YAN Chunchao, XIAO Feng, QIU Tong, ZHANG Xueqiong, ZHAN Siwen, LU Mengli, YANG Mengjia＊, ZHANG Juan

(1. College of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China; 2. Department of Pharmacy, Hubei Provincial Hospital of Traditional Chinese Medicine, Wuhan 430060, China; 3. School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of Technology, Wuhan 430070, China; 4. Maternal and Child Health Hospital of Hubei, Wuhan 430070, China)

Abstract: To synthesize three different grafting ratios of gallic acid (GA)-chitosan (CS) copolymer by a free radical mediated grafting method, which is further applied to the field of antibacterial materials, crosslinking structures of the compound GA-CS copolymer were characterized, fully indicating that gallic acid is resoundingly grafted onto chitosan. The grafting ratios of three copolymers GA-CS are 45.71% (I), 36.12% (II), and 18.96% (III) were determined by UV-Vis spectrophotometer. The minimum inhibitory concentrations of three GA-CS copolymers are 30 µg/mL against Escherichia coli and are ranged from 250 to 550 µg/mL against Staphylococcus aureus. By counting viable bacterial colonies, it can be found that antibacterial property is preferable by increasing the grafting ratio of GA-CS copolymers. Findings of investigation on aforementioned bacteria experiment indicate that the CFU/mL values of GA-CS (I, II, III) are 2.04×106, 7.56×106, 1.48×107 to Staphylococcus aureus, and 2.96×106, 1.01×107, 2.14×107 to Escherichia coli after 12 h treatment. In addition, the interaction process between GA-CS copolymer and bacteria can be observed through a transmission electron microscope. The specific manifestation is that the bacterial cell membranes are ruptured after being treated with the copolymer, which causes the cell contents to flow out, and the cell morphology is shrunk and out of shape.

Key words: gallic acid; chitosan; antibacterial activity; antibacterial material

1 Introduction

Microbial infection is a predisposing factor for a number of diseases, such as influenza, gastroenteritis, dysentery, myocarditis, gynecological inflammation, pneumonia[1,2],etc.Microbial contamination in medical treatment seriously threatens human health, while controlling pathogens is required to shorten the iatrogenic outbreaks and to assure patients a secure, robust and effective treatment and a recovery environment[3,4]. The development of new antibacterial materials to eliminate pathogenic microorganisms is not only the key to the treatment of many diseases, but also is become one of the current research hotspots[5]. Most of the antibacterial materials used in the biomedical field will come into direct contact with organisms, and even need to enter cells or the human bodies. Therefore, the biocompatibility of antibacterial materials should be more strictly. The aim of the scientific researchers is to design and synthesize antibacterial materials with good performance, high biological safety and low cost.

Chitosan (CS) is a natural cationic polysaccharide prepared by deacetylating chitin, which is extremely abundant in nature. It has good antibacterial property and low cytotoxicity, meanwhile it can enhance the body immunity and promote wound healing[6,7]. In vivo, chitosan can be degraded into glucosamine and absorbed by human body. As a multifunctional material with great biocompatibility and non-immunogenicity, chitosan has been widely utilized as a bactericide, antioxidant and antineoplastic[8-11]. Because chitosan contains amine and hydroxyl groups, an additional antibacterial agent can be added by modifying the chemical structure to improve its antibacterial activity, reduce bacterial resistance and expand the application range. Copolymers synthesized in this way have turn into a focus of new antibacterial drugs.

The antibacterial active substance extracted from plants is one of the earliest antibacterial materials used by human beings. Gallic acid (GA), a hydrolysate of plant tannic acid, is a naturally occurring phenolic compound[12]carrying antioxidant properties. Owing to its multiple biological activities, such as good antitumor activity[13], well antiviral toxicity[14], high anti-inflammatory effect[15], and excellent antibacterial behavior[16], especially it is difficult to form drug resistance, gallic acid is widely employed in food and pharmaceutical fields[17-19]. Roberto[20,21]reported that gallic acid exhibited conspicuous antibacterial activity againstHelicobacter pyloriandEscherichia coli, and the minimum inhibitory concentrations (MIC) value againstEscherichia coliwas 3.25 mg/mL. The gallic acid extracted by Chanwitheesuk[22]from Leguminosae showed an excellent inhibitory effect on gram-positive bacteria, and the MIC value againstStaphylococcus aureuswas 1.25 mg/mL. Aforementioned researches testified that gallic acid has a superior application prospect in medical antibacterial materials.

To this day, antibiotic therapy is primarily adopted but it extremely tends to generate a large number of drug-resistant bacteria. However, the appearance of GA-CS copolymer can figure out this circumstance perfectly[23]. At present, conjugation of GA onto chitosan has various means, such as catalyse by using 1-ethyl-3- (dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide[24], free radical mediated grafting method[25]and laccase-mediated functionalization[26]. GA-CS copolymer gets through the strong group of the benzene ring of gallic acid, which could to cut down the intermolecular hydrogen bonding of chitosan. It is possible to improve antioxidant ability and antibacterial activity of chitosan by means of the free radical mediated grafting method, which is green, non-toxic and pollution-free, to synthesize gallic acid onto chitosan[27].

In this work, we grafted gallic acid containing functional phenolic hydroxyl groups and hydrophilic carboxyl groups onto chitosan via a free radical mediated grafting method which is green, environment and non-toxic. And the synthesized product GA-CS copolymer was characterized by UV-Visible (UV-Vis) spectrophotometer, Fourier transform infrared (FT-IR) spectroscopy and proton nuclear magnetic resonance (1H NMR) spectrometer. GA-CS copolymer with well biocompatibility and antibacterial activity can be biodegradedin vivo. In addition, the antibacterial property and the sterilization mechanism of GA-CS copolymer againstEscherichia coliandStaphylococcus aureuswere investigated by MIC value, total number of colonies and TEM. Through this research, we synthesized GA-CS copolymers with low cost, low bacterial resistance and high bio-safety. At the same time, we observed and inferred that the antibacterial mechanism of GA-CS copolymers was through the destruction of bacterial cell membrane. This provides a new theoretical basis for biomedical materials in the field of antibacterial.

2 Experimental

2.1 Materials

Chitosan (CS, deacetylation ≥ 95%, viscosity 100-200 mPa·S, Aladdin), gallic acid (GA, 99%, Aladdin), hydrogen peroxide (H2O2, 30%, Aladdin), ascorbic acid (AsA, ≥ 99%, Aladdin), acetic acid (HOAc, analytical grade, Sinopharm Chemical Reagent Co., Ltd), ethanol (EtOH, ≥ 99.7%, Sinopharm Chemical Reagent Co., Ltd), Folin-phenol reagent (1 mol/L, Sinopharm Chemical Reagent Co., Ltd) , sodium carbonate(Na2CO3, Sinopharm Chemical Reagent Co., Ltd) were used without further purification.

Gram-negative bacterium:Escherichia coli(E. coli, CCTCC AB 93154) and gram-positive bacterium:Staphylococcus aureus(S. aureus, CCTCC AB 91093) used for antibacterial activity were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China) and nourished on Mueller-Hinton Broth (MHB). Glutaraldehyde solution (2.5%, Shanghai yuanye Bio-Technology Co., Ltd), phosphate buffered solution (pH 7.4, HyClone), and glycerol (≥ 99%, Biosharp) were used in the antibiotic tests.

2.2 Instrumentation

Freeze Drier (FD-1A-50, Biocool, Beijing, China), infrared spectrometers (Tensor 27, Brock, Germany),1H NMR (DRX600MHz, Brock, Germany), and ultraviolet spectrophotometers (UV-2600, Shimazu (Hong Kong) Co., Ltd) were used for synthesis and structure analysis.

High speed refrigerated centrifuge (KDC-160HR, HKUST Innovation Co., Ltd.), TEM (Tecnai G2 20 TWIN, FEI, USA), ultramicrotome (EM UC7, Leica, Germany) were tested for antibacterial experiments.

2.3 Synthesis of gallic acid grafted onto chitosan (GA-CS)

The compounds of three kinds of GA-CS copolymers with diverse grafting ratios were performed according to the method of Curcio[25]and Cho[28]with modifications. The chitosan (0.5 g) was placed in a schlenk bottle, afterwards sealed with rubber stopper, vacuumized with a double-row tube and next injected with nitrogen (repeat 3 times). Inject acetic acid (5 mL, 2%,v/v) then stir until the solution is clarified, and put 1 mL of 30% H2O2containing 0.02 g of ascorbic acid. After 30 minutes, gallic acid was dissolved in ethanol and added to the mixture at different molar ratios of gallic acid to chitosan (1:1, 3:1, and 5:1). After stirring the reaction at 25 ℃ for 36 h, air was performed to quench the reaction. Finally, the reaction mixture was transferred to the 3 500 Da dialysis tubing and dialyzed with distilled water for 72 h in order to remove unreacted gallic acid. By way of vacuum freeze-drying to obtain the target compound GA-CS copolymer.

2.4 Analysis and characterization of GA-CS

In order to identify whether gallic acid was successfully grafted onto chitosan, UV-Vis spectrophotometer, FT-IR spectroscopy, and1H NMR spectrometer were adopted to characterize and analyze the GA-CS copolymer.

Chitosan and GA-CS copolymer samples were dissolved with 2% acetic acid solution and separately formulated into a suitable concentration. Using 2% acetic acid solution as blank control, the absorption spectra of GA-CS copolymer were scanned at a wavelength range of 200 - 600 nm with a Shimazu UV-2600 UV-Vis spectrophotometer. The measurements for each sample were repeated three times, and the results were averaged.

Grounded the specimen which was kept dry into powder with KBr, and pressed into a thin sheet, then detected by a Brock Tensor 27 FT-IR spectroscopy in the range of 4 000-400 cm-1. The chromatogram and data were recorded.

Samples were dissolved in CD3COOD /D2O (2%,v/v) for NMR detection under 600 MHz magnetic field using a Brock DRX600MHz NMR spectrometer.

2.5 Determination of GA-CS grafting ratio

The grafting ratio of gallic acid in GA-CS copolymer was determined by an improved Folin-phenol reagent method[29]. Gallic acid solutions of diverse volumes were taken into 10 mL volumetric flask, added 4 mL distilled water and 1 mL Folin-phenol reagent, react at 30 ℃ for 30 minutes in the dark, and then appended 10% sodium carbonate solution (Na2CO3) to 10 mL. The absorbance was measured at 760 nm. The solution without gallic acid solution was used as a control. Each sample was measured in parallel three times and the results were averaged. Process the data and draw a standard curve.

Precisely weighed a certain amount of GA-CS copolymer and formulated it to a concentration suitable for Lambert-Beer’s law. 4 mL of the configured solution was placed in a 10 mL volumetric flask, then 1 mL of Folin-phenol reagent was added. After the reaction was conducted at 30 ℃ for 30 minutes without light, followed by 10% Na2CO3solution to 10 mL. Absorbance was measured at 760 nm using a Shimazu UV-2600 UV-Vis spectrophotometer. Gallic acid contents were calculated according to the standard curve of gallic acid, and the results were expressed as milligrams of gallic acid per gram of dried GA-CS (mg GA/g GACS).

2.6 Bacterial culture

E. coli(CCTCC AB 93154) andS. aureus(CCTCC AB 91093) were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China) and were incubated in MHB for 12 to 24 h at 37 ℃ constant temperature incubator. The centrifugal cells were washed 3 times with sterile PBS and uniformly dispersed in same buffer solution. The two diverse bacterium suspensions were used to experiment the MIC values and to identify the antibacterial activity of GACS copolymer.

2.7 Determination of MIC values

Two individual bacterial suspensions were diluted with sterile PBS, which were adjusted to the cell concentration approximately 1 × 104CFU/mL. Briefly, GA-CS solution was added to test tubes containing MHB which inoculated 1 × 104CFU/mL bacterial suspension. The tubes were cultured in a 37 ℃ constant temperature oscillator at 180 r/min for 24 h. Three parallel groups were set, while sterile PBS (pH 7.4) was used as the blank control.

2.8 Antibacterial activity test

Counting viable bacterial cell colonies were used to evaluate the antibiofilm activity of GA-CS copolymer, which was estimated by the spread plate method in the same incubation time with various grafting ratios of GA-CS. Copolymers of different gallic acid contents were added to MHB medium which inoculated with the test bacteria (E. coliandS. aureus), followed by react for 12 h. In the next moment, 200 μL germ solution that diluted by 12,000 times was evenly coated on MHB solid plate, afterwards cultured at 37 ℃ for 12 h. The solution containing only bacteria was a blank group. The measurements for each sample were repeated three times.

The property of the killing mechanisms of GA-CS copolymer interaction withE. coliandS. aureuswas scanned by transmission electron microscope (TEM). Test bacteria were incubated with GA-CS copolymer and chitosan in MHB for 12 h at 37 ℃. In the back of the incubation, 3 mL bacterial solution was centrifuged at 12,000 rpm for 10 minutes, discarded the culture fluid and fixed overnight with 2.5% glutaraldehyde. Samples were rinsed 3 times with 0.1 M PBS (pH 7.4), fixed with 1% OsO4in 0.1 M PBS (pH 7.4) and placed at room temperature for 2 h, then washed with 0.1 M PBS (pH 7.4) for 3 times. After ethanol gradient dehydration and embedding, the slices were cut into 80 - 100 nm on a Leica (Germany) ultramicrotome, stained with both uranyl acetate and lead citrate. After all these, the thin sections were dried overnight and observed the alterations of cell pattern with TEM.

3 Results and discussion

3.1 Compound and characterization of GACS copolymer

Ascorbic acid exists in the form of dibasic acid (AscH2) in acetic acid solution (2%,v/v), and reacts with hydrogen peroxide to generate hydroxyl radical (HO•) which was used as initiators in the synthesis of chitosan-gallic acid. The reaction mechanism and process are shown in Scheme 1. The whole reaction system meets the requirements of green chemistry, among which hydrogen peroxide can be converted into non-toxic water and oxygen.

Scheme 1 Schematic presentation of the synthesis of GA-CS copolymer: (a) Generation of first-class free radicals; (b) Grafting gallic acid onto chitosan through free radical reaction

The UV-Vis absorbances of chitosan, gallic acid and GA-CS copolymer are shown in Fig.1. Chitosan had no absorption and gallic acid showed the absorption band benzene ring π system at 212 nm and 262 nm, while GA-CS copolymer appeared a new absorption band at 272 nm was due to the covalent bonding of gallic acid to chitosan reduces the energy required forn-π * and π-π * transitions.

Fig.1 UV-Vis absorbance of CS, GA, and GA-CS copolymer in the range of 200-600 nm

Fig.2 shows the FT-IR spectra of chitosan, gallic acid and GA-CS copolymer. As shown in Fig.2, gallic acid which exhibits the characteristic absorption of phenolic substances, provided the following bands in cm−1: 3 550- 3 200 (O-H, ν), 1 366 (O-H, β), 1 600-1 450 (C=C, ν), 1 300-1 200 (C-O, ν). The absorption peaks of chitosan were demonstrated as follows (in cm−1): 3 500-3 200 (N-H, O-H, ν), 1 654 (C=O, ν), 1 600 (R-NH, δ), 1 325 (C-N, ν), 1 157(C-O-C, νas), 1 090 (C-O, ν). The FT-IR spectra of GA-CS copolymer contains the above bands, meanwhile 1 600 (R-NH, δ) cm−1is replaced by 1 536 (R2-NH, δ) cm−1, indicating that gallic acid has been chemically conjugated onto chitosan.

Fig.2 FT-IR spectra of (a) CS, (b) GA-CS copolymer, and (c) GA

The1H NMR spectrum was employed to analyse the hydrogen bonds in the GA-CS copolymer. The1H NMR spectra of blank chitosan and GA-CS polymer are provided in Fig.3. Blank chitosan:1H NMR (600 MHz, D2O, δ): 4.6 (1H, H-1), 2.59 (1H, H-2), 3.46-3.66 (1H, H-3/6), 1.82 (H-Ac), 4.73 (D2O). GA-CS polymer:1H NMR (600 MHz, D2O, δ): 6.93 (phenyl protons of gallic acid, H-a), 4.59 (1H, H-1), 2.56 (1H, H-2), 3.54-3.73 (1H, H-3/6), 1.86 (H-Ac), 4.69 (D2O). A new peak at 6.93 ppm further confirmed that gallic acid accurately grafted onto chitosan.

Fig.3 1H NMR spectra of (a) GA, (b) CS, and (c) GA-CS copolymer

3.2 Determination of GA-CS grafting ratio

The standard curve of gallic acid is shown in Fig.4, and the expression of this straight line is A= 0.128 C+ 0.018, R2= 0.99911. The gallic acid contents in three GA-CS copolymers are indexed on Table 1 and Fig.5: 31.64 mg GA/g GA-CS (I), 70.78 mg GA/g GACS (II), and 104.32 mg GA/g GA-CS (III). Three GACS grafting ratios, which were calculated the ratio of gallic acid content in GA-CS copolymer to the usage amount in preparation, from GA-CS (I), GA-CS (II), and GA-CS (III) were 18.96%, 36.12%, and 45.71%.

Fig.4 The standard curve of gallic acid

Table 1 GA contents and grafting ratios of GA-CS copolymers

Fig.5 Comparison of GA contents and grafting ratios in GA-CS copolymers. The values represent means ± S.D (n=3)

3.3 Antimicrobial activities of GA-CS copolymer

Gallic acid and chitosan both have antibacterial activities. The MIC value indicates the strength of bacteriostatic ability. The lower the MIC value of a drug, the stronger its bacteriostatic ability. We referred to the MIC values of GA for different bacteria reported by Chanwitheesuk[22]and Li Dan[30], and set different concentration ranges after the pre-experiment. The MIC values of GA-CS copolymers with three dissimilar grafting ratios were determined by E. coli and S. aureus (Table 2). The consequences showed that GACS copolymers had better antibacterial activities than blank chitosan. Three GA-CS copolymers showed the MIC values to E. coli were all 40 μg/mL, and to S. aureus were 250, 550, and 550 μg/mL. In Table 3, the MIC values of blank chitosan against the two individual strains were 150 and 1 000 μg/mL, respectively. As described above, the antibacterial activity ranges from high to low was in order of GA-CS (I), GA-CS (II), and GA-CS(III). In addition, GA-CS (I) possessed the highest content of gallic acid in three GA-CS copolymers. Thus, the content of gallic acid in GA-CS copolymer played a vital role in improving the antibacterial activi-ty of chitosan.

Table 2 The MIC values of GA-CS copolymers with three dissimilar grafting ratios

Table 3 The MIC values of blank chitosan against S. aureus and E. coli

By counting viable bacterial cell colonies, it can be found that GA-CS copolymers with different gallic acid contents have a great difference in bacteriostatic effect. The inhibitory effects of chitosan and three groups of GS-CS copolymers onS. aureusandE. coliwere observed in Fig.6. As shown in this figure, the CFU/mL values of chitosan againstS. aureusandE. coliwere 2.32 × 107and 3.18×107, respectively. GACS copolymer grafted with gallic acid had stronger antibacterial activities than chitosan, and the CFU/mL values of GA-CS (I, II, III) were 2.04 × 106, 7.56 × 106, 1.48 × 107toS. aureus, and 2.96×106, 1.01×107, 2.14×107toE. coli. The results proved that the grafting of gallic acid to chitosan had a synergistic effect on antibacterial activity, and the antibacterial activity cumulatively improved with the enhance of gallic acid content.

Fig.6 Antibacterial activity of some copolymers against S. aureus (A) and E. coli (B): (a) pH 7.4 PBS, (b) chitosan, (c) GACS (I), (d) GA-CS (II), and (e) GA-CS(III)

The antibacterial activity of gallic acid may be obtained by destroying the phospholipid bilayer on the cell membrane[31].S. aureusandE. coliprocessed with GA-CS copolymers were observed and analyzed by TEM. As illustrated in Fig.7, compared with the chitosan-treated cells, the cell membranes after GA-CS copolymer treatment were ruptured, which results in the outflow of cell contents and the cells morphology atrophy and abnormal.

Fig.7 TEM image of some copolymers against S. aureus (A) and E. coli (B): (a) pH 7.4 PBS, (b) GA-CS (I), (c) GA-CS (II), and (d) GA-CS(III)

4 Conclusions

The present work, under the protection of nitrogen, conjugated a green, harmless and antibacterial copolymer GA-CS by the radical reaction between gallic acid and chitosan, which used hydrogen peroxide and ascorbic acid REDOX pair as the free radical initiator. Characterization of the compound GA-CS copolymer by means of FT-IR, UV-Vis spectrophotometer and NMR spectrometer. The results figured that GA was triumphantly grafted onto chitosan, and the copolymer was a gray powder with good water solubility. Through antibacterial experiments, it was found that the GA-CS copolymer was highly active againstS. aureusandE. coli. Meanwhile the higher grafting ratios, the stronger ability of GA-CS copolymer to kill bacteria. The bacterial cytoplasmic membrane becomes damaged and functionally disabled during GA-CS copolymer contacts with varying bacterium, which leads to leach out the intracellular components, impacts the cellular normal operation and ensures the bacterium die to accomplish an antibacterial therapeutic effect. The inhibitory effect is more distinct with the growth of GA contents. In conclusion, gallic acid has synergistic effect with chitosan, which increases the antibacterial property of chitosan. GA-CS copolymer expose a tremendous potential application prospect in the sector of antibacterial materials, and may be utilized for medical establishment, biological research institutions, and other fields where require to stick up to antibiotic resistant bacteria.