Rapid UHPLC determination of polyphenols in aqueous infusions of Salvia officinalis L. (sage tea)

Sage tea, the aqueous infusion of dried sage leaves (Salvia officinalis L.), is used as a form of food as well as a form of traditional herbal medicine. Several in vivo and in vitro studies point to sage polyphenols as active principles that may inhibit lipid peroxidation and improve antioxidant defences. This study describes an UHPLC methodology with MS/MS and UV detection, which allows the separation, identification and quantification of the major phenolic constituents in sage tea within 34 min, and was used to characterize 16 commercial brands of sage tea.The quantitatively dominating compounds were either rosmarinic acid (12.2–296 mg/l) or luteolin-7-o-glucuronide (37.9–166 mg/l) [corrected].In general, considerable differences in polyphenolic composition between the brands were detected, leading to the demand for quality standardization and control, especially if these sage teas are to be used for therapeutic purposes.     

Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis)

(+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases.     

Aeromonas detection and characterization using genus-specific PCR and single-strand conformation polymorphism (SSCP)

Based on sequence alignment, oligonucleotide primers targeting the Aeromonas extracellular lipase gene were developed for PCR detection of member of the genus. A pair of primers designed for conserved regions of the gene amplified a 276?bp sequence in all Aeromonas species and tested strains, but did not have a positive result with other Gram-positive and Gram-negative bacteria, showing high specificity and sensitivity. Selective enrichment in alkaline peptone water, followed by centrifugation, and direct usage of cells suspension as template, detected initial populations of 10?c.f.u.?ml(-1). Single-strand conformation polymorphism analysis of the PCR products allowed the characterization of Aeromonas strains with a high discriminatory power (Simpson's index?=?0.988). The method presented here provides a useful tool for the rapid detection of Aeromonas and the characterization of Aeromonas isolates.     

Control of downy mildew (Pseudoperonospora cubensis) of greenhouse grown cucumbers with alternative biological agents

In organic cucumber production infection with downy mildew (Pseudoperonospora cubensis) is a major problem. Plant extracts from Glycyrrhiza glabra L. (licorice), a plant belonging to the family Fabaceae, and Salvia officinalis (sage) as well as cultures of the bacterium Aneurinibacillus migulanus were investigated for efficacy of disease control under commercial growing conditions. Contrary to bioassays, where sage extract and the microorganism showed highest activity, in the trials of 2008 G. glabra extract was more effective than sage extract or A. migulanus against P. cubensis. Parameters such as concentrations of the preparations or application intervals could have been the reason for this. In the following year's trial (2009) the concentration of these agents was therefore increased somewhat and plants were either treated in seven day application intervals or in ten day application intervals. In the semi-commercial trials of 2009 all alternative biological agents showed good efficacies up to around 80% against infection with downy mildew. The application interval seemed to have a marginal effect only. Again, the licorice extract tended to be the best agent.     

Metabolism of Monoterpenes in Cell Cultures of Common Sage (Salvia officinalis) : Biochemical Rationale for the Lack of Monoterpene Accumulation

Leaves of common sage (Salvia officinalis) accumulate monoterpenes in glandular trichomes at levels exceeding 15 milligrams per gram fresh weight at maturity, whereas sage cells in suspension culture did not accumulate detectable levels of monoterpenes (<0.3 nanograms per gram fresh weight) at any stage of the growth cycle, even in the presence of a polystyrene resin trap. Monoterpene biosynthesis from [U-¹⁴C]sucrose was also virtually undetectable in this cell culture system. In vitro assay of each of the enzymes required for the sequential conversion of the ubiquitous isoprenoid precursor geranyl pyrophosphate to (+)-camphor (a major monoterpene product of sage) in soluble extracts of the cells revealed the presence of activity sufficient to produce (+)-camphor at a readily detectable level (>0.3 micrograms per gram fresh weight) at the late log phase of growth. Other monoterpene synthetic enzymes were present as well. In vivo measurement of the ability to catabolize (+)-camphor in these cells indicated that degradative capability exceeded biosynthetic capacity by at least 1000-fold. Therefore, the lack of monoterpene accumulation in undifferentiated sage cultures could be attributed to a low level of biosynthetic activity (relative to the intact plant) coupled to a pronounced capacity for monoterpene catabolism.     

Distribution and essential oils of Salvia pomifera subsp. pomifera (Labiatae) on the island of Crete (S Greece)

The distribution of Salvia pomifera subsp. pomifera (Cretan sage) on the island of Crete is presented. The essential oils of six populations scattered on the island are studied. The essential oil content varies from 2.1–4.2%, whereas the main oil components were in all cases α- and/or β-thujone (27.4–72.3% and 7.1–40.8%, respectively). The comparison of our results to literature data, suggest that S. pomifera can be distinguished from S. fruticosa (Greek sage), on the basis of its essential oil composition.     

Relationship of Camphor Biosynthesis to Leaf Development in Sage (Salvia officinalis)

The camphor content of sage (Salvia officinalis L.) leaves increases as the leaves expand, and the increase is roughly proportional to the number of filled peltate oil glands which appear on the leaf surface during the expansion process. ¹⁴CO₂ is more rapidly incorporated into camphor and its direct progenitors in expanding leaves than in mature leaves, and direct in vitro measurement of the key enzymes involved in the conversion of geranyl pyrophosphate to camphor indicates that these enzymes, including the probable rate-limiting cyclization step, are at the highest levels during the period of maximum leaf expansion. These results clearly demonstrate that immature sage leaves synthesize and accumulate camphor most rapidly.     

Development and Essential Oil Content of Secretory Glands of Sage (Salvia officinalis)

Scanning electron microscopy of sage (Salvia officinalis L.) leaves confirmed the presence of two basic types of glandular trichomes consisting of a capitate stalked form containing a multicellular stalk and surmounted by a unicellular secretory head, and a capitate sessile form containing a unicellular stalk and unicellular, or multicellular, secretory head. In the latter type, secretory activity and filling of the subcuticular cavity may begin at virtually any stage of the division cycle affording fully developed glands containing from one to twelve cells in the secretory head. Gas liquid chromatographic analysis of the oil content of the most numerous gland species (capitate stalked, capitate sessile with one and with eight secretory cells) indicated only minor quantitative differences in essential oil composition. Thus, each gland type is capable of producing the four major monoterpene families (p-menthanes, pinanes, bornanes and thujanes) characteristic of sage.     

Toxicity of plant essential oils to Tetranychus urticae (Acari: Tetranychidae) and Phytoseiulus persimilis (Acari: Phytoseiidae)

Fifty-three plant essential oils were tested for their toxicity against eggs and adults of Tetranychus urticae Koch as well as adults of Phytoseiulus persimilis Athias-Henriot, by using a filter paper diffusion bioassay without allowing direct contact. Responses varied according to oil type and dose, and mite species. In a plastic container (4.5 by 9.5 cm) bioassay at 14 x 10(-3) microl/ml air, caraway seed, citronella java, lemon eucalyptus, pennyroyal, and peppermint oils gave > 90% mortality against adult T. urticae, whereas 82 and 81% mortality was observed with sage and spearmint oils, respectively. With the exception of sage oil, the other six essential oils were highly effective against T. urticae eggs at 9.3 x 10(-3) microl/ml air. Against adult P. persimilis, these six test oils caused > 90% mortality at 7.1 x 10(-3) microl/ml air. Particularly peppermint oil at 4.7 x 10(-3) microl/ml air was highly toxic. In an acrylic cage (30 by 30 by 40 cm ) test, lemon eucalyptus, pennyroyal, peppermint, and spearmint oils were highly effective against adult T. urticae at 1.4 x 10(-3) microl/ml air. These results indicate that the mode of delivery of these essential oils was largely a result of action in the vapor phase via the respiratory system. The essential oils described herein merit further study as potential fumigants for T. urticae control.     

Catabolism of camphor in tissue cultures and leaf disks of common sage (Salvia officinalis).

(+)-Camphor constitutes nearly 30% of the monoterpenes accumulated in the leaves of common sage (Salvia officinalis), and as the plant approaches maturity the content of this monoterpene ketone decreases by roughly half. Although the ability to catabolize camphor has been demonstrated previously in sage leaf disks, tissue cultures proved to be a more suitable system for examining the responsible degradative pathway. Cell suspension cultures were shown to convert (+)-[3-3H2]camphor, in sequence, to 6-hydroxycamphor, 6-oxocamphor, alpha-campholonic acid, and 2-hydroxy-alpha-campholonic acid, and each intermediate of the pathway was identified by chromatographic and spectroscopic means. This oxidative ring opening sequence resembles the pathway for camphor degradation by the soil diphtheroid, Mycobacterium rhodochrous, that ultimately leads to isoketocamphoric as the last defined metabolite that contains all 10 carbons of the original bicyclic nucleus. Studies with both cell cultures and leaf disks also demonstrated that the catabolism of camphor via 1,2-campholide, a metabolite in sage leaves previously described, was a minor degradative pathway. The first step in the metabolism of camphor was demonstrated in cell-free extracts of the cultured sage cells, and several lines of evidence indicated that this microsomal (+)-camphor-6-exo-hydroxylase is a cytochrome P-450-dependent monooxygenase.     

Verticillium wilt of sage (Salvia officinalis L.)

Verticillium dahliae Kleb. was established as the cause of wilt in sage. The disease causes leaf yellowing and necrosis from the base of the plant upwards, followed by defoliation. Microsclerotia found in the bark and sometimes the pith of severely affected stems are regarded as a diagnostic feature. Surveys confirmed the widespread occurrence of the disease in the Vale of Evesham and showed that it was most severe on heavy clay soils and in poorly drained areas but was not related to soil pH, phosphate or potash content. The rate of disease spread and the distribution of affected plants suggested that infected debris was the main source of disease dispersal, particularly as microsclerotia were abundant in many fallen leaves. It was shown that the disease could be disseminated in propagating material, and field evidence suggested that the pathogen could survive in the field in the absence of sage for at least 10 years. Replanted sage in affected areas generally contracted the disease within 1 year. Control measures suggested include careful stock selection, weed control and improved general hygiene.     

Modelling the distribution of a threatened habitat: the California sage scrub

Aim Using predictive species distribution and ecological niche modelling our objectives are: (1) to identify important climatic drivers of distribution at regional scales of a locally complex and dynamic system – California sage scrub; (2) to map suitable sage scrub habitat in California; and (3) to distinguish between bioclimatic niches of floristic groups within sage scrub to assess the conservation significance of analysing such species groups. Location Coastal mediterranean‐type shrublands of southern and central California. Methods Using point localities from georeferenced herbarium records, we modelled the potential distribution and bioclimatic envelopes of 14 characteristic sage scrub species and three floristic groups (south‐coastal, coastal–interior disjunct and broadly distributed species) based upon current climate conditions. Maxent was used to map climatically suitable habitat, while principal components analysis followed by canonical discriminant analysis were used to distinguish between floristic groups and visualize species and group distributions in multivariate ecological space. Results Geographical distribution patterns of individual species were mirrored in the habitat suitability maps of floristic groups, notably the disjunct distribution of the coastal–interior species. Overlap in the distributions of floristic groups was evident in both geographical and multivariate niche space; however, discriminant analysis confirmed the separability of floristic groups based on bioclimatic variables. Higher performance of floristic group models compared with sage scrub as a whole suggests that groups have differing climate requirements for habitat suitability at regional scales and that breaking sage scrub into floristic groups improves the discrimination between climatically suitable and unsuitable habitat. Main conclusions The finding that presence‐only data and climatic variables can produce useful information on habitat suitability of California sage scrub species and floristic groups at a regional scale has important implications for ongoing efforts of habitat restoration for sage scrub. In addition, modelling at a group level provides important information about the differences in climatic niches within California sage scrub. Finally, the high performance of our floristic group models highlights the potential a community‐level modelling approach holds for investigating plant distribution patterns.     

Spatially explicit models of seasonal habitat for greater sage‐grouse at broad spatial scales: Informing areas for management in Nevada and northeastern California

Defining boundaries of species' habitat across broad spatial scales is often necessary for management decisions, and yet challenging for species that demonstrate differential variation in seasonal habitat use. Spatially explicit indices that incorporate temporal shifts in selection can help overcome such challenges, especially for species of high conservation concern. Greater sage‐grouse Centrocercus urophasianus (hereafter, sage‐grouse), a sagebrush obligate species inhabiting the American West, represents an important case study because sage‐grouse exhibit seasonal habitat patterns, populations are declining in most portions of their range and are central to contemporary national land use policies. Here, we modeled spatiotemporal selection patterns for telemetered sage‐grouse across multiple study sites (1,084 sage‐grouse; 30,690 locations) in the Great Basin. We developed broad‐scale spatially explicit habitat indices that elucidated space use patterns (spring, summer/fall, and winter) and accounted for regional climatic variation using previously published hydrographic boundaries. We then evaluated differences in selection/avoidance of each habitat characteristic between seasons and hydrographic regions. Most notably, sage‐grouse consistently selected areas dominated by sagebrush with few or no conifers but varied in type of sagebrush selected by season and region. Spatiotemporal variation was most apparent based on availability of water resources and herbaceous cover, where sage‐grouse strongly selected upland natural springs in xeric regions but selected larger wet meadows in mesic regions. Additionally, during the breeding period in spring, herbaceous cover was selected strongly in the mesic regions. Lastly, we expanded upon an existing joint–index framework by combining seasonal habitat indices with a probabilistic index of sage‐grouse abundance and space use to produce habitat maps useful for sage‐grouse management. These products can serve as conservation planning tools that help predict expected benefits of restoration activities, while highlighting areas most critical to sustaining sage‐grouse populations. Our joint–index framework can be applied to other species that exhibit seasonal shifts in habitat requirements to help better guide conservation actions.     

Differential influences of local subpopulations on regional diversity and differentiation for greater sage‐grouse (Centrocercus urophasianus)

The distribution of spatial genetic variation across a region can shape evolutionary dynamics and impact population persistence. Local population dynamics and among‐population dispersal rates are strong drivers of this spatial genetic variation, yet for many species we lack a clear understanding of how these population processes interact in space to shape within‐species genetic variation. Here, we used extensive genetic and demographic data from 10 subpopulations of greater sage‐grouse to parameterize a simulated approximate Bayesian computation (ABC) model and (i) test for regional differences in population density and dispersal rates for greater sage‐grouse subpopulations in Wyoming, and (ii) quantify how these differences impact subpopulation regional influence on genetic variation. We found a close match between observed and simulated data under our parameterized model and strong variation in density and dispersal rates across Wyoming. Sensitivity analyses suggested that changes in dispersal (via landscape resistance) had a greater influence on regional differentiation, whereas changes in density had a greater influence on mean diversity across all subpopulations. Local subpopulations, however, varied in their regional influence on genetic variation. Decreases in the size and dispersal rates of central populations with low overall and net immigration (i.e. population sources) had the greatest negative impact on genetic variation. Overall, our results provide insight into the interactions among demography, dispersal and genetic variation and highlight the potential of ABC to disentangle the complexity of regional population dynamics and project the genetic impact of changing conditions.     

Water and methanolic extracts of Salvia officinalis protect HepG2 cells from t-BHP induced oxidative damage

Common sage (Salvia officinalis L., Lamiaceae) is an aromatic and medicinal plant well known for its antioxidant properties. Some in vivo studies have shown the biological antioxidant effects of sage. However, the intracellular antioxidant mechanisms of action are still poorly understood. In this study, we evaluated the cytoprotective effects of two sage extracts (a water and a methanolic extract) against tert-butyl hydroperoxide (t-BHP)-induced toxicity in HepG2 cells. The most abundant phenolic compounds present in the extracts were rosmarinic acid and luteolin-7-glucoside. Both extracts, when co-incubated with the toxicant, protected significantly HepG2 cells against cell death. The methanolic extract, with a higher content of phenolic compounds than the water extract, conferred better protection in this in vitro model of oxidative stress with liver cells. Both extracts, tested in a concentration that protects 80% against cell death (IC(80)), significantly prevented t-BHP-induced lipid peroxidation and GSH depletion, but not DNA damage assessed by the comet assay. The ability of sage extracts to reduce t-BHP-induced GSH depletion by 62% was probably the most relevant contributor to the observed cytoprotection. A good correlation between the above cellular effects of sage and the effects of their main phenolic compounds was found. When incubated alone for 5h, sage extracts induced an increase in basal GSH levels of HepG2 cells, which indicates an improvement of the antioxidant potential of the cells. Compounds present in sage extracts other than phenolics may also contribute to this latter effect. Based in these results, it would be of interest to investigate whether sage has protective effects in suitable in vivo models of liver diseases, where it is known that oxidative stress is involved.     

Effects of Protocalliphora braueri (Diptera: Calliphoridae) parasitism and inclement weather on nestling sage thrashers.

Infection with blow fly larvae (Protocalliphora braueri) had no effect on sage thrasher (Oreoscoptes montanus) nestling weight or size at fledging nor on mean fledging age. However, the combination of cold, wet weather and parasite infection did significantly reduce nestling survival and the percent young fledged.     

Seasonal variations of phenolic compounds and biological properties in sage (Salvia officinalis L.)

The aim was to investigate the phenolic content, antioxidant capacity, and antibacterial activity of Dalmatian sage (Salvia officinalis L.) leaves collected during different vegetation periods. Separation and quantification of the individual phenols were performed by reversed-phase (RP)-HPLC coupled with a PDA (photodiode array) detector and using an internal standard, while the contents of total phenols, flavonoids, flavones, and flavonols were determined spectrophotometrically. The antioxidant properties of the sage leaf extracts were evaluated using five different antioxidant assays (FRAP, DPPH, ABTS, Briggs-Rauscher reaction, and β-carotene bleaching). The antimicrobial activity of the extracts was tested against two Gram-positive (Bacillus cereus and Staphylococcus aureus) and two Gram-negative (Salmonella Infantis and Escherichia coli) bacterial reference strains. All extracts were extremely rich in phenolic compounds, and provided good antioxidant and antibacterial properties, but the phenophase in which the leaves were collected affected the phenolic composition of the sage extracts and consequently their biological activity. The May Extract, the richest in total flavonoids, showed the best antioxidant properties and the highest antimicrobial activity. Thus, collection of the plants during May seems the best choice for further use of them in the pharmaceutical and food industry.     

Olfaction and Feeding Behaviour in Juvenile Geese (Anser a. anser and Anser domesticus)

Juvenile geese start reacting to odours at the age of 11 to 20 h. In inexperienced goslings head-shaking is released by the odours of plants on which adult geese would not feed (e.g. lavender, violet etc.). Goslings aged 4 to 9 days show a quantitatively different reaction to different odours, such as sage, dwarf-pine, dill etc. Goslings quickly become habituated to odours, they will not react any more if an odorant is presented too often.     

Restoring Sage‐grouse nesting habitat through removal of early successional conifer

Conifer woodlands have expanded into sagebrush (Artemisia spp.) ecosystems and degrade habitat for sagebrush obligate species such as the Greater Sage‐grouse (Centrocercus urophasianus). Conifer management is increasing despite a lack of empirical evidence assessing outcomes to grouse and their habitat. Although assessments of vegetation recovery after conifer removal are common, comparisons of successional trends with habitat guidelines or actual data on habitat used by sage‐grouse is lacking. We assessed impacts of conifer encroachment on vegetation characteristics known to be important for sage‐grouse nesting. Using a controlled repeated measures design, we then evaluated vegetation changes for 3 years after conifer removal. We compared these results to data from 356 local sage‐grouse nests, rangewide nesting habitat estimates, and published habitat guidelines. We measured negative effects of conifer cover on many characteristics important for sage‐grouse nesting habitat including percent cover of forbs, grasses, and shrubs, and species richness of forbs and shrubs. In untreated habitat, herbaceous vegetation cover was slightly below the cover at local nest sites, while shrub cover and sagebrush cover were well below cover at the nest sites. Following conifer removal, we measured increases in herbaceous vegetation, primarily grasses, and sagebrush height. Our results indicate that conifer abundance can decrease habitat suitability for nesting sage‐grouse. Additionally, conifer removal can improve habitat suitability for nesting sage‐grouse within 3 years, and trajectories indicate that the habitat may continue to improve in the near future.