Holistic quality evaluation of commercial Agastache rugosa by multiple chromatographic and chemometric analysis.

In present study, a comprehensive strategy integrating multiple chromatographic and chemometric methods to simultaneously characterize the volatile and non-volatile components was developed for the holistic quality evaluation of commercial Agastache rugosa (AR), a common edible and medicinal herb, collected in China. The volatile components and the non-volatile components were characterized by GC-MS and UPLC-QTOF-MS/MS, respectively. And the data were analyzed either independently or integratively by multivariate statistical analysis (MVS) for the quality assessment of commercial samples. The results revealed that the commercial AR samples were different in both the composition and the content of volatile components. However, the compositions of non-volatile components in commercial AR were generally similar, whereas the contents of some components were different. All the results indicated that the holistic quality of commercial AR was inconsistent, and the commercial samples collected could be classified into two main groups, the volatile components were majorly responsible for the classification. Whether or not the holistic quality variations affect the efficacy of AR deserves further investigation.

Classification-based strategies to simplify complex traditional Chinese medicine (TCM) researches through liquid chromatography-mass spectrometry in the last decade (2011-2020): Theory, technical route and difficulty.

The difficulty of traditional Chinese medicine (TCM) researches lies in the complexity of components, metabolites, and bioactivities. For a long time, there has been a lack of connections among the three parts, which is not conducive to the systematic elucidation of TCM effectiveness. To overcome this problem, a classification-based methodology for simplifying TCM researches was refined from literature in the past 10 years (2011-2020). The theoretical basis of this methodology is set theory, and its core concept is classification. Its starting point is that "although TCM may contain hundreds of compounds, the vast majority of these compounds are structurally similar". The methodology is composed by research strategies for components, metabolites and bioactivities of TCM, which are the three main parts of the review. Technical route, key steps and difficulty are introduced in each part. Two perspectives are highlighted in this review: set theory is a theoretical basis for all strategies from a conceptual perspective, and liquid chromatography-mass spectrometry (LC-MS) is a common tool for all strategies from a technical perspective. The significance of these strategies is to simplify complex TCM researches, integrate isolated TCM researches, and build a bridge between traditional medicines and modern medicines. Potential research hotspots in the future, such as discovery of bioactive ingredients from TCM metabolites, are also discussed. The classification-based methodology is a summary of research experience in the past 10 years. We believe it will definitely provide support and reference for the following TCM researches.

Lipidomics characterization of the mechanism of Cynomorium songaricum polysaccharide on treating type 2 diabetes.

Although Cynomorium songaricum Rupr. polysaccharide (CSP) has been examined for its effects on glucose regulation, its underlying mechanism is still unclear. To address this issue, a MS-based lipidomics strategy was developed to gain a system-level understanding of the mechanism of CSP on improving type 2 diabetes mellitus (T2DM). UPLC-QTOF/MS and multivariate statistical tools were used to identify the alteration of serum metabolites associated with T2DM and responses to CSP treatment. As a result, 35 potential biomarkers were found and identified in serum, amongst which 26 metabolites were regulated to normal like levels after the administration of CSP. By analyzing the metabolic pathways, glycerophospholipid metabolism was suggested to be closely involved. These results indicated that the intake of CSP exhibited promising anti-diabetic activity, largely due to the regulation of phospholipid metabolism, including phosphatidylcholines, lysophosphatydylcholines, phosphtatidylethanolamines and sphingomyelins.

A metabolomic strategy based on integrating headspace gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry to differentiate the five cultivars of Chrysanthemum flower.

The extreme complexity of the chemical composition of plant extracts requires an unbiased and comprehensive detection methodology to improve the potential of metabolomic study. The present work, taking five closely related cultivars of Chrysanthemum flowers as a typical case, attempts to develop a metabolomic strategy to find more markers of metabolites for precise differentiation based on headspace gas chromatography-mass spectrometry (HSGC-MS) and ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). In detail, 53 batches of Chrysanthemum flower samples were collected and analyzed. The fusion of datasets from HSGC-MS and UHPLC-QTOF/MS was done in two different ways. After comparison, the fusion of the total peak area normalized metabolomic data was performed for multivariate statistical analysis. A total of 21 marker compounds (including 14 volatile and 7 nonvolatile metabolites) were identified, and a heatmap was employed for clarifying the distribution of the identified metabolites among the five cultivars. The results indicated that the integrated platform benefited the metabolomic study of medicinal and edible herbs by providing complementary information through fully monitoring functional constituents.

[Ideas and methods on efficient screening of traditional medicines for anti-osteoporosis activity based on M-Act/Tox integrated evaluation using zebrafish].

The increasingly apparent liver injury problems of bone strengthening Chinese medicines have brought challenges for clinical application, and it is necessary to consider both effectiveness and safety in screening anti-osteoporosis Chinese medicines. Metabolic transformation is closely related to drug efficacy and toxicity, so it is significant to comprehensively consider metabolism-action/toxicity(M-Act/Tox) for screening anti-osteoporosis Chinese medicines. The current evaluation models and the number of compounds(including metabolites) severely restrict efficient screening in vivo. By referring to previous relevant research and domestic and abroad literature, zebrafish M-Act/Tox integrative method was put forward for efficiently screening anti-osteoporosis herb medicines, which has organically integrated zebrafish metabolism model, osteoporosis model and toxicity evaluation method. This method can break through the bottleneck and blind spots that trace compositions can't achieve efficient and integrated in vivo evaluation, and realize both efficient and comprehensive screening on anti-osteoporosis traditional medicines based on in vivo process taking both safety and effectiveness into account, which is significant to accelerate discovery of effective and safe innovative traditional Chinese medicines for osteoporosis.

[Research and application of hepatotoxicity evaluation technique of traditional Chinese medicine].

The safety of traditional Chinese medicine (TCM) has received the widespread attention in recent years. Hepatotoxicity of TCM is one of the key problems of the safety of TCM. This article summarized research progress and application prospect in the mechanism of TCM hepatotoxicity, biomarkers, toxic omics database, prevention of hepatotoxicity of the liver cell lines, subcellular fraction, three-dimensional cultivation models, the model animals, aiming to provide theoretical basis for TCM toxicity evaluation and technical guidelines, thus promoting the development of TCM toxicity studies. Hope for Chinese medicine liver toxicity evaluation method provides the theoretical foundation and technical guidelines, promote the development and improvement of TCM liver toxicity research system.

Rapid identification of herbal compounds derived metabolites using zebrafish larvae as the biotransformation system.

Metabolites derived from herbal compounds are becoming promising sources for discovering new drugs. However, the rapid identification of metabolites from biological matrixes is limited by massive endogenous interference and low abundance of metabolites. Thus, by using zebrafish larvae as the biotransformation system, we herein proposed and validated an integrated strategy for rapid identification of metabolites derived from herbal compounds. Two pivotal steps involved in this strategy are to differentiate metabolites from herbal compounds and match metabolites with their parent compounds. The differentiation step was achieved by cross orthogonal partial least-squares discriminant analysis. Automatic matching analysis was performed on R Project based on a self-developed program, of which the number of matched ionic clusters and its corresponding percentage between metabolite and parent compound were taken into account to assess their similarity. Using this strategy, 46 metabolites screened from incubation water samples of zebrafish treated with total Epimedium flavonoids (EFs) could be matched with their corresponding parent compounds, 37 of them were identified and validated by the known metabolic pathways and fragmentation patterns. Finally, 75% of the identified EFs metabolites were successfully detected in urine samples of rats treated with EFs. These experimental results indicate that the proposed strategy using zebrafish larvae as the biotransformation system will facilitate the rapid identification of metabolites derived from herbal compounds, which shows promising perspectives in providing additional resources for pharmaceutical developments from natural products.

A strategy to identify and quantify closely related adulterant herbal materials by mass spectrometry-based partial least squares regression.

In this study, a new strategy combining mass spectrometric (MS) techniques with partial least squares regression (PLSR) was proposed to identify and quantify closely related adulterant herbal materials. This strategy involved preparation of adulterated samples, data acquisition and establishment of PLSR model. The approach was accurate, sensitive, durable and universal, and validation of the model was done by detecting the presence of Fritillaria Ussuriensis Bulbus in the adulteration of the bulbs of Fritillaria unibracteata. Herein, three different MS techniques, namely wooden-tip electrospray ionization mass spectrometry (wooden-tip ESI/MS), ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and UPLC-triple quadrupole tandem mass spectrometry (UPLC-TQ/MS), were applied to obtain MS profiles for establishing PLSR models. All three models afforded good linearity and good accuracy of prediction, with correlation coefficient of prediction (rp2) of 0.9072, 0.9922 and 0.9904, respectively, and root mean square error of prediction (RMSEP) of 0.1004, 0.0290 and 0.0323, respectively. Thus, this strategy is very promising in tracking the supply chain of herb-based pharmaceutical industry, especially for identifying adulteration of medicinal materials from their closely related herbal species.

An in vitro approach for lipolysis measurement using high-resolution mass spectrometry and partial least squares based analysis.

The elevation of free fatty acids (FFAs) has been regarded as a universal metabolic signature of excessive adipocyte lipolysis. Nowadays, in vitro lipolysis assay is generally essential for drug screening prior to the animal study. Here, we present a novel in vitro approach for lipolysis measurement combining UHPLC-Orbitrap and partial least squares (PLS) based analysis. Firstly, the calibration matrix was constructed by serial proportions of mixed samples (blended with control and model samples). Then, lipidome profiling was performed by UHPLC-Orbitrap, and 403 variables were extracted and aligned as dataset. Owing to the high resolution of Orbitrap analyzer and open source lipid identification software, 28 FFAs were further screened and identified. Based on the relative intensity of the screened FFAs, PLS regression model was constructed for lipolysis measurement. After leave-one-out cross-validation, ten principal components have been designated to build the final PLS model with excellent performances (RMSECV, 0.0268; RMSEC, 0.0173; R2, 0.9977). In addition, the high predictive accuracy (R2 = 0.9907 and RMSEP = 0.0345) of the trained PLS model was also demonstrated using test samples. Finally, taking curcumin as a model compound, its antilipolytic effect on palmitic acid-induced lipolysis was successfully predicted as 31.78% by the proposed approach. Besides, supplementary evidences of curcumin induced modification in FFAs compositions as well as lipidome were given by PLS extended methods. Different from general biological assays, high resolution MS-based method provide more sophisticated information included in biological events. Thus, the novel biological evaluation model proposed here showed promising perspectives for drug evaluation or disease diagnosis.

Global detection and semi-quantification of Fritillaria alkaloids in Fritillariae Ussuriensis Bulbus by a non-targeted multiple reaction monitoring approach.

Methods based on triple quadrupole tandem mass spectrometry have been widely used and reported as highly selective and sensitive methods for quantifying substances of herbal medicines. However, most of them were limited to targeted components, due to the difficulties to optimize the multiple reaction monitoring transitions without authentic standards. This study proposed a novel strategy for non-targeted optimization of multiple reaction monitoring method based on the diagnostic ion guided family classifications, tandem mass spectrometry database establishment, and transitions and collision energy screening. Applying this strategy, 59 Fritillaria alkaloids in Fritillariae Ussuriensis Bulbus have been classified, and 51 of these Fritillaria alkaloids were successfully detected by the optimal multiple reaction monitoring method. For semi-quantification, the easy-to-obtain Fritillaria alkaloids of each type, such as verticinone for cevanine type and peimisine for jervine type, were used as the reference standards to calibrate the other Fritillaria alkaloids in the same type. The method was demonstrated a good linearity (R(2) > 0.998) with satisfactory accuracy and precision, and the lower limits of quantification of verticinone and peimisine were estimated to be 0.076 and 0.216 pg, respectively. In addition, the results suggested that the proposed strategy might obtained high quality metabolomics data in discrimination of Fritillaria unibracteata and Fritillaria ussuriensis.

A direct ionization mass spectrometry-based approach for differentiation of medicinal Ephedra species.

Herein, rapid and efficient identification of species of medicinal Ephedrae Herba was performed using DI-MS (direct ionization-mass spectrometry)-based metabolomics analysis. As a direct ionization technique, DI-MS can provide rapid analysis of samples without sample preparation, so it has been advantageously applied to high-throughput metabolomics analysis. In this flow chart, the MS fingerprints of Ephedrae Herba samples obtained by DI-MS method were firstly pretreated by background subtracts, smooth and center procedures to extract MS features. Then, these MS features were aligned using in a house program written in MATLAB to produce MS dataset. After that, PCA and PLS-DA analysis were performed based on the obtained MS dataset. Finally, the parameter VIP (Variable importance in the Projection) was employed to screen the valuable MS features for discrimination. Using such an approach, three medicinal species of Ephedrae Herba, Ephedra sinica, Ephedra intermedia, and Ephedra equisetina., had been successfully differentiated. Additionally, this method has also been applied to identify the changes in components of Ephedrae Herba after honey treated. In present study, DI-MS in combination with metabolomics was shown to be an efficient and accurate way to identify the sources of herbs.

Identification of effective combinatorial markers for quality standardization of herbal medicines.

Quality standardization of herbal medicines (HMs) is an important task with great challenges. Selection of abundant compounds as markers is currently a major approach for the quality control of HMs; however, such marker compounds are irrelevant to the bioactivities in many cases. Taking Lycoridis Radiatae Bulbus (LRB) as an example, we proposed a universal strategy to identify the effective combinatorial markers (ECMs) that are representative of the bioactivities of HMs, and took them as chemical markers for quality standardization. Fingerprinting and quantification were employed to find out the common components in various batches of medicines. The contribution of each common compound to the overall bioactivity was determined through fingerprint-bioactivity modeling, which based on the absolute quantification of each compound and the acetylcholinesterase (AChE) inhibitory activity of LRB. Two most effective compounds, ungerimine and galanthamine, were therefore proposed as ECMs. Interestingly, these two compounds could synergistically inhibit AChE. This approach demonstrated its strong advantage of the bioactivity relevant quality assessment when compared with conventional methods. And the success of applying this ECMs-based method to the quality assessment of unknown LRB samples proved that our approach was reliable and reproducible. In conclusion, this approach is not only useful for the bioactivity relevant quality control of HMs but also helpful for the discovery of ECMs as new drug candidates.

Rapid identification of plant materials by wooden-tip electrospray ionization mass spectrometry and a strategy to differentiate the bulbs of Fritillaria.

The counterfeit plant products, especially by using incorrect plant materials in pharmaceutical industry, have become a global problem. The plant materials belonging to closely related species but differing in medicinal properties are difficult to be identified. Here, a novel and generally applicable approach to identify the sources of plant materials was developed, which was based on the use of wooden-tip electrospray ionization mass spectrometry (wooden-tip ESI-MS) and multivariate statistical analysis of unidentified MS features (non-targeted). Using this approach, six officinal species of Fritillariae Cirrhosae Bulbus had been successfully differentiated. In addition, Fritillariae Pallidiflorae Bulbus, a common adulterant of Fritillariae Cirrhosae Bulbus, was also identified by using the strategy reported here. Compared with DNA phylogenetic trees, our approach provided finer resolution in distinguishing the closely related Fritillaria species. By combining wooden-tip ESI-MS and multivariate statistical analysis, a useful method was developed here for rapid identification of the sources of herbs, which showed promising perspectives in tracking the supply chain of pharmaceutical suppliers.

Authentication of Bulbus Fritillariae Cirrhosae by RAPD-derived DNA markers.

Bulbus Fritillariae is the most commonly used antitussive herb in China. Eleven species of Fritillaria are recorded as Bulbus Fritillariae in the Chinese Pharmacopoeia. Bulbus Fritillariae Cirrhosae is a group of six Fritillaria species with higher efficiency and lower toxicity derived mainly from wild sources. Because of their higher market price, five other Fritillaria species are often sold deceptively as Bulbus Fritillariae Cirrhosae in the herbal market. To ensure the efficacy and safety of medicinal herbs, the authentication of botanical resources is the first step in quality control. Here, a DNA based identification method was developed to authenticate the commercial sources of Bulbus Fritillariae Cirrhosae. A putative DNA marker (0.65 kb) specific for Bulbus Fritillariae Cirrhosae was identified using the Random Amplified Polymorphic DNA (RAPD) technique. A DNA marker representing a Sequence Characterized Amplified Region (SCAR) was developed from a RAPD amplicon. The SCAR marker was successfully applied to differentiate Bulbus Fritillariae Cirrhosae from different species of Fritillaria. Additionally, the SCAR marker was also useful in identifying the commercial samples of Bulbus Fritillariae Cirrhosae. Our results indicated that the RAPD-SCAR method was rapid, accurate and applicable in identifying Bulbus Fritillariae Cirrhosae at the DNA level.

Direct pharmacokinetic analysis of puqietinone by in vivo microdialysis sampling and turbulent-flow chromatography coupled with liquid chromatography-mass spectrometry.

Sample pretreatment is a key step in bioanalytical process because of possible interference and matrix effects in mass spectrometry analysis. In this work, a novel strategy towards high speed and sensitivity was developed combining in vivo microdialysis (MD) sampling, turbulent-flow chromatography (TFC), and liquid chromatography-mass spectrometry (LC-MS). The procedures of cleanup, preconcentration, and separation were completed on-line in one step within 10min. During the MD optimization procedure, 1% hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was used to improve the relative recovery of the analyte. Untreated MD samples were directly injected, and a TFC precolumn was flushed for 1min with aqueous phase of 4mL/min flow rate to desalt and concentrate biosamples. The retained analytes were then back-flushed by a switching valve onto a fast LC column (4.6mm×50mm, 1.8µm) for separation. Another diverter valve was employed to prevent the HP-ß-CD that interferes with the ESI process from entering the MS. Puqietinone, a lipophilic alkaloid from Fritillaria puqiensis, was used as a case for validation. Results showed that the limit of quantification for puqietinone was 0.10ng/mL, and good linearity (R(2)=0.9993) was maintained over the range of 1.02-200.02ng/mL. Accuracy and precision were satisfactory within the range of the standard curve. This approach was able to effectively eliminate the influences of matrix effect and carry-over as the injection volume increased up to 20µL. The developed method was successfully applied to pharmacokinetic study of puqietinone after intravenous administration to rat. Results demonstrate the potential of using MD with TFC-LC/MS for in vivo monitoring experiments.

Strategies for integral metabolism profile of multiple compounds in herbal medicines: pharmacokinetics, metabolites characterization and metabolic interactions.

Herbal medicines (HMs) are gaining more and more attention all over the world, because of their significant curative effect in treating multi-factorial diseases. Recently, the in vivo and in vitro metabolism study of HMs has become an important issue because these data can help us to better understand the efficacies and toxicities of HMs. However, the integral metabolism profile of HMs is confronted with many challenges: 1) HM is a multi-component system; 2) most components are unknown (nontarget); 3) trace of components in HM. Given the challenges described above, the demand for more powerful bioanalytical tools and strategies that are adequate for integral metabolism profile of HMs' multi-components has increased. In the past few years, newer methods, or adaptations to methods, have been published, and this review will attempt to discuss new improvements in strategies and methodologies for HMs' multi-component ADME evaluation. In particular, improvements have been reported for experimental approaches to pharmacokinetics study of HMs, as well as strategies applied to metabolites characterization of HMs' multi-components, and the metabolic interactions between ingredients in HMs, including advance and proposed strategy: "chemical fishing" based strategy for metabolic interactions of HMs.

Characterization and identification of steroidal alkaloids in Fritillaria species using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC/ESI-QTOF-MS/MS) was performed to study the fragmentation behaviors of steroidal alkaloids from Fritillaria species, the antitussive and expectorant herbs widely used in traditional Chinese medicine. We propose, herein, a strategy that combining key diagnostic fragment ions and the relative abundances and amounts of major fragment ions (the ions exceeding 10% in abundance) to distinguish different sub-classes of Fritillaria alkaloids (FAs). It was found that hydrogen rearrangement and induction effects result in ring cleavage of the basic skeletons occurred in the MS/MS process and produced characteristic fragment ions, which are useful for structural elucidation. This method was finally used to investigate the primary steroidal alkaloids in the extracts of eight major Fritillaria species. As a result, 41 steroidal alkaloids (29 cevanine type, 1 jervine type, 6 veratramine type and 5 secosolanidine type alkaloids) were selectively identified in these Fritillaria species. Twenty-six compounds were unambiguously identified by comparing with the reference compounds and 15 compounds were tentatively identified or deduced according to their MS/MS data. Logical fragmentation pathways for different types of FAs have been proposed and are useful for the identification of these types of steroidal alkaloids in natural products especially when there are no reference compounds available.