Deeper Chemical Perceptions for Better Traditional Chinese Medicine Standards

Traditional Chinese medicines (TCMs), a complex system of natural resources with many diverse components, are widely used as approved medicinal agents in China. Quality control of TCMs is a huge challenge for the government and for testing institutes and is associated with numerous scientific issues. Among these considerations include the following questions: How many components are in TCMs? How can the multiple components in TCMs be comprehensively delineated and subsequently characterized? What is the level and range of these (active) metabolites within these multiple-component TCMs, in order to recommend standards? and What are the qualities required for a marker constituent to be selected, and from a practical perspective, how can these components be assessed with low cost and in a short time? All of these factors require significant and deep thinking in order to understand the individualistic chemistry of TCM in order to develop enhanced TCM quality standards for improved and consistent patient care. In this review, the latest exploratory research in TCM chemistry analytical techniques and methods is summarized in order to begin to develop responses to these scientific issues. Advances in these methods have included multidimensional separation for liquid chromatography–high-resolution mass spectrometry (LC–HRMS), smart triggering data-dependent acquisition of LC–HRMS, target analysis with liquid chromatography–mass spectrometry (LC–MS), supercritical fluid chromatography, and data mining of large mass spectrometry (MS) datasets. In addition, two quality strategies have been introduced in order to save reference standards and the analysis time for a TCM quality standard, including the application of the single standard to determine multi-components (SSDMC) and monomethodheterotrait matrix methods. Finally, a series of future improvements for analytical methods for TCMs are proposed.

An integrated strategy for comprehensive characterization of metabolites and metabolic profiles of bufadienolides from Venenum Bufonis in rats

Comprehensive characterization of metabolites and metabolic profiles in plasma has considerable significance in determining the efficacy and safety of traditional Chinese medicine(TCM)in vivo.However,this process is usually hindered by the insufficient characteristic fragments of metabolites,ubiquitous matrix interference,and complicated screening and identification procedures for metabolites.In this study,an effective strategy was established to systematically characterize the metabolites,deduce the metabolic pathways,and describe the metabolic profiles of bufadienolides isolated from Venenum Bufonis in vivo.The strategy was divided into five steps.First,the blank and test plasma samples were injected into an ultra-high performance liquid chromatography/linear trap quadrupole-orbitrap-mass spectrometry(MS)system in the full scan mode continuously five times to screen for valid matrix compounds and metabolites.Second,an extension-mass defect filter model was established to obtain the targeted precursor ions of the list of bufadienolide metabolites,which reduced approximately 39%of the interfering ions.Third,an acquisition model was developed and used to trigger more tandem MS(MS/MS)fragments of precursor ions based on the targeted ion list.The acquisition mode enhanced the acquisition capability by approximately four times than that of the regular data-dependent acquisition mode.Fourth,the acquired data were imported into Compound Discoverer software for identification of metabolites with metabolic network prediction.The main in vivo metabolic pathways of bufadienolides were elucidated.A total of 147 metabolites were characterized,and the main biotransformation reactions of bufadienolides were hydroxylation,dihydroxylation,and isomerization.Finally,the main prototype bufadienolides in plasma at different time points were determined using LC-MS/MS,and the metabolic profiles were clearly identified.This strategy could be widely used to elucidate the metabolic profiles of TCM preparations or Chinese patent medicines in vivo and provide critical data for rational drug use.

Active constituents and mechanisms of Respiratory Detox Shot, a traditional Chinese medicine prescription, for COVID-19 control and prevention: Network-molecular docking-LC–MS^E analysis

Objective: Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot(RDS), was developed based on a classical prescription of traditional Chinese medicine(TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019(COVID-19), have been shown. However,the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS.Methods: To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein–protein interaction(PPI) network, and cluster analysis;further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS.Results: For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent–target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target–constitu ent–disease network. Twelve disease pathways attributed to the Lung–Large Intestine meridians, with six and five attributed to the Kidney–Urinary Bladder and Stomach–Spleen meridians, respectively. Onehundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-23-chymotrypsin-like protease(3 CLpro), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3 CLproinhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode,the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches.Conclusion: RDS acts primarily in the Lung–Large Intestine, Kidney–Urinary Bladder and Stomach–Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS’s dual effects of healthstrengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.

Current Status and Future Perspective in the Globalization of Traditional Chinese Medicines

Globalization of traditional Chinese medicines started around 1996, which was initiated by the Chinese government. However, substantial progress was only achieved in recent years including the adoption of TCM quality monographs in the western pharmacopoeias(United States Pharmacopoeia and European Pharmacopoeia) and registration in main stream drug regulatory agencies such as US Food and Drug Administration(FDA) and European Medicines Agency(EMA). So far, several TCM herbal quality monographs were adopted by the United States Pharmacopoeia including Chinese Salvia, Ganoderma lucidum and Panax notoginseng, etc. Over 45 TCM quality monographs were recorded in the European Pharmacopoeia with 20 more in progress. After the successful registration of the first TCM product named Diao Xin Xue Kang as traditional medicine via the Medicines Evaluation Board of the Netherlands, several other TCM herbal products are in the registration process in several European member states. So far, there has been still not any TCM product authorized as a drug by the FDA regardless of a few TCM products in phase III or phase II clinical trials. This review summarizes the progress made in the globalization of traditional Chinese medicines in recent years and future issues in this regard.

AN EFFICIENT AND TARGET-ORIENTED SAMPLE ENRICHMENT METHOD FOR PREPARATIVE SEPARATION OF MINOR ALKALOIDS BY PH-ZONE-REFINING COUNTER-CURRENT CHROMATOGRAPHY

A sample enrichment method focusing on the minor targeted components was established to help them to be successfully separated by pH-zone refining CCC.Seven minor indole alkaloids in Uncaria rhynchophylla(Miq.)Miq.ex Havil(UR)were chosen to show the advantage of this method.The sample enrichment and separation were

Chemical Analysis of Xueshuantong Lyophilized Powder by LC-MS Profiling

Objective To elucidate the chemical components of Xueshuantong （XST） Lyophilized Powder and primarily disclose the chemical difference between XST and Panax notoginseng roots. Methods Liquid chromatography coupled with electrospray ionization tandem mass spectrometry （LC-ESI-MSn） was used to profile the saponins in XST and P. notoginseng. Structural elucidation was based on spectral analyses of negative and positive ESI-MS3 data, and the comparison of the retention behaviors. Results The optimized LC-MS profiling approach enabled resolution of major saponins. The negative mode ESI-MS3 fragmentation gave diagnostic information on the nature （neutral loss 162 Da for GIc, 146 Da for Rha, and 132 Da for a pentose） and sequence （priority： terminal 〉 inner） of sugars and sapogenins （m/z 475 for protopanaxatriol; re^z459 for protopanaxadiol）, while the intact glycosyl portion could be characterized by characteristic Zoα, Cnnα＋, and Cnβ＋ （n = 2 or 3） obtained in the positive mode. Ultimately, a total of 30 saponins were characterized from XST. Compared with the roots of P. notoginseng, three malonyl-ginsenosides, ginsenoside Rd, and gyponoside XVII （or its isomer） were almost undetectable, and showed potential significance for their differentiation. Conclusion The established LC-MS profiling approach is powerful for the chemical analysis of P. notoginseng and its preparations such as XST.

Exploration of Bioactive Constituents and Immunoregulatory Mechanisms of a Hanshi-Yufei Formulation for Treating COVID-19

Objective:The objective of this study was to characterize the chemical compounds of a Hanshi-Yufei formulation(HSYF;a modified formulation of a traditional Chinese medicine used for treating COVID-19)to elucidate the mechanism of action and to evaluate potential anti-inflammatory effects of HSYF.Materials and Methods:The chemical constituents of HSYF extract were characterized using UPLC-Q-TOF/MS.Subsequently,a set of TCM network pharmacology methods was applied to identify disease-associated genes and to predict target profiles and pharmacological actions associated with the constituents of HSYF.Then,the antiviral effects of HSYF on H1N1 were assessed in RAW264.7 cells using MTT assays.Expression levels of pro-inflammatory cytokines interleukin(IL)-6 and tumor necrosis factor(TNF)-αfollowing infection of RAW264.7 cells with H1N1 were measured using an enzyme-linked immune sorbent assay(ELISA),and expression levels of inflammatory-related factors were detected using western blotting.Results:In total,165 chemical constituents(including glycosides,tannins,volatile oils,amino acids,triterpenoids,polyphenols,phenylpropanoids,sesquiterpenes,alkaloids,and flavonoids,among others)were tentatively identified in HSYF.Network pharmacology demonstrated that HSYF can regulate immunomodulatory-and anti-inflammatory-related targets of multiple pathways through its active ingredients,suggesting potential anti-COVID-19 effects.Furthermore,cell viability assays and ELISA showed that HSYF significantly inhibited H1N1 replication in RAW64.7 cells and markedly reduced expression of pro-inflammatory cytokines TNF-αand IL-6 at the proteins level.Conclusions:The results of the present study help improve our understanding of the therapeutic effects of HSYF in COVID-19 treatment from multi-level perspectives.

A precise and specific method for quick determination of sulfur fumigation for moutan cortex

Objective: Establish a quick, precise and specific method to determine whether the moutan cortex obtained from market is processed with sulfur; provide a reliable method for the scientific evaluation.Methods: Three methods, including acid-base titration method, high-performance liquid-chromatography(HPLC) method, and ultraperformance liquid chromatography coupled with time-of-flight mass spectrometry(UHPLC-TOF-MS) method were used to detect whether30 batches of samples were fumigated with sulfur.Results: The results of three methods were substantially the same. Fifteen batches were identified to have been processed with sulfur fumigation, while others were not.Conclusions: The HPLC method was found to be most appropriate for the determination of sulfur fumigation for moutan cortex.

DART-MS:A New Research Tool for Herbal Medicine Analysis

Direct analysis in real time(DART) possesses the merits of analyzing sample in its native status with minimal or even no sample pretreatment.In this review, we summarized the recent applications of DART in the field of herbal medicine analysis such as compound detection, species identification, metabolites profiling and initial quantification. DART with the characters of hyper-rapid, easy-hyphenated offers a new research tool for herbal medicines to complete the experimental process in a very simple but still reliable way. It is anticipated that more wide and deep applications of DART in herbal medicine analysis, as rapid quantification, high-throughput active compounds screening, rapid species identification, and fast illegal additives screening will be promising and foreseeable in the near future.

TRADITIONAL CHINESE MEDICINE IS MARCHING TOWARDS EVIDENCE-BASED MEDICINE

Traditional Chinese medicine(TCM)has over 3000 years of history treating diseases in China and has played a pivotal role in the Chinese health system.But the experience-based feature of traditional Chinese medicine requires for its scientific evidence in quality,safety and efficacy to evolve into evidence-based medicine.In the past 20 years or so,great progress has been achieved on the modernization

MHC-2D LC STRATEGY FOR THE HOLISTIC QUALITY CONTROL OF NOTOGINSENG-CONTAINING CHINESE PATENT MEDICINES

Current China Pharmacopoeia(ChP)standards employ diversified and case-dependent assay methods to evaluate the quality of different Chinese patent medicines(CPM)that contain Panax notoginseng as the monarch drug.These conventional HPLC-based approaches utilizing complex sample preparation procedure can easily result in low analytical efficiency and possible component loss.We thereby propose a'monomethodheterotrait matrix'(MHM)