Integrated In Vitro, In Vivo, and In Silico Evaluation of Antioxidant, Anti-Inflammatory, Analgesic, and Anti-Arthritic Activities of Selected Marine Species

Abstract

Marine ecosystems represent a largely untapped reservoir of bioactive compounds with significant pharmacological potential. This study aimed to evaluate the therapeutic properties of ethanol extracts from four marine species: Padina australis, Spatoglossum asperum, Holothuria (Halodeima) atra, and Hypnea valentiae. Phytochemical screening, along with a comprehensive series of in vitro, in vivo, and in silico assays, was performed to evaluate the extracts' pharmacological activities, including antioxidant potential (2,2-diphenyl-1-picrylhydrazyl assay), anti-inflammatory effect (carrageenan-induced paw edema method), analgesic activity (acetic acid-induced writhing and tail immersion tests), and anti-arthritic efficacy (protein denaturation assay). The extracts were found to be rich in flavonoids, tannins, alkaloids, saponins, glycosides, and phenolic compounds, which may underlie the observed bioactivities. In the acetic acid-induced writhing test, Hypnea valentiae at 400 mg/kg exhibited the highest peripheral analgesic activity, producing 82.51% inhibition of writhing (p < 0.001). In the tail immersion assay, Padina australis at doses of 200 and 400 mg/kg showed significant central analgesic effects, as evidenced by increased latency time and percentage of maximum possible effect (MPE). In the carrageenan-induced paw edema model, several treatment groups, including Padina australis, Hypnea valentiae, Spatoglossum asperum, and Holothuria atra, at both tested doses showed marked suppression of inflammation, with some groups achieving complete inhibition (100%; p < 0.001) at 120 min. The ethanol extract of Holothuria atra exhibited the strongest antioxidant and anti-arthritic activities, with an IC₅₀ value of 88.39 µg/mL in the DPPH assay and 81.35% inhibition of protein denaturation. Additionally, the compounds derived from the four marine species exhibited significant binding affinity to the selected target receptors, thereby validating the experimental findings. The marine species studied possess multifaceted pharmacological properties, supporting their potential as natural sources for developing therapeutic agents supporting the blue economy. Further studies are recommended to isolate active compounds and elucidate underlying mechanisms to support future drug development efforts.

Keywords: analgesic; anti-inflammatory; antiarthritic; antioxidant; marine drug; phytochemicals; seaweed.

Figure 1

Evaluation of Analgesic Activity of Marine Sample through Acetic Acid-Induced Writhing Method. Data are expressed as mean number of writhes ± SEM (n = 5). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. p < 0.001 *** compared with the control group. PA = Padina australis, SPT = Spatoglossum asperum, HA = Holothuria atra, HV = Hypnea valentiae.

Figure 2

IC₅₀ Value of Different Marine Samples in the DPPH Radical Scavenging Assay. PA = Padina australis, SPT = Spatoglossum asperum, HA = Holothuria atra, HV = Hypnea valentiae. This assay was conducted as a preliminary screening, and no inferential statistical analysis was applied.

Figure 3

Docking Interactions of Compounds Against Cox-2 Inhibitor (PDB: 6cox): (a). Benzophenone (b). Phytol acetate (c). Methyl arachidonate (d). Diclofenac (Standard).

Figure 4

Docking Interactions of Compounds Against Human COX-1 (PDB: 6y3c): (a). Lucenin 2 (b). Fucosterol (c). Benzophenone (d). Diclofenac (Standard).

Figure 4

Docking Interactions of Compounds Against Human COX-1 (PDB: 6y3c): (a). Lucenin 2 (b). Fucosterol (c). Benzophenone (d). Diclofenac (Standard).

Figure 5

Docking Interactions of Compounds Against Human Glutathione Reductase Inhibitor (PDB: 1xan): (a). Fucosterol (b). Retinoic acid, methyl ester (c). Retinol acetate (d). Ascorbic acid (Standard).

Figure 6

Docking Interactions of Compounds Against TNF-alpha (PDB ID: 2AZ5): (a). Fucosterol (b). Lucenin 2 (c). Retinol acetate (d). Diclofenac (Standard).