Ethanol, PVA (MW_average 72,000 g/mol, 98% hydrolyzed), CS (MW 270,000 g/mol, 85% degree of deacetylation), PEO, tween 80 (polysorbate 80), potassium bromide (KBr), ammonium molybdate, N-(1-naphthyl) ethylenediamine dihydrochloride (NED), hydrogen peroxide (H₂O₂), sulfanilamide, phosphoric acid (H₃PO₄), xylazine, and acetic acid were purchased from Merck Company (Germany). Ketamine and phenytoin cream were procured from Alfasan (Woerden, Netherlands) and Behvazan Company (Iran), respectively. All used compounds and chemicals were of analytical grade.

The T. graminifolius DC. [Asteraceae] was collected in April 2020 from around the Kermanshah province, Iran. The plant name has been checked with the World Flora Online (WFO) site (www.worldfloraonline.org) and approved by the Central Herbarium of Tehran University, School of Biology, College of Science, University of Tehran, Tehran, Iran. The plant voucher number (43603) was stored at the University of Tehran in the Central Department of Botany. The aerial parts of TG were washed and dried in the shadow under suitable conditions. The dried TG was powdered by a mechanical grinder. Then, 100 mg of the dried plant was poured into an Erlenmeyer flask and extracted with 70% ethanol. The resulting mixture was placed in a shaker for about 60 h. In the next step, the mixture is filtered and the resulting solution is concentrated under vacuum by a rotary apparatus (Heidolph Instruments GmbH and Co., Germany) to obtain the desired extract. Three extracts with concentrations of 10, 50, and 90% w/v TG were prepared and tested to obtain the most effective extract (Farzaei et al., 2014b).

To prepare the polymer solution, 0.3 g of CS was dissolved in 10 mL of acetic acid. In the second beaker, 0.2 g of PEO was dissolved in 10 mL of acetic acid. Also, in the third beaker to prepare a PVA solution, 0.6 g of the polymer was added to 4.4 mL of distilled water. In order to prepare the working solution, the ratios of each polymer were chosen in such a way that the final product consisted of 1:1:0.3 g for CS, PVA, and PEO, respectively. The TG extract was then added as 10, 50, and 90% w/v into the final working solution.

After preparing polymer solutions containing TG extracts with concentrations of 10, 50, and 90% (w/v), the desired experiments were performed to achieve the proper nanofibers with an electrospinning device. To attain the best nanofibers, different parameters of the electrospinning such as nozzle-to-collector distance, voltage, and material concentration were thoroughly examined. Notably, distances of 8, 12, and 17 cm and voltages of 8, 12, and 17 kV were investigated and a light microscope was used to find the best fiber. The best nanofibers were obtained at a distance of 12 cm from the nozzle at an injection rate of 0.7 mL per hour, and the working voltage of 17 kV with 50% TG extract. The study was done on room temperature and humidity.

The main characteristics of nanofibers including morphology, diameter distribution, and shape were evaluated utilizing a Scanning Electron Microscopy (SEM) (FEI Model Quanta 450 FEG, Hillsboro, OR, United States), at an operating voltage of 25 kV. All the samples were fixed to an aluminum stub and sputter coated with gold under an argon atmosphere.

To determine the amount of TG extract released from nanofiber, 5 mg of nanofiber was accurately weighed and placed inside the dialysis membrane (Cut-off ∼ 12 kDa), containing 2 mL of phosphate buffer plus 1% v/v Tween 80. The dialysis membrane was then immersed in 50 mL of phosphate buffer (release medium) at pH 7.4 and was stirred at 100 rpm. This process was repeated 3 times and sampling was done at different times (15, 30, 45, 60, 75, 90, 105,120, 150, 180, 210, 240, 300, 360, 420, 1680, 3120, 3360, and 3600 min). The absorbance of the samples was measured by UV-visible apparatus at a wavelength of 263 nm.

To evaluate the swelling of nanofiber, 10 mg of dried samples were immersed in aqueous media containing phosphate buffered saline (PBS). Samples were incubated for 30 min at 37°C. After that, the samples were expelled and then their weights were again accurately measured. The following formula was used to calculate weight changes: Q s   % = W t   –   W 0   /   W 0 ×   100

Q_s = Inflation ratio.

W_t = Weight of swollen nanofibers.

W₀ = Weight of primary dry nanofibers.

To assess the mechanical properties of the PVA/PEO/CS and PVA/PEO/CS/TG, a mechanical tensile testing device was employed (STM-1 DBBP-100, South Korea). The nanofibers were cut to 40 mm × 10 mm and related mechanical properties were tested at room temperature. The tensioning speed was 2 mm/min.

In this research, twenty-four male Wistar rats weighing 220 ± 20 g were selected from the Animal House Unit of the School of Pharmacy, Kermanshah University of Medical Sciences (Kermanshah, Iran), and divided into 4 groups of 6 each. Rats were placed in plastic cages under standard laboratory conditions (25°C, 60% humidity, 12-h/12-h dark cycle) and fed with water and chow. All animal procedures were approved by the animal ethics committee of Kermanshah University of Medical Sciences, Iran (IR.KUMS.REC.1398.673) and were performed in adherence to pertinent guidelines and regulations.

The rats underwent anesthesia through intraperitoneal (i.p.) injections of a combination of ketamine and xylazine (80 mg/kg and 10 mg/kg). Subsequently, the back surface of the rats was shaved and sterilized. The excision wound (20 mm × 20 mm) was made on the back through the removal of a section of skin.

After 1 week of acclimatization, 24 adult male Wistar albino rats were randomly divided into 4 groups (n = 6). The four studied groups included a negative control (untreated), phenytoin cream (as positive control), polymer (PVA/PEO/CS), and drug (nanofiber-containing 50% of TG extract; named PVA/PEO/CS/TG) groups. All treatments were administered topically once daily for 14 days. The initiation of wound creation was regarded as zero, and the commencement of the treatment occurred 24 h after the creation of the wound. The rats were then sacrificed on the 15th day. Prior to the sacrifice on the 15th day, blood samples were taken from rats in order to evaluate the nitrite and CAT serum levels, which serve as biomarkers for the measurement of oxidative stress.

Wound contraction was evaluated through the utilization of a digital camera to capture photographs. Subsequently, the obtained images were subjected to analysis via ImageJ software to measure the size of the wound area. The wound closure rate was then represented as the percentage of reduction in the zero-day wound size. This rate was computed using the following formula:

Wound contraction (%) = [wound size of the induction day − wound size of the specific day (days 0, 3, 7, 10, and 14) after treatment)]/wound size of the induction day × 100.

For histopathological evaluations, on day 15, the rats were anesthetized through i.p. administration of thiopental sodium, and then skin tissue samples from the wound areas were collected and subjected to fixation using a 10% formalin. After preparing tissues, sections measuring 7 μm in thickness were stained with hematoxylin and eosin (H&E). The Olympus CX23 light microscope, Dino-Lite camera, and DinoCapture 2.0 software were used for visualization of the samples. Histopathological investigations were done by a blinded experimenter.

Nitric oxide was measured indirectly because it is unstable. For this reason, we measured its stable metabolites, namely the nitrate and nitrite anions. Nitrite measurement was performed by Griess reaction Griess 1858 (Sun et al., 2003). In this method, deproteinization is necessary and the use of zinc sulfate is the best method compared to other methods. In the Griess reaction, nitrite reacts with sulfanilamide to give an unstable diazonium salt. Then, this salt combines with NED to form purple-colored aromatic compounds that can be used to measure nitrite concentration. This reaction creates a spectrum of purple paint that can be used to measure nitrite concentration. 100 μL of serum was mixed with 50 μL sulfanilamide (dissolved in 5% H₃PO₄). Following 5 min keeping of the prepared sample at room temperature, 50 μL of NED solution (0.1% in water) was added to each sample. A purple complex was then formed, the absorption of which was read at 540 nm with a 630 nm reference filter by an ELISA reader. Finally, the standard curve was drawn and results were reported as a percentage (%) of control (Kumar et al., 2009; Fakhri et al., 2022).

Catalase activity was measured to assess the antioxidant level according to the method of Aebi (1984). Briefly, 20 µL of serum samples were combined via 100 µL of H₂O₂ (65 mM) into 96-well plate wells. Thereupon, incubated at room temperature for 4 min. The reaction was stopped through the addition of 100 µL ammonium molybdate (32.4 mM) and a yellow molybdate and H₂O₂ complex were formed. The absorbance of the sample was subsequently determined at a wavelength of 405 nm using an ELISA reader (Fakhri et al., 2022). Results were reported as a percentage of control (%).

All data were expressed as mean values ± standard deviation (S.D.). Repeated measures of one-way and two-way analysis of variance (ANOVA) and Tukey’s post-hoc analysis were done. The statistical significance was determined by accepting the p-values of ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 with different symbols.

After preparing the optimized nanofibers, their morphology and diameter were analyzed through SEM imaging. SEM images related to optimal nanofibers are shown in Figures 1A, B with different magnifications. SEM images of electrospun nanofibers with 50% TG are illustrated in Figures 2A, B. Comparing the initial fiber (Figure 1C) and after adding the TG extract to the polymeric solution the mean fiber diameter was increased but the overall structure of the nanofiber did not change (Figure 2C). These results confirm that the utilized nanofiber in this study is the suitable system of choice for successfully loading the TG extract.

The release profile of TG extract from the optimal nanofibers (PVA/PEO/CS) at pH 7.4 at different times is shown in Figure 3. The results show that the total extract release from the formulation after 60 h is close to 30%.

The swelling test was conducted three times, and the average swelling percentage of nanofibers was calculated to be 86.88%, which indicated the high ability of water absorption by the nanofibers (Table 1). This property can be very effective in absorbing secretions around the wound and keeping its surface dry. Water angle test studies on the surface of the nanofibers showed that this surface is one of the most hydrophilic surfaces, which could increase cell adhesion and facilitate the wound-healing process.

The stress-strain curve of electrospun nanofibers without extract and with TG extract is presented in Figure 4. According to the results, the modulus of elasticity at all levels of nanofibers with extract is remarkably enhanced in comparison to nanofibers without extract. Both the modulus of elasticity and the tensile strength are higher in nanofibers with extracts than in nanofibers without extracts. Consequently, it is evident from the results that the mechanical strength of the PVA/PEO/CS nanofiber containing the TG extract has increased and as a result, the stress-strain curve has been transferred to higher stresses.

In order to assess the efficacy of nanofibers in promoting wound healing, the size of the wounds was measured in different treated groups on days 0, 3, 7, 10, and 14. According to the results, rats whose wounds were treated with PVA/PEO/CS/TG nanofibers showed a significant reduction in their wound size compared to the normal saline, phenytoin, and polymer groups after day 14. Thus, the wound healing activity of PVA/PEO/CS/TG nanofibers was notably higher than other treated groups at all the time intervals assessed. Figure 5 represents the macroscopic trends of wound healing, and Figure 6 shows associated statistical results on wound contraction.

Histological assessment of tissue samples from the wound area on the final day was conducted using H&E staining. Figure 7 illustrates the magnifications of tissue sections obtained from the studied groups. Additionally, normal skin tissue sections (Figures 7A1, A2) were provided for comparison, and not as main findings of the present study. The structure of healthy skin (Figures 7A1, A2) is a thick tissue, in which different parts of it, such as the epidermis and dermis, encompassing keratinized stratified squamous epithelium, fat glands, hair follicles, types of connective cells, multiple cellular components, and clusters of connective fibers are seen. In the normal saline (negative control) group, the tissue structure was disrupted, the epidermis was not formed on a large surface, and the dermis displayed an irregular and detached structure with a lack of appendages, along with a notable infiltration of inflammatory cells. In addition, in the extracellular matrix, collagen fibers are low-density and irregular and no skin appendages were observed (Figures 7B1, B2). In the PVA/PEO/CS-treated group, skin tissue structure improved and in some cases, necrotic tissue and inflammatory cell accumulations were observed (Figures 7C1, C2). However, in the groups treated with PVA/PEO/CS/TG nanofibers (Figures 7D1, D2) and phenytoin (positive control) (Figures 7E1, E2), the extent of skin tissue damage was reduced to some degree in comparison to the normal saline group. The wrinkled epidermis displayed significant regeneration. Moreover, the dermis represented a high density of collagen fibers from connective tissue, accompanied by the presence of normal cells. Additionally, the percentage of collagen production motility in the PVA/PEO/CS/TG nanofibers group was higher than both phenytoin and PVA/PEO/CS nanofibers groups.

In the microscopic examination of tissue sections, the thickness of the epithelium (Figure 8) and the blood vessel number in the treated groups (Figure 9) were evaluated. Our results were indicative of a significant increase in the regeneration of epithelium thickness (epithelialization) (p < 0.001) and the blood vessel number (p < 0.001) in the group treated with PVA/PEO/CS/TG in comparison to the normal saline group. Moreover, PVA/PEO/CS/TG administration showed a remarkable elevation in comparison to the phenytoin (p < 0.001), and the PVA/PEO/CS nanofibers group (polymer) (p < 0.001) groups. The PVA/PEO/CS/TG group was also associated with an increase in the number of blood vessels compared to the other two groups (p < 0.05). These results showed that PVA/PEO/CS/TG treatment was able to accelerate the wound healing process through stimulation of angiogenesis and epithelialization.

According to the analysis illustrated in Figure 10, the serum levels of nitrite were significantly attenuated in all groups treated with 1% phenytoin cream, PVA/PEO/CS, and PVA/PEO/CS/TG compared to the normal saline group (p < 0.05).

The level of catalase in the serum is regarded as a factor with antioxidant properties, and an elevated level of catalase is deemed favorable. It was found that (Figure 11) all treatments notably elevated CAT activity compared to the normal saline group (p < 0.05).