Ulvan and Ulva oligosaccharides from Ulva sp. attenuate osteoarthritis in a high-fat diet and ligamentous meniscal injury-induced rat model
Sabri Sudirman, Yi-Chia Lin, Yi-Yuh Hwang, Jerrell Felim, Hsiang-Ping Kuo, Deng-Fwu Hwang, Zwe-Ling Kong
TL;DR
This study shows that compounds from seaweed can reduce osteoarthritis symptoms in rats by lowering inflammation and cartilage damage.
Contribution
The study demonstrates the protective effects of Ulva-derived ulvan and oligosaccharides in an osteoarthritis rat model.
Findings
Ulvan and Ulva oligosaccharides reduced inflammation and cartilage degradation in OA rats.
Treatments lowered triglycerides, cholesterol, and pro-inflammatory markers in OA rats.
Ulva hydrolysate showed higher sugar and sulfated group content than the original extract.
Abstract
Osteoarthritis (OA) is a chronic joint disease. It is marked by the progressive deterioration of subchondral bone, articular cartilage, and synovium, with obesity acting as a significant risk factor by promoting inflammation and cartilage degradation. Ulvan and Ulva oligosaccharides derived from Ulva sp. seaweed have shown anti-inflammatory properties that may be beneficial in this context. Therefore, the aim of this research was to determine the protective effect of ulvan and Ulva oligosaccharides from Ulva sp. on monosodium iodoacetate (MIA)-induced inflammation in SW1353 cells and in a high-fat-diet/ACL-meniscus-injury rat model of osteoarthritis. The Ulva extract (UE) was hydrolyzed with cellulase to obtain Ulva hydrolysate (UH). The rats were treated using UE (50 mg/kg) and three different doses of UH (UH1, 50 mg/kg; UH2, 100 mg/kg; UH5, 250 mg/kg). In addition, UH contains higher…
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Taxonomy
TopicsSeaweed-derived Bioactive Compounds · Phytochemical and Pharmacological Studies · Medicinal plant effects and applications
Introduction
Osteoarthritis (OA) is a chronic degenerative disease of the joints indicated by gradual cartilage breakdown and inflammation, resulting in pain, stiffness, and limited mobility (Primorac et al. 2020). The global burden of OA has increased substantially between 1990 and 2021 (Wu et al. 2025), and long-term trends are expected to persist, largely driven by population aging and the increasing prevalence of obesity (Long et al. 2022). Obesity is a well-recognized risk factor for OA, as it increases mechanical loading on weight-bearing joints and hastens the degradation of articular cartilage (Misra et al. 2019). Moreover, obesity-driven inflammation—mediated by adipose-derived cytokines, chemokines, and adipokines—further contributes to OA progression. Elevated circulating pro-inflammatory cytokine levels, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1βhave been documented in animal models and obese people. Obesity also enhances oxidative stress, marked by elevated nitric oxide production and increased inflammatory mediators, such as cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2), which further aggravate chondrocyte dysfunction (Huang et al. 2015; Chan et al. 2016). These inflammatory factors induce the production of matrix metalloproteinases (MMPs), leading to accelerated cartilage degradation (Siwik et al. 2000; Zhang et al. 2017).
Osteoarthritis has been studied using a variety of animal models, encompassing both spontaneous and surgically induced approaches (Kuyinu et al. 2016). A common technique for triggering post-traumatic OA is anterior cruciate ligament transection (ACLT), which destabilizes the knee joint (Anderson et al. 2011; Kao et al. 2016), while total or partial meniscectomy alters normal load distribution and promotes OA-like changes (Song et al. 2008; Little and Fosang 2010). For in vitro studies, the human chondrosarcoma cell line SW1353 is commonly used, and monosodium iodoacetate (MIA) induces chondrocyte death to simulate OA-associated inflammation (Gebauer et al. 2005; Chiu et al. 2016).
Non-steroidal anti-inflammatory medicines (NSAIDs) are frequently used to treat the symptoms of osteoarthritis (OA); nevertheless, long-term usage is frequently associated with side effects, such as gastrointestinal irritation, ulceration, and cardiovascular issues (Domper Arnal et al. 2021; Sohail et al. 2023). Consequently, identifying natural anti-inflammatory compounds with fewer side effects has become a significant research priority. Bioactive substances—including polysaccharides and polyphenols derived from aquatic and terrestrial plants—have shown promise in attenuating inflammation in OA models (Hridayanka et al. 2024; Salehi and Rashidinejad 2025). Previous studies have shown that seaweed polysaccharides, including fucoidan and carrageenan, can reduce inflammation and alleviate OA-associated cartilage degradation (Sudirman et al. 2019; Hwang et al. 2023; Kraiem et al. 2024). Marine polysaccharides, including carrageenan derived from red algae and fucoidan from brown algae, are well known for their immunomodulatory, anti-inflammatory, and antioxidant activities and have been thoroughly investigated for their possible therapeutic uses in conditions involving persistent inflammation (Akter et al. 2024; Jim et al. 2025). Although these compounds exhibit strong bioactivity, ulvan—a sulfated polysaccharide derived from green seaweeds—has been comparatively less studied, despite its unique structural features and promising biological activities.
Ulva sp., commonly referred to as sea lettuce, is an edible green seaweed belonging to the family Ulvaceae. Ulvan exhibits a range of biological properties, such as anti-inflammatory, antihyperlipidemic, and antioxidant (Tanna and Mishra 2019; Li et al. 2020). Structurally, ulvan mainly consists of repeating disaccharide units composed of rhamnose sulfate, xylose, and either iduronic or uronic acid (Mo’o et al. 2020), which differentiates it from other marine polysaccharides, such as fucoidan (rich in fucose) and carrageenan (composed of galactose-based units). Converting seaweed polysaccharides into oligosaccharides is an emerging process that expands their functional applications in food and biochemical industries (Kraiem et al. 2024; Lakhrem et al. 2024). Enzyme-assisted extraction is regarded as an efficient approach for isolating bioactive compounds from seaweeds (Wijesinghe and Jeon 2012; Hung et al. 2021). Earlier studies have reported that ulvan and Ulva-derived oligosaccharides regulate inflammation by inhibiting nuclear factor (NF)-κB activation and suppressing downstream cytokines, such as TNF-α, IL-6, and COX-2 (Amaro et al. 2022; Flórez-Fernández et al. 2023; Ou et al. 2024). Based on this evidence, we hypothesized that ulvan and Ulva oligosaccharides may also alleviate inflammation and suppress OA progression in an in vivo model; however, this specific therapeutic effect has not yet been documented. Thus, the purpose of this study was to evaluate the protective properties of ulvan and Ulva oligosaccharides from Ulva sp. on osteoarthritis progression induced by anterior cruciate ligament transection combined with medial meniscectomy in a high-fat diet rat model. Additionally, this study examined their effects on pro-inflammatory mediators, cytokines, and cartilage-degrading enzymes in both cell and animal models.
Materials and methods
Materials
Green seaweed (Ulva sp.) was hand-harvested from the coastal waters of Keelung, Taiwan, and promptly rinsed with seawater to eliminate adhering debris. The specimen was taxonomically identified and deposited under the supervision of Professor Zwe-Ling Kong at the Department of Food Science, National Taiwan Ocean University (Keelung, Taiwan). Male Sprague–Dawley (SD) rats were purchased from BioLASCO Co. Ltd. (Yilan, Taiwan). The human chondrosarcoma cell line SW1353 was purchased from the Bioresource Collection and Research Center of the Food Industry Research and Development Institute and the American Type Culture Collection (ATCC). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Gibco BRL (New York, USA). LabDiet 5001 Rodent Diet was purchased from PMI Nutrition International (Minnesota, USA). Collagen Type II Alpha 1 (Collagen 2α1), NF-κB p65, IL-1β, IL-6, MMP-3, and TNF-α ELISA kits were purchased from Elabscience (Texas, USA). Total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and triglycerides (TG) kits were purchased from Randox (Crumlin, UK). Every analysis utilizing commercial kits was carried out in compliance with the guidelines provided by the manufacturer.
Sample preparation
The Ulva extract (UE) and Ulva hydrolysate (UH) were prepared following previously reported methods (Li et al. 2023; Malvis Romero et al. 2023; Rodríguez-Iglesias et al. 2024). Briefly, dried Ulva sp. powder (1,500 g) was mixed with 30 L of distilled water and extracted at 121 °C for 20 min. The mixture was subsequently centrifuged at 8,800 × g for 20 min, and the supernatant was collected. Next, 95% ethanol (4× volume) was added to precipitate the polysaccharides. The resulting mixture was concentrated using a rotary evaporator and then freeze-dried to obtain UE. The UE was subsequently hydrolyzed with cellulase at 37 °C for 4 h, followed by incubation in a boiling water bath for 10 min to terminate the reaction. The resulting hydrolysate was then freeze-dried to obtain UH.
Chemical characterization of Ulva samples
The moisture, fat, protein, and ash contents of dried Ulva sp. powder were analyzed according to the Association of Official Analytical Chemist (AOAC 2000). The carbohydrate content was measured by difference method [100 − (protein + moisture + fat + ash)] (McCleary and McLoughlin 2021). The total sugar content of UE and UH were analyzed according to the previous method (Nielsen 2017) and the sulfated groups were analyzed using the Dodgson-Price method (Dodgson and Price 1962; Kanno et al. 2014). Whereas, the monosaccharide compositions of UH were analyzed by thin-layer chromatography (TLC) according to previous methods (Liu et al. 2021; Toschkova 2022). Briefly, UH was separated using silica gel 60 TLC, and pre-coated plates were developed with a mobile phase (n-butanol: acetic acid: distilled water, 2:1:1, v/v/v). A sulfuric acid–anhydrous methanol spray reagent was used to see the separated sugars.
Cell assay
The SW1353 human chondrosarcoma cell line was exposed to monosodium iodoacetate (MIA) to evaluate the chondroprotective effects of Ulva extract (UE) and Ulva hydrolysate (UH), according to previously described methods (Chiu et al. 2016). Cells were maintained at 37 °C in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin in a 5% CO₂ atmosphere. A total of 5 × 10⁵ cells were seeded into 6 cm dishes and allowed to attach for 24 h. After attachment, the cells were incubated with UE or UH at concentrations of 1,000, 2,000, or 3,000 µg/mL, either alone or in combination with 5 µM MIA, for another 24 h. Post-treatment, cellular responses were examined, and levels of IL-6, MMP-3, and Collagen 2α1 were quantified following the respective manufacturer instructions.
Animal experiment
The National Taiwan Ocean University’s Institutional Animal Care and Use Committee examined and approved all experiments (IACUC Approval No. 109026). Fifty-six five-week-old male Sprague–Dawley rats (N = 56) were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). The animals were housed under controlled environmental conditions, with a temperature of 23 ± 1 °C, relative humidity of 40–60%, and a 12 h light/dark cycle. Each rat was housed individually and provided ad libitum access to a standard chow diet (LabDiet 5001) and water. The rats were acclimated for one week and then randomly assigned to two main groups (Fig. 1): a normal group fed a Chow diet (n = 14) and a High-Fat Diet (HFD; Obesity group) (n = 42). The Normal group was further divided into two subgroups after six weeks of feeding: A subgroup underwent anterior cruciate ligament transection with medial meniscus resection (ACLT + MMx) surgery (OA) (n = 7), and the other subgroup had an opened knee-joint capsule without ACLT + MMx surgery (Control) (n = 7). The HFD group underwent ACLT + MMx surgery (n = 42) and was then divided into six subgroups: one subgroup was administered saline (OBOA), one subgroup received UE at 50 mg/kg body weight (OBOA + UE), three subgroups were administered three different doses of UH at 50 mg/kg body weight (OBOA + UH1), 100 mg/kg body weight (OBOA + UH2), and 250 mg/kg body weight (OBOA + UH5), and the last subgroup received glucosamine sulfate at 100 mg/kg body weight as a positive Control (OBOA + GS). The sample were given once daily by oral gavage beginning one week after surgery. The treatment was administered for six weeks, after which the rats were euthanized and blood samples were taken for further analysis.
Fig. 1. The flowchart of green seaweed (Ulva nitidum) treatment on ACLT + MMx-induced osteoarthritis in obesity rats. ACLT + MMx, anterior cruciate ligament transection and medial meniscus resection; GS, glucosamine sulphate, HFD, high-fat diet; UE, Ulva extract; UH, Ulva hydrolysate; OA, osteoarthritis; OB, Obesity
Surgical induction of osteoarthritis
Post-traumatic osteoarthritis (OA) was surgically induced in obese rats by anterior cruciate ligament transection combined with medial meniscectomy (ACLT + MMx), following a previously described method (Sudirman et al. 2019). Briefly, the rats were anesthetized via intraperitoneal administration of Zoletil (25 mg/kg body weight), and the hair covering the right knee was removed using a digital hair clipper. In the Sham (control) group, the knee joint capsule was opened without performing ACLT + MMx. In contrast, rats in the OA and obese OA (OBOA) groups (OBOA, OBOA + UE, OBOA + UH1, OBOA + UH2, OBOA + UH5, and OBOA + GS) underwent ACLT + MMx surgery. After the procedure, the knee joint capsule and skin were sutured using chromic catgut sterile sutures (4–0) and silk braided sterile sutures (3–0; Unik Surgical Sutures Mfg. Co. Ltd., Taipei, Taiwan), respectively. To prevent postoperative infection, rats received intraperitoneal injections of cefazolin (30 mg/kg/day) for 3 consecutive days after surgery.
Blood sample collection and analysis
A heparinized syringe was used to collect blood from the rats’ abdominal aorta on the day of sacrifice. Centrifugation (3,000 rpm for 15 min at 4 °C) using a Kubota Centrifuge 3500 (Kubota Corp., Tokyo, Japan) was used to separate the plasma. The supernatant was carefully collected using a micropipette and stored at − 20 °C for further experiment (Mussbacher et al. 2017).
Plasma and inflammatory marker analysis
Plasma TG, TC, and LDL-cholesterol levels were measured using commercial assay kits. Inflammatory markers, including NF-κB, IL-1β, TNF-α, PGE₂, MMP-3, and C-terminal cross-linked telopeptides of type II collagen (CTX-II), were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits in accordance with the manufacturers’ instructions. Nitric oxide (NO) levels were determined using the Griess reaction according to a previously described method (Sun et al. 2003).
Histological analysis
On the day of sacrifice, the right knee joints were removed and preserved in 4% formaldehyde. After decalcification, 5-µm paraffin-embedded sections were prepared and stained with Safranin-O. The staining was performed by Li Pie Co. Ltd. (Taichung, Taiwan) One section was taken from each right knee joint for histological evaluation. The stained knee joints were subsequently assessed for proteoglycan depletion, morphological changes, and osteoarthritis (OA) cartilage lesions, which were graded following the Osteoarthritis Research Society International (OARSI) guidelines, as previously reported (Pritzker et al. 2006; Sudirman et al. 2019), and are summarized in Table 1.
Table 1. Grading of OA histopathology cartilage according to OARSI gradeGradeDescriptionsGrade 0Normal cartilage; hyaline articular cartilage uninvolved with OAGrade 1Threshold in cartilage for OA and characterized by the retention of the articular cartilage surface layerGrade 2Focal discontinuity of the cartilage superficial zoneGrade 3The extension of matrix cracks into the mid zone to form vertical fissures (clefts)Grade 4Cartilage erosionGrade 5Denudation, the complete erosion of the hyaline cartilage to a level of mineralized cartilage or boneGrade 6Changes in the contour of the cartilage surface (deformation)
Statistical analysis
The mean ± standard deviation (Mean ± SD) is used to display the data and statistical analyses were conducted using SPSS Statistics version 19.0. Statistical differences were evaluated using one-way analysis of variance (ANOVA), followed by Duncan’s post-hoc test. A p-value of less than 0.05 (p < 0.05) was considered statistically significant.
Results
The chemical properties of Ulva samples
The chemical composition of dried Ulva sp. powder is presented in Table 2. The dried Ulva sp. consisted primarily of carbohydrates (60.8%) and ash (20.5%). It also contained 12.1% moisture, 5.1% crude fat, and 1.5% crude protein. The total sugar and sulfated group contents of UE and UH are shown in Table 3. Ulva hydrolysate (UH) had higher levels of total sugar (22.8%) and sulfated groups (18.4%) compared to UE, which contained 19.6% total sugar and 15.5% sulfated groups. The monosaccharide composition of UH is shown in Table 4. UH was composed of rhamnose (retention factor, Rf = 0.61) and glucose (Rf = 0.45).
Table 2. Chemical composition of dried Ulva SpChemical compositionsValues (%)Crude protein1.5 ± 0.3Crude fat5.1 ± 0.4Moisture12.1 ± 0.8Ash20.5 ± 1.2Carbohydrate (by difference)60.8 ± 3.6
Table 3. Total sugar and sulphated group content of UH and UESampleTotal sugar (%)Sulfated groups (%)Ulva extract (UE)19.6 ± 1.015.5 ± 0.1Ulva hydrolysate (UH)22.8 ± 1.318.4 ± 0.2
Table 4. Retention factor of UH and monosaccharides in mobile phaseMobile phaseRf-valuesIdentified monosaccharides and UHButanol: acetic acid: pure water (2:1:1)0.48Arabinose0.45Glucose0.48Mannose0.61Rhamnose0.57Xylose0.60; 0.43Ulva* hydrolysate (UH)*Visualization by spraying with sulfuric acid-anhydrous methanol
Effects of UE and UH on IL-6, MMP-3, and collagen 2α1
The effect of UE and UH on the level of MMP-3, IL-6, and collagen 2α1 in MIA-induced inflammation of SW1353 cells is shown in Fig. 2. An increase in IL-6 and MMP-3 was observed in the MIA-induced group. Treatment with UE and UH successfully decreased IL-6 and MMP-3 levels, with UH significantly (p < 0.05) reducing these levels compared to the UE group. On the other hand, a decrease in Collagen 2α1 levels was detected in the MIA-induced group. Treatment with UE slightly increased Collagen 2α1 levels, while UH significantly (p < 0.05) increased Collagen 2α1 levels compared to UE.
Fig. 2. Effects of UE and UH on the production of IL-6, MMP-3, and Collagen 2α1 in MIA-induced SW1353 cells. Data are presented as mean ± SD (n = 3). ^#^ indicates significant difference at p < 0.05; ^##^ indicates significant difference at p < 0.01. Collagen 2α1, Collagen Type II Alpha 1; IL, interleukin; MIA, monosodium iodoacetate; MMP, matrix metalloproteinase; UE, Ulva extract; UH, Ulva hydrolysate
Effects of UE and UH on body weight and lipid profile
The effects of UE and UH on the body weight of rats are shown in Fig. 3. Body weight was significantly higher (p < 0.05) in the OBOA group compared to the Control. In contrast, there was no significant difference (p > 0.05) in body weight between the medium- and high-dose UH treatment groups (OBOA + UH2 and OBOA + UH5, respectively) and the Control group.
Fig. 3. The bodyweight of rats after 6 weeks of treatment. Data are shown as mean (n = 7). *; indicated a significant difference at p < 0.05 versus Control group
Total adipose tissue weight was significantly higher (p < 0.05) in the OBOA group but was markedly reduced following six weeks of treatment with UE and UH (Table 5). Furthermore, Table 5 shows that the OBOA group had significantly elevated levels of TG, TC, and LDL-C, all of which were significantly reduced (p < 0.05) following six weeks of treatment with UE and UH.
Table 5. Effect of UE and UH on plasma TG, TC, and LDL-C in the animal modelPropertiesControlOAOBOAOBOA + UEOBOA + UH1OBOA + UH2OBOA + UH5OBOA + GSOrgan weight (g) / Body weight (100 g)Total adipose2.8 ± 0.6^a^2.7 ± 0.6^a^5.6 ± 0.9^c^4.1 ± 0.9^b^3.7 ± 0.8^b^3.4 ± 0.8^ab^3.8 ± 0.4^b^4.1 ± 0.5^b^Blood properties (mg/dL)TG156 ± 7^a^157 ± 19^a^324 ± 37^c^155 ± 9^a^155 ± 19^a^198 ± 43^b^161 ± 30^a^189 ± 30^ab^TC74 ± 14^bc^79 ± 13^bc^94 ± 11^d^75 ± 4^bc^78 ± 17^bc^68 ± 8^ab^60 ± 11^a^85 ± 10^cd^LDL-C38 ± 7^a^33 ± 8^a^61 ± 9^b^34 ± 1^a^42 ± 12^a^38 ± 12^a^38 ± 8^a^45 ± 9^a^Data are presented as the mean ± SD (n = 7). Different letters indicate statistically significant differences (p < 0.05) between groups, as determined by one-way ANOVA with Duncan’s post-hoc test. LDL-C, low-density lipoprotein-cholesterol; GS, glucosamine sulfate; OA, osteoarthritis; OB, obesity; UE, Ulva extract; UH, Ulva hydrolysate
Effects of UE and UH on reduce oxidative stress and inflammation
The levels of NO, COX-2, and PGE2 were significantly (p < 0.05) elevated in the OBOA group (Fig. 4). Treatment with UE and UH significantly (p < 0.05) reduced NO and PGE2 levels. In contrast, COX-2 levels were significantly (p < 0.05) reduced by UE and by low- and medium-dose UH treatment (OBOA + UH1 and OBOA + UH2, respectively). Similarly, levels of NF-κB, IL-1β, and TNF-α were significantly (p < 0.05) elevated in the OBOA group (Fig. 5), but were significantly (p < 0.05) reduced after six weeks of treatment with UE and UH. Furthermore, MMP-3 and CTX-II levels were significantly (p < 0.05) higher in the OBOA group (Fig. 6) and were significantly (p < 0.05) reduced following treatment with UE and medium-dose UH (OBOA + UH2).
Fig. 4. Effects of UE and UH on the levels of A nitric oxide (NO), B cyclooxygenase (COX)-2, and C prostaglandin E2 (PGE2) in rats after 6 weeks of treatment. Data are presented as the mean ± SD (n = 7). Different letters (a-e) indicate statistically significant differences (p < 0.05) between groups, as determined by one-way ANOVA with Duncan’s post-hoc test. GS, glucosamine sulfate; OA, osteoarthritis; OB, obesity; UE, Ulva extract; UH, Ulva hydrolysate
Fig. 5. Effects of UE and UH on the expression of A interleukin (IL)-1β, B nuclear factor (NF)-κB, and C tumor necrosis factor (TNF)-α in rats after 6 weeks of treatment. Data are presented as the mean ± SD (n = 7). Different letters (a-e) indicate statistically significant differences (p < 0.05) between groups, as determined by one-way ANOVA with Duncan’s post-hoc test. GS, glucosamine sulfate; OA, osteoarthritis; OB, obesity; UE, Ulva extract; UH, Ulva hydrolysate
Fig. 6. Effects of UE and UH on the expression of A matrix metalloproteinase (MMP)-3 and B C-terminal cross-linked telopeptides of type II collagen (CTX-II) in rats after 6 weeks of treatment. Data are presented as the mean ± SD (n = 7). Different letters (a-c) indicate statistically significant differences (p < 0.05) between groups, as determined by one-way ANOVA with Duncan’s post-hoc test. GS, glucosamine sulfate; OA, osteoarthritis; OB, obesity; UE, Ulva extract; UH, Ulva hydrolysate
UE and UH treatments improve knee-joint histopathology
Representative Safranin-O staining images and Osteoarthritis Research Society International (OARSI) histopathology grading scores for each group are shown in Fig. 7. Normal cartilage structure was observed in the group without anterior cruciate ligament transection and medial meniscus resection (Control group). In contrast, the OBOA group exhibited proteoglycan loss, diminished staining intensity, and a reduced number of chondrocytes in the superficial cartilage zone. This condition was associated with a significantly (p < 0.05) higher OARSI grade compared to the Control group. However, the OA grade was significantly (p < 0.05) reduced following treatment with UE and UH. Notably, the OBOA + UH2 group exhibited a significantly (p < 0.05) lower OA grade compared to the other groups subjected to anterior cruciate ligament transection and medial meniscus resection.
Fig. 7. Knee-joint histopathology of the rats: A Representative Safranin-O staining for each group. B OARSI histopathology grading. Cartilage (orange to red) and nuclei (black). The specimens were observed with 40× magnification. Data are presented as the mean ± SD (n = 7). Different letters (a-e) indicate statistically significant differences (p < 0.05) between groups, as determined by one-way ANOVA with Duncan’s post-hoc test. GS, glucosamine sulfate; OA, osteoarthritis; OB, obesity; UE, Ulva extract; UH, Ulva hydrolysate
Discussion
In this study, Ulvan and Ulva oligosaccharide were successfully extracted from dried Ulva sp. The dried Ulva powder contained a high carbohydrate content (68.83%), as shown in Table 2. Earlier findings have suggested that the carbohydrate content of Ulva sp. ranges from 31.50% to 58.10% (Rasyid 2017; Hung et al. 2021; Barakat et al. 2022). Polysaccharides, the most abundant form of carbohydrates in nature, are composed of monosaccharide units connected by glycosidic bonds (Mohammed et al. 2021). Furthermore, a prior research indicated that Ulva sp. from Kochi, Japan, contains a variety of monosaccharides, such as rhamnose, galactose, glucose, xylose, and mannose (Tsubaki et al. 2014).
The extraction process resulted in UE. The UE contained 19.60% total sugar and 15.50% sulfated group content (Table 3). A previous study reported that Ulva fasciata extract contained 13.75% total sugar and 20.45% sulfate group content (Barakat et al. 2022). The UE was then hydrolyzed to produce UH using cellulase enzyme. Previous studies also used cellulase enzymes in the production of oligosaccharides from seaweed (Cheong et al. 2018) and in the saccharification of seaweed polysaccharides (Hebbale et al. 2019). The UH had higher total sugar (22.80%) and sulfate group content (18.40%) than the UE. Another study also reported that Ulva extract was successfully hydrolyzed using enzymes to produce Ulvan oligosaccharides. This process resulted in an increase in reducing sugar and total phenol content in the Ulvan oligosaccharides compared to the Ulva extract (Hung et al. 2021). Additionally, enzyme-assisted extraction is regarded as an effective technique for obtaining bioactive compounds from seaweeds (Wijesinghe and Jeon 2012). According to the thin-layer chromatography analysis (Table 4), this hydrolysate contained rhamnose and glucose. Therefore, we concluded that UH contained Ulva oligosaccharides, while UE contained ulvan. Ulvan, a type of sulfated polysaccharide found in green seaweed, including Ulva sp., primarily consists of a repeating sequence of disaccharides composed of rhamnose sulfate, xylose, and iduronic or uronic acid (Mo’o et al. 2020). The presence of glucose in the extract indicates that Ulva sp. also contains other monosaccharides in addition to ulvan. A previous study reported that the hydrolysate from Ulva meridionalis and Ulva ohnoi is composed of rhamnose, galactose, xylose, glucose, and mannose (Tsubaki et al. 2014). Moreover, the chemical composition of ulvan has been shown to vary depending on factors, such as the Ulva species used, the harvest season, and the extraction and purification methods (Kidgell et al. 2019; Wahlström et al. 2020).
Increasing MMP-3 and IL-6 levels is observed in MIA-induced inflammation of SW1353 cells (Fig. 2). A previous study also reported an increase in some pro-inflammatory cytokines, such as IL-6 and IL-1β, in MIA-induced inflammation of SW1353 cells (Chiu et al. 2016). Treatment with UE and UH successfully reduced IL-6 and MMP-3 levels. On the other hand, UE and UH increased the production of Collagen 2α1. Based on this information, UE and UH exhibit anti-inflammatory activities in SW1353 cells.
In the animal study, the OBOA group showed significantly higher body weight compared to the Control group (Fig. 3). This condition indicates that obesity was successfully induced in the rats during the experiment using a high-fat diet (HFD). Along with the increase in body weight, total adipose tissue also increased in the OBOA group (Table 5). A previous study reported a significant increase in adipose tissue in HFD-induced obese rats (Woods et al. 2003; Marques et al. 2015). These conditions resulted in elevated levels of TG, TC, and LDL-Cholesterol in the rats’ plasma (Table 5). A previous research also reported increased levels of TG, TC, and LDL-C in HFD-induced obese rats (Jia et al. 2013). However, these levels were significantly decreased after treatment with UE and various doses of UH. Another study reported that seaweed polysaccharides and oligosaccharides have been shown to be promising as anti-obesity and cholesterol-lowering agents (Gao et al. 2024; Li et al. 2024). Additionally, oligosaccharides were suggested to help treat obesity through mechanisms, such as regulating gut microbes, modulating immunity, strengthening the intestinal barrier, and inhibiting pathogens (Bai et al. 2023).
In the case of OA, obesity has been recognized as a risk factor. Obesity increases the mechanical stress on weight-bearing joints and promotes the degradation of articular cartilage. It is also associated with inflammation, primarily driven by adipose tissue, which produces various cytokines, chemokines, and adipokines that regulate the immune response in cartilage (Misra et al. 2019). In the present study, an ACLT + MMx was used to induce osteoarthritis in obese rats. Figure 4 shows an increase in the levels of NO, COX-2, and PGE2 in the untreated OBOA group. Earlier studies reported that obesity contributes to increased oxidative stress, including elevated levels of NO and inflammatory mediators, such as PGE2 and COX-2 (Huang et al. 2015; Naomi et al. 2023). The mechanical stress in the knee joint due to ACLT + MMx is also involved in the increase of oxidative stress (Liu et al. 2022). The levels of NO, COX-2, and PGE2 were reduced after treatment with UE and UH. “Previous research also reported that Ulva polysaccharides reduce NO levels and COX-2 expressions (Amaro et al. 2022). Additionally, seaweed oligosaccharides inhibit NO and PGE2 by downregulating inducible NO synthesis and COX-2, respectively (Luan et al. 2023). Moreover, Ulva prolifera polysaccharide reduced oxidative stress by regulating the Nrf2/Keap1 and MAPK signaling pathways (Flórez-Fernández et al. 2023).
The increasing of some pro-inflammatory cytokines, such as TNF-α and IL-1β also are observed in untreated OBOA group (Fig. 5). This condition due to the higher adipose tissue in obesity condition and result from mechanical insult (Burrage 2006). A previous research also reported that serum or plasma levels of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, produced by macrophages derived from adipose tissue, are elevated in obese individuals and animals (Chan et al. 2016; Chira et al. 2025). As the result, the high expression of NF-κB is observed in untreated OBOA group. As a result of increased NF-κB expression in the OBOA group, MMP-3 also significantly increased in this group compared to the Control group (Fig. 6). A previous research reported that MMP-3 was also observed to be increased in OA patients (Naito et al. 1999). The NF-κB transcription factor is not only activated by pro-inflammatory cytokines but also triggers the production of pro-inflammatory cytokines (Rahmouni et al. 2024; Chira et al. 2025). NF-κB also induces the upregulation of MMPs (Choi et al. 2019). We hypothesized that UE and UH also regulate the mitogen-activated protein kinase (MAPK) pathway. Previous research has reported that MAPKs are crucial signaling pathways that promote inflammation by activating NF-κB and stimulating the release of pro-inflammatory cytokines (Flórez-Fernández et al. 2023).
The CTX-II was also observed to have significantly increased in the untreated OBAO group. A previous study reported that CTX-II is one of the most frequently assessed markers for OA diagnosis (Cheng et al. 2018). In the present study, NF-κB expression, pro-inflammatory cytokines, MMP-3, and CTX-II levels were significantly (p < 0.05) reduced after treatment with UE and various doses of UH. These findings indicate that UE and UH exhibit anti-inflammatory activity in this OA model. Beyond osteoarthritis, these findings highlight the broader potential of ulvan and Ulva oligosaccharides as bioactive compounds in functional foods and nutraceutical formulations, supporting sustainable marine-derived health products. Their anti-inflammatory, antioxidant, and lipid-lowering properties suggest they could contribute to the prevention and management of other chronic inflammatory or metabolic conditions, making them promising candidates for a wide range of health-promoting applications.
The histopathology of knee OA, with Safranin-O staining and OARSI grading, is shown in Fig. 7. The untreated OBOA group exhibits significantly (p < 0.05) higher OARSI grading compared to the Control group. Normal cartilage was observed in the group without anterior cruciate ligament transection and medial meniscus resection (Control group). Conversely, the untreated OBOA group exhibited proteoglycan loss, diminished staining, and lower cell density in the superficial zone of the cartilage. Previous studies have indicated that cartilage degradation is a key feature of OA, playing a significant role in the advancement of joint dysfunction. Healthy cartilage is distinguished by a smooth surface, with the matrix and chondrocytes arranged in well-organized zones (Pritzker et al. 2006). Furthermore, in the untreated OBOA group, we observed cell hypotrophy and surface irregularities in the superficial cartilage layer, whereas treatment with UE and UH helped preserve cartilage integrity and slowed OA progression. Therefore, OARSI grading was significantly (p < 0.05) decreased after treatment with UE and UH. This result indicates that UE and UH successfully protected against cartilage loss in this OA model. Overall, this study demonstrated that UE and UH improved the biochemical factors associated with OA development and mitigated cartilage-related OA damage in obese rats. Some parameters indicate that the reduction effect of the medium dose of UH (UH2) is more effective compared to the high dose (UH5). We hypothesize that this is due to saturation effects, where the biological targets or pathways are fully activated at moderate doses, but higher doses may lead to a plateau in response as the receptors or enzymes become saturated and cannot process further amounts effectively (Mazaleuskaya et al. 2015; Ovacik and Lin 2018). Additionally, inhibitory effects at high concentrations may also play a role. At higher doses, UH could potentially cause feedback inhibition, where an excess of the compound triggers regulatory mechanisms that reduce overall activity, possibly through receptor desensitization or downregulation of key signaling pathways (Sanchez and Demain 2008).
Similar to other sulfated polysaccharides like fucoidan and carrageenan, Ulvan also exhibits anti-inflammatory properties, with potential applications in the treatment of OA. This effect is attributed to the presence of sulfate groups, which interact with the immune system and regulate inflammatory pathways. Fucoidans, which are sulfated polysaccharides containing fucose, have been shown to have diverse biological activities. A previous study highlighted that the nature of sulfation, the number of sulfate groups, the degree of branching, and the monosaccharide composition are key factors influencing the biological activity of fucoidans (Li et al. 2025). Fucoidans are primarily sulfated at the C2 position, with less frequent sulfation at the C4 position (Zaitseva et al. 2022). Moreover, the biological activity of fucoidans is also influenced by their molecular weight (Zayed et al. 2020). Carrageenan, a high molecular weight linear sulfated galactans, consists of repeating disaccharide units containing alternating 1,4-β-galactose and 1,3-α-galactose, as well as 3,6-anhydrogalactose. The sulfate groups are covalently attached via ester bonds to the C-2, C-4, or C-6 atoms of galactose (Zaitseva et al. 2022). The sulfate content in carrageenan can range from 0% to 41% (Khotimchenko et al. 2020). In comparison, sulfated ulvan heteropolysaccharides are intricate hydrocolloids made up of sulfated glucuronoxylomannan units, incorporating xylose, glucuronic acid, glucose, iduronic acid, and their sulfated derivatives. Sulfation in ulvan primarily occurs at the C-3 or C-2 positions (Zaitseva et al. 2022).
Conclusion
In this study, ulvan and Ulva oligosaccharides were successfully extracted from Ulva sp. This extract contained monosaccharides, such as rhamnose and glucose, as well as a sulfated group. The Ulva extract is composed of ulvan, whereas the Ulva hydrolysate consists of Ulva oligosaccharides. Treatment with these compounds effectively reduced interleukin-6 and matrix metalloproteinase-3 levels, while increasing collagen type II alpha 1 levels in MIA-induced inflammation of SW1353 cells. In the animal study, both Ulva extract and Ulva hydrolysate significantly lowered triglyceride, total cholesterol, and low-density lipoprotein-cholesterol levels. These treatments also decreased nitric oxide levels in osteoarthritis rats and inhibited pro-inflammatory cytokines and mediators, including matrix metalloproteinase-3 and C-terminal cross-linked telopeptides of type II collagen. Additionally, treatment reduced proteoglycan loss in cartilage lesions after six weeks, demonstrating a protective effect on articular cartilage and suppression of cartilage degradation in both cell and animal OA models.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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