The impact of different extracts of six Lamiaceae species on deleterious effects of oxidative stress assessed in acellular, prokaryotic and eukaryotic models in vitro

The main objective of this research was to evaluate the impact of methanolic, ethanolic and aqueous extracts of Origanum majorana L., Origanum vulgare L., Teucrium chamaedrys L., Teucrium montanum L., Thymus serpyllum L. and Thymus vulgaris L. (Lamiaceae) on the effects of free radicals using different model systems. The extracts were characterized on the basis of the contents of total phenolics, phenolic acids, flavonoids and flavonols, and also using high-performance liquid chromatography with diode-array detection. Antioxidant activity in vitro was assessed using DPPH assay. The genoprotective properties were tested using plasmid relaxation assay on pUC19 E. coli XL1-Blue, while SOS/umuC assay on Salmonella typhimurium TA1535/pSK1002 and Comet assay on human lung fibroblasts were used to assess the antigenotoxicity of the extracts. Ethanolic extracts had the most phenolics (up to 236.20 mg GAE/g at 0.5 mg/mL), flavonoids (up to 42.47 mg QE/g at 0.5 mg/mL) and flavonols (up to 16.56 mg QE/g at 0.5 mg/mL), and they exhibited the highest DPPH activity (up to 92.16% at 0.25 mg/mL). Interestingly enough, aqueous extracts provided the best protection of plasmid DNA (the lowest IC50 value was 0.17 mg/mL). Methanolic extracts, on the other hand, most efficiently protected the prokaryotic DNA, while all the extracts had a significant impact against genomic damages inflicted on human fibroblasts. O. vulgare extracts are considered to be the most promising in preserving the overall DNA integrity against oxidative genomic damages. Moreover, HPLC-DAD analysis highlighted rosmarinic acid as the most abundant in the investigated samples (551.45 mg/mL in total in all the extracts), followed by luteolin-7-O-glucoside (150.19 mg/mL in total), while their presence correlates with most of the displayed activities. The novelty of this study is reflected in the application of a prokaryotic model for testing the antigenotoxic effects of Lamiaceae species, as no previous reports have yet been published on the genoprotective potential of these species

Optimisation of the microdilution method for detection of minimum inhibitory concentration values in selected bacteria

In this study we investigated the influence of preparation of the bacterial inoculum for a microdilution susceptibility test, e.g., the effect of its optical density, on assessment of the minimum inhibitory concentrations (MIC). The approach employed in the majority of microdilution susceptibility studies is use of the same optical density for preparation of inoculums for different bacterial strains. In the present work, this approach was questioned by determining the ratio between the optical density and the number of bacteria in cultures. We also investigated whether the number of bacteria in inoculums can affect assessment of the MIC value for two antibiotics of broad spectra, rifampicin and streptomycin. The study was performed on four Gram-positive and four Gram-negative bacteria (ATCC collection) commonly used to investigate antimicrobial potential. The ratio between the optical density and number of bacteria in cultures was determined for each strain, and a strong linear correlation was detected. However, it was evident that different bacteria have different cell numbers at the same OD600 value. Based on the obtained results, inoculums for selected strains were prepared to obtain final cell numbers of 103, 104, 105 and 106 /well in the microdilution assay. Two different approaches were used in determining the MIC for rifampicin and streptomycin: approximation of MIC with IC90 and the resazurin reduction assay. Our results indicated that the ratio between optical density and cell numbers is not constant and use of the same OD for inoculums for all strains can therefore lead to misinterpretation of the MIC values. We also observed influence of cell numbers in inoculums in determination of MIC values. For both approaches used (approximation of MIC with IC90 and the resazurin reduction assay), the same trend was detected: antibiotics had the highest potency in experiments with the lowest bacteria cell number (103/well). The lowest cell number (103/well) is not recommended, as it can lead to false susceptibility results and to partial reduction of resazurin, which further complicates MIC determination. A final cell number of 104/well can therefore be recommended as optimal

A comprehensive assessment of the chemical composition, antioxidant, genoprotective and antigenotoxic activities of Lamiaceae species using different experimental models in vitro

This research was aimed to assess the potential of Glechoma hederacea, Hyssopus officinalis, Lavandula angustifolia, Leonurus cardiaca, Marrubium vulgare and Sideritis scardica (Lamiaceae) methanolic, ethanolic and aqueous extracts against the damaging effects of oxidative stress using different experimental models. The chemical characterization was done spectrophotometrically by quantifying total phenolics, phenolic acids, flavonoids and flavonols in the extracts, as well as by employing HPLC-DAD technique. Moreover, DPPH assay was used to assess the extracts' radical scavenging potential. Genoprotective properties of the extracts were evaluated using plasmid pUC19 Escherichia coli XL1-Blue, whereas their antigenotoxic potential was determined using Salmonella typhimurium TA1535/pSK1002 and normal human lung fibroblasts. All of the extracts showed antioxidant activity in DPPH assay. Furthermore, the results have shown that aqueous extracts provided the best protection for plasmid DNA, while alcoholic extracts most effectively contributed to the preservation of prokaryotic DNA. Additionally, each of the tested samples significantly protected the eukaryotic cells against genomic damages. Finally, despite not showing exceptional results in DPPH assay, S. scardica extracts are regarded as the most favorable in maintaining the integrity of DNA, which might be due to high quantities of phenolics such as quercetin (up to 17.95 mg g(-1)), naringin (up to 5.07 mg g(-1)) and luteolin-7-O-glucoside (up to 3.54 mg g(-1)). Overall, this comprehensive concept highlights the ability of these Lamiaceae species to safeguard the DNA from reactive oxygen species, to curtail the inflicted damage and also improve the efficiency of the DNA repair mechanisms, while emphasizing the importance of polyphenols as their active principles

A Study of Phytochemistry, Genoprotective Activity, and Antitumor Effects of Extracts of the Selected Lamiaceae Species

This study was designed to evaluate the genoprotective, antigenotoxic, as well as antitumor potential of methanolic, ethanolic, and aqueous extracts of Melissa officinalis, Mentha x piperita, Ocimum basilicum, Rosmarinus officinalis, Salvia officinalis, and Satureja montana (Lamiaceae), in different model systems. The polyphenols in these extracts were quantified both spectrophotometrically and using HPLC-DAD technique, while DPPH assay was used to assess the antioxidant activity. The genoprotective potential was tested on pUC19 Escherichia coli XL1-blue, and the antigenotoxicity on Salmonella typhimurium TA1535/pSK1002 and human lung fibroblasts, while the antitumor activity was assessed on colorectal cancer cells. Rosmarinic acid, quercetin, rutin, and luteolin-7-O-glucoside were among the identified compounds. Methanolic extracts had the best DPPH-scavenging and SOS-inducing activities, while ethanolic extracts exhibited the highest antigenotoxicity. Additionally, all extracts exhibited genoprotective potential on plasmid DNA. The antitumor effect was mediated by modulation of reactive oxygen species (ROS), nitric oxide (NO) production, and exhibition of genotoxic effects on tumor cells, especially with O. basilicum ethanolic extract. Generally, the investigated extracts were able to provide antioxidant protection for the acellular, prokaryotic, and normal human DNA, while also modulating the production of ROS and NO in tumor cells, leading to genotoxicity toward these cells and their decrease in proliferation