Chitosan-Gallic Acid Conjugate with Enhanced Functional Properties and Synergistic Wound Healing Effect

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Hiatsevich, Kseniya S. Hileuskaya, Viktoryia V. Nikalaichuk, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4982795/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chitosan-gallic acid conjugates were synthesized by carbodiimide method and characterized by physicochemical methods (UV-vis, FTIR, 1 H NMR, TGA). The FTIR and NMR assays confirmed that the chemical interaction occurred solely due to the formation of an amide bond. It was established that by varying the ratio of the components during synthesis it is possible to obtain conjugates with desired conjugation ratio, grafting efficiency and gallic acid content up to 8%, 71% and 80 µg gallic acid/mg chitosan, respectively. Chitosan-gallic acid conjugate with a 5% conjugation ratio demonstrated excellent antioxidant properties: the IC50 value for ABTS radical scavenging activity was 0.0073±0.0001 mg/mL. In vitro tests showed that conjugation of chitosan with phenolic acid provided the antiglycemic activity of the material and its good biocompatibility. A low level of cytotoxicity was recorded in the HaCaT cell line model (IC50 was 1030.4 μg/mL). The received eco-friendly chitosan-gallic acid conjugate effectively inhibited the growth of thermophilic spore-forming bacteria G. thermodenitrificans and the resistant to classical antibiotics strain A. palidus . The results of an in vivo comparative analysis showed that chitosan-gallic acid conjugate had excellent wound healing properties due to the synergism of the polysaccharide and the natural antioxidant. chitosan chitosan-gallic acid conjugate antioxidant activity antibacterial activity cytotoxicity wound healing effect Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Trends in greening the economy make natural-origin molecules from renewable raw materials the most attractive resource for the design of a new generation of multifunctional, biocompatible and stable formulations for therapeutic technologies. In this regard, two classes of compounds have the greatest potential. The first group is polysaccharides (PSs) with high biocompatibility and a wide range of physiological activity. Over the years PSs have become an object of interest for the development of novel drugs and functional biomaterials. In terms of biomedical application, important characteristics of PSs are chelating, film-forming, moisturizing and mucoadhesive properties. Grafting or conjugation with other biomolecules can endow PSs with increased complexity, conformational versatility and enhanced biological activities [ 1 ]. PSs modification is crucial to adjust their properties in accordance with specific applications in the biomedical field. The second group is natural polyphenols, which exhibit a wide range of antioxidant activity and have the potential of anticancer, antidiabetic, antimicrobial and antiviral activities [ 2 ]. Besides, due to their aromatic structure, phenolic acids act as natural UV filters, while the catecholic fragment in a structure of most phenolic acids determines their adhesive properties. However, a serious limitation of their practical application is their high lability, oxidation tendency, low photo- and thermostability, limited solubility and, as a result, low bioavailability [ 3 – 5 ]. Moreover, many polyphenols at high concentrations exhibit pro-oxidant effect [ 6 ]. Pro-oxidant activity is usually catalyzed by metals, especially transition metals, such as Fe and Cu, which are widespread in biological systems [ 7 ]. For example, flavonoids and dihydroxycinnamic acids can nick DNA via the production of radicals in the presence of Cu and oxygen. Phenoxyl radicals can also initiate lipid peroxidation [ 5 ], while the active forms of rosmarinic acid, o -quinone and o -semiquinone, can cause severe DNA oxidative damage even in low concentrations, as the original polyphenol is recovered from the radicals in the presence of NADH [ 8 ]. The antioxidant/pro-oxidant transition is difficult to control in in vitro conditions and almost impossible in ex vivo and in vivo systems [ 4 , 9 ]. The combination of the potential of these two groups of natural compounds, PSs and polyphenols, will allow creating derivatives with improved desired characteristics and overcome some of their limitations. Most PSs are polyanionic (pectin, alginate) or neutral (cellulose, starch) polymers, except chitosan (Chit), which is polycationic. In particular, chitosan molecule contains three types of highly reactive functional groups, including amino/acetamido group, and primary or secondary hydroxyl groups, at C-2, C-3 and C-6 positions, respectively. Chit demonstrates good biocompatibility, non-toxicity, wide availability, low cost, biodegradability, antibacterial, anti-inflammatory and hemostatic properties, which make it an ideal choice for wound healing materials [ 10 ]. In spite of the range of Chit benefits, its limited solubility in water reduces its interaction with the wound area and wound fluids, which may impair its healing ability. Besides, Chit does not have pronounced antioxidant properties, while it plays an important role in wound healing. In comparison with other polymers, modification of Chit macromolecules is relatively simple, taking into account the functional activity of amino and hydroxyl groups. The introduction of additional hydrophilic fragments into the macromolecule is the most common method for obtaining water-soluble Chit derivatives. By this moment, carboxymethylchitosan and quertenized chitosan have become widespread. The last one also exhibits increased antimicrobial activity in comparison with the neat polymer. One of the promising approaches is obtaining of Chit-saccharide conjugates by the Maillard reaction. Such derivatives exhibit enhanced water-solubility and antioxidant activity [ 11 , 12 ]. However, conjugation with phenolic acids provides the production of fundamentally new derivatives with improved functionality [ 13 , 14 ]. The main methods of conjugation are carbodiimide modification, enzyme-mediated method and free radical induced grafting reaction. Advantages and disadvantages of each approach are described in detail in some modern reviews [ 14 – 17 ]. The method of ultrasonic modification can be attributed to non-traditional methods of Chit conjugation with natural phenolic acids [ 18 ]. Oxycoric acids (caffeic and ferulic) and gallic acid (GA) are the typical representatives of natural phenolic acids. We have previously shown [ 19 , 20 ] that conjugation of caffeic and ferulic acids makes it possible to obtain derivatives with enhanced antioxidant effect and improved stability. In addition, based on the comparative IC50 of chitosan-caffeic acid/caffeic acid, chitosan-caffeic acid conjugate unexpectedly exhibited more in 1.3–1.5 times radical scavenging ability. This phenomenon of the excellence IC50 of chitosan-caffeic acid conjugate under pure antioxidant for the radical quenching system suggested the synergistic effect and the role of Chit in retarding the pro-oxidation of phenolic acid. Gallic acid is the most studied representative of the polyphenolic antioxidants. It exhibits strong antiradical and antioxidant activities and, like other phenolic acids, possesses antimicrobial, anticancer, anti-inflammatory [ 21 ] and antiulcer [ 22 ] properties. Unique benefits of GA in terms of its accessibility, low toxicity, low cost and multiple pharmacological effects make it a universal choice in comparison with expensive growth factors, included in wound dressings. Gallic acid, such as other polyphenols with antioxidant activity, may act as a pro-oxidant causing a copper-dependent DNA damage [ 23 , 24 ]. Moreover, it has been reported that GA might induce oxidative stress in the rat liver and affect renal function [ 25 ]. The oxidative stress by reactive oxygen species, •OH in particular, is one of the mechanisms of GA-induced death of vascular smooth muscle cells, the mode of which was different from typical apoptosis. Yen et al. [ 26 ] have shown that the pro-oxidant mechanism for GA is most likely due to the strong reducing power and weak metal-chelating ability. In addition to the high pro-oxidant activity, a significant disadvantage of GA is its poor stability at high temperatures, light and other conditions, which leads to its easy degradation. Modification of GA molecules, including through conjugation with polymers, allows overcoming a number of limitations and creating a material with desired properties and suitable for biomedical applications. Polymer matrices provide film-forming properties and prolonged release. Thus, combination of GA with polylactide was used for coronary stent effective modification, and in vivo (in a porcine coronary restenosis model) it was shown that the coating provided a prolonged release of GA and had a mild suppressive effect on vascular inflammation in the stented arteries [ 27 ]. Functionalized by polyallylamine-GA conjugate surface showed remarkable enhancement in the adhesion, viability, proliferation, migration, and release of nitric oxide of human umbilical vein endothelial cells in comparison with suppressing vascular smooth muscle cell proliferation [ 19 ]. This striking selectivity of polymer-based conjugates of GA may provide a guide for designing the new generation of multifunctional vascular devices. The authors’ data [ 20 ] indicate that surfaces, modified by polymer-GA conjugate, provide a favorable microenvironment for endothelial cell growth. Healthy endothelial cells play a key role in maintaining vascular homeostasis and in regulation of inflammation during the early stages of wound healing [ 28 ]. The carboxyl and phenolic hydroxyl groups of polyphenol presented different influence on the growth behavior of cells. The authors showed [ 29 ] that GA conjugation with formation of the amide bond provides a relative decrease in the cytotoxicity of gallate against endothelial cells in comparison with the derivative based on a Schiff base reaction of quinone groups of GA with the polymer amino groups. In this work, for conjugation with gallic acid, we suggest to use the natural polysaccharide chitosan as a model polymer rich in amino groups. The hypothesis is that conjugation of GA with Chit will ensure the creation of a water-soluble conjugate with low cytotoxicity, good antimicrobial properties and suitable for wound healing on account of the active ester bonding of the primary amine group of Chit with activated carboxylic groups of GA. In the present work, chitosan-gallic acid conjugates (Chit-GA) with different conjugation ratio were prepared by carbodiimide grafting method and characterized by NMR, FTIR and UV-vis spectroscopy. The standard carbodiimide grafting approach allows accurately controlling conjugation process and avoiding GA oligomerization or Schiff bases formation. The synthesized Chit-GA conjugates were evaluated for antioxidant assay by ABTS and Folin-Ciocalteu methods. We demonstrate that conjugation ratio of 5% provides increased antioxidant activity of the Chit-GA conjugate in comparison with the initial polymer and excellent antimicrobial properties. The in vitro inhibition assay of bovine serum albumin advanced glycation end products (BSA-AGE) was used to evaluate the antidiabetic potential of Chit-GA conjugate. For the first time, wound healing properties of Chit, GA and Chit-GA conjugate were evaluated in the in vivo comparative experiment. Compared to the original components, Chit-GA showed synergistic enhancement of the wound healing. Here we also show that due to the film-forming properties, the conjugate is suitable as a wound healing material on its own without additional structure-forming manipulations. 2. Materials and Methods 2.1. Materials Low molecular weight chitosan (Chit, M w ~30 kDa, degree of deacetylation > 90%) was purchased from Glentham Life Sciences (UK). Gallic acid (GA, ≥ 99%) was obtained from Acros Organics (Belgium). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Carl Roth (Germany). 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS, ~ 98%) and bovine serum albumin (BSA, lyophilized powder, ≥ 96%) were obtained from Sigma-Aldrich (Germany). Folin-Ciocalteu reagent was purchased from Merck KGaA (Germany). These and other used reagents from commercial sources were analytical grade and employed without further purification. 2.2. Synthesis of Chit-GA Chitosan-gallic acid conjugates were synthesized by carbodiimide method with preliminary activation of GA carboxyl group by EDC [ 30 ]. GA (5 mg/mL) and EDC were dissolved in dimethylsulfoxide (DMSO), drained in a 1:1 volume ratio and stirred for 2 h in the dark at 20–22℃. EDC was used in a three-fold molar excess over GA. Then, the solution of activated GA was added dropwise to chitosan solution (5 mg/mL) in 0.5% acetic acid and left for 24 h in the dark at 20–22℃ under the constant stirring. The weight ratio of Chit:GA in the reaction mixture was 45:1, 30:1, 15:1, 10:1, 5:1, 2:1 and 1:1. The synthesized Chit-GA conjugates were purified by dialysis (dialysis tubing cellulose membrane, 14 kDa, Sigma Aldrich, Germany) against distilled water for 1 day and lyophilized (Labconco FreeZone 1.0, USA) at -50℃ and 0.03 mbar for 16 h. 2.3. UV-vis spectroscopy The content of GA in the synthesized Chit-GA conjugates was determined by UV-vis spectroscopy. Absorption spectra of the obtained samples were recorded in the range of 200–400 nm using the spectrofluorimeter CM2203 (Solar, Belarus). The amount of GA was calculated from the calibration curve A λ=266 nm = f ( C ), where A is the absorption intensity at 266 nm, C is the concentration of GA in solution. Conjugation ratio ( CR ) of Chit-GA, grafting efficiency ( GE ) and the amount of grafted GA at 1 mg of Chit ( µ ) were calculated using the following equations: $$\:CR=\:\frac{{n}_{\text{G}\text{A}}}{{n}_{{\text{N}\text{H}}_{2}}}\bullet\:100\%$$ 1 where \(\:{n}_{\text{G}\text{A}}\) is the amount of GA in the conjugate (mole) and \(\:{n}_{{\text{N}\text{H}}_{2}}\) is the amount of Chit monomers containing amino groups (mole), $$\:GE=\:\frac{{m}_{\text{g}\text{r}\text{a}\text{f}\text{t}}}{{m}_{\text{i}\text{n}\text{i}\text{t}}}\:\bullet\:100\%$$ 2 where \(\:{m}_{\text{g}\text{r}\text{a}\text{f}\text{t}}\) is the mass of GA in the conjugate (mg) and \(\:{m}_{\text{i}\text{n}\text{i}\text{t}}\) is the mass of initial GA used for synthesis (mg), µ \(\:\:=\:\frac{{m}_{\text{g}\text{r}\text{a}\text{f}\text{t}}}{{m}_{\text{C}\text{h}\text{i}\text{t}}}\:\bullet\:100\%\) (3) where \(\:{m}_{\text{C}\text{h}\text{i}\text{t}}\) is the mass of initial Chit used for synthesis (mg). 2.4. FTIR spectroscopy FTIR spectra of Chit, GA and Chit-GA conjugate were recorded in the range from 400 to 4000 cm − 1 using Tensor-27 spectrometer (Bruker, Germany). Data collection was performed with a 4 cm − 1 spectral resolution and 32 scans. 2.5. NMR spectroscopy 1 H NMR analysis was performed by Bruker Avance 500 MHz high-resolution NMR spectrometer (Bruker, USA). Samples were dissolved in D 2 O to give a concentration of 15 mg/mL. 2.6. TGA A thermogravimetric analysis was carried out to determine the thermal stability of GA, Chit and Chit-GA conjugate using TGA instrument STA 449 F3 (Netzsch, Germany) under the following conditions: oxygen/nitrogen atmosphere, crucible Al 2 O 3 , temperature range was from 20 to 800℃, heating rate was 5 K/min, scanning rate was 10 ℃/min, weight of samples was 11–19 mg. 2.7. Antioxidant assays 2.7.1. ABTS + radical-scavenging activity The ABTS radical scavenging activity was evaluated according to the method described in [ 31 ]. The ABTS + radical was generated by mixing 7 mmol ABTS solution containing 2.45 mmol potassium persulfate in the dark overnight (more than 16 h) at 20–22℃. The ABTS • + stock solution was diluted with water to obtain an absorbance of 0.700 ± 0.030 units at 734 nm. The working ABTS • + solution (1 mL) was mixed with 100 µL of the sample of different concentrations, and after 6 min of incubation at 20–22℃ the absorbance was measured at 734 nm using the spectrofluorimeter CM2203 (Solar, Belarus). The 1 mL of ABTS • + solution with 100 µL of water was used as a control. The scavenging effect was calculated according to the following equation: $$\:Scavenging\:effect=\frac{{A}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}-{A}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}}{{A}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}}\bullet\:100\%$$ 4 where \(\:{A}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\) and \(\:{A}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}\) is the absorption intensity at 734 nm of control and sample respectively. 2.7.2. Reducing power The reducing power of Chit-GA conjugate was quantified by the following method. 0.12 mL of the sample with a conjugate concentration of 1 mg/mL was added in a bottle followed by 0.6 mL of diluted 10 times Folin-Ciocalteu reagent. After 3 min, 0.48 mL of sodium carbonate (7.5 wt.%) was added. The content of the bottle was mixed thoroughly. The mixture was allowed to stand for 1 h at 20–22℃. The absorbance was measured at 765 nm in the spectrofluorimeter CM2203 (Solar, Belarus). The same was repeated for GA at different concentrations (10–150 µg/mL). The equation obtained for GA standard curve y = 10.097 x – 0.0138 and the R 2 value was 0.9995. The reducing power of the samples was expressed in µg-eq GA/mg of the sample. 2.8. Determination of the inhibitory effect on AGEs formation To evaluate the ability of Chit-GA conjugate to inhibit AGEs generated by glucose in comparison with Chit and GA, the method adduced in [ 32 ] with some modifications was used. The reaction mixtures were contained the solutions of BSA (2 mg/mL), glucose (500 mmol) and sodium azide (0.2 mg/mL) in a phosphate buffer (PBS, pH 7.4) and the Chit-GA water solution (0.005-1.5 mg/mL). The final volume of the reaction mixture was 2 mL. The mixtures were incubated for 24 h at 60℃. The fluorescence intensities of the solutions were measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm by the spectrofluorimeter CM2203 (Solar, Belarus). Before the samples, the spectrum of the blank sample (PBS) was recorded to neutralize the effect of the cuvette. The solutions prepared without the test materials were used as the control group. The AGEs formation inhibition ratio was calculated according to the following equation: $$\:Inhibition\:percentage\:of\:AGEs\:formation=1-\:\frac{{A}_{\text{s}}}{{A}_{\text{c}}}\:\bullet\:100\%$$ 5 where \(\:{A}_{\text{s}}\) and \(\:{A}_{\text{c}}\) is the absorbance at 440 nm of the sample and the control group, respectively. 2.9. Antibacterial activity To determine the minimum inhibitory concentration (MIC), 96-well sterile tablets (Sarstedt, USA) were used. Double serial dilutions of Chit, GA and Chit-GA solutions were prepared horizontally in the tablet in a sterile Mueller-Hinton Broth. For this, 50 µL of sterile medium was introduced into the tablet wells from 2 to 9, 100 µL into the well 10, and 100 µL of the drug in the starting concentration (10 mg/mL) into the well 1. Then, 50 µL were taken from the first well and transferred to the second and so on to the eighth hole, from which 50 µL were removed. Thus, double dilutions of drugs were obtained in wells from 1 to 8. Next, 50 µL of bacterial suspension was introduced into wells from 1 to 9. Two types of controls were used in the experiment: well 9 was served as a control of the growth of microorganisms, and well 10 was a control of the sterility of the medium and the tablet. The tablets were incubated in a thermostat for 24 hours at the temperatures of 28°C for B.subtilis , 37°C for E.coli and P.aeruginosa and 50°C for G. thermodenitrificans and A. palidus . Bacterial growth was determined by turbidity and discoloration in the wells by adding 5 µL of 0.2% aqueous solution of dimethylthiazolyl-diphenyltetrazolium bromide (MTT) (Thiazolyl blue tetrazolium bromide). During the metabolism of viable bacteria due to the mitochondrial activity of cells, the tetrazolium salt changes color from yellow to blue when MTT was restored by cellular enzymes oxyreductases. 2.10. Cytotoxicity HaCaT cells were seeded in 96-well cell culture plates at a density of 1.5∙10 4 cells/well, and 100 µL of cell suspension was added to each well and cultured in a constant temperature incubator containing 5% CO 2 at 37℃ for 24 h. The old medium was sucked and discarded, and 100 µL medium (Gibco) was added to each well in the control group, and sample solution prepared with 100 µL medium was added to each well in the sample group, and the cells were incubated for 12 h. And 50 µL MTT solution was added to each well and incubated at 37℃ for 4 h. Then the supernatant was discarded and the absorbance value of each well was detected at 570 nm wavelength. The absorbance value ratio of different groups to the control group was calculated. 2.11. Evaluation of the wound healing effect of Chit-GA in mice The animal experiments were approved by the Ethics Committee of the Institute of Bioorganic Chemistry of the Academy of Sciences of Uzbekistan and conducted in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes [ 33 ]. The experimental animal groups were distributed as follows: 1st group – control group, without treatment; 2nd group – treated with 0.5% Chit solution; 3rd group – treated with 1.0% Chit solution; 4th group – treated with 2.0% Chit solution; 5th group – treated with 0.5% Chit-GA solution; 6th group – treated with 1.0% Chit-GA solution; 7th group – treated with 2.0% Chit-GA solution. For wound formation, rats were anaesthetized by intraperitoneal injection of sodium ethaminal (50 mg/kg). The dorsal region was then depilated and, after antiseptic treatment, a 2.5 cm 2 area of skin was excised along the underlying fascia [ 27 ]. After one day, the treatment was carried out. The wounds were treated with aqueous solutions (200 µL), daily during the entire treatment period. The wounds were left uncovered. Throughout the entire study period, control studies were carried out, which took into account the following parameters of the course of the wound process: the presence and nature of the inflammatory reaction, the condition of the edges and the bottom of the wound, the timing of the wound cleansing from necrotic tissue, the timing of the onset of wound epithelization. To assess the healing process, wound diameter was measured with a caliper. The area of induced wounds was recorded by digital Vernier caliper (HD-5214) on days 3, 6, 9 and 12 of treatment, and the wound healing rate was calculated using the following equation: $$\:Wound\:healing\:rate=\frac{{S}_{0}-S}{{S}_{0}}\bullet\:100\%$$ 6 where \(\:{S}_{0}\) is the area of the initial wound (cm 2 ) and \(\:S\) is the area of the wound after the reproduced pathology (cm 2 ). 2.12. Statistical analysis The obtained results were presented as mean ± standard deviation. The statistical analysis of the data was performed using the one-way analysis of variance (ANOVA) with a significant level of p = 0.05. The value of p < 0.05 was considered to be statistically significant. 3. Results and discussion Chit-GA conjugates were synthesized by carbodiimide method [ 30 , 34 ] due to the formation of an amide bond between amino group of Chit and carboxyl group of GA (Fig. 1 ). The stock solutions of GA and EDC were transparent and colorless. After activation of the carboxyl group, GA solution remained transparent, but had a bright yellow color. Besides, during the synthesis conjugates were acquired a golden color. Meanwhile, a direct dependence of the color intensity on the amount of GA in the reaction mixtures was observed (increase in the color intensity with an increase in the acid content). The formation of Chit-GA conjugates was confirmed by UV-vis, FTIR and NMR spectroscopy and by thermogravimetric analysis (TGA). 3.1. UV-vis spectroscopy Figure 2 depicts the UV-vis absorbance spectra at 200–400 nm of starting materials and Chit-GA. Gallic acid solution had an absorption peak at 266 nm, corresponding to the aromatic ring π-system [ 35 ], while solution of chitosan showed no absorption peaks in the range from 200 to 400 nm. Meanwhile, UV-vis spectra of synthesized Chit-GA conjugates were exhibited the absorption peak from 259 to 262 nm depending on weight ratio in the reaction mixtures. Observed in the conjugate absorption spectrum hypsochromic shift of the GA characteristic peak was indicated the formation of a covalent bond between chitosan amino groups and gallic acid carboxyl groups. The effect of Chit:GA weight ratio in the reaction mixtures on the amount of grafted GA was evaluated. According to the obtained data, an increase in GA content was led to the enhancement in Chit-GA conjugation ratio value by about five times: from 1.50 ± 0.20 to 8.09 ± 1.72% (Table 1 ). As for the grafting efficiency, its value was decreased by almost an order when changing the mass ratio of Chit:GA from 45:1 to 1:1 in the reaction mixtures: from 70.51 ± 9.45 to 7.77 ± 1.49%. It was determined that by varying Chit:GA weight ratio during the synthesis, conjugates with controlled GA amount in the range from 15.7 ± 2.1 to 79.9 ± 2.4 µg GA/mg Chit could be obtained. Table 1 Physicochemical characteristics of the synthesized Chit-GA conjugates Chit:GA weight ratio in reaction mixture CR , % GE , % µ , µg/mg Chit 45:1 1.50 ± 0.20 70.51 ± 9.45 15.7 ± 2.1 30:1 1.79 ± 0.21 55.78 ± 6.52 18.6 ± 2.2 15:1 2.86 ± 0.08 44.56 ± 1.23 29.7 ± 0.8 10:1 3.47 ± 0.16 34.53 ± 2.11 36.2 ± 1.5 5:1 4.90 ± 0.66 25.45 ± 3.45 50.9 ± 6.9 2:1 7.70 ± 0.23 16.01 ± 0.46 79.9 ± 2.4 1:1 8.09 ± 1.72 7.77 ± 1.49 77.5 ± 15.0 Taking into account the obtained data, Chit-GA conjugate synthesized upon Chit:GA weight ratio 5:1 was chosen for the further research. 3.2. FTIR spectroscopy FTIR spectra of Chit, GA and Chit-GA conjugate recorded in the range from 4000 to 400 cm − 1 are presented in Fig. 3 . In the spectrum of GA characteristic bands at 3495 (O-H stretching), 3283 (C-H stretching), 1702, 1615 (C = O stretching), 1430 (C = C aromatic ring stretching), 1316 (C-O stretching), 1265 (COOH stretching), 1222 (C-O and C-C stretching) and 1026 (C-O stretching) cm − 1 were observed [ 36 ]. The spectra of Chit showed main bands at 3441 (O-H symmetric stretching, N-H stretching), 2921 (O-H asymmetric stretching), 1642 (C = O stretching, amide I), 1552 (N-H bending, amide II), 1424 (CH 2 bending), 1381 (C-N stretching, amide III), 1155 (pyranose ring C-O-C bridge asymmetric stretching), 1096 (C-O stretching) and 895 (C-H bending) cm − 1 [ 11 , 29 ]. Observed in the FTIR spectra of Chit-GA conjugate main changes were associated with the formation of a covalent bond between chitosan and gallic acid. First, a decrease in the intensity of O-H stretching vibrations and its shift towards lower wavenumbers from 3441 to 3272 cm − 1 and from 2921 to 2879 cm − 1 compared to the neat Chit were determined. Next, C-O stretching vibrations at 1024 cm − 1 , which was absent in the spectra of native chitosan, was observed in the conjugate spectrum. However, the main changes in the FTIR spectra were occurred in the range of wavenumbers 1600 − 1000 cm − 1 . Firstly, Chit amide I vibrations at 1642 cm − 1 disappeared, indicating the change of the primary amine to the secondary amine due to the reactions at chitosan NH 2 sites [ 29 ]. Secondly, the intensity increase and the shift towards lower wavenumbers from 1552 to 1548 cm − 1 of amide II bending vibrations were registered. Finally, the C-O stretching vibrations shift from 1096 to 1065 cm − 1 with the intensity increase in comparison with Chit was observed. Conjugation could occur due to both amide bonding and ester bonding [ 28 ]. However, the obtained spectra contained no peaks, which could be related to ester bond at the wavenumbers range 1800 − 1700 cm − 1 , which was registered at 1702 cm − 1 in the case of gallic acid. This fact, combined with the presence of an altered peak of amide II, confirms the formation of the conjugate due to an amide bond between the carboxyl group of the acid and the amino group of the polymer. 3.3. NMR spectroscopy 1 H-NMR analysis was used to study Chit-GA structural changes in comparison to starting Chit. The 1 H-NMR spectra of chitosan, gallic acid and Chit-GA conjugate are shown in Fig. 4 . The characteristic peaks at 2.04 ppm (H N−COCH3 , methyl protons of N-acetyl glucosamine), 3.01 ppm (H-2 of glucosamine residues) and the multiplet 3.56–3.76 ppm (H-3–H-6 protons of the pyranose ring) were observed. The peaks at 1.92 ppm and 4.75 ppm, corresponding to chemical shifts of protons in CH 3 COOH and D 2 O respectively, were also registered [ 11 ]. After conjugation, the main structure of the polymer was preserved, but some difference between Chit and Chit-GA spectra was found. A new signal at 6.99 ppm, which represented symmetric phenyl protons of GA, indicated successful grafting of gallic acid to the chitosan polymer chain [ 37 ]. 3.4. TGA TG, DTG and DTA curves of chitosan, gallic acid and Chit-GA are shown in Fig. 5 . On the TG curve of chitosan three consecutive weight loss steps were observed. The first stage was a weight loss about 36.3% below 290℃, attributed to physically adsorbed and hydrogen-bonded water evaporation (Table 2 ). The second stage in the range of 290–498℃ was corresponded to depolymerisation of chitosan and degradation of pyranose rings; the weight loss was about 46.4%. Moreover, the third degradation step from 498 to 800℃ was the thermo-oxidative process and the destruction of chitosan residues with the weight loss of 17.3% [ 11 ]. According to the analysis of DTA curve, an endometric peak at 92℃ and an exometric peak at 319℃ were assigned to the loss of water and the degradation of the chitosan chains, respectively [ 29 ]. On the TG curve of gallic acid, three consecutive weight loss steps were observed: from 20 to 268℃ (14.5% weight loss), from 268 to 377℃ (44.4% weight loss) and from 377 to 800℃ (35.8% weight loss), and the residual weight was 5.3%. Table 2 Thermal degradation steps of Chit, GA and Chit-GA Sample Stage Temperature range, ℃ Weight loss, % Residual weight, % Chit 1st 20–290 36.31 0 2nd 290–498 46.41 3rd 498–800 17.28 GA 1st 20–268 14.45 5.31 2nd 268–377 44.43 3rd 377–800 35.81 Chit-GA 1st 20–243 36.99 2.18 2nd 243–443 41.91 3rd 443–800 18.92 After conjugation, some changes in thermal behavior were established. The endometric peak at 90℃ on DTA curve was shifted toward lower values. It showed that introduction of GA caused the decrease in water holding capacity. At the same time, exometric peak at 279℃ was decreased in comparison with the neat Chit, and the decrease in the thermostability after acid grafting was determined [ 29 ]. The temperature of maximum weight loss was determined by DTG curves and attributed to the decomposition temperature of the sample. For Chit, GA and Chit-GA these values were 289, 291 and 246℃ respectively. The inclusion of acid in the polymer matrix lowered the decomposition temperature, which may be due to the obstruction of the chitosan chain packing after conjugation [ 28 ]. A similar effect of thermal stability reducing was observed by the authors [ 29 ] after GA grafting with chitosan chains using an ascorbic acid/hydrogen peroxide redox pair in inert air. 3.5. Antioxidant activity The ABTS radical scavenging activity detection method was shown, that the antioxidant potential of Chit-GA significantly (up to seven times) higher than that value of the neat chitosan (Fig. 6 ). IC50 value, that displays the concentration of a substance, reducing the number of radicals by 50%, is a good general accepted indicator for quantifying the antioxidant capacity. Therefore, the estimated IC50 values of Chit-GA and neat GA were comparable and amounted 0.0073 ± 0.0001 and 0.0077 ± 0.0002 mg/mL, respectively (Table 3 ). The equivalent IC50 of Chit-GA and GA for the ABTS radical quenching system suggested the synergistic function of chitosan in retarding the pro-oxidation of GA [ 38 ]. Taking this into consideration, the introduction of an H-atom donating group, i.e. gallic acid, onto chitosan is a good strategy to develop a chitosan derivative with robust antioxidant capacity. Table 3 IC50 values of Chit, GA and Chit-GA Sample IC50, mg/mL GA 0.0077 ± 0.0002 Chit-GA 0.0073 ± 0.0001 (standardization by GA concentration) Chit 7.1061 ± 0.0104 Chit-GA 0.0913 ± 0.0012 (standardization by Chit concentration) The most common mechanism of radical-scavenging activity of chitosan and its derivatives is attributed to that amino and hydroxyl groups, which react with unstable free radicals to form stable macromolecule radicals [ 11 ]. At the same time, low antioxidant potential of Chit may be attributed to its strong ion chelating ability because of its nitrogen atom. Improving the chitosan antioxidant activity becomes possible by introducing the H + atom donor group [ 39 ]. GA has been proved to possess greater antioxidant capacity due to its strong hydrogen-donating ability [ 40 ]. Thus, in the case of GA embedding, the antioxidant properties were improved. It should be noted that according to the literature ABTS radical scavenging activity of chitosan-gallic acid conjugates is significantly lower. For example, Chit-GA conjugate with a degree of substitution of 10.3% was characterized by an IC50 value of 219 µg/mL [ 41 ], while we obtained the conjugate with a conjugation ratio of only 5% and 30 times greater antiradical activity with an IC50 of 7.3 µg/mL. The authors' data obtained for the chitosan oligomer (5–10 kDa) conjugate are the closest to our value: the IC50 is 19.30 ± 0.46 µg/mL with a GA conjugation ratio of about 15%. Reducing power assay had also been used to evaluate the ability of antioxidants to donate electrons. The reducing power of Chit, Chit-GA conjugate and Chit/GA mechanical mixture is shown in Fig. 7 . Compared with the neat chitosan, which reducing power was 9.7 ± 0.2 µg-eq GA/mg of the sample, the conjugate value was increased by about 6 times and amounted to 54.6 ± 0.5 µg-eq GA/mg of the sample. At the same time, reducing power values of the conjugate and the mechanical mixture of chitosan with gallic acid was turned out to be practically comparable (53.0 ± 0.8 µg-eq GA/mg of the sample for mechanical mixture). The GA amount in the conjugate, calculated from the absorption spectra (p. 3.1) was amounted to 54.1 ± 1.2 µg-eq GA/mg of the sample, which was consistent with the result obtained by the Folin-Ciocalteu method. Obtained data are consistent with published quantum chemical calculations. Frontier molecular orbitals (FMOs) play a fundamental role in the study of substrate chemical characteristics including ability to absorb light and autoxidative capacity. Highest occupied molecular orbital (HOMO) is an important electronic factor in the description of antioxidant capacity, especially since it can be related to electron transfer reactions [ 42 ]. The higher the HOMO energy, the greater the ability to carry out nucleophilic attacks, thus, it is easy to release electrons and the higher the autoxidation capacity [ 24 ]. According to the Sekkal-Rahal et al. data [ 43 ], the HOMO energy of Chit-GA in water is higher than that of the GA (-6.12 ev and − 6.20 ev, respectively). This predicts that the capacity of Chit-GA to have faster nucleophilic attacks compared to GA, therefore, the abstraction of an electron will be easy in this polyphenol-chitosan conjugate [ 42 ]. 3.6. Antiglycation activity Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia, deficient production of insulin (type 1) or insufficient response of this hormone (type 2). Factors such as high blood glucose levels, increased production of reactive oxygen species (ROS) and formation of advanced glycation end products (AGEs) play a significant role in the development of diabetes mellitus [ 44 ]. Oxygen free radical activity can initiate peroxidation of lipids, which in turn stimulates glycation of protein, inactivation of enzymes and play a role in the long term complications of diabetes. Oxidative stress in diabetes coexists with a reduction in the antioxidant status, which can increase the deleterious effects of free radicals. Changes in glucose metabolism in diabetes mellitus are frequently accompanied by changes in the activities of the enzymes that control glycolysis and gluconeogenesis in liver and muscle. In this context, polyphenols received much attention because of their potent free radical scavenging and antioxidant actions [ 45 ]. The property of polyphenols and flavonoids to inhibit protein glycation and the consequent formation of AGEs is mostly defined by their antioxidant properties, metal-chelating capacity, protein interaction, methylglyoxal trapping and their ability to block the receptor for AGEs [ 44 ]. Gallic acid showed significant antiglycation results in the previous studies [ 29 , 44 , 46 – 48 ]. The study [ 49 ] reported that GA may directly combine with free radicals and lead to their inactivation, that in turn may decrease the intracellular concentration of free radical such as superoxide, peroxyl and hydroxyl radicals. GA ability to prevent the oxidation of Maillard reaction intermediates and to capture dicarbonyls were also shown [ 50 , 51 ]. The structural features and the antiglycation ability of phenolic acids might be associated with the antioxidant function. Antiglycation activity depends on the number of OH groups and their positions [ 52 ]. According to this, the ability of Chit-GA conjugate to inhibit AGEs generated by glucose in comparison with neat Chit and GA was evaluated in the model non-enzymatic reaction of BSA glycation by glucose. The curves of the dependence of glycation inhibition on the concentration of gallic acid ( a ) and chitosan ( b ) are shown in Fig. 8 . In the range of low concentrations, the conjugate showed better antiglycation activity than gallic acid. Compared with chitosan, inhibition was delayed earlier, which may be due to the limited solubility of the polymer under experimental conditions. 3.7. Antibacterial activity The physicochemical properties and biological activities of Chit-GA have been documented and many novel applications have been explored. However, studies of antimicrobial properties and mechanisms of Chit-GA were rare. Here, the minimum inhibitory concentrations (MICs) of GA, Chit and Chit-GA were determined by the serial dilution method. In general, the antibacterial activity of the conjugate is comparable or slightly less compared to the initial Chit (Table 4 ). MIC analysis showed that, compared with the initial GA, Chit-GA conjugate significantly more actively inhibits the growth of gram-negative and gram-positive bacteria, with the exception of P. aeruginosa strain. It should be noted that conjugate could inhibit the growth of thermophilic spore-forming gram-positive bacteria ( G. thermodenitrificans and A. palidus ): MIC values were 78.13 and < 4.88 µg/mL, respectively. The conjugate also exhibited high inhibitory properties against Enterococcus faecalis. These bacteria are highly resistant to high temperatures, acids, salts and to erythromycin and kanamycin. E. faecalis can live in an extreme alkaline environment due to its proton pump activity, which makes it resistant to calcium hydroxide medication. In addition, E. faecalis can form biofilms in medicated root canals in vivo [ 53 ]. Depending on the molecular weight, Chit can have an inhibitory effect on standard and clinical isolates of the strain E. faecalis [ 53 ]. However, the inhibitory activity of chitosan-gallic acid conjugate against this type of bacteria was evaluated for the first time. The mechanism of antimicrobial action of the Chit-GA conjugate is associated with disruption of the bacterial cell membrane, resulting in leakage of cytoplasm and an increase in relative conductivity [ 54 ]. Besides, chitosan derivatives could enter bacterial cells through damaged cell membranes and inhibit DNA synthesis in the nucleus [ 54 ]. The results of Chit-GA antibacterial activity are consistent with the literature data. Lima et al. [ 55 ] evaluated the antimicrobial activity of gallic, caffeic and pyrogallic acids against clinical strains E. coli , P. aeruginosa and S. aureus . According to these authors, the three tested phenolic compounds did not have clinically significant antibacterial activity with values of MIC ≥ 1024 µg/mL. However, the potential of the inhibitory effect of the conjugate against thermophilic bacteria and the resistant strain of E. faecalis is undeniable. Table 4 The MIC values of Chit, GA and Chit-GA Sample MIC for different strains, mg/mL E. coli ( g -) B. subtilis ( g +) P. aeruginosa ( g -) G. thermodenitrificans ( g +) A. palidus ( g +) E. faecalis ( g +) GA 0.62500 1.25000 1.25000 1.25000 0.15625 1.25000 Chit-GA 0.03906 0.62500 1.25000 0.07813 < 0.00488 < 0.00488 Chit 0.01953 0.15625 1.25000 0.03906 < 0.00488 < 0.00488 3.8. Cytotoxicity The cytotoxicity of Chit-GA was tested against HaCaT cells by MTT assay. As shown in Fig. 9 , the proliferation rates of Chit-GA were greater than 80% at low concentrations (up to 250.0 µg/mL), indicating that the cytotoxicity of Chit-GA was substantially absent at low concentrations. After that, the increase in Chit-GA concentration resulted in the significant decrease in cell viability. Notably, 70% of keratinocytes treated with 500.0 µg/mL of Chit-GA survived, while 45% of cells were killed after treated with 1000 µg/mL of Chit-GA. This result revealed that Chit-GA with a concentration below 750 µg/mL was toxic because the cell viability was less than 60%. The IC50 value was 1030.4 µg/mL of Chit-GA or 0.3 mmol of gallic acid. The received data are consistent with the literature. Thus, the authors [ 54 ] demonstrated a significant decrease in cytotoxicity of Chit against human fibroblasts due to modification with gallic acid: Chit-GA with a concentration below 1.0 µg/mL was non-toxic because the cell viability was above 80%. In that way, conjugation of chitosan with gallic acid ensures the production of water-soluble and safe material with antibacterial and pronounced antioxidant activities. 3.9. Evaluation of the wound healing effect of Chit-GA in mice Here, for the first time, we evaluate the wound-healing effect of chitosan conjugate in an in vivo comparative experiment. Taking into account all experimental groups, it was possible to reproduce an experimental model of skin wounds with an area from 2.50±0.06 to 2.70±0.09 cm 2 . Macroscopically after damage was inflicted for 10–20 minutes, changes in the form of accumulation of intercellular fluid and slightly pronounced capillary fullness were noted in the edges of the induced wounds; the edges were also slightly swollen and raised above the surface. The intensity of the manifestations increased with every hour of observation and persisted throughout the day. It is worth noting that neither purulent infiltration nor pronounced hyperemia was found in all groups of experimental animals during the observation period, which would complicate the wound process and lengthen the healing time. This fact makes it possible to assert the reliability of the reproduced wounds model. In the control group of animals the course of regenerative processes in dynamics is reflected in a planimetric reduction in the wound area and an increase in the healing rate of induced wounds. Thus, on the 3rd, 6th, 9th and 12th days the wound area was 2.30±0.10, 1.80±0.06, 1.50±0.06 and 0.90±0.07 cm 2 , and the calculated healing rate was 21, 28, 39 and 65%, respectively. The results obtained in the control group reflect the general pattern of the reparative process phase change after injury from exudation and proliferation to reparative regeneration. Treatment of wound pathology with 0.5%, 1.0% and 2.0% Chit solutions generally led to an acceleration of the reparative process in all animals. Thus, when using 0.5% Chit solution, the wound area was statistically significantly reduced to 1.90±0.07, 1.60±0.03, 1.20±0.08 and 0.70±0.05 cm 2 on days 3, 6, 9 and 12, respectively (Fig. 10 a). There was also a slight difference in the rate of wound healing: in the dynamics on the 3rd, 6th, 9th and 12th days, the difference was 4, 8, 9 and 7% compared to untreated animals. At the same time, the phases of the reparative process, the state of the edges and the bottom of the wound, the timing of purification from necrotic tissues, the formation of wound crusts, and then the onset of wound epithelization in this experimental group proceeded similarly to the control group of animals. Animals treated with 1.0% Chit solution showed a decrease in wound area in dynamics (Fig. 10 b), which was generally reflected in the rate of wound healing: the values were 11, 14, 20 and 14% higher on the 3rd, 6th, 9th and 12th days compared with the control group. On the 3rd and 4th days of the reparative process, cleansing of wounds from necrotic tissues was observed. The process of crusting formation shifted in all treated animals from day 6 to day 5 compared to the control group, while the crusts fell was registered by the 9th day of wound process treatment. The epithelialization process occurred on the 8th and 9th days. Of the used concentrations of Chit solutions as a wound healing agent, 2.0% Chit solution proved to be the most effective: in dynamics on days 3, 6, 9 and 12 the wound area decreased to 1.70±0.08, 1.40±0.10, 0.95±0.08 and 0.58±0.05 cm 2 (Fig. 10 c), and the wound healing rate was 14, 19, 24 and 17% higher compared to untreated animal wounds. The reparative process phases change after damage from exudation and proliferation to reparative regeneration in this group occurred faster: wound cleaning was detected on the 3rd and 4th days, and the crust formation was observed on the 5th day with a fall on the 7th and 8th days. It should be noted that the treatment process, characterized by the area and the rate of wound healing, was significantly improved with an increase in the concentration of used Chit solutions, but did not exceed the experimental groups of animals treated with Chit-GA conjugate solutions in similar concentrations. Chit-GA solutions were statistically significantly superior in the wound healing effect to the neat Chit and led to a faster regeneration process of the wound surface. When treated with 0.5% solution of Chit-GA conjugate, the formation of wound crusts was observed in 100% of animals on the 5th day, with a fall on the 7th and 8th days. The phase changes of the reparative process after damage from exudation and proliferation to reparative regeneration in this group proceeded in the same way as in the experimental group, where treatment was carried out with 1.0% Chit solution. This was also reflected in the close values of the wound healing rate of these experimental groups: when treated with 0.5% conjugate solution, calculated from the area (Fig. 10 a) rate values on days 3, 6, 9 and 12 were 25, 36, 48 and 72%, respectively. When treated with 1.0% Chit-GA solution, the wounds reparative process was expected to be more intense: in dynamics on the 3rd, 6th, 9th and 12th days the area decreased to 1.60±0.07, 1.20±0.07, 0.70±0.06 and 0.45±0.02 cm 2 (Fig. 10 b), while the difference in the wound healing rate compared with untreated animals was 14, 24, 29 and 18%, respectively. Already on the 2nd day of the reparative process course, regeneration without signs of exudative-proliferative reactions was observed during treatment with 1.0% conjugate solution. Further observation on the 3rd day showed that the wounds were completely cleared of necrotic tissues and the healing of damage continues with the formation of crusts on the 5th day with a fall on the 7th day. The wound healing process during treatment with a 2.0% Chit-GA solution proceeded even more intensively compared to previous concentrations: on days 3, 6, 9 and 12, a statistically significant area decrease to 1.50±0.06, 1.00±0.08, 0.50±0.08 and 0.20±0.04 cm 2 was observed (Fig. 10 c). The calculated wound healing rate increased by 2 times in comparison with the control group up to 41, 60 and 81% on days 3, 6 and 9, respectively. The dynamic observation of wound healing processes in this experimental group led to the conclusion that the cleansing of wounds from necrotic tissues, the formation of crusts, and epitalization proceeded in the same way as in the experimental group using a 1.0% solution of Chit-GA conjugate. Thus, all the samples turned out to be potent, but the most effective were solutions of Chit-GA conjugate, which statistically significantly exceeded the original chitosan in wound healing effect and led to a faster regeneration process of the wound surface. The maximum effect was observed when using a 2.0% solution of chitosan-gallic acid conjugate (Fig. 11 ). There is a clear dependence that the addition of gallic acid to chitosan macromolecules accelerates the reparative process of wound healing and has a positive effect on the cleansing of wounds from necrotic tissues, the formation of crusts and epithelialization. At the inflammatory stage of the wound healing process, neutrophils and macrophages secrete a large amount of reactive oxygen species (ROS) along with cytokines and matrix metalloproteases [ 56 ]. Formed ROS play a dual role. From one side, they inhibit the growth of microbial pathogens and promote phagocytosis [ 57 ]. In addition, ROS they can stimulate angiogenesis, division and migration of endothelial cells by expressing vascular endothelial growth factor (VEGF) and promote the formation of blood vessels [ 57 ]. From other side, a high level of ROS leads to oxidative stress, which damages and worsens the condition of neighboring tissues due to hydrolysis of extracellular matrix proteins and function impairment of dermal fibroblasts and keratinocytes [ 58 ]. Thus, normalization of ROS levels is critically important for a successful wound healing process [ 59 ]. That is why antioxidants are considered as one of the new promising components for wound healing [ 56 , 58 ]. In the case of Chit modified with GA, improved wound healing properties compared to neat Chit are probably due to the synergism of the components. Considering the classic four main phases mechanisms of wound healing [ 60 ], we can assume the reasons for synergistic enhancement of wound healing process of Chit-GA. The wound healing properties of chitosan and the mechanism of its action were described in sufficient detail in the Feng et al. review article [ 10 ]. Chitosan is applicable in the first three stages of wound healing. Firstly, due to the hemostatic action of amino groups, Chit promotes platelet and erythrocyte aggregation and inhibits the dissolution of fibrin at the stage of hemostasis. Secondly, chitosan inhibits the growth of bacteria at the stage of inflammation. Finally, chitosan depolymerizes releasing N -acetylglucosamine, which promotes fibroblast proliferation and collagen synthesis (proliferation stage). At the same time, GA promotes wound healing by directly increasing the expression of antioxidant genes, accelerating the migration of keratinocyte and fibroblast cells, activation of focal adhesion kinases (FAK), c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinases (Erk) [ 61 ]. Besides, taking into account the pronounced antioxidant activity, Chit-GA conjugate can neutralize excessive ROS levels during the inflammatory stage of the wound healing process. However, this requires additional detailed research. The results we have obtained revealed the great role of gallic acid as a supportive agent to hasten the wound healing process, which support the date obtained by other researches regarding the wound healing effect of GA and the role of antioxidants in the healing process [ 62 – 65 ]. 4. Conclusion Conjugates of chitosan with gallic acid were synthesized by carbodiimide method with preliminary activation of GA carboxyl groups by EDC. The formation of conjugates was confirmed by physical measurements and spectroscopic methods. It was shown that by varying the ratio of components during synthesis it is possible to obtain conjugates with controlled conjugation ratio from 1.50 ± 0.20 to 8.09 ± 1.72%, grafting efficiency from 7.77 ± 1.49 to 70.51 ± 9.45% and gallic acid content up to 79.9 ± 2.4 µg GA/mg Chit. FTIR and NMR methods proved that conjugation of molecules occurs due to the formation of an amide bond. TGA analysis showed that Chit with grafted GA had reduced thermal stability compared to neat chitosan. The result showed that Chit-GA conjugate had obvious antibacterial activity against E. coli and B. subtilis . For the first time, the inhibitory activity against thermophilic spore-forming bacteria was evaluated using the example of strains G. thermodenitrificans and A. pallidus. The MIC values of Chit-GA against G. thermodenitrificans and A. pallidus were 78.13 and less than 4.88 µg/mL, indicating that gram-positive thermophilic bacterium was more sensitive to Chit-GA than gram-positive B. subtilis (MIC value was 625 µg/mL). In addition, the high inhibitory activity of Chit-GA had been recorded against the resistant to the aminoglycoside antibiotic kanamycin strain E. faecalis. Besides, we demonstrated the outstanding (IC50 of 7.3 µg/mL) antioxidant activity of the conjugate with a degree of substitution of only 5% and correlate with literary quantum calculations. The great ability of Chit-GA conjugate to inhibit AGEs generated by glucose in comparison with neat Chit and GA was evaluated on the model non-enzymatic glycation reaction of BSA by glucose. The Chit-GA showed no significant cytotoxicity at low concentrations against human epidermal keratinocyte. Finally, we prove the enhanced wound healing properties of the modified chitosan compared to the original polymer in an in vivo comparative experiment. Declarations Funding: This work was financially supported by the Belarusian Republican Foundation for Fundamental Research (grant no. X23MN-007) and Mongolian Foundation for Science and Technology (grant no. BLR-2023/03). Conflict of Interest: The authors declare no conflicts of interest. Author Contribution Data curation: K.V. Hiatsevich; Supervision: K.S. Hileuskaya; Conceptualization: K.S. Hileuskaya (lead), A.I. Ladutska (supporting), L. You (supporting); Visualization: O.R. Akhmedov; Formal analysis: K.V. Hiatsevich (lead), V.V. Nikalaichuk (lead), N.N. Abrekova (equal), P. Shao (equal); Investigation: A.I. Ladutska (equal), N.N. Abrekova (equal), P. Shao (equal); Methodology: K.S. Hileuskaya (equal), A.I. Ladutska (equal), L. You (equal); Writing – original draft preparation: K.V. Hiatsevich; Writing – review and editing: K.S. Hileuskaya (equal), V.V. Nikalaichuk (equal), M.M. Odonchimeg (equal); Resources: O.R. Akhmedov (equal), L.You (equal); Project administration: M.M. Odonchimeg. Acknowledgement This work was financially supported by the Belarusian Republican Foundation for Fundamental Research (grant no. X23MN-007) and Mongolian Foundation for Science and Technology (grant no. BLR-2023/03). Data Availability: The data that support the findings of this study are available from the corresponding authors upon reasonable request. References Yazdi MK, Seidi F, Hejna A, Zarrintaj P, Rabiee N, Kucinska-Lipka J, Saeb RM, Bemcherif SA (2024) Tailor-Made Polysaccharides for Biomedical Applications. 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Ann Med Res 26:2720–2727. http://doi.org/10.5455/annalsmedres.2019.05.301 Tamer TM, Valachová K, Hassan MA, Omer AM, El-Shafeey M, Mohy Eldin MS, Šoltés L (2018) Chitosan/hyaluronan/edaravone membranes for anti-inflammatory wound dressing: In vitro and in vivo evaluation studies. Mater Sci Eng C 90:227–235. https://doi.org/10.1016/j.msec.2018.04.053 Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4982795","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":350406578,"identity":"b59519b8-40e2-4d0a-a6d0-a48c67a59d9b","order_by":0,"name":"Katsiaryna V. 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(\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e), Chit-GA (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e) and GA (\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/b64e6d2f02cbba14d390ec76.png"},{"id":65464736,"identity":"8f5ea11e-41c7-4007-a24c-1ad646d9eafd","added_by":"auto","created_at":"2024-09-27 19:15:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":380484,"visible":true,"origin":"","legend":"\u003cp\u003eNMR spectra of GA, Chit and Chit-GA\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/cac4f5a69fb16733fa3a85e3.png"},{"id":65464741,"identity":"43a1d2f0-78ff-4d22-b557-9d9f4a878ebc","added_by":"auto","created_at":"2024-09-27 19:15:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":404367,"visible":true,"origin":"","legend":"\u003cp\u003eTG, DTA and DTG curves obtained for thermal degradation of Chit, GA and Chit-GA\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/65dbe1d0c543f4c112e8232b.png"},{"id":65465127,"identity":"ab8430f1-a368-435d-b770-c8a4b89152c7","added_by":"auto","created_at":"2024-09-27 19:31:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":340473,"visible":true,"origin":"","legend":"\u003cp\u003eDependence of ABTS cation radicals inhibition on the concentration of Chit (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e) and GA (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/ffe884cb5b14b449d015af6a.png"},{"id":65464745,"identity":"4c6a1de4-19f3-4762-abff-34c595c2288c","added_by":"auto","created_at":"2024-09-27 19:15:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":86302,"visible":true,"origin":"","legend":"\u003cp\u003eReducing power of Chit, Chit-GA conjugate and Chit/GA mechanical mixture\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/1998750d35c7d9bae759758d.png"},{"id":65464748,"identity":"62317abe-9197-4b05-80c4-fbe5dd0cafcd","added_by":"auto","created_at":"2024-09-27 19:15:18","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":231577,"visible":true,"origin":"","legend":"\u003cp\u003eDependence of glycation inhibition on the concentration of gallic acid (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e) and chitosan (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/ec864209218af2631fe4de82.png"},{"id":65464910,"identity":"80722508-eb15-4daa-b7e8-29cbd3205cbd","added_by":"auto","created_at":"2024-09-27 19:23:17","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":452436,"visible":true,"origin":"","legend":"\u003cp\u003eViability estimation of HaCaT cells under the action of Chit-GA\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/106d9cfea98f75bcc225c5e0.png"},{"id":65464744,"identity":"312ded14-b4b4-4eee-a216-3ec43629de51","added_by":"auto","created_at":"2024-09-27 19:15:17","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":369711,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of the studied samples of various concentrations on the area of induced wounds: \u003cem\u003eа\u003c/em\u003e – 0.5%, \u003cem\u003eb\u003c/em\u003e – 1.0%, \u003cem\u003ec\u003c/em\u003e – 2.0%\u003c/p\u003e","description":"","filename":"Fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/144f4be5990de9b4a05ebc3f.png"},{"id":65464904,"identity":"4f4dd8a1-c56f-40f5-996d-7b3d95134e8a","added_by":"auto","created_at":"2024-09-27 19:23:16","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":3340191,"visible":true,"origin":"","legend":"\u003cp\u003eWound healing process in different groups of rats: \u003cem\u003ea\u003c/em\u003e – control group, without treatment; \u003cem\u003eb\u003c/em\u003e – treated with 2.0% Chit solution; \u003cem\u003ec\u003c/em\u003e – treated with 2.0% Chit-GA solution\u003c/p\u003e","description":"","filename":"Fig11.png","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/3d49f7ef6abb027026dc8553.png"},{"id":74427977,"identity":"e295ba65-8fd1-49b5-8ba6-81ca6426b35d","added_by":"auto","created_at":"2025-01-22 08:17:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10683377,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/4170014e-2f4b-4482-a4a6-6822b3a6395f.pdf"},{"id":65464902,"identity":"07021633-b64f-4d16-b791-985c0a8ae09c","added_by":"auto","created_at":"2024-09-27 19:23:16","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":402990,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-4982795/v1/35df28974afaab2bc4352366.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chitosan-Gallic Acid Conjugate with Enhanced Functional Properties and Synergistic Wound Healing Effect","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTrends in greening the economy make natural-origin molecules from renewable raw materials the most attractive resource for the design of a new generation of multifunctional, biocompatible and stable formulations for therapeutic technologies. In this regard, two classes of compounds have the greatest potential. The first group is polysaccharides (PSs) with high biocompatibility and a wide range of physiological activity. Over the years PSs have become an object of interest for the development of novel drugs and functional biomaterials. In terms of biomedical application, important characteristics of PSs are chelating, film-forming, moisturizing and mucoadhesive properties. Grafting or conjugation with other biomolecules can endow PSs with increased complexity, conformational versatility and enhanced biological activities [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. PSs modification is crucial to adjust their properties in accordance with specific applications in the biomedical field.\u003c/p\u003e \u003cp\u003eThe second group is natural polyphenols, which exhibit a wide range of antioxidant activity and have the potential of anticancer, antidiabetic, antimicrobial and antiviral activities [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Besides, due to their aromatic structure, phenolic acids act as natural UV filters, while the catecholic fragment in a structure of most phenolic acids determines their adhesive properties. However, a serious limitation of their practical application is their high lability, oxidation tendency, low photo- and thermostability, limited solubility and, as a result, low bioavailability [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, many polyphenols at high concentrations exhibit pro-oxidant effect [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Pro-oxidant activity is usually catalyzed by metals, especially transition metals, such as Fe and Cu, which are widespread in biological systems [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For example, flavonoids and dihydroxycinnamic acids can nick DNA via the production of radicals in the presence of Cu and oxygen. Phenoxyl radicals can also initiate lipid peroxidation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], while the active forms of rosmarinic acid, \u003cem\u003eo\u003c/em\u003e-quinone and \u003cem\u003eo\u003c/em\u003e-semiquinone, can cause severe DNA oxidative damage even in low concentrations, as the original polyphenol is recovered from the radicals in the presence of NADH [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The antioxidant/pro-oxidant transition is difficult to control in \u003cem\u003ein vitro\u003c/em\u003e conditions and almost impossible in \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e systems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe combination of the potential of these two groups of natural compounds, PSs and polyphenols, will allow creating derivatives with improved desired characteristics and overcome some of their limitations. Most PSs are polyanionic (pectin, alginate) or neutral (cellulose, starch) polymers, except chitosan (Chit), which is polycationic. In particular, chitosan molecule contains three types of highly reactive functional groups, including amino/acetamido group, and primary or secondary hydroxyl groups, at C-2, C-3 and C-6 positions, respectively. Chit demonstrates good biocompatibility, non-toxicity, wide availability, low cost, biodegradability, antibacterial, anti-inflammatory and hemostatic properties, which make it an ideal choice for wound healing materials [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn spite of the range of Chit benefits, its limited solubility in water reduces its interaction with the wound area and wound fluids, which may impair its healing ability. Besides, Chit does not have pronounced antioxidant properties, while it plays an important role in wound healing. In comparison with other polymers, modification of Chit macromolecules is relatively simple, taking into account the functional activity of amino and hydroxyl groups. The introduction of additional hydrophilic fragments into the macromolecule is the most common method for obtaining water-soluble Chit derivatives. By this moment, carboxymethylchitosan and quertenized chitosan have become widespread. The last one also exhibits increased antimicrobial activity in comparison with the neat polymer. One of the promising approaches is obtaining of Chit-saccharide conjugates by the Maillard reaction. Such derivatives exhibit enhanced water-solubility and antioxidant activity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, conjugation with phenolic acids provides the production of fundamentally new derivatives with improved functionality [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main methods of conjugation are carbodiimide modification, enzyme-mediated method and free radical induced grafting reaction. Advantages and disadvantages of each approach are described in detail in some modern reviews [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The method of ultrasonic modification can be attributed to non-traditional methods of Chit conjugation with natural phenolic acids [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOxycoric acids (caffeic and ferulic) and gallic acid (GA) are the typical representatives of natural phenolic acids. We have previously shown [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] that conjugation of caffeic and ferulic acids makes it possible to obtain derivatives with enhanced antioxidant effect and improved stability. In addition, based on the comparative IC50 of chitosan-caffeic acid/caffeic acid, chitosan-caffeic acid conjugate unexpectedly exhibited more in 1.3\u0026ndash;1.5 times radical scavenging ability. This phenomenon of the excellence IC50 of chitosan-caffeic acid conjugate under pure antioxidant for the radical quenching system suggested the synergistic effect and the role of Chit in retarding the pro-oxidation of phenolic acid.\u003c/p\u003e \u003cp\u003eGallic acid is the most studied representative of the polyphenolic antioxidants. It exhibits strong antiradical and antioxidant activities and, like other phenolic acids, possesses antimicrobial, anticancer, anti-inflammatory [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and antiulcer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] properties. Unique benefits of GA in terms of its accessibility, low toxicity, low cost and multiple pharmacological effects make it a universal choice in comparison with expensive growth factors, included in wound dressings. Gallic acid, such as other polyphenols with antioxidant activity, may act as a pro-oxidant causing a copper-dependent DNA damage [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, it has been reported that GA might induce oxidative stress in the rat liver and affect renal function [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The oxidative stress by reactive oxygen species, \u0026bull;OH in particular, is one of the mechanisms of GA-induced death of vascular smooth muscle cells, the mode of which was different from typical apoptosis. Yen et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] have shown that the pro-oxidant mechanism for GA is most likely due to the strong reducing power and weak metal-chelating ability.\u003c/p\u003e \u003cp\u003eIn addition to the high pro-oxidant activity, a significant disadvantage of GA is its poor stability at high temperatures, light and other conditions, which leads to its easy degradation. Modification of GA molecules, including through conjugation with polymers, allows overcoming a number of limitations and creating a material with desired properties and suitable for biomedical applications. Polymer matrices provide film-forming properties and prolonged release. Thus, combination of GA with polylactide was used for coronary stent effective modification, and \u003cem\u003ein vivo\u003c/em\u003e (in a porcine coronary restenosis model) it was shown that the coating provided a prolonged release of GA and had a mild suppressive effect on vascular inflammation in the stented arteries [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Functionalized by polyallylamine-GA conjugate surface showed remarkable enhancement in the adhesion, viability, proliferation, migration, and release of nitric oxide of human umbilical vein endothelial cells in comparison with suppressing vascular smooth muscle cell proliferation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This striking selectivity of polymer-based conjugates of GA may provide a guide for designing the new generation of multifunctional vascular devices.\u003c/p\u003e \u003cp\u003eThe authors\u0026rsquo; data [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] indicate that surfaces, modified by polymer-GA conjugate, provide a favorable microenvironment for endothelial cell growth. Healthy endothelial cells play a key role in maintaining vascular homeostasis and in regulation of inflammation during the early stages of wound healing [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The carboxyl and phenolic hydroxyl groups of polyphenol presented different influence on the growth behavior of cells. The authors showed [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] that GA conjugation with formation of the amide bond provides a relative decrease in the cytotoxicity of gallate against endothelial cells in comparison with the derivative based on a Schiff base reaction of quinone groups of GA with the polymer amino groups.\u003c/p\u003e \u003cp\u003eIn this work, for conjugation with gallic acid, we suggest to use the natural polysaccharide chitosan as a model polymer rich in amino groups. The hypothesis is that conjugation of GA with Chit will ensure the creation of a water-soluble conjugate with low cytotoxicity, good antimicrobial properties and suitable for wound healing on account of the active ester bonding of the primary amine group of Chit with activated carboxylic groups of GA. In the present work, chitosan-gallic acid conjugates (Chit-GA) with different conjugation ratio were prepared by carbodiimide grafting method and characterized by NMR, FTIR and UV-vis spectroscopy. The standard carbodiimide grafting approach allows accurately controlling conjugation process and avoiding GA oligomerization or Schiff bases formation. The synthesized Chit-GA conjugates were evaluated for antioxidant assay by ABTS and Folin-Ciocalteu methods. We demonstrate that conjugation ratio of 5% provides increased antioxidant activity of the Chit-GA conjugate in comparison with the initial polymer and excellent antimicrobial properties. The \u003cem\u003ein vitro\u003c/em\u003e inhibition assay of bovine serum albumin advanced glycation end products (BSA-AGE) was used to evaluate the antidiabetic potential of Chit-GA conjugate. For the first time, wound healing properties of Chit, GA and Chit-GA conjugate were evaluated in the \u003cem\u003ein vivo\u003c/em\u003e comparative experiment. Compared to the original components, Chit-GA showed synergistic enhancement of the wound healing. Here we also show that due to the film-forming properties, the conjugate is suitable as a wound healing material on its own without additional structure-forming manipulations.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eLow molecular weight chitosan (Chit, \u003cem\u003eM\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e~30 kDa, degree of deacetylation\u0026thinsp;\u0026gt;\u0026thinsp;90%) was purchased from Glentham Life Sciences (UK). Gallic acid (GA, \u0026ge;\u0026thinsp;99%) was obtained from Acros Organics (Belgium). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Carl Roth (Germany). 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS, ~\u0026thinsp;98%) and bovine serum albumin (BSA, lyophilized powder, \u0026ge;\u0026thinsp;96%) were obtained from Sigma-Aldrich (Germany). Folin-Ciocalteu reagent was purchased from Merck KGaA (Germany). These and other used reagents from commercial sources were analytical grade and employed without further purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis of Chit-GA\u003c/h2\u003e \u003cp\u003eChitosan-gallic acid conjugates were synthesized by carbodiimide method with preliminary activation of GA carboxyl group by EDC [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. GA (5 mg/mL) and EDC were dissolved in dimethylsulfoxide (DMSO), drained in a 1:1 volume ratio and stirred for 2 h in the dark at 20\u0026ndash;22℃. EDC was used in a three-fold molar excess over GA. Then, the solution of activated GA was added dropwise to chitosan solution (5 mg/mL) in 0.5% acetic acid and left for 24 h in the dark at 20\u0026ndash;22℃ under the constant stirring. The weight ratio of Chit:GA in the reaction mixture was 45:1, 30:1, 15:1, 10:1, 5:1, 2:1 and 1:1.\u003c/p\u003e \u003cp\u003eThe synthesized Chit-GA conjugates were purified by dialysis (dialysis tubing cellulose membrane, 14 kDa, Sigma Aldrich, Germany) against distilled water for 1 day and lyophilized (Labconco FreeZone 1.0, USA) at -50℃ and 0.03 mbar for 16 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. UV-vis spectroscopy\u003c/h2\u003e \u003cp\u003eThe content of GA in the synthesized Chit-GA conjugates was determined by UV-vis spectroscopy. Absorption spectra of the obtained samples were recorded in the range of 200\u0026ndash;400 nm using the spectrofluorimeter CM2203 (Solar, Belarus). The amount of GA was calculated from the calibration curve \u003cem\u003eA\u003c/em\u003e\u003csub\u003eλ=266 nm\u003c/sub\u003e = \u003cem\u003ef\u003c/em\u003e(\u003cem\u003eC\u003c/em\u003e), where \u003cem\u003eA\u003c/em\u003e is the absorption intensity at 266 nm, \u003cem\u003eC\u003c/em\u003e is the concentration of GA in solution. Conjugation ratio (\u003cem\u003eCR\u003c/em\u003e) of Chit-GA, grafting efficiency (\u003cem\u003eGE\u003c/em\u003e) and the amount of grafted GA at 1 mg of Chit (\u003cem\u003e\u0026micro;\u003c/em\u003e) were calculated using the following equations:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:CR=\\:\\frac{{n}_{\\text{G}\\text{A}}}{{n}_{{\\text{N}\\text{H}}_{2}}}\\bullet\\:100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{n}_{\\text{G}\\text{A}}\\)\u003c/span\u003e\u003c/span\u003e is the amount of GA in the conjugate (mole) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{n}_{{\\text{N}\\text{H}}_{2}}\\)\u003c/span\u003e\u003c/span\u003e is the amount of Chit monomers containing amino groups (mole),\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:GE=\\:\\frac{{m}_{\\text{g}\\text{r}\\text{a}\\text{f}\\text{t}}}{{m}_{\\text{i}\\text{n}\\text{i}\\text{t}}}\\:\\bullet\\:100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{m}_{\\text{g}\\text{r}\\text{a}\\text{f}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e is the mass of GA in the conjugate (mg) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{m}_{\\text{i}\\text{n}\\text{i}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e is the mass of initial GA used for synthesis (mg),\u003c/p\u003e \u003cp\u003e \u003cem\u003e\u0026micro;\u003c/em\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\:=\\:\\frac{{m}_{\\text{g}\\text{r}\\text{a}\\text{f}\\text{t}}}{{m}_{\\text{C}\\text{h}\\text{i}\\text{t}}}\\:\\bullet\\:100\\%\\)\u003c/span\u003e \u003c/span\u003e (3)\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{m}_{\\text{C}\\text{h}\\text{i}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e is the mass of initial Chit used for synthesis (mg).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. FTIR spectroscopy\u003c/h2\u003e \u003cp\u003eFTIR spectra of Chit, GA and Chit-GA conjugate were recorded in the range from 400 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using Tensor-27 spectrometer (Bruker, Germany). Data collection was performed with a 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e spectral resolution and 32 scans.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. NMR spectroscopy\u003c/h2\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH NMR analysis was performed by Bruker Avance 500 MHz high-resolution NMR spectrometer (Bruker, USA). Samples were dissolved in D\u003csub\u003e2\u003c/sub\u003eO to give a concentration of 15 mg/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. TGA\u003c/h2\u003e \u003cp\u003eA thermogravimetric analysis was carried out to determine the thermal stability of GA, Chit and Chit-GA conjugate using TGA instrument STA 449 F3 (Netzsch, Germany) under the following conditions: oxygen/nitrogen atmosphere, crucible Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, temperature range was from 20 to 800℃, heating rate was 5 K/min, scanning rate was 10 ℃/min, weight of samples was 11\u0026ndash;19 mg.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Antioxidant assays\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. ABTS\u003csup\u003e+\u003c/sup\u003e radical-scavenging activity\u003c/h2\u003e \u003cp\u003eThe ABTS radical scavenging activity was evaluated according to the method described in [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The ABTS\u003csup\u003e+\u003c/sup\u003e radical was generated by mixing 7 mmol ABTS solution containing 2.45 mmol potassium persulfate in the dark overnight (more than 16 h) at 20\u0026ndash;22℃. The ABTS\u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e+\u003c/sup\u003e stock solution was diluted with water to obtain an absorbance of 0.700\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030 units at 734 nm. The working ABTS\u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e+\u003c/sup\u003e solution (1 mL) was mixed with 100 \u0026micro;L of the sample of different concentrations, and after 6 min of incubation at 20\u0026ndash;22℃ the absorbance was measured at 734 nm using the spectrofluorimeter CM2203 (Solar, Belarus). The 1 mL of ABTS\u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e+\u003c/sup\u003e solution with 100 \u0026micro;L of water was used as a control. The scavenging effect was calculated according to the following equation:\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:Scavenging\\:effect=\\frac{{A}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}-{A}_{\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}}{{A}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}}\\bullet\\:100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{A}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{A}_{\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}\\)\u003c/span\u003e\u003c/span\u003e is the absorption intensity at 734 nm of control and sample respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. Reducing power\u003c/h2\u003e \u003cp\u003eThe reducing power of Chit-GA conjugate was quantified by the following method. 0.12 mL of the sample with a conjugate concentration of 1 mg/mL was added in a bottle followed by 0.6 mL of diluted 10 times Folin-Ciocalteu reagent. After 3 min, 0.48 mL of sodium carbonate (7.5 wt.%) was added. The content of the bottle was mixed thoroughly. The mixture was allowed to stand for 1 h at 20\u0026ndash;22℃. The absorbance was measured at 765 nm in the spectrofluorimeter CM2203 (Solar, Belarus). The same was repeated for GA at different concentrations (10\u0026ndash;150 \u0026micro;g/mL). The equation obtained for GA standard curve \u003cem\u003ey\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.097\u003cem\u003ex\u003c/em\u003e \u0026ndash; 0.0138 and the \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e value was 0.9995. The reducing power of the samples was expressed in \u0026micro;g-eq GA/mg of the sample.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Determination of the inhibitory effect on AGEs formation\u003c/h2\u003e \u003cp\u003eTo evaluate the ability of Chit-GA conjugate to inhibit AGEs generated by glucose in comparison with Chit and GA, the method adduced in [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] with some modifications was used. The reaction mixtures were contained the solutions of BSA (2 mg/mL), glucose (500 mmol) and sodium azide (0.2 mg/mL) in a phosphate buffer (PBS, pH 7.4) and the Chit-GA water solution (0.005-1.5 mg/mL). The final volume of the reaction mixture was 2 mL. The mixtures were incubated for 24 h at 60℃. The fluorescence intensities of the solutions were measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm by the spectrofluorimeter CM2203 (Solar, Belarus). Before the samples, the spectrum of the blank sample (PBS) was recorded to neutralize the effect of the cuvette. The solutions prepared without the test materials were used as the control group. The AGEs formation inhibition ratio was calculated according to the following equation:\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:Inhibition\\:percentage\\:of\\:AGEs\\:formation=1-\\:\\frac{{A}_{\\text{s}}}{{A}_{\\text{c}}}\\:\\bullet\\:100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{A}_{\\text{s}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{A}_{\\text{c}}\\)\u003c/span\u003e\u003c/span\u003e is the absorbance at 440 nm of the sample and the control group, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Antibacterial activity\u003c/h2\u003e \u003cp\u003eTo determine the minimum inhibitory concentration (MIC), 96-well sterile tablets (Sarstedt, USA) were used. Double serial dilutions of Chit, GA and Chit-GA solutions were prepared horizontally in the tablet in a sterile Mueller-Hinton Broth. For this, 50 \u0026micro;L of sterile medium was introduced into the tablet wells from 2 to 9, 100 \u0026micro;L into the well 10, and 100 \u0026micro;L of the drug in the starting concentration (10 mg/mL) into the well 1. Then, 50 \u0026micro;L were taken from the first well and transferred to the second and so on to the eighth hole, from which 50 \u0026micro;L were removed. Thus, double dilutions of drugs were obtained in wells from 1 to 8. Next, 50 \u0026micro;L of bacterial suspension was introduced into wells from 1 to 9. Two types of controls were used in the experiment: well 9 was served as a control of the growth of microorganisms, and well 10 was a control of the sterility of the medium and the tablet.\u003c/p\u003e \u003cp\u003eThe tablets were incubated in a thermostat for 24 hours at the temperatures of 28\u0026deg;C for \u003cem\u003eB.subtilis\u003c/em\u003e, 37\u0026deg;C for \u003cem\u003eE.coli\u003c/em\u003e and \u003cem\u003eP.aeruginosa\u003c/em\u003e and 50\u0026deg;C for \u003cem\u003eG. thermodenitrificans\u003c/em\u003e and \u003cem\u003eA. palidus\u003c/em\u003e. Bacterial growth was determined by turbidity and discoloration in the wells by adding 5 \u0026micro;L of 0.2% aqueous solution of dimethylthiazolyl-diphenyltetrazolium bromide (MTT) (Thiazolyl blue tetrazolium bromide). During the metabolism of viable bacteria due to the mitochondrial activity of cells, the tetrazolium salt changes color from yellow to blue when MTT was restored by cellular enzymes oxyreductases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Cytotoxicity\u003c/h2\u003e \u003cp\u003eHaCaT cells were seeded in 96-well cell culture plates at a density of 1.5∙10\u003csup\u003e4\u003c/sup\u003e cells/well, and 100 \u0026micro;L of cell suspension was added to each well and cultured in a constant temperature incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃ for 24 h. The old medium was sucked and discarded, and 100 \u0026micro;L medium (Gibco) was added to each well in the control group, and sample solution prepared with 100 \u0026micro;L medium was added to each well in the sample group, and the cells were incubated for 12 h. And 50 \u0026micro;L MTT solution was added to each well and incubated at 37℃ for 4 h. Then the supernatant was discarded and the absorbance value of each well was detected at 570 nm wavelength. The absorbance value ratio of different groups to the control group was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Evaluation of the wound healing effect of Chit-GA in mice\u003c/h2\u003e \u003cp\u003eThe animal experiments were approved by the Ethics Committee of the Institute of Bioorganic Chemistry of the Academy of Sciences of Uzbekistan and conducted in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The experimental animal groups were distributed as follows: 1st group \u0026ndash; control group, without treatment; 2nd group \u0026ndash; treated with 0.5% Chit solution; 3rd group \u0026ndash; treated with 1.0% Chit solution; 4th group \u0026ndash; treated with 2.0% Chit solution; 5th group \u0026ndash; treated with 0.5% Chit-GA solution; 6th group \u0026ndash; treated with 1.0% Chit-GA solution; 7th group \u0026ndash; treated with 2.0% Chit-GA solution.\u003c/p\u003e \u003cp\u003eFor wound formation, rats were anaesthetized by intraperitoneal injection of sodium ethaminal (50 mg/kg). The dorsal region was then depilated and, after antiseptic treatment, a 2.5 cm\u003csup\u003e2\u003c/sup\u003e area of skin was excised along the underlying fascia [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. After one day, the treatment was carried out. The wounds were treated with aqueous solutions (200 \u0026micro;L), daily during the entire treatment period.\u003c/p\u003e \u003cp\u003eThe wounds were left uncovered. Throughout the entire study period, control studies were carried out, which took into account the following parameters of the course of the wound process: the presence and nature of the inflammatory reaction, the condition of the edges and the bottom of the wound, the timing of the wound cleansing from necrotic tissue, the timing of the onset of wound epithelization. To assess the healing process, wound diameter was measured with a caliper. The area of induced wounds was recorded by digital Vernier caliper (HD-5214) on days 3, 6, 9 and 12 of treatment, and the wound healing rate was calculated using the following equation:\u003cdiv id=\"Equ5\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ5\" name=\"EquationSource\"\u003e\n$$\\:Wound\\:healing\\:rate=\\frac{{S}_{0}-S}{{S}_{0}}\\bullet\\:100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{S}_{0}\\)\u003c/span\u003e\u003c/span\u003e is the area of the initial wound (cm\u003csup\u003e2\u003c/sup\u003e) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:S\\)\u003c/span\u003e\u003c/span\u003e is the area of the wound after the reproduced pathology (cm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe obtained results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The statistical analysis of the data was performed using the one-way analysis of variance (ANOVA) with a significant level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05. The value of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eChit-GA conjugates were synthesized by carbodiimide method [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] due to the formation of an amide bond between amino group of Chit and carboxyl group of GA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The stock solutions of GA and EDC were transparent and colorless. After activation of the carboxyl group, GA solution remained transparent, but had a bright yellow color. Besides, during the synthesis conjugates were acquired a golden color. Meanwhile, a direct dependence of the color intensity on the amount of GA in the reaction mixtures was observed (increase in the color intensity with an increase in the acid content). The formation of Chit-GA conjugates was confirmed by UV-vis, FTIR and NMR spectroscopy and by thermogravimetric analysis (TGA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. UV-vis spectroscopy\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicts the UV-vis absorbance spectra at 200\u0026ndash;400 nm of starting materials and Chit-GA. Gallic acid solution had an absorption peak at 266 nm, corresponding to the aromatic ring π-system [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], while solution of chitosan showed no absorption peaks in the range from 200 to 400 nm. Meanwhile, UV-vis spectra of synthesized Chit-GA conjugates were exhibited the absorption peak from 259 to 262 nm depending on weight ratio in the reaction mixtures. Observed in the conjugate absorption spectrum hypsochromic shift of the GA characteristic peak was indicated the formation of a covalent bond between chitosan amino groups and gallic acid carboxyl groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effect of Chit:GA weight ratio in the reaction mixtures on the amount of grafted GA was evaluated. According to the obtained data, an increase in GA content was led to the enhancement in Chit-GA conjugation ratio value by about five times: from 1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 to 8.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As for the grafting efficiency, its value was decreased by almost an order when changing the mass ratio of Chit:GA from 45:1 to 1:1 in the reaction mixtures: from 70.51\u0026thinsp;\u0026plusmn;\u0026thinsp;9.45 to 7.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49%. It was determined that by varying Chit:GA weight ratio during the synthesis, conjugates with controlled GA amount in the range from 15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 to 79.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 \u0026micro;g GA/mg Chit could be obtained.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical characteristics of the synthesized Chit-GA conjugates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChit:GA weight ratio in reaction mixture\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCR\u003c/em\u003e, %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGE\u003c/em\u003e, %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026micro;\u003c/em\u003e, \u0026micro;g/mg Chit\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e70.51\u0026thinsp;\u0026plusmn;\u0026thinsp;9.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e55.78\u0026thinsp;\u0026plusmn;\u0026thinsp;6.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e18.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e44.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e29.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e34.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e25.45\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e50.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e16.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e79.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e77.5\u0026thinsp;\u0026plusmn;\u0026thinsp;15.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTaking into account the obtained data, Chit-GA conjugate synthesized upon Chit:GA weight ratio 5:1 was chosen for the further research.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. FTIR spectroscopy\u003c/h2\u003e \u003cp\u003eFTIR spectra of Chit, GA and Chit-GA conjugate recorded in the range from 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In the spectrum of GA characteristic bands at 3495 (O-H stretching), 3283 (C-H stretching), 1702, 1615 (C\u0026thinsp;=\u0026thinsp;O stretching), 1430 (C\u0026thinsp;=\u0026thinsp;C aromatic ring stretching), 1316 (C-O stretching), 1265 (COOH stretching), 1222 (C-O and C-C stretching) and 1026 (C-O stretching) cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were observed [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The spectra of Chit showed main bands at 3441 (O-H symmetric stretching, N-H stretching), 2921 (O-H asymmetric stretching), 1642 (C\u0026thinsp;=\u0026thinsp;O stretching, amide I), 1552 (N-H bending, amide II), 1424 (CH\u003csub\u003e2\u003c/sub\u003e bending), 1381 (C-N stretching, amide III), 1155 (pyranose ring C-O-C bridge asymmetric stretching), 1096 (C-O stretching) and 895 (C-H bending) cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Observed in the FTIR spectra of Chit-GA conjugate main changes were associated with the formation of a covalent bond between chitosan and gallic acid. First, a decrease in the intensity of O-H stretching vibrations and its shift towards lower wavenumbers from 3441 to 3272 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and from 2921 to 2879 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e compared to the neat Chit were determined. Next, C-O stretching vibrations at 1024 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was absent in the spectra of native chitosan, was observed in the conjugate spectrum. However, the main changes in the FTIR spectra were occurred in the range of wavenumbers 1600\u0026thinsp;\u0026minus;\u0026thinsp;1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Firstly, Chit amide I vibrations at 1642 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e disappeared, indicating the change of the primary amine to the secondary amine due to the reactions at chitosan NH\u003csub\u003e2\u003c/sub\u003e sites [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Secondly, the intensity increase and the shift towards lower wavenumbers from 1552 to 1548 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of amide II bending vibrations were registered. Finally, the C-O stretching vibrations shift from 1096 to 1065 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with the intensity increase in comparison with Chit was observed. Conjugation could occur due to both amide bonding and ester bonding [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the obtained spectra contained no peaks, which could be related to ester bond at the wavenumbers range 1800\u0026thinsp;\u0026minus;\u0026thinsp;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was registered at 1702 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the case of gallic acid. This fact, combined with the presence of an altered peak of amide II, confirms the formation of the conjugate due to an amide bond between the carboxyl group of the acid and the amino group of the polymer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3. NMR spectroscopy\u003c/h2\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR analysis was used to study Chit-GA structural changes in comparison to starting Chit. The \u003csup\u003e1\u003c/sup\u003eH-NMR spectra of chitosan, gallic acid and Chit-GA conjugate are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The characteristic peaks at 2.04 ppm (H\u003csub\u003eN\u0026minus;COCH3\u003c/sub\u003e, methyl protons of N-acetyl glucosamine), 3.01 ppm (H-2 of glucosamine residues) and the multiplet 3.56\u0026ndash;3.76 ppm (H-3\u0026ndash;H-6 protons of the pyranose ring) were observed. The peaks at 1.92 ppm and 4.75 ppm, corresponding to chemical shifts of protons in CH\u003csub\u003e3\u003c/sub\u003eCOOH and D\u003csub\u003e2\u003c/sub\u003eO respectively, were also registered [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. After conjugation, the main structure of the polymer was preserved, but some difference between Chit and Chit-GA spectra was found. A new signal at 6.99 ppm, which represented symmetric phenyl protons of GA, indicated successful grafting of gallic acid to the chitosan polymer chain [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4. TGA\u003c/h2\u003e \u003cp\u003eTG, DTG and DTA curves of chitosan, gallic acid and Chit-GA are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. On the TG curve of chitosan three consecutive weight loss steps were observed. The first stage was a weight loss about 36.3% below 290℃, attributed to physically adsorbed and hydrogen-bonded water evaporation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The second stage in the range of 290\u0026ndash;498℃ was corresponded to depolymerisation of chitosan and degradation of pyranose rings; the weight loss was about 46.4%. Moreover, the third degradation step from 498 to 800℃ was the thermo-oxidative process and the destruction of chitosan residues with the weight loss of 17.3% [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. According to the analysis of DTA curve, an endometric peak at 92℃ and an exometric peak at 319℃ were assigned to the loss of water and the degradation of the chitosan chains, respectively [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. On the TG curve of gallic acid, three consecutive weight loss steps were observed: from 20 to 268℃ (14.5% weight loss), from 268 to 377℃ (44.4% weight loss) and from 377 to 800℃ (35.8% weight loss), and the residual weight was 5.3%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThermal degradation steps of Chit, GA and Chit-GA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTemperature range, ℃\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWeight loss, %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eResidual weight, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1st\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2nd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e290\u0026ndash;498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3rd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e498\u0026ndash;800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1st\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2nd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e268\u0026ndash;377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3rd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e377\u0026ndash;800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChit-GA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1st\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2nd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e243\u0026ndash;443\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3rd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e443\u0026ndash;800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter conjugation, some changes in thermal behavior were established. The endometric peak at 90℃ on DTA curve was shifted toward lower values. It showed that introduction of GA caused the decrease in water holding capacity. At the same time, exometric peak at 279℃ was decreased in comparison with the neat Chit, and the decrease in the thermostability after acid grafting was determined [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The temperature of maximum weight loss was determined by DTG curves and attributed to the decomposition temperature of the sample. For Chit, GA and Chit-GA these values were 289, 291 and 246℃ respectively. The inclusion of acid in the polymer matrix lowered the decomposition temperature, which may be due to the obstruction of the chitosan chain packing after conjugation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A similar effect of thermal stability reducing was observed by the authors [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] after GA grafting with chitosan chains using an ascorbic acid/hydrogen peroxide redox pair in inert air.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Antioxidant activity\u003c/h2\u003e \u003cp\u003eThe ABTS radical scavenging activity detection method was shown, that the antioxidant potential of Chit-GA significantly (up to seven times) higher than that value of the neat chitosan (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). IC50 value, that displays the concentration of a substance, reducing the number of radicals by 50%, is a good general accepted indicator for quantifying the antioxidant capacity. Therefore, the estimated IC50 values of Chit-GA and neat GA were comparable and amounted 0.0073\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0001 and 0.0077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0002 mg/mL, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The equivalent IC50 of Chit-GA and GA for the ABTS radical quenching system suggested the synergistic function of chitosan in retarding the pro-oxidation of GA [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Taking this into consideration, the introduction of an H-atom donating group, i.e. gallic acid, onto chitosan is a good strategy to develop a chitosan derivative with robust antioxidant capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC50 values of Chit, GA and Chit-GA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC50, mg/mL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChit-GA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0073\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0001 (standardization by GA concentration)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.1061\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChit-GA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0913\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0012 (standardization by Chit concentration)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe most common mechanism of radical-scavenging activity of chitosan and its derivatives is attributed to that amino and hydroxyl groups, which react with unstable free radicals to form stable macromolecule radicals [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. At the same time, low antioxidant potential of Chit may be attributed to its strong ion chelating ability because of its nitrogen atom. Improving the chitosan antioxidant activity becomes possible by introducing the H\u003csup\u003e+\u003c/sup\u003e atom donor group [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. GA has been proved to possess greater antioxidant capacity due to its strong hydrogen-donating ability [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thus, in the case of GA embedding, the antioxidant properties were improved. It should be noted that according to the literature ABTS radical scavenging activity of chitosan-gallic acid conjugates is significantly lower. For example, Chit-GA conjugate with a degree of substitution of 10.3% was characterized by an IC50 value of 219 \u0026micro;g/mL [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], while we obtained the conjugate with a conjugation ratio of only 5% and 30 times greater antiradical activity with an IC50 of 7.3 \u0026micro;g/mL. The authors' data obtained for the chitosan oligomer (5\u0026ndash;10 kDa) conjugate are the closest to our value: the IC50 is 19.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 \u0026micro;g/mL with a GA conjugation ratio of about 15%.\u003c/p\u003e \u003cp\u003eReducing power assay had also been used to evaluate the ability of antioxidants to donate electrons. The reducing power of Chit, Chit-GA conjugate and Chit/GA mechanical mixture is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Compared with the neat chitosan, which reducing power was 9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;g-eq GA/mg of the sample, the conjugate value was increased by about 6 times and amounted to 54.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026micro;g-eq GA/mg of the sample. At the same time, reducing power values of the conjugate and the mechanical mixture of chitosan with gallic acid was turned out to be practically comparable (53.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 \u0026micro;g-eq GA/mg of the sample for mechanical mixture). The GA amount in the conjugate, calculated from the absorption spectra (p. 3.1) was amounted to 54.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 \u0026micro;g-eq GA/mg of the sample, which was consistent with the result obtained by the Folin-Ciocalteu method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eObtained data are consistent with published quantum chemical calculations. Frontier molecular orbitals (FMOs) play a fundamental role in the study of substrate chemical characteristics including ability to absorb light and autoxidative capacity. Highest occupied molecular orbital (HOMO) is an important electronic factor in the description of antioxidant capacity, especially since it can be related to electron transfer reactions [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The higher the HOMO energy, the greater the ability to carry out nucleophilic attacks, thus, it is easy to release electrons and the higher the autoxidation capacity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. According to the Sekkal-Rahal et al. data [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], the HOMO energy of Chit-GA in water is higher than that of the GA (-6.12 ev and \u0026minus;\u0026thinsp;6.20 ev, respectively). This predicts that the capacity of Chit-GA to have faster nucleophilic attacks compared to GA, therefore, the abstraction of an electron will be easy in this polyphenol-chitosan conjugate [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Antiglycation activity\u003c/h2\u003e \u003cp\u003eDiabetes mellitus is a chronic metabolic disease characterized by hyperglycemia, deficient production of insulin (type 1) or insufficient response of this hormone (type 2). Factors such as high blood glucose levels, increased production of reactive oxygen species (ROS) and formation of advanced glycation end products (AGEs) play a significant role in the development of diabetes mellitus [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Oxygen free radical activity can initiate peroxidation of lipids, which in turn stimulates glycation of protein, inactivation of enzymes and play a role in the long term complications of diabetes. Oxidative stress in diabetes coexists with a reduction in the antioxidant status, which can increase the deleterious effects of free radicals. Changes in glucose metabolism in diabetes mellitus are frequently accompanied by changes in the activities of the enzymes that control glycolysis and gluconeogenesis in liver and muscle. In this context, polyphenols received much attention because of their potent free radical scavenging and antioxidant actions [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe property of polyphenols and flavonoids to inhibit protein glycation and the consequent formation of AGEs is mostly defined by their antioxidant properties, metal-chelating capacity, protein interaction, methylglyoxal trapping and their ability to block the receptor for AGEs [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Gallic acid showed significant antiglycation results in the previous studies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The study [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] reported that GA may directly combine with free radicals and lead to their inactivation, that in turn may decrease the intracellular concentration of free radical such as superoxide, peroxyl and hydroxyl radicals. GA ability to prevent the oxidation of Maillard reaction intermediates and to capture dicarbonyls were also shown [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The structural features and the antiglycation ability of phenolic acids might be associated with the antioxidant function. Antiglycation activity depends on the number of OH groups and their positions [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to this, the ability of Chit-GA conjugate to inhibit AGEs generated by glucose in comparison with neat Chit and GA was evaluated in the model non-enzymatic reaction of BSA glycation by glucose. The curves of the dependence of glycation inhibition on the concentration of gallic acid (\u003cb\u003ea\u003c/b\u003e) and chitosan (\u003cb\u003eb\u003c/b\u003e) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. In the range of low concentrations, the conjugate showed better antiglycation activity than gallic acid. Compared with chitosan, inhibition was delayed earlier, which may be due to the limited solubility of the polymer under experimental conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Antibacterial activity\u003c/h2\u003e \u003cp\u003eThe physicochemical properties and biological activities of Chit-GA have been documented and many novel applications have been explored. However, studies of antimicrobial properties and mechanisms of Chit-GA were rare. Here, the minimum inhibitory concentrations (MICs) of GA, Chit and Chit-GA were determined by the serial dilution method. In general, the antibacterial activity of the conjugate is comparable or slightly less compared to the initial Chit (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). MIC analysis showed that, compared with the initial GA, Chit-GA conjugate significantly more actively inhibits the growth of gram-negative and gram-positive bacteria, with the exception of \u003cem\u003eP. aeruginosa\u003c/em\u003e strain. It should be noted that conjugate could inhibit the growth of thermophilic spore-forming gram-positive bacteria (\u003cem\u003eG. thermodenitrificans\u003c/em\u003e and \u003cem\u003eA. palidus\u003c/em\u003e): MIC values were 78.13 and \u0026lt;\u0026thinsp;4.88 \u0026micro;g/mL, respectively. The conjugate also exhibited high inhibitory properties against \u003cem\u003eEnterococcus faecalis.\u003c/em\u003e These bacteria are highly resistant to high temperatures, acids, salts and to erythromycin and kanamycin. \u003cem\u003eE. faecalis\u003c/em\u003e can live in an extreme alkaline environment due to its proton pump activity, which makes it resistant to calcium hydroxide medication. In addition, \u003cem\u003eE. faecalis\u003c/em\u003e can form biofilms in medicated root canals \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Depending on the molecular weight, Chit can have an inhibitory effect on standard and clinical isolates of the strain \u003cem\u003eE. faecalis\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, the inhibitory activity of chitosan-gallic acid conjugate against this type of bacteria was evaluated for the first time. The mechanism of antimicrobial action of the Chit-GA conjugate is associated with disruption of the bacterial cell membrane, resulting in leakage of cytoplasm and an increase in relative conductivity [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Besides, chitosan derivatives could enter bacterial cells through damaged cell membranes and inhibit DNA synthesis in the nucleus [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The results of Chit-GA antibacterial activity are consistent with the literature data. Lima et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] evaluated the antimicrobial activity of gallic, caffeic and pyrogallic acids against clinical strains \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. According to these authors, the three tested phenolic compounds did not have clinically significant antibacterial activity with values of MIC\u0026thinsp;\u0026ge;\u0026thinsp;1024 \u0026micro;g/mL. However, the potential of the inhibitory effect of the conjugate against thermophilic bacteria and the resistant strain of \u003cem\u003eE. faecalis\u003c/em\u003e is undeniable.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe MIC values of Chit, GA and Chit-GA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eMIC for different strains, mg/mL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u003cem\u003eg\u003c/em\u003e-)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u003cem\u003eg\u003c/em\u003e+)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u003cem\u003eg\u003c/em\u003e-)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eG. thermodenitrificans\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u003cem\u003eg\u003c/em\u003e+)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eA. palidus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u003cem\u003eg\u003c/em\u003e+)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u003cem\u003eg\u003c/em\u003e+)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.62500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.25000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.25000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.25000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.15625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.25000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChit-GA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.03906\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.62500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.25000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.07813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.00488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.00488\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.01953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.15625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.25000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.03906\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.00488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.00488\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Cytotoxicity\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of Chit-GA was tested against HaCaT cells by MTT assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the proliferation rates of Chit-GA were greater than 80% at low concentrations (up to 250.0 \u0026micro;g/mL), indicating that the cytotoxicity of Chit-GA was substantially absent at low concentrations. After that, the increase in Chit-GA concentration resulted in the significant decrease in cell viability. Notably, 70% of keratinocytes treated with 500.0 \u0026micro;g/mL of Chit-GA survived, while 45% of cells were killed after treated with 1000 \u0026micro;g/mL of Chit-GA. This result revealed that Chit-GA with a concentration below 750 \u0026micro;g/mL was toxic because the cell viability was less than 60%. The IC50 value was 1030.4 \u0026micro;g/mL of Chit-GA or 0.3 mmol of gallic acid. The received data are consistent with the literature. Thus, the authors [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] demonstrated a significant decrease in cytotoxicity of Chit against human fibroblasts due to modification with gallic acid: Chit-GA with a concentration below 1.0 \u0026micro;g/mL was non-toxic because the cell viability was above 80%. In that way, conjugation of chitosan with gallic acid ensures the production of water-soluble and safe material with antibacterial and pronounced antioxidant activities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Evaluation of the wound healing effect of Chit-GA in mice\u003c/h2\u003e \u003cp\u003eHere, for the first time, we evaluate the wound-healing effect of chitosan conjugate in an \u003cem\u003ein vivo\u003c/em\u003e comparative experiment. Taking into account all experimental groups, it was possible to reproduce an experimental model of skin wounds with an area from 2.50\u0026plusmn;0.06 to 2.70\u0026plusmn;0.09 cm\u003csup\u003e2\u003c/sup\u003e. Macroscopically after damage was inflicted for 10\u0026ndash;20 minutes, changes in the form of accumulation of intercellular fluid and slightly pronounced capillary fullness were noted in the edges of the induced wounds; the edges were also slightly swollen and raised above the surface. The intensity of the manifestations increased with every hour of observation and persisted throughout the day. It is worth noting that neither purulent infiltration nor pronounced hyperemia was found in all groups of experimental animals during the observation period, which would complicate the wound process and lengthen the healing time. This fact makes it possible to assert the reliability of the reproduced wounds model.\u003c/p\u003e \u003cp\u003eIn the control group of animals the course of regenerative processes in dynamics is reflected in a planimetric reduction in the wound area and an increase in the healing rate of induced wounds. Thus, on the 3rd, 6th, 9th and 12th days the wound area was 2.30\u0026plusmn;0.10, 1.80\u0026plusmn;0.06, 1.50\u0026plusmn;0.06 and 0.90\u0026plusmn;0.07 cm\u003csup\u003e2\u003c/sup\u003e, and the calculated healing rate was 21, 28, 39 and 65%, respectively. The results obtained in the control group reflect the general pattern of the reparative process phase change after injury from exudation and proliferation to reparative regeneration.\u003c/p\u003e \u003cp\u003eTreatment of wound pathology with 0.5%, 1.0% and 2.0% Chit solutions generally led to an acceleration of the reparative process in all animals. Thus, when using 0.5% Chit solution, the wound area was statistically significantly reduced to 1.90\u0026plusmn;0.07, 1.60\u0026plusmn;0.03, 1.20\u0026plusmn;0.08 and 0.70\u0026plusmn;0.05 cm\u003csup\u003e2\u003c/sup\u003e on days 3, 6, 9 and 12, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). There was also a slight difference in the rate of wound healing: in the dynamics on the 3rd, 6th, 9th and 12th days, the difference was 4, 8, 9 and 7% compared to untreated animals. At the same time, the phases of the reparative process, the state of the edges and the bottom of the wound, the timing of purification from necrotic tissues, the formation of wound crusts, and then the onset of wound epithelization in this experimental group proceeded similarly to the control group of animals. Animals treated with 1.0% Chit solution showed a decrease in wound area in dynamics (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb), which was generally reflected in the rate of wound healing: the values were 11, 14, 20 and 14% higher on the 3rd, 6th, 9th and 12th days compared with the control group. On the 3rd and 4th days of the reparative process, cleansing of wounds from necrotic tissues was observed. The process of crusting formation shifted in all treated animals from day 6 to day 5 compared to the control group, while the crusts fell was registered by the 9th day of wound process treatment. The epithelialization process occurred on the 8th and 9th days. Of the used concentrations of Chit solutions as a wound healing agent, 2.0% Chit solution proved to be the most effective: in dynamics on days 3, 6, 9 and 12 the wound area decreased to 1.70\u0026plusmn;0.08, 1.40\u0026plusmn;0.10, 0.95\u0026plusmn;0.08 and 0.58\u0026plusmn;0.05 cm\u003csup\u003e2\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec), and the wound healing rate was 14, 19, 24 and 17% higher compared to untreated animal wounds. The reparative process phases change after damage from exudation and proliferation to reparative regeneration in this group occurred faster: wound cleaning was detected on the 3rd and 4th days, and the crust formation was observed on the 5th day with a fall on the 7th and 8th days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt should be noted that the treatment process, characterized by the area and the rate of wound healing, was significantly improved with an increase in the concentration of used Chit solutions, but did not exceed the experimental groups of animals treated with Chit-GA conjugate solutions in similar concentrations. Chit-GA solutions were statistically significantly superior in the wound healing effect to the neat Chit and led to a faster regeneration process of the wound surface.\u003c/p\u003e \u003cp\u003eWhen treated with 0.5% solution of Chit-GA conjugate, the formation of wound crusts was observed in 100% of animals on the 5th day, with a fall on the 7th and 8th days. The phase changes of the reparative process after damage from exudation and proliferation to reparative regeneration in this group proceeded in the same way as in the experimental group, where treatment was carried out with 1.0% Chit solution. This was also reflected in the close values of the wound healing rate of these experimental groups: when treated with 0.5% conjugate solution, calculated from the area (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea) rate values on days 3, 6, 9 and 12 were 25, 36, 48 and 72%, respectively. When treated with 1.0% Chit-GA solution, the wounds reparative process was expected to be more intense: in dynamics on the 3rd, 6th, 9th and 12th days the area decreased to 1.60\u0026plusmn;0.07, 1.20\u0026plusmn;0.07, 0.70\u0026plusmn;0.06 and 0.45\u0026plusmn;0.02 cm\u003csup\u003e2\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb), while the difference in the wound healing rate compared with untreated animals was 14, 24, 29 and 18%, respectively. Already on the 2nd day of the reparative process course, regeneration without signs of exudative-proliferative reactions was observed during treatment with 1.0% conjugate solution. Further observation on the 3rd day showed that the wounds were completely cleared of necrotic tissues and the healing of damage continues with the formation of crusts on the 5th day with a fall on the 7th day. The wound healing process during treatment with a 2.0% Chit-GA solution proceeded even more intensively compared to previous concentrations: on days 3, 6, 9 and 12, a statistically significant area decrease to 1.50\u0026plusmn;0.06, 1.00\u0026plusmn;0.08, 0.50\u0026plusmn;0.08 and 0.20\u0026plusmn;0.04 cm\u003csup\u003e2\u003c/sup\u003e was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec). The calculated wound healing rate increased by 2 times in comparison with the control group up to 41, 60 and 81% on days 3, 6 and 9, respectively. The dynamic observation of wound healing processes in this experimental group led to the conclusion that the cleansing of wounds from necrotic tissues, the formation of crusts, and epitalization proceeded in the same way as in the experimental group using a 1.0% solution of Chit-GA conjugate.\u003c/p\u003e \u003cp\u003eThus, all the samples turned out to be potent, but the most effective were solutions of Chit-GA conjugate, which statistically significantly exceeded the original chitosan in wound healing effect and led to a faster regeneration process of the wound surface. The maximum effect was observed when using a 2.0% solution of chitosan-gallic acid conjugate (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). There is a clear dependence that the addition of gallic acid to chitosan macromolecules accelerates the reparative process of wound healing and has a positive effect on the cleansing of wounds from necrotic tissues, the formation of crusts and epithelialization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the inflammatory stage of the wound healing process, neutrophils and macrophages secrete a large amount of reactive oxygen species (ROS) along with cytokines and matrix metalloproteases [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Formed ROS play a dual role. From one side, they inhibit the growth of microbial pathogens and promote phagocytosis [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In addition, ROS they can stimulate angiogenesis, division and migration of endothelial cells by expressing vascular endothelial growth factor (VEGF) and promote the formation of blood vessels [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. From other side, a high level of ROS leads to oxidative stress, which damages and worsens the condition of neighboring tissues due to hydrolysis of extracellular matrix proteins and function impairment of dermal fibroblasts and keratinocytes [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Thus, normalization of ROS levels is critically important for a successful wound healing process [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. That is why antioxidants are considered as one of the new promising components for wound healing [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the case of Chit modified with GA, improved wound healing properties compared to neat Chit are probably due to the synergism of the components. Considering the classic four main phases mechanisms of wound healing [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], we can assume the reasons for synergistic enhancement of wound healing process of Chit-GA. The wound healing properties of chitosan and the mechanism of its action were described in sufficient detail in the Feng et al. review article [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Chitosan is applicable in the first three stages of wound healing. Firstly, due to the hemostatic action of amino groups, Chit promotes platelet and erythrocyte aggregation and inhibits the dissolution of fibrin at the stage of hemostasis. Secondly, chitosan inhibits the growth of bacteria at the stage of inflammation. Finally, chitosan depolymerizes releasing \u003cem\u003eN\u003c/em\u003e-acetylglucosamine, which promotes fibroblast proliferation and collagen synthesis (proliferation stage).\u003c/p\u003e \u003cp\u003eAt the same time, GA promotes wound healing by directly increasing the expression of antioxidant genes, accelerating the migration of keratinocyte and fibroblast cells, activation of focal adhesion kinases (FAK), c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinases (Erk) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Besides, taking into account the pronounced antioxidant activity, Chit-GA conjugate can neutralize excessive ROS levels during the inflammatory stage of the wound healing process. However, this requires additional detailed research. The results we have obtained revealed the great role of gallic acid as a supportive agent to hasten the wound healing process, which support the date obtained by other researches regarding the wound healing effect of GA and the role of antioxidants in the healing process [\u003cspan additionalcitationids=\"CR63 CR64\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eConjugates of chitosan with gallic acid were synthesized by carbodiimide method with preliminary activation of GA carboxyl groups by EDC. The formation of conjugates was confirmed by physical measurements and spectroscopic methods. It was shown that by varying the ratio of components during synthesis it is possible to obtain conjugates with controlled conjugation ratio from 1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 to 8.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72%, grafting efficiency from 7.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49 to 70.51\u0026thinsp;\u0026plusmn;\u0026thinsp;9.45% and gallic acid content up to 79.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 \u0026micro;g GA/mg Chit. FTIR and NMR methods proved that conjugation of molecules occurs due to the formation of an amide bond. TGA analysis showed that Chit with grafted GA had reduced thermal stability compared to neat chitosan. The result showed that Chit-GA conjugate had obvious antibacterial activity against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e. For the first time, the inhibitory activity against thermophilic spore-forming bacteria was evaluated using the example of strains \u003cem\u003eG. thermodenitrificans\u003c/em\u003e and \u003cem\u003eA. pallidus.\u003c/em\u003e The MIC values of Chit-GA against \u003cem\u003eG. thermodenitrificans\u003c/em\u003e and \u003cem\u003eA. pallidus\u003c/em\u003e were 78.13 and less than 4.88 \u0026micro;g/mL, indicating that gram-positive thermophilic bacterium was more sensitive to Chit-GA than gram-positive \u003cem\u003eB. subtilis\u003c/em\u003e (MIC value was 625 \u0026micro;g/mL). In addition, the high inhibitory activity of Chit-GA had been recorded against the resistant to the aminoglycoside antibiotic kanamycin strain \u003cem\u003eE. faecalis.\u003c/em\u003e Besides, we demonstrated the outstanding (IC50 of 7.3 \u0026micro;g/mL) antioxidant activity of the conjugate with a degree of substitution of only 5% and correlate with literary quantum calculations. The great ability of Chit-GA conjugate to inhibit AGEs generated by glucose in comparison with neat Chit and GA was evaluated on the model non-enzymatic glycation reaction of BSA by glucose. The Chit-GA showed no significant cytotoxicity at low concentrations against human epidermal keratinocyte. Finally, we prove the enhanced wound healing properties of the modified chitosan compared to the original polymer in an \u003cem\u003ein vivo\u003c/em\u003e comparative experiment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the Belarusian Republican Foundation for Fundamental Research (grant no. X23MN-007) and Mongolian Foundation for Science and Technology (grant no. BLR-2023/03).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of Interest:\u003c/strong\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eData curation: K.V. Hiatsevich; Supervision: K.S. Hileuskaya; Conceptualization: K.S. Hileuskaya (lead), A.I. Ladutska (supporting), L. You (supporting); Visualization: O.R. Akhmedov; Formal analysis: K.V. Hiatsevich (lead), V.V. Nikalaichuk (lead), N.N. Abrekova (equal), P. Shao (equal); Investigation: A.I. Ladutska (equal), N.N. Abrekova (equal), P. Shao (equal); Methodology: K.S. Hileuskaya (equal), A.I. Ladutska (equal), L. You (equal); Writing \u0026ndash; original draft preparation: K.V. Hiatsevich; Writing \u0026ndash; review and editing: K.S. Hileuskaya (equal), V.V. Nikalaichuk (equal), M.M. Odonchimeg (equal); Resources: O.R. Akhmedov (equal), L.You (equal); Project administration: M.M. Odonchimeg.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was financially supported by the Belarusian Republican Foundation for Fundamental Research (grant no. X23MN-007) and Mongolian Foundation for Science and Technology (grant no. BLR-2023/03).\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYazdi MK, Seidi F, Hejna A, Zarrintaj P, Rabiee N, Kucinska-Lipka J, Saeb RM, Bemcherif SA (2024) Tailor-Made Polysaccharides for Biomedical Applications. 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Mater Sci Eng C 90:227\u0026ndash;235. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.msec.2018.04.053\u003c/span\u003e\u003cspan address=\"10.1016/j.msec.2018.04.053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"chitosan, chitosan-gallic acid conjugate, antioxidant activity, antibacterial activity, cytotoxicity, wound healing effect","lastPublishedDoi":"10.21203/rs.3.rs-4982795/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4982795/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChitosan-gallic acid conjugates were synthesized by carbodiimide method and characterized by physicochemical methods (UV-vis, FTIR, \u003csup\u003e1\u003c/sup\u003eH NMR, TGA). The FTIR and NMR assays confirmed that the chemical interaction occurred solely due to the formation of an amide bond. It was established that by varying the ratio of the components during synthesis it is possible to obtain conjugates with desired conjugation ratio, grafting efficiency and gallic acid content up to 8%, 71% and 80 µg gallic acid/mg chitosan, respectively. Chitosan-gallic acid conjugate with a 5% conjugation ratio demonstrated excellent antioxidant properties: the IC50 value for ABTS radical scavenging activity was 0.0073±0.0001 mg/mL. \u003cem\u003eIn vitro\u003c/em\u003e tests showed that conjugation of chitosan with phenolic acid provided the antiglycemic activity of the material and its good biocompatibility. A low level of cytotoxicity was recorded in the HaCaT cell line model (IC50 was 1030.4 μg/mL). The received eco-friendly chitosan-gallic acid conjugate effectively inhibited the growth of thermophilic spore-forming bacteria \u003cem\u003eG. thermodenitrificans\u003c/em\u003e and the resistant to classical antibiotics strain \u003cem\u003eA. palidus\u003c/em\u003e. The results of an \u003cem\u003ein vivo\u003c/em\u003e comparative analysis showed that chitosan-gallic acid conjugate had excellent wound healing properties due to the synergism of the polysaccharide and the natural antioxidant.\u003c/p\u003e","manuscriptTitle":"Chitosan-Gallic Acid Conjugate with Enhanced Functional Properties and Synergistic Wound Healing Effect","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-27 19:15:11","doi":"10.21203/rs.3.rs-4982795/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d75b868c-98a9-4995-9871-7f91cef2f407","owner":[],"postedDate":"September 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-22T08:08:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-27 19:15:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4982795","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4982795","identity":"rs-4982795","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}