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.
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 1/2 MIC, showed synergy with antibiotics representing the beta-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 beta-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.
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 lM) and various concentrations of 6-benzylaminopurine (BAP), kinetin, zeatin (1, 2, 4, 8 lM) or tidiazuron (TDZ) (0.2, 0.5, 1 lM). 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 lM 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 lM IAA, when compared to cytokinin-free medium. TDZ at a concentration of 0.5 lM 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 [2,20 -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 lM TDZ demonstrated the strongest antioxidant activity. The results indicate that higher polyphenolic content correlated with greater reducing power and antiradical efficiency.
One of the most effective strategies to enhance metabolite biosynthesis and accumulation in biotechnological systems is the use of elicitation processes. This study assesses the influence of different concentrations of yeast extract (YE) on ginsenoside biosynthesis in Panax quinquefolium (American ginseng) hairy roots cultivated in shake flasks and in a nutrient sprinkle bioreactor after 3 and 7 days of elicitation. The saponin content was determined using HPLC. The maximum yield (20 mg g−1 d.w.) of the sum of six examined ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) in hairy roots cultivated in shake flasks was achieved after application of YE at 50 mg L−1 concentration and 3 day exposure time. The ginsenoside level was 1.57 times higher than that attained in control medium. The same conditions of elicitation (3 day time of exposure and 50 mg L−1 of YE) also favourably influenced the biosynthesis of studied saponins in bioreactor cultures. The total ginsenoside content was 32.25 mg g−1 d.w. and was higher than that achieved in control medium and in shake flasks cultures. Obtained results indicated that yeast extract can be used to increase ginsenoside production in hairy root cultures of P. quinquefolium.
The flavonoid (baicalin, wogonoside, luteolin, luteolin-7-glucoside) and verbascoside contents of Scutellaria altissima in both shoot cultures, and the shoots and roots of micropropagated plants grown in the greenhouse for 12 weeks or in the field for 2 years were determined. The level of secondary metabolites was found to be strongly affected by the age and type of plant organ. A comparative analysis of S. altissima plants propagated in vitro and from seeds revealed no differences in the level of secondary metabolites when plants of the same age were studied. The antioxidant potential of methanolic extracts from shoot cultures, and the shoots and roots of S. altissima plants propagated in vitro, were evaluated using ABTS radical scavenging, FRAP metal reduction power and the lipid peroxidation test, in relation to the content of baicalin, wogonoside, verbascoside, total phenolic and total flavonoid compounds. Extracts from the roots of field-grown regenerated plants at the flowering stage were found to possess the strongest antioxidant activity. Correlation analysis revealed that the antioxidant activity of extracts correlated most closely with their total phenolic content estimated by the Folin-Ciocalteu method.
Hairy root culture of Salvia sclarea L. was established following infection with Agrobacterium rhizogenes LBA 9402. The culture was grown in growth regulator-free half-strength B5 Gamborg medium with 30 g l )1 sucrose and was investigated with respect to its capability of producing diterpenoids and triterpenoids. Four diterpenoids (ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) and two ursenetype triterpenoids (2a,3a-dihydroxy-urs-12-en-28-oic acid and 2a,3a,24-trihydroxy-urs-12-en-28-oic acid) were isolated from the hairy roots. The presence of three sterols (b-sitosterol, stigmasterol and campesterol), as well as oleanolic and ursolic acids was also shown by GC-MS analysis. The quantitative and qualitative differences in diterpenoid and triterpenoid production patterns between hairy roots grown in the light and in the dark were described.
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