Essential oils (EOs) have great potential in inhalation therapy for the treatment of respiratory infections. However, innovative methods for evaluation of antimicrobial activity of their vapors are still needed. The current study reports validation of the broth macrodilution volatilization method for assessment of the antibacterial properties of EOs and shows the growth-inhibitory effect of Indian medicinal plants against pneumonia-causing bacteria in liquid and vapor phase. Among all samples tested, Trachyspermum ammi EO exhibits the strongest antibacterial effect against Haemophilus influenzae, with minimum inhibitory concentrations of 128 and 256 µg/mL in the liquid and vapor phases, respectively. Furthermore, Cyperus scariosus EO is found to be nontoxic to normal lung fibroblasts assessed by modified thiazolyl blue tetrazolium bromide assay. Chemical analysis performed using gas chromatography-mass spectrometry identified α-citral, cyperotundone, and thymol as the main constituents of Cymbopogon citratus, C. scariosus, and T. ammi EOs, respectively. In addition, β-cymene is identified as the major compound of T. ammi EO vapors when analyzed using solid-phase microextraction and gas-tight syringe sampling techniques. This study demonstrates the validity of the broth macrodilution volatilization method for antimicrobial screening of volatile compounds in the vapor phase and suggests the therapeutic potential of Indian medicinal plants in inhalation therapy.

• Keywords
• Cymbopogon citratus, Cyperus scariosus, GC/MS, MTT assay, Trachyspermum ammi, antimicrobial activity, headspace analysis, macrodilution, respiratory infections, vapor phase, volatiles,
• MeSH
• Anti-Bacterial Agents chemistry   MeSH
• Anti-Infective Agents * analysis   MeSH
• Plants, Medicinal * MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Oils, Volatile * pharmacology   chemistry   MeSH
• Gas Chromatography-Mass Spectrometry MeSH
• Pneumonia * MeSH
• Volatilization MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Anti-Infective Agents * MeSH
• Oils, Volatile * MeSH

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (β-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase.

• Keywords
• antimicrobial, cytotoxicity, macrodilution method, respiratory infections, thymohydroquinone, thymoquinone, vapor phase, volatile compound, β-thujaplicin,
• MeSH
• Anti-Bacterial Agents analysis   chemistry   pharmacology   MeSH
• Administration, Inhalation MeSH
• Bacteria drug effects   MeSH
• Benzoquinones administration & dosage   pharmacology   MeSH
• Haemophilus influenzae drug effects   MeSH
• Microbial Sensitivity Tests MeSH
• Monoterpenes administration & dosage   pharmacology   MeSH
• Oils, Volatile chemistry   pharmacology   MeSH
• Staphylococcus aureus drug effects   MeSH
• Streptococcus pneumoniae drug effects   MeSH
• Streptococcus pyogenes drug effects   MeSH
• Volatile Organic Compounds chemistry   pharmacology   MeSH
• Thymol administration & dosage   analogs & derivatives   pharmacology   MeSH
• Tropolone administration & dosage   analogs & derivatives   pharmacology   MeSH
• Volatilization MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Benzoquinones MeSH
• beta-thujaplicin MeSH Browser
• Monoterpenes MeSH
• Oils, Volatile MeSH
• Volatile Organic Compounds MeSH
• thymohydroquinone MeSH Browser
• Thymol MeSH
• thymoquinone MeSH Browser
• Tropolone MeSH