Characterization and Wound-Healing Effects of Fucoidan from Sargassum polycystum Collected in South Sulawesi in a Staphylococcus aureus-Infected Rat Model

Characterization and Wound-Healing Effects of Fucoidan from Sargassum polycystum Collected in South Sulawesi in a Staphylococcus aureus-Infected Rat Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization and Wound-Healing Effects of Fucoidan from Sargassum polycystum Collected in South Sulawesi in a Staphylococcus aureus-Infected Rat Model Fausyiah Sasmitha AB, Frederika Tangdi Lintin, Sulistiawati Sulistiawati, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9573456/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The skin, the body's largest organ, acts as the primary barrier against infections, but this function can be compromised by wounds. While antibiotics are commonly used for treating skin infections, their misuse has led to antibiotic-resistant pathogens, emphasizing the need for safer alternatives. Fucoidan has shown antibacterial and anti-inflammatory properties. However, its biological activity can vary due to differences in species and geographical origin. In this study, fucoidan was isolated from Sargassum polycystum collected in the intertidal zone of South Sulawesi, Indonesia, and its wound-healing potential was tested in Staphylococcus aureus -infected rats. Fucoidan was extracted using hot ethanol-water extraction and CaCl₂-ethanol precipitation, and characterized by FTIR, ¹H NMR, and ¹³C NMR. Toxicity was assessed with a hemolysis assay, and wound healing was evaluated in rats with 6 mm excision wounds infected with S. aureus (0.1 mL, 10⁷ CFU/mL). Results showed that fucoidan was non-toxic and that a 2.4% topical application significantly reduced bacterial load and accelerated healing within 9 days. Histological analysis revealed enhanced fibroblast proliferation, granulation tissue formation, re-epithelialization, and thicker collagen deposition compared to the control group (Vaseline only) (P < 0.05). These findings suggest that fucoidan from S. polycystum is safe and effective in promoting wound healing, supporting its potential as an alternative therapy for infected wounds. Brown Algae Fucoidan Sargassum polycystum Wound Healing Inflammation Staphylococcus aureus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The skin is the largest organ in the human body, serving as an excellent protective barrier. However, this barrier may be compromised when the skin is wounded, either by physical objects, chemicals, excessive heat, or other causes (Wen et al. 2023). Although most wounds can heal naturally within 14–30 days through an organized wound-healing process, which restores the function and anatomical integrity of the skin (Morguette et al. 2023 ), the process may also be impeded, due to bleeding, infection, inflammation, and/or impaired angiogenesis and regeneration. Angiogenesis is a critical process in tissue repair following injury and represents an important intrinsic protective mechanism. The formation of new blood vessels is essential for wound healing, as it supplies oxygen and nutrients to cells at the wound site (Wen et al. 2023). Microbial infections are among the most serious problems, as they may lead to morbidity and mortality. Globally, the increased incidence of skin infections has been associated with higher risks of mortality, with a reported case-fatality rate of 20.3% and an annual mortality rate of 3.4 per 100.000 population (Nurul Fitri Marzaman et al. 2022 ). Infections with S. aureus are among the most common causes of morbidity and mortality, as they can spread to the bloodstream and cause sepsis (Elbatouti et al. 2023 ). One compound that has been extensively studied as a wound-healing agent is fucoidan, a unique water-soluble sulphated polysaccharide produced by brown algae (seaweed), which exhibits a wide range of biological activities, including antitumor, anti-inflammatory, antioxidant, anticoagulant, and wound healing (Manggau et al. 2022 ; Abdel-Latif et al. 2022 ). A study by Lu et al. demonstrated that a gelatine–fucoidan–based hydrogel (Gel–Fuc–TA) can modulate the wound immune response by promoting macrophage polarization toward the M2 phenotype, suppressing pro-inflammatory mediators, and increasing the expression of anti-inflammatory cytokines and transforming growth factor-β (TGF-β), thereby contributing to reduced inflammation during wound-healing process (Lu et al. 2022 ). Other studies also reported that fucoidan enhances angiogenesis by activating the AKT/Nrf2/HIF-1α pathway, which accelerates wound healing (Wen et al. 2023a ). In line with these findings, Wang et al. reported that fucoidan- and alginate-based dressings enhanced skin tissue regeneration in animal models, as indicated by accelerated wound closure, reduced inflammatory response, and increased collagen deposition, possibly through activation of the TGF-β1/Smad pathway (Liang et al. 2025 ). Recent studies have also demonstrated that incorporating fucoidan into gelatine and oxidized carboxymethyl cellulose–based hydrogels support wound closure by modulating inflammation. This effect is characterized by suppression of inducible nitric oxide synthase (iNOS) and reduced nitric oxide production in macrophages, as well as enhanced collagen synthesis in human skin fibroblasts, thereby confirming the potential of fucoidan-loaded hydrogels as an effective delivery system for tissue regeneration (Jeong et al. 2025 ). Fucoidan has also been reported to exhibit significant anti-inflammatory and antibacterial activities, which contribute substantially to its therapeutic potential as a wound-healing agent. These effects are mediated by the suppression of pro-inflammatory mediators, particularly tumour necrosis factor-α (TNF-α), and by inhibition of bacterial growth and colonization. Numerous studies have demonstrated that the application of fucoidan-based wound dressings can modulate the inflammatory response while reducing bacterial burden, thereby creating a favourable microenvironment that promotes accelerated tissue regeneration and enhanced antibacterial efficacy in vivo , particularly in infected wounds (Lu et al. 2022 ; Jiang et al. 2024 ). In line with these findings, fucoidans isolated from Fucus vesiculosus have demonstrated bacteriostatic effects against multiple bacterial species, including E. coli and S. aureus . Microscopic analyzes revealed preservation of bacterial cell integrity accompanied by alterations in cell size and surface morphology, effects that were more pronounced in Gram-positive bacteria (Ayrapetyan et al. 2021 ). However, fucoidan is a mixture of structurally complex sulphated polysaccharides that vary significantly depending on the species of seaweed and the geological location, which may also affect its biological properties. Despite these advances, the molecular mechanisms underlying the pro-angiogenic effects of fucoidan during wound healing, particularly in relation to the spatial distribution of neovascularization within the wound area, remain poorly understood. In this study, we evaluated the wound-healing property of fucoidan isolated from the brown algae Sargassum polycystum collected in the intertidal zone of South Sulawesi, Indonesia, using a wound model in rats infected with S. aureus . To the best of our knowledge, this was the first in vivo study in S. aureus -infected rats with histopathological assessments conducted across the inflammatory, proliferative, and remodelling phases. In addition, the anti-inflammatory and antibacterial activities of the fucoidan against S. aureus were systematically investigated. As Indonesia contributes approximately 38% of global seaweed production and ranks as the world’s second-largest producer, the results underscore the immense potential of Indonesian seaweed as source of fucoidan, which can be developed as natural medicines to treat infected wounds (Manggau et al. 2022 ). Materials and Methods Harvesting brown algae The brown algae ( S. polycystum ) were collected from the coast of Takalar Regency, South Sulawesi (5°34'39.8"S 119°25'37.7"E), at a water depth of approximately 5 m. Characterization of Brown Algae Identification of the Brown Algae Identification of the brown algae sample was done at the Environmental and Marine Science Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Hasanuddin University, using microscopy and morphological evaluations (Sundari et al. 2023 ). Isolation of Fucoidan from S. polycystum The brown algae were first washed with seawater, then rinsed with running freshwater to remove contaminants such as sand and salt. Further, it was cleaned with distilled water to eliminate organoleptic properties such as odour and taste. After cleaning, the brown algae were dried at room temperature. Once dried, the brown algae were chopped into small pieces and ground into a fine powder, then sieved using a 100-mesh sieve. Fine brown algae powder (50 g) was then extracted with EtOH (1 L). The mixture was stirred and left for 12 h at room temperature. It was then centrifuged at 2,000 rpm for 10 min. The resulting precipitate was dried overnight at room temperature. Next, the dried powder (5 g) was mixed with water (100 mL) at 65°C for 1 h. The mixture was centrifuged at 15,000 rpm for 10 min. To obtain the supernatant, the solution is mixed with 1% CaCl₂ and left overnight at 4°C to precipitate the alginic acid. The precipitate was then centrifuged at 15,000 rpm for 10 min, and the supernatant was collected and mixed with 99% EtOH to reach a final concentration of 30%. The solution was stored at 4°C for 4 h, then centrifuged at 15,000 rpm for 10 min (Palanisamy et al. 2017 ). Finally, the sample was freeze-dried and ground into a fine powder. The yield percentage was calculated using the following formula (Kim et al. 2021 ). $$\:Fucoidan\:\left(\%\right)=\:\frac{Weight\:of\:dry\:fucoidan\:obtained\:as\:a\:result\:of\:ethanol\:precipitation}{biomass\:weight}\:X\:100%$$ (1) Spectroscopy Analysis Purified fucoidan was analyzed using a Shimadzu® IRPrestige-21 FTIR spectrometer in ATR mode. Spectra were collected over the wavenumber range of 4000–500 cm⁻¹ at a resolution of 4 cm⁻¹, with a scan duration of 32 min (14,15). 1 H and 13 C NMR spectra were acquired using a Bruker Avance III 700 MHz. Fucoidan Hemolytic Activity Test A haemolysis assay was conducted to evaluate the cytotoxicity of fucoidan. Whole blood was collected from Wistar rats and transferred into sterile 50 mL Falcon™ tubes. The samples were centrifuged at 2000 rpm for 10 min to separate plasma from red blood cells (RBCs). The plasma supernatant was discarded, and the RBC pellet was washed four times with phosphate-buffered saline (PBS). The washed RBCs were resuspended in PBS by vortexing for 3 min and then diluted with PBS to obtain a 10% (v/v) RBC suspension (Stephanie et al. 2024 ; Sapiun et al. 2024 ). Then, the mixture was generated in three concentrations of 10, 100, and 1000 µg/ml. For each test, 100 µL of fucoidan solution was mixed with 900 µL of the 10% RBC suspension. The mixtures were incubated at 37°C for 60 min and then centrifuged at 2000 rpm for 10 min. The absorbance of free haemoglobin in the supernatant was measured at 540 nm using a UV–Vis spectrophotometer. RBCs in PBS served as the negative control, whereas RBCs in distilled water served as the positive controlc (Stephanie et al. 2024 ). The hemolytic percentage was calculated according to the following equation (Fontelo et al. 2022 ). $$\:\text{H}\text{e}\text{m}\text{o}\text{l}\text{y}\text{t}\text{i}\text{c}\:\:\left(\text{%}\right)=\:\frac{\left(\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}-\text{N}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}{\text{P}\text{o}\text{s}\text{i}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{N}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\:\text{X}\:100$$ 2 In Vivo Fucoidan Activity Test Study Preparation of Topical Fucoidan from S. polycystum Topical 1.2% and 2.4% fucoidan samples were prepared by mixing fucoidan (120 mg and 240 mg, respectively) with Vaseline base to a total amount of 10 g. Acclimatization of Experimental Animals (Rattus norvegicus) Male rats ( Rattus norvegicus ), aged 3–4 months and weighing 200–250 grams, obtained from the Wisbalb breeder in Indonesia, were used. Before the experiment, the rats underwent a 7–14 days acclimatization period to adapt to the laboratory environment of Pharmacology-Toxicology, Faculty of Pharmacy, Hasanuddin University. The animals were monitored daily to ensure their health. Animals were also weighed weekly to determine their body weight and development. The animals were placed in cages with standard AD II feed and drinking water ad libitum. The animal cages were cleaned twice a week to ensure the level of humidity, comfort, cleanliness, and safety of the animals. The laboratory environment is set with temperatures ranging from 25–30°C, as well as 12 h of light and darkness (dos Santos et al. 2022 ). Preparation of S. aureus Bacterial Suspension All equipment used in the preparation of bacterial suspensions was sterilized using an autoclave at 121°C for 30 min. Each equipment was covered with cotton and aluminium foil to maintain sterility. The medium is prepared by dissolving Nutrient Agar (0.39 g) in distilled water (10 mL) in an Erlenmeyer flask, then heating to dissolve the components fully. Next, the medium is transferred to a test tube and left in an inclined position until it forms agar. S. aureus is then inoculated into the test tube and incubated at 37°C for 24 h. The turbidity of the bacterial suspension was measured by comparing it with a McFarland 1 standard solution using a McFarland densitometer. If the suspension turbidity exceeded the specified standard, 0.9% sterile NaCl solution was added until the appropriate turbidity was achieved (Sartini 2020 ). Acute Wound Infection with S. aureus Fifteen rats were anesthetized using ketamine (0.3 mL) intraperitoneally. The rat dorsal region was shaved until completely clean from hairs. Then, four wounds were made using a biopsy punch on the dorsal surface of each rat with a diameter of 6 mm. The wounds, coded with F1, F2, F3 and F4, were infected with S. aureus (in 0.1 mL bacterial suspension) intradermally. The wounds were then covered using gauze and square parafilm. After the procedure, the animals were placed back into their cages (Elbatouti et al. 2023 ). Experimental Treatment This study used 15 rats, which were divided into 3 groups of 5 rats each. Each rat received specific treatments for four different wound areas: F1 (Nisagon ® wound healing cream was used as positive control), F2 (Vaseline as negative control), F3 (1.2% Vaseline-based fucoidan extract) and F4 (2.4% Vaseline-based fucoidan extract). The treatments were administered daily at noon to all part of the wounds. The treatment was carried out by applying 10 mg of the preparation using a sterile cotton swab. It was also carried out by measuring the initial wound diameter until the healing measurement at the end, for 10 consecutive days, until the experimental animals were analyzed for wounds. 5 experimental animals to represent the wound condition in the inflammatory phase. Group I was made on day 3 and tissue was taken to check the inflammation phase process in macrophages, leucocytes, and ulcers. 5 experimental animals to represent the wound condition in the inflammatory phase. Group I was made on day 6 and tissue was taken to check the proliferation phase process in angiogenesis and fibroblast. 5 experimental animals to represent the wound condition in the inflammatory phase. Group I was made on day 3 and tissue was taken to check the maturation phase process in collagen and re-epithelialization. S. aureus colony counting Bacterial samples were taken using an inoculating loop, transferred into a 5 mL Eppendorf tube containing 0.5 mL of sterile distilled water, and vortexed for 1 min. The suspension (0.1 mL) was transferred into an Eppendorf tube containing 0.4 mL of distilled water and then diluted to 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , and 10 7 concentrations, then vortexed for 1 minute. A sample of 100 µl from each dilution was taken and streaked into a petri dish with sterile count plate agar (PCA) media. Then, the media was incubated for 24 h at 37°C. After 24 hours, the number of colonies was observed in two ways: counting using a colony count and manually counting using a closed petri dish. The colonies were photographed and counted visually from the back of the petri dish. (Makrai et al. 2023 ). Fucoidan Activity in Wound Healing A biopsy punch wound model in rats was used to evaluate the wound-healing activity of fucoidan. Before creating the wound, the rat was weighed and anaesthetized with ketamine intraperitoneally in an appropriate dose 0.3 mL/kg BW. Subsequently, four circular wounds with a diameter of 6 mm each were made and photographed before dressing (Ishi et al. 2023 ; Sari et al. 2024 ). The Wound healing diameter was measured at three different points to ensure accuracy and consistency in wound size on day 0, 3, 6, and 9. Wound contraction was calculated as the percentage reduction in wound area, as shown in the formula below (Moglad et al. 2020 ). \(\:\text{W}\text{o}\text{u}\text{n}\text{d}\:\text{H}\text{e}\text{a}\text{l}\text{i}\text{n}\text{g}\:\left(\text{%}\right)=\:\frac{\left(\text{W}\text{o}\text{u}\text{n}\text{d}\:\text{a}\text{r}\text{e}\text{a}\:\text{o}\text{n}\:\text{t}\text{h}\text{e}\:\text{f}\text{i}\text{r}\text{s}\text{t}\:\text{d}\text{a}\text{y}-\text{T}\text{h}\text{e}\:\text{e}\text{x}\text{t}\text{e}\text{n}\text{t}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{w}\text{o}\text{u}\text{n}\text{d}\:\text{o}\text{n}\:\text{t}\text{h}\text{e}\:\text{l}\text{a}\text{s}\text{t}\:\text{d}\text{a}\text{y}\right)}{\text{F}\text{i}\text{r}\text{s}\text{t}\:\text{w}\text{o}\text{u}\text{n}\text{d}\:\text{a}\text{r}\text{e}\text{a}}\:\text{X}\:100\) % Histopathological Analysis The excised skin tissue was processed through a series of steps and histopathologic examination was performed using light microscope. Fixation: Tissues were fixed using 10% formalin buffer solution with pH 7.0–7.4 for 12 to 18 h. Dehydration: Tissues were immersed gradually in ethanol solutions of increasing concentration, starting with 70% EtOH, followed by 90%, 95%, and finally 100% EtOH. Immersion: Tissues were immersed in first stage xylol (xylol I) for 1 h, then transferred to second stage xylol (xylol II) and incubated for 30 min to 1 h. Immersion: Tissues were embedded in paraffin with a low melting point of approximately 56–59°C for 30 min. Embedding : Tissue was placed in a mould where the surface to be sectioned was oriented downward. The mould was then filled with molten paraffin and cooled in a refrigerator until solidified, after which the paraffin block was removed and sectioned. Cutting and Gluing: Tissues were thinly sectioned and glued onto glass slides in preparation for further analysis (Elbatouti et al. 2023 ). The rats were euthanized by neck dislocation, and the skin was rinsed with 0.9% NaCl solution then weighed and placed in 10% neutral buffered formalin and after 24 hours replaced with 70% alcohol for histopathological studies. Table 1 Scoring of wound tissue changes (modified scoring method) (Guarro et al. 2021 ) Stage Characteristic Score Indicator Inflammation Ulcer 0 Absent 1 Low number of cells 2 Moderate number of cells 3 Abundant number of cells Leukocytes 0 Absent 1 60% extensive leukocyte infiltration Macrophage 0 Absent 1 Low number of cells 2 Moderate number of cells 3 Abundant number of cells Proliferation Angiogenesis 0 Absent 1 Mild 2 Moderate 3 Extensive Fibroblast 0 Absent 1 Present only in the perivascular area 2 Present in 50% of wound tissue Maturation Collagen 0 Absent 1 Present around blood vessels 2 Moderate collagen deposition 3 Extensive collagen deposition Re-epithelialization 0 Absent 1 Partial 2 Complete but thin 3 Complete and mature Data Availability All data supporting the findings of this study are available within the manuscript and its supplementary files. Raw data related to participant characteristics, study measurements, and research analyses are available from the corresponding author upon reasonable request, with due consideration for participant confidentiality and ethical approval. Results Characteristics of Fucoidan in S. polycystum Determination of Brown Algae Collected from South Sulawesi The identification of the brown algae sample was carried out macroscopically by looking at morphological characteristics. The algae are brown in colour, and in their environment, they attached to hard substrates. Stipules are cylindrical and rigid and may erect along the thallus. The main branch stiffly sends out secondary branches, which are growing alternately. The branches bear leaf-like structures and a cylindrical thallus with small spines. The leaves are flat, wavy, and irregularly serrated along the edges, with a smooth, slightly stiff surface. Numerous round, fruit-like air bladders are present and usually occur in clusters. The algae have a short main axis with dense primary branching. Previous studies have reported that S. polycystum typically has a slender thallus and diverse branching patterns, with round air bladders and leaf-like structures that vary in shape, including oval forms with jagged or uneven edges (Fernando et al. 2020 ). Based on its morphology described above, we concluded that the collected brown algae are S. polycystum . Fucoidan Structural Characterization by FTIR and NMR The identity of the isolated brown algae compound was confirmed by FTIR and NMR. The obtained FTIR spectrum showed a broad peak at 3481–3442 cm -1 associated with the presence of O-H (hydroxyl) groups, consistent with the chemical structure of fucoidan and with the spectrum of standard compound (Fig. 1 A) (Mike D. Stuart 2004 ). In addition, the spectrum also showed frequencies of the carbonyl group (C = O) around 1620–1622 cm -1 , and various C-H vibrations of polysaccharides around 1419–1427 cm -1 (Palanisamy et al. 2017 ). The presence of sulphate ester groups as a characteristic of fucoidan is shown by S = O stretching vibrations at 1259–1257 cm -1 (Lutfia et al. 2020 ). Finally, the main component in the fucoidan polysaccharide structure (C-O-S group) showed a characteristic fingerprint frequency at 825 cm -1 (in the isolated fucoidan) and 845 cm -1 (in standard fucoidan), which are consistent with those reported in the literature (815–845 cm -1 ) (Stuart B 2004 ). The 1 H NMR spectrum of the isolated fucoidan showed resonances around 1.1–1.2 ppm, indicating the presence of methyl protons (Fig. 1 B). Resonances around 3.5–4.5 ppm further reflect the characteristic features of fucoidan. The complex signals also indicate the heterogeneity of sulphated fucose units related to linkage patterns and sugar composition. The presence of resonances around 5.0–5.3 ppm reflects anomeric protons arising from glycosidically bonded L-fucose and related sugar residues. The 13 C NMR spectrum of the isolated fucoidan showed a signal around 15 ppm, attributed to the C-6 methyl group of the L-fucose residue, whereas signals in the 65–80 ppm region are associated with the hydroxylated carbons (Fig. 1 C). The presence of signals at 15 ppm and around 100–102 ppm further confirms the identity of the isolated fucoidan. These resonances are also consistent with those reported in the literature (Yue et al. 2025 ). In Vitro Hemolysis Test of the Isolated Fucoidan Toxicological assessment is a fundamental requirement in the development of novel pharmaceutical products. One of the most used preliminary methods for assessing cytotoxicity is the hemolysis assay (Omidian and Dey Chowdhury 2025 ). A hemolytic rate below 5% is considered safe (Elbatouti et al. 2023 ). To assess the hemolysis-inducing potential of fucoidan, a hemolytic activity assay was performed using erythrocytes obtained from Wistar rats (Greco et al. 2020 ). The results showed that fucoidan from S. polycystum had no hemolytic effect on red blood cells (Fig. 2 ). In Vivo Fucoidan Activity Test Wound Healing Activity of the Isolated Fucoidan Wound healing is a complex physiological process involving a coordinated series of events and interactions between various cells, tissues, and molecules to restore homeostasis and tissue integrity (Chinko and Precious-Abraham 2024 ). These phases involve complex cellular biochemical processes, as well as components of the inflammatory and coagulation pathways. Various cells, including fibroblasts, keratinocytes, endothelial cells, and immune cells (monocytes, macrophages, and lymphocytes), play an active role in this process (Tottoli et al. 2020 ). To evaluate the wound healing property of the isolated fucoidan, in vivo mouse experiments with 6 mm excision skin wounds infected with S. aureus (0.1 mL, 10⁷ CFU/mL) were performed (Fig. 3 A). The specific characteristics of the aligned membrane, especially the most optimal diameter range to support tissue regeneration, require further verification through experimental research. Smaller diameters and narrower size intervals improve the mechanical stability of the aligned membrane, which may contribute to the prevention of scar tissue formation during the wound-healing process of the skin (Wang et al. 2022 ). Figure 3 B presents the results of the nine-day wound healing with and without fucoidan and the positive control Nisagon®. While each treatment group showed an increase in the percentage of wound healing, those treated with fucoidan (G3 and G4) showed more significant progress ( p < 0.05) during the first several days than the G1 and G2. This finding confirms the potential benefit of fucoidan as a topical treatment for wound healing. Analysis of the wound healing effects of fucoidan using a single wound model in rats showed significant wound healing acceleration on days 3, 6, and 9 compared to those treated with the negative and positive controls. Effects of the Isolated Fucoidan on S. aureus Growth Elimination of microbes in a wound is necessary for the healing process to occur more quickly. To examine if the isolated fucoidan also has antibacterial activity against S. aureus in the animal wounds, we determined the number of bacteria in the wound samples using a colony counting method on plates (Olivia et al.)(Martín et al. 2021 ). Each group was analyzed on day 0 and day 9 and the colony counts were recorded (Fig. 4 ). The results showed that wounds treated with 1.2% and 2.4% fucoidan contained fewer bacteria after 9 days, similar to those treated with the positive control Nisagon®. A reduction of bacterial colonies was also observed in the negative control after 9 days, most likely due to the animal innate immunity. Nevertheless, the results showed that in addition to its wound healing property, fucoidan may have strong antibacterial activity against S. aureus , validating its potential application to support the healing process of infected wounds. Effects of Fucoidan on Modulating Inflammatory Responses Histopathological changes in wound tissue were assessed using a modified scoring system (Table 1 ), which included parameters related to inflammation, proliferation, and maturation phases of wound healing. Wound healing generally goes through three main phases: the inflammation phase, the proliferation phase, and the remodelling phase. The inflammation phase, indicated by the presence of lymphocytes, leukocytes, and macrophages, aims to rid the wound of microorganisms and cellular debris. Macrophages are widely distributed in various body tissues and serve as key cells inducing inflammatory immune responses. They play a dual role, contributing to injury and supporting tissue repair (Yu et al. 2022 ). Previous studies have shown that macrophages play diverse roles at different stages of the skin recovery process (Pham et al. 2025 ). To assess the impact of fucoidan on modulating inflammatory responses in S. aureus -induced wounds, ulceration, leukocyte infiltration, and macrophage activity were examined. Day 3 post-injury was selected for evaluation, as it represents a critical juncture in the inflammatory phase preceding the proliferative stage. Histopathological scoring revealed that the F1 exhibited the highest mean ulcer scores (Fig. 5 A), indicating extensive tissue damage. In contrast, groups treated with F3 and F4. Fucoidan demonstrated significantly lower ulcer scores compared to the F1, suggesting that fucoidan administration may expedite healing by mitigating ulcer severity. Notably, the group F4 showed no statistically significant difference ( p < 0.05) in ulcer scores when compared to the group F2, implying comparable efficacy to standard therapy in reducing tissue damage. Analysis of leukocyte infiltration (Fig. 5 B) indicated elevated scores in the group F1, reflecting a heightened inflammatory response, potentially due to uncontrolled infection. Conversely, the fucoidan-treated groups (F3 and F4) exhibited moderately reduced leukocyte scores relative to the F1, pointing to fucoidan's potential anti-inflammatory properties in curbing excessive inflammation. Similarly, the group F2 displayed decreased leukocyte infiltration, with no significant statistical difference ( p < 0.05) between F2 and F4, suggesting that fucoidan's effect on leukocyte modulation is on par with standard treatment. The results are consistent with a previous study by Olsthoorn, who reported that fucoidan can modulate local immune responses by reducing excessive leukocyte infiltration and optimizing macrophage numbers (Olsthoorn et al. 2021 ). Fucoidan also contributes to decreased tissue damage (ulceration) resulting from bacterial infection, likely through its antibacterial and anti-inflammatory effects. Furthermore, Velnar et al. emphasized that effective regulation of the inflammatory phase is crucial for a seamless transition to the proliferative phase. Fucoidan is believed to function by downregulating pro-inflammatory cytokines, such as TNF-α and IL-6, while promoting the secretion of growth factors by macrophages (Fig. 5 C), thereby facilitating early granulation tissue formation. Our results showed that F4 was superior in reducing ulcer scores and modulating immune responses (leukocytes and macrophages) compared to F3. Although fucoidan-treated groups outperformed the negative control, F4 provided a more controlled inflammatory response, indicating the wound's readiness to progress to the proliferative phase. Effects of Fucoidan on Cell Proliferation The proliferation phase is characterized by the formation of granulation tissue and angiogenesis, followed by collagen synthesis and re-epithelization that serve to close the wound. Therefore, an adequate blood supply provides the necessary basis for granulation tissue growth, which is a key factor in the wound healing process (Sun et al. 2024 ). To assess the impact of fucoidan on proliferation and tissue repair in S. aureus -infected wounds, we evaluated the formation of angiogenesis and fibroblast in wound tissues collected from day 6 (Fig. 6 A and B). The histopathological assessment of angiogenesis showed that samples from group F4 (fucoidan 2.4%) had the highest average of angiogenesis score (blood vessel formation). This finding suggests that fucoidan has the potential to accelerate the wound healing process through stimulating the formation of new blood vessels. However, there was no statistically significant difference between the F4 group and the F3, F2, and F1 groups ( p < 0.05). These results indicate that the F4 formulation has efficacy comparable to that of other formulations in supporting angiogenesis. Previous studies have shown that on day four to twenty-one after injury, angiogenesis and granulation tissue increases (Čoma et al. 2021 ). Myofibroblasts, fibroblasts, inflammatory cells, and endothelial cells contribute to the formation of new extracellular matrix components and continue to develop until the wound closure process begins (Cialdai et al. 2022 ). Histopathological analysis on day 6 showed that the wound was in the active proliferation phase. The negative control group (F1) showed the lowest fibroblast proliferation and new tissue formation. In contrast, the positive control group (F2) showed greater tissue formation than the F1 group. In the fucoidan treatment group, formulation F3 (1.2%) showed a lower healing response than F4 (2.4%), whereas F4 exhibited higher fibroblast proliferation and tissue formation. These findings indicate an increase in wound healing response with increasing fucoidan concentration (Fig. 6 B). Effects of Fucoidan on Wound Maturation Cutaneous wound healing remodeling is an important phase in wound healing, characterized by maturation and restructuring of the extracellular matrix (ECM) to restore tissue integrity. Following fibroblast proliferation, collagen synthesis and deposition is an important part of the process of replacing damaged tissue (Choudhary et al. 2024 ). One week after the wound occurs in the maturation phase in wound healing can increase collagen back to normal (Murali et al. 2025 ). A study conducted by Zixuan et al. in 2024 revealed that collagen regeneration in wounds is an important indicator in the skin regeneration process. This finding suggests that optimal collagen formation can accelerate wound healing and improve the function of damaged skin tissue (Zhang et al. 2024 ). Collagen had the potential to increase the percentage of epithelialization after seven days of therapy. This suggests that collagen plays an important role in supporting the wound healing process by accelerating the formation of a new epithelial layer. Collagen administration can repair damaged skin integrity, increase epithelial cell proliferation, and accelerate tissue recovery (Benito-Martínez et al. 2022 ). To evaluate the effect of fucoidan on would healing maturation, we analyzed collagen formation on tissue samples collected from day 9. Day 9 was chosen as the evaluation point, as it is a critical phase in collagen formation and epithelialization. The results showed that tissue samples from the group receiving fucoidan 2.4% (group F4) had the highest average collagen histopathological score, followed by the group that received Nisagon ® (Fig. 6 C). On the other hand, the negative control (group F1) and the group receiving fucoidan 1.2% (F3) showed lower scores. This result suggests that fucoidan 2.4% can accelerate wound healing process by stimulating the formation of collagen. Similarly, tissue samples from the group receiving fucoidan 2.4% (group F4) had the highest average re-epithelialization score, followed by the group that received Nisagon® (Fig. 6 D), whereas administration of fucoidan 1.2% (F3) did not show significantly better re-epithelialization scores compared to the negative control. The high re-epithelialization score for fucoidan 2.4% suggests that the administration of fucoidan at higher concentrations can accelerate the re-epithelialization process in S. aureus infected wound healing. Conclusion The brown algae ( S. polycystrum ) from the sea around Takalar Regency, South Sulawesi – Indonesia, is a rich source of fucoidan. The identity of the isolated fucoidan was confirmed by FTIR and NMR. The compound showed no cytotoxicity activity in a red blood cell hemolysis test. We found that fucoidan at a concentration of 2.4% significantly accelerated wound healing. It also improved the inflammatory, proliferation, and maturation phases. We conclude that 2.4% of fucoidan may be used as an effective and safe anti-inflammatory and antibacterial agent in wound healing therapy. Declarations Ethics Approval The animal ethics guidelines were followed and approved by the Research Ethics Committee, Faculty of Pharmacy, Hasanuddin University ( 913/UN4.17/KEP/2024). Consent for Publication The authors approve processing this manuscript for publication. Competing interests The authors declare no competing interests. Author Contribution Fausyiah Sasmitha AB Writing – review and editing, Writing – original draft, Methodology, Conceptualization, Data curation, Formal analysis, Investigation. Frederika Tangdi Lintin Writing – review and editing, Conceptualization, Investigation, Methodology, Visualization. Sulistiawati Validation, Software. Sitti Nur Fatimah Software. Stephanie Investigation, Formal analysis, Writing – original draft. Muhammad Nur Amir Conceptualization. Syamsiah Conceptualization. Ngoc Dan Thuy Nguyen Investigation, Data curation. Taifo Mahmud Conceptualization, Supervision, Resources, Funding acquisition. Muhammad Nasrum Massi Writing – review and editing, Resources, Project administration, Methodology, Conceptualization. Marianti A. Manggau Writing – review and editing, Supervision, Resources, Funding acquisition, Conceptualization Acknowledgments For the technical support and help in performing some techniques, we want to expresses gratitude to the Ministry of Health of the Republic of Indonesia for providing a scholarship for a master’s degree in pharmaceutical science. Fausyiah Sasmitha AB. was financially supported by the Ministry of Health of the Republic of Indonesia to pursue a Master’s degree in Pharmaceutical Sciences (Ref. No. HK.01.07/F.I/5666/2023). Data availability No data was used for the research described in the article. Code Availability Not applicable. References Abdel-Latif HMR, Dawood MAO, Alagawany M et al (2022) Health benefits and potential applications of fucoidan (FCD) extracted from brown seaweeds in aquaculture: An updated review. 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Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 20 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers invited by journal 07 May, 2026 Editor assigned by journal 06 May, 2026 Submission checks completed at journal 06 May, 2026 First submitted to journal 30 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9573456","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":636570710,"identity":"cb1ae047-30c2-47e5-a314-44329b447889","order_by":0,"name":"Fausyiah Sasmitha AB","email":"data:image/png;base64,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","orcid":"","institution":"Hasanuddin University","correspondingAuthor":true,"prefix":"","firstName":"Fausyiah","middleName":"Sasmitha","lastName":"AB","suffix":""},{"id":636570720,"identity":"ca53405f-cc0e-4c9d-bebb-86afc817fb1c","order_by":1,"name":"Frederika Tangdi Lintin","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Frederika","middleName":"Tangdi","lastName":"Lintin","suffix":""},{"id":636570730,"identity":"1a0a8615-76ac-4c8c-b91d-d1f34b180d09","order_by":2,"name":"Sulistiawati Sulistiawati","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Sulistiawati","middleName":"","lastName":"Sulistiawati","suffix":""},{"id":636570731,"identity":"73113641-ee48-476d-9bd1-7e7c648c89b7","order_by":3,"name":"Siti Nur Fatimah","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Siti","middleName":"Nur","lastName":"Fatimah","suffix":""},{"id":636570732,"identity":"e3f85be1-48ef-4746-9378-7623140d2754","order_by":4,"name":"Stephanie Stephanie","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Stephanie","suffix":""},{"id":636570735,"identity":"5f3c2f7c-e895-450e-8cfd-b9d9f1e9d576","order_by":5,"name":"Muhammad Nur Amir","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Nur","lastName":"Amir","suffix":""},{"id":636570737,"identity":"39b1df03-285b-4371-9a49-2cd48fbc423a","order_by":6,"name":"syamsiah Syamsiah","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"syamsiah","middleName":"","lastName":"Syamsiah","suffix":""},{"id":636570739,"identity":"c5b358c2-d39e-4dc1-a5b6-1621b1484f91","order_by":7,"name":"Ngoc Dan Thuy Nguyen","email":"","orcid":"","institution":"Oregon State University","correspondingAuthor":false,"prefix":"","firstName":"Ngoc","middleName":"Dan Thuy","lastName":"Nguyen","suffix":""},{"id":636570746,"identity":"cbda4b38-8288-4fce-a64d-2c91d2931477","order_by":8,"name":"Taifo Mahmud","email":"","orcid":"","institution":"Oregon State University","correspondingAuthor":false,"prefix":"","firstName":"Taifo","middleName":"","lastName":"Mahmud","suffix":""},{"id":636570747,"identity":"4b54c0ff-6acc-4a5c-ad8f-585247180e0c","order_by":9,"name":"Muhammad Nasrum Massi","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Nasrum","lastName":"Massi","suffix":""},{"id":636570748,"identity":"2b285d3b-f350-40e2-a5c4-55c23f583fe8","order_by":10,"name":"Marianti A. Manggau","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Marianti","middleName":"A.","lastName":"Manggau","suffix":""}],"badges":[],"createdAt":"2026-04-30 07:24:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9573456/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9573456/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109446408,"identity":"735016d5-ab87-4d27-8727-1b0ad9301111","added_by":"auto","created_at":"2026-05-18 08:22:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88415,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of purified fucoidan from \u003cem\u003eS. polycystum\u003c/em\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) FTIR spectrum, (\u003cstrong\u003eb\u003c/strong\u003e) \u003csup\u003e1\u003c/sup\u003eH NMR spectrum; and (\u003cstrong\u003eb\u003c/strong\u003e) \u003csup\u003e13\u003c/sup\u003eC NMR spectrum.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9573456/v1/36b25db72b3e151cf183c159.png"},{"id":109760165,"identity":"af313801-2d6a-451f-99f1-4843c2581bf3","added_by":"auto","created_at":"2026-05-22 07:28:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267284,"visible":true,"origin":"","legend":"\u003cp\u003eHemolysis test of the isolated fucoidan.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9573456/v1/40d2968061819926d497b4c2.png"},{"id":109759671,"identity":"963ce0fd-1a51-4f54-b602-27dfd12a929d","added_by":"auto","created_at":"2026-05-22 07:27:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":457850,"visible":true,"origin":"","legend":"\u003cp\u003eWound healing property of the isolated fucoidan. (\u003cstrong\u003ea\u003c/strong\u003e) Pictures of wound healing process from Day 0 to Day 9 across all treatment groups, and (\u003cstrong\u003eb\u003c/strong\u003e) the percentage of wound healing measured on days 3, 6, and 9 (mean ± SD).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9573456/v1/7f71309a21881e6db52facbd.png"},{"id":109446409,"identity":"a59dcc21-28e8-40cf-8b66-f342e8e73a5e","added_by":"auto","created_at":"2026-05-18 08:22:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73050,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of G3 and G4 \u003cem\u003ein vitro\u003c/em\u003e against the viability of \u003cem\u003eS. aureus\u003c/em\u003ebacteria, with positive and negative controls.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9573456/v1/26cb665909273b936ee6b4d3.png"},{"id":109446412,"identity":"66360926-56ae-41d6-9670-3819857734a3","added_by":"auto","created_at":"2026-05-18 08:22:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1508033,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological assessment results with scoring in the inflammatory phase: (\u003cstrong\u003ea\u003c/strong\u003e) ulceration severity, (\u003cstrong\u003eb\u003c/strong\u003e) leucocyte infiltration, and (\u003cstrong\u003ec\u003c/strong\u003e) macrophage presence, (\u003cstrong\u003ed\u003c/strong\u003e) Histological of skin tissue in the inflammatory, proliferation and maturation phases (F1 F2, F3 and F4).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9573456/v1/048708b2ad9ac876da51e377.png"},{"id":109446413,"identity":"25074ac9-3f03-4502-82d1-c14813a4a258","added_by":"auto","created_at":"2026-05-18 08:22:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46403,"visible":true,"origin":"","legend":"\u003cp\u003eHistological photomicrographs of skin tissue in proliferation and maturation phases. (\u003cstrong\u003ea\u003c/strong\u003e) angiogenesis, (\u003cstrong\u003eb\u003c/strong\u003e) fibroblast, (\u003cstrong\u003ec\u003c/strong\u003e) collagen, and (\u003cstrong\u003ed\u003c/strong\u003e) re-epithelialization.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9573456/v1/c81937e6199c10acaa56d9b7.png"},{"id":109765107,"identity":"1e7bc725-eb52-41e7-b353-c7618e81d143","added_by":"auto","created_at":"2026-05-22 07:39:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2517420,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9573456/v1/e46622bb-d3de-4f67-9f57-8499af231396.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization and Wound-Healing Effects of Fucoidan from Sargassum polycystum Collected in South Sulawesi in a Staphylococcus aureus-Infected Rat Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe skin is the largest organ in the human body, serving as an excellent protective barrier. However, this barrier may be compromised when the skin is wounded, either by physical objects, chemicals, excessive heat, or other causes (Wen et al. 2023). Although most wounds can heal naturally within 14\u0026ndash;30 days through an organized wound-healing process, which restores the function and anatomical integrity of the skin (Morguette et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the process may also be impeded, due to bleeding, infection, inflammation, and/or impaired angiogenesis and regeneration. Angiogenesis is a critical process in tissue repair following injury and represents an important intrinsic protective mechanism. The formation of new blood vessels is essential for wound healing, as it supplies oxygen and nutrients to cells at the wound site (Wen et al. 2023). Microbial infections are among the most serious problems, as they may lead to morbidity and mortality. Globally, the increased incidence of skin infections has been associated with higher risks of mortality, with a reported case-fatality rate of 20.3% and an annual mortality rate of 3.4 per 100.000 population (Nurul Fitri Marzaman et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Infections with \u003cem\u003eS. aureus\u003c/em\u003e are among the most common causes of morbidity and mortality, as they can spread to the bloodstream and cause sepsis (Elbatouti et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eOne compound that has been extensively studied as a wound-healing agent is fucoidan, a unique water-soluble sulphated polysaccharide produced by brown algae (seaweed), which exhibits a wide range of biological activities, including antitumor, anti-inflammatory, antioxidant, anticoagulant, and wound healing (Manggau et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Abdel-Latif et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A study by Lu \u003cem\u003eet al.\u003c/em\u003e demonstrated that a gelatine\u0026ndash;fucoidan\u0026ndash;based hydrogel (Gel\u0026ndash;Fuc\u0026ndash;TA) can modulate the wound immune response by promoting macrophage polarization toward the M2 phenotype, suppressing pro-inflammatory mediators, and increasing the expression of anti-inflammatory cytokines and transforming growth factor-β (TGF-β), thereby contributing to reduced inflammation during wound-healing process (Lu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Other studies also reported that fucoidan enhances angiogenesis by activating the AKT/Nrf2/HIF-1α pathway, which accelerates wound healing (Wen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). In line with these findings, Wang \u003cem\u003eet al.\u003c/em\u003e reported that fucoidan- and alginate-based dressings enhanced skin tissue regeneration in animal models, as indicated by accelerated wound closure, reduced inflammatory response, and increased collagen deposition, possibly through activation of the TGF-β1/Smad pathway (Liang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Recent studies have also demonstrated that incorporating fucoidan into gelatine and oxidized carboxymethyl cellulose\u0026ndash;based hydrogels support wound closure by modulating inflammation. This effect is characterized by suppression of inducible nitric oxide synthase (iNOS) and reduced nitric oxide production in macrophages, as well as enhanced collagen synthesis in human skin fibroblasts, thereby confirming the potential of fucoidan-loaded hydrogels as an effective delivery system for tissue regeneration (Jeong et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFucoidan has also been reported to exhibit significant anti-inflammatory and antibacterial activities, which contribute substantially to its therapeutic potential as a wound-healing agent. These effects are mediated by the suppression of pro-inflammatory mediators, particularly tumour necrosis factor-α (TNF-α), and by inhibition of bacterial growth and colonization. Numerous studies have demonstrated that the application of fucoidan-based wound dressings can modulate the inflammatory response while reducing bacterial burden, thereby creating a favourable microenvironment that promotes accelerated tissue regeneration and enhanced antibacterial efficacy \u003cem\u003ein vivo\u003c/em\u003e, particularly in infected wounds (Lu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In line with these findings, fucoidans isolated from \u003cem\u003eFucus vesiculosus\u003c/em\u003e have demonstrated bacteriostatic effects against multiple bacterial species, including \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. Microscopic analyzes revealed preservation of bacterial cell integrity accompanied by alterations in cell size and surface morphology, effects that were more pronounced in Gram-positive bacteria (Ayrapetyan et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, fucoidan is a mixture of structurally complex sulphated polysaccharides that vary significantly depending on the species of seaweed and the geological location, which may also affect its biological properties. Despite these advances, the molecular mechanisms underlying the pro-angiogenic effects of fucoidan during wound healing, particularly in relation to the spatial distribution of neovascularization within the wound area, remain poorly understood.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the wound-healing property of fucoidan isolated from the brown algae \u003cem\u003eSargassum polycystum\u003c/em\u003e collected in the intertidal zone of South Sulawesi, Indonesia, using a wound model in rats infected with \u003cem\u003eS. aureus\u003c/em\u003e. To the best of our knowledge, this was the first \u003cem\u003ein vivo\u003c/em\u003e study in \u003cem\u003eS. aureus\u003c/em\u003e-infected rats with histopathological assessments conducted across the inflammatory, proliferative, and remodelling phases. In addition, the anti-inflammatory and antibacterial activities of the fucoidan against \u003cem\u003eS. aureus\u003c/em\u003e were systematically investigated. As Indonesia contributes approximately 38% of global seaweed production and ranks as the world\u0026rsquo;s second-largest producer, the results underscore the immense potential of Indonesian seaweed as source of fucoidan, which can be developed as natural medicines to treat infected wounds (Manggau et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eHarvesting brown algae\u003c/h2\u003e\n\u003cp\u003eThe brown algae (\u003cem\u003eS. polycystum\u003c/em\u003e) were collected from the coast of Takalar Regency, South Sulawesi (5\u0026deg;34'39.8\"S 119\u0026deg;25'37.7\"E), at a water depth of approximately 5 m.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCharacterization of Brown Algae\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eIdentification of the Brown Algae\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eIdentification of the brown algae sample was done at the Environmental and Marine Science Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Hasanuddin University, using microscopy and morphological evaluations (Sundari et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eIsolation of Fucoidan from S. polycystum\u003c/h3\u003e\n\u003cp\u003eThe brown algae were first washed with seawater, then rinsed with running freshwater to remove contaminants such as sand and salt. Further, it was cleaned with distilled water to eliminate organoleptic properties such as odour and taste. After cleaning, the brown algae were dried at room temperature. Once dried, the brown algae were chopped into small pieces and ground into a fine powder, then sieved using a 100-mesh sieve. Fine brown algae powder (50 g) was then extracted with EtOH (1 L). The mixture was stirred and left for 12 h at room temperature. It was then centrifuged at 2,000 rpm for 10 min. The resulting precipitate was dried overnight at room temperature.\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eNext, the dried powder (5 g) was mixed with water (100 mL) at 65\u0026deg;C for 1 h. The mixture was centrifuged at 15,000 rpm for 10 min. To obtain the supernatant, the solution is mixed with 1% CaCl₂ and left overnight at 4\u0026deg;C to precipitate the alginic acid. The precipitate was then centrifuged at 15,000 rpm for 10 min, and the supernatant was collected and mixed with 99% EtOH to reach a final concentration of 30%. The solution was stored at 4\u0026deg;C for 4 h, then centrifuged at 15,000 rpm for 10 min (Palanisamy et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Finally, the sample was freeze-dried and ground into a fine powder. The yield percentage was calculated using the following formula (Kim et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equa\" class=\"mathdisplay\"\u003e$$\\:Fucoidan\\:\\left(\\%\\right)=\\:\\frac{Weight\\:of\\:dry\\:fucoidan\\:obtained\\:as\\:a\\:result\\:of\\:ethanol\\:precipitation}{biomass\\:weight}\\:X\\:100%$$\u003c/div\u003e\n\u003cdiv class=\"mathdisplay\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"mathdisplay\"\u003e(1)\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eSpectroscopy Analysis\u003c/h2\u003e\n\u003cp\u003ePurified fucoidan was analyzed using a Shimadzu\u0026reg; IRPrestige-21 FTIR spectrometer in ATR mode. Spectra were collected over the wavenumber range of 4000\u0026ndash;500 cm⁻\u0026sup1; at a resolution of 4 cm⁻\u0026sup1;, with a scan duration of 32 min (14,15). \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were acquired using a Bruker Avance III 700 MHz.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eFucoidan Hemolytic Activity Test\u003c/h3\u003e\n\u003cp\u003eA haemolysis assay was conducted to evaluate the cytotoxicity of fucoidan. Whole blood was collected from Wistar rats and transferred into sterile 50 mL Falcon\u0026trade; tubes. The samples were centrifuged at 2000 rpm for 10 min to separate plasma from red blood cells (RBCs). The plasma supernatant was discarded, and the RBC pellet was washed four times with phosphate-buffered saline (PBS). The washed RBCs were resuspended in PBS by vortexing for 3 min and then diluted with PBS to obtain a 10% (v/v) RBC suspension (Stephanie et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sapiun et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Then, the mixture was generated in three concentrations of 10, 100, and 1000 \u0026micro;g/ml. For each test, 100 \u0026micro;L of fucoidan solution was mixed with 900 \u0026micro;L of the 10% RBC suspension. The mixtures were incubated at 37\u0026deg;C for 60 min and then centrifuged at 2000 rpm for 10 min. The absorbance of free haemoglobin in the supernatant was measured at 540 nm using a UV\u0026ndash;Vis spectrophotometer. RBCs in PBS served as the negative control, whereas RBCs in distilled water served as the positive controlc (Stephanie et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). The hemolytic percentage was calculated according to the following equation (Fontelo et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equ1\" class=\"mathdisplay\"\u003e$$\\:\\text{H}\\text{e}\\text{m}\\text{o}\\text{l}\\text{y}\\text{t}\\text{i}\\text{c}\\:\\:\\left(\\text{%}\\right)=\\:\\frac{\\left(\\text{S}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\:\\text{a}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}-\\text{N}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}{\\text{P}\\text{o}\\text{s}\\text{i}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\text{N}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\:\\text{X}\\:100$$\u003c/div\u003e\n\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eIn Vivo Fucoidan Activity Test Study\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003ePreparation of Topical Fucoidan from S. polycystum\u003c/h2\u003e\n\u003cp\u003eTopical 1.2% and 2.4% fucoidan samples were prepared by mixing fucoidan (120 mg and 240 mg, respectively) with Vaseline base to a total amount of 10 g.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eAcclimatization of Experimental Animals (Rattus norvegicus)\u003c/h2\u003e\n\u003cp\u003eMale rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e), aged 3\u0026ndash;4 months and weighing 200\u0026ndash;250 grams, obtained from the Wisbalb breeder in Indonesia, were used. Before the experiment, the rats underwent a 7\u0026ndash;14 days acclimatization period to adapt to the laboratory environment of Pharmacology-Toxicology, Faculty of Pharmacy, Hasanuddin University. The animals were monitored daily to ensure their health. Animals were also weighed weekly to determine their body weight and development. The animals were placed in cages with standard AD II feed and drinking water ad libitum. The animal cages were cleaned twice a week to ensure the level of humidity, comfort, cleanliness, and safety of the animals. The laboratory environment is set with temperatures ranging from 25\u0026ndash;30\u0026deg;C, as well as 12 h of light and darkness (dos Santos et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003ePreparation of S. aureus Bacterial Suspension\u003c/h2\u003e\n\u003cp\u003eAll equipment used in the preparation of bacterial suspensions was sterilized using an autoclave at 121\u0026deg;C for 30 min. Each equipment was covered with cotton and aluminium foil to maintain sterility. The medium is prepared by dissolving Nutrient Agar (0.39 g) in distilled water (10 mL) in an Erlenmeyer flask, then heating to dissolve the components fully. Next, the medium is transferred to a test tube and left in an inclined position until it forms agar. \u003cem\u003eS. aureus\u003c/em\u003e is then inoculated into the test tube and incubated at 37\u0026deg;C for 24 h. The turbidity of the bacterial suspension was measured by comparing it with a McFarland 1 standard solution using a McFarland densitometer. If the suspension turbidity exceeded the specified standard, 0.9% sterile NaCl solution was added until the appropriate turbidity was achieved (Sartini \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eAcute Wound Infection with S. aureus\u003c/h2\u003e\n\u003cp\u003eFifteen rats were anesthetized using ketamine (0.3 mL) intraperitoneally. The rat dorsal region was shaved until completely clean from hairs. Then, four wounds were made using a biopsy punch on the dorsal surface of each rat with a diameter of 6 mm. The wounds, coded with F1, F2, F3 and F4, were infected with \u003cem\u003eS. aureus\u003c/em\u003e (in 0.1 mL bacterial suspension) intradermally. The wounds were then covered using gauze and square parafilm. After the procedure, the animals were placed back into their cages (Elbatouti et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eExperimental Treatment\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThis study used 15 rats, which were divided into 3 groups of 5 rats each. Each rat received specific treatments for four different wound areas: F1 (Nisagon\u003csup\u003e\u0026reg;\u003c/sup\u003e wound healing cream was used as positive control), F2 (Vaseline as negative control), F3 (1.2% Vaseline-based fucoidan extract) and F4 (2.4% Vaseline-based fucoidan extract). The treatments were administered daily at noon to all part of the wounds. The treatment was carried out by applying 10 mg of the preparation using a sterile cotton swab. It was also carried out by measuring the initial wound diameter until the healing measurement at the end, for 10 consecutive days, until the experimental animals were analyzed for wounds.\u003c/p\u003e\n\u003c/div\u003e\n\u003col\u003e\n\u003cli\u003e\n\u003cp\u003e5 experimental animals to represent the wound condition in the inflammatory phase. Group I was made on day 3 and tissue was taken to check the inflammation phase process in macrophages, leucocytes, and ulcers.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e5 experimental animals to represent the wound condition in the inflammatory phase. Group I was made on day 6 and tissue was taken to check the proliferation phase process in angiogenesis and fibroblast.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e5 experimental animals to represent the wound condition in the inflammatory phase. Group I was made on day 3 and tissue was taken to check the maturation phase process in collagen and re-epithelialization.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ol\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eS. aureus colony counting\u003c/h2\u003e\n\u003cp\u003eBacterial samples were taken using an inoculating loop, transferred into a 5 mL Eppendorf tube containing 0.5 mL of sterile distilled water, and vortexed for 1 min. The suspension (0.1 mL) was transferred into an Eppendorf tube containing 0.4 mL of distilled water and then diluted to 10\u003csup\u003e1\u003c/sup\u003e, 10\u003csup\u003e2\u003c/sup\u003e, 10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e, 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e6\u003c/sup\u003e, and 10\u003csup\u003e7\u003c/sup\u003e concentrations, then vortexed for 1 minute. A sample of 100 \u0026micro;l from each dilution was taken and streaked into a petri dish with sterile count plate agar (PCA) media. Then, the media was incubated for 24 h at 37\u0026deg;C. After 24 hours, the number of colonies was observed in two ways: counting using a colony count and manually counting using a closed petri dish. The colonies were photographed and counted visually from the back of the petri dish. (Makrai et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eFucoidan Activity in Wound Healing\u003c/h2\u003e\n\u003cp\u003eA biopsy punch wound model in rats was used to evaluate the wound-healing activity of fucoidan. Before creating the wound, the rat was weighed and anaesthetized with ketamine intraperitoneally in an appropriate dose 0.3 mL/kg BW. Subsequently, four circular wounds with a diameter of 6 mm each were made and photographed before dressing (Ishi et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sari et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe Wound healing diameter was measured at three different points to ensure accuracy and consistency in wound size on day 0, 3, 6, and 9. Wound contraction was calculated as the percentage reduction in wound area, as shown in the formula below (Moglad et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{W}\\text{o}\\text{u}\\text{n}\\text{d}\\:\\text{H}\\text{e}\\text{a}\\text{l}\\text{i}\\text{n}\\text{g}\\:\\left(\\text{%}\\right)=\\:\\frac{\\left(\\text{W}\\text{o}\\text{u}\\text{n}\\text{d}\\:\\text{a}\\text{r}\\text{e}\\text{a}\\:\\text{o}\\text{n}\\:\\text{t}\\text{h}\\text{e}\\:\\text{f}\\text{i}\\text{r}\\text{s}\\text{t}\\:\\text{d}\\text{a}\\text{y}-\\text{T}\\text{h}\\text{e}\\:\\text{e}\\text{x}\\text{t}\\text{e}\\text{n}\\text{t}\\:\\text{o}\\text{f}\\:\\text{t}\\text{h}\\text{e}\\:\\text{w}\\text{o}\\text{u}\\text{n}\\text{d}\\:\\text{o}\\text{n}\\:\\text{t}\\text{h}\\text{e}\\:\\text{l}\\text{a}\\text{s}\\text{t}\\:\\text{d}\\text{a}\\text{y}\\right)}{\\text{F}\\text{i}\\text{r}\\text{s}\\text{t}\\:\\text{w}\\text{o}\\text{u}\\text{n}\\text{d}\\:\\text{a}\\text{r}\\text{e}\\text{a}}\\:\\text{X}\\:100\\)\u003c/span\u003e \u003c/span\u003e%\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eHistopathological Analysis\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe excised skin tissue was processed through a series of steps and histopathologic examination was performed using light microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003col\u003e\n\u003cli\u003e\n\u003cp\u003eFixation: Tissues were fixed using 10% formalin buffer solution with pH 7.0\u0026ndash;7.4 for 12 to 18 h.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eDehydration: Tissues were immersed gradually in ethanol solutions of increasing concentration, starting with 70% EtOH, followed by 90%, 95%, and finally 100% EtOH.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eImmersion: Tissues were immersed in first stage xylol (xylol I) for 1 h, then transferred to second stage xylol (xylol II) and incubated for 30 min to 1 h.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eImmersion: Tissues were embedded in paraffin with a low melting point of approximately 56\u0026ndash;59\u0026deg;C for 30 min.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eEmbedding : Tissue was placed in a mould where the surface to be sectioned was oriented downward. The mould was then filled with molten paraffin and cooled in a refrigerator until solidified, after which the paraffin block was removed and sectioned.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eCutting and Gluing: Tissues were thinly sectioned and glued onto glass slides in preparation for further analysis (Elbatouti et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe rats were euthanized by neck dislocation, and the skin was rinsed with 0.9% NaCl solution then weighed and placed in 10% neutral buffered formalin and after 24 hours replaced with 70% alcohol for histopathological studies.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eScoring of wound tissue changes (modified scoring method) (Guarro et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eStage\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCharacteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eScore\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIndicator\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"12\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eInflammation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eUlcer\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLow number of cells\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eModerate number of cells\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbundant number of cells\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eLeukocytes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;30% leukocyte infiltration\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30\u0026ndash;60% moderate leukocyte infiltration\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;60% extensive leukocyte infiltration\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eMacrophage\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLow number of cells\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eModerate number of cells\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbundant number of cells\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"8\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eProliferation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eAngiogenesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMild\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eModerate\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eExtensive\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eFibroblast\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePresent only in the perivascular area\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePresent in \u0026lt;\u0026thinsp;50% of wound tissue\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePresent in \u0026gt;\u0026thinsp;50% of wound tissue\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"8\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMaturation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eCollagen\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePresent around blood vessels\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eModerate collagen deposition\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eExtensive collagen deposition\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eRe-epithelialization\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePartial\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eComplete but thin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eComplete and mature\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the manuscript and its supplementary files. Raw data related to participant characteristics, study measurements, and research analyses are available from the corresponding author upon reasonable request, with due consideration for participant confidentiality and ethical approval.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacteristics of Fucoidan in\u003c/b\u003e \u003cb\u003eS. polycystum\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Brown Algae Collected from South Sulawesi\u003c/h2\u003e \u003cp\u003eThe identification of the brown algae sample was carried out macroscopically by looking at morphological characteristics. The algae are brown in colour, and in their environment, they attached to hard substrates. Stipules are cylindrical and rigid and may erect along the thallus. The main branch stiffly sends out secondary branches, which are growing alternately. The branches bear leaf-like structures and a cylindrical thallus with small spines. The leaves are flat, wavy, and irregularly serrated along the edges, with a smooth, slightly stiff surface. Numerous round, fruit-like air bladders are present and usually occur in clusters. The algae have a short main axis with dense primary branching. Previous studies have reported that \u003cem\u003eS. polycystum\u003c/em\u003e typically has a slender thallus and diverse branching patterns, with round air bladders and leaf-like structures that vary in shape, including oval forms with jagged or uneven edges (Fernando et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Based on its morphology described above, we concluded that the collected brown algae are \u003cem\u003eS. polycystum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFucoidan Structural Characterization by FTIR and NMR\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe identity of the isolated brown algae compound was confirmed by FTIR and NMR. The obtained FTIR spectrum showed a broad peak at 3481\u0026ndash;3442 cm\u003csup\u003e-1\u003c/sup\u003e associated with the presence of O-H (hydroxyl) groups, consistent with the chemical structure of fucoidan and with the spectrum of standard compound (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) (Mike D. Stuart \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In addition, the spectrum also showed frequencies of the carbonyl group (C\u0026thinsp;=\u0026thinsp;O) around 1620\u0026ndash;1622 cm\u003csup\u003e-1\u003c/sup\u003e, and various C-H vibrations of polysaccharides around 1419\u0026ndash;1427 cm\u003csup\u003e-1\u003c/sup\u003e (Palanisamy et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The presence of sulphate ester groups as a characteristic of fucoidan is shown by S\u0026thinsp;=\u0026thinsp;O stretching vibrations at 1259\u0026ndash;1257 cm\u003csup\u003e-1\u003c/sup\u003e (Lutfia et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Finally, the main component in the fucoidan polysaccharide structure (C-O-S group) showed a characteristic fingerprint frequency at 825 cm\u003csup\u003e-1\u003c/sup\u003e (in the isolated fucoidan) and 845 cm\u003csup\u003e-1\u003c/sup\u003e (in standard fucoidan), which are consistent with those reported in the literature (815\u0026ndash;845 cm\u003csup\u003e-1\u003c/sup\u003e) (Stuart B \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of the isolated fucoidan showed resonances around 1.1\u0026ndash;1.2 ppm, indicating the presence of methyl protons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Resonances around 3.5\u0026ndash;4.5 ppm further reflect the characteristic features of fucoidan. The complex signals also indicate the heterogeneity of sulphated fucose units related to linkage patterns and sugar composition. The presence of resonances around 5.0\u0026ndash;5.3 ppm reflects anomeric protons arising from glycosidically bonded L-fucose and related sugar residues. The \u003csup\u003e13\u003c/sup\u003eC NMR spectrum of the isolated fucoidan showed a signal around 15 ppm, attributed to the C-6 methyl group of the L-fucose residue, whereas signals in the 65\u0026ndash;80 ppm region are associated with the hydroxylated carbons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The presence of signals at 15 ppm and around 100\u0026ndash;102 ppm further confirms the identity of the isolated fucoidan. These resonances are also consistent with those reported in the literature (Yue et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eIn Vitro Hemolysis Test of the Isolated Fucoidan\u003c/h2\u003e \u003cp\u003eToxicological assessment is a fundamental requirement in the development of novel pharmaceutical products. One of the most used preliminary methods for assessing cytotoxicity is the hemolysis assay (Omidian and Dey Chowdhury \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). A hemolytic rate below 5% is considered safe (Elbatouti et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To assess the hemolysis-inducing potential of fucoidan, a hemolytic activity assay was performed using erythrocytes obtained from Wistar rats (Greco et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The results showed that fucoidan from \u003cem\u003eS. polycystum\u003c/em\u003e had no hemolytic effect on red blood cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eIn Vivo Fucoidan Activity Test\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eWound Healing Activity of the Isolated Fucoidan\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWound healing is a complex physiological process involving a coordinated series of events and interactions between various cells, tissues, and molecules to restore homeostasis and tissue integrity (Chinko and Precious-Abraham \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These phases involve complex cellular biochemical processes, as well as components of the inflammatory and coagulation pathways. Various cells, including fibroblasts, keratinocytes, endothelial cells, and immune cells (monocytes, macrophages, and lymphocytes), play an active role in this process (Tottoli et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To evaluate the wound healing property of the isolated fucoidan, \u003cem\u003ein vivo\u003c/em\u003e mouse experiments with 6 mm excision skin wounds infected with \u003cem\u003eS. aureus\u003c/em\u003e (0.1 mL, 10⁷ CFU/mL) were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe specific characteristics of the aligned membrane, especially the most optimal diameter range to support tissue regeneration, require further verification through experimental research. Smaller diameters and narrower size intervals improve the mechanical stability of the aligned membrane, which may contribute to the prevention of scar tissue formation during the wound-healing process of the skin (Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB presents the results of the nine-day wound healing with and without fucoidan and the positive control Nisagon\u0026reg;. While each treatment group showed an increase in the percentage of wound healing, those treated with fucoidan (G3 and G4) showed more significant progress (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) during the first several days than the G1 and G2. This finding confirms the potential benefit of fucoidan as a topical treatment for wound healing. Analysis of the wound healing effects of fucoidan using a single wound model in rats showed significant wound healing acceleration on days 3, 6, and 9 compared to those treated with the negative and positive controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eEffects of the Isolated Fucoidan on S. aureus Growth\u003c/h2\u003e \u003cp\u003eElimination of microbes in a wound is necessary for the healing process to occur more quickly. To examine if the isolated fucoidan also has antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e in the animal wounds, we determined the number of bacteria in the wound samples using a colony counting method on plates (Olivia et al.)(Mart\u0026iacute;n et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Each group was analyzed on day 0 and day 9 and the colony counts were recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The results showed that wounds treated with 1.2% and 2.4% fucoidan contained fewer bacteria after 9 days, similar to those treated with the positive control Nisagon\u0026reg;. A reduction of bacterial colonies was also observed in the negative control after 9 days, most likely due to the animal innate immunity. Nevertheless, the results showed that in addition to its wound healing property, fucoidan may have strong antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e, validating its potential application to support the healing process of infected wounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eEffects of Fucoidan on Modulating Inflammatory Responses\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHistopathological changes in wound tissue were assessed using a modified scoring system (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which included parameters related to inflammation, proliferation, and maturation phases of wound healing. Wound healing generally goes through three main phases: the inflammation phase, the proliferation phase, and the remodelling phase. The inflammation phase, indicated by the presence of lymphocytes, leukocytes, and macrophages, aims to rid the wound of microorganisms and cellular debris. Macrophages are widely distributed in various body tissues and serve as key cells inducing inflammatory immune responses. They play a dual role, contributing to injury and supporting tissue repair (Yu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Previous studies have shown that macrophages play diverse roles at different stages of the skin recovery process (Pham et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo assess the impact of fucoidan on modulating inflammatory responses in \u003cem\u003eS. aureus\u003c/em\u003e-induced wounds, ulceration, leukocyte infiltration, and macrophage activity were examined. Day 3 post-injury was selected for evaluation, as it represents a critical juncture in the inflammatory phase preceding the proliferative stage. Histopathological scoring revealed that the F1 exhibited the highest mean ulcer scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), indicating extensive tissue damage. In contrast, groups treated with F3 and F4. Fucoidan demonstrated significantly lower ulcer scores compared to the F1, suggesting that fucoidan administration may expedite healing by mitigating ulcer severity. Notably, the group F4 showed no statistically significant difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) in ulcer scores when compared to the group F2, implying comparable efficacy to standard therapy in reducing tissue damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of leukocyte infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) indicated elevated scores in the group F1, reflecting a heightened inflammatory response, potentially due to uncontrolled infection. Conversely, the fucoidan-treated groups (F3 and F4) exhibited moderately reduced leukocyte scores relative to the F1, pointing to fucoidan's potential anti-inflammatory properties in curbing excessive inflammation. Similarly, the group F2 displayed decreased leukocyte infiltration, with no significant statistical difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) between F2 and F4, suggesting that fucoidan's effect on leukocyte modulation is on par with standard treatment. The results are consistent with a previous study by Olsthoorn, who reported that fucoidan can modulate local immune responses by reducing excessive leukocyte infiltration and optimizing macrophage numbers (Olsthoorn et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Fucoidan also contributes to decreased tissue damage (ulceration) resulting from bacterial infection, likely through its antibacterial and anti-inflammatory effects. Furthermore, Velnar \u003cem\u003eet al.\u003c/em\u003e emphasized that effective regulation of the inflammatory phase is crucial for a seamless transition to the proliferative phase. Fucoidan is believed to function by downregulating pro-inflammatory cytokines, such as TNF-α and IL-6, while promoting the secretion of growth factors by macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), thereby facilitating early granulation tissue formation. Our results showed that F4 was superior in reducing ulcer scores and modulating immune responses (leukocytes and macrophages) compared to F3. Although fucoidan-treated groups outperformed the negative control, F4 provided a more controlled inflammatory response, indicating the wound's readiness to progress to the proliferative phase.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Fucoidan on Cell Proliferation\u003c/h2\u003e \u003cp\u003eThe proliferation phase is characterized by the formation of granulation tissue and angiogenesis, followed by collagen synthesis and re-epithelization that serve to close the wound. Therefore, an adequate blood supply provides the necessary basis for granulation tissue growth, which is a key factor in the wound healing process (Sun et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the impact of fucoidan on proliferation and tissue repair in \u003cem\u003eS. aureus\u003c/em\u003e-infected wounds, we evaluated the formation of angiogenesis and fibroblast in wound tissues collected from day 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). The histopathological assessment of angiogenesis showed that samples from group F4 (fucoidan 2.4%) had the highest average of angiogenesis score (blood vessel formation). This finding suggests that fucoidan has the potential to accelerate the wound healing process through stimulating the formation of new blood vessels. However, there was no statistically significant difference between the F4 group and the F3, F2, and F1 groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). These results indicate that the F4 formulation has efficacy comparable to that of other formulations in supporting angiogenesis. Previous studies have shown that on day four to twenty-one after injury, angiogenesis and granulation tissue increases (Čoma et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMyofibroblasts, fibroblasts, inflammatory cells, and endothelial cells contribute to the formation of new extracellular matrix components and continue to develop until the wound closure process begins (Cialdai et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Histopathological analysis on day 6 showed that the wound was in the active proliferation phase. The negative control group (F1) showed the lowest fibroblast proliferation and new tissue formation. In contrast, the positive control group (F2) showed greater tissue formation than the F1 group. In the fucoidan treatment group, formulation F3 (1.2%) showed a lower healing response than F4 (2.4%), whereas F4 exhibited higher fibroblast proliferation and tissue formation. These findings indicate an increase in wound healing response with increasing fucoidan concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Fucoidan on Wound Maturation\u003c/h2\u003e \u003cp\u003eCutaneous wound healing remodeling is an important phase in wound healing, characterized by maturation and restructuring of the extracellular matrix (ECM) to restore tissue integrity. Following fibroblast proliferation, collagen synthesis and deposition is an important part of the process of replacing damaged tissue (Choudhary et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). One week after the wound occurs in the maturation phase in wound healing can increase collagen back to normal (Murali et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). A study conducted by Zixuan et al. in 2024 revealed that collagen regeneration in wounds is an important indicator in the skin regeneration process. This finding suggests that optimal collagen formation can accelerate wound healing and improve the function of damaged skin tissue (Zhang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Collagen had the potential to increase the percentage of epithelialization after seven days of therapy. This suggests that collagen plays an important role in supporting the wound healing process by accelerating the formation of a new epithelial layer. Collagen administration can repair damaged skin integrity, increase epithelial cell proliferation, and accelerate tissue recovery (Benito-Mart\u0026iacute;nez et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo evaluate the effect of fucoidan on would healing maturation, we analyzed collagen formation on tissue samples collected from day 9. Day 9 was chosen as the evaluation point, as it is a critical phase in collagen formation and epithelialization. The results showed that tissue samples from the group receiving fucoidan 2.4% (group F4) had the highest average collagen histopathological score, followed by the group that received Nisagon\u003csup\u003e\u0026reg;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). On the other hand, the negative control (group F1) and the group receiving fucoidan 1.2% (F3) showed lower scores. This result suggests that fucoidan 2.4% can accelerate wound healing process by stimulating the formation of collagen.\u003c/p\u003e \u003cp\u003eSimilarly, tissue samples from the group receiving fucoidan 2.4% (group F4) had the highest average re-epithelialization score, followed by the group that received Nisagon\u0026reg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), whereas administration of fucoidan 1.2% (F3) did not show significantly better re-epithelialization scores compared to the negative control. The high re-epithelialization score for fucoidan 2.4% suggests that the administration of fucoidan at higher concentrations can accelerate the re-epithelialization process in \u003cem\u003eS. aureus\u003c/em\u003e infected wound healing.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe brown algae (\u003cem\u003eS. polycystrum\u003c/em\u003e) from the sea around Takalar Regency, South Sulawesi \u0026ndash; Indonesia, is a rich source of fucoidan. The identity of the isolated fucoidan was confirmed by FTIR and NMR. The compound showed no cytotoxicity activity in a red blood cell hemolysis test. We found that fucoidan at a concentration of 2.4% significantly accelerated wound healing. It also improved the inflammatory, proliferation, and maturation phases. We conclude that 2.4% of fucoidan may be used as an effective and safe anti-inflammatory and antibacterial agent in wound healing therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics Approval\u003c/strong\u003e \u003cp\u003eThe animal ethics guidelines were followed and approved by the Research Ethics Committee, Faculty of Pharmacy, Hasanuddin University (\u003cb\u003e913/UN4.17/KEP/2024).\u003c/b\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003e The authors approve processing this manuscript for publication.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eFausyiah Sasmitha AB Writing \u0026ndash; review and editing, Writing \u0026ndash; original draft, Methodology, Conceptualization, Data curation, Formal analysis, Investigation. Frederika Tangdi Lintin Writing \u0026ndash; review and editing, Conceptualization, Investigation, Methodology, Visualization. Sulistiawati Validation, Software. Sitti Nur Fatimah Software. Stephanie Investigation, Formal analysis, Writing \u0026ndash; original draft. Muhammad Nur Amir Conceptualization. Syamsiah Conceptualization. Ngoc Dan Thuy Nguyen Investigation, Data curation. Taifo Mahmud Conceptualization, Supervision, Resources, Funding acquisition. Muhammad Nasrum Massi Writing \u0026ndash; review and editing, Resources, Project administration, Methodology, Conceptualization. Marianti A. Manggau Writing \u0026ndash; review and editing, Supervision, Resources, Funding acquisition, Conceptualization\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eFor the technical support and help in performing some techniques, we want to expresses gratitude to the Ministry of Health of the Republic of Indonesia for providing a scholarship for a master\u0026rsquo;s degree in pharmaceutical science. Fausyiah Sasmitha AB. was financially supported by the Ministry of Health of the Republic of Indonesia to pursue a Master\u0026rsquo;s degree in Pharmaceutical Sciences (Ref. No. HK.01.07/F.I/5666/2023).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eNo data was used for the research described in the article.\u003c/p\u003e\u003ch2\u003eCode Availability\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel-Latif HMR, Dawood MAO, Alagawany M et al (2022) Health benefits and potential applications of fucoidan (FCD) extracted from brown seaweeds in aquaculture: An updated review. Fish Shellfish Immunol 122:115\u0026ndash;130\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAyrapetyan ON, Obluchinskaya ED, Zhurishkina EV et al (2021) Antibacterial properties of fucoidans from the brown algae fucus vesiculosus l. of the barents sea. 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Int J Biol Macromol 283. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2024.138039\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2024.138039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Brown Algae, Fucoidan, Sargassum polycystum, Wound Healing, Inflammation, Staphylococcus aureus","lastPublishedDoi":"10.21203/rs.3.rs-9573456/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9573456/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe skin, the body's largest organ, acts as the primary barrier against infections, but this function can be compromised by wounds. While antibiotics are commonly used for treating skin infections, their misuse has led to antibiotic-resistant pathogens, emphasizing the need for safer alternatives. Fucoidan has shown antibacterial and anti-inflammatory properties. However, its biological activity can vary due to differences in species and geographical origin. In this study, fucoidan was isolated from \u003cem\u003eSargassum polycystum\u003c/em\u003e collected in the intertidal zone of South Sulawesi, Indonesia, and its wound-healing potential was tested in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e-infected rats. Fucoidan was extracted using hot ethanol-water extraction and CaCl₂-ethanol precipitation, and characterized by FTIR, \u0026sup1;H NMR, and \u0026sup1;\u0026sup3;C NMR. Toxicity was assessed with a hemolysis assay, and wound healing was evaluated in rats with 6 mm excision wounds infected with \u003cem\u003eS. aureus\u003c/em\u003e (0.1 mL, 10⁷ CFU/mL). Results showed that fucoidan was non-toxic and that a 2.4% topical application significantly reduced bacterial load and accelerated healing within 9 days. Histological analysis revealed enhanced fibroblast proliferation, granulation tissue formation, re-epithelialization, and thicker collagen deposition compared to the control group (Vaseline only) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings suggest that fucoidan from \u003cem\u003eS. polycystum\u003c/em\u003e is safe and effective in promoting wound healing, supporting its potential as an alternative therapy for infected wounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Characterization and Wound-Healing Effects of Fucoidan from Sargassum polycystum Collected in South Sulawesi in a Staphylococcus aureus-Infected Rat Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 08:22:53","doi":"10.21203/rs.3.rs-9573456/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-20T13:23:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43892574592323245003836320884807120318","date":"2026-05-08T03:52:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259266601480484251148023794040152719105","date":"2026-05-08T00:45:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270605917674909729637640093275181069551","date":"2026-05-07T22:49:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-07T20:51:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-06T10:16:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-06T10:15:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biotechnology","date":"2026-04-30T07:15:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"84564f3e-38e8-4fb0-8167-2e57cb493168","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-20T13:23:24+00:00","index":32,"fulltext":""},{"type":"reviewerAgreed","content":"43892574592323245003836320884807120318","date":"2026-05-08T03:52:11+00:00","index":31,"fulltext":""},{"type":"reviewerAgreed","content":"259266601480484251148023794040152719105","date":"2026-05-08T00:45:23+00:00","index":30,"fulltext":""},{"type":"reviewerAgreed","content":"270605917674909729637640093275181069551","date":"2026-05-07T22:49:59+00:00","index":29,"fulltext":""},{"type":"reviewersInvited","content":"21","date":"2026-05-07T20:51:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-06T10:16:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-06T10:15:55+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T08:22:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 08:22:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9573456","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9573456","identity":"rs-9573456","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}