Monoterpene synthases of three closely related sage species (Salvia officinalis, S. fruticosa and S. pomifera, Lamiaceae).

The diversity of plant monoterpenes is largely based on the catalytic activity of monoterpene synthases. Additionally, copy number variation of monoterpene synthase genes may contribute to the quantity of transcripts and hence to the essential oil profile. This study used whole-genome sequencing and digital PCR for the measurement of copy number variation and quantification of gene expression in three closely related Salvia species, namely Salvia officinalis, Salvia pomifera and Salvia fruticosa. Twelve, 13 and 15 monoterpene synthase-encoding open-reading frames were predicted for Salvia officinalis, Salvia pomifera and Salvia fruticosa, respectively. In Salvia officinalis, one of the open reading frames was disrupted indicating a pseudogene. Monoterpene synthase genes were generally single copy per haploid genome, only a few were double or triple copy genes. Expression levels of monoterpene synthases in leaves corresponded generally well with essential oil composition. In some cases, a higher expression level of a certain monoterpene synthase could be explained by its duplication or triplication. The very high content of thujones in Salvia pomifera, for example, was accompanied by gene duplication and increased gene expression of (+)-sabinene synthase responsible for the thujone precursor sabinene. In Salvia officinalis, three individuals different in their essential oil profile showed significant differences in their monoterpene synthase expression levels corresponding roughly to the profile of the essential oils. Transcript expression of monoterpene synthase genes were measured in leaf, calyx and corolla. The corolla differed significantly from leaves, while calyces usually showed a profile intermediary between leaf and corolla.

Intraspecific Genetic Diversity of Cistus creticus L. and Evolutionary Relationships to Cistus albidus L. (Cistaceae): Meeting of the Generations?

Cistus (Cistaceae) comprises a number of white- and purple-flowering shrub species widely distributed in the Mediterranean basin. Within genus Cistus, many taxa are subject to various taxonomic uncertainties. Cistus creticus, a prominent member of the purple-flowered clade, is a prime case of the current taxonomic troubles. Floras and databases approve different species names and utilise different or additional/fewer synonyms. Various intraspecific classification systems based on subspecies or varieties are in use. The inconsistent determination of plant material makes it difficult to compare literature regarding the phytochemical diversity and biological activities of plant material and impedes a systematic utilization of the manifold medicinal properties of C. creticus. In the present investigation, we used DNA sequence data from one nuclear region (ITS) and two chloroplast regions (trnL-trnF, rpl32-trnL) to test the intraspecific genetic diversity of C. creticus and its evolutionary relationships to the closely related C. albidus. The combined DNA data confirmed C. creticus as a rather heterogeneous species that integrates two major evolutionary lineages with clearly different genetic characteristics. The 'Eastern Mediterranean clade' seems to represent old and ancestral characteristics. This lineage exhibits a close relationship to the geographically distant C. albidus, expressed by very closely related ribotypes and an interspecifically shared chlorotype. The 'Western Mediterranean clade' is characterized by a distinctive ITS polymorphism (co-occurring paralogous ribotypes) and more distantly related chlorotypes. The formation of the genetically complex 'Western Mediterranean clade' seems to have involved hybridization and recurrent formation or migration movements.

A phylogenetic analysis of the wild Tulipa species (Liliaceae) of Kosovo based on plastid and nuclear DNA sequence.

In Kosovo, the genus Tulipa is represented by eight taxa, most of which form a species complex surrounding Tulipa scardica. To investigate the phylogenetic relationship of these Tulipa species a Bayesian analysis was undertaken using the ITS nuclear marker and trnL-trnF, rbcL and psbA-trnH plastid markers. The resulting phylogenetic trees show that Kosovarian Tulipa species consistently group into two main clades, the subgenera Eriostemones and Tulipa. Furthermore, our analyses provide some evidence that the subspecies of Tulipa sylvestris are genetically distinguishable, however not significantly enough to support their reclassification as species. In contrast, the markers provide some novel information to reassess the species concepts of the T. scardica complex. Our data provide support for the synonymisation of Tulipa luanica and Tulipa kosovarica under the species Tulipa serbica. Resolution and sampling limitations hinder any concrete conclusion about whether Tulipa albanica and T. scardica are true species, yet our data do provide some support that these are unique taxa and therefore should continue to be treated as such until further clarification. Overall, our work shows that genetic data will be important in determining species concepts in this genus, however, even with a molecular perspective pulling apart closely related taxa can be extremely challenging.

Erratum: A phylogenetic analysis of the wild Tulipa species (Liliaceae) of Kosovo based on plastid and nuclear DNA sequence.

[This corrects the article DOI: 10.1002/ggn2.202100016.].

What Else Is in Salviae officinalis folium? Comprehensive Species Identification of Plant Raw Material by DNA Metabarcoding.

Quality control of drugs consists of identifying the raw material to avoid unwanted admixtures or exchange of material as well as looking for abiotic and biotic contaminations. So far, identity and microbial contamination are analyzed by separate processes and separate methods. Species identification by their DNA ("DNA barcoding") has the potential to supplement existing methods of identification. The introduction of next-generation sequencing methods offers completely new approaches like the identification of whole communities in one analysis, termed "DNA metabarcoding". Here we present a next-generation sequencing assessment to identify plants and fungi of two commercial sage samples (Salvia officinalis) using the standard DNA barcoding region "internal transcribed spacer" consisting of internal transcribed spacer 1 and internal transcribed spacer 2, respectively. The main species in both samples was identified as S. officinalis. The spectrum of accompanying plant and fungal species, however, was completely different between the samples. Additionally, the composition between internal transcribed spacer 1 and internal transcribed spacer 2 within the samples was different and demonstrated the influence of primer selection and therefore the need for harmonization. This next-generation sequencing approach does not result in quantitative species composition but gives deeper insight into the composition of additional species. Therefore, it would allow for a better knowledge-based risk assessment than any other method available. However, the method is only economically feasible in routine analysis if a high sample throughput can be guaranteed.

DNA-based identification of Calendula officinalis (Asteraceae).

PREMISE OF THE STUDY: For the economically important species Calendula officinalis, a fast identification assay based on high-resolution melting curve analysis was designed. This assay was developed to distinguish C. officinalis from other species of the genus and other Asteraceae genera, and to detect C. officinalis as an adulterant of saffron samples. METHODS AND RESULTS: For this study, five markers (ITS, rbcL, 5' trnK-matK, psbA-trnH, trnL-trnF) of 10 Calendula species were sequenced and analyzed for species-specific mutations. With the application of two developed primer pairs located in the trnK 5' intron and trnL-trnF, C. officinalis could be distinguished from other species of the genus and all outgroup samples tested. Adulterations of Calendula DNA in saffron could be detected down to 0.01%. CONCLUSIONS: With the developed assay, C. officinalis can be reliably identified and admixtures of this species as adulterant of saffron can be revealed at low levels.

Essential oil diversity of European Origanum vulgare L. (Lamiaceae).

This investigation focused on the qualitative and quantitative composition of essential oil compounds of European Origanum vulgare. Extracts of 502 individual O. vulgare plants from 17 countries and 51 populations were analyzed via GC. Extracts of 49 plants of 5 populations of Israeli Origanum syriacum and 30 plants from 3 populations of Turkish Origanum onites were included to exemplify essential oil characteristics of 'high-quality' oregano. The content of essential oil compounds of European O. vulgare ranged between 0.03% and 4.6%. The monoterpenes were primarily made up of sabinene, myrcene, p-cymene, 1,8-cineole, ß-ocimene, γ-terpinene, sabinene hydrate, linalool, α-terpineol, carvacrol methyl ether, linalyl acetate, thymol and carvacrol. Among the sesquiterpenes ß-caryophyllene, germacrene D, germacrene D-4-ol, spathulenol, caryophyllene oxide and oplopanone were often present in higher amounts. According to the proportions of cymyl-compounds, sabinyl-compounds and the acyclic linalool/linalyl acetate three different main monoterpene chemotypes were defined. The cymyl- and the acyclic pathway were usually active in plants from the Mediterranean climate whereas an active sabinyl-pathway was a characteristic of plants from the Continental climate.

DNA-based identification of Peucedanum ostruthium specimens and detection of common adulterants by high-resolution melting curve analysis.

Masterwort (Peucedanum ostruthium, syn. Imperatoria ostruthium, Apiaceae) is an old economic plant in Alpine countries cultivated as ornamental plant and used for spirits and in folk medicine. P. ostruthium is a species that has often been confused with related Apiaceae species or morphologically similar roots or tubers resulting in products of minor quality. Masterwort can be distinguished from other Apiaceae species by nrDNA (ITS1 and ITS2). The analysed chloroplast markers (trnK 5' intron, trnT-trnL, and psbA-trnH), however, showed no species-specific mutations. With the application of two primer pairs amplifying parts of ITS and developed for high-resolution melting curve analysis (HRM) the target species was distinguishable from the other Peucedanum and Apiaceae species of our reference set. A multiplex PCR/HRM was developed to detect adulterations with Gentiana spp., Aconitum napellus and Veratrum album.

Polyacetylenes from Radix et Rhizoma Notopterygii incisi with an inhibitory effect on nitric oxide production in vitro.

Notopterygium roots (Qiang Huo) have been used in traditional Chinese medicine for treating colds, inflammatory diseases like rheumatoid arthritis, and as an analgesic. The anti-inflammatory activity of the roots of Notopterygium incisum has been evaluated by testing the inhibitory activity on nitric oxide production by inducible nitric oxide synthase. The apparent authenticity of the sample was checked by DNA sequence comparison. Using activity-guided isolation, different compounds were isolated and structurally characterized by means of NMR and mass spectroscopy. Eight polyacetylenes could be identified and were tested on their inhibitory activity on nitric oxide production in RAW 264.7 mouse macrophages using the Griess assay. Different 3-hydroxy allyl polyacetylenes exhibited significant activity (IC50: 8-acetoxyfalcarinol, 20.1 µM; falcarindiol, 9.2 µM; 9-epoxyfalcarindiol, 8.8 µM; and crithmumdiol, 23.6 µM).

Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and ß-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/ß-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively).

Quantitative high-resolution melting analysis for detecting adulterations.

Admixtures of different plant species are a common problem in raw materials for medicinal use. Two exemplary assays were developed to admixtures in Helleborus niger with high-resolution melting analysis. HRM proved to be a very sensitive tool in detecting admixtures, able to detect a ratio of 1:1000 with unknown species, and of 1:200,000 with Veratrum nigrum. The example proves the ability of HRM for quantification in multiplex PCR. The method is not limited to detecting adulterations. It can also be used to quantify a specific target by integrating a second amplicon in the assay as internal standard.

DNA-based identification of Helleborus niger by high-resolution melting analysis.

Hellbori nigri rhizoma is a drug that is difficult to distinguish from other species of the genus Helleborus. In this communication we present a DNA-based identification by high-resolution melting analysis (HRM) that is able to differentiate between Helleborus niger and other species of the genus. HRM is a very specific, time- and labour-saving method for identifying DNA sequence variations and is ideally suitable for routine PCR analysis. The HRM assay developed is specific for the genus Helleborus. This method not only detects the presence of the target species H. niger but also, to a certain extent, identifies other Helleborus species by their different melting curve shapes. Markers were developed based on the trnL-trnF intergenic spacer and on the matK sequence. For an unambiguous identification of Helleborus niger, melting curves of both markers should be used.

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.

Validation of a quantitative assay of arbutin using gas chromatography in Origanum majorana and Arctostaphylos uva-ursi extracts.

INTRODUCTION: Arbutin is a skin-whitening agent that occurs naturally in the bark and leaves of various plants. It is commonly quantified in plant extracts and skin-whitening products by HPLC. OBJECTIVE: To develop an alternative gas chromatographic method for the separation and quantification of arbutin in Origanum majorana and Arctostaphylos uva-ursi extracts. METHODOLOGY: N,O-Bis(trimethylsilyl)acetamide and trimethylchlorosilane were used as silylation reagents, and the gas chromatographic separation of silylated extracts and standards was performed using a DB-5 narrow bore column. GC-MS was used for the compound identification, and the quantification was carried out by GC-FID. The quantitative results were compared with those of HPLC analysis. RESULTS: The developed method gave a good sensitivity with linearity in the range 0.33-500 mg/mL and recovery >98%, allowing the quantification of arbutin in O. majorana and A. uva-ursi extracts. The relative standard deviations (RSD) relating to intra-day and inter-day precision were <0.002% and <4.8%, respectively. The GC results correlated well with those obtained by HPLC analysis. CONCLUSION: The analysis of marjoram and bearberry samples showed that the established GC method was rapid, selective, and demonstrated that arbutin could be screened alternatively by gas chromatography.

Composition of essential oil compounds from different Syrian populations of Origanum syriacum L. (Lamiaceae).

The chemical compositions of the essential oil compounds of 117 individual plants belonging to 11 Syrian populations of Origanum syriacum L. (Lamiaceae) were studied by GC-FID and GC-MS. The composition was dominated by carvacrol and/or thymol with a high degree of polymorphism in the occurrence of these two compounds between the different populations. In three populations carvacrol was dominating, with thymol being present only in minor amounts, whereas in only one population thymol was the main compound, with carvacrol only in traces. In all other populations both carvacrol and thymol were present as major compounds. No geographical pattern could be detected for the occurrence of the chemotypes. Thymoquinone, a promising anticancer candidate, was present in the extracts in a wide range between 0.04 and 23.7%.