Cytotoxic Activity, Seasonal Variation and GC/MS-based Metabolomics of Three Callistemon species Coupled to Chemometrics

Genus Callistemon, commonly known as bottlebrush, is recognized in folk medicine for its anti-bronchitis, anti-cough, and insecticidal effects. The essential oils have been used as antimicrobial and antifungal agents. In this study, interspecies and seasonal variations in the essential oil composition of three Callistemon species was assessed using GC-MS coupled to multivariate data analysis. The essential oils obtained by hydrodistillation of the fresh leaves of C. citrinus, C. rigidus and C. viminalis (Myrtaceae) were characterized by GC and GC/MS analysis. A total of 31 compounds were annotated accounting for 93.37% to 98.68% of the whole volatile constituents in four seasons. Eucalyptol represented the major component in the three Callistemon species (51.62% – 80.72%), followed by α-pinene (1.11% – 20.75%), linalool (0%-13.8%) and α-terpineol (2.13% – 10.27%). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied as pattern recognition techniques in an attempt to discriminate the three species (three replicates for each species) based on their total ion content to investigate their metabolic variations for each season individually. The PCA plot was able to discriminate between the three Callistemon species in different seasons. They were clustered into three main groups, where each species was positioned in a separate quadrant. The loading plot showed that the main discriminating makers were eucalyptol, α-pinene and linalool, showing the highest influence on the segregation between the samples. The dendogram obtained by applying HCA confirmed the results of PCA. Furthermore, PCA was able to partially segregate individual species in the different seasons. Moreover, cytotoxic activity of Callistemon essential oils was assessed using MTT assay on Caco-2 cell lines. The results revealed that C. citrinus essential oil showed very potent cytotoxicity with IC₅₀ equals 0.80 µg/mL followed by C. rigidus and C. viminalis, exhibiting IC₅₀ values of 2.0, and 2.84 µg/ml, respectively.

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