Bixin from Bixa orellana L.: Analytical Method Validation, Physicochemical Characterization, and Selective Antifungal Activity against Candida spp

Abstract

Bixin is a lipophilic apocarotenoid abundant in the aril of Bixa orellana L. seeds. Widely used as a natural colorant in food, textile, and cosmetic industries, its pharmacological potential remains underexplored. This study aimed to extract and purify bixin, characterize its physicochemical properties, develop and validate an HPLC method for quantification, and evaluate its antifungal activity, cytotoxicity, and selectivity in vitro. Extraction was performed by Soxhlet using hexane and chloroform, followed by recrystallization in acetone. Characterization (TG/DTA, FTIR, and ¹H NMR) confirmed predominance of the 9'-cis isomer. The HPLC method (λ=470 nm) was validated per ICH Q2-(R2), showing specificity, linearity (R² = 0.9993), low LOD/LOQ (0.638 and 1.934 μg mL^-1), and high precision and accuracy (RSD < 2%; recovery 99.4-100.8%). Antifungal assays against eight Candida strains revealed moderate activity, with MICs from 2 to 256 μg mL^-1. The best results were observed for C. glabrata and C. tropicalis (MIC = 2 and 4 μg mL^-1). Bixin also inhibited virulence factors inC. albicans Cytotoxicity on Vero cells showed a CC₅₀ of 30.71 μg mL^-1, with selectivity indices of 15.36 and 7.68 forC. glabrata andC. tropicalis, respectively. These results support bixin's potential as a natural antifungal agent.

1

Chromatogram of the bixin standard. Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm particle size), detector wavelength: 470 nm, column temperature: 40 °C, mobile phase: acetonitrile and 2% acetic acid in an 80:20 v/v, under isocratic conditions, flow rate: 1.2 mL min^–1.

2

Chromatogram of the purified bixin sample obtained by extraction and recrystallization. Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm particle size), UV–vis detector wavelength: 470 nm, column temperature: 40 °C, mobile phase: acetonitrile and 2% acetic acid in an 80:20 v/v ratio, under isocratic conditions, flow rate: 1.2 mL min^–1.

3

Chemical structure of bixin.

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TG and DTA curves of bixin obtained under a dynamic N₂ atmosphere, with a flow rate of 50 mL·min^–1 and a heating rate of 10 °C min^–1.

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Fourier transform infrared (FTIR) spectrum of bixin.

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¹H NMR spectrum in CDCl₃ (400 MHz) for bixin at a temperature of 27 °C.

7

Graphical representation of the fungal death curve (Log₁₀ CFU mL^–1) for C. glabrata ATCC 2001 (A) and C. tropicalis ATCC 28707 (B) under the action of bixin (1024 μg mL^–1, ■), nystatin (positive control, 0.25 μg mL^–1, ●), and no antifungal (growth control, ▲) over time.

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Effect of bixin (8 μg mL^–1, 16 μg mL^–1, and 32 μg mL^–1) on the yeast-to-hypha transition in C. albicans ATCC 18804 incubated for 24, 48, and 72 h. Fluconazole (2 μg mL^–1) was used as a positive control.

9

Graphical representation of the effect of bixin and fluconazole at concentrations of 16–512 μg mL^–1 on mature biofilms formed by C. albicans ATCC 18804 after 24 and 48 h. Three asterisks (***) indicate a statistically significant difference from the control (p < 0.0001). The symbol (#) indicates a statistically significant difference from the wells treated with fluconazole (p < 0.01), and (##) indicates p < 0.001.

10

Graphical representation of the quantitative assessment of cell viability. % of viable cells observed in Vero cells after 48 h of bixin contact with the cell layer at concentrations of 0–200 μg mL^–1. Three asterisks (***) indicate a statistically significant difference in the reduction of the % of viable cells compared to the concentration of 3 μg mL-1 (p < 0.0001).