Abstract
Introduction: Frondanol, a non-polar lipid extract derived from the edible sea cucumber Cucumaria frondosa, has shown promising anti-inflammatory properties.
Methods: This study investigated its molecular mechanisms in modulating inflammation using lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages.
Results: Frondanol was found to be non-cytotoxic at the tested dilutions (1:80,000 to 1:10,000). Co-treatment with LPS and Frondanol significantly reduced the production of inflammatory mediators. Nitric oxide (NO) levels were decreased by up to 30% (p < 0.05), while iNOS protein and mRNA expression were reduced by approximately 45% (p < 0.05) and 80% (p < 0.0001), respectively, at a 1:10 K dilution. Prostaglandin E2 (PGE2) levels were suppressed by nearly 40% (p < 0.0001), accompanied by a 60% reduction in COX-2 protein (p < 0.01) and a 70% decrease in COX-2 mRNA expression (p < 0.05). The pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interleukin (IL)-6 were also significantly attenuated by Frondanol treatment. Mechanistically, Frondanol inhibited LPS-induced NF-κB activation by reducing IκBα phosphorylation and preventing nuclear translocation of NF-κB p65. Furthermore, Frondanol significantly downregulated the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38.
Conclusion: These results suggest that Frondanol exerts its anti-inflammatory effects through suppression of both NF-κB and MAPK signalling pathways, leading to reduced production of inflammatory mediators and cytokines. Given its efficacy and lack of cytotoxicity, Frondanol may hold strong potential as a nutraceutical agent for the prevention and management of chronic inflammatory diseases.
Introduction
Inflammation is a physiological response of the immune system to various agents, including infections and toxins, involving the overproduction of inflammatory cytokines (Che et al., 2018). Although this response is crucial in regulating tissue repair and inflammation, the excess generation of these mediators contributes to the development of inflammatory diseases (Soliman and Barreda, 2022; Kany et al., 2019). Therefore, it is essential to control inflammation to prevent and treat such conditions. Macrophages play a significant role in the host defence mechanism and are commonly activated in inflamed tissues following exposure to interferon-γ (IFN-γ), tumor necrosis factor (TNF)-α, or microbial lipopolysaccharides (LPS) (Klein et al., 2019; Chawla et al., 2011). Once activated, macrophages produce pro-inflammatory cytokines such as TNF-α, interleukin (IL)-1β, and interleukin (IL)-6, as well as inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and leukotriene (LT)-B4 (Parisi et al., 2018). Their synthesis and release at high levels are implicated in the development of chronic diseases such as atherosclerosis (Kong et al., 2022), rheumatoid arthritis (Bedeković et al., 2023), inflammatory bowel disease (IBD) (Neurath, 2014), and cancer (Singh et al., 2019).
A key factor in macrophage activation is LPS, which are endotoxins derived from Gram-negative bacteria. LPS are large amphipathic glycoconjugates consisting of a hydrophobic lipid domain attached to a core oligosaccharide and a distal polysaccharide. LPS stimulate macrophages through Toll-like receptor 4 (TLR4), triggering the activation of several inflammatory signalling pathways, including nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK). This cascade results in the excessive production of inflammatory mediators and cytokines, culminating in abnormal inflammatory responses that contribute to the pathogenesis of various inflammatory diseases (Kuzmich et al., 2017). Under basal conditions, NF-κB is kept inactive in the cytoplasm by its inhibitor, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκB-α). Upon exposure to LPS or other inflammatory stimuli, IκB-α is degraded, allowing NF-κB to translocate to the nucleus and initiate the transcription of genes involved in the inflammatory response (Liu et al., 2017). In addition to NF-κB, kinases such as MAPK—including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK—along with the PI3K/Akt pathway, contribute to the regulation of cytokine expression and further amplify NF-κB activation in macrophages (Dorrington and Fraser, 2019; Moens et al., 2013).
Given the critical role of these inflammatory pathways in diseases such as atherosclerosis, rheumatoid arthritis, IBD, and cancer, targeting these mechanisms has become a promising strategy for developing new or adjunct anti-inflammatory therapies (Zhao et al., 2021). Marine-sourced natural products, particularly polysaccharides derived from marine microorganisms, have shown significant potential in modulating inflammatory pathways (Cheung et al., 2016; Notarte et al., 2018; Notarte et al., 2017). These bioactive compounds can inhibit the activation of NF-κB and other inflammatory signalling pathways, making them attractive candidates for the prevention and treatment of chronic inflammatory diseases (Cheung et al., 2016; Yasir et al., 2024).
Sea cucumbers and their extracts are known for their high nutritional value and various potential health benefits, including anti-inflammatory effects. For centuries, they have been used as customary foods and in folk medicine in countries including Japan, Indonesia, Korea, and China (Ghelani et al., 2022). Frondanol, a nutraceutical non-polar extract of the edible sea cucumber Cucumaria frondosa, is a US-patented agent reported to possess potent anti-inflammatory activity, observed in animal and human studies without signs of toxicity (Ghelani et al., 2023; Subramanya et al., 2018; Collin, 2002). In a murine model of colitis, Frondanol administration resulted in significant reductions in colonic inflammation and inflammatory cytokines, suggesting immunomodulatory effects within the gut mucosa (Subramanya et al., 2018). Moreover, in vitro studies have shown that Frondanol inhibits the 5-lipoxygenase (5-LOX) and 12-lipoxygenase (12-LOX) pathways, reducing the production of lipid mediators such as 12-hydroxyeicosatetraenoic acid (12-HETE), 5-hydroxyeicosatetraenoic acid (5-HETE), and leukotriene B4 (LTB4) in human neutrophils (Collin, 2002).
Building on these findings, a randomized, double-blind, placebo-controlled clinical trial was designed to evaluate Frondanol’s efficacy in adults with IBD (Ghelani et al., 2023). This published protocol outlines the study design, inclusion criteria, and endpoints, and unpublished preliminary findings support potential anti-inflammatory effects in patients with IBD, suggesting clinical relevance of the observations seen in colitis model of IBD. While the existing literature supports Frondanol’s anti-inflammatory potential, the mechanism(s) by which it exerts these effects remains largely unknown, particularly in macrophage-driven inflammation. Therefore, this study aimed to investigate the molecular mechanisms underlying the anti-inflammatory activity of Frondanol on a cellular model of LPS-induced RAW 264.7 macrophages. We hypothesized that Frondanol attenuates LPS-induced inflammatory responses by modulating key signaling pathways, specifically NF-κB and MAPK. To test this, we evaluated its effects on the production of inflammatory mediators, including nitric oxide and prostaglandin E2, the expression of inducible enzymes such as iNOS and COX-2, and the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), as well as the phosphorylation status of key proteins involved in NF-κB and MAPK pathways.
Download the full article as PDF here Anti-inflammatory effects of Frondanol, a nutraceutical extract from Cucumaria frondosa, via modulation of NF-κB and MAPK pathways in LPS-induced RAW 264.7 cells
or read more here
2 Materials and methods
2.1 Preparation of Frondanol
Frondanol is a trademarked nutraceutical lipid extract (developed and marketed by Coastside BioResources, Stonington, ME, United States) derived from the gut material of edible Atlantic Sea cucumber (Cucumaria frondosa). The extraction process involves using hexane as an organic solvent to isolate lipids from the intestinal tissues of the sea cucumber. This extract is used in various dietary supplements and health products due to its purported health benefits. Frondanol, in the form of oil, is encapsulated within soft-gel capsules. For the current investigation, the oil contained in the capsules was initially dissolved in dimethyl sulfoxide (DMSO) to prepare a working solution. This DMSO-dissolved oil was then further diluted in culture media to produce a series of dilutions: 1:10,000, 1:20,000, 1:40,000, and 1:80,000, corresponding to final concentrations of 0.01%, 0.005%, 0.0025%, and 0.00125% (v/v), respectively. To achieve maximum solubility, each dilution step was carried out using warm (37 °C) conditions, followed by vigorous mixing and brief sonication to ensure the Frondanol was completely solubilized.
Hardik Ghelani, Kerat Hanspal, Lana Talo, Sami Talo, Hala Altaher, Hadil Sarsour, Marah Tabbal, Sally Badawi, Peter Collin, Thomas E. Adrian, Reem K. Jan, Anti-inflammatory effects of Frondanol, a nutraceutical extract from Cucumaria frondosa, via modulation of NF-κB and MAPK pathways in LPS-induced RAW 264.7 cells, ORIGINAL RESEARCH article, Front. Pharmacol., 30 October 2025, Sec. Ethnopharmacology, Volume 16 – 2025 | https://doi.org/10.3389/fphar.2025.1683630
Read also our introduction article on Softgel Capsules here:
