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Purpose <p>The escalating global health crisis of antimicrobial resistance underscores the urgent need for safer and effective therapeutic alternatives to conventional synthetic antibiotics. This study investigated a novel approach combining lysozyme, a natural enzyme with potent antimicrobial properties, and curcumin, a polyphenolic compound known for its diverse pharmacological effects. We aimed to propose and substantiate an RP-HPLC strategy for the synchronous quantification of lysozyme and curcumin, addressing their limitations, particularly curcumin's poor bioavailability.&#xa0;Additionally, we sought to provide molecular-level insights into their interaction to understand the complex's potential for enhanced therapeutic activity.</p> Methods <p> The separation was performed on a Zorbax 300 SB-C18 column maintained at 50°C. An optimized gradient elution program was employed, using a mobile phase composed of 0.1% trifluoroacetic acid in water (Solution A) and 0.1% trifluoroacetic acid in acetonitrile: water (80:20, v/v) (Solution B). Detection was carried out at 250 nm with a flow rate of 1.1 mL/min.&#xa0;To complement the analytical development, molecular dynamics (MD) simulations and density functional theory (DFT) calculations were conducted to investigate the structural dynamics and electronic interactions within the lysozyme-curcumin complex.</p> Results <p> Our method achieved a correlation coefficient (R) exceeding 0.999 over a range of concentrations from 3 to 50 µg/mL, confirming excellent linearity. In terms of lysozyme, the limit of detection and quantification was 2.15 µg/mL and 5.53 µg/mL, and in terms of curcumin, it was 1.76 µg/mL and 3.34 µg/mL, respectively, with recovery rates of 100.91% for lysozyme and 100.26% for curcumin.&#xa0;The MD simulations revealed that curcumin undergoes significant structural deformation upon binding to lysozyme, while the DFT analysis indicated a concomitant reduction in its HOMO-LUMO energy gap. These computational findings suggest a potential mechanism for the activation of curcumin within the protein complex.</p> Conclusions <p> The novel method adheres to greenness and blueness concepts such as green energy and risk mitigation. More importantly, this integrated experimental and computational study provides a validated analytical tool for quality control and delivers fundamental molecular insights into the lysozyme-curcumin interaction.&#xa0;The findings establish a robust analytical and molecular foundation, supporting the future development of the lysozyme-curcumin complex as a promising therapeutic alternative.</p>

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Green and White Analytical Evaluation of an Innovative RP-HPLC Approach for Quantification of Lysozyme and Curcumin in a Co-formulation Prototype: Molecular Insights for Safer Therapeutic Development

  • 期刊论文

  • 发表日期：

    2026年3月18日

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  • Samar M. Mahgoub,
  • Lateefa A. Al-Khateeb,
  • Rehab Mahmoud,
  • Rami Shafei,
  • Omkulthom Al kamaly,
  • Amr A. Essawy,
  • Mahmoud A. Mohamed

摘要

Purpose

The escalating global health crisis of antimicrobial resistance underscores the urgent need for safer and effective therapeutic alternatives to conventional synthetic antibiotics. This study investigated a novel approach combining lysozyme, a natural enzyme with potent antimicrobial properties, and curcumin, a polyphenolic compound known for its diverse pharmacological effects. We aimed to propose and substantiate an RP-HPLC strategy for the synchronous quantification of lysozyme and curcumin, addressing their limitations, particularly curcumin's poor bioavailability. Additionally, we sought to provide molecular-level insights into their interaction to understand the complex's potential for enhanced therapeutic activity.

Methods

The separation was performed on a Zorbax 300 SB-C18 column maintained at 50°C. An optimized gradient elution program was employed, using a mobile phase composed of 0.1% trifluoroacetic acid in water (Solution A) and 0.1% trifluoroacetic acid in acetonitrile: water (80:20, v/v) (Solution B). Detection was carried out at 250 nm with a flow rate of 1.1 mL/min. To complement the analytical development, molecular dynamics (MD) simulations and density functional theory (DFT) calculations were conducted to investigate the structural dynamics and electronic interactions within the lysozyme-curcumin complex.

Results

Our method achieved a correlation coefficient (R) exceeding 0.999 over a range of concentrations from 3 to 50 µg/mL, confirming excellent linearity. In terms of lysozyme, the limit of detection and quantification was 2.15 µg/mL and 5.53 µg/mL, and in terms of curcumin, it was 1.76 µg/mL and 3.34 µg/mL, respectively, with recovery rates of 100.91% for lysozyme and 100.26% for curcumin. The MD simulations revealed that curcumin undergoes significant structural deformation upon binding to lysozyme, while the DFT analysis indicated a concomitant reduction in its HOMO-LUMO energy gap. These computational findings suggest a potential mechanism for the activation of curcumin within the protein complex.

Conclusions

The novel method adheres to greenness and blueness concepts such as green energy and risk mitigation. More importantly, this integrated experimental and computational study provides a validated analytical tool for quality control and delivers fundamental molecular insights into the lysozyme-curcumin interaction. The findings establish a robust analytical and molecular foundation, supporting the future development of the lysozyme-curcumin complex as a promising therapeutic alternative.