In an effort to develop biopolymer-based antioxidant and antibacterial materials, a chitosan-phloroglucinol conjugate was prepared and cellular antioxidant activity and minimum inhibitory concentration against foodborne pathogens and methicillin-resistant
Chitosan is a naturally occurring mucopolysaccharide and the second most abundant biopolymer, exhibiting versatile biological properties including biodegradability, biocompatibility, and a less toxic nature. These properties make chitosan attractive for a wide variety of pharmaceutical, biomedical, food industry, health and agricultural applications (Felt et al., 1998; Kim et al., 2007; Lin et al., 2009). Moreover, chitosan has been used for the development of new physiologically bioactive materials because it exhibits versatile biological properties, including antioxidant, antibacterial, anti-cancer, antiinflammatory and immunostimulatory activities (Jeon and Kim, 2001; Park et al., 2004a; 2004b; Lee et al., 2009a, 2009b, 2011). However, its water-insolubility is a major limiting factor. Therefore, there is growing interest in developing novel chitosan derivatives with desired characteristics, including enhanced water solubility. Consequently, methods to improve not only the water solubility but also the biological activities of chitosan have been developed by using both chemical and enzymatic modifications. Typically, appropriate moieties are conjugated onto the chitosan backbone. Recently, our group developed a gallic acid-
Marine-derived bioactive molecules have been isolated and their bioavailability characterized. Phloroglucinol is a phytochemical derived from edible brown algae with antioxidant, tyrosinase-inhibiting, cytoprotective and anti-inflammatory activities (Kang et al., 2004, 2006; Heo et al., 2005; Yoon et al., 2009; Kim and Kim, 2010). Previously, we demonstrated successful conjugation of phloroglucinol onto the chitosan backbone, and the chitosan-phloroglucinol conjugate showed higher antioxidant activity
Chitosan (average MW, 310 kDa; degree of deacetylation, 90%) was donated by Kitto Life Co. (Seoul, Korea). Phloroglucinol, Folin-Ciocalteau phenol reagent, and hydrogen peroxide were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Monobromobimane (mBBr), diphenyl-1-pyrenylphosphine (DPPP), and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Molecular Probes Inc. (Eugene, OR, USA). The other materials required for cell culture were purchased from Gibco BRL, Life Technologies (Grand Island, NY, USA). All other chemicals and reagents used in this study were of analytical grade and commercially available.
The bacterial strains tested for antibacterial activity were purchased from the Korean Collection for Type Cultures (KCTC; Daejeon, Korea) and 15 clinical isolates of MRSA were provided by Dong-A University Hospital (Busan, Korea). All strains were grown aerobically at 37℃ in Mueller-Hinton broth (Difco, Detroit, MI, USA) and subsequently used for assays of antibacterial activity.
The chitosan-phloroglucinol conjugate was prepared according to our previous method with a slight modification (Woo and Je, 2013). Briefly, chitosan (0.25 g) was dissolved in 25 mL of 2% acetic acid, and then 0.5 mL of 1.0 M hydrogen peroxide containing 0.054 g of ascorbic acid was added. After 30 min, phloroglucinol (18.43 mg) was added to the mixture, and then allowed to rest at room temperature for 24 h. Unreacted phloroglucinol was removed by dialysis for 48 h using distilled water. Unmodified chitosan was also prepared without the addition of phloroglucinol. Molar ratios of repeating units of chitosan to phloroglucinol were 1:0.1, which was optimal.
To confirm successful synthesis, 1H NMR analysis was 230conducted and the results were compared to the report by Woo and Je (2013). Unmodified chitosan: 1H NMR (400 MHz, D2O) δ: 4.90 (1H, H-1), 3.14 (1H, H-2), 3.41-4.30 (1H, H-3/6), 2.00 (H-Ac), 5.0 (D2O). Chitosan-phloroglucinol conjugate: 1H NMR (400 MHz, D2O) δ: 6.30 and 7.58 (aromatic protons of phloroglucinol), 4.90 (1H, H-1), 3.25 (1H, H-2), 3.65-4.36 (1H, H-3/6), 2.12 (H-Ac), 5.0 (D2O).
The phloroglucinol content of the chitosan-phloroglucinol conjugate determined using the Folin-Ciocalteau method was 29.28 ± 0.90 mg phloroglucinol/g chitosan-phloroglucinol conjugate.
The twofold serial dilution method was used to determine the chitosan-phloroglucinol conjugate MIC against MRSA and foodborne pathogens as described by the National Committee for Clinical Laboratory Standards (2004). The MIC was defined as the lowest concentration that demonstrated no visible growth after incubation at 37℃ for 24 h.
All results are expressed as means ± standard deviation (SD) of three determinations. Differences between the means of each group were assessed by one-way analysis of variance (ANOVA) followed by Duncan’s test using the statistical software, PASW Statistics 19.0 (SPSS Inc., Chicago, IL, USA). A value of
Cytotoxicity was first determined in RAW264.7 macrophage cells at the desired concentration (50, 100, and 200 μg/ mL) by MTT assay; the results confirmed that the chitosanphloroglucinol conjugate did not exhibit any cytotoxic effect (Fig. 1). Cellular ROS scavenging activities of the chitosanphloroglucinol conjugate and the unmodified chitosan were first evaluated using a fluorescent probe, DCFH-DA (Fig. 2). DCFH-DA freely penetrated into the cells and was hydrolyzed by intracellular esterases to DCFH and trapped inside the cells. DCFH was further oxidized to DCF by ROS, emitting
[Fig. 2.] Inhibition of intracellular reactive oxygen species (ROS) formation of unmodified chitosan and chitosan-phloroglucinol conjugate in mouse macrophage cells. Mouse macrophage cells were labeled with a non-toxicfluorescence dye, DCFH-DA, and treated with different concentrations of chitosan-phloroglucinol conjugate. The fluorescence intensities of DCF due to oxidation of DCFH by cellular ROS (generated by H2O2) were detected (λexcitation = 485 nm, λemission = 528 nm). All assays were done in triplicate and data are expressed as means ± SE. *P < 0.05 vs. control. a,bThe values with different subscripts indicate significant difference at the same concentration (P < 0.05).
fluorescence. Pre-treatment with the unmodified chitosan and the chitosan-phloroglucinol conjugate decreased DCF fluorescence in a dose-dependent manner. At 200 μg/mL unmodified chitosan, a 26.29% ROS scavenging activity was observed, whereas the chitosan-phloroglucinol conjugate exhibited 62.29% ROS scavenging activity at the same concentration; this difference was significant (
Excessive production of ROS may lead to a number of de-generative processes, such as cancer, cardiovascular and neurodegenerative conditions, and premature aging (Halliwell and Gutteridge, 1999; Finkel and Holbrook, 2000). Additionally, a crucial step in these ROS-mediated effects is DNA damage (Halliwell and Gutteridge, 1999). Thus, prevention of ROS-induced oxidative stress may help to maintain human health, and consuming dietary antioxidants from natural sources may
[Fig. 3.] Effect of chitosan-phloroglucinol conjugate on membrane lipid peroxidation inhibition as assessed by DPPP fluorescence assay. Mouse macrophage cells, treated with different concentrations of chitosan-phloroglucinol conjugate, were exposed to AAPH to initiate membrane lipid peroxidation. DPPP oxide fluorescence emitted due to the oxidation of DPPP by lipid hydroperoxides was compared with an AAPH non-treated blank group and an AAPH alone-treated control. The results are the mean ± SE of three independent experiments. a-eThe values with different subscripts indicate significant difference at P < 0.05.
[Fig. 4.] Effect of chitosan-phloroglucinol conjugate on expression of glutathione (GSH) level in mouse macrophage cells.The cells were treated with predetermined concentrations of chitosan-phloroglucinol conjugate and incubated for 30 min. Cellular GSH levels weredetermined using mBBr as a thiol-staining reagent according to the method described in the text, by measuring mBBr-GSH fluorescence intensity (λexcitation = 360 nm, λemission = 465 nm). The results are the mean ± SE of three independent experiments. a-dThe values with different subscripts indicate significant difference at P < 0.05.
have health-promoting effects. In this study, we demonstrated that the chitosan-phloroglucinol conjugate effectively scavenged intracellular ROS.
Overproduction of ROS results in an attack of not only DNA, but also other cellular components including the polyunsaturated fatty acid residues of phospholipids, which are highly sensitive to oxidation (Siems et al., 1995). Therefore, unsaturated fatty acids in cell membranes are susceptible to free radical-mediated oxidation. The DPPP fluorescent probe was used to evaluate lipid peroxidation in the cells (Fig. 3). After exposure to AAPH, considerable lipid peroxidation was observed in the control group (3.06-fold) compared to the non-treatment group. However, pre-treatment with the chitosan-phloroglucinol conjugate significantly inhibited lipid peroxidation in a dose-dependent manner (
GSH is the major soluble antioxidant in cell compartments and is a key cellular reductant, reducing numerous oxidizing compounds, including ROS and lipid peroxides, and is oxidized to GSH disulfide and other mixed disulfides (Kadiska et al., 2000). The main protective roles of GSH against oxidative stress include acting as a cofactor of GSH peroxidase, which detoxifies hydrogen peroxide and lipid peroxides, directly scavenging •OH and singlet oxygen, and regenerating vitamin C and E
Chitosan exhibits antibacterial activity against a broad spectrum of foodborne pathogens; this activity is influenced by the type of chitosan, molecular weight and several other physiochemical properties (Park et al., 2004a). To date, many chitosan derivatives exhibiting antibacterial activity have been developed by introducing specific functional groups, indicating that modification of chitosan is a good strategy for developing antibacterial biopolymers (Tang et al., 2010; Xiao et al., 2011). Additionally, we previously demonstrated that gallic acid-
for MSSA and fourfold for standard and clinical MRSA isolates compared to unmodified chitosan. Similar results were observed for foodborne pathogens (Table 1). Unmodified chitosan showed an MIC of 128 μg/mL for gram-positive bacteria (