Comparative Studies of Phytochemical Screening, HPLC-PDA-ESI-MS/MS-LC/HR-ESI-MS Analysis, Antioxidant Capacity and in Vitro Fermentation of Officinal Sage (Salvia officinalis L.) Cultivated in Different Biotopes of Northwestern Tunisia.

We aimed in the present study to investigate the chemical composition, the antioxidant capacities as well as the in vitro fermentation properties of Salvia officinalis leaves aqueous extract (SOLAE) grown in four regions of northwestern Tunisia. Our data firstly indicated a spatial variation (P<0.05) in condensed tannins, total lipids, polyphenols and flavonoids contents. The HPLC-PDA-ESI-MS/MS-LC/HR-ESI-MS technique allowed to the identification of 13 phenolic compounds and showed that protocatechuic acid is the major constituent of the plant leaves grown in Tabarka, Ain Draham and Testour. The SOLAE of the plant grown in Tabarka presents the most potent scavenging activity against DPPH radical and had the highest percentage of inhibition. More importantly, we found in the present study that the digestibility of dry matter and in vitro fermentation showed a significant variation between the regions and the animal species. Also, we showed a very positive correlation between antioxidant properties and phenolic compounds contents. In conclusion, we suggest that SOLAE had potential beneficial effects owing in part to its antioxidant and ROS scavenging activities. Therefore, S. officinalis can be proposed as an additive food for animals' nutrition and health.

Seed germination of medicinal sage is affected by gibberellic acid, magnetic field and laser irradiation.

Proper priming techniques are among the most important methods for increasing seed germination and seedling growth. Three experiments were conducted to investigate the effects of plant hormone (500 and 1000 mg/L gibberellic acid (GA)), magnetic field (3, 15, 30 mili Tesla (mT)) and laser irradiation at 650 nm (200 mW) on the germination and the growth of Salvia officinalis. We examined the plumule and radical length, plumule and radical fresh weight, plumule and radical dry weight, germination percentage, germination rate and seed vigor. The two concentrations of GA significantly increased seed germination and seedling growth. The magnetic field at 15 mT significantly increased radical length. The effect of laser irradiation was also significant on plumule length, and fresh and dry weight, radicle fresh weight, germination percentage and rate and seed vigor. Such results may be of practical use in the field, especially in arid and semiarid areas, but more research must determine the response of medicinal sage, treated with the priming techniques tested in our experiments, under stress conditions.

Genetic Diversity and Demographic History of Wild and Cultivated/Naturalised Plant Populations: Evidence from Dalmatian Sage (Salvia officinalis L., Lamiaceae).

Dalmatian sage (Salvia officinalis L., Lamiaceae) is a well-known aromatic and medicinal Mediterranean plant that is native in coastal regions of the western Balkan and southern Apennine Peninsulas and is commonly cultivated worldwide. It is widely used in the food, pharmaceutical and cosmetic industries. Knowledge of its genetic diversity and spatiotemporal patterns is important for plant breeding programmes and conservation. We used eight microsatellite markers to investigate evolutionary history of indigenous populations as well as genetic diversity and structure within and among indigenous and cultivated/naturalised populations distributed across the Balkan Peninsula. The results showed a clear separation between the indigenous and cultivated/naturalised groups, with the cultivated material originating from one restricted geographical area. Most of the genetic diversity in both groups was attributable to differences among individuals within populations, although spatial genetic analysis of indigenous populations indicated the existence of isolation by distance. Geographical structuring of indigenous populations was found using clustering analysis, with three sub-clusters of indigenous populations. The highest level of gene diversity and the greatest number of private alleles were found in the central part of the eastern Adriatic coast, while decreases in gene diversity and number of private alleles were evident towards the northwestern Adriatic coast and southern and eastern regions of the Balkan Peninsula. The results of Ecological Niche Modelling during Last Glacial Maximum and Approximate Bayesian Computation suggested two plausible evolutionary trajectories: 1) the species survived in the glacial refugium in southern Adriatic coastal region with subsequent colonization events towards northern, eastern and southern Balkan Peninsula; 2) species survived in several refugia exhibiting concurrent divergence into three genetic groups. The insight into genetic diversity and structure also provide the baseline data for conservation of S. officinalis genetic resources valuable for future breeding programmes.

Influence of sodium salicylate on rosmarinic acid, carnosol and carnosic acid accumulation by Salvia officinalis L. shoots grown in vitro.

OBJECTIVES: To evaluate sodium salicylate (NaSA) as an elicitor of rosmarinic acid (RA) and phenolic diterpenes, carnosol (C) and carnosic acid (CA) production, in a culture of Salvia officinalis shoots. RESULTS: In sage shoots grown in vitro, 28 polyphenolic compounds (phenolic acids, flavonoids, and phenolic diterpenes) were identified. In shoots treated for 1 week with increasing NaSA concentrations, the content of C increased from 2.3 in control to 5.7 mg g(-1) DW in shoots treated with 500 µM NaSA. In shoots that were recovered on basal medium for 3 weeks, the maximal amount of C (14 mg/g(-1) DW) was with 150 µM NaSA treatment. In treated and recovered shoots, the increase in C was accompanied with a decrease in CA, resulting in 1.9-fold increase in the C/CA ratio. Accumulation of RA was not affected by the NaSA treatment. However, elicitation by NaSA was accompanied with growth retardation. CONCLUSIONS: NaSA can improve C production in sage shoot culture, probably by stimulating the conversion of CA to C.

Fungal communities in soils along a vegetative ecotone.

We investigated the community composition and diversity of soil fungi along a sharp vegetative ecotone between coastal sage scrub (CSS) and nonnative annual grassland habitat at two sites in coastal California. USA- We pooled soil samples across 29 m transects on either side of the ecotone at each of the two sites, and. using clone libraries of fungal ribosomal DNA, we identified 280 operational taxonomic units (OTUs) from a total 40 g soil. We combined information from partial LSU and ITS sequences and found that the majority of OTUs belonged to the phylum Ascomycota, followed by Basidiomycota. Within the Ascomycota. a quarter of OTUs were Sordariomycetes. 17% were Leotiomycet.es, 16% were Dothideomycetes and the remaining OTUs were distributed among the classes Eurotiomycetes, Pezizomycetes, Lecanoromycetes, Orbiliomycetes and Arthoniomycetes. Within the Basidiomycota. all OTUs but one belonged to the subphylum Agaricomycotina. We also sampled plant communities at the same sites to offer a point of comparison for patterns in richness of fungal communities. Fungal communities had higher alpha and beta diversity than plant communities; fungal communities were approximately 20 times as rich as plant communities and the majority of OTUs were found in single soil samples. Soils harbored a unique mycoflora that did not reveal vegetative boundaries or site differences. High alpha and beta diversity and possible sampling artifacts necessitate extensive sampling to reveal differentiation in these fungal communities.

Assessing and monitoring semi-arid shrublands using object-based image analysis and multiple endmember spectral mixture analysis.

Arid and semi-arid shrublands have significant biological and economical values and have been experiencing dramatic changes due to human activities. In California, California sage scrub (CSS) is one of the most endangered plant communities in the US and requires close monitoring in order to conserve this important biological resource. We investigate the utility of remote-sensing approaches--object-based image analysis applied to pansharpened QuickBird imagery (QBPS/OBIA) and multiple endmember spectral mixture analysis (MESMA) applied to SPOT imagery (SPOT/MESMA)--for estimating fractional cover of true shrub, subshrub, herb, and bare ground within CSS communities of southern California. We also explore the effectiveness of life-form cover maps for assessing CSS conditions. Overall and combined shrub cover (i.e., true shrub and subshrub) were estimated more accurately using QBPS/OBIA (mean absolute error or MAE, 8.9 %) than SPOT/MESMA (MAE, 11.4 %). Life-form cover from QBPS/OBIA at a 25 × 25 m grid cell size seems most desirable for assessing CSS because of its higher accuracy and spatial detail in cover estimates and amenability to extracting other vegetation information (e.g., size, shape, and density of shrub patches). Maps derived from SPOT/MESMA at a 50 × 50 m scale are effective for retrospective analysis of life-form cover change because their comparable accuracies to QBPS/OBIA and availability of SPOT archives data dating back to the mid-1980s. The framework in this study can be applied to other physiognomically comparable shrubland communities.

Physiological changes, phenolic content and antioxidant activity of Salvia officinalis L. grown under saline conditions.

BACKGROUND: Hydroponic culture was used to investigate the effect of NaCl concentrations on the growth, nutrient uptake, phenolic content and antioxidant activity of Salvia officinalis L. leaves. The antioxidant capacity of the methanolic extract of S. officinalis was evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test and ß-carotene-linoleic acid bleaching assay. Physiological and biochemical parameters of S. officinalis were assessed after 4 weeks of salt treatment with 0, 25, 50, 75 and 100 mmol L(-1) NaCl. RESULTS: Plant growth exhibited a reduction of 61% at 100 mmol L(-1) NaCl. Assessment of Na(+), K(+) and Ca(2+) and water contents of shoots and roots showed that S. officinalis is able to regulate Na(+) concentration by active compartmentation in vacuoles. Salvia officinalis phenolics were increased in response to salinity at the threshold of 75 mmol L(-1) NaCl. This herb was also found to be able to achieve important DPPH(•) quenching activity and to inhibit the ß-carotene-linoleic acid bleaching notably enhanced by salt treatment. It is interesting to highlight the correlation between the phenolic and antioxidant activity, suggesting the involvement of these compounds in this activity. CONCLUSION: Salvia officinalis treated with 75 mmol L(-1) NaCl constitutes a potential source for production of secondary metabolites useful in several applications.

Kinetin applications alleviate salt stress and improve the antioxidant composition of leaf extracts in Salvia officinalis.

A pot experiment was carried out under glasshouse conditions with common sage (Salvia officinalis L.) to investigate the interactive effects of salt stress and kinetin on growth attributes and the abundance of pigments, ions, phenolic diterpenes and α-tocopherol in leaf extracts of this species. The plants were subjected to the following four treatments: (i) control (nutrient solution), (ii) control + 10 µM kinetin, (iii) salt stress (nutrient solution + 100 mM NaCl), and (iv) salt stress + 10 µM kinetin. Kinetin was applied as a foliar fertilizer. Salt stress reduced water contents, photosynthetic activity and pigment contents of sage leaves. In addition, it increased Na(+) contents, and reduced those of Ca(2+) and K(+) in leaves. Salt stress reduced carnosic acid and 12-O-methyl carnosic acid contents in leaves, while it did not affect carnosol and α-tocopherol contents. Foliar applications of kinetin seemed to counterbalance or alleviate the stress symptoms induced by salinity, improving ion and pigment contents, while leaf phenolic diterpene (mainly carnosol) and α-tocopherol contents also increased in both control and NaCl-treated plants; still this effect was much more obvious in salt-treated plants. A similar effect was also obtained when plants were sprayed with KNO(3) or Ca(NO(3))(2), thus suggesting that kinetin effects were at least partly due to an improvement of ion homeostasis. Kinetin applications resulted in increased transcript levels of the isoprenoid and tocopherol biosynthetic genes, DXPRI and VTE2 and VTE4 in control plants, but not in NaCl-treated plants. We conclude that kinetin can alleviate the negative impact of salt on sage plants cultivated under arid environments with salinity problems.

Influence of ionic interactions on essential oil and phenolic diterpene composition of Dalmatian sage (Salvia officinalis L.).

The potential of four essential cations (K(+), Ca(2+), Mg(2+) and Fe(2+)) to alleviate salt toxicity was studied in sage (Salvia officinalis L.) plants grown in pots. Two concentrations of the following chloride salts: KCl, CaCl2, MgCl2 and FeCl3, were used together with 100mM NaCl to study the effects of these nutrients on plant growth, leaf essential oils (EOs) and phenolic diterpenes composition. The sage plants accumulated Na(+) in their leaves (includers); this has affected secondary metabolites' biosynthesis. Treatment with 100mM NaCl slightly decreased borneol and viridiflorol, while increased manool concentrations. Addition of KCl, CaCl2 and MgCl2 increased considerably in a dose-dependent manner the oxygen-containing monoterpenes (1.8-cineole, camphor, ß-thujone and borneol) in 100mM NaCl-treated sage. Whereas, the contents of viridiflorol decreased further with the addition of KCl in 100mM NaCl-treated sage. Our results suggest that the changes in EOs composition were more related to K(+) and Ca(2+) availability than to Na(+) toxicity. Furthermore, treatment with NaCl decreased by 50% carnosic acid (CA), a potent antioxidant, content in the leaves. K(+) and Ca(2+) promoted the accumulation of CA and its methoxylated form (MCA) in the leaves. The concentration of CA was positively correlated with leaf K(+) (r=0.56, P=0.01) and Ca(2+) (r=0.44, P=0.05) contents. It appears that different salt applications in combination with NaCl treatments had a profound effect on EOs and phenolic diterpene composition in sage. Therefore, ionic interactions may be carefully considered in the cultivation of this species to get the desired concentrations of these secondary metabolites in leaf extracts.

Effects of foliar fertilization and arbuscular mycorrhizal colonization on Salvia officinalis L. growth, antioxidant capacity, and essential oil composition.

BACKGROUND: The effect of foliar fertilization and Glomus intraradices inoculation on the growth, qualitative and quantitative pattern of essential oil in Salvia officinalis was determined. Sage plants were grown in a glass house on a soil/sand mixture (w/w = 3:1). Agroleaf total, N:P:K = 20:20:20 + microelements, was used at the whole vegetative growth stage as a 0.3% solution. Inoculation with Glomus intraradices was done at the sowing stage. RESULTS: Application of foliar fertilization and/or mycorrhizal colonization improved dry biomass accumulation and increased the content of antioxidant metabolites (ascorbate and reduced glutathione). Applied treatments lowered the activities of the antioxidants enzymes catalase, ascorbate peroxidase and superoxide dismutase, while guaiacol peroxidase increased. The relative quantity of essential oil pattern was also altered as a result of the applied treatments. Combined application (FF + Gi) significantly promoted 1,8-cineole and alpha-thujone, mycorrhizal colonization enhanced bornyl acetate, 1,8-cineole, alpha- and beta-thujones, while foliar fertilization increased bornyl acetate and camphor. The favorable effect of root colonization by Glomus intraradices was determined both on quantitative and qualitative pattern of sage essential oil. CONCLUSION: We conclude that inoculation with Glomus intraradices resulted in improved essential oil yield and quality, while combined application of foliar fertilizer and mycorrhizal fungi predominantly enhanced shoot biomass accumulation.

Ação de potenciais hidrogeniônicos no crescimento e produtividade de sálvia (Salvia officinalis L. ) / Action of potentials of hydrogen on growth and productivity of sage (Salvia officinalis L. )

O potencial hidrogeniônico (pH) do solo é um parâmetro que indica o nível de acidez ou alcalinidade do mesmo. Sabe-se que as plantas respondem de diferentes maneiras às mudanças no pH dos solos. Poucos artigos relacionam o crescimento e produtividade de plantas medicinais à variação do pH dos solos. O objetivo deste experimento foi avaliar o efeito de diferentes potenciais hidrogeniônicos, em solo Latossolo Vermelho Distroférrico, no crescimento e produção de sálvia. Com o auxílio de curva de neutralização da acidez do solo foram aplicadas diferentes doses de CaCO3 para atingir pH de 4,5; 5,0; 5,5; 6,0 e 6,5. O experimento foi conduzido em casa de vegetação durante 18 meses, perfazendo as fases de semeadura, transplantio e três cortes. Os parâmetros avaliados foram altura de plantas, massa seca de folhas, raízes e da parte aérea (soma das massas secas das folhas, ramos e caule), área foliar e volume das raízes. O crescimento e produção das plantas de sálvia foram diretamente afetados com a variação do pH do solo. A manutenção de pH > 5,0 foi suficiente para aumentar significativamente a área foliar, a massa seca das folhas e da parte área das plantas de sálvia em 52,2 por cento, 60,5 por cento e 42,4 por cento, respectivamente, e não afetou o desenvolvimento das raízes de sálvia cultivadas em solos com diferentes pHs.

The soil potential of hydrogen (pH) is a parameter that indicates the acidity or alkalinity level. Plants are known to react differently to changes in the soil pH. Few studies have related growth and productivity of medicinal plants to the soil pH variation. The aim of this experiment was to evaluate the effect of different potentials of hydrogen in Oxisol on sage growth and productivity. With the aid of a soil acidity neutralization curve, different CaCO3 levels were applied to reach pH values of 4.5, 5.0, 5.5, 6.0 and 6.5. The experiment was carried out in a greenhouse during 18 months, covering sowing, transplanting and three-pruning phases. Plant height, leaf, root and shoot dry matter (sum of leaf, branch and stem dry matter), leaf area and root volume were evaluated. Sage growth and productivity were directly affected by the soil pH variation. The maintenance of pH > 5.0 was enough to significantly increase leaf area, and leaf and shoot dry matter of sage plants at 52.2 percent, 60.5 percent and 42.4 percent, respectively, and did not affect root development of sage plants grown in soils presenting different pH levels.

Influence of gibberellin and daminozide on the expression of terpene synthases and on monoterpenes in common sage (Salvia officinalis).

Common sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants, with antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, composed mainly of the monoterpenes 1,8-cineole, alpha-thujone, beta-thujone and camphor, is responsible for some of these effects. Gibberellins regulate diverse physiological processes in plants, such as seed germination, shoot elongation and cell division. In this study, we analyzed the effect of exogenously applied plant growth regulators, namely gibberellic acid (GA(3)) and daminozide, on leaf morphology and essential oil formation of two leaf stages during the period of leaf expansion. Essential oil content increased with increasing levels of gibberellins and decreased when gibberellin biosynthesis was blocked with daminozide. With increasing levels of gibberellins, 1,8-cineole and camphor contents increased. Daminozide blocked the accumulation of alpha- and beta-thujone. GA(3) at the highest level applied also led to a significant decrease of alpha- and beta-thujone. Monoterpene synthases are a class of enzymes responsible for the first step in monoterpene biosynthesis, competing for the same substrate geranylpyrophosphate. The levels of gene expression of the three most important monoterpene synthases in sage were investigated, 1,8-cineole synthase leading directly to 1,8-cineole, (+)-sabinene synthase responsible for the first step in the formation of alpha- and beta-thujone, and (+)-bornyl diphosphate synthase, the first step in camphor biosynthesis. The foliar application of GA(3) increased, while daminozide significantly decreased gene expression of the monoterpene synthases. The amounts of two of the end products, 1,8-cineole and camphor, were directly correlated with the levels of gene expression of the respective monoterpene synthases, indicating transcriptional control, while the formation of alpha- and beta-thujone was not transcriptionally regulated.

Comparative in vitro study on the anti-herpetic effect of phytochemically characterized aqueous and ethanolic extracts of Salvia officinalis grown at two different locations.

Aqueous and ethanolic extracts of Salvia officinalis (Lamiaceae) from two different locations (Garden and Swabian Mountains) were examined in vitro on RC-37 cells for their antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) using a plaque reduction assay. The 50% inhibitory concentrations (IC50) of the extracts for HSV plaque formation were determined in dose-response studies. All extracts tested revealed a high virucidal activity against free HSV-1 and HSV-2. The experimental data exhibited a significant higher sensitivity of HSV against the extracts derived from Garden in comparison with those from Swabian Mountains. The most active one was the Garden 20% ethanol extract with IC50 values of 0.18 microg/ml for HSV-1 and 0.04 microg/ml for HSV-2. In order to identify the mode of antiviral action, the extracts were added to the host cells (RC-37) or viruses at different stages of infection. Independently of the location, both types of herpes viruses were considerably inactivated after treatment with the extracts prior to cell infection. Plaque formation was significantly reduced by >90% for HSV-1 and by >99% for HSV-2. Pretreatment of the host cells with both Garden and Swabian Mountains 20% and 40% ethanolic extracts prior to virus infection revealed a strong reduction of HSV-2 plaque formation by 94% and 70% (Garden) and 99% and 45% (Swabian Mountains), respectively. In time-activity studies with free HSV-1 over a period of 2h, a clearly time-dependent activity was demonstrated whereby the ethanolic extracts of both locations revealed a much higher activity than the aqueous ones. The 20% ethanolic extracts of both locations are of special interest and were effective when added to host cells and free virus. A topical application with a dual mode of action would be ideal against recurrent herpes infections.

[Study on characteristics of seed germination of Salvia officinalis].

OBJECTIVE: To investigate the influences of lightness and temperature on seeds germination of Salvia officinalis, and offer the basis for the standardized cultivation of S. officinalis. METHOD: The morphological characters, 1 000-grains weight and rate of water absorption of the seed were observed, the germination capacity, percent of germination power and germination index at the five different degrees: 10, 15, 20, 25, 30 degrees C were also measared. RESULT: 1 000-grains weight of the seeds was 8.03 g, the rate of water absorption is 49% within 24 h, the highest rate of germination capacity reached 85.33%, the germination index is 45.56. CONCLUSION: The lightness and 25 degrees C are the most suitable parameters for the seed germination of S. officinalis.

Assessment of wool waste and hair waste as soil amendment and nutrient source.

A field and two container experiments were conducted to assess uncomposted wool and hair wastes as a nutrient source for crops and to evaluate their potential to improve soil biological and chemical properties. Overall, addition of wool or hair waste to soil increased yields of basil (Ocimum basilicum L. 'Trakia'), thorn apple (Datura innoxia Mill. 'Inka'), peppermint (Mentha x piperita L. 'Black Mitchum'), and garden sage (Salvia officinalis L. 'Desislava'), increased NH(4)-N and NO(3)-N in soil, increased total N (and protein) content in plant tissue, stimulated soil microbial biomass, and decreased mycorrhizae colonization of plant roots of thorn apple but not in basil. Wool and hair waste additions to soil altered slightly the content and composition of plant secondary metabolites (essential oils or alkaloids); however, overall the constituents remained within the "typical" range for the respective crops. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis demonstrated that wool and hair wastes decompose slowly under field or greenhouse conditions, and act as a slow release S, N, P, and K fertilizer. These results, along with the measured concentrations of NO(3)-N in soil at harvest, suggest that the addition of wool or hair waste of only 3.3 g kg(-1) of soil may support two to five harvests or crops under greenhouse conditions and two to four field seasons in field production systems, and would improve soil biological and chemical characteristics. Further research is needed to optimize the rate of application of these waste materials to the nutrient requirements of specific crops to avoid nitrate leaching into the ground water. In addition, the effect of wool and hair waste on other environmental end points should also be further investigated before specific recommendations for growers are provided.

Biomass segregation in sage cell suspension culture.

The biomass of sage (Salvia officinalis L.) cell suspension culture was composed of single cells and cell aggregates. The development of aggregated cell culture from a single-cell suspension was monitored by particle size distribution for four particle size classes. Particle size distribution was compared between the biomass grown in bioreactor and shake flasks. The size of the particles had a strong influence on content of secondary metabolite, ursolic acid (UA). The single cell biomass fraction accumulated up to 7.7 mg UA g(-1) DW which was up to 50 times higher compared to aggregated biomass fractions.

Essential oils produced by in vitro shoots of sage (Salvia officinalis L.).

In vitro shoots of sage (Salvia officinalis L.) were established under eight different hormonal supplementations and proliferated by subculture of nodal shoot segments. The respective essential oils, obtained by hydrodistillation, were composed of more than 75 compounds, 65 of which were identified. The 10 major compounds were, by order of retention time, alpha-pinene (4.1-5.4%), camphene (6-7.1%), beta-pinene (9.3-14.5%), limonene (2-2.3%), 1,8-cineole (3.6-5.6%), (-)-thujone (13.2-16.1%), (+)-isothujone (6.6-7.4%), camphor (19.8-24%), alpha-humulene (5.1-6.8%), and manool (4.2-7.7%). Notwithstanding the eight different hormonal supplementations tested, the percentage composition of the shoot essential oils were kept in a narrow range of variation. However, the type and concentration of growth regulators apparently influenced the accumulation of essential oils. The highest accumulation of essential oils and the highest shoot biomass growth were obtained with 2.0 mg/L kinetin and 0.05 mg/L 2,4-dichlorophenoxyacetic acid.