Farm or Lab? A Comparative Study of Oregano's Leaf and Callus Volatile Isolates Chemistry and Cytotoxicity

Abstract

Oregano (Origanum vulgare, Lamiaceae plant family) is a well-known aromatic herb with great commercial value, thoroughly utilized by food and pharmaceutical industries. The present work regards the comparative assessment of in vitro propagated and commercially available oregano tissue natural products. This study includes their secondary metabolites' biosynthesis, antioxidant properties, and anticancer activities. The optimization of callus induction from derived oregano leaf explants and excessive oxidative browning was performed using various plant growth regulators, light conditions, and antioxidant compounds. The determination of oregano callus volatiles against the respective molecules in maternal herbal material was performed using gas chromatography-mass spectrometry (GC/MS) analysis. In total, the presence of twenty-seven phytochemicals was revealed in both leaf and callus extracts, from which thirteen molecules were biosynthesized in both tissues studied, seven compounds were present only in callus extracts, and seven metabolites only in leaf extracts. Carvacrol and sabinene hydrate were the prevailing volatiles in all tissues exploited, along with alkanes octacosane and triacontane and the trimethylsilyl (TMS) derivative of carvacrol that were detected in significant amounts only in callus extracts. The MTT assay was employed to assess the in vitro cytotoxic properties of oregano extracts against the epithelial human breast cancer MDA-MB-231 and the human neuroblastoma SK-N-SH cell lines. The extracts displayed concentration and time-dependent responses in cell proliferation rates.

Keywords: Origanum vulgare; callus culture; cancer cytotoxicity; epithelial human breast cancer MDA-MB-231; human neuroblastoma SK-N-SH; volatile compounds.

Figure 1

Evaluation of conditions affecting oregano callus induction from leaf explants and callus browning rate. B = MS + 0.5 mg/L 2,4 D + 3 mg/L BAP, K = MS + 2 mg/L 2,4 D + 2 mg/L Kin, A = ascorbic acid (10 mg/mL), L = 16 light, D = constant dark. Letters (a, b, c) indicate no statistical difference in callus induction among treatments.

Figure 2

Origanum vulgare leaf-derived calluses. (a) Healthy calluses. (b) Brown calluses due to oxidation.

Figure 3

Chemical structures of carvacrol and sabinene hydrate.

Figure 4

Venn diagram presenting differences and similarities in metabolite content of oregano leaves and leaf-derived calluses.

Figure 5

Percentage of ΜDA-ΜΒ-231 cells’ viability after treatment with the extracts derived from oregano callus and leaves at concentrations of 2.5, 5, 10, 25, 50, 100, and 250 μg/mL for 24 h and 48 h. Average results from replicate experiments ± SD (n = 6). The * indicates statistically significant differences from the control for the 24 h incubation, ≠ indicates statistically significant differences from the control for the 48 h incubation, and ▪ indicates statistically significant callus between callus and leaf extract when administered at the same concentration (yellow line for the 24 h incubation and blue line for the 48 h). Significant differences (Student’s t-test) between normalized cell responses * ▪ p < 0.05, ** ▪▪ p < 0.01, *** p < 0.001.

Figure 6

Percentage of SK-N-SH cells’ viability after treatment with extracts derived from oregano callus and leaves at concentrations of 2.5, 5, 10, 25, 50, 100, and 250 μg/mL, for 24 h and 48 h. Average results from replicate experiments ± SD (n = 6). The * indicates statistically significant differences from the control for the 24 h incubation, ≠ indicates statistically significant differences from the control for the 48 h incubation, and ▪ indicates statistically significant differences between callus and leaf extract when administered at the same concentration (yellow line for the 24 h incubation and blue line for the 48 h). Significant differences (Student’s t-test) between normalized cell responses * ▪ p < 0.05, ** ≠≠ p < 0.01, *** ≠≠≠ ▪▪▪ p < 0.001.

Figure 7

(a) Origanum vulgare seedlings growing in vitro. (b) Origanum vulgare leaves inoculated for callus induction.