Transcriptome sequencing of medical herb Salvia Rosmarinus (Rosemary) revealed the phenylpropanoid biosynthesis pathway genes and their phylogenetic relationships.

BACKGROUND: The Salvia rosmarinus spenn. (rosemary) is considered an economically important ornamental and medicinal plant and is widely utilized in culinary and for treating several diseases. However, the procedure behind synthesizing secondary metabolites-based bioactive compounds at the molecular level in S. rosmarinus is not explored completely. METHODS AND RESULTS: We performed transcriptomic sequencing of the pooled sample from leaf and stem tissues on the Illumina HiSeqTM X10 platform. The transcriptomics analysis led to the generation of 29,523,608 raw reads, followed by data pre-processing which generated 23,208,592 clean reads, and de novo assembly of S. rosmarinus obtained 166,849 unigenes. Among them, nearly 75.1% of unigenes i.e., 28,757 were interpreted against a non-redundant protein database. The gene ontology-based annotation classified them into 3 main categories and 55 sub-categories, and clusters of orthologous genes annotation categorized them into 23 functional categories. The Kyoto Encyclopedia of Genes and Genomes database-based pathway analysis confirmed the involvement of 13,402 unigenes in 183 biochemical pathways, among these unigenes, 1,186 are involved in the 17 secondary metabolite production pathways. Several key enzymes involved in producing aromatic amino acids and phenylpropanoids were identified from the transcriptome database. Among the identified 48 families of transcription factors from coding unigenes, bHLH, MYB, WRKYs, NAC, C2H2, C3H, and ERF are involved in flavonoids and other secondary metabolites biosynthesis. CONCLUSION: The phylogenetic analysis revealed the evolutionary relationship between the phenylpropanoid pathway genes of rosemary with other members of Lamiaceae. Our work reveals a new molecular mechanism behind the biosynthesis of phenylpropanoids and their regulation in rosemary plants.

Variation in photosynthetic capacity of Salvia przewalskii along elevational gradients on the eastern Qinghai-Tibetan Plateau, China.

Elevational variation in plant growing environment drives diversification of photosynthetic capacity, however, the mechanism behind this reaction is poorly understood. We measured leaf gas exchange, chlorophyll fluorescence, anatomical characteristics, and biochemical traits of Salvia przewalskii at elevations ranging from 2400 m to 3400 m above sea level (a.s.l) on the eastern Qinghai-Tibetan Plateau, China. We found that photosynthetic capacity showed an initial increase and then a decrease with rising elevation, and the best state observed at 2800 m a.s.l. Environmental factors indirectly regulated photosynthetic capacity by affecting stomatal conductance (gs), mesophyll conductance (gm), maximum velocity of carboxylation (Vc max), and maximum capacity for photosynthetic electron transport (Jmax). The average temperature (T) and total precipitation (P) during the growing season had the highest contribution to the variation of photosynthetic capacity of S. przewalskii in subalpine areas, which were 25% and 24%, respectively. Photosynthetic capacity was mainly affected by diffusional limitations (71%-89%), and mesophyll limitation (lm) played a leading role. The variation of gm was attributed to the effects of environmental factors on the volume fraction of intercellular air space (fias), the thickness of cell wall (Tcw), the surface of mesophyll cells and chloroplasts exposed to intercellular airspace (Sm, Sc), and plasma membrane intrinsic protein (PIPs, PIP1, PIP2), independent of carbonic anhydrase (CA). Optimization of leaf tissue structure and adaptive physiological responses enabled plants to efficiently cope with variable climate conditions of high-elevation areas, and the while maintaining high levels of carbon assimilation.

Improvement of Bioactive Polyphenol Accumulation in Callus of Salvia atropatana Bunge.

Callus cultures of the Iranian medicinal plant Salvia atropatana were initiated from three-week-old seedlings on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and various cytokinins. Although all tested hormonal variants of the medium and explant enabled callus induction, the most promising growth was noted for N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU)-induced calli. Three lines obtained on this medium (cotyledon line-CL, hypocotyl line-HL, and root line-RL) were preselected for further studies. Phenolic compounds in the callus tissues were identified using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry) and quantified with HPLC (high-performance liquid chromatography). All lines exhibited intensive growth and contained twelve phenolic acid derivatives, with rosmarinic acid predominating. The cotyledon-derived callus line displayed the highest growth index values and polyphenol content; this was exposed to different light-emitting diodes (LED) for improving biomass accumulation and secondary metabolite yield. Under LED treatments, all callus lines exhibited enhanced RA and total phenolic content compared to fluorescent light, with the highest levels observed for white (48.5-50.2 mg/g dry weight) and blue (51.4-53.9 mg/g dry weight) LEDs. The selected callus demonstrated strong antioxidant potential in vitro based on the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) tests. Our findings confirm that the S. atropatana callus system is suitable for enhanced rosmarinic acid production; the selected optimized culture provide high-quality plant-derived products.

Anti-Leishmania effect of icetexanes from Salvia procurrens.

BACKGROUND AND PURPOSE: Leishmaniasis is a globally prevalent vector-borne disease caused by parasites of the genus Leishmania. The available chemotherapeutic drugs present problems related to efficacy, emergence of parasite resistance, toxicity and high cost, justifying the search for new drugs. Several classes of compounds have demonstrated activity against Leishmania, including icetexane-type diterpenes, previously isolated from Salvia and other Lamiaceae genera. Thus, in this study, compounds of Salvia procurrens were investigated for their leishmanicidal and immunomodulatory activities. METHODS: The exudate of S. procurrens was obtained by rapidly dipping the aerial parts in dichloromethane. The compounds were isolated by column and centrifugal planar chromatography over silica gel. The effects on L. amazonensis growth, survival, membrane integrity, reactive oxygen species (ROS) generation, mitochondrial membrane potential and cytotoxicity of the compounds towards human erythrocytes, peripheral blood mononuclear cells and macrophages were evaluated. The effects on intracellular amastigote forms, nitric oxide (NO) and TNF-α production were also investigated. RESULTS: The exudate from the leaves afforded the novel icetexane 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2), fruticulin A (3) and demethylfruticulin A (4). The compounds (1-4) were tested against promastigotes of L. amazonensis and showed an effective inhibition of the parasite survival (IC50 = 4.08-16.26 µM). In addition, they also induced mitochondrial ROS production, plasma membrane permeability and mitochondrial dysfunction in treated parasites, and presented low cytotoxicity against macrophages. Furthermore, all diterpenes tested reduced the number of parasites inside macrophages, by mechanisms involving TNF-α, NO and ROS. CONCLUSION: The results suggest the potential of 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2),fruticulin A (3) and demethylfruticulin A (4) as candidates for use in further studies on the design of anti-leishmanial drugs.

Microencapsulation of Essential Oils Using Faba Bean Protein and Chia Seed Polysaccharides via Complex Coacervation Method.

The aim of this study was to develop microcapsules containing juniper or black pepper essential oils, using a combination of faba bean protein and chia seed polysaccharides (in ratios of 1:1, 1:2, 2:1). By synergizing these two polymers, our goal was to enhance the efficiency of essential oil microencapsulation, opening up various applications in the food industry. Additionally, we aimed to investigate the influence of different polymer mixing ratios on the properties of the resulting microcapsules and the course of the complex coacervation process. To dissolve the essential oils and limit their evaporation, soybean and rapeseed oils were used. The powders resulting from the freeze-drying of coacervates underwent testing to assess microencapsulation efficiency (65.64-87.85%), density, flowability, water content, solubility, and hygroscopicity. Additionally, FT-IR and DSC analyses were conducted. FT-IR analysis confirmed the interactions between the components of the microcapsules, and these interactions were reflected in their high thermal resistance, especially at a protein-to-polysaccharide ratio of 2:1 (177.2 °C). The water content in the obtained powders was low (3.72-7.65%), but it contributed to their hygroscopicity (40.40-76.98%).

Distinction of chia varieties in vivo and in vitro based on the flow cytometry and rosmarinic acid production.

Flow cytometry has made a significant contribution to the study of several complex fundamental mechanisms in plant cytogenetics, becoming a useful analytical tool to understand several mechanisms and processes underlying plant growth, development, and function. In this study, the genome size, DNA ploidy level, and A-T/G-C ratio were measured for the first time for two genotypes of chia, Salvia hispanica, an herbaceous plant commonly used in phytotherapy and nutrition. This study also evaluated, for the first time by flow cytometry, the capacity to produce organic acids of tissues stained with LysoTracker Deep Red after elicitation with either yeast extract or cadmium chloride. Rosmarinic acid content differed between the two chia varieties treated with different elicitor concentrations, compared with non-elicited plant material. Elicited tissues of both varieties contained a higher content of rosmarinic acid compared with non-elicited cultures, and cadmium chloride at 500 µM was much better than that at 1000 µM, which led to plant death. For both genotypes, a dose-response was observed with yeast extract, as the higher the concentration of elicitor used, the higher rosmarinic acid content, resulting also in better results and a higher content of rosmarinic acid compared with cadmium chloride. This study demonstrates that flow cytometry may be used as a taxonomy tool, to distinguish among very close genotypses of a given species and, for the first time in plants, that this approach can also be put to profit for a characterization of the cytoplasmic acid phase and the concomitant production of secondary metabolites of interest in vitro, with or without elicitation. KEY POINTS: • Genome size, ploidy level, A-T/G-C ratio, and cytoplasm acid phase of S. hispanica • Cytometry study of cytoplasm acid phase of LysoTracker Deep Red-stained plant cells • Yeast extract or cadmium chloride elicited rosmarinic acid production of chia tissues.

Consumption of a Byproduct of Chia Seed Oil Extraction by Cold Pressing Ameliorates Cardiovascular Risks Factors in an Experimental Model of Metabolically Unhealthy Normal Weight.

The byproduct of Salvia hispanica (chia) seed oil extraction by cold pressing, also known as expeller, possesses a high nutritional value. It is rich in proteins, fibers, minerals, and has a residual oil content of 7-11%, which is rich in omega 3 linolenic acid (ALA). However, this byproduct has been historically undervalued. Thus, the aim of current work was to study the effects of consuming of a rich in chia expeller diet on a rabbit model of metabolically unhealthy normal weight to validate their use as a functional food. Rabbits were fed different diets for a period of 6 weeks: a standard diet (CD), a high-fat diet (HFD), a rich in expeller CD (Exp-CD) and a rich in expeller HFD (Exp-HFD). The Exp-HFD attenuated the rise in basal glucose, TyG index, triglycerides, cholesterol and non-HDL cholesterol induced by the HFD. Both rich in expeller diets reduced mean arterial blood pressure (MAP) and increase liver and fat ALA levels compared to their respective controls. Furthermore, the angiotensin converting enzyme (ACE) activity was lower in the lungs of animals fed on rich in expeller diets compared to their respective controls. In vitro studies showed that ALA inhibited ACE activity. The evaluation of vascular reactivity revealed that rich in expeller diets improved angiotensin II affinity and reduced contractile response to noradrenaline. In conclusion, the consumption of rich in expeller diets showed beneficial effects in preventing cardiovascular risk factors such as insulin resistance, dyslipidemia and MAP. Therefore, its use as functional ingredient holds significant promise.

Effect of Salvia hispanica (Chia seeds) and Foeniculum vulgare (Fennel seeds) against weight-loss and lipid profile in obese human subjects.

Increased levels of bad cholesterol in the body result in increasing blood pressure and weight gain. The rate of mortality in people, especially who are obese, is increasing due to absence of organic sources of fiber in their diets. Chia and fennel seeds are rich sources of fiber. The objective of this study was to evaluate the combined effect of Salvia hispanica (Chia seeds) and Foeniculum vulgare (Fennel seeds) against weight-loss and lipid profile in obese human subjects. The research was conducted on obese people aged 25 to 40 years at the Jinnah Hospital Lahore. The study design was randomized control trial (RCT). The sample size was calculated and was divided in-to two groups. With the duration of study being 3 months, pre-testing of all the participants was done. Group 1 was control group, given placebo treatment and Group 2 was an intervention group and given chia and fennel seeds. Post-testing was done and data were analyzed. Results showed that chia and fennel seeds have significant effect (p <0.05) on BMI and lipid profile hence, both are beneficial for lowering body weight and improving LDL, HDL, serum triglycerides and total cholesterol levels.

Chemometric Analysis Evidencing the Variability in the Composition of Essential Oils in 10 Salvia Species from Different Taxonomic Sections or Phylogenetic Clades.

Essential oil (EO) of Salvia spp. has been widely used for culinary purposes and in perfumery and cosmetics, as well as having beneficial effects on human health. The present study aimed to investigate the quantitative and qualitative variations in EOs in wild-growing and cultivated pairs of samples from members in four Salvia sections or three clades, namely S. argentea L. (Sect. Aethiopis; Clade I-C), S. ringens Sm. (Sect. Eusphace; Clade I-D), S. verticillata L. (Sect. Hemisphace; Clade I-B), S. amplexicaulis Lam., and S. pratensis L. (Sect. Plethiosphace; Clade I-C). Furthermore, the natural variability in EO composition due to different genotypes adapted in different geographical and environmental conditions was examined by employing members of three Salvia sections or two phylogenetic clades, namely S. sclarea L. (six samples; Sect. Aethiopis or Clade I-C), S. ringens (three samples; Sect. Eusphace or Clade I-D), and S. amplexicaulis (five samples; Sect. Plethiosphace or Clade I-C). We also investigated the EO composition of four wild-growing species of two Salvia sections, i.e., S. aethiopis L., S. candidissima Vahl, and S. teddii of Sect. Aethiopis, as well as the cultivated material of S. virgata Jacq. (Sect. Plethiosphace), all belonging to Clade I-C. The EO composition of the Greek endemic S. teddii is presented herein only for the first time. Taken together, the findings of previous studies are summarized and critically discussed with the obtained results. Chemometric analysis (PCA, HCA, and clustered heat map) was used to identify the sample relationships based on their chemical classes, resulting in the classification of two distinct groups. These can be further explored in assistance of classical or modern taxonomic Salvia studies.

Investigating the simultaneous effect of chitosan and arbuscular mycorrhizal fungi on growth, phenolic compounds, PAL enzyme activity and lipid peroxidation in Salvia nemorosa L.

Considering the importance of Salvia nemorosa L. in the pharmaceutical and food industries, and also beneficial approaches of arbuscular mycorrhizal fungi (AMF) symbiosis and the use of bioelicitors such as chitosan to improve secondary metabolites, the aim of this study was to evaluate the performance of chitosan on the symbiosis of AMF and the effect of both on the biochemical and phytochemical performance of this plant and finally introduced the best treatment. Two factors were considered for the factorial experiment: AMF with four levels (non-inoculated plants, Funneliformis mosseae, Rhizophagus intraradices and the combination of both), and chitosan with six levels (0, 50, 100, 200, 400 mg L-1 and 1% acetic acid). Four months after treatments, the aerial part and root length, the levels of lipid peroxidation, H2O2, phenylalanine ammonia lyase (PAL) activity, total phenol and flavonoid contents and the main secondary metabolites (rosmarinic acid and quercetin) in the leaves and roots were determined. The flowering stage was observed in R. intraradices treatments and the highest percentage of colonization (78.87%) was observed in the treatment of F. mosseae × 400 mg L-1 chitosan. Furthermore, simultaneous application of chitosan and AMF were more effective than their separate application to induce phenolic compounds accumulation, PAL activity and reduce oxidative compounds. The cluster and principal component analysis based on the measured variables indicated that the treatments could be classified into three clusters. It seems that different treatments in different tissues have different effects. However, in an overview, it can be concluded that 400 mg L-1 chitosan and F. mosseae × R. intraradices showed better results in single and simultaneous applications. The results of this research can be considered in the optimization of this medicinal plant under normal conditions and experiments related to abiotic stresses in the future.

Antifungal activity of a trypsin inhibitor from Salvia hispanica L. (chia) seeds against fluconazole-resistant strains of Candida spp. and evaluation of its toxicity in vitro.

The incidence of Candida species resistant to traditional antifungal drugs is increasing globally. This issue significantly impacts patients' lives and increases healthcare expenses, confirming the need to develop novel therapeutic strategies. Recently, a thermostable trypsin inhibitor named ShTI (11.558 kDa), which has antibacterial effects on Staphylococcus aureus, was isolated from Salvia hispanica L. (chia) seeds. This study aimed to assess the antifungal effect of ShTI against Candida species and its synergism with fluconazole and to evaluate its mode of action. Preliminary toxicological studies on mouse fibroblasts were also performed. ShTI exhibited antifungal effects against C. parapsilosis (ATCC® 22,019), C. krusei (ATCC® 6258), and six clinical fluconazole-resistant strains of C. albicans (2), C. parapsilosis (2), and C. tropicalis (2). The minimum inhibitory concentration (MIC) values were 4.1 µM (inhibiting 50% of the isolates) and 8.2 µM (inhibiting 100% of the isolates). Additionally, when combined with fluconazole, ShTI had a synergistic effect on C. albicans, altering the morphological structure of the yeast. The mode of action of ShTI against C. krusei (ATCC® 6258) and C. albicans involves cell membrane permeabilization, the overproduction of reactive oxygen species, the formation of pseudohyphae, pore formation, and consequently, cell death. In addition, ShTI (8.65 and 17.3 µM) had noncytotoxic and nongenotoxic effects on L929 mouse fibroblasts. These findings suggest that ShTI could be a promising antimicrobial candidate, but further research is necessary to advance its application as a novel antifungal agent.

Metabolomic Profiling and DNA-Fingerprinting of Newly Recorded White-Flowered Populations of Salvia lanigera Poir. in Egypt.

Salvia lanigera Poir. is a small herbaceous perennial species with violet flowers that grows in low-altitude deserts, and sandy loam. During the collection of S. lanigera, unusual populations with white flowers were found. Therefore, the two populations (violet- and white-flowered) were subjected to comparative investigations, including DNA fingerprinting, chemical composition, and biological evaluation. The two populations showed DNA variations, with 6.66 % polymorphism in ISSR and 25 % in SCoT markers. GC/MS and UHPLC/HRMS of aqueous methanol extracts, led to the tentative identification of 43 and 50 compounds in both populations. In addition, the structures of nine compounds, including four first-time reported compounds in the species, were confirmed by NMR. Furthermore, the total extracts exhibited weak radical scavenging activity against DPPH and a lower inhibitory effect towards acetylcholinesterase. In conclusion, the obtained data suggested that the white-colored flower could be an additional important character record for the Egyptian S. lanigera.

Nutritional profiling, fiber content and in vitro bioactivities of wheat-based biscuits formulated with novel ingredients.

This study evaluated the nutritional profile and fiber content of innovative formulations of wheat-based biscuits enriched with chia seeds, carob flour and coconut sugar. The in vitro antioxidant, cytotoxic, anti-inflammatory and antimicrobial activities were also investigated to understand the potential health advantages of the incorporation of these new ingredients. The novel biscuits demonstrated significant improvements in protein and mineral content, with increases of 50% and 100% in chia biscuits, and up to 20% and 40% in carob biscuits, respectively. Fiber also notably increased, particularly in samples containing 10% carob flour, which increased four times as compared to wheat-based samples. The new ingredients exhibited antibacterial and antifungal activity, particularly against Yersinia enterocolitica (minimum inhibitory concentration 1.25 mg mL-1 in coconut sugar) and Aspergillus fumigatus (minimum inhibitory concentration/minimum fungicidal concentrations 2.5/5 mg mL-1 in chia seeds). However, the final biscuits only displayed antifungal properties. Carob flour and chia seeds had a remarkably high capacity to inhibit the formation of TBARS and promoted greater antioxidant activity in biscuit formulations, with EC50 values decreasing from 23.25 mg mL-1 (control) to 4.54 mg mL-1 (15% defatted ground chia seeds) and 1.19 mg mL-1 (10% carob flour). Only chia seeds exhibited cellular antioxidant, anti-inflammatory and cytotoxic activity, attributes that were lost when seeds were added into the biscuits. These findings highlight the potential health benefits of these ingredients, particularly when incorporated in new wheat-based formulations.

Structure, Absolute Configuration, Antiproliferative and Phytotoxic Activities of Icetexane and Abietane Diterpenoids from Salvia carranzae and Chemotaxonomic Implications.

From the aerial parts of Salvia carranzae Zamudio and Bedolla, three new icetexane-type diterpenoids were isolated. Their structures were established through spectroscopic methods and named the following: salvicarranzanolide (1), 19-deoxo-salvicarranzanolide (2) and 19-deoxo-20-deoxy-salvicarranzanolide (3). In addition, the known icetexane-type diterpenoids, 6,7,11,14-tetrahydro-7-oxo-icetexone (4), iso-icetexone (5), 19-deoxo-iso-icetexone (6), icetexone (7), 19-deoxo-icetexone (8) and 7α-acetoxy-6,7-dihydroicetexone (9), were also isolated, along with the abietanes sessein (10) and ferruginol (11). α-Tocopherol was also identified. Compounds 5, 6 and 8 were tested for their antiproliferative activity using the sulforhodamine B assay on six cancer and one normal human cell lines. Diterpenoids 5 and 6 showed noteworthy antiproliferative activity, exhibiting an IC50 (µM) = 0.43 ± 0.01 and 1.34 ± 0.04, respectively, for U251 (glioblastoma), an IC50 (µM) = 0.45 ± 0.01 and 1.29 ± 0.06 for K5621 (myelogenous leukemia), 0.84 ± 0.07 and 1.03 ± 0.10 for HCT-15 (colon cancer), and 0.73 ± 0.06 and 0.95 ± 0.09 for SKLU-1 (lung adenocarcinoma) cell lines. On the other hand, the phytotoxicity of compounds 5-7 and 9-10 was evaluated on seed germination and root growth in some weeds such as Medicago sativa, Panicum miliaceum, Amaranthus hypochondriacus and Trifolium pratense as models. While compounds 5 and 10 exhibited a moderate inhibitory effect on the root growth of A. hypochondriacus and T. pratense at 100 ppm, the diterpenoids 6, 7 and 9 were ineffective in all the plant models. Taxonomic positions based on the chemical profiles found are also discussed.

Two Undescribed Germacrane-Type Sesquiterpenoids from Salvia cavaleriei var. simplicifolia Stib. and Their Anti-Alzheimer's Disease Activity.

Two undescribed germacrane-type sesquiterpenoids, salcasins A (1) and B (2), together with three known compounds (3-5) were isolated and identified from the whole plant of Salvia cavaleriei var. simplicifolia Stib. The structures of the undescribed compounds were elucidated on the basis of spectroscopic methods, such as HR-ESI-MS, 1D and 2D NMR data. The relative configurations of 1 and 2 were established by analyzing their NOESY spectra as well as by 13C NMR calculations with DP4+ probability analyses. The absolute configurations of 1 and 2 were determined by comparing experimental and calculated ECD spectra. Furthermore, the in vivo anti-Alzheimer's disease activities of 1-5 were evaluated using Caenorhabditis elegans AD pathological model. Among all isolated compounds, salcasin A (1) significantly delayed AD-like symptoms of worm paralysis, which may be a potential anti-AD candidate agent.

Lactobacillus bulgaricus-loaded and chia mucilage-rich gum arabic/pullulan nanofiber film: An effective antibacterial film for the preservation of fresh beef.

In recent years, the development of probiotic film by incorporating probiotics into edible polymers has attracted significant research attention in the field of active packaging. However, the influence of the external environment substantially reduces the vitality of probiotics, limiting their application. Therefore, to improve the probiotic activity, this study devised a novel nanofiber film incorporating chia mucilage protection solution (CPS), gum arabic (GA), pullulan (PUL), and Lactobacillus bulgaricus (LB). SEM images indicated the successful preparation of the nanofiber film incorporating LB. CPS incorporation significantly improved the survival ability of LB, with a live cell count reaching 7.62 log CFU/g after 28 days of storage at 4 °C - an increase of 1 log CFU/g compared to the fiber film without CPS. The results showed that the fiber film containing LB inhibited Escherichia coli and Staphylococcus aureus. Finally, the novel probiotic nanofiber film was applied to beef. The results showed that the shelf life of the beef during the experiments was extended for 2 days at 4 °C. Therefore, the novel probiotic film containing LB was suitable for meat preservation.

Comparative transcriptomics of two Salvia subg. Perovskia species contribute towards molecular background of abietane-type diterpenoid biosynthesis.

Tanshinones, are a group of diterpenoid red pigments present in Danshen - an important herbal drug of Traditional Chinese Medicine which is a dried root of Salvia miltiorrhiza Bunge. Some of the tanshinones are sought after as pharmacologically active natural products. To date, the biosynthetic pathway of tanshinones has been only partially elucidated. These compounds are also present in some of the other Salvia species, i.a. from subgenus Perovskia, such as S. abrotanoides (Kar.) Sytsma and S. yangii B.T. Drew. Despite of the close genetic relationship between these species, significant qualitative differences in their diterpenoid profile have been discovered. In this work, we have used the Liquid Chromatography-Mass Spectrometry analysis to follow the content of diterpenoids during the vegetation season, which confirmed our previous observations of a diverse diterpenoid profile. As metabolic differences are reflected in different transcript profile of a species or tissues, we used metabolomics-guided transcriptomic approach to select candidate genes, which expression possibly led to observed chemical differences. Using an RNA-sequencing technology we have sequenced and de novo assembled transcriptomes of leaves and roots of S. abrotanoides and S. yangii. As a result, 134,443 transcripts were annotated by UniProt and 56,693 of them were assigned as Viridiplantae. In order to seek for differences, the differential expression analysis was performed, which revealed that 463, 362, 922 and 835 genes indicated changes in expression in four comparisons. GO enrichment analysis and KEGG functional analysis of selected DEGs were performed. The homology and expression of two gene families, associated with downstream steps of tanshinone and carnosic acid biosynthesis were studied, namely: cytochromes P-450 and 2-oxoglutarate-dependend dioxygenases. Additionally, BLAST analysis revealed existence of 39 different transcripts related to abietane diterpenoid biosynthesis in transcriptomes of S. abrotanoides and S. yangii. We have used quantitative real-time RT-PCR analysis of selected candidate genes, to follow their expression levels over the vegetative season. A hypothesis of an existence of a multifunctional CYP76AH89 in transcriptomes of S. abrotanoides and S. yangii is discussed and potential roles of other CYP450 homologs are speculated. By using the comparative transcriptomic approach, we have generated a dataset of candidate genes which provides a valuable resource for further elucidation of tanshinone biosynthesis. In a long run, our investigation may lead to optimization of diterpenoid profile in S. abrotanoides and S. yangii, which may become an alternative source of tanshinones for further research on their bioactivity and pharmacological therapy.

Electroencephalographic profile of Salvia amarissima Ortega and amarisolide A in the absence and presence of PTZ-induced seizures in mice.

Salvia amarissima Ortega is a plant used in traditional medicine to treat CNS's affections. Despite its depressant properties in anxiety and fibromyalgia, there is no scientific evidence about its capability to control seizure activity. This study aimed to investigate the effects of the S. amarissima aqueous extract (SAAE) and its metabolite amarisolide A (AMA) on the electrocorticographic (ECoG) activity. The ECoG profiles were previously and concurrently analyzed to the pentylenetetrazole (85 mg/kg, i.p.)-induced seizure behavior after thirty min of the administration of several doses of the SAAE (1, 10, 30, and 100 mg/kg, i.p.) and two doses of AMA (0.5 and 1 mg/kg, i.p.). A dosage of AMA (1 mg/kg,i.p.) was selected to explore a possible mechanism of action by using antagonists of inhibitory receptors such as GABAA (picrotoxin, 1 mg/kg, i.p.) or 5-HT1A of serotonin (WAY100635, 1 mg/kg, i.p.). Significant changes in the frequency bands and the spectral power were observed after the treatment alone. Additionally, SAAE and AMA produced significant and dose-dependent anticonvulsant effects by reducing the incidence and severity of seizures and increasing latency or survival. Both antagonists prevented the effects of AMA in the severity score of seizures and survival during the tonic-clonic seizures. In conclusion, our preclinical data support that S. amarissima possesses anticonvulsant properties, in part due to the presence of amarisolide A, mediated by different inhibitory mechanisms of action. Our scientific evidence suggests that this Salvia species and amarisolide A are potential neuroprotective alternatives to control seizures in epilepsy therapy.

Lack of pollinators selects for increased selfing, restricted gene flow and resource allocation in the rare Mediterranean sage Salvia brachyodon.

Range contraction and habitat fragmentation can cause biodiversity loss by creating conditions that directly or indirectly affect the survival of plant populations. Fragmented habitats can alter pollinator guilds and impact their behavior, which may result in pollen/pollinator limitation and selection for increased selfing as a mechanism for reproductive assurance. We used Salvia brachyodon, a narrowly distributed and endangered sage from eastern Adriatic, to test the consequences of range contraction and habitat fragmentation. Molecular data indicate a severe and relatively recent species range reduction. While one population is reproductively almost completely isolated, moderate gene flow has been detected between the remaining two populations. The high pollen-to-ovule ratio and the results of controlled hand pollination indicate that S. brachyodon has a mixed mating system. Quantitative and qualitative differences in the community and behaviour of flower visitors resulted in limited pollination services in one population where no effective pollinator other than pollen and nectar robbers were observed. In this population, self-pollination predominated over cross-pollination. Various environmental factors, in which plant-pollinator interactions play a pivotal role, have likely created selection pressures that have led to genetic and phenotypic differentiation and different resource allocation strategies among populations.