The essential oils obtained by hydrodistillation of dried aerial parts of Salvia sclarea L. plants, regenerated in vitro and reproduced from seeds, were analyzed by GC and GC-MS. The oils from in vitro and in vivo plants were compared in respect to their chemical composition as well as antimicrobial and cytotoxic activities. The chemical profiles of both oils were very similar, although the yield of essential oil from in vitro plants was lower (0.1%, v/w) than the oil yield isolated from in vivo S. sclarea plants (0.2%, v/w). Both oils showed antimicrobial and cytotoxic activity. The oil from in vitro regenerated plants of S. sclarea exhibited stronger cytotoxic action against NALM-6 cell lines in comparison with the essential oil from in vivo plants.

The present study evaluates the effects of various cytokinins on Scutellaria alpina shoot proliferation and production of polyphenolic metabolites (baicalin, wogonoside, luteolin, luteolin 7-O-glucoside, verbascoside). The shoots were induced from shoot tips on MS medium supplemented with IAA (indole-3-acetic acid, 0.57 µM) and various concentrations of 6-benzylaminopurine (BAP), kinetin, zeatin (1, 2, 4, 8 µM) or tidiazuron (TDZ) (0.2, 0.5, 1 µM). Among the cytokinins tested, BAP was the most effective for shoot induction, and the highest number of shoots (25 per explant) was achieved with 2 and 4 µM BAP. Maximum biomass production was also achieved on these media. Significantly higher baicalin, wogonoside and verbascoside contents were recorded in treatments containing cytokinins combined with 0.57 µM IAA, when compared to cytokinin-free medium. TDZ at a concentration of 0.5 µM was the most effective for polyphenol production. However, supplementation with cytokinins often results in the reduction of luteolin and its 7-O-glucoside production in the shoot culture of S. alpina. ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] and ferric reducing antioxidant power assays were used to identify the antioxidant potential of methanolic extracts from shoots cultured in the presence of different types and concentrations of cytokinins. In both tests, the shoots from medium supplemented with 0.5 µM TDZ demonstrated the strongest antioxidant activity. The results indicate that higher polyphenolic content correlated with greater reducing power and antiradical efficiency.

Two diterpenoids, salvipisone (Salv) and aethiopinone (Aeth), isolated from hairy roots of Salvia sclarea, were tested with respect to their activity against methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis (MRSE) strains, cultured as planktonic cells or as adherent biofilms. The standard CLSI method, MTT reduction assay or confocal laser scanning microscopy (CLSM) were used for this purpose and also applied for testing the susceptibility to oxacillin, vancomycin, linezolid and their potential synergy with diterpenoids (evaluated as a fractional inhibitory concentration (FIC) index). Salv and Aeth were shown to be bactericidal or bacteriostatic against S. aureus and S. epidermidis planktonic cultures. Both diterpenoids, at the concentrations of œ MIC, showed synergy with antibiotics representing the β-lactam, glycopeptide and oxazolidinone groups. None of the antibiotics used at a high concentration killed the staphylococcal biofilms. On the contrary, Salv and Aeth decreased the number of live biofilm cells by 45.7 - 77.1 % and slightly reduced the biofilm inhibitory concentration of oxacillin. Diterpenoids also changed the parameters of biofilm morphology, as shown by the CLSM image processing package (PHLIP). It was concluded that salvipisone and aethiopinone (relatively highly lipophilic, log P respectively = 3.4; 4.8) synergized the action of β-lactam antibiotics towards MRSA and MRSE probably by alteration of cell surface hydrophobicity and cell wall/membrane permeability, but not by changing penicillin-binding protein, PBP2a expression and penicillinase production or by direct binding to the cell wall peptidoglycan and teichoic acids. Aeth:aethiopinone ATCC:American Type Culture Collection CAM:calcein-AM CLSI:Clinical and Laboratory Standards Institute CLSM:confocal laser scanning microscopy CSH:cell surface hydrophobicity DMSO:dimethyl sulfoxide FIC:fractional inhibitory concentration MHB:Mueller-Hinton broth MIC:minimal inhibitory concentration MRSA/MSSA:methicillin resistant/susceptible Staphylococcus aureus MRSE/MSSE:methicillin resistant/susceptible S. epidermidis MTT:3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide OD:optical density PBP:penicillin binding protein PG:peptidoglycan PI:propidium iodide Salv:salvipisone SAT:salt aggregation test TA:teichoic acids TSB:tryptic soy broth