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 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).

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.