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Preparation and characterization of catechin-grafted chitosan with antioxidant and antidiabetic potential
International Journal of Biological Macromolecules 70 (2014) 150–155
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Preparation and characterization of catechin-grafted chitosan with antioxidant and antidiabetic potential Weili Zhu a,∗ , Zhanjun Zhang b a b
Department of Blood Transfusion, Subei People’s Hospital of Jiangsu Province, Yangzhou 225001, Jiangsu, China College of Biological and Chemical Engineering, Yangzhou Vocational University, Yangzhou 225009, Jiangsu, China
a r t i c l e
i n f o
Article history: Received 26 April 2014 Received in revised form 6 June 2014 Accepted 19 June 2014 Available online 1 July 2014 Keywords: Conjugate Chitosan Catechin Antioxidant Antidiabetic
a b s t r a c t In the present study, the preparation, characterization, antioxidant and antidiabetic activities of catechingrafted chitosan (catechin-g-chitosan) were investigated. The graft of catechin onto chitosan was achieved by redox system and confirmed using various instrumental methods. Proton nuclear magnetic resonance spectroscopy indicates that catechin has been successfully grafted onto chitosan. The morphology observation shows that chitosan changes to a softened nature with porous surface after grafting. Catechin-g-chitosan also exhibits reduced thermal stability and enhanced crystallinity compared to chitosan. Moreover, catechin-g-chitosan shows 0.51 of reducing power, 46.81% of hydroxyl radical-scavenging activity and 67.08% of DPPH radical-scavenging activity at 1 mg/ml, which are much higher than that of chitosan. The antidiabetic activity in vitro assays shows that the ␣-glucosidase inhibitory effect decreases in the order of catechin-g-chitosan > catechin > acarbose > chitosan, and the ␣amylase inhibitory effect decreases in the order of acarbose > catechin-g-chitosan > catechin > chitosan. The improved antioxidant and antidiabetic activities of catechin-g-chitosan are attributed to the phenolic groups in the catechin residues. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chitosan, the cationic (1-4)-2-amino-2-deoxy--d-glucan partly acetylated to the typical extent close to 0.25, is industrially produced from marine chitin [1]. Due to its nontoxicity, good biocompatibility and susceptibility to chemical modification, chitosan has been shown to be a reactive and functional polymer with a wide range of applications in biomedicine, pharmacology and agriculture [2,3]. The cationic character, along with the presence of reactive functional groups in chitosan, has given it particular properties for incorporation with many phenolic compounds. Previous study has shown that encapsulation of (−)-epigallocatechin-3-gallate (EGCG) using caseinophosphopeptide and chitosan nanoparticles could be a potential approach to enhance its antioxidant activity in biological systems [4]. Francesko et al. [5] reported that collagen, collagen/hyaluronic acid (HA) and collagen/HA/chitosan sponges loaded with EGCG, catechin and gallic acid efficiently inhibited the myeloperoxidase activity.
∗ Corresponding author. Tel.: +86 514 87373646. E-mail address: [email protected] (W. Zhu). http://dx.doi.org/10.1016/j.ijbiomac.2014.06.047 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Graft copolymerization of phenolic compounds onto chitosan can introduce desired properties, such as antioxidant, antimicrobial and antidiabetic activity, as well as enlarge the field of the potential applications of chitosan [6–9]. In recent years, a number of initiator systems have been developed to initiate grafting copolymerization. Redox systems, such as ceric ammonium nitrate, potassium persulfate, ascorbic acid (Vc) and hydrogen peroxide (H2 O2 ), have been frequently used to produce free radical sites on chitosan backbones [9–11]. Till now, many studies have been carried out on the graft copolymerization of phenolic acids, including gallic acid [6,9,12], ferulic acid [7,13] and caffeic acid [6,8] onto chitosan. However, only few studies have been focused on the graft copolymerization of other phenolic compounds, such as flavonoids [14] and tannins [15] onto chitosan. Catechin is one of the biologically effective flavonoids present in the human diet, particularly in wine and tea [16]. Various biological activities of catechin, including antioxidant, antimutagenic, anticarcinogenic, antidiabetic, antiinflammatory and antimicrobial properties, have been reported [17–19]. In order to improve the antioxidant and antidiabetic activities of chitosan, the grafting of catechin onto chitosan using Vc and H2 O2 as redox initiator in acetic acid solution was investigated in this study. The obtained catechingrafted chitosan (catechin-g-chitosan) was characterized by many
W. Zhu, Z. Zhang / International Journal of Biological Macromolecules 70 (2014) 150–155
instrumental methods to confirm the conjugation. The antioxidant and antidiabetic activities of catechin-g-chitosan were also determined. This study provides novel information on the structure and bioactivities of catechin-g-chitosan. 2. Materials and methods 2.1. Reagents and chemicals Chitosan with an average molecular weight (Mw ) of 250 kDa was purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The degree of deacetylation of chitosan was determined to be 72% by proton nuclear magnetic resonance (1 H NMR). (+)-Catechin, deuterium oxide (D2 O), ascorbic acid (Vc), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 4-nitrophenyl␣-d-glucopyranoside (pNPG), ␣-glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20), porcine pancreatic ␣-amylase (PPA; EC 3.2.1.1) and azure starch were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acarbose was obtained from Bayer HealthCare Co. Ltd. (Bejing, China). All other reagents were of analytical grade. 2.2. Preparation of catechin-g-chitosan Catechin was grafted onto chitosan by redox pair system according to the method of Liu et al. [20] with some modifications. Firstly, chitosan (0.5 g) was dissolved in 50 ml of 2% acetic acid solution (v/v) in a two-necked round-bottom flask. The flask was then placed in a water bath (20 ◦ C) and bubbled by a slow stream of nitrogen for 30 min to remove the dissolved oxygen. Then, 0.1 g of Vc, 0.5 g of catechin and 2 ml of 5 M H2 O2 were added into the flask to initiate the reaction. The grafting process was carried out at 20 ◦ C for 24 h. The nitrogen atmosphere was maintained throughout the reaction period. Finally, the reaction mixture was dialyzed against distilled water for 3 days and lyophilized to obtain catechin-gchitosan. The grafting ratio of catechin-g-chitosan was determined by the Folin–Ciocalteu method and expressed as mg of catechin equivalents per g (mgCAE/g) of the conjugate [21]. 2.3. Characterization of catechin-g-chitosan To verify the conjugation, catechin-g-chitosan was characterized by 1 H NMR, field-emission scanning electron microscopy (FESEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). 1 H NMR spectra were obtained at 298 K on AVANCE-600 spectrometer (Bruker Inc., Germany). The morphology observation was performed on S-4800 FESEM (Hitachi Ltd., Japan) at an accelerating voltage of 15 kV. Samples were mounted on a metal stub and sputter-coated with gold. Thermal gravimetric analysis was conducted with Pyris 1 TGA (Perkin-Elmer Ltd., USA). Each sample (2.0 mg) was heated from 30 to 800 ◦ C in a platinum pan at a heating rate of 10 ◦ C/min under nitrogen flow of 20 ml/min. Powder XRD measurements were performed on D8 Advance X-ray diffractometer (Bruker AXS, Germany). The powder samples were placed on low-background quartz sample holders. XRD patterns from 10◦ to 80◦ (2) were recorded at room temperature using Cu K␣ radiation. 2.4. Determination of antioxidant activity in vitro 2.4.1. Reducing power assay Reducing power assay was carried out according to the method of Oyaizu [22]. Various concentrations of sample solutions (0.025–1 mg/ml, 2.5 ml) were mixed with 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide (w/v). The mixture was incubated at 50 ◦ C for 20 min. After 2.5 ml of 10% trichloroacetic acid (w/v) was added, the mixture was centrifuged at 5000 rpm for 10 min. The upper layer (5 ml) was mixed
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with 5 ml of deionized water and 1 ml of 0.1% ferric chloride (w/v), and the absorbance was measured at 700 nm. Higher absorbance indicates higher reducing power. 2.4.2. Hydroxyl radical-scavenging activity assay The hydroxyl radical-scavenging activity was determined according to the method of Jen with some modifications [23]. Various concentrations of sample solutions (0.025–1 mg/ml, 1 ml) were mixed with 1 ml of 9 mM FeSO4 solution and 1 ml of 9 mM salicylic acid–ethanol solution. The reaction was initiated by the addition of 1 ml H2 O2 (8.8 mM) to the mixture and carried out at 37 ◦ C for 1 h. Then the absorbance was determined at 510 nm. The hydroxyl radical-scavenging activity was calculated by the following equation:
Hydroxyl radical scavenging activity (%) = 1 −
A1 − A2 A0
× 100 (1)
where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample and A2 is the absorbance of the sample only (salicylic acid–ethanol solution instead of FeSO4 and H2 O2 solutions). 2.4.3. DPPH radical-scavenging activity assay The DPPH radical-scavenging activity was assayed according to the method of Qiao with some modifications [24]. Briefly, 0.2 ml of DPPH solution (0.4 mM DPPH in dehydrated alcohol) was mixed with 1.0 ml of the sample (0.1–4 mg/ml) and 2.8 ml of distilled water. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. The absorbance of the mixture was measured at 517 nm. The DPPH radical-scavenging activity was calculated by the following equation:
DPPH radical scavenging activity (%) = 1 −
A1 − A2 A0
× 100
(2)
where A0 is the absorbance of the control (water instead of the sample), A1 is the absorbance of the sample and A2 is the absorbance of the sample only (water instead of DPPH). 2.5. Determination of antidiabetic activity in vitro 2.5.1. ˛-Glucosidase inhibitory effect assay ␣-Glucosidase inhibitory effect assay was performed according to the method of Kim with some modifications [25]. The reaction mixture contained 1 ml of 0.1 M potassium phosphate buffer (pH 6.8), 1 ml of substrate solution (2.5 mM pNPG in 0.1 M potassium phosphate buffer), 1 ml of sample (0.05–1 mg/ml) and 1 ml of ␣glucosidase (0.2 U/ml in 0.1 M potassium phosphate buffer). After incubation at 37 ◦ C for 15 min, 1 ml of 0.2 M Na2 CO3 was added to stop the reaction. Then, the absorbance of the reaction mixture was determined at 405 nm. ␣-Glucosidase inhibitory effect was calculated by the following equation:
␣-Glucosidase inhibitory effect (%) = 1 −
A1 − A2 A0
× 100
(3)
where A0 is the absorbance of the control (phosphate buffer instead of the sample), A1 is the absorbance of the sample and A2 is the absorbance of the blank (phosphate buffer instead of pNPG). 2.5.2. ˛-Amylase inhibitory effect assay ␣-Amylase inhibitory effect assay was carried out using the method of Wang with some modifications [26]. Starch azure (10 mg) as the substrate was suspended in 1 ml of 0.1 M Tris–HCl buffer (pH 6.9) containing 0.01 M CaCl2 and incubated at 95 ◦ C for 20 min. The reaction mixture contained 1 ml of the sample
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(0.5–10 mg/ml), 1 ml of PPA solution (1.4 U/ml in Tris–HCl buffer) and 1 ml of cooled starch azure solution. After incubation at 37 ◦ C for 10 min, 1 ml of 50% acetic acid (v/v) was added to stop the reaction. The mixture was centrifuged at 5000 rpm for 30 min, and the absorbance of the supernatant was determined at 595 nm. ␣-Amylase inhibitory activity was calculated by the following equation:
␣-Amylase inhibitory effect (%) = 1 −
A1 − A2 A0
Chitosan
× 100
(4)
where A0 is the absorbance of the control (Tris–HCl buffer instead of the sample), A1 is the absorbance of the sample and A2 is the absorbance of the blank (Tris–HCl buffer instead of starch azure). 2.6. Statistical analysis Catechin-g-chitosan
Data were expressed as mean ± standard deviation (SD) and evaluated by one-way analysis of variance (ANOVA) followed by Duncan’s multiple-range tests. All statistical analyses were carried out by using SAS for Windows. Difference was considered to be significant if p < 0.05.
10.0
9.0
8.0
Fig. 1.
1
7.0
6.0
5.0 ppm
4.0
3.0
2.0
1.0
0
H spectra of chitosan and catechin-g-chitosan.
3. Results and discussion 3.1. Characterization of catechin-g-chitosan In order to confirm the conjugation, the product was characterized by chemical and instrumental methods. The grafting ratio of catechin-g-chitosan is 65.89 mgCAE/g determined by Folin–Ciocalteu method. 1 H NMR spectra of chitosan and cateching-chitosan are shown in Fig. 1. Chitosan shows a peak at 1.9 ppm for the methyl protons of acetylated glucosamine residues. Peaks between 3.3 and 3.7 ppm are attributed to the protons of C–3, C–4, C–5 and C–6 of the pyranose ring. And peaks at 2.9 and 4.4 ppm are assigned to protons of C-2 and C-1 of the glucosamine residues, respectively. In the case of catechin-g-chitosan, a new peak appeared at 4.6 ppm is attributed to the protons of C-2 of
catechin residues. This result confirms the successful grafting of catechin with chitosan. However, no other proton peaks for catechin is observed in the conjugate. Vittorio et al. found peaks at 6.6–6.9 and 8.8–9.2 ppm in the 1 H NMR spectra of dextran–catechin conjugate, assigning to the aromatic and phenolic protons of catechin residues, respectively. They indicated that H-6/H-8 of catechin (A ring) and H-2 /H-5 of catechin (B ring) were the potential grafting positions [27]. Liu et al. [28] suggested that H-6/H-8 of catechin (A ring) were the grafting positions in inulin–catechin conjugate. Our results show that the linkage occurred between amino groups of chitosan and protons of catechin without any site specificity. These further suggest that the grafting positions between catechin and polysaccharides are mainly depended on the property of polysaccharides rather than catechin.
Fig. 2. FESEM micrographs of chitosan (a, b) and catechin-g-chitosan (c, d) at different magnifications.
W. Zhu, Z. Zhang / International Journal of Biological Macromolecules 70 (2014) 150–155
(a)
153
100
60 40
Intensity
Weight (%)
80
Chitosan
20
Catechin-g-chitosan
Catechin-g-chitosan
Chitosan
0 0
100
200
300
400
500
600
700
800
Temperature (°C)
0
(b)
20
40
60
80
2 (deg) Fig. 4. XRD spectra of chitosan and catechin-g-chitosan.
DTG(%/min)
Chitosan
3.2. Antioxidant activity in vitro of catechin-g-chitosan
Catechin-g-chitosan
0
200
400
600
800
Temperature (°C) Fig. 3. TGA (a) and DTG (b) curves of chitosan and catechin-g-chitosan.
The morphology of chitosan and catechin-g-chitosan are observed by FESEM. As shown in Fig. 2, the FESEM image of chitosan shows a flaky nature with smooth surface due to stronger interaction between the chitosan molecules. However, a distinguished change is observed in the surface morphology after grafting. Catechin-g-chitosan exhibits a softened nature with porous surface. In addition, the needle-like crystals observed in the conjugate should be attributed to catechin residues [28]. The change in morphology of the conjugate indicates that intermolecular and intramolecular hydrogen bonds of chitosan have been greatly decreased during the grafting process. The thermograms of chitosan and catechin-g-chitosan are shown in Fig. 3. The thermogram of chitosan exhibits two stages. One in the range of 30–180 ◦ C with maximum decomposition rate at 170 ◦ C is associated with loss water, the other in the range of 200–420 ◦ C with maximum decomposition rate at 310 ◦ C is ascribed to a complex process including dehydration of the saccharide rings, depolymerization and decomposition of the polymer [29]. However, the thermogram of catechin-g-chitosan shows three degradation stages. The first stage ranges between 80 and 200 ◦ C with maximum decomposition rate at 140 ◦ C is due to the weight loss of adsorbed and bound water. The second and third stages are contributed to the decomposition of catechin-g-chitosan. Notably, chitosan degrades more slowly than catechin-g-chitosan, indicating that the thermal stability of chitosan is higher than catechin-g-chitosan. The powder X-ray diffractograms of chitosan and catechin-gchitosan are shown in Fig. 4. Chitosan exhibits a major peak at around 20◦ due to 100 and 110 reflections [30]. However, XRD spectra of catechin-g-chitosan show a crystalline area in the region of 10–50◦ due to the grafting of catechin onto chitosan backbones. This indicates that the introduction of catechin onto chitosan has increased the crystallinity of chitosan.
As shown in Fig. 5a, the reducing power of all samples increases with increasing concentrations. At the concentration of 0.1 mg/ml, the reducing power of chitosan, catechin-g-chitosan, catechin and Vc are 0.10, 0.37, 1.56 and 1.60, respectively. It has been reported that the reducing power is generally associated with the presence of reductones because of their hydrogen-donating ability [31]. Among all samples tested, chitosan has the lowest reducing power because the strong intramolecular hydrogen bonds weakened the hydrogen-donating ability of hydroxyl and amino groups. Cateching-chitosan exhibits an excellent reducing power than chitosan. This is probably due to the grafted catechin moieties destroys the hydrogen bonds of chitosan and increases the hydrogen-donating ability of the conjugate. As shown in Fig. 5b, the hydroxyl radical-scavenging activities of chitosan, catechin-g-chitosan, catechin and Vc at the concentration of 1 mg/ml are 30.74, 46.81, 62.02 and 100%, respectively. Compared with chitosan, catechin-g-chitosan exhibits a higher hydroxyl radical-scavenging activity. This suggests that the scavenging ability of chitosan was enhanced after grafting by inhibiting the generation of hydroxyl radical via a Fenton-like reaction. DPPH radical-scavenging activities of each sample are presented in Fig. 5c. At a concentration of 1 mg/ml, the DPPH-scavenging activities of chitosan, catechin-g-chitosan, catechin and Vc are 36.67, 67.08, 72.50 and 99.58%, respectively. The DPPH radical-scavenging activity of catechin-g-chitosan is a little lower than that of catechin. It is well known that catechin is a potent antioxidant, which acts by scavenging free radicals and ROS [32]. In this study, we attempt the conjugation of catechin with chitosan in order to improve the antioxidant ability of chitosan. The above results show that the conjugate exhibited stronger antioxidant activities than the unmodified chitosan. Therefore, it suggests that conjugation of antioxidant phenolics onto chitosan is a useful approach for generating of novel range of polymeric antioxidants. 3.3. Antidiabetic activity in vitro of catechin-g-chitosan The ␣-glucosidase inhibitory effects of chitosan and catechin-gchitosan are shown in Fig. 6a. At the concentration of 1 mg/ml, the ␣-glucosidase inhibitory effects of chitosan, catechin-g-chitosan, catechin and acarbose are 27.06, 72.45, 58.86 and 36.65%, respectively. Acarbose, the drug frequently used for the treatment of type 2 diabetes, exhibits lower ␣-glucosidase inhibitory effect than catechin. This is consistent with previous study that flavonoids are effective ␣-glucosidase inhibitors [33]. In addition, the ␣glucosidase inhibitory effect of catechin-g-chitosan is much higher
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W. Zhu, Z. Zhang / International Journal of Biological Macromolecules 70 (2014) 150–155
(a)
1.8
100
1.5
Inhibitory effect (%)
Absorbance at 700 nm
(a)
1.2 0.9 0.6 0.3
40 20
0 0.2
0
0.4
0.6
0.8
(b) Inhibitory effect (%)
120
90
60
0.6
0.8
1
100 80 60 40 20
30 0 0
2
4
6
8
10
Concentration (mg/ml) 0
0.2
0.4
0.6
0.8
1 Fig. 6. ␣-Glucosidase (a) and ␣-amylase (b) inhibitory effects of chitosan (--), catechin-g-chitosan (--), catechin (--) and acarbose (-♦-). Data are presented as means ± SD of triplicates.
Concentration (mg/ml)
Scavenging activity (%)
0.4
Concentration (mg/ml)
0
(c)
0.2
1
Concentration (mg/ml)
Scavenging activity (%)
60
0
0
(b)
80
120
the treatment of type 2 diabetes, because of its potent inhibitory effect against ␣-glucosidase and mild inhibitory effect against ␣amylase.
90
60
4. Conclusions
30
Our results suggest that catechin can be successfully grafted with chitosan by redox system. Antioxidant and antidiabetic activities in vitro of chitosan can be greatly enhanced by grafting with catechin. Catechin-g-chitosan can be explored as a promising antioxidant and antidiabetic agent in pharmaceutical industry.
0 0
0.2
0.4
0.6
0.8
1
Concentration (mg/ml) References Fig. 5. The reducing power (a), hydroxyl radical (b) and DPPH radical-scavenging activities (c) of chitosan (--), catechin-g-chitosan (--), catechin (--) and Vc (-♦-). Data are presented as means ± SD of triplicates.
than catechin and chitosan, indicating that conjugation of catechin with chitosan can enhance the ␣-glucosidase inhibitory effect of chitosan. The ␣-amylase inhibitory effects of chitosan and catechin-gchitosan are shown in Fig. 6b. At the concentration of 10 mg/ml, the ␣-amylase inhibitory effects of chitosan, catechin-g-chitosan, catechin and acarbose are 17.65, 36.47, 32.35 and 62.94%, respectively. Among all samples tested, acarbose exhibits the highest ␣-amylase inhibitory effect. Catechin shows much lower ␣amylase inhibitory effect than acarbose. This result is in agreement with that of previous study, which has demonstrated that plantderived phenolics have low inhibitory activity on ␣-amylase and high inhibition potential against ␣-glucosidase [34]. Moreover, catechin-g-chitosan shows higher ␣-glucosidase and ␣-amylase inhibitory effects than catechin and chitosan. This suggests that some synergistic action exists between catechin and chitosan on ␣glucosidase and ␣-amylase inhibitory effect. Therefore, our results indicate that catechin-g-chitosan has the potential to contribute to
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Preparation and characterization of catechin-grafted chitosan with antioxidant and antidiabetic potential Weili Zhu a,∗ , Zhanjun Zhang b a b
Department of Blood Transfusion, Subei People’s Hospital of Jiangsu Province, Yangzhou 225001, Jiangsu, China College of Biological and Chemical Engineering, Yangzhou Vocational University, Yangzhou 225009, Jiangsu, China
a r t i c l e
i n f o
Article history: Received 26 April 2014 Received in revised form 6 June 2014 Accepted 19 June 2014 Available online 1 July 2014 Keywords: Conjugate Chitosan Catechin Antioxidant Antidiabetic
a b s t r a c t In the present study, the preparation, characterization, antioxidant and antidiabetic activities of catechingrafted chitosan (catechin-g-chitosan) were investigated. The graft of catechin onto chitosan was achieved by redox system and confirmed using various instrumental methods. Proton nuclear magnetic resonance spectroscopy indicates that catechin has been successfully grafted onto chitosan. The morphology observation shows that chitosan changes to a softened nature with porous surface after grafting. Catechin-g-chitosan also exhibits reduced thermal stability and enhanced crystallinity compared to chitosan. Moreover, catechin-g-chitosan shows 0.51 of reducing power, 46.81% of hydroxyl radical-scavenging activity and 67.08% of DPPH radical-scavenging activity at 1 mg/ml, which are much higher than that of chitosan. The antidiabetic activity in vitro assays shows that the ␣-glucosidase inhibitory effect decreases in the order of catechin-g-chitosan > catechin > acarbose > chitosan, and the ␣amylase inhibitory effect decreases in the order of acarbose > catechin-g-chitosan > catechin > chitosan. The improved antioxidant and antidiabetic activities of catechin-g-chitosan are attributed to the phenolic groups in the catechin residues. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chitosan, the cationic (1-4)-2-amino-2-deoxy--d-glucan partly acetylated to the typical extent close to 0.25, is industrially produced from marine chitin [1]. Due to its nontoxicity, good biocompatibility and susceptibility to chemical modification, chitosan has been shown to be a reactive and functional polymer with a wide range of applications in biomedicine, pharmacology and agriculture [2,3]. The cationic character, along with the presence of reactive functional groups in chitosan, has given it particular properties for incorporation with many phenolic compounds. Previous study has shown that encapsulation of (−)-epigallocatechin-3-gallate (EGCG) using caseinophosphopeptide and chitosan nanoparticles could be a potential approach to enhance its antioxidant activity in biological systems [4]. Francesko et al. [5] reported that collagen, collagen/hyaluronic acid (HA) and collagen/HA/chitosan sponges loaded with EGCG, catechin and gallic acid efficiently inhibited the myeloperoxidase activity.
∗ Corresponding author. Tel.: +86 514 87373646. E-mail address: [email protected] (W. Zhu). http://dx.doi.org/10.1016/j.ijbiomac.2014.06.047 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Graft copolymerization of phenolic compounds onto chitosan can introduce desired properties, such as antioxidant, antimicrobial and antidiabetic activity, as well as enlarge the field of the potential applications of chitosan [6–9]. In recent years, a number of initiator systems have been developed to initiate grafting copolymerization. Redox systems, such as ceric ammonium nitrate, potassium persulfate, ascorbic acid (Vc) and hydrogen peroxide (H2 O2 ), have been frequently used to produce free radical sites on chitosan backbones [9–11]. Till now, many studies have been carried out on the graft copolymerization of phenolic acids, including gallic acid [6,9,12], ferulic acid [7,13] and caffeic acid [6,8] onto chitosan. However, only few studies have been focused on the graft copolymerization of other phenolic compounds, such as flavonoids [14] and tannins [15] onto chitosan. Catechin is one of the biologically effective flavonoids present in the human diet, particularly in wine and tea [16]. Various biological activities of catechin, including antioxidant, antimutagenic, anticarcinogenic, antidiabetic, antiinflammatory and antimicrobial properties, have been reported [17–19]. In order to improve the antioxidant and antidiabetic activities of chitosan, the grafting of catechin onto chitosan using Vc and H2 O2 as redox initiator in acetic acid solution was investigated in this study. The obtained catechingrafted chitosan (catechin-g-chitosan) was characterized by many
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instrumental methods to confirm the conjugation. The antioxidant and antidiabetic activities of catechin-g-chitosan were also determined. This study provides novel information on the structure and bioactivities of catechin-g-chitosan. 2. Materials and methods 2.1. Reagents and chemicals Chitosan with an average molecular weight (Mw ) of 250 kDa was purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The degree of deacetylation of chitosan was determined to be 72% by proton nuclear magnetic resonance (1 H NMR). (+)-Catechin, deuterium oxide (D2 O), ascorbic acid (Vc), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 4-nitrophenyl␣-d-glucopyranoside (pNPG), ␣-glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20), porcine pancreatic ␣-amylase (PPA; EC 3.2.1.1) and azure starch were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acarbose was obtained from Bayer HealthCare Co. Ltd. (Bejing, China). All other reagents were of analytical grade. 2.2. Preparation of catechin-g-chitosan Catechin was grafted onto chitosan by redox pair system according to the method of Liu et al. [20] with some modifications. Firstly, chitosan (0.5 g) was dissolved in 50 ml of 2% acetic acid solution (v/v) in a two-necked round-bottom flask. The flask was then placed in a water bath (20 ◦ C) and bubbled by a slow stream of nitrogen for 30 min to remove the dissolved oxygen. Then, 0.1 g of Vc, 0.5 g of catechin and 2 ml of 5 M H2 O2 were added into the flask to initiate the reaction. The grafting process was carried out at 20 ◦ C for 24 h. The nitrogen atmosphere was maintained throughout the reaction period. Finally, the reaction mixture was dialyzed against distilled water for 3 days and lyophilized to obtain catechin-gchitosan. The grafting ratio of catechin-g-chitosan was determined by the Folin–Ciocalteu method and expressed as mg of catechin equivalents per g (mgCAE/g) of the conjugate [21]. 2.3. Characterization of catechin-g-chitosan To verify the conjugation, catechin-g-chitosan was characterized by 1 H NMR, field-emission scanning electron microscopy (FESEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). 1 H NMR spectra were obtained at 298 K on AVANCE-600 spectrometer (Bruker Inc., Germany). The morphology observation was performed on S-4800 FESEM (Hitachi Ltd., Japan) at an accelerating voltage of 15 kV. Samples were mounted on a metal stub and sputter-coated with gold. Thermal gravimetric analysis was conducted with Pyris 1 TGA (Perkin-Elmer Ltd., USA). Each sample (2.0 mg) was heated from 30 to 800 ◦ C in a platinum pan at a heating rate of 10 ◦ C/min under nitrogen flow of 20 ml/min. Powder XRD measurements were performed on D8 Advance X-ray diffractometer (Bruker AXS, Germany). The powder samples were placed on low-background quartz sample holders. XRD patterns from 10◦ to 80◦ (2) were recorded at room temperature using Cu K␣ radiation. 2.4. Determination of antioxidant activity in vitro 2.4.1. Reducing power assay Reducing power assay was carried out according to the method of Oyaizu [22]. Various concentrations of sample solutions (0.025–1 mg/ml, 2.5 ml) were mixed with 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide (w/v). The mixture was incubated at 50 ◦ C for 20 min. After 2.5 ml of 10% trichloroacetic acid (w/v) was added, the mixture was centrifuged at 5000 rpm for 10 min. The upper layer (5 ml) was mixed
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with 5 ml of deionized water and 1 ml of 0.1% ferric chloride (w/v), and the absorbance was measured at 700 nm. Higher absorbance indicates higher reducing power. 2.4.2. Hydroxyl radical-scavenging activity assay The hydroxyl radical-scavenging activity was determined according to the method of Jen with some modifications [23]. Various concentrations of sample solutions (0.025–1 mg/ml, 1 ml) were mixed with 1 ml of 9 mM FeSO4 solution and 1 ml of 9 mM salicylic acid–ethanol solution. The reaction was initiated by the addition of 1 ml H2 O2 (8.8 mM) to the mixture and carried out at 37 ◦ C for 1 h. Then the absorbance was determined at 510 nm. The hydroxyl radical-scavenging activity was calculated by the following equation:
Hydroxyl radical scavenging activity (%) = 1 −
A1 − A2 A0
× 100 (1)
where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample and A2 is the absorbance of the sample only (salicylic acid–ethanol solution instead of FeSO4 and H2 O2 solutions). 2.4.3. DPPH radical-scavenging activity assay The DPPH radical-scavenging activity was assayed according to the method of Qiao with some modifications [24]. Briefly, 0.2 ml of DPPH solution (0.4 mM DPPH in dehydrated alcohol) was mixed with 1.0 ml of the sample (0.1–4 mg/ml) and 2.8 ml of distilled water. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. The absorbance of the mixture was measured at 517 nm. The DPPH radical-scavenging activity was calculated by the following equation:
DPPH radical scavenging activity (%) = 1 −
A1 − A2 A0
× 100
(2)
where A0 is the absorbance of the control (water instead of the sample), A1 is the absorbance of the sample and A2 is the absorbance of the sample only (water instead of DPPH). 2.5. Determination of antidiabetic activity in vitro 2.5.1. ˛-Glucosidase inhibitory effect assay ␣-Glucosidase inhibitory effect assay was performed according to the method of Kim with some modifications [25]. The reaction mixture contained 1 ml of 0.1 M potassium phosphate buffer (pH 6.8), 1 ml of substrate solution (2.5 mM pNPG in 0.1 M potassium phosphate buffer), 1 ml of sample (0.05–1 mg/ml) and 1 ml of ␣glucosidase (0.2 U/ml in 0.1 M potassium phosphate buffer). After incubation at 37 ◦ C for 15 min, 1 ml of 0.2 M Na2 CO3 was added to stop the reaction. Then, the absorbance of the reaction mixture was determined at 405 nm. ␣-Glucosidase inhibitory effect was calculated by the following equation:
␣-Glucosidase inhibitory effect (%) = 1 −
A1 − A2 A0
× 100
(3)
where A0 is the absorbance of the control (phosphate buffer instead of the sample), A1 is the absorbance of the sample and A2 is the absorbance of the blank (phosphate buffer instead of pNPG). 2.5.2. ˛-Amylase inhibitory effect assay ␣-Amylase inhibitory effect assay was carried out using the method of Wang with some modifications [26]. Starch azure (10 mg) as the substrate was suspended in 1 ml of 0.1 M Tris–HCl buffer (pH 6.9) containing 0.01 M CaCl2 and incubated at 95 ◦ C for 20 min. The reaction mixture contained 1 ml of the sample
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(0.5–10 mg/ml), 1 ml of PPA solution (1.4 U/ml in Tris–HCl buffer) and 1 ml of cooled starch azure solution. After incubation at 37 ◦ C for 10 min, 1 ml of 50% acetic acid (v/v) was added to stop the reaction. The mixture was centrifuged at 5000 rpm for 30 min, and the absorbance of the supernatant was determined at 595 nm. ␣-Amylase inhibitory activity was calculated by the following equation:
␣-Amylase inhibitory effect (%) = 1 −
A1 − A2 A0
Chitosan
× 100
(4)
where A0 is the absorbance of the control (Tris–HCl buffer instead of the sample), A1 is the absorbance of the sample and A2 is the absorbance of the blank (Tris–HCl buffer instead of starch azure). 2.6. Statistical analysis Catechin-g-chitosan
Data were expressed as mean ± standard deviation (SD) and evaluated by one-way analysis of variance (ANOVA) followed by Duncan’s multiple-range tests. All statistical analyses were carried out by using SAS for Windows. Difference was considered to be significant if p < 0.05.
10.0
9.0
8.0
Fig. 1.
1
7.0
6.0
5.0 ppm
4.0
3.0
2.0
1.0
0
H spectra of chitosan and catechin-g-chitosan.
3. Results and discussion 3.1. Characterization of catechin-g-chitosan In order to confirm the conjugation, the product was characterized by chemical and instrumental methods. The grafting ratio of catechin-g-chitosan is 65.89 mgCAE/g determined by Folin–Ciocalteu method. 1 H NMR spectra of chitosan and cateching-chitosan are shown in Fig. 1. Chitosan shows a peak at 1.9 ppm for the methyl protons of acetylated glucosamine residues. Peaks between 3.3 and 3.7 ppm are attributed to the protons of C–3, C–4, C–5 and C–6 of the pyranose ring. And peaks at 2.9 and 4.4 ppm are assigned to protons of C-2 and C-1 of the glucosamine residues, respectively. In the case of catechin-g-chitosan, a new peak appeared at 4.6 ppm is attributed to the protons of C-2 of
catechin residues. This result confirms the successful grafting of catechin with chitosan. However, no other proton peaks for catechin is observed in the conjugate. Vittorio et al. found peaks at 6.6–6.9 and 8.8–9.2 ppm in the 1 H NMR spectra of dextran–catechin conjugate, assigning to the aromatic and phenolic protons of catechin residues, respectively. They indicated that H-6/H-8 of catechin (A ring) and H-2 /H-5 of catechin (B ring) were the potential grafting positions [27]. Liu et al. [28] suggested that H-6/H-8 of catechin (A ring) were the grafting positions in inulin–catechin conjugate. Our results show that the linkage occurred between amino groups of chitosan and protons of catechin without any site specificity. These further suggest that the grafting positions between catechin and polysaccharides are mainly depended on the property of polysaccharides rather than catechin.
Fig. 2. FESEM micrographs of chitosan (a, b) and catechin-g-chitosan (c, d) at different magnifications.
W. Zhu, Z. Zhang / International Journal of Biological Macromolecules 70 (2014) 150–155
(a)
153
100
60 40
Intensity
Weight (%)
80
Chitosan
20
Catechin-g-chitosan
Catechin-g-chitosan
Chitosan
0 0
100
200
300
400
500
600
700
800
Temperature (°C)
0
(b)
20
40
60
80
2 (deg) Fig. 4. XRD spectra of chitosan and catechin-g-chitosan.
DTG(%/min)
Chitosan
3.2. Antioxidant activity in vitro of catechin-g-chitosan
Catechin-g-chitosan
0
200
400
600
800
Temperature (°C) Fig. 3. TGA (a) and DTG (b) curves of chitosan and catechin-g-chitosan.
The morphology of chitosan and catechin-g-chitosan are observed by FESEM. As shown in Fig. 2, the FESEM image of chitosan shows a flaky nature with smooth surface due to stronger interaction between the chitosan molecules. However, a distinguished change is observed in the surface morphology after grafting. Catechin-g-chitosan exhibits a softened nature with porous surface. In addition, the needle-like crystals observed in the conjugate should be attributed to catechin residues [28]. The change in morphology of the conjugate indicates that intermolecular and intramolecular hydrogen bonds of chitosan have been greatly decreased during the grafting process. The thermograms of chitosan and catechin-g-chitosan are shown in Fig. 3. The thermogram of chitosan exhibits two stages. One in the range of 30–180 ◦ C with maximum decomposition rate at 170 ◦ C is associated with loss water, the other in the range of 200–420 ◦ C with maximum decomposition rate at 310 ◦ C is ascribed to a complex process including dehydration of the saccharide rings, depolymerization and decomposition of the polymer [29]. However, the thermogram of catechin-g-chitosan shows three degradation stages. The first stage ranges between 80 and 200 ◦ C with maximum decomposition rate at 140 ◦ C is due to the weight loss of adsorbed and bound water. The second and third stages are contributed to the decomposition of catechin-g-chitosan. Notably, chitosan degrades more slowly than catechin-g-chitosan, indicating that the thermal stability of chitosan is higher than catechin-g-chitosan. The powder X-ray diffractograms of chitosan and catechin-gchitosan are shown in Fig. 4. Chitosan exhibits a major peak at around 20◦ due to 100 and 110 reflections [30]. However, XRD spectra of catechin-g-chitosan show a crystalline area in the region of 10–50◦ due to the grafting of catechin onto chitosan backbones. This indicates that the introduction of catechin onto chitosan has increased the crystallinity of chitosan.
As shown in Fig. 5a, the reducing power of all samples increases with increasing concentrations. At the concentration of 0.1 mg/ml, the reducing power of chitosan, catechin-g-chitosan, catechin and Vc are 0.10, 0.37, 1.56 and 1.60, respectively. It has been reported that the reducing power is generally associated with the presence of reductones because of their hydrogen-donating ability [31]. Among all samples tested, chitosan has the lowest reducing power because the strong intramolecular hydrogen bonds weakened the hydrogen-donating ability of hydroxyl and amino groups. Cateching-chitosan exhibits an excellent reducing power than chitosan. This is probably due to the grafted catechin moieties destroys the hydrogen bonds of chitosan and increases the hydrogen-donating ability of the conjugate. As shown in Fig. 5b, the hydroxyl radical-scavenging activities of chitosan, catechin-g-chitosan, catechin and Vc at the concentration of 1 mg/ml are 30.74, 46.81, 62.02 and 100%, respectively. Compared with chitosan, catechin-g-chitosan exhibits a higher hydroxyl radical-scavenging activity. This suggests that the scavenging ability of chitosan was enhanced after grafting by inhibiting the generation of hydroxyl radical via a Fenton-like reaction. DPPH radical-scavenging activities of each sample are presented in Fig. 5c. At a concentration of 1 mg/ml, the DPPH-scavenging activities of chitosan, catechin-g-chitosan, catechin and Vc are 36.67, 67.08, 72.50 and 99.58%, respectively. The DPPH radical-scavenging activity of catechin-g-chitosan is a little lower than that of catechin. It is well known that catechin is a potent antioxidant, which acts by scavenging free radicals and ROS [32]. In this study, we attempt the conjugation of catechin with chitosan in order to improve the antioxidant ability of chitosan. The above results show that the conjugate exhibited stronger antioxidant activities than the unmodified chitosan. Therefore, it suggests that conjugation of antioxidant phenolics onto chitosan is a useful approach for generating of novel range of polymeric antioxidants. 3.3. Antidiabetic activity in vitro of catechin-g-chitosan The ␣-glucosidase inhibitory effects of chitosan and catechin-gchitosan are shown in Fig. 6a. At the concentration of 1 mg/ml, the ␣-glucosidase inhibitory effects of chitosan, catechin-g-chitosan, catechin and acarbose are 27.06, 72.45, 58.86 and 36.65%, respectively. Acarbose, the drug frequently used for the treatment of type 2 diabetes, exhibits lower ␣-glucosidase inhibitory effect than catechin. This is consistent with previous study that flavonoids are effective ␣-glucosidase inhibitors [33]. In addition, the ␣glucosidase inhibitory effect of catechin-g-chitosan is much higher
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(a)
1.8
100
1.5
Inhibitory effect (%)
Absorbance at 700 nm
(a)
1.2 0.9 0.6 0.3
40 20
0 0.2
0
0.4
0.6
0.8
(b) Inhibitory effect (%)
120
90
60
0.6
0.8
1
100 80 60 40 20
30 0 0
2
4
6
8
10
Concentration (mg/ml) 0
0.2
0.4
0.6
0.8
1 Fig. 6. ␣-Glucosidase (a) and ␣-amylase (b) inhibitory effects of chitosan (--), catechin-g-chitosan (--), catechin (--) and acarbose (-♦-). Data are presented as means ± SD of triplicates.
Concentration (mg/ml)
Scavenging activity (%)
0.4
Concentration (mg/ml)
0
(c)
0.2
1
Concentration (mg/ml)
Scavenging activity (%)
60
0
0
(b)
80
120
the treatment of type 2 diabetes, because of its potent inhibitory effect against ␣-glucosidase and mild inhibitory effect against ␣amylase.
90
60
4. Conclusions
30
Our results suggest that catechin can be successfully grafted with chitosan by redox system. Antioxidant and antidiabetic activities in vitro of chitosan can be greatly enhanced by grafting with catechin. Catechin-g-chitosan can be explored as a promising antioxidant and antidiabetic agent in pharmaceutical industry.
0 0
0.2
0.4
0.6
0.8
1
Concentration (mg/ml) References Fig. 5. The reducing power (a), hydroxyl radical (b) and DPPH radical-scavenging activities (c) of chitosan (--), catechin-g-chitosan (--), catechin (--) and Vc (-♦-). Data are presented as means ± SD of triplicates.
than catechin and chitosan, indicating that conjugation of catechin with chitosan can enhance the ␣-glucosidase inhibitory effect of chitosan. The ␣-amylase inhibitory effects of chitosan and catechin-gchitosan are shown in Fig. 6b. At the concentration of 10 mg/ml, the ␣-amylase inhibitory effects of chitosan, catechin-g-chitosan, catechin and acarbose are 17.65, 36.47, 32.35 and 62.94%, respectively. Among all samples tested, acarbose exhibits the highest ␣-amylase inhibitory effect. Catechin shows much lower ␣amylase inhibitory effect than acarbose. This result is in agreement with that of previous study, which has demonstrated that plantderived phenolics have low inhibitory activity on ␣-amylase and high inhibition potential against ␣-glucosidase [34]. Moreover, catechin-g-chitosan shows higher ␣-glucosidase and ␣-amylase inhibitory effects than catechin and chitosan. This suggests that some synergistic action exists between catechin and chitosan on ␣glucosidase and ␣-amylase inhibitory effect. Therefore, our results indicate that catechin-g-chitosan has the potential to contribute to
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