70)
1(4+1)
3(90)
15.99
9
3(2)
3(1:80)
2(5+1)
1(70)
k1
0.1375
0.1283
0.1296
0.1160
k2
0.1309
0.1442
0.1632
0.1477
k3
0.1498
0.1457
0.1253
0.1545
R
0.0189
0.0174
0.0379
0.0385
Best conditions
cr
an
A3
B3
C2
D3
A3 B3 C2 D3
M
Best levels
D> C >A > B
15.65
us
The sequence of the factors
ip t
4
*4+1 refers to alkali extraction for 4 h and subsequent ultrasonic-assisted extraction
244
for 1 h; the remainders are similar.
245
247 248 249 250 251
3.2. GPC and FTIR
Ac ce p
246
te
d
243
The ULP was separated through a stepwise precipitation with ethanol solution. Two
fractions with highest content (ULP1, ULP2) in the crude polysaccharide were obtained with 70% and 80% ethanol, respectively. The percentage of ULP1 and ULP2 was 33.44% and 15.05%, respectively. The GPC curves revealed that each fraction was represented by a broad and symmetrical peak (Fig.1). According to the
252
calibration with dextran standards, the number-average molecular weights of ULP1
253
and ULP2 were 189kDa and 230kDa, respectively. The molecular weights of ULPs
254
were in the range of reported molecular weights (5.3×105 to 3.6×106 g/mol) of
255
polysaccharides from genera UlVa seaweeds [27]. The polydispersity index was 1.31 12
Page 12 of 25
and 1.69 for ULP1 and ULP2, respectively, indicating that the polysaccharides were
257
relatively homogeneous in molecular weight distribution.
258 259 260
(Figure 1)
an
us
cr
ip t
256
M
261
d
a: ULP1 b: ULP2
266
Ac ce p
te
c: acidized ULP1
267
IR spectra showed no significant difference between ULP1 and ULP2. The strong
268
absorption band at 3445cm-1 was the O-H stretching vibration of sugar, resulting from
269
intermolecular and intramolecular hydrogen bonding. The peak near 2930 cm-1 was
270
due to C-H stretching vibration, while that near 1418cm-1 was due to the shear
262 263 264 265
(Figure 2)
The FTIR spectra of ULP1, ULP2, and acidized ULP1 are shown in Fig. 2, where
acidized ULP was obtained after acidifying ULP1 with diluted hydrochloric acid. The
13
Page 13 of 25
vibration of CH2. The peak at 1070cm-1 was attributed to the stretching vibration of C-
272
O-C. The absorption at 1635cm-1 was due to the asymmetric stretch vibration of COO-
273
. A new peak showed up at 1720cm-1 after acidification, which further verified that
274
the ULP contained carboxylic groups. The absorption peaks at 1260 cm-1 and 850 cm-
275
1
276
3.3. Monosaccharide analysis
277
The sugar content of the polysaccharides was determined using a PMP-derivation
278
method. ULP1 and ULP2 were hydrolyzed with 2mol/L TFA, subsequently modified
279
with PMP, and then monitored by HPLC. The HPLC spectra are shown in Fig. 3A.
280
Compared to the standard sugars, both ULP1 and ULP2 were composed of rhamnose,
281
xylose, glucose, and glucuronic acid. The contents of the sugars were listed in Table
282
2. It is observed that rhamnose was the main composition of the both fractions. The
283
content of rhamnose, xylose, glucose, glucuronic acid was 51%, 12%, 20%, 17%,
284
respectively, for ULP1, and 61%, 14%, 8%, 17%, respectively, for ULP2. ULP1 has a
286 287 288
cr us
an
M
d
te
Ac ce p
285
corresponded to the –SO3H and C-O-S groups, respectively [28].
ip t
271
lower content of rhamnose and sulfate than ULP2, while a higher content of glucose than ULP2. The contents of uronic acid were determined by chemical analysis by the sulfuric-carbazole reaction[18], which were consistent with those from HPLC. The sulfate content of ULP1 and ULP2 was 12.0% and 26.8%, respectively.
289
14
Page 14 of 25
ip t cr
290
(Figure 3)
us
291 292
Furthermore, the hydrolyzed products were derived by acetic anhydride and
294
detected by GC. The GC spectra of ULPs and the standard sugars are shown in Fig.
295
3B. It was verified that ULP1 and ULP2 were composed of rhamnose, glucose, and
296
xylose. Glucuronic acid was absent in Fig. 3B owing to its high boiling point. The
297
compositions of ULPs are similar with those of Ulva polysaccharides from other
298
species, with rhamnose, xylose, and glucuronic acid being the main components.
299
However, in the reported Ulva polysaccharides, the content of the sugars are in the
301 302 303
M
d
te
Ac ce p
300
an
293
range of rhamnose (16.8-45.0%), xylose (2.1-12.0%), glucose (0.5-6.4%), uronic acid (6.5-19.0%), and sulfate (16.0-23.2%) [29]. ULPs have a higher amount of rhamnose than the reported Ulva polysaccharides, which might give ULPs special properties.
304 305 306 307
15
Page 15 of 25
Table 2. The monosaccharide compositions of ULPs (%) fractions
Rha
Xyl
Glu
GAa
GAb
sulfate
ULP1
51.2
12.3
20.1
16.4
16.8
12.0
ULP2
60.8
14.2
8.2
16.8
16.8
26.8
ip t
308
a: obtained through PMP-HPLC method; b: obtained through barium chloride-gelatin
310
method.
us
cr
309
311
Periodate oxidation followed by Smith degradation with 2mol/L CF3CO2H at
313
105°C for 4 h was carried out on ULP1 and ULP2. The by-products from the
314
degradation were measured by GC using inositol as the internal standard. The spectra
315
are shown in Fig. 3C. As observed, only glycerol and rahmnose were detected after
316
the degradation, while erythritol is absent. The results indicated that there were no
317
1→4 and 1→4,6 linked residues in the polysaccharides. The structure is different
318
from those reported polysaccharides from Ulva, which contain 1→4 gycosidic bonds
322
Ac ce p
te
d
M
an
312
323
connected by oxidative glycosidic bonds 1→, 1→6, 1→2, or 1→2, 6, and unoxidized
324
glycosidic bonds [30].
319 320 321
for most sugars. In ULPs, rahmnose was present with a 1→4 linkage, which contained a sulfate at the 3 position[22]. There was formic acid formed after periodate oxidation, which revealed that there were 1→ and 1→6 glycosidic bonds. According to the amount of NaIO4 consumed and formic acid generated, the other residues were
325 326 327 16
Page 16 of 25
328
3.5. NMR analysis
us
cr
ip t
329
331
(Figure 4)
332
1
H-NMR and
13
an
330
C-NMR spectra of ULP1 and ULP2 were recorded in D2O at
room temperature. The spectra of ULP 1 and ULP2 were similar. The spectra of ULP2
334
are shown in Fig. 4. According to the resonance signals of anomeric H-1 at 4.5~5.5
335
ppm in the 1H-NMR spectra of ULP2 (Fig.4A), ULP2 contained both α-type and β-
336
type glycosidic bonds. The chemical shift at 4.92 ppm was due to the anomeric H-1 of
337
α-L-Rha [31]. The peak at 4.64 ppm was due to the H-1 signal of β-D-xylose, and that
339 340 341
d
te
Ac ce p
338
M
333
at 4.44 ppm due to the H-1 of β-D-xylose. The amount of glucose was low which resulted in invisible signal of H-1 proton NMR. The strong resonance signal owing to –CH3 at 1.31 ppm confirmed the presence of L-Rhamnose [32,33]. The
13
C-NMR spectrum of ULP2 is shown in Fig. 4B. The signal at 102.0 ppm
342
was attributed to the C-1 of D-GlcA, and that at 98.7 ppm attributed to C-1 of α-
343
(1→4)-L-Rha. The peaks at 77.6, 72.6 wee owing to C-4 and C-2,3 of glucouronic
344
acid. Those peaks at 76.9, 67.7, 66.9 ppm were the signals from C-4, C-2, and C-5 of
345
rhamnose. The peak at 61.2 ppm was due to C-5 of xylose. The peak at 56.2 ppm was
17
Page 17 of 25
346
due to the methyl of rhamonse. The signal at 174.17 ppm was assigned to –C=O of D-
347
glucuronic acid. The peak at 15.62 ppm was the characteristic signal of C6 of Rha
348
[34]. Those small signals not assigned are derived from xylose and glucose.
ip t
349
3.6. Surface properties
351
The surface tension of ULP1 and ULP2 in water were measured at room temperature,
352
with the sample concentration being 5mg/mL. The data was measured in triplicate at
353
the same conditions. ULP1 and ULP2 showed reduced water surface tensions, being
354
51.63mN/m and 57.37mN/m, respectively. ULP1 showed better ability than ULP2 as
355
evidenced by the fact that the water surface tension decreased. It was also observed
356
that equilibrium was not easy to achieve during the measurement due to large
357
molecular size and high viscosity. Therefore, steady-state fluorescence was used to
358
measure the critical micellular concentration (CMC) of the polysaccharides.
360 361 362 363
us
an
M
d
te
Pyrene was used as the fluorescence probe to provide indirect measurement of the
Ac ce p
359
cr
350
surface properties of the polysaccharides has been amply demonstrated previously [35]. The ratio of intensities of the first and third peaks I373/I383 reveals the polarity level of the microenvironment where pyrene is located [28]. The plots of fluorescence I373/I383 ratio versus logarithm of ULPs concentration in aqueous solution are shown
364
in Fig. 4. In Fig. 4, a continuous decrease in the fluorescence intensity I373/I383 with an
365
increase in ULPs concentration occurred. The decrease of fluorescence intensity
366
I373/I383 indicated that pyrene was sensing a more hydrophobic environment, which
367
resulted from the aggregation of ULPs and incorporation of pyrene into the
18
Page 18 of 25
hydrophobic area of the ULPs micelles. It is widely accepted that pyrene I373/I383 for
369
surfactants is well fitted by a Boltzmann function [36]. Function 1 and function 2
370
were the Boltzmann functions of ULP1 and ULP2, respectively. According to the
371
Boltzmann functions, the CMC of ULP1 and ULP2 was about 1.01 mg/mL (5.3×10-
372
3
373
good surface activities.
374
Y1
375
Y2
ip t
368
1.23
(1)
0.84 1.05 1 e ( x0.48) / 0.46
(2)
an
1 e
( x 0.44 ) / 0.41
us
0.66
cr
mmol/L) and 1.14mg/mL (5.0×10-3mmol/L), respectively, indicating ULPs have
376
M
377 378
d
2.0
—■—ULP1
1.6
Ac ce p
I1/I3
—◆—ULP2 1.4
te
1.8
1.2
1.0
-2.0
379 380 381
-1.5
-1.0
-0.5
0.0
0.5
1.0
logC
(Figure 5)
382
The self-aggregation of ULPs in aqueous solution was due to their amphiphilic
383
structures, in which they contain hydrophilic –OHs, -SO3Na and hydrophobic methyls
384
of rhamnose, in accordance with the structural results. ULP1 and ULP2 have high
385
contents of rhamnose (51.2% and 60.8%), which gives them good surface properties.
19
Page 19 of 25
Surface activity is affected by the chemical structure of the samples, especially the
387
balance of hydrophilic and hydrophobic groups. For ULPs, methyl of rhamnose is the
388
hydrophobic group, while sulfate plays important role on the hydrophilicity. ULP1
389
has lower rhamnose content and much lower sulfate content (shown in Table 2, 51.2%
390
rha, 12.0% sulfate) than ULP2 (60.8% rha, 26.8% sulfate), resulting in ULP1
391
expressing better surface activity than ULP2. There are no reports on the surface
392
activities of algae polysaccharides until now. The natural origin and good surface
393
properties of Ulva polysaccharides makes them potentially useful as surfactants in
394
food pharmaceutical and cosmetic applications.
395
4. Conclusions
M
an
us
cr
ip t
386
Two sulfated polysaccharides were extracted from Ulva lactuca L. collected
397
from the South China Sea through ultrasonic-assistant extraction, and followed by
398
stepwise ethanol precipitation. The polysaccharides consisted of rhamnose, xylose,
399
glucose, glucuronic acid and sulfate. The polysaccharides contain 1→, 1→6, 1→2,
401 402 403
te
Ac ce p
400
d
396
1→2,6, or unoxidized glycosidic bonds, different from the reported polysaccharides from other Ulva species. The present polysaccharides have good surface properties. Surface properties of natural polysaccharides were first reported, which might inspire more concerns on polysaccharides and make them potential feedstocks as food,
404
cosmetic, and pharmaceutical components. The specific information of sugar
405
sequence could not be obtained due to the unsuccessful methylation analysis of ULPs.
406
Further work is required to specify the structures. And the relationship between the
407
amphiphilic structure and the surface properties should be more detailed.
20
Page 20 of 25
408
Acknowledgments
410
The authors acknowledge the National Science Foundation of China (Project No.
411
21264007, 21466011), Key Scientific and Technological Project of Haikou city
412
(2010-084), and the Young Researcher Project of Hainan University (qnjj1221) for
413
financial support. We also thank Prof L.A. Lucia for helpful discussions regarding the
414
preparation of the manuscript.
us
cr
ip t
409
an
415
Figure Captions
417
Fig. 1 The GPC distributions of ULPs
418
Fig. 2 The FTIR spectra of ULPs
419
Fig. 3 HPLC spectra of PMP modified sugars (A), GC chromatograms of sugar after
420
acetylation (B), and GC chromatograms of ULPs products from Smith
421
degradation (C)
422
Fig. 4 The 1H-NMR (A) and 13C-NMR (B) spectra of ULP2
423
Ac ce p
te
d
M
416
424 425 426 427 428
Fig. 5 The plot fluorescence I373/I383 ratio versus logarithm ULPs concentration in aqueous solution
References[1] Nikapitiya C, De Zoysa M, Jeon Y J, et al. Isolation of sulfated
429
anticoagulant compound from fermented red seaweed Grateloupia filicina, Journal
430
of the World Aquaculture Society. 38 (2007) 407-417.
431 432
[2] Pujol C A, Errea M I, Matulewicz M C, et al, Antiherpetic activity of S1, an algal derived sulphated galactan, Phytotherapy Research. 10 (1996) 410-413.
433
[3] Rebello S., Asok A. K., Mundayoor S., Jisha M. S., Surfactants: toxicity,
434
remediation and green surfactants, Environmental Chemistry Letter. 12 (2014) 21
Page 21 of 25
435 436 437
275–287. [4] Lourith, N., Kanlayavattanakul, M., Natural surfactants used in cosmetics: glycolipids, International Journal of Cosmetic Science. 31 (2009) 255–261. [5] Zhang, T., Marchan, R. E., Novel Polysaccharide Surfactants: The Effect of
439
Hydrophobic and Hydrophilic Chain Length on Surface Active Properties, J.
440
Colloid Interface Sci. 177 (1996) 419–426.
ip t
438
[6] Melo, M. R. S., Feitosa, J. P. A., Freitas, A. L. P., De Paula, R. C. M., Isolation and
442
characterization of soluble sulfated polysaccharide from the red seaweed
443
Gracilaria cornea. Carbohydr. Polym. 49 (2002) 491-498.
us
445
[7] Yanishlieva, N. V, Marinova, E., Pokorny, J., Natural antioxidants from herbs and spices, Eur. J. Lipid Sci. Technol. 108 (2006) 776–793.
an
444
cr
441
[8] Percival, E., Wold, J. K., The acid polysaccharide from the green seaweed Ulva
447
lactuca. Part II. The site of the ester sulphate, J. Chem. Soc. 1040 (1963) 5459-
448
5468.
M
446
[9] Brading, J. W., Georg-Plant, M. M. T., Hardy, D. M. The polysaccharide from the
450
alga Ulva lactuca. Purification, hydrolysis, and methylation of the polysaccharide,
451
J. Chem. Soc. (1954) 319-324.
452
[10] Nagaoka, M., Shibata, H., Kimura-Takagi, I., Hashimoto, S., Aiyama, R.,
453
Ac ce p
te
d
449
454 455 456 457 458
Ueyama, S., Yokokura, T. Anti-ulcer effects and biological activities of polysaccharides from marine algae, Biofactors. 12 (2000) 267-274.
[11] Costa C., Alves A., Pinto P. R., Sousa R. A., Silva E. A. B., Reis R. L., Rodrigues A. E, Characterization of ulvan extracts to assess the effect of different steps in the extraction procedure, Carbohydr. Polym. 88 (2012) 537- 546.
[12] Hernández-Garibay, E., Zertuche-González, J. A., Pacheco-Ruíz, I, Isolation and
459
chemical characterization of algal polysaccharides from the green seaweed Ulva
460
clathrata (Roth) C. Agardh, J. Appl. Phycol. 23 (2011) 537-542.
461
[13] Qi, H., Huang, L., Liu, X., Liu, D., Zhang, Q., Liu, S. Antihyperlipidemic
462
activity of high sulfate content derivative of polysaccharide extracted from Ulva
463
pertusa(Chlorophyta), Carbohydr. Polym. 87 (2012) 1637-1640.
464
[14] Qi, H., Zhang, Q., Zhao, T., Chen, R., Zhang, H., Niu, X., Li, Z. Antioxidant 22
Page 22 of 25
activity of different sulfate content derivatives of polysaccharide extracted from
466
Ulva pertusa(Chlorophyta) in vitro, Int. J. Biol. Macromol. 37 (2005) 195-199.
467
[15] Jaulneau, V., Lafitte, C., Corio-Costet, M. F., Stadnik, M. J., Salamagne, S.,
468
Briand, X., Dumas, B., An Ulva armoricana extract protects plants against three
469
powdery mildew pathogens, European journal of plant pathology. 131 (2011) 393-
470
401.
ip t
465
[16] Shao, P., Chen, M., Pei, Y., & Sun, P., In intro antioxidant activities of different
472
sulfated polysaccharides from chlorophytan seaweeds Ulva fasciata, Int. J. Biol.
473
Macromol. 59 (2013) 295-300.
us
cr
471
[17] Navarini, L., Gilli, R., Gombac, V., Abatangelo, A., Bosco, M., Toffanin, R.,
475
Polysaccharides from hot water extracts of roasted Coffea arabica beans: isolation
476
and characterization, Carbohydr. Polym. 40 (1999) 71-81.
an
474
[18] Huang, X., Wang, D., Hu, Y., Lu, Y., Guo, Z., Kong, X., Sun, J., Effect of
478
sulfated astragalus polysaccharide on cellular infectivity of infectious bursal
479
disease virus, Int. J. Biol. Macromol. 42 (2008) 166-171.
d
M
477
[19] Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P., & Smith, F., Colorimetric
481
method for determination of sugars and related substances, Anal. Chem. 28 (1956)
482
350-356.
484 485 486 487 488 489 490
Ac ce p
483
te
480
[20] Lv, Y., Yang, X., Zhao, Y., Ruan, Y., Yang, Y., Wang, Z., Separation and quantification of component monosaccharides of the tea polysaccharides from Gynostemma pentaphyllum by HPLC with indirect UV detection, Food Chem. 112 (2009) 742-746.
[21] Hsu, W. K., Hsu, T. H., Lin, F. Y., Cheng, Y. K., Yang, J. P. W., Separation, purification, and α-glucosidase inhibition of polysaccharides from Coriolus versicolor LH1 mycelia, Carbohydr. Polym. 92 (2013) 297-306. [22] Sun, H. H., Mao, W. J., Chen, Y., Guo, S. D., Li, H. Y., Qi, X. H., Xu, J.,
491
Isolation,
492
polysaccharides from marine fungus Penicillium sp. F23-2, Carbohydr. Polym. 78
493
(2009) 117-124.
494
chemical
characteristics
and
antioxidant
properties
of
the
[23] Gutiérrez, A., Prieto, A., Martínez, A. T., Structural characterization of 23
Page 23 of 25
495
extracellular polysaccharides produced by fungi from the genus Pleurotus,
496
Carbohydr. Res. 281 (1996) 143-154. [24] Souza, Bartolomeu W.S., et al, Chemical characterization and antioxidant activity
498
of sulfated polysaccharide from the red seaweed Gracilaria birdiae, Food
499
Hydrocolloids. 27 (2012) 287–292.
503 504 505
cr
502
Biochem. 4 (1962) 330-334.
[26] Mathias, J. H., Rosen, M. J., Davenport, L., Fluorescence study of premicellar aggregation in cationic gemini surfactants, Langmuir. 17 (2001) 6148-6154.
us
501
[25] Bitter, T., Muir, H. M., A modified uronic acid carbazole reaction, Anal.
[27] Alves, A., Sousa, R., Reis, R., A practical perspective on ulvan extracted from green algae, Journal of Applied Phycology. 25 (2013) 407-424.
an
500
ip t
497
[28] Yang, B., Jiang, Y., Zhao, M., Chen, F., Wang, R., Chen, Y., & Zhang, D.,
507
Structural characterisation of polysaccharides purified from longan (Dimocarpus
508
longan Lour.) fruit pericarp, Food Chem. 115 (2009) 609-614.
M
506
[29] Lahaye, M., Robic, A., Structure and functional properties of ulvan, a
510
polysaccharide from green seaweeds, Biomacromolecules. 8 (2007) 1765-1774.
511
[30] Baird, J. K., Holroyde, M. J., Ellwood, D. C., Analysis of the products of Smith
512
degradation of polysaccharides by glc of the acetylated, derived aldononitriles and
513
alditols, Carbohydr. Res. 27 (1973) 464-467.
515 516 517 518 519 520 521
te
Ac ce p
514
d
509
[31] Lahaye, M., Inizan, F., Vigoureux, J., NMR analysis of the chemical structure of ulvan and of ulvan-boron complex formation. Carbohydr Polym. 36 (1998) 239249.
[32] Guentas, L., Pheulpin, P., Michaud, P., Heyraud, A., Gey, C., Courtois, B., Courtois, J., Structure of a polysaccharide from a Rhizobium species containing 2deoxy-β-d- arabino-hexuronic acid, Carbohydr. Res. 332 (2001) 167-173. [33] I, Uhliariková, et al, Positional specifity of acetylxylan esterases on natural polysaccharide: an NMR study, Biochim Biophys Acta. 1830 (2013) 3365–3372.
522
[34] D, Zhang, Three sulphated polysaccharides isolated from the mucilage of mud
523
snail, Bullacta exarata philippi: Characterization and antitumour activity, Food
524
Chem. 138 (2013) 306–314. 24
Page 24 of 25
525
[35] G, Basu Ray, Pyrene absorption can be a convenient method for probing critical
526
micellar concentration (cmc) and indexing micellar polarity, J Colloid Interface
527
Sci. 294 (2006) 248–254. [36] Aguiar, J., et al, On the determination of the critical micelle concentration by the
529
pyrene 1:3 ratio method, Journal of Colloid & Interface Science. 258 (2003) 116–
530
122.
ip t
528
Ac ce p
te
d
M
an
us
cr
531
25
Page 25 of 25