Preparation, characterization and antioxidant activity of silk peptides grafted carboxymethyl chitosan

Accepted Manuscript Title: Preparation, characterization and antioxidant activity of silk peptides grafted carboxymethyl chitosan Authors: Meng Liu, Lian Min, Chen Zhu, Ziqie Rao, Liangling Liu, Wenyan Xu, Pengfeng Luo, Lihong Fan PII: DOI: Reference:

S0141-8130(17)31682-3 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.06.071 BIOMAC 7751

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

11-5-2017 13-6-2017 14-6-2017

Please cite this article as: Meng Liu, Lian Min, Chen Zhu, Ziqie Rao, Liangling Liu, Wenyan Xu, Pengfeng Luo, Lihong Fan, Preparation, characterization and antioxidant activity of silk peptides grafted carboxymethyl chitosan, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.06.071 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.

Preparation, characterization and antioxidant activity of silk peptides grafted carboxymethyl chitosan

Meng Liu, Lian Min, Chen Zhu, Ziqie Rao, Liangling Liu, Wenyan Xu, Pengfeng Luo, Lihong Fan*

School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of Technology, Wuhan 430070, China

*

Corresponding author. Tel.: +86 27 87859019; fax: +86 27 87859019

E-mail addresses: [email protected] (L. Fan)

Abstract Free radicals are closely related to the occurrence and development of aging, cancer and inflammation, the ability to scavenge free radicals is an important indicator of the antioxidant activity. In this study, we prepared a water soluble, free radical scavenging and biocompatible copolymer for regenerative therapy. Carboxymethyl chitosan (CMC) was modified with silk peptide (SP) by microbial transglutaminase (MTGase), FT-IR and NMR spectroscopy were used to confirm the successful grafting of SP to CMC. The degree of substitution was determined by ultraviolet spectrophotometry. In vitro antioxidant activity assays demonstrated that, 1

within the scope of study the highest scavenging activity of DPPH was 24.86%, 91% of hydroxyl radical and 36.8% of H2O2. Finally, no relevant cytotoxicity against NIH-3T3 mouse fibroblasts was found for the copolymers. Briefly, our results suggested the potential application of CMC-SP as an antioxidant for regenerative therapy.

Key words: carboxymethyl chitosan; silk peptides; microbial transglutaminase; antioxidant

1. Introduction The free radicals are independently existing chemical substances that have one or more unpaired electrons. In order to maintain their own stability, they may attack cells to capture electrons, which resulting in function damage of cells, and triggering a series of lesions [1]. Scientific experiments have shown that the overproduction of free radicals were the root cause of many diseases, it disrupted the oxidant/antioxidant balance of cells, which while resulting in aging, inflammation, slow tissue regeneration and wound healing [2-5]. In order to avoid these damages, advanced studies were carried out to find new materials with antioxidant activity used for repairing process [6]. To this issue, many nature macromolecules and their derivatives with antioxidant property were employed to scavenge free radicals, which including polysaccharides, bioactive peptides and so on [7-8]. 2

Chitosan (CS) is the most important product of chitin and is obtained by the deacetylation of chitin in alkaline medium [9]. As a nature polymer material, CS has many excellent properties, including the film forming ability, antimicrobial activity and interaction with different substances, which makes it widely used in many fields, such as medicine, pharmaceuticals, food and cosmetics [9-10]. Various chemical modifications of CS (such as graft, carboxylation, acylation and quaternization) are possible due to the reactive hydroxyl and amine group of the polymer chain [11-12]. One of the most important derivatives is carboxymethyl chitosan (CMC). The modified CMC not only retains the original antitumor, radical scavenging activity, antimicrobial properties of CS, but also exhibits improved water solubility and enhanced biological activities because of the introduction of carboxymethyl group [13-14]. The antioxidant activity of CMC was directly related to the amino and hydroxyl groups in the polymer chain [15], they could abstract hydrogen atoms easily from free radicals to form stable macromolecule free radicals [16]. In order to further improve the antibacterial, antioxidant, biocompatibility and other biological properties of CMC, various modification of CMC have been investigated in recent years [17-18]. However, to date, the use of silk peptide (SP) modified CMC has not been reported. Silk peptide (SP) is the hydrolysate of silk protein, is an important ingredient of cocoons [19-20]. The component of SP is similar to proteins such as keratin, collagen and elastin [21]. SP have a good absorption mechanism, and have some incomparable physiological functions, such as hypoglycemic effect, lowing cholesterol, antioxidant 3

activity, improve intestinal physiological function, and prevent aging [20]. At present, the mechanism of antioxidant activity of peptide has not yet formed a theory. But preliminary studies have shown that the antioxidant activity was closely related to the numbers, species, orders and hydrophobicity of amino acid. In addition, studies have shown that the antioxidant property was also related to the specific groups in peptide that could react with free radicals: hydrogen donor [22]. Because of these excellent characteristics, SP possess high development value in the fields of functional food, cosmetics and medicine. Enzymatic methods are one of the ways to improve or develop new functional substitutes of polysaccharide. Among them, microbial transglutaminase (MTGase) is a promising and efficient choice. MTGase can catalyze an acyl transfer reaction. The biological reaction of MTGase has high selectivity, specificity and without change in their own structure [23], it has been reported to catalyze macromolecular grafting and crosslink proteins [24-25]. Therefore the purpose of this work was to prepare CMC grafting SP with MTGase as biocatalyst. In this study, we reported the preparation, physic characterization, antioxidant property and biocompatibility evaluation of the novel enzymatic grafted CMC-SP copolymer, which have all theoretical premises to be used as an antioxidant to promote wound healing.

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2. Experimental 2.1 Materials Silk peptides (Mw 700) and microbial transglutaminase were purchased from Huashun Biological Technology CO. Ltd, Wuhan, China. Chitosan powder (Mw 520000, 92% deacetylated) was supplied by the Golden-Bell (Cochin, India). Isopropyl alcohol, monochloro acetic acid, sodium nitrite, sodium bisulfite, sodium hydroxide and all other solvents and reagents were of analytical grade and were used without further purification. They were purchased from Sinopharm Group Chemical Reagent Corp. 2.2 Purification of MTGase First, preparation of 0.2 mol/L NaH2PO4 buffer solution (PBS, pH=6.0), then a certain amount of crude MTGase powder was dissolved in the PBS buffer solution, the solution was centrifuged at 3000 r/min for 10 min, and the supernatant was vacuum filtered twice, finally the filtrate was dialyzed for three days and then freeze dried to obtain MTGase lyophilized powder and stored at 4°C. 2.3 Synthesis of CMC Carboxymethyl chitosan (CMC) was synthesized according to methods described in the literature [26]. Briefly, 10 g of chitosan powder was added to the previously prepared 10 mL of 50% sodium hydroxide solution, stirred evenly, cooled and then put into the refrigerator to freeze for 24 h. Remove a certain amount of alkalized chitosan placed in a three-necked flask, added 200 mL of isopropyl alcohol as 5

dispersion medium, stirred at room temperature for 1 h. After heating the mixture to 60°C, 9 g of monochloro acetic acid was divided into two groups and added to the mixture, the carboxymethylation reaction was continuously stirred for 5 h. The resulting CMC was rinsed with absolute ethanol and filtered for three times. The crude carboxymethyl chitosan was dissolved in distilled water, dialyzed for three days, and then concentrated by rotary evaporation, finally dried at constant temperature to obtain purified carboxymethyl chitosan. 2.4 Synthesis of CMC-SP copolymers with MTGase treatment 1 g CMC was dissolved in phosphate buffer solution (PBS) at pH value of 6.0, meanwhile a certain amount of SP an MTGase powder were respectively dissolved in the PBS (pH=6.0), all these solution were mixed in a 250mL three-necked flask, after a period of reaction at 40°C, the temperature was raised to 100°C for 15 min and later cooled to room temperature. The modified CMC product was filtered, dialysis for 3 days, the product was freeze-dried and CMC-SP copolymers were obtained. The synthesize procedure was shown in Fig.1. 2.5 Measurement of the degree of substitution The degree of substitution (DS) was defined as the number of amino groups substituted for each repeating structural unit on the CMC skeleton. In this study, the maximum absorption wavelength of SP was determined by ultraviolet spectrophotometry, and the standard curve of SP was measured to calculate the content of SP. The reaction conditions were optimized against temperature, reaction 6

time and mass ratio of SP to CMC. A certain concentration of SP solution was prepared, and UV scan was performed in the wavelength range from 185 to 500 nm to determine the maximum absorption wavelength of SP. As shown in Fig.2 the maximum absorption wavelength was at 190nm. The concentration of SP between 0.001 g/L to 0.02 g/L was linear relation with absorbance at 190 nm by ultraviolet spectrophotometry. The linear relation was described as Eq. (1). The DS of CMC-SP was determined by Eq. (2). 𝐴 = 48.6161𝐶 + 0.03452 𝐷𝑆 =

209C 13.66−683C

𝑅2 = 0.99446

(1)

(2)

2.6 Characterization 2.6.1 FT-IR analysis Fourier transform infrared spectroscopy (FT-IR) was done on a Nicolet 5700 Fourier transform infrared spectrometer (USA) using KBr discs in the range of 4000-500 cm-1. 2.6.2 1H NMR characterization The 1H NMR spectra of CMC and CMC-SP were recorded on a Bruker AMX-500 NMR spectrometer at an ambient temperature. The samples were dissolved in 1% DCl D2O and D2O. 2.7 Studies on antioxidant activity in vitro 2.7.1 Solution preparation To provide a comprehensive test system, the antioxidant activity of CMC-SP was 7

evaluated by various biochemical assays, including DPPH; hydroxyl radical; H2O2 scavenging activity and reducing power assay. First of all the copolymers of different DS were treated with distilled water at concentration of 0.5、1.0、1.5、2.0 and 2.5 mg/mL，these solution were used for the study on antioxidant property of copolymers. 2.7.2 Assay of DPPH radical scavenging activity The DPPH (1-1-diphenyl 2-picrylhydrazyl) radical scavenging activity was carried out as the method reported by Liu with some modifications [27]. Briefly, 2 mL of DPPH solution (0.1 mM in ethanol) was mixed with 2.0 mL of sample (0.5-2.5 mg/mL). 2.0 mL DPPH mixed in 2.0 mL distilled water was used as control. The 2.0 mL sample in 2.0 mL ethanol was served as blank. The mixture was shaken vigorously and incubated at 25°C for 30 min in dark. The absorbance was measured at 517 nm using ultraviolet spectrophotometry. The DPPH radical scavenging activity of the test sample was determined as the following formula: Scavenging activity (%) = (1 −

𝐷𝑠 − 𝐷𝑏 ) × 100 𝐷𝑐

(3)

Where Ds, Db and Dc was the absorbance of the test sample, the blank and the control group respectively. 2.7.3 Assay of hydroxyl radical scavenging activity The hydroxyl radical scavenging activity was determined according to the method of Li with some modifications [28]. The mixture containing 2.0 mL of sample (0.5-2.5 mg/mL), 2.0 mL of 1.5 mM FeSO4, 2.0 mL of 0.03% H2O2 and 2.0 mL of 1.5 Mm 1,10-phenanthroline-ethanol solution was shaken vigorously and incubated at 8

37°C for 1 h. Using the water instead of sample as the control group, the water instead of H2O2 as the blank group. The absorbance was measured at 536 nm using ultraviolet spectrophotometry. The hydroxyl radical scavenging activity was calculated by the following formula: Scavenging activity (%) = (

𝐴𝑠 − 𝐴𝑛 ) × 100 𝐴𝑏 − 𝐴𝑛

(4)

Where As, Ab and An was the absorbance of the test sample, the blank and the control group respectively. 2.7.4 Assay of H2O2 scavenging activity The H2O2 scavenging activity was determined according to the method of Liu with some modifications [27] The mixture containing 1.0 mL of sample (0.5-2.5 mg/mL), 6.0 mL of phosphate buffer (0.1M, pH=7.4) and 1.0 mL of 40 mM H2O2 was shaken vigorously and incubated at 25°C for 10 min. The water instead of sample was used as control, phosphate buffer instead of H2O2 was served as blank. The absorbance was measured at 230 nm using ultraviolet spectrophotometry. The H2O2 scavenging activity was calculated by the following formula: Scavenging activity (%) = (1 −

𝐴𝑠 − 𝐴𝑏 ) × 100 𝐴𝑐

(5)

Where As, Ab and Ac was the absorbance of the test sample, the blank and the control group respectively. 2.7.5 Assay of reducing power The reducing power was determined by the description of a literature report of Li [29]. Generally, The mixture containing 2.0 mL of sample (0.5-2.5 mg/mL), 2.5 mL 9

potassium hexacyanoferrate solution (1%) was shaken vigorously and incubated at 50°C for 30 min, after that 1.5 mL trichloroacetic acid (10%) was added, the mixture was centrifuged at 3500 r/min. The upper layer 2 mL was mixed with 2.0 mL distilled water and 0.5 mL FeCl3 solution (0.1%). Finally, the absorbance was recorded at 700 nm. The higher absorbance indicated the stronger reducing power. 2.8 Cytotoxic assessment using MTT assay 2.8.1. Preparation of the cell suspension The NIH-3T3 cell strain was cultured with DMEM culture medium (contain 10% Fetal Bovine Serum and 100U/m penicillin, 100 μg/mL streptomycin). After vigorously grown in a humidified atmosphere contain 5% CO2 at 37 °C for 2-3 d, cells were digested with 0.25% trypsin and replaced with fresh culture medium, the suspension was used for further experiment. 2.8.2. MTT assay In vitro cytotoxicity of CMC-SP was investigated by the MTT assay using NIH-3T3 cells, because fibroblasts cells played an important role in wound repair. 6000 cells per well were seeded in 96 well plate and cultured till 80% confluence. The culture fluid was discarded and the cells were washed with PBS for 3 times, fresh medium was added. Then samples dissolved by PBS with different concentrations (10, 50, 100, 500 and 1000 ppm) were added to each well. Cells added with the same volume PBS acted as control group. After incubation for 24 h, the cells were washed with PBS 3 times, and the medium was renewed. Subsequently, 20 μL of MTT (5 10

mg/mL) was added to each well, incubated for another 4 h, followed by removal of the medium, 100 μL DMSO was added into each well to dissolve the MTT formazan purple crystals. The absorbance of the solution was measured at 490 nm by a microplate reader. All experiments were done in triplicate. The relative cell viability (RCV) was calculated with the following equation. RCV (%) =

𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒 × 100 𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙

Where ODsampl was the average absorbance of test group; ODcontral was the average absorbance of control group. 3. Results and discussion 3.1 Characterization of SP grafted CMC 3.1. 1 FT-IR spectra The FT-IR spectra of chitosan (DD=92%), CMC and CMC-SP were shown in Fig. 2. For chitosan, broad peaks at 3407 cm-1 and 1035-1150 cm-1 were observed, corresponding of the stretching vibration of O-H, the extension vibration of N-H, and C-O bonds, respectively. The band at 1598 cm-1 was assigned to the N-H bending of the primary amine. In addition, absorption peaks at 1654 and 1322 cm-1 were attribute to the C=O stretching and C-N stretching of the residual N-acetyl groups (Fig. 3 a). This indicated that the chitosan was not fully deacetylated [30]. Different from the chitosan, CMC showed a strong absorption peak at 1407 cm-1 which could be assigned to the symmetrical vibration of COO-. The asymmetrical stretching vibration of COOC- group was overlapped with the deforming vibration of -NH2 to obtain a 11

strong peak at 1597 cm-1(Fig. 3 b) [17]. In comparison with CMC, the most striking differences with CMC-SP were observed at 1646 and 1545 cm-1, they were assigned to the typical amide Ι and Ⅱ bands. And the increasing width of peak at 3432 cm-1 reflected the presence of additional -OH and -NH2 groups originating from SP, this was similar to some of the previous studies [31]. Therefore, the combination of SP and CMC might be accomplished by the formation of amide bonds (Fig. 3 c). 3.1. 2 1H-NMR studies It was an efficient technique to character polymers with 1H-NMR spectroscopy. The 1H-NMR spectra of CMC and CMC grafted SP dissolved in D2O were shown in Fig. 4. CMC exhibited a small single peak at 4.35 ppm, which was attributed to the protons of (-CH2-COO-), multiple peaks at 3.22-3.79 ppm were related to (CH) and (CH2) at C3 to C6, the signals at 2.40 and 1.89 ppm referred to the presence of (CH, C-2) and (CH3) of acetylamino respectively [32]. As compared to CMC, CMC-SP showed all the characteristic peaks of CMC, furthermore it has showed a new peak at 5.16 ppm belonging to the rotational conformer due to hinder rotation around the N-CO bond [33]. And the multiple peaks at 0.9-1.4 ppm might be the (CH3) and (CH2) because the introduction of SP. These results further confirmed the successful grafting of SP onto CMC. 3.2 Optimization of reaction condition The SP was grafted on the CMC to prepare an amide bond-bound glycopeptide polymer. The DS depends on many factors, including reaction time, reaction 12

temperature and mass ratio of CMC to SP. Fig. 5(a) showed the effect of time on DS. The DS increased with the increasing of the reaction time from 0.268 to 0.361, but when the reaction time was more than 4 h, the DS declined sharply. That might be: in the early reaction, increasing the reaction time could accelerate the contact rate of CMC and SP with the MTGase activity center, after 9 h of reaction, the contact of enzyme with substrate reached a saturated state. Fig. 5(b) showed the effect of temperature on DS. From which we found that when the temperature increased from 20°C to 40°C, the DS increased from 0.328 to 0.361, but continue to increase the temperature, the DS was slightly reduced to 0.319. That might be: increasing the reaction temperature could accelerate the collision between molecules to speed up the catalytic reaction rate, but when the temperature was further increased, MTGase partial denatured to be inactive, resulting in the decrease of DS. Fig. 5(c) showed the effect of mass ratio of SP to CMC on DS. When the ratio increased from 0.6 to 1.2, DS increased significantly from 0.211 to 0.393, when the mass ratio was further increased, the increase of DS was not obvious. That might be the steric hindrance and electrostatic repulsion between the reactant, the reaction was maintained within a dynamic equilibrium. With the progress of reaction, the relative reduction of the amount of CMC, made the catalytic reaction of amino groups reduced, easily lead to a catalytic reaction between the SP. The highest DS (0.393) was 13

obtained in the condition of 40°C, mass ratio 1.2 and reacted for 4 h, four kinds of CMC-SP with different DS was prepared for further experimentation as show in Table 1. 3.3 Antioxidant activities of CMC-SP 3.3.1 DPPH radical scavenging activity In the DPPH assay, the copolymer was able to reduce the stable DPPH radical, make it gradually faded, and its absorption peak at 517 nm gradually weakened. The activity of the scavenger could be evaluated by measuring the degree of attenuation of the absorption. As demonstrated in Fig. 6 (a), with the increase of the concentration from 0.5 mg /mL to 2.5 mg/mL, the scavenging activities of SP, CMC and its grafted copolymers (CMC-SP) enhanced significantly for all the samples. At the concentration of 2.5 mg/mL, the DPPH scavenging activities increased with the increase of DS. When the degree of substitution was 0.393, the DPPH scavenging activity was 24.6%, at the same concentration, the DPPH scavenging activity was 28.2% of SP. Statistical analysis showed that the DPPH scavenging activity of CMC-SP was closely related to the concentration and DS. Mainly due to the antioxidant effect of SP [34], with the increasing of concentration and DS, the content of SP also increased, and thus the DPPH scavenging activity enhanced. 3.3.2 Hydroxyl radical scavenging activity Hydroxyl radicals could cause lipid peroxidation, nucleic acid cleavage, decomposition of proteins and polysaccharides [35]. The hydroxyl radicals were 14

produced by the Fenton reaction (Fe2++H2O2→Fe3++·OH+OH-)[36]. The hydroxyl radicals could oxidize Fe2+ to Fe3+, and only Fe2+ could bind to 1,10,-phenanthroline to form a red complex, the scavenging activities could be reflected by the degree of discoloration of the test solution[37]. From Fig. 6 (b), it could be seen that the scavenging activities of all samples increased with the increase of concentrations, but the change was not particularly obvious. When the degree of DS was 0.393, the scavenging rate of CMC-SP at the concentration of 0.5-2.5 mg/mL was 10.6%-90.5%, and the scavenging rate of SP in the same concentration range was 20.5%-92.0%. It could be seen that the CMC-SP has a good hydroxyl radical scavenging ability when the concentration reaches a certain degree. 3.3.3 H2O2 scavenging activity H2O2 was not classified as free radicals in the structure, but its damage in human body was similar to the free radicals, it could react with superoxide anion radical and Fe2+ to form hydroxyl radical [38]. Therefore, H2O2 was also regarded as a free radical in free radical damage studies. As depicted in Fig. 6 (c), the scavenging activity was increased with the increase of concentration of all samples. At the concentration of 2.5 mg/mL, the scavenging activity for CMC, CMC-SP (DS=0.211), CMC-SP (DS=0.345), CMC-SP (DS=0.393) and SP were 25.0%, 26.5%， 27.8%， 36.3% and 50.4%, respectively. In addition, the H2O2 scavenging activity of SP was higher than other samples, this also showed that the SP could be used to improve the free radical scavenging effect of materials. 15

3.3.4 Total reducing power The reducing power was an important indicator of compounds. The assay was based on the principle that antioxidant could react with potassium ferricyanide (K3Fe3+(CN)6) to form potassium ferrocyanide (K4Fe2+(CN)6), it could further reacted with ferric chloride to form Prussian blue (Fe4[Fe(CN)6]3), that has a maximum absorbance at 700 nm, and the reducing power of the samples could be judged according to the absorbance. As shown in Fig. 6 (d), the absorbance at 700 nm of all samples was increased with the increase of the concentration, and the reducing power of CMC-SP was stronger than CMC. When the concentration increased to 2.5 mg/mL, the absorbance of CMC-SP (0.820, DS=0.393) was even more than SP (0.801). The reducing power assay of all samples suggested that the reducing power of CMC was greatly enhanced by grafting it with SP. The enhanced biological activity might be due to the introduction of SP，which has been reported to have a good antioxidant activity [34]. 3.4 In vitro cell toxicity of CMC-SP To evaluate the biological safety of a biomedical material, cytotoxicity was one of the most important detection methods [39]. Our results demonstrated that all the materials were highly biocompatible, the cell viability rate of the materials were almost more than 70%. As presented in Fig. 7, at the concentration of 50 ppm, the cell viability rate of NIH-3T3 mouse fibroblasts cultured with CMC and CMC-SP was almost ＞95%. Meanwhile it was detected that the cell viability rate was increased 16

from 97.41% to 105.26% as DS increased. At a higher concentration of 1000 ppm, the viability rate had a slight decline, but still great than 70%. The viability rate of CMC was decreased with the concentration increased, but when it grafted SP, the viability rate has increased in varying degrees. The mechanism of such increase was not completely resolved, it might be due to the rich amino acids in SP, which has good cell compatibility, could support a variety of cells adhesion, proliferation and differentiation, the excellent blood compatibility and no potential immunogenicity might be also one of the reasons. Further research needs to confirm this hypothesis. 4. Conclusion Our results indicated that carboxymethyl chitosan modified by silk peptide was successfully synthesized by using MTGase as bio-catalyst. The covalent bond was confirmed by FT-IR spectra and 1H-NMR analysis. Additionally, several factors could be involved in to affect the DS of SP, the optimal conditions with the DS =0.393 were found: reaction time 4 h; reaction temperature 40°C and mass ratio of SP to CMC was 1.2:1.0. Compared to CMC, it was found that CMC-SP showed strong antioxidant activity in various tests, including DPPH, hydroxyl radical, H2O2 scavenging activity and total reducing power analysis. The enhanced bioactivities were probably due to the grafted SP on CMC could provide more hydrogen atoms or transfer electrons to react with free radicals. The in vitro cell toxicity assay indicated that CMC-SP was non-toxic to fibroblast cells. Overall, the results obtained in the present investigation suggested that CMC-SP might be excellent candidates for biomedical materials. 17

Acknowledgements The work was supported by the National Natural Science Foundation of China (Foundation No. 51173143, 51273156), The Special Funds Project of Major New Products of Hubei Province (Foundation No. 20132h0040), University-Industry Cooperation Projects of the Ministry of Education of Guangdong Province (Foundation No. 2012B091100437), the Innovation Fund Project of the Ministry of Science and Technology of Small and Medium-Sized Enterprises (Foundation No. 11C26214202642, No. 11C26214212743), Zhuhai Science and Technology Plan Projects (Foundation No. 2011B050102003),Wuhan Science and Technology Development (Foundation No. 201060623262), and the Fundamental Research Funds for the Central Universities (Foundation No. 2014-zy-220), Hubei-NOST KLOS & KLOBME.

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Figure Captions Fig. 1. Synthesis of CMC modified with SP by using MTGase as catalyst. CMC: carboxymethyl chitosan; SP:silk peptide; MTGase: microbial transglutaminase. Fig. 2. Full wavelength scan to determine the maximum absorption peak of SP. Fig. 3. FT-IR spectra of the chitosan (a), CMC (b) and CMC-SP (c). Fig. 4. 1H spectrum of CMC (a), CMC-SP (b). Fig. 5. The influence of reaction conditions on the substitution degree CMC modified with SP. Fig. 6. The DPPH radical, hydroxyl radical, H2O2 scavenging activities and reducing power of CMC-SP. Fig. 7. The relative cell viability of CMC-SP as a function of DS and concentration.

25

NH2 O

OH O

O

NH2

CH2OH O OH

O

NaOH,ClCH2COOH

O

OR1 O CH2OR1

Isopropanol NH2

CH2OH

n

CH2OH O OH

O

O

NH2

n

R1=H or CH2COONa

Silk peptide NH2 O

OR1

CH2OH O OH

O

O

MTGase

O

C

NH2

CH2OR1

NH3

n

O

OR1 O

+ NH2 C

O

N H O

CH2OH O OH

CH2OR1

NH2

O R1=H or CH2COONa

Silk peptide

Fig. 1. Synthesis of CMC modified with SP by using MTGase as catalyst. CMC: carboxymethyl chitosan; SP: silk peptide; MTGase: microbial transglutaminase.

0.35

190nm

0.30

Absorbance

0.25 0.20 0.15 0.10 0.05 0.00 -0.05 200

250

300

350

400

450

500

wavelength(nm)

Fig. 2. Full wavelength scan to determine the maximum absorption peak of SP.

26

O

n

Transmittance (%)

1654 1598

1322

3407

1407 1597 3440 1545 1646

3432

4000

3500

3000

2500

2000

1500

-1 Wavenumber (cm ) Fig.3. FT-IR spectra of the chitosan (a), CMC (b) and CMC-SP (c).

Fig. 4. 1H spectrum of CMC (a), CMC-SP (b).

27

1000

500

0.36

0.34

DS

0.32

0.30

0.28

0.26 2

3

4

5

6

50

60

1.2

1.4

time(h)

(a) 0.37

0.36

0.35

DS

0.34

0.33

0.32

0.31 20

30

40

temperature （ °C）

(b) 0.45

0.40

DS

0.35

0.30

0.25

0.20 0.6

0.8

1.0

mass ration of SP/CMC

(c) 28

Fig. 5. The influence of reaction conditions on the substitution degree CMC modified with SP: (a) The influence of reaction time on DS (T=40°C, mSPS/mCMC=1.0); (b) The influence of reaction temperature on DS (t=4h, mSPS/mCMC=1.0); (c) The influence of the mass ratio of SP to CMC on DS (T=40°C, t=4h).

30

DPPH scavenging rate (%)

25

20

0.5 mg/ml 1.0 mg/ml 1.5 mg/ml 2.0 mg/ml 2.5 mg/ml

15

10

5

0 0

0.211

0.345

0.393

SP

0.393

SP

DS

(a) 0.5 mg/ml 1.0 mg/ml 1.5 mg/ml 2.0 mg/ml 2.5 mg/ml

·OH scavenging rate (%)

100

80

60

40

20

0 0

0.211

0.345

DS

(b)

29

0.5 mg/ml 1.0 mg/ml 1.5 mg/ml 2.0 mg/ml 2.5 mg/ml

H2O2 scavenging rate (%)

50

40

30

20

10

0 0

0.211

0.345

0.393

SP

DS

(c)

0 0 .2 1 1

0 .7 5 A b so rb a n c e a t 7 0 0 n m

0 .3 4 5 0 .3 9 3 SP

0 .6 0

0 .4 5

0 .3 0 0 .5

1 .0

1 .5

2 .0

2 .5

c o n c e n t r a t i o n （ m g /m L )

(d) Fig. 6. The DPPH radical (a), hydroxyl radical (b), H2O2 scavenging activities (c) and reducing power (d) of CMC and CMC-SP with different DS.

30

120

DS=0 DS=0.211 DS=0.345 DS=0.393

100

RCV (%)

80

60

40

20

0 10

50

100

500

1000

Concentration (ppm)

Fig. 7. The relative cell viability of CMC-SP as a function of DS and concentration.

31

Table 1 Different DS of CMC-SP prepared by different reaction conditions used for further experimentation.

Samples time(h) temperature(°C) mSPS/mCMC DS

S0 \ \ \ 0

S1 4 40 0.6 0.211

S2 4 30 1.0 0.345

32

S3 4 40 1.2 0.393