Antiinflammatory efficacy of n-Hexadecanoic acid from a mangrove plant Excoecaria agallocha L. through in silico, in vitro and in vivo Ramamoorthy Purushothaman * , Ganapathy Vishnuram , Thirugnanasambandam Ramanathan * Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu 608502, India A R T I C L E INFO Keywords: Excoecaria agallocha N-Hexadecanoic acid Anti-inflammatory Molecular docking Paw edema ABSTRACT The various parts of Excoecaria agallocha L. have been scientifically evaluated for their role in inflammatory reactions. This study aimed to validate the potent anti-inflammatory efficacy of n-Hexadecanoic acid (HDA) isolated from Excoecaria agallocha using in silico, in vitro, and in vivo approaches. GC-MS and NMR analyses were employed for compound identification, revealing 30 phytochemical constituents. Molecular docking studies demonstrated strong inhibitory interactions between HDA and key inflammatory mediators, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). In vitro, assays confirmed a dose-dependent anti-inflammatory response, including protein denaturation, hypotonicity-induced hemolysis, and heat-induced hemolysis tests. Furthermore, in vivo evaluation using a carrageenan-induced Wistar rat model showed significant attenuation of paw edema in HDA-treated groups compared to the control. These findings collectively establish the potent anti- inflammatory properties of n-Hexadecanoic acid and its potential as a therapeutic candidate for inflammation- related disorders. 1. Introduction Inflammation is the body’s natural defense mechanism, triggered in response to foreign pathogens, tissue injury, or chemical irritation. It plays a crucial role in eliminating injurious stimuli and initiating the healing process, which is typically rapid and self-limiting [1]. Both in- fectious and non-infectious stimuli can activate inflammatory pathways, leading to an acute inflammatory response that helps restore physio- logical homeostasis. However, when inflammation becomes prolonged, it can turn chronic, exacerbating tissue damage and contributing to various chronic inflammatory diseases [2,3]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to alleviate pain and inflammation by inhibiting the metabolism of arachidonic acid via cyclooxygenase enzymes (COX-1 and COX-2), thereby reducing prostaglandin production. However, prolonged NSAID use is associated with adverse side effects, necessitating the search for safer alternatives. Medicinal plants have long been recognized for their potent anti-inflammatory properties with minimal side effects. Mangrove plants, known for their rich phytochemical diversity, have been extensively studied for their anti-inflammatory potential. Excoe- caria agallocha L., a mangrove tree belonging to the Euphorbiaceae family, grows up to 15 m in height and holds significant ecological, economic, and medicinal value. Various bioactive compounds isolated from different parts of E. agallocha have demonstrated anti- inflammatory effects in traditional medicine [4]. In addition to its anti-inflammatory activity, the plant exhibits a range of pharmacolog- ical properties, including antioxidant, antimicrobial, analgesic, anti- ulcer, anticancer, antihistamine-release, antidiabetic, and antitumor activities. N-hexadecanoic acid (HDA), commonly known as palmitic acid, is a long-chain saturated fatty acid with the molecular formula CH₃(CH₂)₁₄COOH. It is widely found in both plant and animal sources and is synthesized during fatty acid metabolism (lipogenesis). Studies have shown that palmitic acid possesses antioxidant properties, con- tributes to the prevention of atherosclerosis in animal models, and ex- hibits antibacterial activity against both Gram-positive and Gram- negative bacterial strains [5]. The present study aims to evaluate the anti-inflammatory efficacy of n-Hexadecanoic acid derived from E. agallocha L. using in silico, in vitro, and in vivo approaches. Computational molecular docking techniques were employed to assess its binding affinity with key inflammatory mediators, while in vitro assays were conducted to evaluate its anti- inflammatory potential. Additionally, in vivo experiments using a carrageenan-induced inflammation model in Wistar rats were * Corresponding authors. E-mail addresses: purushothaman.r1997@gmail.com (R. Purushothaman), drtrcasmb@gmail.com (T. Ramanathan). Contents lists available at ScienceDirect Pharmacological Research - Natural Products journal homepage: www.sciencedirect.com/journal/prenap https://doi.org/10.1016/j.prenap.2025.100203 Received 23 August 2024; Received in revised form 9 March 2025; Accepted 12 March 2025 Pharmacological Research - Natural Products 7 (2025) 100203 Available online 19 March 2025 2950-1997/© 2025 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.