International Journal of Biological Macromolecules 69 (2014) 39–45
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
O-acetylation of low-molecular-weight polysaccharide from Enteromorpha linza with antioxidant activity Zhongshan Zhang a,∗ , Xiaomei Wang a , Mingxing Zhao a , Huimin Qi b a b
Department of Pharmacology, Huzhou Teachers College, Huzhou 313000, China School of Pharmaceutical and Biological Sciences, Weifang Medical University, Weifang 261053, China
a r t i c l e
i n f o
Article history: Received 29 January 2014 Received in revised form 1 April 2014 Accepted 17 April 2014 Available online 19 May 2014 Keywords: Enteromorpha linza Acetylation Antioxidant activities
a b s t r a c t Polysaccharide extracted from green algae Enteromorpha linza (EP) is a sulfated polysaccharide, which possesses excellent antioxidant activities. In present study, the acetylated derivatives of low-molecularweight polysaccharide (LEP) was prepared with the method of response surface quadratic model. And then the antioxidant activities of the derivatives were investigated including scavenging effects of superoxide and hydroxyl radicals. The results of chemical analysis and FT-IR spectrum showed the acetylation was successful. And in addition, certain derivative with different degree of substitution (DS) exhibited different antioxidant activity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Marine algae have been used widely for centuries in the world as food and pharmaceutical supplements, the production of valuable chemicals [1,2]. They are rich in vitamins, minerals, proteins and polysaccharides with a documented antioxidant activity which would elevatetheir value in the human diet [3–5]. Up to now, there were a large number of studies about the brown and red alga. However, recently researchers have paid great attention to green algae, which are the most diverse group of algae, with more than 7000 species growing in a variety of habitats [6]. Preliminary studies on Enteromorpha sp. cell-wall polysaccharides have been carried out during sixties and seventies [7–9]. Among the species of green alga, Enteromorpha linza is a common one. It is reported that it could tolerate a wide range of salinities and has often been used as a heavy metal monitor [10]. Ganesan et al. found the different solvent extracts total phenol obtained from edible species of E. lina exhibited good antioxidant activities [11]. However, to our knowledge, little information is available on the polysaccharides from this alga. It was reported in our previous study that polysaccharide from E. linza was isolated, degraded and evaluated as a novel antioxidant and humectant [12]. But little was known about chemistry modification of this polysaccharide and its effects on water solubility.
Polysaccharides and their derivatives have been found numerous applications in a variety of fields including food, chemical and pharmaceutical industries. Xing et al. found that the sulfated derivatives of chitosan showed stronger scavenging activity on superoxide radical and reducing power [13]. Beside the sulfation, acetylation of polysaccharide was also a method to broad range of functional and physicochemical properties. It was suggested the presence and the position of O-acetyl groups in the polysaccharide chain may influence the immune response [14]. In addition, the importance of the O-acetylation for polysaccharide immunogenicity was shown for Neisseria meningitidis, group B streptococci, Staphylococcus aureus, Salmonella typhi, Bletilla striata and Cryptococcus neoformans polysaccharides [15–18]. In the present study, an acetylation method was developed to improve water solubility of polysaccharide from E. linza, with acetic anhydride (AA) in formamide (FA) as reaction solvent. Relative viscosity and antioxidant activity of the resulted acetylated derivative were analyzed to evaluate its water solubility. The relationship between the nature of functionalized groups and the chemically modified derivatives and their antioxidant activity were also discussed. 2. Materials and methods 2.1. Chemicals
∗ Corresponding author. Tel.:+86 572 2321166; fax: +86 572 2321166. E-mail addresses: [email protected], [email protected] (Z. Zhang). http://dx.doi.org/10.1016/j.ijbiomac.2014.04.058 0141-8130/© 2014 Elsevier B.V. All rights reserved.
E. linza was collected on the coast of Ningbo in October 2011. The fresh alga was soon washed; sun dried and kept in plastic bags at room temperature for use.
40
Z. Zhang et al. / International Journal of Biological Macromolecules 69 (2014) 39–45
Table 1 Box–Behnken experimental design with the independent variables. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
X1 /temperature (◦ C)
−1 (60) 1 (100) −1 1 −1 1 −1 1 0 (80) 0 0 0 0 0 0 0 0
X2 /time (h)
−1 (6) −1 1 (8) 1 0 (10) 0 0 0 −1 1 −1 1 0 0 0 0 0
0 (20) 0 0 0 −1(15) −1 1 (25) 1 −1 −1 1 1 0 0 0 0 0
All chemicals and reagents, unless otherwise specified, were of analytical grade. Dialysis membranes were produced by Spectrum Co., and molecular weight was cut off at 3600 Da. Sulfate content was determined by barium chloride method [19]. Total sugar content was determined by phenol–sulfuric acid method [20] using rhamnose as standard. The acetyl content was determined by the modified hydroxylamine–ferric trichloride method [21]. The degree of substitution (DS) of acetylation was obtained as the following equation: DS =
DS of acetyl group
X3 /ratio of acetic anhydride to raw (mL/g)
162 × W % 43 − 42 × W %
where W% is the content of acetyl group. The Fourier transform-infrared (FT-IR) spectra were recorded on a Bruker Vector 22 instrument with a resolution of 4 cm−1 in the 4000–400 cm−1 region. 2.2. Polysaccharide and degradation The nature polysaccharide was extracted from E. linza in hot water with the method of previous study [12]. A solution of nature polysaccharide (10 mg/mL) was added in 10 mM ascorbic acid and 10 mM·H2 O2 , stirred for 2 h, and then precipitated to give the degraded product named after LEP [22]. 2.3. Acetylation of polysaccharide A solution of LEP in formamide (FA) (12.5 mg/mL) was added in to acetic anhydride (designed volume) and 1% of Nbromosuccinimide (NBS) (in designed volume of acetic anhydride) at designed tempreture. After designed reaction time, the mixture was added in to 20 mL of distilled water, and precipitated with 75% ethanol. The solution of obtained precipitate was dissolved in 100 mL distilled water and dialyzed, concentrated and lyophilized to give the product acetylated polysaccharide (AEP).
Actual vatue
Predicted vatue
0.14 0.34 0.18 0.35 0.13 0.33 0.15 0.29 0.21 0.27 0.26 0.29 0.31 0.32 0.32 0.31 0.29
0.14 0.33 0.19 0.35 0.11 0.32 0.16 0.30 0.23 0.28 0.25 0.27 0.31 0.31 0.31 0.31 0.31
reaction temperature (X1 , A), reaction time (X2 , B) and ratio of acetic anhydride to raw material LEP (X3 , C) while the response variable was the yield of AEP. In Table 1, these three factors were prescribed into three levels coded +1, 0 and −1 for high, medium and low. In order to predict the optimized conditions, experimental data were analysed using software Design-Expert and fitted to an empirical second-order polynomial regression model: Y = ˇ0 +
k i−1
ˇi X1 +
k
ˇii Xi2 +
i−1
k
ˇij Xi Xj
i>j
where Y is the estimated response (DS of acetyl); ˇ0 is the constant, ˇi , ˇii , and ˇij are the regression coefficients for linearity, square, and interaction, respectively; and Xi and Xj are the independent variables. And k equals to the number of the tested factors (k = 3). 2.5. Relative viscosity determination The intrinsic viscosity (r ) of reaction mixtures was determined in an Ubbelohde viscosimeter at 25 ◦ C. The result was measured as the following equation [r = (ln t/t0 )/c], where t is the solution flow time (s), t0 the solvent flow time (s) and c is the concentration of solution in distilled water (g/mL). 2.6. Antioxidant activity 2.6.1. Superoxide radical assay The superoxide radical scavenging abilities of all samples were assessed by the modified method of Nishimiki et al. [23]. In this experiment, the sample (0.5–50.0 g/mL) in 4.5 mL of Tris–HCl buffer (16 mM, pH 8.0) was added into 0.5 mL of NBT (300 M) solution, 0.5 mL of NADH (468 M) and 0.5 mL of PMS (60 M). The reaction mixture was incubated at room temperature for 5 min and the absorbance was read at 560 nm by a spectrophotometer against blank samples. The capability of scavenging the superoxide anion radicals was calculated using the following equation:
1 − Asample 560
2.4. Experimental design of RSM
scavenging effect (%) =
After determining the preliminary range of the extraction parameters by a single-factor experiment for the above acetylation of polysaccharide, a Box–Behnken design with three variables was used for the optimization of AEP. Based on the results of preliminary experiments, three independent variables considered were
2.6.2. Hydroxyl radical assay The reaction mixture, containing all different derivatives (0.6–7.0 mg/mL), was incubated with EDTA-Fe2+ (2.0 mM), saffron (360 g/mL), and H2 O2 (3%) in potassium phosphate buffer
Acontrol 560
× 100
Z. Zhang et al. / International Journal of Biological Macromolecules 69 (2014) 39–45
41
Table 2 Analysis of variance of the Box–Behnken design experimental results. Variables
Sum of squares
DF
Mean square
F value
P value
Model X1 X2 X3 X1 X2 X1 X3 X2 X3 X12 X22 X32 Residue Lack of fit Pure error Total
0.084 0.0630 0.0025 0.0003 0.0002 0.0009 0.0002 0.0009 0.0007 0.0067 0.019 0.0013 0.0006 0.086
9 1 1 1 1 1 1 1 1 1 7 3 4 16
0.0094 0.0630 0.0025 0.0003 0.0002 0.0009 0.0002 0.0009 0.0007 0.0067 0.0003 0.0004 0.0002
34.14 229.14 9.91 1.14 0.82 3.27 0.82 31.00 2.39 24.50
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
O-acetylation of low-molecular-weight polysaccharide from Enteromorpha linza with antioxidant activity Zhongshan Zhang a,∗ , Xiaomei Wang a , Mingxing Zhao a , Huimin Qi b a b
Department of Pharmacology, Huzhou Teachers College, Huzhou 313000, China School of Pharmaceutical and Biological Sciences, Weifang Medical University, Weifang 261053, China
a r t i c l e
i n f o
Article history: Received 29 January 2014 Received in revised form 1 April 2014 Accepted 17 April 2014 Available online 19 May 2014 Keywords: Enteromorpha linza Acetylation Antioxidant activities
a b s t r a c t Polysaccharide extracted from green algae Enteromorpha linza (EP) is a sulfated polysaccharide, which possesses excellent antioxidant activities. In present study, the acetylated derivatives of low-molecularweight polysaccharide (LEP) was prepared with the method of response surface quadratic model. And then the antioxidant activities of the derivatives were investigated including scavenging effects of superoxide and hydroxyl radicals. The results of chemical analysis and FT-IR spectrum showed the acetylation was successful. And in addition, certain derivative with different degree of substitution (DS) exhibited different antioxidant activity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Marine algae have been used widely for centuries in the world as food and pharmaceutical supplements, the production of valuable chemicals [1,2]. They are rich in vitamins, minerals, proteins and polysaccharides with a documented antioxidant activity which would elevatetheir value in the human diet [3–5]. Up to now, there were a large number of studies about the brown and red alga. However, recently researchers have paid great attention to green algae, which are the most diverse group of algae, with more than 7000 species growing in a variety of habitats [6]. Preliminary studies on Enteromorpha sp. cell-wall polysaccharides have been carried out during sixties and seventies [7–9]. Among the species of green alga, Enteromorpha linza is a common one. It is reported that it could tolerate a wide range of salinities and has often been used as a heavy metal monitor [10]. Ganesan et al. found the different solvent extracts total phenol obtained from edible species of E. lina exhibited good antioxidant activities [11]. However, to our knowledge, little information is available on the polysaccharides from this alga. It was reported in our previous study that polysaccharide from E. linza was isolated, degraded and evaluated as a novel antioxidant and humectant [12]. But little was known about chemistry modification of this polysaccharide and its effects on water solubility.
Polysaccharides and their derivatives have been found numerous applications in a variety of fields including food, chemical and pharmaceutical industries. Xing et al. found that the sulfated derivatives of chitosan showed stronger scavenging activity on superoxide radical and reducing power [13]. Beside the sulfation, acetylation of polysaccharide was also a method to broad range of functional and physicochemical properties. It was suggested the presence and the position of O-acetyl groups in the polysaccharide chain may influence the immune response [14]. In addition, the importance of the O-acetylation for polysaccharide immunogenicity was shown for Neisseria meningitidis, group B streptococci, Staphylococcus aureus, Salmonella typhi, Bletilla striata and Cryptococcus neoformans polysaccharides [15–18]. In the present study, an acetylation method was developed to improve water solubility of polysaccharide from E. linza, with acetic anhydride (AA) in formamide (FA) as reaction solvent. Relative viscosity and antioxidant activity of the resulted acetylated derivative were analyzed to evaluate its water solubility. The relationship between the nature of functionalized groups and the chemically modified derivatives and their antioxidant activity were also discussed. 2. Materials and methods 2.1. Chemicals
∗ Corresponding author. Tel.:+86 572 2321166; fax: +86 572 2321166. E-mail addresses: [email protected], [email protected] (Z. Zhang). http://dx.doi.org/10.1016/j.ijbiomac.2014.04.058 0141-8130/© 2014 Elsevier B.V. All rights reserved.
E. linza was collected on the coast of Ningbo in October 2011. The fresh alga was soon washed; sun dried and kept in plastic bags at room temperature for use.
40
Z. Zhang et al. / International Journal of Biological Macromolecules 69 (2014) 39–45
Table 1 Box–Behnken experimental design with the independent variables. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
X1 /temperature (◦ C)
−1 (60) 1 (100) −1 1 −1 1 −1 1 0 (80) 0 0 0 0 0 0 0 0
X2 /time (h)
−1 (6) −1 1 (8) 1 0 (10) 0 0 0 −1 1 −1 1 0 0 0 0 0
0 (20) 0 0 0 −1(15) −1 1 (25) 1 −1 −1 1 1 0 0 0 0 0
All chemicals and reagents, unless otherwise specified, were of analytical grade. Dialysis membranes were produced by Spectrum Co., and molecular weight was cut off at 3600 Da. Sulfate content was determined by barium chloride method [19]. Total sugar content was determined by phenol–sulfuric acid method [20] using rhamnose as standard. The acetyl content was determined by the modified hydroxylamine–ferric trichloride method [21]. The degree of substitution (DS) of acetylation was obtained as the following equation: DS =
DS of acetyl group
X3 /ratio of acetic anhydride to raw (mL/g)
162 × W % 43 − 42 × W %
where W% is the content of acetyl group. The Fourier transform-infrared (FT-IR) spectra were recorded on a Bruker Vector 22 instrument with a resolution of 4 cm−1 in the 4000–400 cm−1 region. 2.2. Polysaccharide and degradation The nature polysaccharide was extracted from E. linza in hot water with the method of previous study [12]. A solution of nature polysaccharide (10 mg/mL) was added in 10 mM ascorbic acid and 10 mM·H2 O2 , stirred for 2 h, and then precipitated to give the degraded product named after LEP [22]. 2.3. Acetylation of polysaccharide A solution of LEP in formamide (FA) (12.5 mg/mL) was added in to acetic anhydride (designed volume) and 1% of Nbromosuccinimide (NBS) (in designed volume of acetic anhydride) at designed tempreture. After designed reaction time, the mixture was added in to 20 mL of distilled water, and precipitated with 75% ethanol. The solution of obtained precipitate was dissolved in 100 mL distilled water and dialyzed, concentrated and lyophilized to give the product acetylated polysaccharide (AEP).
Actual vatue
Predicted vatue
0.14 0.34 0.18 0.35 0.13 0.33 0.15 0.29 0.21 0.27 0.26 0.29 0.31 0.32 0.32 0.31 0.29
0.14 0.33 0.19 0.35 0.11 0.32 0.16 0.30 0.23 0.28 0.25 0.27 0.31 0.31 0.31 0.31 0.31
reaction temperature (X1 , A), reaction time (X2 , B) and ratio of acetic anhydride to raw material LEP (X3 , C) while the response variable was the yield of AEP. In Table 1, these three factors were prescribed into three levels coded +1, 0 and −1 for high, medium and low. In order to predict the optimized conditions, experimental data were analysed using software Design-Expert and fitted to an empirical second-order polynomial regression model: Y = ˇ0 +
k i−1
ˇi X1 +
k
ˇii Xi2 +
i−1
k
ˇij Xi Xj
i>j
where Y is the estimated response (DS of acetyl); ˇ0 is the constant, ˇi , ˇii , and ˇij are the regression coefficients for linearity, square, and interaction, respectively; and Xi and Xj are the independent variables. And k equals to the number of the tested factors (k = 3). 2.5. Relative viscosity determination The intrinsic viscosity (r ) of reaction mixtures was determined in an Ubbelohde viscosimeter at 25 ◦ C. The result was measured as the following equation [r = (ln t/t0 )/c], where t is the solution flow time (s), t0 the solvent flow time (s) and c is the concentration of solution in distilled water (g/mL). 2.6. Antioxidant activity 2.6.1. Superoxide radical assay The superoxide radical scavenging abilities of all samples were assessed by the modified method of Nishimiki et al. [23]. In this experiment, the sample (0.5–50.0 g/mL) in 4.5 mL of Tris–HCl buffer (16 mM, pH 8.0) was added into 0.5 mL of NBT (300 M) solution, 0.5 mL of NADH (468 M) and 0.5 mL of PMS (60 M). The reaction mixture was incubated at room temperature for 5 min and the absorbance was read at 560 nm by a spectrophotometer against blank samples. The capability of scavenging the superoxide anion radicals was calculated using the following equation:
1 − Asample 560
2.4. Experimental design of RSM
scavenging effect (%) =
After determining the preliminary range of the extraction parameters by a single-factor experiment for the above acetylation of polysaccharide, a Box–Behnken design with three variables was used for the optimization of AEP. Based on the results of preliminary experiments, three independent variables considered were
2.6.2. Hydroxyl radical assay The reaction mixture, containing all different derivatives (0.6–7.0 mg/mL), was incubated with EDTA-Fe2+ (2.0 mM), saffron (360 g/mL), and H2 O2 (3%) in potassium phosphate buffer
Acontrol 560
× 100
Z. Zhang et al. / International Journal of Biological Macromolecules 69 (2014) 39–45
41
Table 2 Analysis of variance of the Box–Behnken design experimental results. Variables
Sum of squares
DF
Mean square
F value
P value
Model X1 X2 X3 X1 X2 X1 X3 X2 X3 X12 X22 X32 Residue Lack of fit Pure error Total
0.084 0.0630 0.0025 0.0003 0.0002 0.0009 0.0002 0.0009 0.0007 0.0067 0.019 0.0013 0.0006 0.086
9 1 1 1 1 1 1 1 1 1 7 3 4 16
0.0094 0.0630 0.0025 0.0003 0.0002 0.0009 0.0002 0.0009 0.0007 0.0067 0.0003 0.0004 0.0002
34.14 229.14 9.91 1.14 0.82 3.27 0.82 31.00 2.39 24.50
