Quality evaluation of medicinal products and health foods containing chaste berry (Vitex agnus-castus) in Japanese, European and American markets.

The aim of present study was to evaluate the qualities of chaste berry (fruit of Vitex agnus-castus L.) preparations using HPLC fingerprint analysis. Seven medicinal products 1 from Japan and 6 from Europe, and 17 health foods, 6 from Japan and 11 from the United States were analyzed. HPLC profile and 26 authentic peaks were compared medicinal products and health foods. Whereas medicinal products had similar HPLC profiles, health foods had various profiles and each peak was also greatly different. The measured amounts of two markers in 5 traditional medicinal products, agnuside and casticin specified in the European Pharmacopoeia (EP), the U.S. Pharmacopoeia (USP) or the WHO monographs of chaste berry, were much lower than those in 2 medicinal products defined as "well-established use" by the European Medicines Agency. The amounts of two markers for 17 health foods differed in a great deal from 14-5054% and 3-1272%, respectively. Furthermore the amount ratios of two markers, agnuside/casticin, in about half of the health foods were remarkably larger than the standard crude drug and the ratios were closer to one of the related Chinese herbs, Vitex negundo L. It is concluded that a combination of HPLC fingerprints and the amount ratios of the marker compounds of chaste berry preparations serves as a useful tool to evaluate the qualities of these preparations.

7-deoxyloganetic acid synthase catalyzes a key 3 step oxidation to form 7-deoxyloganetic acid in Catharanthus roseus iridoid biosynthesis.

Iridoids are key intermediates required for the biosynthesis of monoterpenoid indole alkaloids (MIAs), as well as quinoline alkaloids. Although most iridoid biosynthetic genes have been identified, one remaining three step oxidation required to form the carboxyl group of 7-deoxyloganetic acid has yet to be characterized. Here, it is reported that virus-induced gene silencing of 7-deoxyloganetic acid synthase (7DLS, CYP76A26) in Catharanthus roseus greatly decreased levels of secologanin and the major MIAs, catharanthine and vindoline in silenced leaves. Functional expression of this gene in Saccharomyces cerevisiae confirmed its function as an authentic 7DLS that catalyzes the 3 step oxidation of iridodial-nepetalactol to form 7-deoxyloganetic acid. The identification of CYP76A26 removes a key bottleneck for expression of iridoid and related MIA pathways in various biological backgrounds.

Evaluation of the botanical origin of black cohosh products by genetic and chemical analyses.

Despite the increasing sales of black cohosh (the dried rhizome and root of Cimicifuga racemosa L.) in the world herbal market, these products have continuous adulteration issues. The botanical authenticity of the black cohosh products is the first important step for ensuring their quality, safety and efficacy. In this study, we genetically identified the botanical sources of 10 black cohosh products and 5 Cimicifuga Rhizome crude drugs of Japanese Pharmacopoeia grade, and analyzed the metabolic profiling of 25 black cohosh products using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Consequently, we found that C. dahurica and possibly C. foetida are misused as sources of the black cohosh products and in some cases, the extracts of black cohosh were adulterated with the plant materials of C. dahurica. We demonstrated that these three species can be distinguished by three marker compounds in a specific mass range. These results must be helpful in establishing regulations for the safe use of the black cohosh products.

A 7-deoxyloganetic acid glucosyltransferase contributes a key step in secologanin biosynthesis in Madagascar periwinkle.

Iridoids form a broad and versatile class of biologically active molecules found in thousands of plant species. In addition to the many hundreds of iridoids occurring in plants, some iridoids, such as secologanin, serve as key building blocks in the biosynthesis of thousands of monoterpene indole alkaloids (MIAs) and many quinoline alkaloids. This study describes the molecular cloning and functional characterization of three iridoid glucosyltransfeases (UDP-sugar glycosyltransferase6 [UGT6], UGT7, and UGT8) from Madagascar periwinkle (Catharanthus roseus) with remarkably different catalytic efficiencies. Biochemical analyses reveal that UGT8 possessed a high catalytic efficiency toward its exclusive iridoid substrate, 7-deoxyloganetic acid, making it better suited for the biosynthesis of iridoids in periwinkle than the other two iridoid glucosyltransfeases. The role of UGT8 in the fourth to last step in secologanin biosynthesis was confirmed by virus-induced gene silencing in periwinkle plants, which reduced expression of this gene and resulted in a large decline in secologanin and MIA accumulation within silenced plants. Localization studies of UGT8 using a carborundum abrasion method for RNA extraction show that its expression occurs preferentially within periwinkle leaves rather than in epidermal cells, and in situ hybridization studies confirm that UGT8 is preferentially expressed in internal phloem associated parenchyma cells of periwinkle species.

Transcriptome analysis based on next-generation sequencing of non-model plants producing specialized metabolites of biotechnological interest.

Plants produce a vast array of specialized metabolites, many of which are used as pharmaceuticals, flavors, fragrances, and other high-value fine chemicals. However, most of these compounds occur in non-model plants for which genomic sequence information is not yet available. The production of a large amount of nucleotide sequence data using next-generation technologies is now relatively fast and cost-effective, especially when using the latest Roche-454 and Illumina sequencers with enhanced base-calling accuracy. To investigate specialized metabolite biosynthesis in non-model plants we have established a data-mining framework, employing next-generation sequencing and computational algorithms, to construct and analyze the transcriptomes of 75 non-model plants that produce compounds of interest for biotechnological applications. After sequence assembly an extensive annotation approach was applied to assign functional information to over 800,000 putative transcripts. The annotation is based on direct searches against public databases, including RefSeq and InterPro. Gene Ontology (GO), Enzyme Commission (EC) annotations and associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps are also collected. As a proof-of-concept, the selection of biosynthetic gene candidates associated with six specialized metabolic pathways is described. A web-based BLAST server has been established to allow public access to assembled transcriptome databases for all 75 plant species of the PhytoMetaSyn Project (www.phytometasyn.ca).