Chemical profiling and cytotoxicity screening of agarwood essential oil (Aquilaria sinensis) in brine shrimp nauplii and cancer cell lines

Abstract

Agarwood essential oil (AEO) has gained attention from healthcare industries due to its numerous pharmacological properties. However, a comprehensive understanding of the chemical composition and its cytotoxic property is lacking. The objective of this study was to investigate the chemical profile as well as the cytotoxic concentration range of AEO derived from Aquilaria sinensis agarwood. Gas chromatography-mass spectrometry (GC-MS) was employed to identify the AEO components. Results showed that sesquiterpenes and sesquiterpenoids constitute 95.85% of the AEO. Among the major compounds identified are allo-aromadendrene (13.04%), dihydro-eudesmol (8.81%), α-eudesmol (8.48%), bulnesol (7.63%), τ-cadinol (4.95%), dehydrofukinone (3.83%), valerenol (3.54%), cis-nerolidol (2.75%), agarospirol (2.72%), dehydrojinkoh-eremol (2.53%), selina-3,11-dien-9-al (2.36%), guaiol (2.12%) and caryophyllene oxide (2.0%). The presence of volatile quality marker compounds such as 10-epi-ϒ-eudesmol, aromadendrane, β-agarofuran, α-agarofuran, γ-eudesmol, agarospirol and guaiol, with no contaminants detected, indicates that the extracted AEO is of high purity. Interestingly, the AEO displayed moderate to high toxicity in brine shrimp lethality test (BLST). All studied tumor cell lines (MDA-MB-231, HepG2, B16F10) exhibited varying degrees of sensitivity to AEO, which resulted in time and dose-dependent reduction of cell proliferation. Moreover, flow cytometry analysis revealed that AEO could induce apoptosis in treated HepG2 cells. Our findings showed that AEO contains bioactive components that may be exploited in future studies for the development of anti-cancer therapeutics.

Fig 1. Cultivated agarwood (A. sinensis) tree.

Fig 2

Illustrations of (A) Cultivated agarwood (A. sinensis) plantation; (B) Agarwood powder; (C) Agarwood powder in distillation tank; (D) AEO collected at the bottom of the separation funnel; (E) Final AEO.

Fig 3. Chromatogram of the analysis by GC for AEO obtained from A. sinensis.

Fig 4. Evaluation of acute toxicity effect of AEO on brine shrimp at different treatment time-points.

Fig 5. Evaluation of effect of AEO on cancer cell lines by MTT assay.

Percentages of viable cells of (A) B16F10; (B) MDA-MB-231; (C) HepG2 at 24 h time-point after AEO treatments. Each experiment was repeated for a minimum of 3 times and plotted as bar graphs with error bars. Two-way ANOVA was conducted and the p value was calculated between control and AEO treated samples, where, *: p value < 0.05.

Fig 6. Morphology changes of B16F10, MDA-MB-231, HepG2 cells after AEO treatment (61.5 μg/mL) for 24 h.

Treated cells were rounded up and cell to cell adhesion was lost. Images showing 40 x magnification. Black bar in each image represents 100 μm.

Fig 7. Effect of AEO on the induction of apoptosis.

HepG2 cells were cultured with (A) control solvent; (B) 50 μg/mL of AEO; (C) 100 μg/mL of AEO for 16 h. Treated cells were then stained with Annexin V and PI, followed by flow cytometry analysis.