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 (CH2 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 NH2 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
1H-NMR analysis was used to study Chit-GA structural changes in comparison to starting Chit. The 1H-NMR spectra of chitosan, gallic acid and Chit-GA conjugate are shown in Fig. 4. The characteristic peaks at 2.04 ppm (HN−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 CH3COOH and D2O 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 cm2. 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 cm2, 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 cm2 on days 3, 6, 9 and 12, respectively (Fig. 10a). 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. 10b), 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 cm2 (Fig. 10c), 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. 10a) 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 cm2 (Fig. 10b), 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 cm2 was observed (Fig. 10c). 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].