Sensitive quantitation of ultra-trace toxic aconitines in complex matrices by perfusion nano-electrospray ionization mass spectrometry combined with gas-liquid microextraction.

The accurate analysis of ultra-trace (e.g. <10-4 ng/mL) substances in complex matrices is a burdensome but vital problem in pharmaceutical analysis, with important implications for precise quality control of drugs, discovery of innovative medicines and elucidation of pharmacological mechanisms. Herein, an innovative constant-flow perfusion nano-electrospray ionization (PnESI) technique was developed firstly features significant quantitative advantages in high-sensitivity ambient MS analysis of complex matrix sample. More importantly, double-labeled addition enrichment quantitation strategies of gas-liquid microextraction (GLME) were proposed for the first time, allowing highly selective extraction and enrichment of specific target analytes in a green and ultra-efficient (>1000-fold) manner. Using complex processed Aconitum herbs as example, PnESI-MS directly enabled the qualitative and absolute quantitative analysis of the processed Aconitum extracts and characterized the target toxic diester alkaloids with high sensitivity, high stability, wide linearity range, and strong resistance to matrix interference. Further, GLME device was applied to obtain the highly specific enrichment of the target diester alkaloids more than 1000-fold, and accurate absolute quantitation of trace aconitine, mesaconitine, and hypaconitine in the extracts of Heishunpian, Zhichuanwu and Zhicaowu was accomplished (e.g., 0.098 pg/mL and 0.143 pg/mL), with the quantitation results well below the LODs of aconitines from any analytical instruments available. This study built a systematic strategy for accurate quantitation of ultra-trace substances in complex matrix sample and expected to provide a technological revolution in many fields of pharmaceutical research.

Multitarget Protective Effects of JUB on Aß-Induced Neurotoxicity and the Mechanism Predication Using Network Pharmacology Analysis.

Amyloid-ß (Aß) is one of the core factors in the pathogenesis of Alzheimer's disease (AD), and the accumulation of its aggregates in the brain can form age-related plaques, leading to brain cell damage and intellectual decline, which may be the common intersection of all causes of neurotoxicity. Jujuboside B (JUB) has many characteristics such as hypnosis, sedation, antianxiety, and antioxidant stress. However, it is still unclear whether JuB can alleviate the neurotoxicity caused by Aß. Our study demonstrates that JUB improves learning and memory deficits in the nematode model. At the same time, JUB increases the antioxidant activity, prevents excessive accumulation of lipid synthesis, and resists endogenous lipofuscin deposition, thereby inhibiting the toxic effect of Aß. In vitro, JUB can improve Aß1-42-induced neuronal apoptosis level through the Bax/Bcl-2/caspase-3 signaling pathway and restore mitochondrial function in SH-SY5Y cells. The network pharmacology has been used to predict the potential neuroprotective mechanism of JUB. In summary, JUB exhibits neuroprotective properties employing both a neural cell and a nematode, which provides a basis for screening candidate ingredients for preventing AD.

Functional diversity of diterpene synthases in Aconitum plants.

Members of the Aconitum genus within the Ranunculaceae family are known to accumulate a broad array of medicinal and toxic diterpenoid alkaloids (DAs). Historically, ent-copalyl diphosphate (ent-CPP) was considered the sole precursor in DAs biosynthesis. However, the recent discovery of ent-8,13-CPP synthase in A. gymnandrum Maxim., which participates in ent-atiserene biosynthesis, raises the question of whether this gene is conserved throughout the Aconitum genus. In this study, RNA sequencing and PacBio Iso-sequencing were employed to identify diterpene synthases (diTPSs) in four additional Aconitum species with distinct DA compositions. In vitro and in vivo analyses functionally characterized a diverse array of 10 class II and 9 class I diTPSs. In addition to the identification of seven class II diTPSs as ent-CPP synthases, three other synthases generating ent-8,13-CPP, 8,13-CPP, and 8α-hydroxy-CPP were also discovered. Four class I kaurene synthases-like (KSLs) were observed to react with ent-CPP to yield ent-kaurene. Three KSLs not only reacted with ent-CPP but also ent-8,13-CPP to produce ent-atiserene. AsiKSL2-1 was found to react with 8α-hydroxy-CPP to produce Z-abienol and AsiKSL2-2 exhibited no activity with any of the four intermediates. This research delineates the known diterpene biosynthesis pathways in six Aconitum species and explores the highly divergent diterpene synthases within the genus, which are consistent with their phylogeny and may be responsible for the differential distribution of diterpenoid alkaloids in root and aerial parts. These findings contribute valuable insights into the diversification of diterpene biosynthesis and establish a solid foundation for future investigation into DA biosynthetic pathways in Aconitum.

Genome-Wide Characterization and Expression of the Hsf Gene Family in Salvia miltiorrhiza (Danshen) and the Potential Thermotolerance of SmHsf1 and SmHsf7 in Yeast.

Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese herb with significant medicinal value. The yield and quality of Danshen are greatly affected by climatic conditions, in particular high temperatures. Heat shock factors (Hsfs) play important regulatory roles in plant response to heat and other environmental stresses. However, little is currently known about the role played by the Hsf gene family in S. miltiorrhiza. Here, we identified 35 SmHsf genes and classified them into three major groups: SmHsfA (n = 22), SmHsfB (n = 11), and SmHsfC (n = 2) using phylogenetic analysis. The gene structure and protein motifs were relatively conserved within subgroups but diverged among the different groups. The expansion of the SmHsf gene family was mainly driven by whole-genome/segmental and dispersed gene duplications. The expression profile of SmHsfs in four distinct organs revealed its members (23/35) are predominantly expressed in the root. The expression of a large number of SmHsfs was regulated by drought, ultraviolet, heat and exogenous hormones. Notably, the SmHsf1 and SmHsf7 genes in SmHsfB2 were the most responsive to heat and are conserved between dicots and monocots. Finally, heterologous expression analysis showed that SmHsf1 and SmHsf7 enhance thermotolerance in yeast. Our results provide a solid foundation for further functional investigation of SmHsfs in Danshen plants as a response to abiotic stresses.

Rapid authentication of different herbal medicines by heating online extraction electrospray ionization mass spectrometry.

The rapid and accurate authentication of traditional Chinese medicines (TCMs) has always been a key scientific and technical problem in the field of pharmaceutical analysis. Herein, a novel heating online extraction electrospray ionization mass spectrometry (H-oEESI-MS) was developed for the rapid and direct analysis of extremely complex substances without the requirement for any sample pretreatment or pre-separation steps. The overall molecular profile and fragment structure features of various herbal medicines could be completely captured within 10-15 s, with minimal sample (<0.5 mg) and solvent consumption (<20 µL for one sample). Furthermore, a rapid differentiation and authentication strategy for TCMs based on H-oEESI-MS was proposed, including metabolic profile characterization, characteristic marker screening and identification, and multivariate statistical analysis model validation. In an analysis of 52 batches of seven types of Aconitum medicinal materials, 20 and 21 key compounds were screened out as the characteristic markers of raw and processed Aconitum herbal medicines, respectively, and the possible structures of all the characteristic markers were comprehensively identified based on Compound Discoverer databases. Finally, multivariate statistical analysis showed that all the different types of herbal medicines were well differentiated and identified (R2X > 0.87, R2Y > 0.91, and Q2 > 0.72), which further verified the feasibility and reliability of this comprehensive strategy for the rapid authentication of different TCMs based on H-oEESI-MS. In summary, this rapid authentication strategy realized the ultra-high-throughput, low-cost, and standardized detection of various complex TCMs for the first time, thereby demonstrating wide applicability and value for the development of quality standards for TCMs.

[Chemical reaction mechanism of decoction of traditional Chinese medicines: a review].

Complicated chemical reactions occur in the decoction of traditional Chinese medicines(TCMs) which features complex components, influencing the safety, efficacy, and quality controllability of TCMs. Therefore, it is particularly important to clarify the chemical reaction mechanism of TCMs in the decoction. This study summarized eight typical chemical reactions in the decoction of TCMs, such as substitution reaction, redox reaction, isomerization/stereoselective reaction, complexation, and supramolecular reaction. With the "toxicity attenuation and efficiency enhancement" of aconitines and other examples, this study reviewed the reactions in decoction of TCMs, which was expected to clarify the variation mechanisms of key chemical components in this process and to help guide medicine preparation and safe and rational use of medicine in clinical settings. The current main research methods for chemical reaction mechanisms of decoction of TCMs were also summed up and compared. The novel real-time analysis device of decoction system for TCMs was found to be efficient and simple without the pre-treatment of samples. This device provides a promising solution, which has great potential in quantity evaluation and control of TCMs. Moreover, it is expected to become a foundational and exemplary research tool, which can advance the research in this field.

Profiling aromatic constituents of Chimonanthus nitens Oliv. leaf granule using mass spectrometry.

RATIONALE: The chemical constituents of Chinese patent medicine are usually different from those of crude medicine because of specific preparation processes. Chimonanthus nitens Oliv. leaf granule is widely used for prevention against COVID-19 in China. However, no research has been reported on the chemical constituents of the granule and their variation during the preparation process. METHODS: Fragmentation patterns of reference compounds were investigated using electrospray ionization mass spectrometry, and the new gas-phase reaction was demonstrated by electronic and steric effects and calculated chemistry. Then, a strategy based on new fragmentation patterns was used to profile aromatic constituents. In addition, based on untargeted metabolomics analytical workflow, a comparison was made on the chemical constituents of the leaf and granule. RESULTS: New fragmentation patterns related to two competing reactions, ring-opening and ring-closing reactions for coumarin, have been proposed and investigated in depth. The newly established diagnostic ion at m/z 81.0331 worked strongly in the assignment of OH-7 and substituent at C-8 of coumarin. McLafferty rearrangement occurring in coumarin glycoside while sugar group locating at C-4 was first observed, and new diagnostic ions at m/z 147.0440, 119.0488, and 91.0543 were constructed. CONCLUSIONS: Aromatic constituents of the granule were first profiled. A total of 114 aromatic compounds were identified; of these 85 compounds were identified first. Kaempferol-7-O-neohesperidoside and its homologues were mostly enriched in the granule. Considering their reported bioactivities, these analogues possibly contribute greatly to clinical efficacy. Our results provided a new fragmentation theory for coumarins and a new material basis for the quality control of the granule.

Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production.

Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32 mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.

Research progress of quality control for the seed of Ziziphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Suan-Zao-Ren) and its proprietary Chinese medicines.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen Ziziphi Spinosae (SZS), the seed of Ziziphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Chinese name Suan-Zao-Ren), is widely distributed in China, Laos, Myanmar, and Iran. It is a classic traditional Chinese medicine with sedative and sleeping effects. In clinical practice, there are more than 155 proprietary Chinese medicines containing SZS. However, many commercial SZS products are difficult to qualify using current methods. Moreover, there is a scarcity of quality standards for SZS in proprietary Chinese medicines. AIM OF THE STUDY: The purpose of this study was to clearly reveal the quality indicators during the entire production process of SZS and its products. MATERIALS AND METHODS: This study reviewed more than 230 articles and related books on the quality control of SZS and its proprietary Chinese medicines published over the last 40 years (from January 1979 to October 2022). Moreover, where available, information on the quality of SZS and its proprietary Chinese medicines was also collected from websites for comparison, including online publications (e.g. PubMed, CNKI, Google Scholar, and Web of Science), the information at Yaozhi website and China Medical Information Platform, along with some classic books on Chinese herbal medicine. The literature and information search were conducted using keywords such as "Suan-Zao-Ren", " Ziziphus jujuba" and "quality control", and the latest results from various databases were combined to obtain valid information. The active components, which in vivo exposure, and Q-markers were also summarized. RESULTS: The jujuboside A, jujuboside B, and spinosin were revealed as the key Q-markers for SZS. Moreover, the advancements and prospects of the quality control for SZS and its extract, proprietary Chinese medicines, health foods, and adulterants were comprehensively summarized. The high-performance liquid chromatography-UV/evaporative light scattering detection and fingerprint analysis were found to be the mainstream methods for the SZS quality control. In particular, the novel quality evaluation method based on the unit content was applied for SZS and its proprietary Chinese medicines. Significant fluctuations were found in the contents of Q-markers. Moreover, the mass transfer rule of Q-markers was comprehensively clarified based on the entire production process, including production origins, ripening time, primary process, processing, compatibility decoction/extract, and storage. Ultimately, the crushing and compatibility of SZS were found to be the key steps affecting the active components. CONCLUSIONS: In short, this study provides solid evidences to reveal quality indicators for the entire production process of developing rational quality standards for SZS and its products. Moreover, this study also provides a template quality control overview, which could be extended to other traditional Chinese medicines.

Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids.

O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, Corydalis yanhusuo, a plant used in traditional Chinese medicine due to the analgesic effects of its BIA-active compounds, was employed to analyze the catalytic characteristics of O-methyltransferases in the formation of BIA diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an O-methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4'- and 6-O-methylations of 1-BIAs was found. CyOMT6 was found to perform two sequential methylations at both 9- and 2-positions of the essential intermediate of tetrahydroprotoberberines, (S)-scoulerine. Two O-methyltransferases (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with the 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2-O-methyltransferase) and the 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, results of integrated phylogenetic molecular docking analysis and site-directed mutation suggested that residues at sites 172, 306, 313, and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at the 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of the 6-position and to expand substrate scopes. This work provides insight into O-methyltransferases in producing BIA diversity in C. yanhusuo and genetic elements for producing BIAs by metabolic engineering and synthetic biology.

Compatibility of Fuzi and Ginseng Significantly Increase the Exposure of Aconitines.

The herb-pair ginseng-Fuzi (the root of Aconitum carmichaelii) is the material basis of Shenfu prescriptions and is popular in traditional Chinese medicine for the treatment of heart failure, and even shock with severe-stage of COVID-19. A narrow therapeutic window of Fuzi may cause significant regional loss of property and life in clinics. Therefore, systemic elucidation of active components is crucial to improve the safety dose window of Shenfu oral prescriptions. A high performance liquid chromatography-mass spectrometry method was developed for quantification of 10 aconitines in SD rat plasma within 9 min. The limit of detection and the limit of quantification were below 0.032 ng/ml and 0.095 ng/ml, respectively. Furthermore, a systemic comparison with their pharmacokinetic characteristics after oral administration of a safe dosage of 2 g/kg of Fuzi and ginseng-Fuzi decoction for 24 h was conducted. Eight representative diester, monoester, and non-ester aconitines and two new active components (i.e., songorine and indaconitine) were all adopted to elucidating the differences of the pharmacokinetic parameters in vivo. The compatibility of Fuzi and ginseng could significantly increase the in vivo exposure of active components. The terminal elimination half-life and the area under the concentration-time curve of mesaconitine, benzoylaconitine, benzoylmesaconitine, benzoylhypaconitine, and songorine were all increased significantly. The hypaconitine, benzoylmesaconitine, and songorine were regarded as the main active components in vivo, which gave an effective clue for the development of new Shenfu oral prescriptions.

Discovery of the directionally detoxification effect and chemical mechanism of Ginseng-Fuzi co-decoction based on real-time online filtration electrospray ionization mass spectrometry.

BACKGROUND: The synergic action of compound prescriptions is an important feature and core advantage of traditional medicine. Ginseng-Fuzi decoction is a classic compatible phytomedicine in China, of which Ginseng can effectively reduce the toxicity of Fuzi in clinical, but the detoxification chemical mechanism is still unclear. PURPOSE: Develop a novel method for real-time tracking and monitoring of complex substances in the decoction system of traditional Chinese medicine to uncover the detoxification effect Ginseng on Fuzi and explore the possible chemical reaction mechanism of Ginseng-Fuzi co-decoction. METHODS: A novel real-time monitoring system, online filtration electrospray ionization mass spectrometry, was developed for extremely complex substances analysis in the decoction of traditional medicine compounds to uncover the directionally detoxification effect and the mechanism of compatibility interaction. RESULTS: Nine key alkaloids and 7 ginsenosides in Ginseng-Fuzi decoction were simultaneously in-situ monitoring in positive ion mode or negative ion mode respectively. Both types of targeted analytes had satisfactory MS signal response for real-time qualitative and quantitative analysis with high precision (RSD < 14.04%) and low LLODs (0.002 ng/ml-10 ng/ml). Through long-term tracking analysis, the exact detoxification and synergistic effect of Ginseng-Fuzi decoction were confirmed as the concentration of main toxic alkaloids decreased (e.g. the content of mesaconitine has been reduced by about 38%) and the main active monoester alkaloids increased obviously. More importantly, the possible molecular mechanism of the detoxification effect of Ginseng compatibility was revealed for the first time, which was the nucleophilic substitution reaction of diester alkaloids catalyzed by fatty acids. CONCLUSION: This study revealed the exact effect of co-decoction of Ginseng and Fuzi at the molecular level and the chemical reaction mechanism of fatty acid-catalyzed degradation of toxic diester-type alkaloids. The comprehensive multi-component real-time monitoring strategy for complex traditional medicine compounds developed and implemented here has important demonstration significance for revealing the scientific connotation of the compatibility of compound traditional medicine.

Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases.

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2-7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.

[Construction of quality evaluation strategy for Chinese medicine based on ambient mass spectrometry].

Quality is the guarantee for the clinical safety and effectiveness of Chinese medicine. Accurate quality evaluation is the key to the standardization and modernization of Chinese medicine. Efforts have been made in improving Chinese medicine quality and strengthening the quality and safety supervision in China, but rapid and accurate quality evaluation of complex Chinese medicine samples is still a challenge. On the basis of the development of ambient mass spectrometry and the application in quality evaluation of complex Chinese medicine systems in recent years, the authors developed the multi-scenario Chinese medicine quality evaluation strategies. A systematic methodology was proposed in specific areas such as real-time monitoring of the quality of complex Chinese medicine decoction system, rapid toxicity grading of compound Chinese patent medicine, and evaluation of bulk medicinals of Chinese patent medicine. Allowing multi-scenario analysis of Chinese medicine, it is expected to provide universal research ideas and technical methods for rapid and accurate quality evaluation of Chinese medicine and boost the high-quality development of Chinese medicine industry.

Molecular differentiation of Panax notoginseng grown under different conditions by internal extractive electrospray ionization mass spectrometry and multivariate analysis.

Panax notoginseng is a highly valuable and widely used herb in traditional Chinese medicine. The quality and efficacy of Panax notoginseng grown under different conditions can greatly vary due to the differences in chemical composition. The analysis of chemical composition in Panax notoginseng typically involves various experimental steps including extraction, chromatographic separation and characterization, which can be time- and labor-consuming. Therefore, the efficient quality assessment and control of Panax notoginseng requires the development of more rapid methods for the chemical characterization and classification of Panax notoginseng. In this study, a method based on internal extractive electrospray ionization mass spectrometry (iEESI-MS) was developed to characterize chemical components of Panax notoginseng samples under different growth conditions (e.g., place of origin, soil quality, growth season) at the speed of 0.5 min per sample, without sample pretreatment and chromatographic separation. A total of 35 chemical components, including sugars, saponins, organic acids, etc., were identified in Panax notoginseng samples. Clear separation was observed in the multivariate analysis of the iEESI-MS data from Panax notoginseng samples grown under different conditions. The difference in the content of sucrose, fructose, Rg1, Rf, Rb1, Noto-R1, malonyl-Rb1, malonyl-Rg1, malonyl-Rf, Rd, Re, linoleic acid, palmitic acid and malic acid can be used as key characteristic indicators to discriminate origin, commercial specifications, and cultivation conditions of Panax notoginseng samples. The results of our study indicate the high power of iEESI-MS for the rapid molecular characterization and classification of Panax notoginseng under different growth conditions, which can be used for the quality assessment of traditional herbal medicines as well as in pharmaceutical and clinical analysis.

Functional identification of the terpene synthase family involved in diterpenoid alkaloids biosynthesis in Aconitum carmichaelii.

Aconitum carmichaelii is a high-value medicinal herb widely used across China, Japan, and other Asian countries. Aconitine-type diterpene alkaloids (DAs) are the characteristic compounds in Aconitum. Although six transcriptomes, based on short-read next generation sequencing technology, have been reported from the Aconitum species, the terpene synthase (TPS) corresponding to DAs biosynthesis remains unidentified. We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the A. carmichaelii transcriptome. Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b, TPS-c, and TPS-e/f subfamilies were identified. In vitro enzyme reaction analysis functional identified two sesqui-TPSs and twelve diTPSs. Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate ent-copalyl diphosphate. Five AcKSLs separately reacted with ent-CPP to produce ent-kaurene, ent-atiserene, and ent-13-epi-sandaracopimaradie: a new diterpene found in Aconitum. AcTPSs gene expression in conjunction DAs content analysis in different tissues validated that ent-CPP is the sole precursor to all DAs biosynthesis, with AcKSL1, AcKSL2s and AcKSL3-1 responsible for C20 atisine and napelline type DAs biosynthesis, respectively. These data clarified the molecular basis for the C20-DAs biosynthetic pathway in A. carmichaelii and pave the way for further exploration of C19-DAs biosynthesis in the Aconitum species.

Chemical-activity-based quality marker screening strategy for Viscum articulatum.

Viscum articulatum Burm. f. is a parasitic plant rich in flavonoids, triterpenoids, and catechins and has a high nutritional value. It has been reported that consuming V. articulatum can prevent cardiac diseases. In this study, six bioactive compounds, including catechins, triterpenoids, and phenylpropanoid glycosides, were determined in alcohol extracts of the plant using HPLC. The anti-inflammatory and antioxidant activities of three catechins, two triterpenoids, and three combination drugs were measured in cardiomyocytes, and the results showed that the anti-inflammatory activity was significantly enhanced while retaining strong antioxidant activity when epicatechin and ursolic acid were used in combination. The main quality markers epicatechin and ursolic acid were screened based on the specificity of the genuine herb and a potent synergistic effect, and the lowest limitation contents of V. articulatum which could discriminate it from some other taxonomically similar materials were accordingly determined. This self-built lowest limitation content of the two screened quality markers could quickly and accurately reflect the efficacy in terms of chemical composition and reverse the disorderly market use of nongenuine herbs or confusing species for adulteration. This study is of some significance for market regulation, drug development, and clinical medication.

Two New Alkaloids from Fuzi and Their Metabolites Study.

Former study suggests alkaloids from herbs of Aconitum genus plants possess excellent bioactivities, which exert great value for related deeper chemical constituent investigation. Herein, chemical isolation was performed and four alkaloids were isolated from Fuzi, of which two were new ones, and the other two were reported NMR data for the first time. Their chemical structures were identified by NMR data, high resolution MS, UV and IR analysis. Additionally, the MS fragmentation patterns were explored, formerly, that of hetisane alkaloid was rarely reported, and fragmentation mechanism of the diagnostic ion was proposed. Based on these fragment pathway, metabolites and metabolic pathways of four compounds were investigated in rat liver microsomes using UPLC-Q/TOF-MS, and dehydrogenation product was firstly found from metabolites of hetisane alkaloid.

The global profiling of alkaloids in Aconitum stapfianum and analysis of detoxification material basis against Fuzi.

Aconitum alkaloids are versatile in chemical structures and are well known for their bioactivity and toxicity. Cases of analogs with closely similar structures or positional isomers are widespread in herbs of the Aconitum genus. It is still challenging to rapidly identify unknown compounds via mass spectrometry, especially positional isomers. Herein, to profile the alkaloids of Aconitum stapfianum that possess bioactivity against intoxication by the lateral root of Aconitum carmichaelii (Fuzi), a strategy was developed by carefully determining the fragmentation pathways of authentic standards. A series of rules was summarized and involved charge site effects, hydrogen bonding effects, competitive channels between charge-remote reactions and charge migration reactions, and fragment patterns exhibiting a "diamond shape". Accordingly, a total of 124 alkaloids in A. stapfianum were tentatively characterized, including 85 potential new compounds and 24 sets of isomers. On the other hand, to explore the material basis of detoxification, a chemical constituent comparison was made between A. stapfianum and Fuzi, and principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to identify markers that were different between the two . In total, 25 characteristic markers were identified to discriminate between these two herbal medicines, of which 14 compounds were specific for A. stapfianum and most of them were characteristic for a para-substituted benzoic acid ester at C-14.

Online discovery of the molecular mechanism for directionally detoxification of Fuzi using real-time extractive electrospray ionization mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum carmichaelii Debeaux, a famous traditional medicinal herb for collapse, rheumatic fever, and painful joints, always raises global concerns about its fatal toxicity from toxic alkaloids when improperly processed. Therefore, it is urgent to clarify the internal molecular mechanism of processing detoxification on Aconitum and develop simple and reliable approaches for clinical application, which is also of great significance to the rational medicinal use of Aconitum. AIM OF THE STUDY: The study aimed at developing a complete molecular mechanism exploration strategy in complex medicinal herb decocting system, clarifying the internal molecular mechanism of processing detoxification on Aconitum, and exploring valid approaches for detoxification. MATERIALS AND METHODS: Aconiti Lateralis Radix Praeparata (Fuzi) was selected as the model for exploring the complex Aconitum detoxification mechanism using an advanced online real-time platform based on extractive electrospray ionization mass spectrometry. The methods realized the sensitive capture of dynamic trace intermediates, accurate qualitative and quantitative analysis, and real-time and long-term monitoring of multi-components with satisfactory accuracy and resistance to complex matrices. RESULTS: Components in the complex Aconitum decocting system were real-timely characterized and fat meat was discovered and verified to directionally detoxify Aconitum while reserving the therapy effect. More importantly, the dynamic detoxification mechanism in the chemically complex Aconitum decoction was molecularly profiled. A novel reaction pathway based on nucleophilic substitution reaction mechanism was proposed. As confirmed by the theoretic calculations at DFT B3LYP/6-31G (d) levels, fatty acids (e.g., palmitic acid) acted as a green, cheap, and high-performance catalyst and promote the decomposition of toxic diester alkaloids to non-toxic and active benzoyl-monoester alkaloids through the discovered mechanism. CONCLUSION: The study exposed a novel detoxification molecular mechanism of Aconitum and provided an effective method for the safe use of Aconitum, which could effectively guide the development of traditional processing technology and compatibility regulation of the toxic herb and had great value to the modernization and standardization development of traditional medicine.