Preparation, Structural Characterization, and Property Investigation of Gallic Acid-Grafted Fungal Chitosan Conjugate

Abstract

Oxidative stress is the cause of numerous diseases in humans; therefore, there has been a continuous search for novel antioxidant molecules. Fungal chitosan is an attractive molecule that has several applications (antifungal, antibacterial, anticancer and antiparasitic action) owing to its unique characteristics; however, it exhibits low antioxidant activity. The aim of this study was to obtain fungal chitosan (Chit-F) from the fungus Rhizopus arrhizus and synthesize its derivative, fungal chitosan-gallic acid (Chit-FGal), as a novel antioxidant chitosan derivative for biomedical use. A low molecular weight Chi-F (~3.0 kDa) with a degree of deacetylation of 86% was obtained from this fungus. Chit-FGal (3.0 kDa) was synthesized by an efficient free radical-mediated method using hydrogen peroxide (H₂O₂) and ascorbic acid. Both Chit-F and Chit-FGal showed similar copper chelating activities; however, Chit-FGal was more efficient as an antioxidant, exhibiting twice the total antioxidant capacity than Chi-F (p < 0.05). Furthermore, H₂O₂ (0.06 M) promoted a 50% decrease in the viabilities of the 3T3 fibroblast cells. However, this effect was abolished in the presence of Chit-FGal (0.05-0.25 mg/mL), indicating that Chit-FGal protected the cells from oxidative damage. These results suggest that Chit-FGal may be a promising agent to combat oxidative stress.

Keywords: Rhizopus arrhizus; antioxidant; fungal chitosan; gallic acid; oxidative stress.

Figure 1

Morphology of R. arrhizus. (A,B). Sporangiophore with sporangium and rhizoid. (C). Sporangiophore with sporangium. (D). Sporangiophore with columella. (E). Rhizoid. (F). Sporangiospores. Scale bars: (A,B) = 100 μm, (C–E) = 50 μm, (F) = 20 μm.

Figure 2

Scheme of the conjugation process of gallic acid (GA) and fungal chitosan (Chit-F). Step 1 is the addition of the redox pair and the formation of macroradicals. The red circle shows the radical. Step 2 is the addition of GA to the solution and the formation of the conjugated molecule. The blue circle shows the position of linked gallic acid. R—GA or hydrogen.

Figure 3

Fourier transformed infrared spectroscopy (FTIR). Chit-F (black) and fungal chitosan-gallic acid (Chit-FGal) (red) spectra. These spectra are representative of three independently performed analyses.

Figure 4

Proton-nuclear magnetic resonance (¹H-NMR) spectra of Chit-F and Chit-FGal. (A) Chit-F spectra (B) Chit-FGal spectra (C) 7.57 Chit-FGal sign highlighted indicates regions where aromatic compounds are found. These spectra are representative of three independently performed analyses.

Figure 5

Microbiological biopolymers from R. arrizhus URM 8111—SEM electromicrographies of (A) Chit-F at 3000× magnification, (B) Chit-FGal at 3000× magnification. The measurement bar = 30 µm. Each short section corresponds to 3 µm.

Figure 6

Chit-F and Chit-FGal copper chelation. The copper chelation tests were performed with GA at a concentration of 0.5 mg/mL, which did not show copper chelation activity under the evaluated conditions. *** indicates the significant difference between the controls and samples (p < 0.05). # indicates the significant difference between the same concentrations of different samples (p < 0.05).

Figure 7

Total antioxidant capacity (TAC) test of Chit-F and Chit-FGal. * indicates significant difference between the samples (p < 0.05).

Figure 8

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reducing activity of the 3T3 fibroblast cells incubated with Chit-FGal for 24 h. Values are expressed as the mean ± standard deviation at concentrations of 0.05, 0.1, and 0.25 mg/mL. *** indicates the significant difference between the control and samples (p < 0.05).

Figure 9

MTT reducing activity of the 3T3 fibroblast cells incubated with Chit-FGal and hydrogen peroxide (H₂O₂) for 6 h. Values are expressed as the mean ± standard deviation at concentrations of 0.05, 0.1, and 0.25 mg/mL. *** indicates the significant difference between the control and samples (p < 0.05). ^a indicates significant difference between the control + peroxide and the tested samples (p < 0.05).

Figure 10

Morphological changes of 3T3 cells incubated with Chit-FGal and hydrogen peroxide (H₂O₂) for 6 h followed by DAPI staining. (A) Fluorescence microscope photographs of untreated cells; (B) cells treated with H₂O_2; (C) cells treated with 0.05 mg/mL Chit-FGal + H₂O₂; (D) cells treated with 0.1 mg/mL Chit-FGal + H₂O₂; (E) cells treated with 0.25 mg/mL Chit-FGal + H₂O₂; (F) DAPI staining quantification—Images were captured from ten different fields and about 100 cells were counted for each field. White arrows indicate nuclear fragmentation and/or chromatin condensation. Yellow arrows indicate cell division. Magnification ×400. *** indicates the significant difference between the control and samples (p < 0.05).