Characterization of Carboxymethylcellulose and Alginate-Based Dressings Incorporated with Plant Extract

Abstract

In this study, hydrogel dressings based on alginate and carboxymethylcellulose were developed, supplemented with extracts from Tagetes nelsonii, Agave americana, and Aloe vera gel, for the treatment and healing of wounds. For this purpose, the physical and mechanical characterization of the films was carried out using different concentrations of the crosslinker, calcium chloride. Additionally, T. nelsonii was the extract that exhibited the highest antioxidant capacity as well as in vivo wound-healing activity. Subsequently, plant extracts were added, the dressings were characterized, and antibacterial activity was determined by the Kirby–Bauer method against Staphylococcus aureus and Pseudomonas aeruginosa. The results indicated that the prepared dressings have potential for use in wound treatment and healing, with the dressing containing T. nelsonii extract being the only one with antibacterial activity. Therefore, all of them can be used for acute wounds on body parts such as the palms of the hands, knees, elbows, and soles of the feet.

1. Introduction

Skin wounds can result from physical and/or mechanical damage, such as cuts, bites, or burns, and may also occur due to chronic conditions associated with systemic diseases [1]. These wounds can be susceptible to bacterial infections if not properly cleaned, which can delay the healing process and exacerbate symptoms such as pain, inflammation, excessive exudate, or sepsis [2,3]. In Mexico, the treatment of traumatic wounds represents a significant public health challenge, with annual costs at approximately 2 million pesos, and wound management is estimated to reach USD 18.7 billion worldwide by 2027 [4,5]. These wounds can severely affect an individual’s quality of life and hinder their ability to perform basic daily activities [4].

Therefore, there is an urgent need for effective alternatives that protect wounds from infections, reduce inflammation, and contribute to healing. Wound dressing is a material designed to facilitate rapid wound closure, prevent infections, and aid in healing with minimal discomfort for the patient [6]. An ideal dressing should possess appropriate mechanical properties, high liquid absorption capacity, biocompatibility, biodegradability, durability, a strong barrier against infectious microorganisms, ease of removal, and cost-effectiveness [7,8]. Hydrogels, for instance, can absorb wound exudates due to their high swelling capacity, semi-permeability, ability to maintain a moist environment, facilitate gas exchange, and provide thermal insulation [2]. Moreover, hydrogels can be combined with therapeutic agents to enhance their biotechnological potential, making them highly promising candidates for wound treatment [9].

To date, various wound dressing materials have been reported that feature high liquid absorption capacity, tensile strength, and good moisture vapor transmission. Examples include alginate (Alg) [8], carboxymethylcellulose (CMC) [10], chitosan (QS) [11], and polyvinyl alcohol (PVA) [11], among others. Alginate is a polysaccharide derived from red, green, and brown macroalgae, composed of blocks of D-mannuronic acid (M) and L-guluronic acid (G) in varying proportions. It is compatible with other natural and synthetic polymers, biodegradable, biocompatible, and has good liquid absorption capacity [12]. In contrast, CMC is a polysaccharide obtained through the chemical modification of cellulose, endowing it with hydrophilic, biocompatible, biodegradable, and non-toxic properties, making it an ideal material for wound and burn dressings [13].

Although polysaccharides such as Alg and CMC offer biocompatibility, biodegradability, and water retention, they may not fully meet all the criteria of an ideal wound dressing. Their combination has been widely studied as a base matrix due to their synergistic gelling behavior. However, many hydrogel formulations reported in the literature lack the incorporation of bioactive plant extracts or have not been evaluated in both in vitro and in vivo wound models [14,15,16]. Recent research has emphasized the potential of enriching hydrogel-based dressings with plant-derived compounds to enhance wound healing through antioxidants, antimicrobial, and anti-inflammatory actions. The present study addresses this gap by incorporating extracts from three medicinal plants traditionally used in Mexico: Tagetes nelsonii, Agave americana, and Aloe vera. Tagetes species are rich in flavonoids, steroids, and terphenes, compounds known for their antimicrobial and antioxidant properties [17]. These bioactivities are of particular interest in wound healing, especially under conditions of oxidative stress. Although Tagetes nelsonii has not yet been specifically studied in this context, its traditional medicinal use suggests potential therapeutic value [17,18,19]. Agave americana contains steroidal saponins and phenolic acids that exhibit anti-inflammatory and antimicrobial effects. Aloe vera gel is well documented for its polysaccharides (e.g., acemannan), vitamins, and glycoproteins that promote fibroblast proliferation, collagen synthesis, and hydration of the wound bed [19,20].

This study proposes a novel formulation combining alginate and CMC with these plant extracts, aiming to produce a hydrogel dressing capable of accelerating wound healing. To the best of our knowledge, this is the first report to integrate T. nelsonii extract into a hydrogel matrix and evaluate its effects through both in vitro and in vivo assays.

2. Materials and Methods

2.1. Materials

Sodium alginate (CAS No. 9005-38-3, MW~20–40 kDa), sodium carboxymethylcellulose (CAS No. 9004-32-4), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-Tris(2-pyridil)-s-triazine (TPTZ), sodium acetate, ferric chloride, glacial acetic acid and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich, Merck KGaA, Darmstadt, Germany. Calcium chloride and methanol were purchased from Fermont, Monterrey, Mexico. Glycerol and phosphate-buffered saline (PBS, pH 7.4; 100 mM K₂HPO₄-KH₂PO₄, 0.14 M NaCl) were obtained from MEYER, Mexico City, Mexico. Mueller-Hinton agar was provided by DIBICO, Mexico City, Mexico. Distilled water with a pH of 7.7 was obtained using a Millipore system. All reagents were of analytical-grade quality.

2.2. Preparation of Plant Extracts

The plant specimens were collected from different locations in Chiapas: Tagetes nelsonii Greenm in San Cristobal de las Casas (16°43′22.7″ N 92°37′05.6″ W), Aloe vera (L.) Burm. f. in Ocozocuautla de Espinoza (16°37′37.6″ N 93°20′19.7″ O), and Agave americana (L.) in Comitan de Dominguez (16°23′58.2″ N 92°12′46.6″ W). The botanical identification voucher numbers (56564, 55137, and 55136) were supplied by the CHIP herbarium, which belongs to the Dr Faustino Miranda Botanical Garden. The leaves of Tagetes nelsonii and Agave americana were dried in an oven at 40 °C and macerated in methanol (1:10) for 48 h. Afterward, they were subjected to sonication (VEVOR, Las Vegas, NV, USA) at 20 °C for 2 h. The extracts were subjected to filtration, centrifuged at 3500 rpm for 15 min using a HERMLE Labortechnik centrifuge (HERMLE Labortechnik, Wehingen, Germany), and concentrated under reduced pressure with a rotary evaporator (BUCHI-R210, Labortechnik AG, Flawil, Switzerland) at 40 °C. For the extraction of Aloe vera gel, leaves were cut crosswise to obtain the inner parenchymatous gel. The gel was homogenized with an electric blender (TAURUS, Oliana, Spain) and then centrifuged at 3500 rpm for 15 min. Finally, the extracts were stored in 50 mL conical tubes wrapped in aluminum foil until further use [20].

2.3. DPPH• Free Radical Scavenging

The antioxidant activity of the methanolic extract of T. nelsonii was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical discoloration method, according to the methodology described by Castañeda-Valbuena et al. [21]. A 0.08 M DPPH solution in methanol was prepared, and its absorbance was adjusted to between 0.700 and 0.900 at a wavelength of 517 nm, as a reference value for the antioxidant assay. The extract was mixed in a 1:29 µL ratio with the DPPH solution and then stored in the dark for 30 min at 24 °C. Subsequently, a calibration curve for Trolox (0 to 300 ppm) was prepared as a positive control, and methanol was used as a blank. Absorbance was measured using a Jenway 6715 UV/Vis scanning spectrophotometer (Cole Parmer, Vernon Hills, IL, USA) at 515 nm. The antioxidant activity was expressed as mg equivalents of Trolox per mL of extract.

2.4. ABTS^•+ Radical Scavenging

The ABTS scavenging activity was evaluated using the ABTS solution, which was previously activated with 40 µL of a 70 mM potassium persulfate solution for 16 h in the dark. Before use, the solution was adjusted to an absorbance between 0.700 and 0.800 at 734 nm. A mixture of 990 µL of the active ABTS solution and 10 µL of the extract was allowed to react for 6 min at room temperature [21]. A Trolox calibration curve (0 to 600 ppm) was prepared as a positive control, and PBS was used as a blank. The antioxidant activity was expressed as mg equivalents of Trolox per mL of extract.

2.5. Ferric Ion Reducing Antioxidant Power (FRAP)

The FRAP assay was performed using FRAP reagent (10:1:1) prepared with 300 mM acetate buffer (pH 3.6), 20 mM ferric chloride hexahydrate, and 10 mM 4,6-tris(2-pyridyl)-s-triazine (TPTZ). Subsequently, 150 µL of the extract was mixed with 2850 µL of FRAP reagent at 37 °C and allowed to react for 8 min in the dark. Absorbance was then measured at 593 nm [22]. A Trolox calibration curve (0 to 100 ppm) was prepared as a positive control, and the FRAP solution was used as a blank. The antioxidant activity was expressed as mg equivalents of Trolox per mL of extract.

2.6. Preparation of Alg and CMC Dressings

The formulation of the dressings was carried out by preparing a base of 2% w/v alginate, 1.5% w/v carboxymethylcellulose, and 10% w/v glycerol in distilled water, stirred at 900 rpm at 80 °C for 2 h. Since the crosslinking agent determines the degree of polymer crosslinking, which is a key parameter for water absorption and vapor transmission of the dressings, three concentrations of calcium chloride (CaCl₂) at 0.09, 0.27, and 0.45 M were used. The CaCl₂ was added dropwise until translucency was observed in the mixture. The resulting formulation was then sonicated for 2 h and degassed using a VEVOR mechanical vacuum pump (VEVOR, Las Vegas, NV, USA) for 15 min. To remove unbound calcium ions and non-crosslinked polymer residues, the hydrogel films were gently washed with deionized water. Finally, 100 mL was poured into square plates of 10 cm² and was dried in an oven at 40 °C for 24 h.

2.7. Preparation of Alg/CMC/Extract Dressings

For the formulations of the dressings with the different extracts, they were added at the minimum inhibitory concentrations previously reported by the research group [20]. The combinations are shown in

Table 1. Initial formulations for the preparation of dressings.

Treatment	Alg (% w/v)	CMC (% w/v)	Glycerol (% v/v)	CaCl2 (M)	Active Component (AC)	Concentration AC * (mg/mL)
Alg/CMC/Tag	2	1.5	10	0.45	T. nelsonii	25
Alg/CMC/AgAv	2	1.5	10	0.45	A. americana + A. vera gel	25/30 **
Alg/CMC/CP	2	1.5	10	0.45	Chloramphenicol	0.256
Alg/CMC/CN	2	1.5	10	0.45	-	-

* Minimum bactericidal concentration [20]; ** A. vera gel concentration % v/v.

. Alginate, CMC, and glycerol were separately dissolved in distilled water under stirring at 900 rpm at 80 °C for 2 h. Once cooled, the extracts of T. nelsonii and the combined extracts of A. americana and A. vera gel were added at a concentration of 3% w/v. The mixtures were sonicated for 2 h and degassed using a mechanical vacuum pump for 15 min. Subsequently, the hydrogel formulations were gently washed with deionized water to remove unbound calcium ions and non-crosslinked polymer residues, and they were dried at 40 °C to obtain films with a uniform thickness. The process was repeated for the positive and negative controls.

2.8. Physical Characterization of Dressings

2.8.1. Thickness

The thickness of the dressings was measured using a digital micrometer at 10 different points on each dressing.

2.8.2. Scanning Electron Microscopy (SEM)

The surface morphology of the film was examined using scanning electron microscopy (SEM) with a JEOL JSM-6010PLUS (JEOL Ltd., 3-1-2, Musashino, Akishima-Shi, Tokyo, Japan) under low vacuum conditions, employing secondary electrons at magnifications of 200× and operating voltages ranging from 3 kV to 15 kV.

2.8.3. Moisture Vapor Transmission Rate (MVTR)

The moisture vapor transmission rate of each dressing was measured using the wet cup method in accordance with ASTM E96-95. The cups used had a transmission area of 0.0149 m². The dressings were cut to a diameter of 68 mm, and their initial weight was recorded before being placed in permeability cups containing 10 mL of distilled water. The cups were then placed at 20 °C in a desiccator containing silica gel to create a relative humidity gradient of 100%. Water vapor transfer through the dressing area was determined based on the weight loss of the cup at intervals of 3, 6, 8, 12, and 24 h over time. Measurements were performed in triplicate, and the MVTR was calculated using the following equation:

$$\mathrm{M}\mathrm{V}\mathrm{T}\mathrm{R}={\displaystyle \frac{\mathrm{W}/\mathrm{t}\times 24}{\mathrm{A}}}$$

(1)

where W/t represents the change in the weight of the container with distilled water per unit of time (g day⁻¹), and A is the exposed surface area of the film (m²).

2.8.4. Light Transmission and Opacity

The dressings were cut into strips approximately 4 × 1 cm in size and read using a UV-Vis spectrophotometer with a scan range from 200 to 800 nm. Opacity was calculated according to the following formula:

$$\mathrm{Opacity}={\displaystyle \frac{{\mathrm{A}}_{200–800}}{\mathrm{T}}}$$

(2)

where A_200–800 is the absorbance (nm) and T is the thickness of the film (mm).

2.8.5. Water Absorption

The dressings were cut into 2 cm × 2 cm squares, and their initial weight was recorded. They were then immersed in 20 mL of distilled water, excess surface water was removed with filter paper, and the wet weight was measured at 3, 5, 10, 20, and 40 min. Measurements were taken in triplicate for each sample. The water absorption capacity was calculated as follows:

$$\mathrm{Water}\mathrm{Absorption}(\%)=\frac{\mathrm{Wi}-\mathrm{Wf}}{\mathrm{Wi}}\times 100$$

(3)

where W_i is the initial weight of the dry dressings and W_f is the equilibrium wet weight of the dressings.

2.8.6. Swelling

The swelling capacity of the dressings was determined by cutting the dressings into 2 cm × 2 cm squares and then immersing them in 20 mL of a phosphate buffer solution (PBS) at pH 7.4 at 37 °C. Excess surface water was removed with filter paper, and the wet weight was measured at 3, 5, 10, 20, and 40 min. Measurements were performed in triplicate for each sample. The swelling percentage was calculated using the following equation:

$$\mathrm{Swelling}(\%)=\frac{\mathrm{Ws}-\mathrm{Wd}}{\mathrm{Wd}}\times 100$$

(4)

where W_d is the mass of the dry dressings and W_s is the mass of the swollen dressings.

2.9. Mechanical Characterization of Dressings

The tensile strength and elongation at break were determined using a Shimadzu Corporation EZ-SX texture analyzer (Shimadzu Corporation, Kyoto, Japan) at 25 °C in triplicate. First, 48 h before the analysis, the films were stored in a desiccator with a relative humidity of 58%. The dressings were cut to a size of 8 × 1.5 cm for each formulation. The tensile strength was expressed as the maximum breaking force per unit area of the initial cross-sectional area of the dressing, and the elongation at break was expressed as a percentage of the original length [23].

2.10. Antibacterial Activity of Wound Dressings with Extract

Antibacterial tests were performed using the Kirby–Bauer method. The dressings were aseptically cut into discs with a diameter of 0.8 cm and placed in Petri dishes containing Mueller-Hinton agar, which were previously inoculated with Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) at a concentration of 1 × 10⁸ CFU mL⁻¹, according to the McFarland 0.5 scale. The dishes were incubated at 37 °C for 24 h, and the inhibition zone was measured using a vernier caliper [23].

2.11. Wound Healing In Vitro

The experiments were conducted using adult male BALB/c mice (25–30 g). The animals were housed in individual cages under laboratory conditions with room temperature control and a 12-h light-dark cycle, with free access to food and water. The experimental study was carried out in accordance with the Official Mexican Standard NOM-062-ZOO-1999 and was approved by the Institutional Committee of the National Technological Institute of Mexico-Technological Institute of Tuxtla Gutiérrez. The mice were anesthetized via an intraperitoneal injection of pentobarbital (Laboratorio Aranda, Queretaro, Mexico) (0.3–0.6 mL/10 kg). Under anesthesia, the dorsal skin was shaved and disinfected with 70% alcohol. On both sides of the shaved dorsum of each mouse, a dorsal wound measuring 0.7 mm in length was created using a sterile No. 11 scalpel.

The mice were then divided into two groups of five:

• Group I: Mice treated with base gel (negative control)
• Group II: Mice treated with T. nelsonii extract in gel (100 mg/kg)

The gel was applied every 48 h directly to the wound site in direct contact with the lesion. The skin of all mice was inspected daily, and the wounds were photographed from a fixed distance. Wound areas were calculated from each photo using ImageJ software (version 1.50, Bethesda, MD, USA) [24,25]. Data experiments were analyzed using GraphPad Prism™ software (version 5.01, La Jolla, CA, USA). The percentage of wound closure was calculated using the following equation:

$$\mathrm{W}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{c}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{e}\left(\mathrm{\%}\right)=\frac{\mathrm{Wi}\times \mathrm{Wf}}{\mathrm{Wi}}\times 100$$

(5)

where Wo is the initial wound area at 0 h; Wf wound area at days 3, 6, 9, 12, and 15.

2.12. Statistical Analysis

Statistical analysis was conducted using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Data are presented as mean ± standard deviation (SD), with a significance level of p < 0.05.

3. Results

3.1. Antioxidant Capacity of the Extracts

Table 2. Antioxidant capacity through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ABTS radical scavenging assays, and the Ferric Reducing Antioxidant Power (FRAP) assay of the methanolic extracts of T. nelsonii, A. americana, and A. vera gel.

Extract	DPPH	ABTS	FRAP
T. nelsonii	11.238 ± 0.033 a	8.624 ± 0.368 a	16.283 ± 0.299 a
A. americana	0.589 ± 0.012 b	0.596 ± 0.012 b	0.806 ± 0.020 b
A. vera	0.011 ± 0.001 c	0.041 ± 0.015 c	0.013 ± 0.003 c

Units expressed as mg equivalents of Trolox per mL of extract. Different letters indicate statistically significant differences among the extracts with respect to each assay according to Tukey’s multiple range test (p < 0.05).

shows the antioxidant activity values quantified by the DPPH, ABTS, and FRAP methods for each extract. Statistical analysis revealed that the antioxidant capacity, expressed as Trolox equivalents, was higher in the extract of T. nelsonii compared to the extract of A. americana across all three methods used.

3.2. Physical Characterization of Dressings

Figure 1 presents digital images and SEM images of the different formulations, analyzing the effect of CaCl₂, were reveals a smooth surface with minimal irregularity, well-defined layers, and no roughness, attributed to the homogeneous distribution of the two polymers.

The physicochemical and mechanical characterization of the dressings at varying concentrations of the crosslinker (

Table 3. Physical and mechanical characterization of the dressings.

Treatment	CaCl2 (%)	TN (mm)	OP (%)	TR (%)	WA (%)	SW (%)	TS (N/mm2)	EB (%)
A	1	0.936 a	0.41 c	41.714 c	34.969 c	56.428 b	0.442 a	79.226 c
B	3	0.891 a	0.29 b	53.703 b	53.778 b	81.38 a	0.338 b	97.544 b
C	5	0.996 a	0.13 a	74.711 a	72.915 a	92.03 a	0.248 c	167.696 a
Tukey		0.082	0.051	6.912	13.975	16.309	0.054	14.794

TN, thickness; OP, opacity; TR, transmittance; WA, water absorption; SW, swelling; TS, tensile strength; EB, elongation at break. Different letters indicate statistically significant differences among the treatments for each characterization, according to Tukey’s multiple range test (p < 0.05).

) indicated no significant difference in thickness among the treatments, confirming the uniformity of the dressings.

The opacity percentage, in relation to transmittance (which measures light passage through the material), is inversely proportional, showing statistically significant differences among the treatments. Treatment C exhibited the lowest opacity and highest transparency and water absorption capacity. Regarding water absorption capacity (WA) and swelling (SW), the results revealed a marked improvement with higher CaCl₂ concentrations. Treatment C presented the highest WA and SW values. The tensile strength (TS) decreased significantly as the CaCl₂ concentration increased. Treatment A showed the highest TS, while treatment C had the lowest and the highest elongation at break (EB).

3.3. Characterization of Dressings with Extract

The dressings containing 3% individual or combined plant extract showed a smooth surface without heterogeneous roughness in their macroscopic appearance and demonstrated uniform coloration throughout the entire area (Figure 2).

The physicochemical and mechanical characteristics of the dressings with 3% T. nelsonii, A. americana extract, and Aloe vera gel, both individually and in combination, were analyzed. The results of this analysis are presented in

Table 4. Physical and mechanical characterization of the dressings with plant extracts.

Dressing	TN (mm)	OP (%)	TR (%)	MVTR (g/m2d)	WA (%)	SW (%)	TS (N/mm2)	EB (%)
Alg/CMC/Tag	1.022 a	0.85 c	14.928 d	395.167 ab	156.934 a	127.869 a	0.506 a	395.167 ab
Alg/CMC/AgAv	0.902 ab	0.43 ab	40.676 b	320.859 b	106.064 b	125.148 b	0.460 ab	320.859 b
Alg/CMC/CP	0.963 ab	0.53 b	30.555 c	330.309 ab	84.009 bc	124.703 bc	0.363 bc	330.309 ab
Alg/CMC/CN	0.981 b	0.28 a	52.62 a	421.691 a	79.583 c	98.371 c	0.266 c	421.691 a
Tukey	0.091	0.172	5.445	98.626	17.780	21.763	0.113	10.161

TN, thickness; OP, opacity; TR, transmittance; MVTR: Moisture Vapor Transmission Rate; WA, water absorption; SW, swelling; TS, tensile strength; EB, elongation at break. Alg/CMC/Tag, Alginate, carboxymethylcellulose, and T. nelsonii extract dressing; Alg/CMC/AgAv, Alginate, carboxymethylcellulose, and A. americana extract and A. vera gel dressing; Alg/CMC/CP, Alginate, carboxymethylcellulose, and antibiotic dressing; Alg/CMC/CN, Alginate and carboxymethylcellulose dressing. Different letters indicate statistically significant differences among the dressings for each characterization, according to Tukey’s multiple range test (p < 0.05).

. Statistical evaluation of the thickness values indicated significant differences among treatments, with the Alg/CMC/Tag dressing displaying the greatest thickness.

The physical and mechanical properties of the dressings varied significantly depending on the type of plant extract incorporated, according to statistical analysis. The Alg/CMC/Tag formulation demonstrated the greatest thickness, opacity, water absorption, swelling, and tensile strength, along with the lowest light transmittance. This low light transmittance suggests a beneficial ability of the dressing to protect wounds from harmful light exposure (Figure 3). Conversely, the Alg/CMC/CN dressing exhibited higher transparency and elongation at break but showed significantly lower absorption capacity and mechanical resistance.

3.4. Antibacterial Activity of Dressings with Extract

The antibacterial activity of the dressings was evaluated to confirm that the incorporation of extracts at the previously reported minimum inhibitory concentration [19] retained their bacteriostatic effect without interference from interactions with the polymeric matrix components. To assess this, the disk diffusion test was performed, and the results are summarized in

Table 5. Antibacterial activity of the dressings with plant extracts.

Bacteria	Alg/CMC/Tag	Alg/CMC/AgAv	Alg/CMC/CP	Alg/CMC/CN
	Inhibition Zone (mm)
S. aureus	19.333 a	0 c	19.666 a	0 c
P. aeruginosa	17.733 b	0 c	18.033 a	0 c

Different letters indicate statistically significant differences among the dressings with respect to each bacterium according to Tukey’s multiple range test (p < 0.05).

. Analysis of the inhibition zones revealed no statistically significant differences between the effects of the Alg/CMC/Tag and Alg/CMC/CP dressings against S. aureus. Notably, the Alg/CMC/Tag dressing maintained its inhibitory effect for 48 h, and the resulting inhibition halo exceeded the previously reported 17.33 mm for T. nelsonii extract (Figure 4) [20].

3.5. Wound Healing In Vivo

Given the previously reported antimicrobial activity of T. nelsonii extract (TGEXT) [20] and its high antioxidant capacity, its potential wound-healing effects were evaluated in an in vivo model. Figure 5 illustrates the progression of wound closure following the application of a gel containing T. nelsonii extract (TGEXT) and the gel base (GelNC) as a negative control.

Upon application of TGEXT and GelNC, wound closure commenced on day six and day 12, respectively. Statistical analysis revealed that, by day six, TGEXT achieved a wound closure of 71.21 ± 6.16%, whereas GelNC reached 43.24 ± 2.03% (Figure 6). By day 12, wound closure rates increased to 95.34 ± 3.59% and 89.97 ± 2.80%, respectively. By day 15, no significant differences were observed between treatments, as both resulted in fully healed wounds with closure rates exceeding 98%, as shown in Figure 6.

4. Discussion

4.1. Antioxidant Capacity of the Extracts

Oxidative stress plays a critical role in delaying tissue repair and promoting microbial proliferation in wounds; therefore, compounds with high antioxidant capacity contribute to the development of treatments that can both protect tissues from oxidative damage and enhance healing processes [26]. Furthermore, antioxidants can modulate inflammatory responses, creating a more favorable environment for tissue regeneration and infection control [26]. Statistical analysis revealed that the antioxidant capacity was higher in the extract of T. nelsonii compared to other extracts. The differences observed between the extracts and the antioxidant activity methods can be attributed to the distinct composition of each extract, as well as the molecular interactions among the phenolic compounds, depending on the method applied. It has been reported that the most significant phytochemicals responsible for antioxidant capacity are polyphenols, particularly flavonoids, alongside certain fatty acids, pigments, and vitamins. In this context, Burlec et al. [27] indicated that the phenolic compounds found in the Tagetes genus, in synergy with terpenes and sterols, enhance antioxidant effects. Although, to the best of our knowledge, there are no studies on the antioxidant activity of T. nelsonii, there are studies on its genus. Lopez et al. [28] evaluated the antioxidant activity of Tagetes patula, which reported values of 16.07 mg TE/mL for DPPH and 33.18 mg TE/mL for ABTS. On the other hand, T. patula L. flower essential oil exhibited an EC₅₀ of 0.02985 mg/mL in the DPPH assay and 0.03022 mg/mL in the FRAP assay [29]. The activity of the T. nelsonii extract underscores its high potential for radical scavenging, supporting its use as a therapeutic agent in the healing process, particularly in instances where oxidative stress hinders healing [30].

4.2. Physical Characterization of Dressings

The results show that there were no statistically significant differences in thickness (TN) among the treatments (p > 0.05), indicating that the concentration of crosslinking agent did not affect this parameter. All formulations exhibited thickness values appropriate for skin application, as they were thinner than human skin (0.5 to 4.0 mm), making them suitable for use as wound dressings. The opacity percentage was inversely proportional, showing statistically significant differences among the treatments. Treatment C exhibited the lowest opacity and highest transparency, allowing for better visualization of wound closure when applied as a dressing [31,32]. A dressing must absorb exudates during the healing process; therefore, treatment C, which demonstrated the highest water absorption capacity, is ideal for preventing tissue maceration and facilitating oxygen transfer [6,33]. Variations in water absorption and swelling can be associated with the hydrophilicity and affinity of the ions dissolved in the buffer with the polymers [34,35]. The absorption capacity of a material depends on its degree of crosslinking; thus, treatments A and B, which showed low absorption capacity, can be linked to the lower percentages of CaCl₂, resulting in reduced crosslinking between the polymers [36,37] and a greater availability of hydrophilic groups, which creates an excess of hydrogen bonds that led to high solubility of the dressing. The increase in swelling, on the other hand, is attributed to the salts present in the BFS, which interact with the polymer matrix [38].

Calcium chloride has been reported as a crosslinker that enhances the bonding of various polymers [38,39,40]. Promotes the interaction of Ca²⁺ ions with the guluronic acid blocks in alginate to form stable “egg-box” structures. This crosslinking significantly influences the physical and mechanical properties and structural stability of the resulting hydrogel films. When combined with carboxymethylcellulose and alginate, the presence of the divalent cation promotes gel formation, as calcium becomes lodged between polymer chains, achieving either permanent or temporary stability [41]. The addition of 0.45 M calcium chloride creates a greater number of crosslinking points between carboxymethylcellulose and alginate, resulting in dressings with lower opacity, higher transmittance, and greater swelling [42,43,44]. This also enhances flexibility and extensibility, which are crucial properties for their application, as the dressings must withstand handling on the skin [34,45]. Analysis of the tensile strength and elongation at break of the dressings confirmed the influence of the crosslinker. Lower tensile strength values resulted in greater flexibility, likely due to covalent bonds and hydrogen bridges, as well as the proportional relationship between the crosslinking agent, alginate, and CMC [46].

Determining the mechanical properties of wound dressings is essential. These materials must endure external damage during storage and use, mainly flexible for skin application, and prevent matrix collapse [34,45]. An ideal dressing should possess a high percentage of elongation at break and high tensile strength, serving as indicators of flexibility and extensibility, vital for determining its application and handling [45,47]. In this study, TS values ranged from 0.266 to 0.506 MPa and from 114.36% to 175.22% for EB. Although these parameters depend on thickness, the polymers used, and the type of crosslinker, the relationship between mechanical properties and the crosslinking agent has been previously explored [43]. Statistical analysis of these property data indicated a significant difference, with the treatment containing 0.45 M crosslinking agent displaying the lowest tensile strength and highest elongation at break, with values of 0.248 N/mm² and 167.696%, respectively. These values fall within the reported ranges for skin application, which are 0.001227 to 854 N/mm² and 2 to 200%, respectively [48,49,50]. These results suggest that higher Ca²⁺ concentration enhances crosslinking density, promoting stronger covalent and ionic interactions within the polymer matrix [44]. In contrast, films prepared with lower CaCl₂ concentrations (0.09 and 0.27 M) exhibited reduced structural integrity and a tendency to partially dissolve during testing, likely due to insufficient crosslinking. Therefore, 0.45 M CaCl₂ was selected as the optimal concentration, offering a balanced combination of mechanical resistance and controlled swelling behavior.

4.3. Characterization of Dressings with Extract and Antibacterial Activity

Alg/CMC/CN revealed no significant statistical difference in opacity compared to the Alg/CMC/AgAv dressing, which demonstrated a medium percentage of transmittance due to the addition of A. vera gel, which induces light retention [51]. Furthermore, analysis of the transmittance curve of the dressings in ultraviolet and visible light ranges indicated that the dressing with the lowest percentage of transmittance was Alg/CMC/Tag (Figure 5). This reduction in transmittance can be attributed to the various bioactive compounds present in the extract, suggesting that the dressing can decrease radiation incidence on the tissue and protect it from oxidative damage. An ideal dressing should maintain skin water loss due to evaporation at an optimal rate. The moisture vapor transmission rate (MVTR) should not be too low, as this can lead to exudate accumulation and delay the healing process, nor should it be too high, to avoid excessive dehydration. Various studies suggest that the MVTR for an ideal dressing should be 204 g/m²/day for normal skin, 279 g/m²/day for injured skin, 1800–2300 g/m²/day for first-degree burns, and 2500 and 5138 g/m²/day for chronic wounds. In this regard, the dressings obtained in this study had an MVTR suitable for injured skin or acute wounds [52]. It is well known that an increase in the hydrophilic nature of a polymer matrix tends to enhance moisture vapor permeability, making the combination of alginate and carboxymethylcellulose responsible for the MVTR of the dressings [53].

The analysis of water absorption percentages disclosed a statistically significant difference among the dressings, with the Alg/CMC/Tag dressing exhibiting the highest water absorption percentage (Table 3). This is likely due to the affinity between the polymeric matrix and the phytochemical compounds found in the T. nelsonii extract, which may enhance the plasticization of the polymers, resulting in lower solubility and greater water absorption [54].

The analysis of swelling percentages demonstrated a statistically significant difference among the dressings, with the Alg/CMC/Tag dressing exhibiting the highest BFS absorption percentage. The swelling capacity depends on the selected fluid, as it can ionically interact with the polymeric matrix and enhance this property [23]. Authors such as Brás et al. [35] and Valor et al. [54] mentioned that the salts in BFS contribute to greater stability in the crosslinking of polymeric matrices. This occurs because carboxymethylcellulose and alginate are polyanionic polymers. When in a saline solution, Na⁺ ions in the solution exchange with the Ca⁺² ions that are nonspecifically bound to the COO-groups of the polymers. This results in electrostatic repulsion among the carboxyl groups, causing chain relaxation and promoting gel swelling [55].

Regarding mechanical properties, these characteristics play a fundamental role in the functionality of wound dressing as they indicate its flexibility and durability. Consequently, wound dressings must exhibit high tensile strength (TS) and a high percentage of elongation at break (EB) [45,56]. In this study, tensile strength values ranged from 0.266 to 0.506 MPa for TS and from 114.36% to 175.22% for EB. Similar values have been reported for dressings made with alginate and carboxymethylcellulose [34,57]. The Alg/CMC/Tag and Alg/CMC/AgAv dressings displayed greater resistance compared to the controls, likely due to the incorporation of the extracts. The functional groups of the compounds in each extract interacted with the polymers constituting the dressing, enhancing its plasticity and crosslinking while reducing its solubility. Ramos et al. [58] noted that a decrease in the percentage of elongation at break is likely due to the strength of hydrogen bonds between phenolic compounds and the extracellular matrix, which restricts molecular chain movement during stretching.

Wound dressings should also exhibit physical properties similar to those of the skin, which has a tensile strength ranging from 4 to 30 MPa and an elongation at break between 35% and 115%, varying according to age and body location [34]. In this context, the Alg/CMC/Tag dressing was the closest in properties to the skin, followed by the Alg/CMC/AgAv and Alg/CMC/CP dressings. These dressings were sufficiently flexible to be applied to challenging areas such as knees and elbows [59].

On the other hand, the antibacterial activity of the Tagetes genus has been reported and is primarily attributed to phenolic compounds, saponins, tannins, and coumarins, among others [17,18]. The antibacterial mechanism of the phenolic compounds and terpenes present in the extract involves the interaction of their hydroxyl groups with the amino acids in the bacterial plasma membrane. This interaction destabilizes the integrity and permeability of the membrane, leading to damage to DNA and proteins, leading to bacterial death.

4.4. Wound Healing In Vivo

Given that T. nelsonii exhibited the highest antioxidant capacity among the evaluated extracts, its wound-healing potential was further assessed in vivo. TGEXT demonstrated a significantly higher percentage of wound closure compared to the negative control, supporting its regenerative potential. Specifically, wound closure began earlier and progressed faster in the TGEXT-treated group, reaching 71.21 ± 6.16% closure by day 6 compared to 43.24 ± 2.03% in the gel control. By day 12, closure rates increased to 95.34 ± 3.59% for TGEXT and 89.97 ± 2.80% for the control. By day 15, both treatments achieved full wound closure with no significant differences. These results indicate that TGEXT can particularly be relevant during the early and intermediate stages of wound healing, when oxidative stress and microbial colonization can delay tissue regeneration. These findings are consistent with previous studies, which showed that the extract promotes increased proliferation and migration of keratinocytes—cells located in the dermis that play a key role in tissue repair [20]. The gel-based nature of TGEXT allowed direct contact with the wound, potentially enhancing the diffusion of bioactive components into the dermis and thereby improving the healing process. While this study demonstrated that the plant extract significantly enhances wound closure in mice, attributing these effects to a single compound remains challenging. The observed benefits may stem from the action of an individual metabolite or the synergistic effects of multiple bioactive compounds identified in Tagetes nelsonii. In our previous chemical characterization by GC-MS [20], several compounds with known biological activity were detected, including limonene, eugenol, phytol, squalene, 11,14,17-eicosatrienoic acid, palmitic acid, 7-hydroxy-2H-1-benzopyran-2-one (a coumarin), and 2,4-bis-(1,1-dimethylethyl) phenol (structures shown in Figure 7). These metabolites have been widely reported to possess antimicrobial, antioxidant, anti-inflammatory, and pro-regenerative properties [60,61,62]. Additionally, these compounds may act by upregulating the mRNA expression of factors involved in keratinocyte proliferation and migration, including keratinocyte growth factor (KGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor-beta (TGF-β).

To contextualize the properties of our carboxymethylcellulose and alginate-based dressings incorporated with Tagetes nelsonii extract, a comparative analysis was conducted with similar plant-based hydrogel systems reported in the literature.

Table 6. Comparative analysis of antimicrobial and wound healing activity of alginate and/or CMC-based dressings incorporated with plant extracts or antimicrobial agents.

Type of Wound Dressing	Extract	Antibacterial Activity	Wound Healing Activity	Advantages	Disadvantages	References
Alg-CMC	Aqueous extract of Capparis sepieria	Strong against Gram-positive and negative	Stimulates fibroblast migration and proliferation	Suitable for burns; good cell viability	No in vivo animal testing	[63]
BC *-CMC *	Curcumin	Moderate (E. coli, S. aureus)	Antioxidant, promotes cell viability	Dual antioxidant and antibacterial activity	Only in vitro characterization; lacks in vivo testing	[14]
Alg *-PVA * hydrogel	Moringa oleifera extract	Antibacterial vs S. aureus, E. faecalis, E. coli	Promototed granulation and ephitelialization un mouse wound model	Good biocompatibility, swelling, moisture retention	Lacks testing vs other strains, limited mechanical/degradation data	[64]
Alg-films based on sodium alginate	Moringa oleifera powder/essential oil (10–30%)	Not analyzed	Suggested for wound types; high swelling/moisture retention	Homogeneous matrix, prolonged release, TS ≈ 0.248 MPa, elongation ≈ 31%, 4800% swelling	No in vivo data; antimicrobial not strain-specific; no degradation analysis	[15]
CMC	Leaf extract of Syzygium cumini	Inhibition zones: S. aureus (15 mm) E. coli (11 mm)	83% wound closure L929 cell line full healing in 7 days (in vivo)	Antioxidant activity (63%)	No long-term mechanical/stability data; needs broader validation	[65]
Pullulan/PVA hydrogel	Calendula officinalis extract	Inhibition zones: S. aureus (13 mm) P. aeruginosa (15 mm)	70% DPPH antioxidant activity	High loading bioadhesiveness; tunable swelling and mechanics	No in vivo data; decreased mechanical strength at higher loading; cytotoxicity at high dose	[16]
Alginate hydrogel	Leaf extract of Lilium candidum	Not quantified	Promotes fibroblast and endothelial migration; better wound closure and scar formation than extract or hydrogel alone (in vitro)	Antioxidant, anti-inflammatory, cell migration support	No antimicrobial quantification	[66]

* BC: Bacterial Cellulose; CMC: Carboxymethylcellulose; Alg: Alginate; PVA: Polyvinyl alcohol.

summarizes the antimicrobial and wound-healing activities of selected formulations using natural extracts such as Hypericum perforatum, Calendula officinalis, Syzygium cumini, and Lilium candidum. While these studies demonstrated notable antimicrobial or antioxidant potential, most relied solely on in vitro assays, lacked in vivo validation, or did not quantify healing outcomes in terms of wound closure percentage. In contrast, our study integrates both in vitro and in vivo evidence, demonstrating a faster onset of healing (71.2% closure by day 6) and nearly complete re-epithelialization by day 12. Moreover, the incorporation of T. nelsonii into a gel-based system ensured enhanced diffusion, biocompatibility, and effective delivery of bioactives at the wound site. These findings support the superior regenerative performance of our formulation and its potential as a viable and natural alternative for wound management.

5. Conclusions

The addition of plant extracts to the dressings improved the crosslinking of alginate and carboxymethylcellulose, resulting in enhanced physicochemical and mechanical properties, including greater water absorption, swelling, and tensile strength. All treatments presented adequate thickness and moisture vapor transmission rate values for application to acute wounds in different parts of the body.

Among the different formulations tested, the Alg/CMC/Tag dressing—supplemented with Tagetes nelsonii extract—exhibited the most favorable properties, including the highest water absorption (156.93%), swelling (127.87%), and antioxidant capacity (16.28 ± 0.29 mg TE/mL by FRAP). Moreover, this formulation was the only one that demonstrated antibacterial activity (inhibition zones of 19.33 mm against S. aureus and 17.73 mm against P. aeruginosa) and significantly accelerated wound healing in vivo (98% closure by day 15). Therefore, the Alg/CMC/Tag dressing shows the greatest potential as an effective wound-healing biomaterial, especially in cases where oxidative stress and microbial infections may impair the healing process.