Aconitum Diterpenoid Alkaloid Profiling to Distinguish between the Official Traditional Chinese Medicine (TCM) Fuzi and Adulterant Species Using LC-qToF-MS with Chemometrics.

The lateral roots of Aconitum carmichaelii, known in Chinese as fuzi, are officially recognized as a materia medica in the Chinese Pharmacopoeia and used culinarily to prepare herbal soups. A strategy combining UPLC-qToF-MS analysis of A. carmichaelii and its intraspecies and interspecies chemometrics study was developed to examine the distribution of Aconitum marker metabolites. Four diterpenoid alkaloids were recognized to be important markers in fuzi, and another 15 markers were identified to differentiate A. carmichaelii from adulterant species. The detected fuzi markers, mesaconitine (47) and hypaconitine (51), are known to be the principal toxins in this herb, while fuziline (6) and benzoylmesaconine (25) are associated with its medicinal properties. Additional marker compounds have been detected in other Aconitum species that are useful for identifying adulteration. This study provides a useful resource for detecting traditional Chinese medicine (TCM) adulterants and assisting in the quality control of botanical products in TCM and beyond.

Recurrent gene duplication in the angiosperm tribe Delphinieae (Ranunculaceae) inferred from intracellular gene transfer events and heteroplasmic mutations in the plastid matK gene.

The study of intracellular gene transfer may allow for the detection of interesting evolutionary processes such as ancient polyploidization. We compared 24 plastid genomes (plastomes) from tribe Delphinieae, one from tribe Nigelleae and one from tribe Ranunculeae, including five newly sequenced genomes. The functional transfers of the plastids rpl32 and rps16 to the nucleus in tribe Delphinieae were identified. Unexpectedly, we discovered multiple divergent copies of the nuclear-encoded plastid rpl32 in the genus Aconitum. Phylogenetic and synonymous substitution rate analyses revealed that the nuclear-encoded plastid rpl32 underwent two major duplication events. These ancient gene duplication events probably occurred via multiple polyploidization events in Aconitum between 11.9 and 24.7 Mya. Furthermore, our sequence rate analysis indicated that the eight plastid-encoded rpl subunits in Aconitum had a significantly accelerated evolutionary rate compared to those in other genera, suggesting that highly divergent paralogs targeted to the plastid may contribute to an elevated rate of evolution in plastid rpl genes. In addition, heteroplasmy of the plastid matK from two Aconitum species suggested the existence of potentially functional plastid maturases in its plastome. Our results provide insight into the evolutionary history of the tribe Delphinieae.

Development of genomic microsatellite markers for Aconitum gymnandrum (Ranunculaceae) by next generation sequencing (NGS).

Mating plays key roles in the demographic and genetic dynamics of populations. Estimates of mating portfolios and system based on progeny array (PA) method required highly polymorphic genetic markers, of which microsatellite is a good choice. In this study, we reported 19 polymorphic microsatellite loci for Aconitum gymnandrum. The number of alleles per locus ranged from 2 to 12. Observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.219 to 0.842, respectively. Seven loci showed significant deviation from Hardy-Weinberg equilibrium. These markers will provide a useful tool for pollination ecology and population genetic studies of A. gymnandrum in Qinghai-Tibet plateau.

Micro-morphological, environmental and phytochemical investigation of herbal medicines by using LM and SEM: A quality control tool.

The use of quality control tool for authentication of Jadwar (Delphinium denudatum Wall. ex Hook.f. & Thomson), a folk herbal drug used for the treatment of different ailments, was studied. People face problems of adulteration for this drug at global, regional, national and local levels. Two different plant species are commercially marketed in the Indo-Pak Subcontinent under the same trade name of Jadwar. One is D. denudatum Wall. ex Hook.f. & Thomson and the other is Aconitum heterophyllum Wall. ex Royle. To focus on this problem, a marketable available drug sample of Jadwar was authenticated by using basic microscopy tools (LM) and advanced chemo-taxonomic markers. Authentication, quality and standardization of this drug was achieved using morphology, organoleptography, UV and IR analyses, scanning electron microscopy of pollen and anatomical investigations. The techniques used for authentication marked the clear difference between the studied plants. Microscopic studies, chemotaxonomic investigation and other techniques used in this project provided the basis for the authentication of this species.

Comparative Analysis of the Complete Chloroplast Genomes of Four Aconitum Medicinal Species.

Aconitum (Ranunculaceae) consists of approximately 400 species distributed in the temperate regions of the northern hemisphere. Many species are well-known herbs, mainly used for analgesia and anti-inflammatory purposes. This genus is well represented in China and has gained widespread attention for its toxicity and detoxification properties. In southwestern China, several Aconitum species, called 'Dula' in the Yi Nationality, were often used to control the poisonous effects of other Aconitum plants. In this study, the complete chloroplast (cp) genomes of these species were determined for the first time through Illumina paired-end sequencing. Our results indicate that their cp genomes ranged from 151,214 bp (A. episcopale) to 155,769 bp (A. delavayi) in length. A total of 111⁻112 unique genes were identified, including 85 protein-coding genes, 36⁻37 tRNA genes and eight ribosomal RNA genes (rRNA). We also analyzed codon usage, IR expansion or contraction and simple sequence repeats in the cp genomes. Eight variable regions were identified and these may potentially be useful as specific DNA barcodes for species identification of Aconitum. Phylogenetic analysis revealed that all five studied species formed a new clade and were resolved with 100% bootstrap support. This study will provide genomic resources and potential plastid markers for DNA barcoding, further taxonomy and germplasm exploration of Aconitum.

De Novo RNA Sequencing and Expression Analysis of Aconitum carmichaelii to Analyze Key Genes Involved in the Biosynthesis of Diterpene Alkaloids.

Aconitum carmichaelii is an important medicinal herb used widely in China, Japan, India, Korea, and other Asian countries. While extensive research on the characterization of metabolic extracts of A. carmichaelii has shown accumulation of numerous bioactive metabolites including aconitine and aconitine-type diterpene alkaloids, its biosynthetic pathway remains largely unknown. Biosynthesis of these secondary metabolites is tightly controlled and mostly occurs in a tissue-specific manner; therefore, transcriptome analysis across multiple tissues is an attractive method to identify the molecular components involved for further functional characterization. In order to understand the biosynthesis of secondary metabolites, Illumina-based deep transcriptome profiling and analysis was performed for four tissues (flower, bud, leaf, and root) of A. carmichaelii, resulting in 5.5 Gbps clean RNA-seq reads assembled into 128,183 unigenes. Unigenes annotated as possible rate-determining steps of an aconitine-type biosynthetic pathway were highly expressed in the root, in accordance with previous reports describing the root as the accumulation site for these metabolites. We also identified 21 unigenes annotated as cytochrome P450s and highly expressed in roots, which represent candidate unigenes involved in the diversification of secondary metabolites. Comparative transcriptome analysis of A. carmichaelii with A. heterophyllum identified 20,232 orthogroups, representing 30,633 unigenes of A. carmichaelii, gene ontology enrichment analysis of which revealed essential biological process together with a secondary metabolic process to be highly enriched. Unigenes identified in this study are strong candidates for aconitine-type diterpene alkaloid biosynthesis, and will serve as useful resources for further validation studies.

The Complete Chloroplast Genome Sequences of Aconitum pseudolaeve and Aconitum longecassidatum, and Development of Molecular Markers for Distinguishing Species in the Aconitum Subgenus Lycoctonum.

Aconitum pseudolaeve Nakai and Aconitum longecassidatum Nakai, which belong to the Aconitum subgenus Lycoctonum, are distributed in East Asia and Korea. Aconitum species are used in herbal medicine and contain highly toxic components, including aconitine. A. pseudolaeve, an endemic species of Korea, is a commercially valuable material that has been used in the manufacture of cosmetics and perfumes. Although Aconitum species are important plant resources, they have not been extensively studied, and genomic information is limited. Within the subgenus Lycoctonum, which includes A. pseudolaeve and A. longecassidatum, a complete chloroplast (CP) genome is available for only one species, Aconitum barbatum Patrin ex Pers. Therefore, we sequenced the complete CP genomes of two Aconitum species, A. pseudolaeve and A. longecassidatum, which are 155,628 and 155,524 bp in length, respectively. Both genomes have a quadripartite structure consisting of a pair of inverted repeated regions (51,854 and 52,108 bp, respectively) separated by large single-copy (86,683 and 86,466 bp) and small single-copy (17,091 and 16,950 bp) regions similar to those in other Aconitum CP genomes. Both CP genomes consist of 112 unique genes, 78 protein-coding genes, 4 ribosomal RNA (rRNA) genes, and 30 transfer RNA (tRNA) genes. We identified 268 and 277 simple sequence repeats (SSRs) in A. pseudolaeve and A. longecassidatum, respectively. We also identified potential 36 species-specific SSRs, 53 indels, and 62 single-nucleotide polymorphisms (SNPs) between the two CP genomes. Furthermore, a comparison of the three Aconitum CP genomes from the subgenus Lycoctonum revealed highly divergent regions, including trnK-trnQ, ycf1-ndhF, and ycf4-cemA. Based on this finding, we developed indel markers using indel sequences in trnK-trnQ and ycf1-ndhF. A. pseudolaeve, A. longecassidatum, and A. barbatum could be clearly distinguished using the novel indel markers AcoTT (Aconitum trnK-trnQ) and AcoYN (Aconitum ycf1-ndhF). These two new complete CP genomes provide useful genomic information for species identification and evolutionary studies of the Aconitum subgenus Lycoctonum.

The complete chloroplast genome sequence of Aconitum coreanum and Aconitum carmichaelii and comparative analysis with other Aconitum species.

Aconitum species (belonging to the Ranunculaceae) are well known herbaceous medicinal ingredients and have great economic value in Asian countries. However, there are still limited genomic resources available for Aconitum species. In this study, we sequenced the chloroplast (cp) genomes of two Aconitum species, A. coreanum and A. carmichaelii, using the MiSeq platform. The two Aconitum chloroplast genomes were 155,880 and 157,040 bp in length, respectively, and exhibited LSC and SSC regions separated by a pair of inverted repeat regions. Both cp genomes had 38% GC content and contained 131 unique functional genes including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene order, content, and orientation of the two Aconitum cp genomes exhibited the general structure of angiosperms, and were similar to those of other Aconitum species. Comparison of the cp genome structure and gene order with that of other Aconitum species revealed general contraction and expansion of the inverted repeat regions and single copy boundary regions. Divergent regions were also identified. In phylogenetic analysis, Aconitum species positon among the Ranunculaceae was determined with other family cp genomes in the Ranunculales. We obtained a barcoding target sequence in a divergent region, ndhC-trnV, and successfully developed a SCAR (sequence characterized amplified region) marker for discrimination of A. coreanum. Our results provide useful genetic information and a specific barcode for discrimination of Aconitum species.

Phylogeny and reclassification of Aconitum subgenus Lycoctonum (Ranunculaceae).

Phylogenetic analyses were performed using multiple nuclear (ITS and ETS) and chloroplast regions (ndhF-trnL, psbA-trnH, psbD-trnT, and trnT-trnL) to test the monophyly of Aconitum subgen. Lycoctonum (Ranunculaceae) and reconstruct the phylogenetic relationships within the subgenus. The subgenus as currently circumscribed is revealed to be polyphyletic. To achieve its monophyly, sect. Galeata and sect. Fletcherum, both being unispecific and each having a unique array of characters (the latter even having the aberrant base chromosome number of x = 6), must be removed from the subgenus. The subgenus Lycoctonum should thus be redefined to include only two sections, the unispecific sect. Alatospermum and the relatively species-rich sect. Lycoctonum. The section Alatospermum, which is both morphologically and karyologically in the primitive condition, is resolved as the first diverging lineage of the subgenus Lycoctonum clade. The monophyly of sect. Lycoctonum is strongly supported, but all the ten series currently recognized within the section are revealed to be para- or poly-phyletic. Five major clades are recovered within the section. We propose to treat them as five series: ser. Crassiflora, ser. Scaposa, ser. Volubilia, ser. Longicassidata, and ser. Lycoctonia. Thus, a formal reclassification of subgen. Lycoctonum is presented, which involves segregating both sect. Galeata and sect. Fletcherum from the subgenus as two independent subgenera within the genus Aconitum, reinstating one series (ser. Crassiflora) and abolishing six series (ser. Laevia, ser. Longibracteolata, ser. Micrantha, ser. Ranunculoidea, ser. Reclinata, and ser. Umbrosa) within sect. Lycoctonum. The series affiliation of some species within the section is adjusted accordingly.

Discovery of characteristic chemical markers for classification of aconite herbs by chromatographic profile and probabilistic neural network.

Most Aconitum species, also known as aconite, are extremely poisonous, so it must be identified carefully. Differentiation of Aconitum species is challenging because of their similar appearance and chemical components. In this study, a universal strategy to discover chemical markers was developed for effective authentication of three commonly used aconite roots. The major procedures include: (1) chemical profiling and structural assignment of herbs by liquid chromatography with mass spectrometry (LC-MS), (2) quantification of major components by LC-MS, (3) probabilistic neural network (PNN) model to calculate contributions of components toward species classification, (4) discovery of minimized number of chemical markers for quality control. The MS fragmentation pathways of diester-, monoester-, and alkyloyamine-diterpenoid alkaloids were compared. Using these rules, 42 aconite alkaloids were identified in aconite roots. Subsequently, 11 characteristic compounds were quantified. A component-species modeling by PNN was then established combining the 11 analytes and 26-batch samples from three aconite species. The contribution of each analyte to species classification was calculated. Selection of fuziline, benzoylhypaconine, and talatizamine, or a combination of more compounds based on a contribution order, can be used for successful categorization of the three aconite species. Collectively, the proposed strategy is beneficial to selection of rational chemical markers for the species classification and quality control of herbal medicines.

Identification of four Aconitum species used as "Caowu" in herbal markets by 3D reconstruction and microstructural comparison.

Authentication is the first priority when evaluating the quality of Chinese herbal medicines, particularly highly toxic medicines. The most commonly used authentication methods are morphological identification and microscopic identification. Unfortunately, these methods could not effectively evaluate some herbs with complex interior structures, such as root of Aconitum species with a circular conical shape and an interior structure with successive changes. Defining the part that should be selected as the standard plays an essential role in accurate microscopic identification. In this study, we first present a visual 3D model of Aconitum carmichaeli Debx. constructed obtained from microscopic analysis of serial sections. Based on this model, we concluded that the point of largest root diameter should be used as the standard for comparison and identification. The interior structure at this point is reproducible and its shape and appearance can easily be used to distinguish among species. We also report details of the interior structures of parts not shown in the 3D model, such as stone cells and cortical thickness. To demonstrate the usefulness of the results from the 3D model, we have distinguished the microscopic structures, at their largest segments, of the other three Aconitum species used for local habitat species of Caowu. This work provides the basis for resolution of some debate regarding the microstructural differences among these species. Thus, we conclude that the 3D model composed of serial sections has enabled the selection of a standard cross-section that will enable the accurate identification of Aconitum species in Chinese medicine.

Diversity in aconitine alkaloid profile of Aconitum plants in Hokkaido contrasts with their genetic similarity.

Aconite tuber is a representative crude drug for warming the body internally in Japanese Kampo medicine and Chinese traditional medicine. The crude drug is used in major prescriptions for the aged. Varieties of Aconitum plants are distributed throughout the Japanese Islands, especially Hokkaido. With the aim of identifying the medicinal potential of Aconitum plants from Hokkaido, 107 specimens were collected from 36 sites in the summer of 2011 and 2012. Their nuclear DNA region, internal transcribed spacer (ITS), and aconitine alkaloid contents were analyzed. Phylogenic analysis of ITS by maximum parsimony analysis showed that the majority of the specimens were grouped into one cluster (cluster I), separated from the other cluster (cluster II) consisting of alpine specimens. The aconitine alkaloid content of the tuberous roots of 76 specimens showed 2 aspects-specimens from the same collection site showed similar aconitine alkaloid profiles, and cluster I specimens from different habitats showed various alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profile of these genetically similar specimens to diversify.

[ISSR analysis for genetic polymorphism of Aconitum leucostomum from different habitats].

OBJECTIVE: To investigate the genetic diversities and variations of Aconitum leucostomum,and to supply essential characteristics for identifying Aconitum crude drugs. METHODS: Plant genome extraction kit was applied to extract DNA,and ultraviolet spectrophotometer was used to detect the concentrations and purity of DNA. 60 ISSR primers were screened to analyze the DNA of Aconitum leucostomum from 10 habitats. Biosoftwares including POPGEN32 and NTSYS-PC were used to analyze the polymorphic bands obtained, and hence to yield the genetic similarity coefficient of the 10 habitats and map the related graphics, and cluster analysis were performed by UPGMA method. RESULTS: 11 primers selected from 60 ISSR primers were used for amplification and a total of 101 DNA bands were obtained, including 89 polymorphic bands,the average percentage of polymorphic bands (PPB) was 88.1%. Shannon information index (I) was 0.5298, the genetic similarity coefficient (H) was 0.3648, observed number of alleles was 1.8911, and effective number of alleles was 1.6555. The genetic identity was from 0.4950 to 0.6931, and the genetic distances were from 0.3666 to 0.7031. According to cluster analysis result of ISSR, the 10 habitats of Aconitum leucostomum were classified into five groups. CONCLUSION: Germplasm resources of Aconitum leucostomum show abundant polymorphism and higher genetic variation, which might supply molecular level basis, and provide basis for building DNA fingerprint.

[Study on ITS sequences of Aconitum vilmorinianum and its medicinal adulterant].

OBJECTIVE: To analyze and compare the ITS sequences of Aconitum vilmorinianum and its medicinal adulterant Aconitum austroyunnanense. METHODS: Total genomic DNA were extracted from sample materials by improved CTAB method, ITS sequences of samples were amplified using PCR systems, directly sequenced and analyzed using software DNAStar, ClustalX1.81 and MEGA 4.0. RESULTS: 299 consistent sites, 19 variable sites and 13 informative sites were found in ITS1 sequences, 162 consistent sites, 2 variable sites and 1 informative sites were found in 5.8S sequences, 217 consistent sites, 3 variable sites and 1 informative site were found in ITS2 sequences. Base transition and transversion was not found only in 5.8S sequences, 2 sites transition and 1 site transversion were found in ITS1 sequences, only 1 site transversion was found in ITS2 sequences comparting the ITS sequences data matrix. By analyzing the ITS sequences data matrix from 2 population of Aconitum vilmorinianum and 3 population of Aconitum austroyunnanense, we found a stable informative site at the 596th base in ITS2 sequences, in all the samples of Aconitum vilmorinianum the base was C, and in all the samples of Aconitum austroyunnanense the base was A. CONCLUSION: Aconitum vilmorinianum and Aconitum austroyunnanense can be identified by their characters of ITS sequences, and the variable sites in ITS1 sequences are more than in ITS2 sequences.

Genus identification of toxic plant by real-time PCR.

Some plants have toxicities that are dangerous for humans. In the case of poisoning by toxic plants, a rapid and easy screening test is required for accurate medical treatment or forensic investigation. In this study, we designed specific primer pairs for identification of toxic plants, such as subgenus Aconitum, genus Ricinus, genus Illicium, and genus Scopolia, by internal transcribed spacer sequences of nuclear ribosomal DNA. Allied species of target plants, foods, and human DNA were not detected, but each primer pair provided a specific PCR product from the target plant using real-time PCR. This method can detect the subgenus Aconitum, genus Ricinus, and genus Scopolia with template DNA of 10 pg, respectively, and genus Illicium with 1 pg. Furthermore, each primer pair provided the exact PCR product from digested target plants in artificial gastric fluid. When a trace unknown plant sample in forensic investigation is collected from stomach contents, this PCR assay may be useful for screening toxic plants.

An expeditious HPLC method to distinguish Aconitum kusnezoffii from related species.

An optimized HPLC-DAD method aided by similarity and hierarchical clustering analysis (HCA) was applied for identification of four species of the roots of Aconitum. The unique properties of this HPLC fingerprints and HCA were validated by analyzing Aconitum kusnezoffii Rchb. (AKR) samples and related herbs including A. karacolicum Rapcs. (AKP) samples, A. austroyunnanense W.T.Wang (AAW) samples and A. contortum Finet & Gagnepain (ACF). The results revealed that the chromatographic fingerprint combining similarity measurement and hierarchical cluster analysis could efficiently identify and distinguish AKR samples from its related species.

Norditerpenoid alkaloids from the roots of Aconitum heterophyllum Wall with antibacterial activity.

Two new aconitine-type norditerpenoid alkaloids 6-dehydroacetylsepaconitine (1) and 13-hydroxylappaconitine (2), along with three known norditerpenoid alkaloids lycoctonine, delphatine and lappaconitine were isolated from the roots of the Aconitum heterophyllum Wall. These compounds exhibited significant antibacterial activity. The structure of compound 1 and 2 were deduced on the basis of their spectral data.

Quantitative determination of diterpenoid alkaloids in four species of Aconitum by HPLC.

A high performance liquid chromatography (HPLC) method has been developed for the determination of five principal alkaloids (benzoylmesaconine, mesaconitine, aconitine, hypaconitine and deoxyaconitine) found in four species of genus Aconitum. The five alkaloids were analyzed simultaneously with an XTerraRP18 column by gradient elution using 0.03 M ammonium hydrogen carbonate-acetonitrile as mobile phase. The recovery of the method was 94.6-101.9%, and all the alkaloids showed good linearity (gamma = 0.9999) in a relatively wide concentration range. The results indicated that contents of alkaloids in Aconitum varied significantly from species to species; hence quality control of Aconitum drugs is very necessary.

Aconitum napellus: veterinary materia medica

Aconitum napellus is an important homoeopathic medicine. Its botany and toxicology are described and traditional and homoeopathic medical indications discussed. The veterinary drug picture is presented, with the major therapeutic indications including fever and acute respiratory disease. Case studies from 6 species are given