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Objective: In this study, we aimed to describe the influence of natural terpenes on the antimycobacterial activity of first-line tuberculostatic drugs against isolated Mycobacterium tuberculosis. Materials and Methods: The natural terpenes used in this study were R-limonene, S-limonene, myrcene, sabinene, α-pinene, and β-elemene. The values of the minimum inhibitory concentration (MIC) for these terpenes, as well as for combinations of terpenes with tuberculostatic antibiotics (ethambutol, isoniazid, and rifampicin), were determined using a tube log₂ dilution method in the range of 125-0.059 µg/mL. Results:S-limonene had a strong synergistic effect with all tested antibiotics (MIC decreased from 16 to 0.475 µg/mL for ethambutol, from 16 to 0.237 µg/mL for rifampicin, and from 32 to 0.475 µg/mL for isoniazid). Combinations of myrcene, R-limonene, β-elemene, and sabinene with tuberculostatic antibiotics resulted in a decreased MIC of the antibiotics (from 3.9 to 0.475 µg/mL for ethambutol, from 15 to 0.475 µg/mL for isoniazid, and from 0.475 to 0.237 µg/mL for rifampicin) while combinations of α-pinene with ethambutol and isoniazid resulted in increased MIC values (from 16 to 125 µg/mL for ethambutol, and from 32 to 125 µg/mL for isoniazid). Rifampicin had a synergistic increase in activity with all the tested compounds. Conclusions: Our study showed that terpenes enhance the activity of tuberculostatic antibiotics.

Background: On account of emergence of multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains, combinations of drugs with natural compounds were tested to search for antibiotic activity enhancers. In this work we studied terpenes (α-pinene, bisabolol, β-elemene, (R)-limonene, (S)-limonene, myrcene, sabinene), which are the main constituents of essential oil obtained from Mutellina purpurea L., a plant with described antitubercular activity, to investigate their interactions with antibiotics against reference Mtb strains and multidrug-resistant clinical isolates. Methods: The serial dilution method was used to evaluate the minimal inhibitory concentration (MIC) of tested compounds, while the fractional inhibitory concentration index (FICI) was calculated for characterization of interactions. Moreover, IC50 values of tested compounds were determined using monkey kidney epithelial cell line (GMK). Results: The combinations of all studied terpenes with ethambutol or rifampicin resulted in a synergistic interaction. Bisabolol and (R)-limonene decreased the MIC for rifampicin at least two-fold for all tested strains, however no synergistic action was observed against virulent strains. The tested terpenes showed slight (bisabolol) or no cytotoxic effect against normal eukaryotic cells in vitro. Conclusions: The obtained enhanced activity (FICI < 0.5) of ethambutol and rifampicin against H37Ra strain under the influence of the studied terpenes may be correlated to the capability of essential oil constituents to modify bacterial resistance mechanisms in general. The observed differences in avirulent and virulent bacteria susceptibility to terpenes tested separately and in combinations with antibiotics can be correlated with the differences in the cell wall structure between H37Ra mutant and all virulent strains.

Synthesis of cheap and natural resources is an important topic in green chemistry.

The most commonly used plant source of β-elemene is Curcuma wenyujin Y. H. Chen & C. Ling (syn. of Curcuma aromatic Salisb.) with its content in supercritical CO2 extract up to 27.83%. However, the other rich source of this compound is Nigella damascena L. essential oil, in which β-elemene accounts for 47%. In this work, the effective protocol for preparative isolation of β-elemene from a new source—N. damascena essential oil—using high performance counter-current chromatography HPCCC was elaborated. Furthermore, since sesquiterpens are known as potent antimicrobials, the need for finding new agents designed to combat multi-drug resistant strains was addressed and the purified target compound and the essential oil were tested for its activity against a panel of Gram-positive and Gram-negative bacteria, fungi, and mycobacterial strains. The application of the mixture of petroleum ether, acetonitrile, and acetone in the ratio 2:1.5:0.5 (v/v) in the reversed phase mode yielded β-elemene with high purity in 70 min. The results obtained for antimicrobial assay clearly indicated that N. damascena essential oil and isolated β-elemene exert action against Mycobacterium tuberculosis strain H37Ra.