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.

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.

[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.

Exploring the resources of the genus Viscum for potential therapeutic applications.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Viscum comprises approximately 100 species that are mainly distributed across Africa, Asia and Europe. The extracts and preparations of Viscum species are widely used as common complementary and alternative medicines in the treatment of rheumatism and cancer. AIM OF THE REVIEW: This review aims to explore the medicinal properties of twelve species belonging to the genus Viscum for potential therapeutic applications. MATERIALS AND METHODS: We collected online information (including PubMed, CNKI, Google Scholar, and Web of Science) from January 1915 to April 2021 and knowledge from classical books on Chinese herbal medicines available for 12 species of the genus Viscum, including Viscum coloratum (Kom.) Nakai, Viscum album L., Viscum articulatum Burm. f., Viscum liquidambaricola Hayata, Viscum ovalifolium DC., Viscum capitellatum Sm., Viscum cruciatum Sieber ex Boiss., Viscum nudum Danser, Viscum angulatum B.Heyne ex DC., Viscum tuberculatum A.Rich., Viscum multinerve Hayata, and Viscum diospyrosicola Hayata. RESULTS: At least 250 different compounds have been reported across twelve Viscum species, including amino acid and peptides, alkaloids, phenolic acids, flavonoids, terpenoids, carbohydrates, fatty acids, lipids, and other types of compounds. In particular, for Viscum coloratum (Kom.) Nakai and Viscum album L., the plants, preparations, and bioactive components have been thoroughly reviewed. This has allowed to elucidate the role of active components, including lectins, viscotoxins, flavonoids, terpenoids, phenolic acids, and polysaccharides, in multiple bioactivities, such as anti-cancer, anti-rheumatism arthralgia, anti-inflammation, anti-cardiovascular diseases, enhancing immunity, and anti-chemotherapy side effects. We also evaluated quality control methods based on active compounds, in vivo exposure compounds, and discriminated chemical markers. CONCLUSIONS: This is the first report to systematically review the pharmaceutical development history, chemical composition, clinical evidence, pharmacological activity, discriminated chemical markers, in vivo exposure, and quality control on twelve distinct species of Viscum plants with medicinal properties. The significant safety and efficacy, along with the minor side effects are constantly confirmed in clinics. The genus Viscum is thus an important medicinal resource that is worth exploring and developing in future pharmacological and chemical studies.

[Research progress on identification methods of growth years of traditional Chinese medicinal materials].

The growth years of traditional Chinese medicinal materials are closely related to their quality, which directly affects the efficacy and safety of clinical medication. Therefore, it is particularly important to establish an identification method for the growth years of traditional Chinese medicinal materials. In this review, the identification methods for the growth years of traditional Chinese medicinal materials were summarized systematically, and were divided into four types according to the identification principles and methods: traditional identification, molecular identification, physical/chemical identification, and integrated identification. Relying on rich experience, objective molecular markers, various physical/chemical methods and integrated identification techniques(including infrared spectroscopy, nuclear magnetic resonance spectroscopy, high performance liquid chromatography, gas chromatography, mass spectrometry, bionic identification technology and their tandem technologies, etc.), the differences of characters or chemical fingerprints were compared in depth. The growth years of traditional Chinese medicinal materials were quickly identified or predicted by the appearance and characters, the whole fingerprint information or the content of specific chemical markers, and their content ratios. Through the case analysis of mature varieties, we intend to promote the establishment of a perfect technology system for the identification of the growth years of traditional Chinese medicinal materials, and to provide a reference for other perennial herbal materials, finally resulting in the accurate and precise quality control of traditional Chinese medicinal materials.

Expansion within the CYP71D subfamily drives the heterocyclization of tanshinones synthesis in Salvia miltiorrhiza.

Tanshinones are the bioactive nor-diterpenoid constituents of the Chinese medicinal herb Danshen (Salvia miltiorrhiza). These groups of chemicals have the characteristic furan D-ring, which differentiates them from the phenolic abietane-type diterpenoids frequently found in the Lamiaceae family. However, how the 14,16-epoxy is formed has not been elucidated. Here, we report an improved genome assembly of Danshen using a highly homozygous genotype. We identify a cytochrome P450 (CYP71D) tandem gene array through gene expansion analysis. We show that CYP71D373 and CYP71D375 catalyze hydroxylation at carbon-16 (C16) and 14,16-ether (hetero)cyclization to form the D-ring, whereas CYP71D411 catalyzes upstream hydroxylation at C20. In addition, we discover a large biosynthetic gene cluster associated with tanshinone production. Collinearity analysis indicates a more specific origin of tanshinones in Salvia genus. It illustrates the evolutionary origin of abietane-type diterpenoids and those with a furan D-ring in Lamiaceae.

Quality tracing evaluation strategies of compatible materials in Aconitum proprietary Chinese medicines.

The proprietary Chinese medicine (PCM) has become a significant supplement of modern medicine. Nevertheless, the absence of quality control standard of compatible materials in PCM has led to serious adulteration, which has an extremely bad effect on safety of drug use and clinical efficacy. Here, a quality tracing evaluation strategy of compatible materials in 32 Aconitum proprietary Chinese medicines (APCMs) was established, including data normalization, model development, model verification, and unknown prescription cracking. The model was delimited based on the weighted content of total 9 key alkaloids in 24 APCMs, which were 5.65-57.10 µg/g for extract medicines and 42.62-380.61 µg/g for powder medicines. Three newly published commercial APCMs, including Wangbi Tablet, Wangbi Granule, and Fengshigutong Capsule, were used to verify its reliability and the results proved to be positive. Moreover, a novel prescription cracking approach was proposed to decode the content of each material in five unknown prescriptions including Yaoxitong Capsule, Tongrendahuoluo Pill, Xinbao Pill, Dahuoluo Capsule, and Mugua Pill. Ultimately, the single or two compatible Aconitum materials in APCMs was successfully decoded and the processed level of the materials were effectively judged. This study for the first time established a practical strategy for supervision and cracking of compatible materials in PCMs and is of great significance to improve the quality control of PCMs.

Limitation standard of toxic aconitines in Aconitum proprietary Chinese medicines using on-line extraction electrospray ionization mass spectrometry.

Development of rapid analytical methods and establishment of toxic component limitation standards are of great importance in quality control of traditional Chinese medicine. Herein, an on-line extraction electrospray ionization mass spectrometry (oEESI-MS) coupled with a novel whole process integral quantification strategy was developed and applied to direct determination of nine key aconitine-type alkaloids in 20 Aconitum proprietary Chinese medicines (APCMs). Multi-type dosage forms (e.g., tablets, capsules, pills, granules, and liquid preparation) of APCM could be determined directly with excellent versatility. The strategy has the characteristics of high throughput, good tolerance of matrix interference, small amount of sample (∼0.5 mg) and reagent (∼240 µL) consumption, and short analysis time for single sample (<15 min). The results were proved to be credible by high performance liquid chromatography-mass spectrometry (LC-MS) and electrospray ionization mass spectrometry, respectively. Moreover, the limitation standard for the toxic aconitines in 20 APCMs was established based on the holistic weight toxicity (HWT) evaluation and the Chinese Pharmacopoeia severally, and turned out that HWT-based toxicity evaluation results were closer to the real clinical applications. Hence, a more accurate and reliable APCM toxicity limitation was established and expected to play an important guiding role in clinics. The current study extended the power of ambient MS as a method for the direct quantification of molecules in complex samples, which is commonly required in pharmaceutical analysis, food safety control, public security, and many other disciplines.

Real-time toxicity prediction of Aconitum stewing system using extractive electrospray ionization mass spectrometry.

Due to numerous obstacles such as complex matrices, real-time monitoring of complex reaction systems (e.g., medicinal herb stewing system) has always been a challenge though great values for safe and rational use of drugs. Herein, facilitated by the potential ability on the tolerance of complex matrices of extractive electrospray ionization mass spectrometry, a device was established to realize continuous sampling and real-time quantitative analysis of herb stewing system for the first time. A complete analytical strategy, including data acquisition, data mining, and data evaluation was proposed and implemented with overcoming the usual difficulties in real-time mass spectrometry quantification. The complex Fuzi (the lateral root of Aconitum)-meat stewing systems were real-timely monitored in 150 min by qualitative and quantitative analysis of the nine key alkaloids accurately. The results showed that the strategy worked perfectly and the toxicity of the systems were evaluated and predicated accordingly. Stewing with trotters effectively accelerated the detoxification of Fuzi soup and reduced the overall toxicity to 68%, which was recommended to be used practically for treating rheumatic arthritis and enhancing immunity. The established strategy was versatile, simple, and accurate, which would have a wide application prospect in real-time analysis and evaluation of various complex reaction systems.