Hibiscus Sabdariffa Topical Extract Ointment Inhibits Inammatory Signals and Exhibits Antibacterial Activity on the Rat Burn Injury: Emerging Approach for New Application in the Supercial Burn Care

Wound healing comprises organized events involving tissue repair and regeneration. The discovery of Toll-like receptors (TLRs) sheds recent light on the mechanisms involved in initiating inammatory responses throughout the healing cascades. Hibiscus sabdariffa (HS) components may exhibit a wound healing action owing to their antioxidant and anti-inammatory activities. This study designed to investigate the effect of HS loaded in an ointment base on the rat burn model compared to that of the standard Iruxol® ointment. A burn injury model was used to evaluate the wound healing potency of the preparation. Rats were treated with ointments three times through 24 hrs. Findings revealed that strong antioxidant property of the HS-loaded ointment augmented the skin healing potential by stimulating biomarkers required for skin regeneration. HS repressed the burning-induced inammation by the effective reduction in the levels of tumor necrosis factor α (TNF-α) and IL-6 through TLR4 protein inhibition. Topical HS inhibited hypertrophic scarring by downregulating transforming growth factor-beta (TGF-β) levels. HS extract possesses a potential bactericidal activity against highly resistant clinical isolates of Pseudomonas aeruginosa. Altogether, study proclaims that HS-loaded topical preparations could be a valuable product that serve as adjuvants to accelerate burn wound healing through inactivating the TLR4 pathway. group showed a signicant elevation in the intensity of the burn compared with that of the normal unexposed group. The Iruxol-treated group showed that the intensity was signicantly decreased to 40% compared to the burn group, whereas it was signicantly increased only to 20% compared to the normal group. The HS-treated group showed that the intensity was signicantly decreased by 50% compared to the burn group and was signicantly increased to 30% compared with the normal group.


Introduction
Wounds are physical injuries caused by opening or breaking of the skin. This skin injury causes disturbance in the anatomy and function, resulting in the discontinuity of the epithelium with or without the loss of the skin connective tissue. The process of wound healing comprises a cascade of biochemical and cellular events that include tissue repair and renewal. The stages of wound healing involve four processes: hemostasis, in ammation, proliferation, and maturation [1,2].
In the in ammatory stage, impaired cells, bacteria, and pathogens are detached from the wound space. Nutrients, enzymes, white blood cells, and growth factors cause swelling, ache, and ush that are generally observed through this phase of wound healing. In ammation, as a natural component of the healing process, is problematic only when it is persistent or excessive [3]. In ammatory cytokines stimulate growth factors such as TGF-β and a group of broblast growth factors, leading to in ltration of stimulated broblasts to the wounded area. Although an accurate level of cytokines is vital for healing, abnormal levels of proin ammatory cytokines lead to disproportionate broblast production, thereby initiating hypertrophic scarring, which lead to substantial deformity in skin tissue. Therefore, proin ammatory cytokines and growth factors play a pivotal part in the consequences of in ammation [4]. In spite of the substantial role of macrophages in wound healing, as they produce more proin ammatory cytokines (TNF-α) and interleukin (IL-6) in early wounds and less TGF-β, whereas the opposite is observed in later stage of wound healing [5].
All TLRs have an extremely homologous cytoplasmic toll/interleukin (IL)-1 receptor domain (TIR), a transmembrane domain, and an extracellular domain. TLR1, 2, 4, 5, 6, 10, and 11 locate on the exterior of the cell membrane, whereas TLR3, 7,8,9,12, and 13 are extant on the membranes of intracellular compartments such as endoplasmic reticulum and lysosomes. TLR4 signaling is one of the vital mechanisms for in ammation [5,6], and is a major receptor involved in different physiological circumstances, including wounds, shock, and autoimmunity. TLR4 has been explored as a target to inhibit interactions, deliver signaling molecules that initiate in ammatory responses, sustain epithelial cell integrity, and encourage recovery from injury [7]. Furthermore, new insights have shown that TLR4 stimulation enhances TGF-β activity. Tissue damage caused by wounds leads to resident accumulation of endogenous TLR4 ligands named "damage-associated molecular patterns (DAMPs)" such as bronectin-EDA and tenascin-C. DAMPs in sequence stimulate innate immune signaling in local broblasts via TLR4, resulting in augmented matrix and TGF-β production [8].
Wounds and burns are easily infected with microbes [11,12]. Microbial infections of wounds slow down the healing process and may badly deteriorate to systemic infections. Topical antiseptics and antimicrobial therapy are crucial to control microbial colonization thus preventing the development of invasive infection [13]. P. aeruginosa is a leading cause of burn wound infection leading to fatal issues if not properly treated. P. aeruginosa is inherently resistant to most antibiotics [14,15] which represents further challenge to manage burn wounds infected with such bacteria.
Hibiscus sabdariffa L. (roselle) (HS), belonging to the Malvaceae family, grows in several countries. This plant is commonly used in traditional remedies for its abundant content of phytochemicals such as polyphenols. HS is used in the treatment of several progressive diseases such as hyperlipidemia, hypertension, cancer and in ammatory diseases of the kidney and liver. HS active constituents are known to have a preventive effect against chronic and degenerative diseases associated with oxidative stress [16]. Its polyphenol content, particularly anthocyanins, may be responsible for its antioxidant capability.
Among its anthocyanins, 85% comprise delphinidin-3-sambubioside (Dp3-Sam), which is the major source of the antioxidant effect of roselle extract. Dp3-Sam is isolated from dried calices, could inhibit proliferation of human leukemia cells in vitro by inducing apoptosis, which was characterized by the activation of caspase-3, -8, and -9 and cell morphology, DNA fragmentation, and inactivation of poly (ADP) ribose polymerase (PARP). In addition, recent studies show that anthocyanin aglycons, especially cyanidin (Cy) and delphinidin (Dp), could reverse the in vitro expression of in ammatory mediators such as prostaglandin (PG) E2 and cyclooxygenase (COX-2) [17]. The antibacterial potential of HS extracts was previously evaluated against broad spectrum of bacterial strains [18,19], however it is necessary to test this activity against P. aeruginosa clinical isolates obtained from wound burn infection.
Emergency of burn cases is crucial in the rst few hours and would in fact account for morbidity and mortality. This study investigates whether roselle extract has the potential to perform as an active wound healing agent, and to test whether such potential is antioxidant, anti-in ammatory activities in in vivo model and evaluate the bactericidal activity against wound isolates of P. aeruginosa.

Collection of plant and preparation of the extract
Fresh owers of HS obtained from Aswan cultivars, and then were seeded in the botanical garden of the Faculty of Pharmacy, Delta University for Science and Technology. Extraction was performed as described by [20]. Brie y, owers were shade-dried at 25°C, milled using a grinder, and sieved. Next, 372.58 g of ne ower powder were suspended in 1000 ml of 70% ethyl alcohol for 48 hours at RT. The residue was removed by ltration (1-mm pore size), and then the extract was effectively concentrated using a rotary evaporator for 1 h. The solid concentration was measured, then the extract was aliquoted and kept at −20°C for further experiments.

Chemicals and reagents
Liquid para n and white soft para n were supplied from Pharmaceutics Department, Delta University for Science and Technology. Iruxol® ointment (1.2 IU, Smith nephew, Knoll Farmaceutica SpA, Muggio, Italy) was used as a standard ointment for comparing the wound healing potential of the plant extract.
Analytical grade chemicals and reagents were used.

Formulation of the ointment
The ointment formulation (10% (w/w)) was prepared by infusing 10 g of the extract into 100 g of a simple ointment base (British Pharmacopoeia) [22]. It was prepared using a simple ointment base (liquid para n (33.32 g), white soft para n (144.44 g), to which 22.22 g of the extract was added. First, all the extract and liquid para n were supplemented and mixed with constant shaking in a water bath at 60°C to conquer a gel-like uniformity. White soft para n was allowed to melt, then the drug and liquid para n mixture were added portion-by-portion onto the melted white soft para n and mixed with constant stirring.

Evaluation of ointment stability
The freshly prepared cooled ointment was evaluated for color and appearance by visualization, and its consistency and homogeneity were also evaluated by rubbing within the fore and rst ngers. Each ointment sample were located in a wide-mouthed plastic container and kept at −5°C, 27°C (RT), and 40°C.
The homogeneity, color/appearance and consistency of these samples were checked daily for 14 days for any alterations. The pH values of the freshly prepared ointment were determined using a pH meter (accumet research AR10, Singapore). Ointment samples were put in storage at 27°C (RT), and their pH were checked daily for 14 days. Next, 5 g of each ointment sample were relocated into a 10-ml beaker for experimentation.
2.6 Testing for acute skin irritation and toxicity of the ointment The ointment was used for evaluating acute irritation and toxicity of the skin at 10% w/w according to the OECD guideline 402. The ointment was applied after shaving dorsal hair of rats. The treated region was checked for adverse dermal reactions such as erythema, irritation, in ammation, and itching compared to the control group treated with the ointment base but without the drug (placebo).

Animals and experimental protocols
Experimental protocols followed the ARRIVE guidelines of the National Centre for the Replacement, Re nement and Reduction of Animals in Research (NC3Rs). Adult male Wistar rats, with a typical weight of 200-250 g, were obtained from the animal facility at the faculty of pharmacy, Delta University (FPDU), and were accommodated in polypropylene cages with ad-lib access to basal diet and water. Animals allowed to acclimatize for one week.. Furthermore, all procedures were approved by the Institutional Animal Care and Use Committee at the FPDU. Rats were anesthetized with thiopental anesthesia (40 mg/kg body weight) before commencing experimental wounds and surgical interventions were aseptically performed. Only healthy animals free from infections were used. Wound models were commonly used to evaluate the wound healing activity [23].

Induction of burn
Burn wounds were done as described by Cai et al. [24]. Brie y, the dorsal back of rats was smoothly shaved. A 100-g cylindrical stainless steel rod (1cm-diameter) was heated to 100°C in boiling water. Contact burns were induced by placing the heated rod on the dorsum for 8 sec, the full 1 cm-diameter wounded tissue was collected including epidermis, dermis and hypodermis, in which it was only one wound per animal. This procedure has been con rmed histologically to induce full-thickness injuries.
Animals were divided into four groups of six each as follows: Group I: Normal topically applied with the ointment base (Negative control, placebo).
Group II: Burn-wounded topically applied with the ointment base (Positive control, placebo).
Group III: Wounded topically applied with the standard Iruxol® ointment.
Group IV: Wounded topically applied with the prepared roselle extract ointment.
The respective ointments were applied to the entire wound area three times through 24hrs.

Collection of tissues
Tissues from control and test groups were harvested after 24-hrs exposure to the wound and treatments.
Tissues were washed in cold saline (0.9% NaCl) to remove blood. The harvested cylinder-like tissue was cut longitudinally into two halves. One portion was xed in 10% formalin for 24 h for histopathological examinations. The second half was stored at −80°C until further determination of antioxidant and in ammatory markers. To determine antioxidant and in ammatory markers, the harvested skin tissue was homogenized in Tris Buffered Saline (TBS) [50 mM Tris-HCl: 150 mM NaCl, 1:2] at pH 7.4 in Ultra-Turax homogenizer. The homogenates were then centrifuged at 4°C (5500 g, 15 min).

Assessment of burn intensity
Burn intensity was assessed based on a collective arbitrary scale depending on the following aspects: blistering, swelling, redness, crust, secretion, and scar tissue formed. A score was given to each clinical feature when being established as follows: None, 0; mild, 1; moderate, 2; and severe, 3. The scoring criteria were blindly visualized by a physician who is an experienced dermatologist in the eld of skin care. The mean of a scoring criterion was calculated for each experimental group and added to the mean of every other individual scoring criterion to produce the total burn intensity value. In other words, the burn intensity value in a rat group is the total of the means of the scoring of every clinical feature as designated by the dermatologist.

Histological and immunohistochemical analysis
Standard histological technique was conducted. Tissue samples were stored in 10% formalin buffer solution, dehydrated, and then blocked with para n wax. Each blocked skin sample was cut into 5-µmthick sections and stained with hematoxylin and eosin (H&E) stain. The skin sections were checked under light microscope, and microphotographs were captured. Tissues obtained from the test group animals were also stained with Masson's trichrome, which is used to identify collagen deposition. Areas were quanti ed as area (A) % using the Image J 1.52n software. The extent of collagen production was expressed as the % of stained area relative to the total area. IL-6 immunolabeling was developed using IL-6 antibodies (NB600-1131, Novus Biologicals, Littleton CO, USA). The immunoreactivity was indicated by calculating the immunopositive cells/ 1000 cell count.

Determination of total antioxidant capacity
Total antioxidant capacity (TAC) was determined using an enzymatic colorimetric method. All measurements followed the instructions of the manufacturer of the kit (Biodiagnostic, Egypt). The antioxidant capacity was determined based on the reaction of antioxidants in the sample with a de ned amount of exogenously provided H 2 O 2 . The antioxidants present in the sample reduce a certain H 2 O 2 amount. The remaining H 2 O 2 is calorimetrically assayed in an enzymatic reaction that comprises the conversion of 3,5-dichloro-2-hydroxybenzenesulfonate into a colored product.

Determination of in ammatory markers
The protein concentrations of TNF-α and TGF-β were quanti ed using ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Absorbance was read against blank in a Spectrum Max Plus 384 spectrophotometer (Sunnyvale, CA, USA), at dual wave lengths of 450 nm and 570 nm. Cytokine's concentrations were calculated using standard curves obtained from recombinant cytokine activities.

Western blot analysis for TLR4 expression
Protein extraction from the skin tissues was performed using Radioimmunoprecipitation assay buffer (RIPA) (Bio Basic Inc., Markham Ontario, Canada). Protein concentrations were estimated using the Bradford method. For each lane, 20 µg of protein was used and separated by 10% Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis SDS-PAGE (Bio-Rad Laboratories, TNC, USA) and then transferred electrophoretically to pol-vinylidene uoride (PVDF) membranes (Bio-Rad Laboratories, Inc.).
The membranes were then incubated at RT for 2 hrs with a blocking solution consisting of 5% non-fat dried milk in TBST (10 mM Tris-Cl, 100 mM NaCl, and 0.1% Tween 20 (pH 7.5)). Next, the membranes were incubated overnight at 4°C with monoclonal antibodies speci c for TLR4 and GAPDH and then with rabbit anti-rat secondary antibody conjugated to horseradish peroxidase (Thermo Fisher Scienti c Inc., USA) at RT for 2 hrs. This was followed by washing four times with TBST at RT after each incubation. A chemiluminescent substrate (Clarity™ Western ECL substrate, BIO-RAD, USA) was used to detect chemiluminescence according to the manufacturer's instructions. Then, the chemiluminescent signals were captured using a CCD camera-based imager, followed by image analysis to read the band intensity of the target proteins against the control sample after normalization by GAPDH on the Chemi Doc MP imager.
In vitro antibacterial activity of HS ethanolic extract was determined using cut-well agar diffusion [26]. Brie y, each bacterial isolate was grown in in 20 mL of tryptone soya broth medium (TSB) at 37 •C for 2 days with constant shaking at 150 rpm. 100 uL of 0.5 McFarland turbidity standards (1.0 × 108 CFU/mL) of each strain were streaked over a Mueller-Hinton agar plate using a cotton swab and wells of 6 mm were prepared. Then, 150 µL HS ethanolic extract at a concentration of 250 mg/mL was loaded to the well. The plate was incubated for 24 h at 37°C. The experiment was done in triplicates and the mean diameter of the zone of inhibition around the well was scored.

Statistical analysis
Data were expressed as the mean ± standard deviation (SD). Statistical analysis was conducted using the GraphPad Prism version 6 (GraphPad Software Inc., La Jolla, CA, USA). Differences between groups were examined using one-way ANOVA, followed by Tukey's Kramer multiple comparison test. Scores of burn intensity were expressed as the median ± interquartile range and were compared using Kruskal-Wallis followed by Dunn's multiple comparison test.

Effect of HS on the intensity of 2nd degree burn wound
The effect of HS on the intensity of 2nd degree burn is presented in Table 1 and Fig. 1. After 24 hrs of exposure to damage, the burn group showed a signi cant elevation in the intensity of the burn compared with that of the normal unexposed group. The Iruxol-treated group showed that the intensity was signi cantly decreased to 40% compared to the burn group, whereas it was signi cantly increased only to 20% compared to the normal group. The HS-treated group showed that the intensity was signi cantly decreased by 50% compared to the burn group and was signi cantly increased to 30% compared with the normal group.

Effect of HS on collagen deposition
The effect of HS on collagen deposition is shown in Fig. 2. Collagen deposition signi cantly increased in the burn group compared with that of the normal group. Collagen deposition was signi cantly decreased in the Iruxol-treated group compared to burn group (p < 0.01). Additionally, the HS-treated group showed a signi cant decrease in collagen deposition compared to that of the burn group (p < 0.05).

Histopathological examination of skin tissues
The photomicrographs of the H&E and the Masson's trichrome staining revealed normal epidermal layer formed of strati ed epithelium with marked keratinization and normal dermal layer contained numerous hair follicles in normal skin group. The 2nd degree burn group exhibited minimal degree of epithelialization with subepithelial edema. However, treatment with either Iruxol or HS resulted in marked epithelialization degree and normal dermal hair follicle appendages. Masson's trichrome staining indicated that the normal dermal layer consisted primarily of parallel collagen. Burn group was associated with dermal collagen formation. In contrast, Iruxol or HS treatments displayed minimal dermal collagen formation and resulted in a marked epithelialization degree with normal dermal hair follicle appendages Fig. 3.

Immunostaining of IL-6
A marked expression of IL-6 within the dermal layer of the burn group as indicated upon calculation of the % immunopositive cells/ 1000 counted cell was clearly displayed. While the treated groups with either Iruxol or HS showed a marked decrease in the expression of IL-6 Fig. 4.

Effect of HS on total antioxidant capacity (TAC)
The burn group showed a signi cant (P < 0.05) decrease in the level of TAC compared to the normal group. In the Iruxol-treated groups, there was a non-signi cant change in the levels of TAC compared to the burn group. However, in the HS-treated groups, there was a signi cant increase in the levels of TAC compared to the burn group (P < 0.05) Fig. 5.

Effect of HS on TNF-α
The burn group showed a signi cant increase in the levels of TNF-α (P < 0.05) compared to the normal group. The Iruxol-or HS-treated group of rats exhibited a signi cant decrease in TNF-α level (P < 0.05) compared to the burn group Fig. 6.

Effect of HS on TGF-β
The burn group showed a signi cant increased levels of TGF-β compared to the normal group. The Iruxoltreated group showed non-signi cant change in the level of TGF-β compared to that of the burn. However, the HS-treated group showed a signi cant decrease those levels compared to the burn group (P < 0.05)

Effect of HS on TLR4 expression in different groups
The burn group showed a signi cant (P < 0.05) increase (8-fold) in the relative expression of TLR4 compared with that of the normal group. The Iruxol-or HS-treated groups showed that TLR4 expression was signi cantly decreased (P < 0.05) compared to the burn group. However, the HS-treated group exhibited a signi cant decrease (P < 0.05) in the expression of TLR4 compared to that of the Iruxoltreated group Fig. 8.

Antibacterial activity of HS on burn wound isolates of P. aeruginosa
We nally evaluated the bactericidal activity of HS extract against 2 clinical isolates of P. aeruginosa obtained from burn wound exudates. While the 2 isolates exhibited prominent resistance against different categories of antibiotics that target the bacterial cell wall, the bacterial ribosomes, or the bacterial DNA replicating enzymes, HS ethanolic extract at a concentration of 250 mg/ml clearly exhibits a bactericidal effect against wound isolates of P. aeruginosa giving zone of inhibition ranging from (24 ± 5) to (33 ± 3.6) mm in diameter Fig. 9.

Discussion
Burn wound healing requires a well-organized cascade of biochemical and cellular events that involve tissue repair and regeneration. HS (roselle) is a widely used commercial traditional medicine, due to which it was primarily selected in our study. This study proposed that the content of polyphenols, especially anthocyanins, in roselle may be responsible for its antioxidant and anti-in ammatory properties essential for burn wound healing [27]. We intended to evaluate the effect of HS alcoholic extract loaded in an ointment base on healing based on its potential to regulate the link between TLR4 and TGF-β signaling pathways. Inhibition of these pathways may stimulate the antioxidant and antiin ammatory effects of HS which may be relevant to the observed skin regeneration in rats with super cial skin excision wounds.
Iruxol® is a topical collagenase preparation used for enzymatic debridement of necrotic tissue caused by ulcers and burns, and hence it was used for the purpose of comparison as a standard treatment in this study. Iruxol® contains the collagenase proteolytic enzyme derived from Clostridium histolyticum. Collagenase digests denatured collagen, cleanses necrotic tissues, and destroys the strands of endogenous collagen, which attach the necrotic residues to the wound bed. Using Iruxol® in uncomplicated wounds is considered to be well tolerated [28]. In the present study, after 24 hrs of Iruxol® treatment, the super cial burns exhibited dramatic healing percentages (40%) with the absence of charring and eschar due to its collagenase activity, which corresponded to the percentage of collagen deposition as evaluated using the Image J software.
In the case of HS ointment, healing occurred signi cantly but at a lower degree than that of Iruxol® treatment. This may be due to the autolytic debridement caused by topically applied natural products such as roselle. Our ndings converge with the autolytic debridement with support from topically applied natural products such as honey, which is believed to go in the same way of the self-healing process to cleanse the wound from tissue debris. The healing activity of honey is attributed to its acidity, suppressing destructive proteases, and osmotic effect on the wound bed [29].
A pivotal crosstalk has been recognized between TLR4 and TGF-β signaling cascades and wound healing. Till now, HS is the rst suggestion to be used an inhibitor for both TLR4, TGF-β and collagen deposition upon tissue damage. Hence, it is recognized that oxidative stress has serious role in the origin and development of wounds. Burning wounds in rats are in close proximity to human wound injuries in terms of histopathological features, characterized by a minimal degree of epithelialization, which signi cantly contributes to the progression of subepithelial edema through the production of dermal collagen [30]. Moreover, tissue injury correlates with strong local immune response that is connected to augmented production of cytokines, particularly TNF-α that can trigger oxidative burst. Effects of both Iruxol® and HS on burn wound healing were con rmed by histopathological examination, which revealed marked epithelialization degree, normal dermal hair follicle appendages and minimal dermal collagen formation in both groups compared with these in burn untreated group.
Emerging evidence indicates that TLR4 participates in the stimulation of innate immune response by triggering signaling cascades along with the release of several in ammatory cytokines such as TNF-α.
Here, we found that HS ointment downregulates TNF-α to <50 pg/ml, as the normal skin TNF-α level is <50 pg/mL [31]. Comparison between Iruxol® and HS treatment results showed that HS treatment resulted in less tissue TNF-α levels than those obtained with Iruxol® ointment treatment, as the latter value was >50 pg/ml. This implied that roselle extract has an anti-in ammatory effect that helps in burn healing. A previous study reported that the levels of IL-6, nitric oxide (NO), and TNF-α in in ammatory cells could be signi cantly reduced by HS treatment.
These in ammatory cytokines lead to the initiation of growth factors release leading to the in ltration of stimulated broblasts to the wounded area. Although the correct level of each cytokine is important for healing, abnormal levels of proin ammatory cytokines lead to extreme broblast proliferation, resulting in hypertrophic scarring, which lead to a robust scarring [32].
In this study, TGF-β level was found to signi cantly decrease after 24 hrs of application of either Iruxol® or HS ointments. Previously, studies reported that new tissue development and scar formation are two crucial steps in the healing process. Fibroblasts are the primary cells in the healing process since they migrate to the wound where they proliferate. Then, these cells produce the extracellular collagen matrix. The functions of broblasts are regulated by growth factors [33]. Recently, there has been a focus on the double-edged weapon of TGF-β level through hypertrophic scarring, which is a common occurrence in burn injuries, with an incidence of 70% [34]. Moreover, the importance of TGF-β level in keeping the vascular activity has been established in several studies, which have shown that increased TGF-β level and its receptors may impair vessels formation [35].
However, the present data justi ed that HS has a greater antioxidant effect than Iruxol® ointment when topically applied three times/day. Numerous in vitro and in vivo reports con rmed that extracts of roselle possess actual antioxidant potential [36]. Wound progression is concomitant with oxidative stress that arises because of the disturbance in the balance between ROS production and endogenous antioxidant protection mechanism, which disturbs the biomolecules functioning in a reversible or irreversible manner [37]. It was established that ordinary cellular antioxidants such as superoxide dismutase and catalase participate in oxygen-protection responses in the tissue matrix by producing water and molecular dioxygen from superoxide. Increasing glutathione level in the tissue matrix results in enhanced glutathione peroxidase which by turn reduces peroxides [38]. The present study results indicate a signi cant increase in TAC in the HS-ointment-applied group as evidenced by the neutralization of additional free radicals in the wound, owing to the phenolic nature of HS, which ultimately encourage a synergistic outcome in wound healing [39].
It has been described that such effects were addressed for both alcoholic and aqueous HS extracts of owers, seeds, or leaves through shielding effect against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage. This activity of HS is due to the inhibition of xanthine oxidase activity [40], cytoprotection from impairment by lipid peroxidation, inhibition of Cu2+-mediated oxidation of LDL and the creation of thio-barbituric acid reactive substances (TBARs), maintaining the formation of malondialdehyde content, reduction of GSH exhaustion, and the increase in SOD and CAT activities [41].
Microbial contamination of wounds and burns greatly slows down the recovery, interferes with the healing process, complicates the management, and may lead to invasive infection. P. aeruginosa is a gram-negative bacterium that is commonly infecting wounds and burns leading to invasive burn sepsis which requires prompt therapeutic intervention [42]. One of the key aspects of burn care guidelines is to prevent infection and colonization of P. aeruginosa, however this pathogen is rapidly developing antimicrobial resistance against most used antibiotics. Broad spectrum antibacterial activity of HS ethanolic extract was previously addressed [43], but our study demonstrated a potential bactericidal activity of HS extract in tolerable concentrations against burn wound isolates of P. aeruginosa that exhibit multidrug resistance against different categories of antibiotics. Taken together, HS extract exhibits a dual role in wound healing through the anti-in ammatory activity which is necessary for the skin regeneration and the antibacterial effect which prevents microbial colonization and invasive infection.

Conclusions
HS ointment has an important role in healing wounds. HS extract enhanced the skin healing capacity by accelerating the healing process itself and stimulating the levels of skin regeneration biomarkers through its strong antioxidant and anti-in ammatory effects. Moreover, HS topical treatment signi cantly reduced hypertrophic scarring through its effect against TGF-β levels. Therefore, it would be encouraging to discover natural medications that have the possibility to control the link between these TLR4 and TGF-β signaling pathways. In addition, HS extract has a remarkable bactericidal activity against wound pathogens. Altogether, this study suggests that HS is a valuable extract to be used in cosmetics and alternative medicine, which may also be considered for use in combination with Iruxol® as a synergistic product to accelerate wound healing. Data availability: The data obtained after analysis in the current study are available from the relevant authors upon reasonable request. Funding: This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.
Ethics approval and consent to participate: The study was conducted according to the ARRIVE guidelines of the National Centre for the Replacement,  Figure 1 Intensity of 2nd degree burn in different groups HS: Hibiscus sabdariffa, +, P < 0.05 vs normal group; *, P < 0.05 vs 2nd degree burn group; * *, P < 0.01 vs 2nd degree burn group. Differences between groups were examined by Kruskal-Wallis followed by Dunn's multiple comparison test. n= 6 Figure 2 Percentage of collagen deposition HS: Hibiscus sabdariffa, +, (p < 0.05) vs normal group; *, (p < 0.05) vs 2nd degree burn group; **, (p < 0.01) vs 2nd degree burn group; @, (p < 0.05) vs 2nd degree burn/Iruxol-treated group. Differences between groups were examined by ANOVA/Tukey's Kramer multiple comparison test. n= 6 Figure 3 Histopathological examination of skin tissues from different groups by H&E staining

Figures
The left panel (A) shows histopathological examination of skin tissues from different groups by H&E staining, ×200. Normal epidermal layer formed of strati ed epithelium with marked keratinization and normal dermal layer, contained numerous hair follicles in the normal skin group (arrow). The 2nd degree burn group exhibited minimal degree of epithelialization (arrow) with subepithelial edema (arrowhead). In contrast, treatment with Iruxol resulted in marked epithelialization degree (arrow) and normal dermal hair follicle appendages (arrowhead). Treatment with HS resulted in marked epithelialization (arrowhead). Bar = 100 µm. The right panel (B) illustrates the Masson's trichrome staining, ×200. Masson's trichrome staining indicated normal dermal layer of primary parallel collagen (arrow). The 2nd degree burn group was associated with massive dermal collagen formation (arrow). In contrast, treatment with Iruxol resulted in minimal dermal collagen formation (arrow). Treatment with HS resulted in minimal dermal collagen formation. Bar = 100 µm.

Figure 4
Immunohistochemical detection of IL-6 in skin tissues Immunohistochemical detection of IL-6 in skin tissues, ×200. Arrows indicate expression of IL-6 within the dermal layers of different groups. Marked immunolabeling was detected in the 2nd degree burn group of rats, while decreased expression was detected in the treatment groups. Bar = 100 µm. +, P < 0.05 vs normal group; *, P < 0.05 vs 2nd degree burn group. Antibacterial activity of HS ethanolic extract against multidrug resistant P. aeruginosa A-Antibiotic sensitivity by DDM of 2 clinical isolates of P. aeruginosa isolated from burn wound exudates against different antibiotics (bacterial isolate was de ned as multidrug resistant when resistance against