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Purpose <p>This study was explored the extraction, characterization, and biological properties of low molecular weight carboxymethyl chitosan (LMW-CMC) derived from cuttlebone of <i>Sepiella inermis</i>.</p> Methods <p>Cuttlebone was processed to obtain 43.53% chitosan (CH), which upon carboxymethylation yielded 75% LMW-CMC. Structural characterization of LMW-CMC was performed using FT-IR, MALDI-TOF/MS, and NMR spectroscopy. The degree of substitution (DS) and molecular weight were determined from NMR analysis. Solubility studies were conducted under neutral, acidic, and alkaline conditions. Antioxidant activity was evaluated using DPPH radical scavenging assay. The in vivo effects of LMW-CMC were assessed in zebrafish larvae (ZFL), focusing on oxidative stress (OS) parameters, antioxidant enzyme levels, neuroprotective markers, and toxicity at varying concentrations (up to 10 µM).</p> Results <p>NMR confirmed successful carboxymethylation of CH, yielding LMW-CMC with a DS of 0.82 and molecular weight of 861 Da. LMW-CMC exhibited high water solubility (72%) but reduced solubility under acidic and alkaline conditions. Antioxidant analysis (in vitro) revealed strong radical scavenging activity, achieving 80.8% inhibition of DPPH at 50 µM. In ZFL, LMW-CMC enhanced antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), restored redox balance by elevating glutathione (GSH) levels, and reduced reactive oxygen species (ROS), conferring protection against OS. Neuroprotective effects were demonstrated through restoration of acetylcholinesterase (AChE) activity and reduction of nitric oxide (NO), indicating anti-inflammatory potential. The effects were dose-dependent, with the highest activity observed at 5 µM. Toxicity evaluation showed LMW-CMC to be biocompatible, with no mortality in ZFL at lower concentrations, minimal developmental impact at 5 µM, but developmental abnormalities at 10 µM.</p> Conclusion <p>LMW-CMC derived from cuttlebone exhibits significant antioxidant, anti-inflammatory, and neuroprotective properties. Its ability to restore cellular redox balance, enhance antioxidant enzymes, and modulate neuroprotective pathways highlights its therapeutic potential in managing oxidative stress. Biocompatibility at lower concentrations further supports its promise for biomedical applications. However, dose-dependent toxicity underscores the need for cautious therapeutic dosing, and further studies are warranted to investigate long-term safety and mechanistic pathways underlying its neuroprotective effects.</p>

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Low Molecular Weight Carboxymethyl Chitosan Derived from Cuttlebone of Sepiella inermis: Alleviating Oxidative Stress in Zebrafish Model

  • 期刊论文

  • 发表日期：

    2025年10月20日

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  • Vignesh Sakthivel,
  • Harshavardhini Parthiban,
  • Ramya Raman,
  • Bindhu Rajendran,
  • Thabitha Aavula,
  • Saravanan Ramachandran

摘要

Purpose

This study was explored the extraction, characterization, and biological properties of low molecular weight carboxymethyl chitosan (LMW-CMC) derived from cuttlebone of Sepiella inermis.

Methods

Cuttlebone was processed to obtain 43.53% chitosan (CH), which upon carboxymethylation yielded 75% LMW-CMC. Structural characterization of LMW-CMC was performed using FT-IR, MALDI-TOF/MS, and NMR spectroscopy. The degree of substitution (DS) and molecular weight were determined from NMR analysis. Solubility studies were conducted under neutral, acidic, and alkaline conditions. Antioxidant activity was evaluated using DPPH radical scavenging assay. The in vivo effects of LMW-CMC were assessed in zebrafish larvae (ZFL), focusing on oxidative stress (OS) parameters, antioxidant enzyme levels, neuroprotective markers, and toxicity at varying concentrations (up to 10 µM).

Results

NMR confirmed successful carboxymethylation of CH, yielding LMW-CMC with a DS of 0.82 and molecular weight of 861 Da. LMW-CMC exhibited high water solubility (72%) but reduced solubility under acidic and alkaline conditions. Antioxidant analysis (in vitro) revealed strong radical scavenging activity, achieving 80.8% inhibition of DPPH at 50 µM. In ZFL, LMW-CMC enhanced antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), restored redox balance by elevating glutathione (GSH) levels, and reduced reactive oxygen species (ROS), conferring protection against OS. Neuroprotective effects were demonstrated through restoration of acetylcholinesterase (AChE) activity and reduction of nitric oxide (NO), indicating anti-inflammatory potential. The effects were dose-dependent, with the highest activity observed at 5 µM. Toxicity evaluation showed LMW-CMC to be biocompatible, with no mortality in ZFL at lower concentrations, minimal developmental impact at 5 µM, but developmental abnormalities at 10 µM.

Conclusion

LMW-CMC derived from cuttlebone exhibits significant antioxidant, anti-inflammatory, and neuroprotective properties. Its ability to restore cellular redox balance, enhance antioxidant enzymes, and modulate neuroprotective pathways highlights its therapeutic potential in managing oxidative stress. Biocompatibility at lower concentrations further supports its promise for biomedical applications. However, dose-dependent toxicity underscores the need for cautious therapeutic dosing, and further studies are warranted to investigate long-term safety and mechanistic pathways underlying its neuroprotective effects.