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.

Polyphenol Diversity and Antioxidant Activity of European Cistus creticus L. (Cistaceae) Compared to Six Further, Partly Sympatric Cistus Species.

This investigation focused on the qualitative and quantitative composition of polyphenolic compounds of Mediterranean northern shore Cistus creticus and six further, partly sympatric Cistus species (C. albidus, C. crispus, C. ladanifer, C. monspeliensis, C. parviflorus, C. salviifolius). Aqueous extracts of 1153 individual plants from 13 countries were analyzed via high performance liquid chromatography (HPLC). The extracts of C. creticus were primarily composed of two ellagitannins (punicalagin and punicalagin gallate) and nine flavonol glycosides (myricetin and quercetin glycosides, with m-3-O-rhamnoside as the dominant main compound). Differences in the proportions of punicalagin derivatives and flavonol glycosides allowed the classification into two chemovariants. Plants containing punicalagin derivatives and flavonol glycosides were especially abundant in the western and central Mediterranean areas and in Cyprus. From Albania eastwards, punicalagin and punicalagin gallate were of much lesser importance and the predominant chemovariant there was a nearly pure flavonol type. With its two chemovariants, C. creticus takes a central position between the flavonol-rich, purple-flowered clade (besides C. creticus, here represented by C. albidus and C. crispus) and the more ellagitannin-rich, white- or whitish-pink-flowered clade (here represented by C. ladanifer, C. monspeliensis, C. parviflorus and C. salviifolius). The median antioxidative capacity of C. creticus plant material was, with 166 mg Trolox equivalents/g dry wt, about half of the antioxidative capacity of C. ladanifer (301 mg te/g dry wt), the species with the highest antioxidative potential.

Differentiation between Cistus L. (Sub-) Species (Cistaceae) Using NMR Metabolic Fingerprinting.

The genus Cistus is taxonomically complex, as taxonomic classification of individual species based on morphological criteria is often difficult and ambiguous. However, specific species contain valuable natural products, especially terpenoids and polyphenols, which exert various biological effects and might therefore be used for treatment of a broad array of disorders. Hence, a fast and reliable method for clear identification of different Cistus (sub-) species is required. Approaches for analysis of secondary metabolite profiles, e.g., with NMR, might remedy the challenging classification of Cistus (sub-) species and help to identify specific markers for differentiation between them. In the present study, 678 samples from wild-growing Cistus populations, including 7 species and 6 subspecies/varieties thereof, were collected in 3 years from populations in 11 countries all over the Mediterranean basin. Samples were extracted with buffered aqueous methanol and analysed with NMR. From the resulting 1D-1H-NOESY and J-Res profile spectra, marker signals or spectral regions for the individual (sub-) species were identified with multivariate statistical tools. By examining the NMR profiles of these extracts, we were able to identify discriminators and specific markers for the investigated Cistus (sub-) species. Various influencing factors, like (sub-) species, wild harvestings of different populations from several countries, numerous collection sites, different years, and cultivation in greenhouses have been considered in this work. As the here identified markers are independent from these influencing factors, the results can be considered a robust model and might be used for future differentiation between Cistus (sub-) species.

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.

Phytogenic pigments in animal nutrition: potentials and risks.

Phytogenic pigments are secondary plant compounds responsible for coloring effects in plant tissues. In particular, phenolic flavonoids and terpenoid carotenoids, but also rare compounds like curcumin and betalain, form this group of biochemical agents used in animal nutrition. From the perspective of ecological mutuality between plants and animals, these compounds are of crucial importance because they serve as visual attraction for herbivores but also signal nutritional and/or health-promoting values. This review focuses on the properties of phytogenic pigments which are likely to impact feed intake and preferences of livestock. Also natural prophylactic and/or therapeutic properties and, in particular, the potential of pigments to enhance quality and health value of animal products for human consumption are important issues. Nevertheless, reasonable limits of use due to possible adverse indications have been suggested recently. Pathways of digestion, metabolism and excretion in animals play a crucial role not only in the evaluation of effectiveness but also in the prediction of potential risks for human consumption. The popularity of natural feed additives is growing; therefore, more research work is needed to better understand metabolic pathways in the animal's body and to better estimate the potentials and risks of pigmenting plant compounds used in animal nutrition.

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.

Essential oil composition variability among natural populations of Pinus mugo Turra in Kosovo.

Pinus mugo Turra, is a native pine species in central and southern Europe, growing in high mountains area (altitudes 1.800-2.300 m.a.s.l.). In Kosovo, it is one of the native pines too, distributed in high altitudes in the Sharri Mountains and Albanian Alps Mountains. Its populations represent an important wealth of essential oil resources available, which make this species very important in terms of economic values. The chemical composition and yields of the essential oils of dwarf pine (Pinus mugo Turra) needles, twigs and cones from six wild populations in Kosovo were investigated with the aim to assess their natural variability. The identity of P. mugo was confirmed by morphology and DNA barcoding. Sixty-two compounds were identified representing 69-95 % of the total identified compounds. The yield ranged from 0.3-0.8 % v/w in needles, 1.0-2.4 % v/w in twigs and 0.1-0.5 % v/w in cones, depending on the origin of plant material and plant organs. α-Pinene (needles: 16.9-24.5 %; twigs: 4.5-8.8 %; cones: 3.1-5.6 %), ß-pinene (needles: 1.5-5.4 %; twigs: 2.2-15.4 %; cones: 1.3-14.2 %), δ-3-carene (needles: 15.4-27.8 %; twigs: 24.0-51.6 %; cones: 10.5-31.5 %), limonene + ß-phellandrene (needles: 1.9-5.9 %; twigs: 12.6-24.2 %; cones: 2.1-9.3 %), (E)-caryophyllene (needles: 4.4-8.9 %; twigs: 4.0-10.8 %; cones: 10.3-26.9 %) and germacrene D (needles: 4.0-8.3 %; twigs: 0.2-6.19 %; cones: 0.1-12.4 %) were the major components of the essential oil. Principal component analysis (PCA) and hierarchical cluster analyses (HCA) suggests that the population of P. mugo clustering is not related to their geographic location, but rather seemed to be linked to local selective forces acting on chemotype diversity. Low variability related to their geographic location has an economic importance since samples originating from different locations in Kosovo can treated with same standards.

The complete chloroplast genome of Origanum vulgare L. (Lamiaceae).

Oregano (Origanum vulgare L., Lamiaceae) is a medicinal and aromatic plant maybe best known for flavouring pizza. New applications e.g. as natural antioxidants for food are emerging due to the plants' high antibacterial and antioxidant activity. The complete chloroplast (cp) genome of Origanum vulgare (GenBank/EBML/DDBJ accession number: JX880022) consists of 151,935 bp and includes a pair of inverted repeats (IR) of 25,527 bp separated by one small and one large single copy region (SSC and LSC) of 17,745 and 83,136 bp, respectively. The genome with an overall GC content of 38% hosts 114 genes that covering 63% of the genome of which 8% were introns. The comparison of the Origanum cp genome with the cp genomes of two other core lamiales (Salvia miltiorrhiza Bunge and Sesamum indicum L.) revealed completely conserved protein-coding regions in the IR region but also in the LSC and SSC regions. Phylogenetic analysis of the lamiids based on 56 protein-coding genes give a hint at the basic structure of the Lamiales. However, further genomes will be necessary to clarify this taxonomically complicated order. The variability of the cp within the genus Origanum, studied exemplarily on 16 different chloroplast DNA regions, demonstrated that in 14 regions analyzed, the variability was extremely low (max. 0.7%), while only two regions showed a moderate variability of up to 2.3%. The cp genome of Origanum vulgare contains 27 perfect mononucleotide repeats (number of repeats>9) consisting exclusively of the nucleotides A or T. 34 perfect repeats (repeat lengths>1 and number of repeats>3) were found, of which 32 were di-, and 2 were trinucleotide repeats.

Genomic characterization, molecular cloning and expression analysis of two terpene synthases from Thymus caespititius (Lamiaceae).

The identification, isolation and functional characterization of two genes encoding two monoterpene synthases-γ-terpinene synthase (Tctps2) and α-terpineol synthase (Tctps5)-from three chemically distinct Thymus caespititius (Lamiaceae) genotypes were performed. Genomic exon-intron structure was also determined for both terpene synthase genes, revealing an organization with seven exons and six introns. The cDNA of Tctps2 was 2,308 bp long and had an open reading frame of 1,794 bp encoding for a protein with 598 amino acids. Tctps5 was longer, mainly due to intron sequences, and presented high intraspecific variability on the plants analyzed. It encoded for a protein of 602 amino acids from an open reading frame of 1,806 bp comprising a total of 2,507 bp genomic sequence. The amino acid sequence of these two active Tctps genes shared 74 % pairwise identity, ranging between 42 and 94 % similarity with about 50 known terpene synthases of other Lamiaceae species. Gene expression revealed a multi-product Tctps2 and Tctps5 enzymes, producing γ-terpinene and α-terpineol as major components, respectively. These enzymatic results were consistent with the monoterpene profile present in T. caespititius field plants, suggesting a transcriptional regulation in leaves. Herewith reported for the first time for this species, these two newly characterized Tctps genes improve the understanding of the molecular mechanisms of reaction responsible for terpene biosynthesis and chemical diversity found in T. caespititius.

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.

Identification of Verbena officinalis based on ITS sequence analysis and RAPD-derived molecular markers.

Verbenae herba is a widely used drug and consists of the aerial parts of Verbena officinalis (Verbenaceae). Until now, the identification has been performed based on morphological and phytochemical analyses, which are not reliable enough to distinguish Verbena officinalis from other relevant species of the genus Verbena. Hence, impurities and adulterants, negatively influencing the therapeutic effect of the drug, may remain undetected. In an attempt to generate an accurate authentication method we used two different DNA-based approaches: comparison of ITS sequences and molecular markers (RAPD). Both approaches generally enabled discrimination of V. officinalis from the rest of the genus despite the intraspecific variation existing within V. officinalis. The application of the two independent methods, supporting each other, increases the security of identification. For better reproducibility and faster analysis, however, a SCAR marker and primers for HRM were derived from the RAPD results. The SCAR marker could distinguish V. officinalis from all other verbena species except its closest relative V. hastata, while discrimination of V. officinalis even from V. hastata was unproblematic with HRM.

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%.

Oregano: a source for peroxisome proliferator-activated receptor gamma antagonists.

Peroxisome proliferator-activated receptors (PPARs) are drug targets for several perturbations of metabolic syndrome, defined as the coexistence of obesity, hyperglycemia, hypertension, and hyper/dyslipidemia. In this study, PPAR activation by oregano (e.g., Origanum vulgare) and its components was tested. Oregano extracts bind but do not transactivate PPARgamma, and binding affinity differs among different oregano extracts. The extracts contain PPARgamma antagonists (e.g., quercetin, luteolin, rosmarinic acid, and diosmetin), selective PPARgamma modulators (e.g., naringenin and apigenin), and PPARgamma agonists (e.g., biochanin A). Oregano extract and isolated compounds in the extract antagonize rosiglitazone-mediated DRIP205/TRAP220 recruitment to PPARgamma, pointing to oregano extracts as putative food supplements for weight reduction. Rosmarinic acid and biochanin A, PPARalpha agonists, may ameliorate the lipid profile. By endothelial nitric oxide synthase activation, oregano extract could prevent atherosclerosis. The results warrant further investigation of oregano extract for its potential to prevent and ameliorate metabolic syndrome and its complications.

A strategy to setup codominant microsatellite analysis for high-resolution-melting-curve-analysis (HRM).

BACKGROUND: High resolution melting curve analysis (HRM) is a technique that measures exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double stranded DNA. The measurement is immediately following PCR in a one-step, closed-tube method. The shape of the melting curve depends on the GC content, length and sequence of the amplicon. Hence it is a powerful, fast and cheap method to detect Single Nucleotide Polymorphisms (SNPs) and other mutations. RESULTS: Here we present a strategy to set up microsatellite analysis for HRM including the correct assignment of heterozygous samples by comparative analysis and artificial mixtures of samples. The approach is demonstrated on two Simple Sequence Repeat (SSR) loci of different complexity in the genus Origanum. Following this strategy all alleles of our sample sets could be classified correctly. CONCLUSION: HRM can be used in microsatellite analysis and other codominant marker systems implementing a protocol of comparative melting curve assignment with artificial mixtures of samples to overcome difficulties in correctly assigning heterozygous samples. The method is faster, more sensitive and cheaper than standard protocols for microsatellite analysis.

Identification and characterization of simple sequence repeat markers from a glandular Origanum vulgare expressed sequence tag.

Oregano (Origanum vulgare) and marjoram (Origanum majorana) are two sensorial distinct spices within the genus Origanum (Lamiaceae). Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) of essential oil glands of O. vulgare. Thirteen EST-SSR loci were evaluated using 20 individual plants of O. vulgare and 19 plants of Origanum majorana. The number of alleles per locus ranged from one to four. All loci developed from O. vulgare successfully cross-amplified in O. majorana.