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Isopentenyl diphosphate isomerase (IDI), a key enzyme in the biosynthetic pathway of diterpenoid alkaloids (DAs), plays an essential regulatory role in the synthesis and accumulation of DAs. In this study, the coding sequence (CDS) of AcIDI1 was isolated from the mother roots of Aconitum carmichaelii Debx. (GeneBank accession number OR915879). Bioinformatics analysis showed that the CDS of AcIDI1 was 894 bp, encoding a protein with 297 amino acids and the putative protein localized in the chloroplast. AcIDI1 exhibited significant homology with sequences encoding IDI in other species, and was most closely related to Aconitum vilmorinianum. Furthermore, the fusion protein has been successfully expressed in Escherichia coli (E. coli), providing a basis for future functional studies of AcIDI1. The expression pattern of AcIDI1 was analyzed by real-time quantitative PCR (qPCR), which demonstrates that AcIDI1 is a tissue-specific gene in the roots of A. carmichaelii and exhibits high expression in both daughter and mother roots. By comparing the expression levels of AcIDI1 in three tissues of the roots of A. carmichaelii at different growth stages, we propose that the mother roots (MRs) are the centers of resources allocation. The roots of A. carmichaelii continuously absorb the energy from external environment, while resources transfer behavior from MRs to both daughter roots (DRs) and axillary buds (ABs) occurs as the plant grows. This study establishes a foundation for applying the IDI gene to regulate the biosynthesis and accumulation of DAs in A. carmichaelii., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, we conducted an assembly and analysis of the organelle genomes of Aconitum carmichaelii. Our investigation encompassed the examination of organelle genome structures, gene transfer events, and the environmental selection pressures affecting A. carmichaelii. The results revealed distinct evolutionary patterns in the organelle genomes of A. carmichaelii. Especially, the plastome exhibited a more conserved structure but a higher nucleotide substitution rate (NSR), while the mitogenome displayed a more complex structure with a slower NSR. Through homology analysis, we identified several instances of unidirectional protein-coding genes (PCGs) transferring from the plastome to the mitogenome. However, we did not observe any events which genes moved from the mitogenome to the plastome. Additionally, we observed multiple transposable element (TE) fragments in the organelle genomes, with both organelles showing different preferences for the type of nuclear TE insertion. Divergence time estimation suggested that rapid differentiation occurred in Aconitum species approximately 7.96 million years ago (Mya). This divergence might be associated with the reduction in CO 2 levels and the significant uplift of the Qinghai-Tibet Plateau (QTP) during the late Miocene. Selection pressure analysis indicated that the dN/dS values of both organelles were less than 1, suggested that organelle PCGs were subject to purification selection. However, we did not detect any positively selected genes (PSGs) in Subg. Aconitum and Subg. Lycoctonum. This observation further supports the idea that stronger negative selection pressure on organelle genes in Aconitum results in a more conserved amino acid sequence. In conclusion, this study contributes to a deeper understanding of organelle evolution in Aconitum species and provides a foundation for future research on the genetic mechanisms underlying the structure and function of the Aconitum plastome and mitogenome., (© 2024. The Author(s).)

Diterpenoid alkaloids (DAs) have been often utilized in clinical practice due to their analgesic and anti-inflammatory properties. Natural DAs are prevalent in the family Ranunculaceae, notably in the Aconitum genus. Nevertheless, the evolutionary origin of the biosynthesis pathway responsible for DA production remains unknown. In this study, we successfully assembled a high-quality, pseudochromosome-level genome of the DA-rich species Aconitum vilmorinianum (A. vilmorinianum) (5.76 Gb). An A. vilmorinianum-specific whole-genome duplication event was discovered using comparative genomic analysis, which may aid in the evolution of the DA biosynthesis pathway. We identified several genes involved in DA biosynthesis via integrated genomic, transcriptomic, and metabolomic analyses. These genes included enzymes encoding target ent-kaurene oxidases and aminotransferases, which facilitated the activation of diterpenes and insertion of nitrogen atoms into diterpene skeletons, thereby mediating the transformation of diterpenes into DAs. The divergence periods of these genes in A. vilmorinianum were further assessed, and it was shown that two major types of genes were involved in the establishment of the DA biosynthesis pathway. Our integrated analysis offers fresh insights into the evolutionary origin of DAs in A. vilmorinianum as well as suggestions for engineering the biosynthetic pathways to obtain desired DAs., (© 2023 Institute of Botany, Chinese Academy of Sciences.)

Diterpene alkaloids (DAs) are characteristic compounds in Aconitum , which are classified into four skeletal types: C 18 , C 19 , C 20 , and bisditerpenoid alkaloids. C 20 -DAs are thought to be the precursor of the other types. Their biosynthetic pathway, however, is largely unclear. Herein, we combine metabolomics and transcriptomics to unveil the methyl jasmonate (MJ) inducible biosynthesis of DAs in the sterile seedling of A. gymnandrum , the only species in the Subgenus Gymnaconitum (Stapf) Rapaics. Target metabolomics based on root and aerial portions identified 51 C 19 -DAs and 15 C 20 -DAs, with 40 inducible compounds. The highest content of C 20 -DA atisine was selected for further network analysis. PacBio Isoform sequencing integrated with RNA sequencing not only provided the full-length transcriptome but also their response to induction, revealing 1994 genes that exhibited up-regulated expression. Further, 38 genes involved in terpenoid biosynthesis were identified, including 7 diterpene synthases. In addition to the expected function of the four diterpene synthases, AgCPS5 was identified to be a new ent -8,13-CPP synthase in Aconitum and could also combine with AgKSL1 to form the C 20 -DAs precursor ent -atiserene. Combined with multiple network analyses, six CYP450 and seven 2-ODD genes predicted to be involved in the biosynthesis of atisine were also identified. This study not only sheds light on diterpene synthase evolution in Aconitum but also provides a rich dataset of full-length transcriptomes, systemic metabolomes, and gene expression profiles, setting the groundwork for further investigation of the C 20 -DAs biosynthesis pathway.

Aconitum, as "the first drug of choice for invigorating Yang and saving lives", has been widely used for the treatment of heart failure. However, toxic components of Aconitum can easily lead to serious arrhythmia, even death (Y. CT., 2009; Zhang XM., 2018). In this study, a High Performance Liquid Chromatography (HPLC) method for the determination of aconitine (AC), mesaconitine (MA) and hypaconitine (HA) was established; The effect of Glycyrrhiza on CYP3A1 / 2 mRNA expression was detected by RT-PCR; SD rats were given Aconitum and compatibility of Glycyrrhizae and Aconitum by gavage respectively, the blood concentration of toxic components were determined by LC-MS / MS; The CHF rat model was established by intraperitoneal injection of adriamycin (2.5 mg / kg), and were randomly divided into model, Aconitum, the compatibility of Glycyrrhizae and Aconitum and Captopril group, 5 mice/group. After 4 weeks of gavage, the corresponding indexes were detected by ELISA and HPLC. The results showed that Ketoconazole significantly inhibited the metabolites of AC, MA and HA; Glycyrrhiza induced CYP3A gene expression; The level of ALD in the compatibility of Glycyrrhizae and Aconitum group was significantly lower than that in Aconitum group. After intervention with the compatibility of Glycyrrhizae and Aconitum, ATP increased, ADP decreased significantly. In conclusion, we found Glycyrrhiza promoted the metabolism of toxic components of Aconitum by up regulating the expression of CYP3A, and reduced the content of BNP, Ang II and ALD, improved the energy metabolism disorder of myocardium, alleviated the development of CHF., Competing Interests: The authors have declared that no competing interests exist.

In recent decades, herbal medicines have played more and more important roles in the healthcare system in the world because of the good efficacy. However, with the increasing use of herbal medicines, the toxicity induced by herbal medicines has become a global issue. Therefore, it is needed to investigate the mechanism behind the efficacy and toxicity of herbal medicines. In this study, using Aconiti Lateralis Radix Praeparata (Fuzi) as an example, we adopted a systems pharmacology approach to investigate the mechanism of Fuzi in treating rheumatoid arthritis and in inducing cardiac toxicity and neurotoxicity. The results showed that Fuzi has 25 bioactive compounds that act holistically on 61 targets and 27 pathways to treat rheumatoid arthritis, and modulation of inflammation state is one of the main mechanisms of Fuzi. In addition, the toxicity of Fuzi is linked to 32 compounds that act on 187 targets and 4 pathways, and the targets and pathways can directly modulate the flow of Na + , Ca 2+ , and K + . We also found out that non-toxic compounds such as myristic acid can act on targets of toxic compounds and therefore may influence the toxicity. The results not only reveal the efficacy and toxicity mechanism of Fuzi, but also add new concept for understanding the toxicity of herbal medicines, i.e., the compounds that are not directly toxic may influence the toxicity as well., (© 2021. The Author(s).)

The chemical constituents from the roots of Aconitum kongboense were studied. Twenty-five diterpenoid alkaloids were isolated from the 95% methanol extract of the roots of A. kongboense by silica gel, reverse-phase silica gel and basic alumina column chromatography. They included a new aconitine-type diterpenoid alkaloid, named as kongboensenine(1), and twenty-four known ones(2-25), i.e., acotarine F(2), acotarine G(3), 14-acetyltalatisamine(4), talatisamine(5), indaconitine(6), yunaconitine(7), chasmanine(8), 6-epi-foresticine(9), homochasmanine(10), 8-deacetyl-yunaconitine(11), chasmaconitine(12), ajaconine(13), franchetine(14), ezochasmanine(15), crassicautine(16), 14-O-deacylcrassicausine(17), genicunine A(18), falconeridine(19), sachaconitine(20), liljestrandisine(21), 8-methyl-14-acetyltalatisamine(22), kongboendine(23), 14-benzoylchasmanine(24) and pseudaconine(25). Their structures were elucidated by common spectroscopic methods including high-resolution electrospray ionization mass spectrometry(HR-ESI-MS) and nuclear magnetic resonance(NMR) techniques. Compounds 2-4, 10, 13, 15-19 and 21-22 were isolated from this plant for the first time. Experimental results showed that all compounds did not have a significant inhibitory activity against acetylcholinesterase(AChE).

Natural plants must actively allocate their limited resources for survival and reproduction. Although vegetative growth, sexual reproduction, asexual reproduction and defense are all basic processes in the life cycle of plants, the strategies used to allocate resources between these processes are poorly understood. These processes are conspicuous in naturally grown Aconitum kusnezoffii Reichb., which makes it a suitable study subject. Here, the morphology, dry matter, total organic carbon, total nitrogen and aconitum alkaloid levels of shoot, principal root (PR) and lateral roots were measured throughout the growing season. Then, transcriptome and metabolite content analyses were performed. We found that vegetative growth began first. After vegetative growth ceased, sexual development began. Flower organ development was accompanied by increased photosynthesis and the PR consumed temporarily stored resources after flower formation. Asexual propagule development initiated earlier than sexual reproduction and kept accumulating resources after that. Development was slow before flower formation, mainly manifesting as increasing length; then, after flower formation it accelerated via enhanced material transport and accumulation. Defense compounds were maintained at low levels before flowering. In particular, the turnover of defense compounds was enhanced before and after flower bud emergence, providing resources for other processes. After flower formation, defense compounds were accumulated. The pattern found herein provides a vivid example for further studies on resource allocation strategies. The exciting finding that the PR, as a more direct storage site for photosynthate, is a buffer unit for resources, and that defense compounds can be reused for other processes, suggests a need to explore potential mechanisms., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Endophytes have been recognized as a source for structurally novel and biologically active secondary metabolites. Among the host plants for endophytes, some medicinal plants that produce pharmaceuticals have been reported to carry endophytes, which could also produce bioactive secondary metabolites. In this study, the medicinal plant Aconitum carmichaeli was selected as a potential source for endophytes. An endophytic microorganism, Aureobasidium pullulans AJF1, harbored in the flower of Aconitum carmichaeli , was cultured on a large scale and extracted with an organic solvent. Extensive chemical investigation of the extracts resulted in isolation of three lipid type compounds ( 1 - 3 ), which were identified to be (3 R ,5 R )-3,5-dihydroxydecanoic acid ( 1 ), (3 R ,5 R )-3-(((3 R ,5 R )-3,5-dihydroxydecanoyl)oxy)-5-hydroxydecanoic acid ( 2 ), and (3 R ,5 R )-3-(((3 R ,5 R )-5-(((3 R ,5 R )-3,5-dihydroxydecanoyl)oxy)-3-hydroxydecanoyl)oxy)-5-hydroxydecanoic acid ( 3 ) by chemical methods in combination with spectral analysis. Compounds 2 and 3 had new structures. Absolute configurations of the isolated compounds ( 1 - 3 ) were established using modified Mosher's method together with analysis of NMR data for their acetonide derivatives. All the isolates ( 1 - 3 ) were evaluated for antibiotic activities against Escherichia coli , Staphylococcus aureus , Bacillus subtilis , Pseudomonas aeruginosa, and their cytotoxicities against MCF-7 cancer cells. Unfortunately, they showed low antibiotic activities and cytotoxic activities., Competing Interests: The authors declare no conflict of interest.

Songorine isolated from Aconitum brachypodum Diels possesses prominent activity of inhibiting G protein-coupled receptors (GPCRs) in the early screening process. In this paper, a series of Songorine derivatives were synthesized and their inhibitory activities on GPCRs were also evaluated by using the Double Antibody Sandwich ELISA (DAS-ELISA) in vitro. Among them, three derivatives (3a, 4, 7) exhibited significant inhibitory activity against GPCRs with IC 50 values of 0.08-0.29 nM. Moreover, the structure-activity relationships (SARs) of songorine derivatives were discussed in detail. They have great potentials as novel GPCRs antagonists in the future., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Trans -ferulic acid-4-β-glucoside (C 16 H 20 O₉, TFA-4β-G) is a monomer extracted from the Chinese medicine called radix aconiti carmichaeli (Fuzi). To date, research on this substance is lacking. Here, we found that trans -ferulic acid-4-β-glucoside effectively promoted cold acclimatization in mice via increased heat production and alleviation of oxidative stress in a cold environment. Thus, our work indicates that ferulic acid-4-β-glucoside is a potential therapeutic candidate for prevention and treatment of cold stress injury.

Aconitum carmichaelii has long been used as a traditional Chinese medicine, and its processed lateral roots are known commonly as fuzi. Aconitine-type C 19 -diterpenoid alkaloids accumulating in the lateral roots are some of the main toxicants of this species, yet their biosynthesis remains largely unresolved. As a first step towards understanding the biosynthesis of aconitine-type C 19 -diterpenoid alkaloids, we performed de novo transcriptome assembly and analysis of rootstocks and leaf tissues of Aconitum carmichaelii by next-generation sequencing. A total of 525 unigene candidates were identified as involved in the formation of C 19 -diterpenoid alkaloids, including those encoding enzymes in the early steps of diterpenoid alkaloids scaffold biosynthetic pathway, such as ent-copalyl diphosphate synthases, ent-kaurene synthases, kaurene oxidases, cyclases, and key aminotransferases. Furthermore, candidates responsible for decorating of diterpenoid alkaloid skeletons were discovered from transcriptome sequencing of fuzi, such as monooxygenases, methyltransferase, and BAHD acyltransferases. In addition, 645 differentially expressed genes encoding transcription factors potentially related to diterpenoid alkaloids accumulation underground were documented. Subsequent modular domain structure phylogenetics and differential expression analysis led to the identification of BAHD acyltransferases possibly involved in the formation of acetyl and benzoyl esters of diterpenoid alkaloids, associated with the acute toxicity of fuzi. The transcriptome data provide the foundation for future research into the molecular basis for aconitine-type C 19 -diterpenoid alkaloids biosynthesis in A. carmichaelii., (Copyright © 2018. Published by Elsevier Ltd.)

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., Competing Interests: The authors declare no conflict of interest.

A simple and sensitive method for determination of three aconitum alkaloids and their metabolites in human plasma was developed using matrix solid-phase dispersion combined with vortex-assisted dispersive liquid-liquid microextraction and high-performance liquid chromatography with diode array detection. The plasma sample was directly purified by matrix solid-phase dispersion and the eluate obtained was concentrated and further clarified by vortex-assisted dispersive liquid-liquid microextraction. Some important parameters affecting the extraction efficiency, such as type and amount of dispersing sorbent, type and volume of elution solvent, type and volume of extraction solvent, salt concentration as well as sample solution pH, were investigated in detail. Under optimal conditions, the proposed method has good repeatability and reproducibility with intraday and interday relative standard deviations lower than 5.44 and 5.75%, respectively. The recoveries of the aconitum alkaloids ranged from 73.81 to 101.82%, and the detection limits were achieved within the range of 1.6-2.1 ng/mL. The proposed method offered the advantages of good applicability, sensitivity, simplicity, and feasibility, which makes it suitable for the determination of trace amounts of aconitum alkaloids in human plasma samples., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The tuberous roots of Aconitum carmichaeli are largely used in traditional Chinese medicine and widely grown in Jiangyou, Sichuan, China. During the growth process, this medicinal plant releases a large amount of allelochemicals into soil, which retard the growth and development of near and late crops. Therefore, a pure culture experiment was thus carried out by seed soaking to study the allelopathic effects of extracts from tuberous roots of A. carmichaeli (ETR) on the seed germination and young seedling growth of Lolium perenne, Trifolium repens, and Medicago sativa, the late pasture grasses after cultivation of A. carmichaeli. The results showed that three pasture grasses varied significantly in seed germination and young seedling growth in response to ETR concentrations. Seed germination of M. sativa was stimulated by low ERT concentration (0.01 x g(-1)), while all of pasture grass seeds germinated poorly in solution with 1.00 g x L(-1). Seed soaking with 1.00 g x L(-1) also inhibited significantly the growth of pasture young seedlings, with M. sativa showing the highest seedling height reduction of 42.05% in seeding height, followed by T. repens (40.21%) and L. perenne with about 11%. Cultivation of L. perenne could thus be beneficial to increase whole land productivity in A. carmichaeli-pasture grass cropping systems. In addition, hydrolysis of protein, starch, and inositol phosphates was blocked and free amino acids, soluble sugars and phosphorus were decreased in seeds by seed soaking with ETR, which could be one of the reason for the inhibition of seed germination. There was a significant reduction in root vigor, nitrate reductase, and chlorophyll after the seed treatment with ETR, indicating the suppression of nutrient uptake, nitrate assimilation, and photosynthesis by allelopathic chemicals in ETR, which could lead to the slow growth rate of pasture grass seedlings.

To authenticate Ayurvedic medicinal plants Ativisha (Aconitum heterophyllum) and Musta (Cyperus rotundus) at the raw drug source and in prepared herbal products, nrDNA ITS sequence based SCAR markers were designed and validated spp.-specific SCAR primers gave amplicon of 415 bp and 134 bp, respectively, in authentic species. The SCAR primers (Cyr-FP and Cyr-RP) could identify tissue sample containing 750 μg to 4.76 mg/100mg of Musta in complex mixtures of DNA extracted from commercial herbal drugs. Ativisha could not be identified through SCAR markers suggesting that authentic species may not been used to prepare herbal drugs despite its being labelled as one of the ingredients in formulations. Analysis of individual tubers of Ativisha and Musta assures the presence of admixtures in raw drug trade of Ativisha, indicates the need to monitor the basic raw material supply and concludes, supplying plant materials through cultivation to manufacturing industries can minimize the risks of adulteration., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: Metabolomics is an 'omics' approach that aims to comprehensively analyse all metabolites in a biological sample, and has great potential for directly elucidating plant metabolic processes. Increasing evidence supports the view that plants produce a broad range of low-molecular-weight secondary metabolites responsible for variation from species to species, thus enabling the use of secondary metabolite profiling in the chemotaxonomy., Objective: To gain deeper insights into the metabolites to increasing plant diversity, we performed systematic untargeted metabolite profiling to exploit the different parts and species of Aconitum as a case study., Method: Application of ultraperformance liquid chromatography-quadrupole time-of-flight-high-definition mass spectrometry (UPLC-QTOF-HDMS) equipped with electrospray ionisation and coupled with pattern recognition analyses to study constituents in the root of two kinds of Aconitum species., Results: Twenty-two metabolites between the mother root of Aconitum carmichaelii Debx (CHW) and lateral root of Aconitum carmichaelii Debx (SFZ) and 13 metabolites between the CHW and root of Aconitum kusnezoffii Reichb (CW) have been identified. Of note, songorine, carmichaeline and isotalatizidine did not exist in CW, whereas they are present in the SFZ and CHW., Conclusion: Metabolomics based UPLC-QTOF-HDMS with multivariate statistical models was effective for analysis of constituents in the root of two kinds of Aconitum species., (Copyright © 2012 John Wiley & Sons, Ltd.)

Aconitum heterophyllum is a high altitude medicinal plant that has become endangered due to overexploitation for their aconitins. The most effective, conventional propagation method for any plant species is by seed. However, in Aconitum seed germination is erratic, and seedling survival is low. In the present study results have been discussed on the possible implication of ethanol treatment on removal of barriers on radical emergence in terms of protein changes. Eighty seven percent of seed germination was achieved in Aconitum with ethanol treatment. Comparative 2-DE analysis of ethanol treated and untreated seed protein profiles in Phase II of germination revealed 40 differentially expressed proteins. Twenty-seven out of 40 proteins were induced, 5 were increased and 8 were repressed. Mass spectrometry and subsequent identification confirmed that these proteins were involved in metabolism, DNA regulation, stress tolerance and plasmamembrane/cell wall biosynthesis/extension processes. These protein changes might be responsible for physiological and physical changes, respectively, resulted in increase in germination percentage. Further, characterization of these proteins will be of great help in understanding the molecular mechanism lying behind enhanced germination in response to ethanol treatment., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The lateral root of Aconitum carmichaelii Debx is named "Fuzi" which is widely distributed across Asia and North America and has been used to relieve joint pain and treat rheumatic diseases for over two thousand years. However, it has very narrow therapeutic ranges and despite the toxicological risk, its usage remains very high. A traditional Chinese processing approach (Paozhi, detoxifying measure) is necessary to remove the poisonous Aconitum alkaloids mainly deriving from the diester diterpene alkaloids (DDAs) including aconitine, mesaconitine and hypaconitine. They can be decomposed into less or non-toxic derivatives through Paozhi that plays an essential role in detoxification. Processed Fuzi is mainly focused on the three main forms of Yanfuzi (YFZ), Heishunpian (HSP) and Baifupian (BFP) which are highly desirable in order to guarantee the clinical safety and their low toxicity in decoctions. The difference in metabolomic characters between Fuzi and its processed preparations is still completely unclear. Therefore, this paper was designed to investigate a comprehensive metabolome of Fuzi and its processed products by ultra-performance liquid-chromatography/electrospray-ionization synapt high-definition mass spectrometry (UPLC-Q-TOF-HDMS) combined with pattern recognition methods. The difference in metabolic profiles between Fuzi and its processed preparations was well observed by the principal component analysis (PCA) of the MS spectra. Significant changes of 19 metabolite biomarkers were detected in the Fuzi samples and three preparations. The underlying regulations of Paozhi-perturbed metabolic pathways were also discussed according to the identified metabolites. The present study proves that UPLC-Q-TOF-HDMS based metabolomic analysis greatly contributes to the investigation of Fuzi metabolism through Paozhi techniques, and provides useful information to further comprehensively understand the pharmacological activity and potential toxicity of processed Fuzi in a clinical environment., (This journal is © The Royal Society of Chemistry 2012)

Context: In traditional Chinese medicine, Aconitum (Ranunculaceae) roots are only applied after processing. Nevertheless, several cases of poisoning by improperly processed aconite roots have been reported., Objective: The aim of this study was to develop a reliable analytical method to assess the amount of toxic aconite alkaloids in commercial aconite roots, and to compare this method with the commonly used total alkaloid content determination by titration., Materials and Methods: The content of mesaconitine, aconitine, and hypaconitine in 16 commercial samples of processed aconite roots was determined by an HPLC method and the total alkaloid content by indirect titration. Five samples were selected for in vivo toxicological investigation., Results: In most of the commercial samples, toxic alkaloids were not detectable, or only traces were found. In four samples, we could detect >0.04% toxic aconite alkaloids, the highest with a content of 0.16%. The results of HPLC analysis were compared with the results obtained by titration, and no correlation was found between the two methods. The in vivo results reassured the validity of the HPLC determination., Discussion and Conclusion: Samples with mesaconitine, aconitine, and hypaconitine content below the HPLC detection limit still contained up to 0.2% alkaloids determined by titration. Since titration of alkaloids gives no information selectively on the aconitine-type alkaloid content and toxicity of aconite roots this method is not appropriate for safety assessment. The HPLC method developed by us provides a quick and reliable assessment of toxicity and should be considered as a purity test in pharmacopoeia monographs.

Objective: To study the polysaccharides contents and monosaccharide compositions in parent root, daughter root and rootlet of Aconitum carmichaeli., Method: The conversion coefficient of A. carmichaeli polysaccharide to glucose was obtained by refined polysaccharides, and then the contents of crude polysaccharides in parent root, daughter root and rootlet were determined by sulfuric-phenol spectrometry method; analysis of monosaccharide compositions in polysaccharides from A. carmichaeli was carried out by pre-column derivatization high performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone (PMP)., Result: The contents of polysaccharides in parent root, daughter root and rootlet were 22.02%, 33.53% and 6.10%, respectively. Parent root, daughter root and rootlet mainly contained glucose, and in addition they contained a small amount of galacturonic acid, galactose and arabinose. Daughter root contained mannose yet, and rootlet still contained mannose, rhamnose and xylose., Conclusion: The method is simple, rapid, and accurate. The content of polysaccharide in rootlet is lowest, and monosaccharide compositions in rootlet are significantly different from parent root and daughter root.

Intensity fading matrix-assisted laser desorption/ionization (IF-MALDI) mass spectrometry has become an alternative screening approach for the affinity-binding analysis of proteins and peptides with ligands. In this study, an attempt has been made to study the interaction of alpha 1-acid glycoprotein (AGP) with aconitum alkaloids by IF-MALDI Fourier transform ion cyclotron resonance mass spectrometry (IF-MALDI-FT-MS). Compared with the nonbinding internal standard, clear reduction in the ion abundances of the target alkaloids was observed with the addition of AGP. Relative binding affinities of different alkaloids towards the protein could also be estimated using IF-MALDI-FT-MS. The binding affinity was also investigated by using ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization mass spectrometry (ultrafiltration LC-DAD/ESI-MS), and results were consistent with that of IF-MALDI-FT-MS., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objective: To study the effects of Fe, Zn, B and Mn fertilizer with different ratio on the yield and quality of Aconitum carmichaeli., Method: Field experiment with the uniform design was applied, the yield and the contents of the total alkaloids and diester-alkaloids were measured., Result: Fe, Zn, B and Mn fertilizer of appropriate ratio could promote the growth of vegetative organs, increase the biomass, the content of alkaloids and the yield of Aconite significantly. Fe, Zn fertilizer of highly concentrated ratio increased the proportion of first sub-roots, but inhibited the growth of other vegetative organs, the number of roots was less than that with other treatments, so it was not conducive to the formation of production. High concentration of Mn was not conducive to the growth of underground of Aconite, its number of sub-roots was fewer, but the number of third sub-roots was more than that with other treatments, the yield was low. The yield treated with low concentration of B was 10% higher than that with high concentration, and the high concentration of B was not conducive to increase the content of the alkaloids. Among these treatments, The fourth treatment was the optimal combination, of which the volume of sub-roots was the largest and the most homogeneous, the growth of the vegetative organs was better and the accumulation of dry matters was more, the yield of this treatment was 10,754.7 kg x hm(-2), which was increased by 14.9%, and the content of alkaloid was increased by 13.9%., Conclusion: The ratio of 4 is the best treatment for high yield and quality cultivation of Aconite.

Processed Bushi powder for ethical dispensing, called TJ-3022, is a herbal drug of processed Aconiti tuber (Aconitum carmichaeli Debeaux) and TJ-3023 is newly developed to contain a higher proportion of diester alkaloid of aconitine (Aconitum carmichaeli Debeaux and Aconitum japonicum Thunberg). Safety of TJ-3022 and TJ-3023 was evaluated by measuring the level of aconitum alkaloids in healthy adult volunteers. Forty subjects were assigned to one of two groups (each 20 subjects): TJ-3022 group or TJ-3023 group. The subjects received the powdered processed Aconiti tuber 3 g/day and the blood concentrations of aconitum alkaloids were measured at 90 min and 72 h after taking the study drug. The serum concentrations of aconitum alkaloids after 90 min and 72 h in the TJ-3023 group were higher than those in the TJ-3022 group. As for the chronological changes in the serum concentration, a significant decrease was observed in the TJ-3022 group, while no significant decrease was seen in the TJ-3023 group, which suggests that an analgesic effect in TJ-3023 was stronger than in TJ-3022. Aconitum alkaloids, which always have been believed to have the blood concentration below the measurement limit in human, were detected for the first time, although the detected amounts were minute. The results suggest that TJ-3023 shows sufficient analgesic effect with smaller dose than TJ-3022.

Oxidative stress and mitochondrial dysfunction are acute consequences of status epilepticus (SE). However, the role of mitochondrial oxidative stress and genomic instability during epileptogenesis remains unknown. Using the kainate animal model of temporal lobe epilepsy, we investigated oxidative mitochondrial DNA (mtDNA) damage and changes in the mitochondrial base excision repair pathway (mtBER) in the rat hippocampus for a period of 3 months after SE. Acute seizure activity caused a time-dependent increase in mitochondrial, but not nuclear 8-hydroxy-2-deoxyguanosine (8-OHdG/2dG) levels and a greater frequency of mtDNA lesions. This was accompanied by increased mitochondrial H2O2 production and a transient decrease in mtDNA repair capacity. The mtBER proteins 8-oxoguanine glycosylase (Ogg1) and DNA polymerase gamma (Pol gamma) demonstrated elevated expression at mRNA and protein levels shortly after SE and this was followed by a gradual improvement in mtDNA repair capacity. Recurrent seizures associated with the chronic phase of epilepsy coincided with the accumulation of mtDNA damage, increased mitochondrial H2O2 levels, decreased expression of Ogg1 and Pol gamma and impaired mtDNA repair capacity. Together, increased oxidative mtDNA damage, mitochondrial H2O2 production and alterations in the mtBER pathway provide evidence for mitochondrial oxidative stress in epilepsy and suggest that mitochondrial injury may contribute to epileptogenesis.

We have previously shown that seizures induce the formation of F(2)-isoprostanes (F(2)-IsoPs), one of the most reliable indices of oxidative stress in vivo. Isofurans (IsoFs) are novel products of lipid peroxidation whose formation is favored by high oxygen tensions. In contrast, high oxygen tensions suppress the formation of F(2)-IsoPs. The present study determined seizure-induced formation of IsoFs and its relationship with cellular oxygen levels (pO2). Status epilepticus (SE) resulted in F(2)-IsoP and IsoF formation, with overlapping but distinct time courses in hippocampal subregions. IsoF, but not F(2)-IsoP formation coincided with mitochondrial oxidative stress. SE resulted in a transient decrease in hippocampal pO2 measured by in vivo electron paramagnetic resonance oximetry suggesting an early phase of seizure-induced hypoxia. Seizure-induced F(2)-IsoP formation coincided with the peak hypoxia phase, whereas IsoF formation coincided with the 'reoxygenation' phase. These results demonstrate seizure-induced increase in IsoF formation and its correlation with changes in hippocampal pO2 and mitochondrial dysfunction.

The Aconitum species (Ranunculaceae) are widely distributed in northern Asia and North America. Their roots are popularly used in herbal medicines in China and Japan. Many cases of accidental, suicidal and homicidal intoxication with this plant have been reported; some of these were fatal because the toxicity of Aconitum is very high. It is thus important to detect and quantify Aconitum alkaloids in body fluids, with high sensitivity. We have developed a simple and sensitive method for measuring four kinds of Aconitum alkaloids (aconitine, hypaconitine, jesaconitine and mesaconitine) by LC/electrospray (ESI)-time-of-flight (TOF)-MS. For all of them, only molecular ions were observed at an orifice voltage of 75 V; at 135 V, base peaks corresponding to [M - 60 + H]+ ions were observed. These four compounds and methyllycaconitine (internal standard) in human plasma samples were purified by solid-phase extraction. The four extracted compounds were completely separated in mass chromatograms; the calibration curves showed good linearity in the range 10-300 ng/ml, and the detection limits were estimated to be 0.2-0.5 ng/ml. Using our method, we also determined the amounts of these compounds in tuber samples. The present method is applicable in clinical and forensic toxicology., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Salmonella typhimurium possesses at least three inducible transport systems for the tricarboxylic acids (citric, isocitric, cis-aconitic, and tricarballylic). The first system was induced by citrate, isocitrate, or cis-aconitate, and transported citric acid and isocitric acid. The second system was also induced by the same acids as in the first system and transported cis-aconitic acid. This system required Mg(2+) ions and was stable at pH 8.4 but unstable at pH 7.0. The metal ion was replaced with Sr(2+) or Ca(2+) ions but not with Ba(2+) ions. The third system was induced by tricarballylate and transported citric acid, cis-aconitic acid, and tricarballylic acid.