Anti‐inflammatory and healing effect of the polysaccharidic extract of Opuntia ficus‐indica cladodes in cutaneous excisional wounds in rats

Abstract

This study aimed to investigate the anti‐inflammatory and wound healing effects of the polysaccharide extract from Opuntia ficus‐indica cladodes (TPL‐Ofi) using a rat cutaneous wound model. After anaesthesia, four 7‐mm‐diameter dorsal wounds per animal (n = 6/group for each experimental day of evaluation) were created in female Wistar rats using a surgical punch. The animals were treated topically twice daily with TPL‐Ofi (0.01–1%; treated group) or sterile saline (control group) for a period of 21 days. Ulcerated tissue was collected for analysis of histological parameters (inflammation score, number of polymorphonuclear, mononuclear, fibroblast/myofibroblasts and blood vessels), immunohistochemical (fibroblast growth factor 2 [FGF‐2]) and oxidative stress markers (myeloperoxidase [MPO] and glutathione [GSH]). After 21 days of treatment, body weight, net organ weight and plasma biochemical levels were measured. TPL‐Ofi, containing a total carbohydrate content of 65.5% and uronic acid at 2.8%, reduced oedema on the second day and increased the nociceptive threshold on the second and third days. TPL‐Ofi reduced mononuclear infiltrate on the second and MPO activity on the fifth day. TPL‐Ofi increased GSH levels on the second day, as well as fibroblast/myofibroblasts counts, neoangiogenesis and FGF‐2 levels on the fifth and seventh days. No changes were observed in body weight, net organ weight or toxicology assessment. Topical application of TPL‐Ofi exhibited anti‐inflammatory and antinociceptive effects, ultimately improving wound healing in cutaneous wounds.

1. INTRODUCTION

Wounds are traumatic events to the skin or mucosa resulting in the loss of integrity of the body's first defence barrier. They can arise from accidents and are also commonly associated with other conditions such as diabetes or pressure ulcers. A wound that fails to heal can serve as entry point for a multitude of pathogens, potentially leading to more severe illnesses, prolonged pain and even sepsis. ¹

Wounds initiate a local inflammatory response that relies on the mechanisms of innate and cellular immunity to resolve. These mechanisms are typically divided into four overlapping phases during the healing time course: homeostasis, inflammation, proliferation and tissue remodelling. An impairment in any of these phases can lead to a wound that fails to heal properly. ² Non‐healing wounds are estimated to have a prevalence of 1.67 per 1000 population, and the number of cases is anticipated to rise due to the aging of the world population, thus becoming a significant concern to clinical patient care. ³

Currently, numerous therapeutical options are being employed to treat non‐healing wounds, primarily in the form of dressings or various ointments for topical use, varying in composition. ⁴ In addition to these, anti‐inflammatory drugs, both steroidal and non‐steroidal, can be prescribed to alleviate pain, a common symptom. ² But none of those options seems to reduce the burden of wound healing treatments on health‐care systems worldwide, thus leading researchers to continue seeking for improved treatment compounds.

The diversity of flora around the world stimulates the search for alternatives to compounds already used therapeutically. In this perspective, plant polysaccharides have emerged as an alternative due to their proven pharmacological activities: anti‐inflammatory and antinociceptive, ⁵ , ⁶ antioxidant ⁶ and healing. ⁷ , ⁸ , ⁹ Opuntia ficus‐indica (Cactaceae) is a species of cactus endemic to the northeast region of Brazil, and its use is documented in folk medicine to treat pelvic inflammatory processes and rheumatism ¹⁰ Its polysaccharides have anti‐inflammatory, ¹¹ , ¹² , ¹³ wound healing, ¹⁴ , ¹⁵ gastroprotective and antioxidant effects. ¹⁶ , ¹⁷

Cactaceae cladodes are particularly rich in carbohydrate‐containing polymers with several sugar residues such as arabinose, galactose, rhamnose, xylose and galacturonic acid. ¹⁸ These polysaccharides exhibit anti‐inflammatory, ¹¹ , ¹² wound healing, ¹⁴ , ¹⁵ gastroprotective and antioxidant properties. ¹⁶ , ¹⁹ Furthermore, they can serve as mucoprotective agents due to their ability to form a molecular network that maintains moisture in the wound bed, thus acting as a protective layer and accelerating the healing. ¹⁶

In the search for new molecules capable of optimizing current treatments, we aimed to investigate the healing, anti‐inflammatory and antinociceptive activities of the polysaccharide extract of O. ficus‐indica in the model of cutaneous wounds in rats.

2. MATERIALS AND METHODS

2.1. Chemicals

Ketamine and xylazine were supplied by König S/A (Argentina). A 0.12% chlorhexidine gluconate solution was obtained from Colgate‐Palmolive (Brazil). Kits for evaluating Fibroblast Growth Factor 2 (FGF‐2) and myeloperoxidase (MPO) activities were purchased from Abcam® (Cambridge, MA‐EUA). Reagents for measuring GSH (glutathione), MDA (malondialdehyde) and galactose, as well as acid D‐glucuronic and albumin serum bovine, were obtained from Sigma Chemical Co. (St. Louis, MO‐ EUA).

2.2. Animals

Female Wistar rats (180–200 g, approximately 60 days old) were kept under standard conditions (25°C; 12/12 h light/dark cycle) with access to water and food ad libitum until the experiments. The protocol was approved (CEUA/UECE N^o. 5814678/2017), in accordance with the guidelines of the Brazilian Council of Control in Animal Experimentation (CONCEA).

2.3. Plant material

O. ficus‐indica was identified, and a voucher specimen (n°. 61,760) was deposited at the Herbarium Prisco Bezerra – Federal University of Ceará. Cladode samples were collected in May 2018 from the Riacho Verde region in Quixadá, Ceará, Brazil.

2.4. Polysaccharides extraction from O. ficus‐indica cladodes

Two cladodes (250 g), cleaned of the thorns and washed with distilled water, were macerated in natura in a blender, producing two distinct homogenates: a liquid one and a solid one. The extraction of polysaccharides was carried out on three different combinations of the homogenates: (I) using only the liquid portion, (II) using only the solid portion and (III) using both portions.

Each solution was processed in the same way: they were suspended and homogenized in 0.1 M NaOH (750 mL, 6 h, 90°C), filtered and centrifuged (15 min, 3500 rpm, 25°C). The resulting supernatant was collected, concentrated (60°C), neutralized with 1 M HCl and precipitated with ethanol (four volumes, 12 h, 4°C). The precipitate was centrifuged (30 min, 3500 rpm, 25°C), dialyzed against distilled water, re‐centrifuged (15 min, 3500 rpm, 25°C), with trichloroacetic acid (TCA, 4 h, 4°C), ¹⁹ re‐centrifuged again and supernatant was dialysed and lyophilized to obtain an extract rich in O. ficus‐indica polysaccharides (TPL‐Ofi). ²⁰

2.5. Chemical‐structural analysis

The chemical analysis of TPL‐Ofi included spectrophotometry for quantification of total carbohydrate (A_{490 nm}) using the phenol‐sulphuric acid reaction, ²¹ uronic acid (A_{525 nm}) measured by the carbazole method ²² and soluble proteins (A_{595 nm}) determined by the Bradford method, ²³ using galactose, acid D‐glucuronic and albumin serum bovine as standards, respectively.

For structural analysis, Fourier‐transform infrared spectroscopy (FT‐IR) and gel permeation chromatography (GPC) were performed. In brief, for the FT‐IR analysis, spectra were measured from 2 mg of TPL‐Ofi macerated and pressed (8 tonnes) in KBr tablets (1:100). After pressing, spectra were registered in a Perkin Elmer (model 16 PC) on intervals of 4000 to 400 cm⁻¹ in a 4 cm⁻¹ resolution. ²⁴ For the GPC analysis, polysaccharide samples were prepared in deionized water (0.2%), filtered through 0.45 μm membranes (Milipore®) and injected (20 μL) in a linear Ultrahydrogel column (7.8 × 300 mm). The chromatographic profile was determined on a chromatograph SHIMADZU LC‐10 AD with a refractive index detector (RID‐10A) at 40°C. The analysis was performed using a column Ultrahydrogel linear (7.8 × 300 mm) and a mobile phase of 0.1 M NaNO₃ (1.0 mL/min flow). The average molar mass value (Mw) was obtained from the equation logMw = −0.96Ve + 13.49; r ² = 0.9987.

2.6. Wound model and tissue collection

Animals were anaesthetised through intraperitoneal injection with 8% ketamine (90 mg/kg) and 2% xylazine (15 mg/kg). Prior to the procedure, animals had their dorsal region shaved and disinfected with 2% chlorhexidine gluconate. Four circular, full‐thickness wounds were created using a biopsy punch (diameter of 8 mm), exposing the panniculus carnosus. After the procedure, animals were kept in individual cages. ²⁵

Daily topical treatment consisted of direct application (100 μL), twice a day, of either saline (0.9% NaCl) or TPL‐Ofi (dissolved in saline at concentrations of 0.01%–1%). The evaluation of clinical signs of inflammation, nociceptive, macroscopical, biochemical, histological and immunohistochemical parameters was conducted on days 2, 5, 7, 10, 14 and 21 post‐ulceration. ⁷

2.7. Clinical signs of inflammation

The signs of hyperaemia, oedema and exudate were evaluated in the wound region according to the following scoring system: (0) absent, (1) mild, (2) moderate and (3) intense. Crust detachment (fissures, fragility) and the presence of scar tissue were reported as relative frequencies (f%) of the signal appearance. Scar tissue was differentiated from the surrounding normal skin by its characteristics: fragile and pink tissue, located between the normal skin and the injured area, and absence of pigmentation. ⁷

Hypernociception was evaluated using an electronic algesimeter (Insight Equipamentos, Brazil) equipped with a polypropylene tip (4.1 mm²). The tip was applied to the wound edges to elicit behavioural responses (wincing and/or writhing) at 12 h and 1, 2, 3, 5, 7, 10 and 14 days. ²⁵

2.8. Histopathological analysis and redox markers evaluation

Wound biopsies were fixed in 10% v/v formaldehyde and embedded in paraffin to prepare H&E‐stained slides (3 μm thickness). A blind evaluation was conducted based on the stages of the wound healing process, and scores were assigned by the presence or absence of ulcer, ranging from 0 to 4: (0) absence of ulcer/fibrosis and mononuclear inflammatory infiltrate, (1) absence of ulcer/fibrosis with mild mononuclear inflammatory infiltrate, (2) presence of ulcer/fibrosis with moderate mononuclear inflammatory infiltrate, (3) presence of ulcer/intense mononuclear inflammatory infiltrate and (4) presence of ulcer/acute process (dilated vessels, mixed inflammatory infiltrate with neutrophils). ⁷

To quantify polymorphonuclear, mononuclear and fibroblast/myofibroblast cells, as well as blood vessels, H&E‐stained slides were photographed immediately below the ulcer/reepithelization area of each animal (five fields per slide) (40× magnification; Nikon Eclipse H550S microscope; Japan) and images were analysed using the “Cell Counter” plugin in ImageJ software (National Institutes of Health, EUA). ⁷

Homogenates of cutaneous wound biopsies were used to determine tissue levels of the inflammatory redox markers GSH (A_{412 nm}) and MPO (A_{450 nm}) through enzyme‐linked immunosorbent assay specific to each biomolecule. ²⁶ , ²⁷ In brief, for the GSH quantification, the samples were homogenized in phosphate‐buffered saline (PBS) and then centrifuged. The supernatant was discarded, and the pellet was resuspended in ddH₂O and 50% trichloroacetic acid (TCA) and then centrifuged (5000 rpm, 15 min, 4°C). Subsequently, Tris–HCl 0.4 M (pH 8.9) and DTNB 0.01 M were added, and the samples proceeded to acquisition in a plate reader at an optical density of 412 nm. Glutathione concentration was expressed in nanograms of GSH per gram of tissue. ²⁶ For the MPO quantification, the samples were homogenized with hexadecyltrimethylammonium bromide (0.5%, 50 mg of tissue/mL) and then centrifuged (4500g, 15 min, 4°C). The supernatant was collected and incubated with 200 μL of the detection solution (5 mg o‐Dianisidine, H₂O₂ 1%, PBS 1×, ddH₂O) in 96‐well plate for 10 minutes. The optical density of the reaction was measured at 450 nm in a plate reader, and data were expressed in MPO per mg of tissue. ²⁷

2.9. Immunohistochemistry of FGF‐2 expression

Wound biopsies containing the wounds were cut into 2.5‐μm‐thick sections, placed on silanized slides and processed. Slides were subjected to immunoreaction using the primary antibody against FGF‐2 (1:1250) (ABCAM®). They were subsequently incubated with a ready‐to‐use biotinylated, monoclonal anti‐rabbit IgG secondary antibody at room temperature for 30 min (K4065, Dako™) followed by streptoavidin peroxidase (ABCAM®). Next, the slides were exposed to 3,3 diaminobenzidine chromogen (DAB; Dako™ K3469) for 10 min. Mayer's haematoxylin was used as a counterstain, after which the specimens were dehydrated (using ethanol and xylene) and were cover‐slipped using a permanent mounting medium. Paired sections were subjected to treatment with the control IgG in place of the primary antibody serving as a negative control. Staining intensity was measured according to the following scores for positive cells: (0) no cells; (1) mild, 1%–33% cells; (2) moderate, 34%–66% cells; and (3) 67%–100% cells. ²⁸ Representative photographs were obtained using a Leica microscope (40× magnification).

2.10. Preliminary assessment of systemic toxicity

For the assessment of systemic toxicity, four animals (n = 2/group) were topically treated for 21 days as follows: two received saline and two received 0.1% TPL‐Ofi. After 21 days, peripheral blood was collected before euthanasia for the quantification of the following parameters: plasma levels of urea and creatinine (renal function); total proteins; activity of alanine transaminase – ALT and aspartate transaminase – AST (liver function markers) and complete blood count parameters (haematocrit, haemoglobin, erythrocytes, platelets, lymphocytes, eosinophils, monocytes, neutrophils and basophils). ²⁹ Following euthanasia, the brain, heart, lungs, spleen, kidneys and liver were removed, and their weights were measured (wet weight in g/100 g of body weight).

2.11. Statistical analysis

Parametric data were presented as mean ± SEM and analysed using t‐test or one−/two‐way ANOVA, followed by Bonferroni's test. Clinical signs of inflammation, histopathological findings and immunohistochemical data were expressed as Median (minimum, maximum) and analysed by the Mann–Whitney's or Kruskal–Wallis's test, followed by the Dunn's test. Categorical data (absent/present) were presented as relative frequency (%f) and analysed using the Chi‐Square test. A significance level of p < .05 was considered statistically significant.

3. RESULTS

3.1. O. ficus‐indica extract is mainly polysaccharide

The extraction of polysaccharides from the cladodes was performed using three different combinations (extractions I, II and III), each demonstrating a distinct analytic profile. Extraction II exhibited the highest total carbohydrate content (65.5%) but had the lowest percentage of uronic acid (2.8%) compared to the other similar extractions. It also achieved a yield of 0.33%, which was higher than that of extractions I and III. Notably, all extractions did not show the presence of protein contaminants after TCA deproteinization (Table 1).

TABLE 1.

Biochemical content of the different Opuntia ficus‐indica extractions.

Extraction	Yield a (%)	Total carbohydrates b (%)	Uronic acid c (%)	Proteins d (%)
I (liquid part)	0.21	29.6	5.2	n.d.
II (solid part)	0.33	65.5	2.8	n.d.
III (solid and liquid parts)	0.18	42.0	9.8	n.d.

Abbreviation: n.d., not detected.

^a From 250 g of solid parts from the cladodes in natura.

^b DUBOIS et al., 1956.

^c DISCHE, 1947.

^d BRADFORD, 1976.

Given that extraction II had the highest content of polysaccharides, it was selected for further analysis by FT‐IR and GPC (Figure 1) and for use in treating cutaneous wounds. From here on, extraction II will be referred to as TPL‐Ofi.

FIGURE 1.

Biochemical profiling of Opuntia ficus‐indica extract. The extract was subjected to infrared spectroscopy (FT‐IR) and gel permeation chromatography (GPC) to determine its chemical composition. (A) Peaks obtained in FT‐IR reveal the presence of carbohydrates (wavenumbers 3404 and 1041 cm⁻¹) and free carboxyl groups (wavenumber 1645). (B) GPC analysis.

The FT‐IR analysis revealed the presence of polymeric structure characteristics of polysaccharides, with peaks at wavenumber 3404 and 1041 cm⁻¹, both indicating the presence of carbohydrates in TPL‐Ofi. Additionally, peaks at 2930 cm⁻¹ and 1645 cm⁻¹ were also detected, characterizing the presence of methyl and carboxyl groups.

The GPC analysis provided a comprehensive characterization of the polysaccharidic extract offering insights into its thermodynamic and physical properties. The extract has an average molecular weight (Mw) of 227,781 and a number average molecular weight (Mn) of 139,622, resulting in an Mw/Mn of 1.6 (Figure 1). These values are considered discrete values for polysaccharides, which is often associated with biodegradable polymers.

Collectively, these findings strongly suggest that TPL‐Ofi is a carbohydrate‐rich compound that does not contain proteins, being therefore considered a polysaccharidic compound.

3.2. TPL‐Ofi attenuates clinical signs of inflammation

Animals were subjected to treatment with three different doses of TPL‐Ofi (0.01, 0.1 and 1%), and the clinical signs of inflammation were evaluated up to the 10th‐day post‐wounding (d.p.w.). On the second‐day post‐wounding (d.p.w.), all tested doses (0.01%–1%) demonstrated a significant reduction in oedema compared to saline‐treated animals (data not shown). Nonetheless, the study proceeded with the 0.1% dose, as it presented effectiveness during the inflammatory phase of wound healing, reducing both oedema and hypernociception (Table 2).

TABLE 2.

Wounds clinical and histological parameters evaluated in a 14‐day time course.

Day post‐wounding
Parameters	Groups	2	5	7	10	14
Hypernociception a	Saline	106	104	121	133	161
	TPL‐Ofi	123*	121	123	137	167
Oedema b	Saline	1 (0;2)	0 (0;2)	0 (0;0)	0 (0;2)	–
	TPL‐Ofi	0.5 (0;2)*	0 (0;1)	0 (0;1)	0 (0;2)	–
Hyperaemia b	Saline	1 (0;3)	0 (0;2)	0 (0;3)	1 (0;3)	–
	TPL‐Ofi	1 (0;2)	0 (0;2)	0 (0;2)	0 (0;3)	–
Exudate b	Saline	0.5 (0;2)	0 (0;1)	0 (0;1)	0 (0;2)	–
	TPL‐Ofi	0 (0;2)	0 (0;2)	0 (0;1)	0 (0;3)	–
Crust detachment (f%) c	Saline	–	93	41	82	8
	TPL‐Ofi	–	93	59	82	33
Granulation tissue (f%) c	Saline	–	–	41	100	100
	TPL‐Ofi	–	5	36	100	100
Histopathological scores b	Saline	3.5 (3;4)	4 (3;4)	2.5 (1;3)	1 (1;1.5)	1 (0;1)
	TPL‐Ofi	4 (3;4)	4 (3;4)	2 (1;2)	1 (1;2)	0 (0;1)

^a Mean ± S.E.M. (n = 6/group; *p < .05 vs. saline). Two‐way ANOVA and Bonferroni post‐test. Clinical signs of inflammation scores: 0 – absent, 1 – mild, 2 – moderate, 3 – intense (median [minimum, maximum] and histopathological scores): (0) absence of ulcer/fibroses and mononuclear inflammatory infiltrate; (1) absence of ulcer/fibrosis with mild mononuclear inflammatory infiltrate; (2) presence of ulcer/fibrosis with moderate mononuclear inflammatory infiltrate; (3) presence of ulcer/intense mononuclear inflammatory infiltrate; (4) presence of ulcer/acute process (dilated vessels, mixed inflammatory infiltrate with neutrophils).

^b Mann–Whitney and Dunn's test (n = 6/group; *p < .05 vs. saline). Hypernociception was evaluated by electronic von Frey.

^c Frequency (f%) (chi‐square; n = 6/group/experimental day. Crust detachment and granulation tissue were evaluated by presence or absence, *p < .05 vs. saline).

Starting on the 5th d.p.w. and persisting up to the 14th d.p.w. the wounds presented a crust with granulation tissue beneath. By the 7th d.p.w., crust detachment was more pronounced in TPL‐Ofi‐treated animals, although the percentage value (59% vs. 41% for saline‐treated animals) did not reach statistical significance. Granulation tissue was present in both groups beginning at the 7th d.p.w.

Regarding nociception evoked by the inflammatory process, there was a significant increase in the mechanical threshold in TPL‐Ofi‐treated animals on the second (123.6 ± 6.24 g vs. saline = 106 ± 4.49 g) (Table 2), suggesting the modulation of inflammation at the injury site by the treatment.

3.3. TPL‐Ofi changes inflammatory histological profile in cutaneous wounds

Microscopical analysis of the cutaneous wounds did not reveal significant differences between the groups during the healing time course. Both groups exhibited intense inflammatory infiltrate (mainly polymorphonuclear and mononuclear cells), oedema, dilated vessels and focal necrotic areas at the 2nd and 5th d.p.w. The histopathological median scores were identical for both groups on both days. By the seventh d.p.w., the TPL‐Ofi‐treated group presented fibrosis with a moderate mononuclear inflammatory infiltrate and some degree of re‐epithelization, whereas the saline group exhibited poor re‐epithelization and lacked fibrosis, indicating a potentially better healing outcome for the TPL‐Ofi‐treated group, although statistical significance was not achieved on the histopathological score analysis. At the 10th and 14th d.p.w., both groups presented complete re‐epithelization with fibrosis and mild mononuclear infiltrate (Table 2, Figure 2).

FIGURE 2.

Histological profile of cutaneous wound at different time‐points (days 2, 5, 7, 10 and 14 post‐wounding). Red arrowheads indicate the epithelium, and the black dashed area indicates the cicatricial tissue formation (Nikon Eclipse H550S microscope; 100× magnification).

Throughout the days evaluated (2nd, 5th and 7th d.p.w.), there was a significant presence of mononuclear cells and a less intense infiltrate of polymorphonuclear cells (Figure 3A). Fibroblasts/myofibroblasts and blood vessels were counted to assess tissue remodelling. At the 5th d.p.w., there was a significant increase in both number of fibroblast/myofibroblast cells (2.4×) and blood vessels (1.9×) in animals treated with TPL‐Ofi (Figure 3B,C).

FIGURE 3.

Inflammatory profile (histopathological cells profile and oxidative stress markers) following treatment with TLP‐Ofi. (A) Absolute number of mononuclear cells and polymorphonuclear cells, counted on slides stained with H&E. Cells were counted on 6–8 fields per slide (n = 6 slides/group). (B) Absolute number of fibroblast/myofibroblast. (C) Quantification of new blood vessels. (D) Quantification of myeloperoxidase (MPO) in wound samples. (E) Quantification of glutathione in wound samples and (F) Fibroblast growth factor 2 (FGF‐2) staining score. One‐way ANOVA and t‐test. *p < .05 vs. saline.

Despite no observed differences in the number of polymorphonuclear cells, MPO activity quantification was performed due to the reduction in oedema and more efficient healing in TPL‐Ofi animals. This reduction suggests that the injury site may be subjected to fewer pro‐inflammatory molecules. A significant reduction in MPO was found at the 5th d.p.w. (27.6 ± 11 vs. saline: 110 ± 58.3 mg/g), and although not statistically different, there was also a marked reduction on the 2nd d.p.w. (73.6 ± 50.3 vs. saline: 156.1 ± 49.5 mg/g) (Figure 3D). Furthermore, the levels of GSH (Figure 3E), an antioxidant product, were increased in the TPL‐Ofi‐treated animals' cutaneous wounds at the 2nd d.p.w.

Finally, fibroblasts/myofibroblast and blood vessels were elevated at the 5th d.p.w. (fibroblasts/myofibroblasts: 37.8 ± 9.6 vs. saline 15.8 ± 4.5; blood vessels: 25.25 ± 4 vs. saline: 13.2 ± 2.5) (Figure 3B,C). Consequently, there was a significant immunostaining for FGF‐2 in the connective tissue cells of the cutaneous wounds of the animals treated with TPL‐Ofi at the 5th and 7th d.p.w. (Figures 3F and 4).

FIGURE 4.

Immunohistochemistry of fibroblast growth factor 2 (FGF‐2) in cutaneous wounds at different time‐points (days 5, 7 and 10). Red dashed circles show positive FGF‐2 cells (in brown) clusters (Leica Microscope; 400× magnification).

3.4. TPL‐Ofi does not affect body homeostasis

To assess the biological safety of TPL‐Ofi treatment, haematological and biochemical tests were conducted on plasma samples collected from animals treated with TPL‐Ofi topically for 21 days, twice a day (Table 3). The numbers of red blood cells, leukocytes and lymphocytes remained unaltered, as did platelet counts. Biochemical parameters were unaffected by TPL‐Ofi treatment, except for glucose levels, which were reduced in treated animals, although this change did not reach statistical significance.

TABLE 3.

Haematological and biochemical parameters evaluated on day 28 post‐treatment to assess TPL‐Ofi biological safety.

Parameters	Saline a	TPl‐Ofi a
Red blood cells (106/mm3)	7.77 ± 0.26	7.72 ± 0.29
Haemoglobin (g/dL)	14.37 ± 0.44	13.00 ± 0.45
Haematocrit (%)	45.18 ± 0.64	45.62 ± 1.21
Leukocytes	9.93 ± 2.85	7.40 ± 2.10
Lymphocytes	7.23 ± 1.73	7.02 ± 2.75
Total cell count (Monocyte, basophile and eosinophil)	0.68 ± 0.43	0.45 ± 0.26
Granulocytes	2.01 ± 0.72	0.88 ± 0.08
Lymphocytes (%)	74.83 ± 3.91	66.75 ± 5.85
a Medium cells (%)	5.76 ± 2.16	5.50 ± 1.50
Granulocytes (%)	19.40 ± 3.00	18.00 ± 3.29
Total platelet count	811.0 ± 93.82	791.0 ± 165.0
Platelets (%)	0.56 ± 0.06	0.43 ± 0.18
ALT (U/L)	55.5 ± 2.5	49.5 ± 5.5
AST (U/L)	163.5 ± 133.5	164.5 ± 84.5
Creatinine (mg/dL)	0.32 ± 0.01	0.22 ± 0.01
Cholesterol (mg/dL)	132.5 ± 8.5	141 ± 15.0
Triglycerides (mg/dL)	147.5 ± 21.5	141.5 ± 61.5
Glucose (mg/dL)	128.5 ± 8.5	84 ± 32
Amylase (U/L)	773.5 ± 36.5	817 ± 17.0
Albumin (g/dL)	3.05 ± 0.16	2.76 ± 0.01
Total proteins (g/dL)	6.22 ± 0.12	6.28 ± 0.01
Uric acid (mg/dL)	3.42 ± 0.97	3.08 ± 0.01
Urea (mg/dL)	62.5 ± 2.5	38 ± 10

Abbreviations: ALT, alanine transaminase enzyme activity; AST, aspartate transaminase.

^a Mean ± S.E.M. (n = 6/group; *p < .05). Two‐way ANOVA and Bonferroni post‐test.

4. DISCUSSION

This study has demonstrated that the polysaccharide extract from O. ficus‐indica cladodes (TPL‐Ofi) can attenuate inflammation and nociception in a rat cutaneous wound model by improving the redox balance and enhancing the proliferative phase of repair.

Polysaccharides obtained from natural sources have gained significant attention in the development of wound dressings due to their abundant availability in nature, biocompatibility, stability and biodegradability. ³⁰ They are particularly useful in dressings that provide a scaffold for epithelial cells and support revascularization, allowing wounds to heal more rapidly. ³¹ Numerous studies have demonstrated that biochemical components of Opuntia plants specieshave anti‐inflammatory, immunomodulatory, anti‐tumour and antioxidant properties. ³² , ³³ , ³⁴ , ³⁵ However, no prior research has specifically examined the role of polysaccharides from O. ficus‐indica in these processes. To the best of our knowledge, our study is the first to demonstrate modulatory effects of O. ficus‐indica polysaccharide extract on the inflammatory and healing process.

The carbohydrate composition of TPL‐Ofi was analysed using two physicochemical analytical methods: FT‐IR and GPC. The results confirmed the nature of our extract, which is rich in carbohydrates and hydroxyl groups. ³⁶ , ³⁷ This classification places TPL‐Ofi in proximity to biodegradable polymers.

A substantial body of research has provided compelling evidence that plant‐derived polysaccharides can effectively modulate oxidative stress, inhibit tumour growth and improve the inflammatory profile by reducing the production of myeloperoxidase (MPO) and inflammatory cytokines. ⁶ , ³⁸ , ³⁹ In our study, TPL‐Ofi treatment resulted in the reduction of clinical signs of inflammation in wounds, particularly oedema and nociception. These findings are in accordance with other studies that also used polysaccharides extracts obtained from plants, such as Pereira et al., ⁷ Silva‐Leite et al. ⁴⁰ and Park et al. ¹³

Park et al. ¹³ demonstrated that an ethanolic extract of O. ficus‐indica cladodes administered orally reduced oedema induced by carrageenan in the paw of rats. Regarding nociception, our study revealed a decrease in early hypernociception, which may be associated with the reduction in clinical signs of inflammation such as oedema, observed on the second‐day post‐wounding, followed by a decrease in MPO activity on day 5. The lack of difference in polymorphonuclear cell numbers could be attributed to the chosen time‐point and experimental approach, as the infiltration of these cells greatly decreases by the fifth day of an inflammatory process. ² Moreover, the histology methods used to identify them are not as specific as MPO quantification. Our findings suggest that the topical treatment could also be modulating the oxidative stress by increasing GSH on the second day, favouring the environment for the reduction of clinical signs of inflammation and the reduction of the early activity of the myeloperoxidase on the fifth day.

The recruitment of leukocytes marks the onset of the inflammatory phase, with neutrophils being the first cells to arrive at the injured site, followed by macrophage infiltration. ² These cells play crucial roles in the wound healing process via stimulating reactive oxygen species (ROS) production by neutrophils, ⁴¹ cytokine secretion and phagocytosis of cellular debris and bacteria. Macrophages, in particular, promote angiogenesis through the release of tumour necrosis factor‐alpha (TNF‐α) and FGF which are mitogenic factors for fibroblasts, endothelial cells and other mesenchymal cells, all of which are important for tissue repair. ⁴⁰

In our study, TPL‐Ofi decreased MPO activity and increased GSH at the second and fifth days post‐wound, indicating a modulation of the oxidative stress. It also increased fibroblast/myofibroblast proliferation, blood vessels and FGF release on day 5 post‐wounding. The absence of FGF signalling can significantly impair wound healing. ⁴² , ⁴³ Collectively, these findings suggest a role for polysaccharides in modulating the proliferation phase of wound healing. These results align with previous study with polysaccharide‐rich extracts obtained from plants such as Caesalpinia ferrea Mart, which increased angiogenesis and the expression of transforming growth factor‐beta 1 (TGF‐β) in cutaneous wounds, ⁵ and with the study by Zhang et al., ⁴⁴ which showed that oral administration of a polysaccharide extract from Sanguisorba officinalis L. in mice accelerated angiogenesis through increased vascular endothelial growth factor (VEGF) production.

While plant components are widely used by folk medicine, it is important to consider their potential toxicity. In our study, we did not observe any changes in body mass, organ mass, haematological or biochemical parameters after 21 days of treatment with TPL‐Ofi. These results are consistent with existing literature, emphasizing the safety of such compounds. ⁶ , ⁴⁵

5. CONCLUSION

The topical treatment with the polysaccharide‐rich extract obtained from O. ficus‐indica cladodes has beneficial effects in the inflammatory and proliferative phases of wound healing by reducing oxidative stress and increasing the presence of blood vessels, fibroblast/myofibroblast and FGF‐2 immunoexpression.

5.1. Limitations

This study has some limitations that must be highlighted. First, the study was exclusively conducted in female rats, raising the question of potential gender‐specific effects of the polysaccharide extract on males. Second, the assessment of toxicity could benefit from a more comprehensive approach, incorporating morphological studies and a broader array of biochemical parameters. Finally, further research involving chronic wound models would greatly contribute to our understanding and validation of the polysaccharide extract's potential as a component in topical formulations for wound healing.

AUTHOR CONTRIBUTIONS

Conceptualization, methodology, investigation, data curation, writing – original draft preparation: Beatriz Lima Adjafre and Iásly Costa Lima. Investigation, data curation, visualization: Ana Paula Negreiros Nunes Alves, Rafael Aires Lessa and Arcelina Pacheco Cunha. Data curation, methodology, writing – original draft preparation, formal analysis: Maria Gonçalves Pereira. Conceptualization, supervision, validation, writing – reviewing and editing: Mário Rogério Lima Mota and Ana Maria Sampaio Assreuy.

FUNDING INFORMATION

This research was supported by CAPES, CNPq and FUNCAP. Ana Maria S. Assreuy is senior investigator of CNPq (Process No. 308433/2017‐3).

CONFLICT OF INTEREST STATEMENT

The authors declare no competing financial interest.

Adjafre BL, Lima IC, Alves APNN, et al. Anti‐inflammatory and healing effect of the polysaccharidic extract of Opuntia ficus‐indica cladodes in cutaneous excisional wounds in rats. Int J Exp Path. 2024;105:33‐44. doi: 10.1111/iep.12498