Exploring the phytochemical variation of non-volatile metabolites within three South African Salvia species using UPLC-MS fingerprinting and chemometric analysis.

The South African Salvia species, Salvia africana-lutea, S. lanceolata and S. chamelaeagnea, are widely used to treat fever and inflammation associated with skin and lung infections. The aim of this study was to explore the non-volatile secondary metabolites and the phytochemical variation within these lesser known species, to support product development and commercialisation. Chemical profiles of the methanol extracts of 81 wild-harvested samples were obtained using ultra performance-quadrupole-Time-of-Flight-mass spectrometry (UPLC-qToF-MS). Forty-one compounds, including caffeic acid, rosmarinic acid, carnosol, carnosic acid and ursolic acid, were detected and confirmed across the three species. Nineteen compounds were tentatively identified of which 14 have not been reported in these species. Principal component analysis revealed distinct clusters corresponding to the three species, confirming chemical differences. Marker compounds for each species were revealed using orthogonal projection to latent structures-discriminant analysis. Further chemometric analysis reflected a degree of intraspecies variation, although the chemistry within populations was mostly conserved. Potential chemotypes for each species were identified through unique compounds associated with each group. The concentrations of medicinally important metabolites, namely, rosmarinic acid, carnosol, carnosic acid and ursolic acid, were determined, using validated UPLC-PDA methods. Ursolic acid was present at levels up to 38.2 mg/g, confirming that these species are a rich source of this compound. No similar studies combining liquid chromatography with chemometric analysis, and utilising a large sample size from various habitats, have been reported for these three Salvia species. The results will guide selection of cultivars with the best attributes for the intended therapeutic application, thereby protecting wild populations from over-exploitation.

Aroma Profiles Analysis of Two Populations of Salvia palaestina Benth. Collected from Mediterranean and Irano-Turanian Zones in Jordan.

The volatile principles emitted from different aerial organs of two S. palaestina Benth. populations (Mediterranean (Med) and Irano-Turanian (IrT)) growing wild in Jordan were extracted by Solid Phase Micro-Extraction (SPME) and analysed by GC/MS technique. Sesquiterpene hydrocarbons dominated stems (59.38%, 49.67%) and leaves (93.28%, 32.39%) emissions from Med and IrT zones, respectively while monoterpene hydrocarbons had the major contribution to the aroma of pre-flowering buds (78.62%, 74.96%), opened flowers (76.12%, 59.99%) and petals (69.57%, 54.28%) and were mostly represented by sabinene (in Med zone) and ociemene isomers (Z & E) in IrT zone. Multivariate analysis classified the two populations into two different clusters based on their origin and indicated the occurrence of two ecotypes of this species. Different organs from the same collection site showed emission profiles of similar chemical composition.

Characterization of Chemical Components in Organs Emissions of Two Salvia dominica L. Populations from Jordan.

The current investigation reports the chemical composition of volatile organic compounds emitted from the different aerial organs of two populations of Salvia dominica L. from Jordan collected from Mediterranean and Irano-Turanian bio-geographical zones. Oxygenated monoterpenes dominated the emission profiles of most organs from the two populations but with variable qualitative and quantitative differences. Mediterranean samples contained higher content of oxygenated monoterpenes (stems: 88.37%, leaves 89.95%, pre-flowering buds 67.14%, fully opened flowers 79.43%, sepals 90.93% and petals 92.25%) as compared to those from Irano-Turanian origin (range 39.85% to 75.06%). trans-Sabinene hydrate dominated the emission profiles of all organs from Irano-Turanian zone (range 38.54% to 73.24%) in addition to the stem, sepal and petal samples from the Mediterranean zone (51.37% to 86.98%). The other organs from the Mediterranean zone were dominated by α-terpenyl acetate (27.66-54.87%). Cluster and Principle Component statistical analysis classified the two populations into two clusters based on their origin. The current study evidenced the different VOCs composition in the two populations, that was mainly related to climatic and environmental conditions and suggested the presence of two ecotypes of S. dominica L.in Jordan.

Anticancer Drug Targets of Salvia Phytometabolites: Chemistry, Biology and Omics.

BACKGROUND: Salvia displays diverse anticancer properties, which are attributable to their diterpene and phenolic contents. There is no comprehensive review on the anticancer diversity and molecular targets of Salvia components. OBJECTIVE: We investigate the diversity and molecular targets of Salvia phytometabolites responsible for the prevention and treatment of cancer and sarcoma. RESULTS AND CONCLUSION: Traditional therapeutic knowledge suggests that Salvia species can be used to develop anticancer drugs. Lots of concerns have been raised for tanshinone (Tan) IIA and cryptotanshinone. Some Salvia compounds disturb cell cycle and induce apoptosis of tumor cells or enhance immune activities, while others inhibit the proliferation, invasion, angiogenesis, and metastasis, or reverse the multi-drug resistance of cancer cells. Salvia phytometabolites regulate most cancer hallmarks defined by Hanahan and Weinberg. The same class of phytometabolite could exert the anticancer activity via multiple pathways. ADME/T properties and pharmacokinetic bebaviors of some phytometabolites have been revealed. Fluorescent probes are powerful tools for screening substrates, inhibitors or inducers of drug metabolizing enzymes/transporters from Salvia phytometabolites. Omics platform will greatly help mining more potentially useful phytometabolites from Salvia plants. More Salvia plants have application potential in pharmaceutical industry and clinical cancer therapy.

Identification of Salvia species using high-performance liquid chromatography combined with chemical pattern recognition analysis.

Salvia miltiorrhiza, also known as Danshen, is a widely used traditional Chinese medicine for the treatment of cardiovascular diseases and hematological abnormalities. The root and rhizome of Salvia przewalskii and Salvia yunnanensis have been found as substitutes for Salvia miltiorrhiza in the market. In this study, the chemical information of 14 major compounds in Salvia miltiorrhiza and its substitutes were determined using a high-performance liquid chromatography method. Stepwise discriminant analysis was adopted to select the characteristic variables. Partial least squares discriminant and hierarchical cluster analysis were performed to classify Salvia miltiorrhiza and its substitutes. The results showed that all of the samples were correctly classified both in partial least squares discriminant analysis and hierarchical cluster analysis based on the four compounds (caffeic acid, rosmarinic acid, salvianolic acid B, and salvianolic acid A). This method can not only distinguish Salvia miltiorrhiza and its substitutes, but also classify Salvia przewalskii and Salvia yunnanensis. The method can be applied for the quality assessment of Salvia miltiorrhiza and identification of unknown samples.

[Advances on chemical constituents of Tibetan Salvia genus].

Salvia is the largest genus of Labiatae family, and there are more than 1 000 species around the world. Our country is rich in the resources of Salvia plants. The plants of this genus contain multiple chemical components, including sesquiterpenoids, diterpenoids, triterpenoids and phenols, et al. In order to develop better Tibetan plants of Salvia genus, this article reviewed and summarized the constituents from Tibetan Salvia genus.

Time to split Salvia s.l. (Lamiaceae) - New insights from Old World Salvia phylogeny.

AIMS: Salvia L. is widely known as the largest genus in the mint family. A morphological modification of the androecium (lever-like stamens) was used to support this genus. However, molecular data revealed that Salvia is polyphyletic. Since phylogenetic studies largely underrepresented Old World Salvia species, we filled this gap and combined new data with existing sequences. The aim of our study was the identification of well-supported clades that provide the basis for evolutionary and taxonomic conclusions. METHODS: We included ITS data (internal transcribed spacer) from 220 Salvia species, 86 of which were sequenced for the first time. Additionally, the highly variable plastid marker rpl32-trnL was sequenced, providing new data for 100 Salvia species. These sequences were combined with the accessions available from GenBank. Old World Salvia is represented herein with 57% of its species. The two datasets were analyzed separately using BI and ML approaches. RESULTS: Our data confirm that Salvia is polyphyletic with four distinct evolutionary lineages (Clade I-IV), including five additional genera. The clades strongly reflect the geographical distribution, i.e., Clade IV (East Asia), Clade III (Southwest Asia to Northern Africa), and Clade II (America). The origin of Salvia s.s. (Clade I) is most likely Southwest Asia. A high degree of parallel character evolution was identified in most of the Old World sections. Based on our results, we reconstructed the evolution and biogeography of Salvia s.l. and propose to split this large group into six genera, each supported by geographical distribution, morphology, and karyology. CONCLUSION: Salvia s.l. is a polyphyletic group that was originally regarded as a genus because its species share a derived stamen structure. However, phylogenetic data clearly indicate that this floral trait and other morphological characters evolved in parallel. Our study illustrates that the combination of different data sets allows a comprehensive reconstruction of taxa and characteristic evolution, both of which are a precondition for future revision.

Rapid Species-level Identification of Salvias by Chemometric Processing of Ambient Ionisation Mass Spectrometry-derived Chemical Profiles.

INTRODUCTION: The Salvia genus contains numerous economically important plants that have horticultural, culinary and nutraceutical uses. They are often similar in appearance, making species determination difficult. Species identification of dried Salvia products is also challenging since distinguishing plant morphological features are no longer present. OBJECTIVE: The development of a simple high-throughput method of analysis of fresh and dried Salvia leaves that would permit rapid species-level identification and detection of diagnostic biomarkers. METHODOLOGY: Plant leaves were analysed in their native form by DART-MS without the need for any sample preparation steps. This furnished chemical fingerprints characteristic of each species. In the same experiment, in-source collision-induced dissociation was used to identify biomarkers. Biomarker presence was also independently confirmed by GC-MS. Chemometric processing of DART-MS profiles was performed by kernel discriminant analysis (KDA) and soft independent modelling of class analogy (SIMCA) to classify the fingerprints according to species. RESULTS: The approach was successful despite the occurrence of diurnal cycle and plant-age related chemical profile variations within species. In a single rapid experiment, the presence of essential oil biomarkers such as 3-carene, α-pinene, ß-pinene, ß-thujone, ß-caryophyllene, camphor and borneol could be confirmed. The method was applied to rapid identification and differentiation of Salvia apiana, S. dominica, S. elegans, S. officinalis, S. farinacea and S. patens. CONCLUSION: Species-level identification of Salvia plant material could be accomplished by chemometric processing of DART-HRMS-derived chemical profiles of both fresh and dried Salvia material. Copyright © 2016 John Wiley & Sons, Ltd.

Salvia divinorum: toxicological aspects and analysis in human biological specimens.

The identification and quantitation of the main psychoactive component of Salvia divinorum (salvinorin A) in biological specimens are crucial in forensic and clinical toxicology. Despite all the efforts made, its uncontrolled abuse has increased quickly, exposing its users' health to serious risks both in the short and long term. The use of alternative biological matrices in toxicological analyzes can be advantageous as complementary postmortem samples, or in situations when neither blood nor urine can be collected; they may be useful tools in those determinations, providing important information about prior exposure. The aim of this article is to present a brief summary of legal aspects of Salvia divinorum and salvinorin A, including the methods used for the determination of the latter in biological matrices.

Fatty Acid Patterns of Seeds of Some Salvia Species from Iran--A Chemotaxonomic Approach.

In this study, the seed oil content and fatty acid (FA) profile of 21 populations from 16 wild Salvia species of Iran were analyzed by GC. Patterns of chemical variations of the oils among species were identified via numerical analyses and also the taxonomic status of the infrageneric grouping was outlined in the genus. Salvia species were scored based on the contents of main FAs using principal coordinate analysis (PCO). The results showed that the total oil content in the seeds varied significantly, and ranged from 6.68 to 38.53% dry weight. α-Linolenic (18:3ω3, 1.69 - 53.56%), linoleic (18:2ω6, 13.04 - 60.64%), oleic (18:1ω9, 6.15 - 27.06%), palmitic (16:0, 3.77 - 9.27%), and stearic (18:0, 1.78 - 3.05%) acid were identified as five major FAs in the oils. The amount of ω-3 and ω-6 FAs ranged between 1.90 - 53.80% and 13.46 - 60.83% of total FAs in the seed oils, respectively. The results confirmed that FA profiles were distinctive among the species and that they can be used as chemotaxonomic markers. The discrimination of Salvia species according to their botanical classification at intersectional level was supported. In general, seed oils of Salvia species were rich sources of polyunsaturated FAs, except in linoleic and α-linolenic acid, and may be valuable for food and pharmaceutical industries.

Determination of volatile organic compounds in the dried leaves of Salvia species by solid-phase microextraction coupled to gas chromatography mass spectrometry.

Salvia spp. are used throughout the world both for food and pharmaceutical purposes. In this study, a method involving headspace solid-phase microextraction combined with gas chromatography-mass spectrometry was developed, to establish the volatiles profile of dried leaves of four Iranian Salvia spp.: Salvia officinalis L., Salvia leriifolia Benth, Salvia macrosiphon Boiss. and two ecotypes of Salvia reuterana Boiss. A total of 95 volatiles were identified from the dried leaves of the five selected samples. Specifically, α-thujone was the main component of S. officinalis L. and S. macrosiphon Boiss. (34.40 and 17.84%, respectively) dried leaves, S. leriifolia Benth was dominated by ß-pinene (27.03%), whereas α-terpinene was the major constituent of the two ecotypes of S. reuterana Boiss. (21.67 and 13.84%, respectively). These results suggested that the proposed method can be considered as a reliable technique for isolating volatiles from aromatic plants, and for plant differentiation based on the volatile metabolomic profile.

Optimization of Micellar Electrokinetic Chromatography Method for the Simultaneous Determination of Seven Hydrophilic and Four Lipophilic Bioactive Components in Three Salvia Species.

A micellar electrokinetic chromatography (MEKC) method was developed for the simultaneous determination of seven hydrophilic phenolic acids and four lipophilic tanshinones in three Salvia species. In normal MEKC mode using SDS as surfactant, the investigated 11 compounds could not be well separated. Therefore, several buffer modifiers including ß-cyclodextrins (ß-CD), ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4) and organic solvents have been added to the buffer solution to improve the separation selectivity. Under the optimized conditions (BGE, 15 mM sodium tetraborate with 10 mM SDS, 5 mM ß-CD, 10 mM [bmim]BF4 and 15% ACN (v/v) as additives; buffer pH, 9.8; voltage, 20 kV; temperature, 25 °C), the 11 investigated analytes could achieve baseline separation in 34 min. The proposed MEKC was additionally validated by evaluating the linearity (R(2) ≥ 0.9965), LODs (0.27-1.39 µg·mL(-1)), and recovery (94.26%-105.17%), demonstrating this method was reproducible, accurate and reliable. Moreover, the contents of the 11 compounds in three Salvia species, including S. miltiorrhiza, S. przewalskii and S. castanea were analyzed. The result showed that the established MEKC method was simple and practical for the simultaneous determination of the hydrophilic and lipophilic bioactive components in Salvia species, which could be used to effectively evaluate the quality of these valued medicinal plants.

The inhibition the Tet(K) efflux pump of tetracycline resistant Staphylococcus epidermidis by essential oils from three Salvia species.

UNLABELLED: The inhibition of efflux pumps is an attractive and powerful response to the emergence of bacteria resistant to antibiotics. Essential oils (EOs) from Salvia fruticosa, Salvia officinalis and Salvia sclarea reduce the minimal inhibition concentration of tetracycline, decrease efflux of antibiotic and decrease the expression of tet(K) gene in tetracycline resistant clinical isolates of Staphylococcus epidermidis. In all the cases S. fruticosa was the best one. By using checkerboard and time-killing methods, we found synergistic interactions of EOs with tetracycline. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data from molecular and functional analyses of inhibitory effect of Salvia's essential oils, namely from S. fruticosa, on Tet(K) pump of Staphylococcus epidermidis and from modulatory studies may be the starting point for consecutive study of pharmacokinetic and pharmacodynamic parameters and their perspective use in combination therapy. Combination of antibiotic with efflux pump inhibitor would be expected to re-establish susceptibility of the bacteria to antibiotics that became no longer effective due to bacterial resistance through the efflux pumps. The inhibition of an efflux pump can potentially improve the clinical efficacy of an antibiotic and simultaneously decrease the selection of resistant mutants.

[Anatomical characteristics of laminae and petioles of 11 species of Salvia and their taxonomic significance].

Anatomical features of the lamina and petiole of 11 species of Salvia had been compared. The microstructures of 11 species of Salvia were observed by paraffin method, and indexes such as the epidermis, xylems and phloems were measured. The data set was analyzed by principal components analysis method and cluster analysis method. The results indicated that the laminae of 11 species of Salvia were bifacial leaf, and the surface of laminae and petioles were covered with villous. The upper epidermis and lower epidermis of the laminae of 11 species of Salvia were constituted by a dense cuticular layer without stratum corneum. Differences existed in the structure of cross sectio mn of laminae and petioles of 11 species of Salvia. Among them, upper epidermal thickness of nine different micromorphological characters could serve as one of the criteria to identify species of taxonomic Salvia. The identification of genetic relationship of these characters in Salvia were discussed in the paper to provide an evidence of anatomy in Salvia.

Vegetative anatomy and micromorphology of Salvia divinorum (Lamiaceae) from Mexico, combined with chromatographic analysis of salvinorin A.

Salvia divinorum--a species traditionally cultivated in Oaxaca, Mexico--possesses hallucinogenic properties. It is legally recognized as a controlled substance and prohibited in many countries. The proper identification of the plant, both in fresh and dried forms, is an important issue in crime-prevention campaigns. This paper provides a thorough anatomical description of leaves, petioles, and stems of S. divinorum. Detailed investigation of foliar trichomes was performed and illustrated. In addition, chromatographic analyses, including TLC and HPLC, were applied to fresh and dried plant material, together with the standard reference salvinorin A. A comprehensive identification method for S. divinorum based on a thorough anatomical examination is proposed, combined with chemical analysis for proper plant recognition.

[Identification of Salvia shandongensis new species based on sequences of the plastid psbA-trnH intergenic region].

To identify Salvia shandongensis and its relatives at molecular level, the psbA-trnH intergenic region of three species including Salvia shandongensis, Salvia miltiorrhiza and S. miltiorrhiza f. alba were amplified and sequenced. Sequences were assembled with CodonCode Aligner. The K2P genetic distances between Salvia shandongensis and its relatives were calculated and UPGMA tree was performed by MEGA5.0. The results indicated that the lengths of psbA-trnH regions of Salvia shandongensis were about 391 bp, while the lengths of psbA-trnH regions of Salvia miltiorrhiza and S. miltiorrhiza f. alba were about 386 bp. The psbA-trnH sequences showed considerable variations between species and thus were revealed as a promising candidate for barcoding of Salvia shandongensis and its relatives. The intra-specific genetic distances of Salvia shandongensis were 0, while the intra-specific genetic distances of Salvia miltiorrhiza and S. miltiorrhiza f. alba were 0.002 and 0.001 respectively. Additionally, the genetic distance of Salvia shandongensis and Salvia miltiorrhiza ranged from 0.034 to 0.04, and the genetic distance of Salvia shandongensis and S. miltiorrhiza f. alba ranged from 0.005 to 0.008, the intra-specific genetic distances of Salvia shandongensis were much smaller than that of Salvia miltiorrhiza and S. miltiorrhiza f. alba; clustering results showed that there were obvious differences between Salvia shandongensis, Salvia miltiorrhiza and S. miltiorrhiza f. alba, which was consistent with morphological characteristics. This study not only firstly provides the scientific basis for establishing the taxonomy position in molecular level and revealing their genetic relationships of S. shandongensis, S. miltiorrhiza and S. miltiorrhiza f. alba; but also provides DNA molecular identification scientific basis for the development of new medicinal plant resources of Salvia shandongensis. Our results suggest that the psbA-trnH intergenic spacer region can be used as a barcoding to identify Salvia shandongensis, Salvia miltiorrhiza and S. miltiorrhiza f. alba.

A gDNA microarray for genotyping salvia species.

Salvia is an important genus from the Lamiaceae with approximately 1,000 species. This genus is distributed globally and cultivated for ornamental, culinary, and medicinal uses. We report the construction of the first fingerprinting array for Salvia species enriched with polymorphic and divergent DNA sequences and demonstrate the potential of this array for fingerprinting several economically important members of this genus. In order to generate the Salvia subtracted diversity array (SDA) a suppression subtractive hybridization (SSH) was performed between a pool of Salvia species and a pool of angiosperms and non-angiosperms to selectively isolate Salvia-specific sequences. A total of 285-subtracted genomic DNA (gDNA) fragments were amplified and arrayed. DNA fingerprints were obtained for fifteen Salvia genotypes including three that were not part of the original subtraction pool. Hierarchical cluster analysis indicated that the Salvia-specific SDA was capable of differentiating S. officinalis and S. miltiorrhiza from their closely related species and was also able to reveal genetic relationships consistent with geographical origins. In addition, this approach was capable of isolating highly polymorphic sequences from chloroplast and nuclear DNA without preliminary sequence information. Therefore, SDA is a powerful technique for fingerprinting non-model plants and for identifying new polymorphic loci that may be developed as potential molecular markers.

Phylogeny of New World Salvia subgenus Calosphace (Lamiaceae) based on cpDNA (psbA-trnH) and nrDNA (ITS) sequence data.

Salvia subgenus Calosphace (Lamiaceae) is economically and ethnomedicinally significant and comprised of more than 500 species. Although strongly supported as monophyletic, it has received no comprehensive systematic research since the initial establishment of 91 taxonomic sections in 1939. Representative taxa of 73 sections of Calosphace were sampled to investigate the phylogenetic relationships and identify major lineages using chloroplast (intergenic spacer psbA-trnH) and nuclear ribosomal DNA (internal transcribed spacer). Phylogenetic analysis of the combined data sets established monophyly of seven sections (Blakea, Corrugatae, Erythrostachys, Hastatae, Incarnatae, Microsphace, and Sigmoideae) and four major lineages (S. axillaris, "Hastatae clade", "Uliginosae clade", and "core Calosphace"). Sections spanning two or more centers of diversity are not supported by our results; rather, supported relationships exhibit significant geographic structure. Mexico is supported as the geographic origin of Calosphace, and no more than seven dispersal events to South America are required to account for current disjunct distributions.

Chemical classification of the essential oils of the Iranian Salvia species in comparison with their botanical taxonomy.

The essential oils of eight Salvia species collected from different localities in Iran were analyzed by gas chromatography/mass spectrometry (GC/MS). The analytical results were compared with those previously published for related Iranian sage species in order to identify chemical markers for these species. Salvia eremophila, S. hypoleuca, and S. reuteriana are endemic, while S. atropatana, S. chloroleuca, S. santolinifolia, S. aegyptiaca, and S. macrosiphon also grow wild in neighboring countries. We categorized the Iranian Salvia species into four main chemotypes according to their essential-oil constituents: those which are dominated by 1) monoterpenes, 2) mono- and sesquiterpenes, or 3) sesquiterpenes as the major constituents, and 4) those containing low-molecular-weight acids, aldehydes, and esters, and green-leaf volatiles (GLVs). Likely due to the chemical diversity of different Salvia chemotypes, this categorization was supported by principal component analysis (PCA) for the group sampled here, but not for the values reported in the literature. We identified the following chemical markers: α-pinene, ß-pinene, 1,8-cineol, linalool, and borneol in monoterpene-rich species, or ß-caryophyllene, germacrene D, bicyclogermacrene, spathulenol, and caryophyllene oxide in sesquiterpene-rich species. Among these, α-pinene, ß-caryophyllene, and germacrene D are the most common and abundant in the Salvia species investigated. In accordance with their close biological taxonomy, the chemical similarity of the essential oils of S. santolinifolia and S. eremophila is so high that we may consider them chemically identical.

Chemical constituents of two sages with free radical scavenging activity.

From the aerial parts of Salvia trichoclada Bentham and S. verticillata L. one new and two known phenolic acids, 3-(3',4'-dihydroxyphenyl)-2-hydroxymethyl propionic acid (1), 3-(3',4'-dihydroxyphenyl) lactic acid (2), and rosmarinic acid (3); two flavonoids, apigenin 4'-methyl ether 7-O-glucuronide (4), and luteolin 7-O-beta glucuronide (5); two lupan type triterpene aglycones, lupeol (6), and 30-hydroxylup-20 (29)-en-3-on (7); an oleanane-type triterpene acid, oleanolic acid (8); and an ursan-type triterpene acid, ursolic acid (9) were isolated. The structures of the compounds were elucidated by spectroscopic analysis. Different extracts of the plants were examined for their free radical scavenging activities by DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay. Some of the polar extracts showed high free radical scavenging activity.