Effect of Different Processing Methods on the Chemical Constituents of Scrophulariae Radix as Revealed by 2D NMR-Based Metabolomics

Scrophulariae Radix (SR) is one of the oldest and most frequently used Chinese herbs for oriental medicine in China. Before clinical use, the SR should be processed using different methods after harvest, such as steaming, “sweating”, and traditional fire-drying. In order to investigate the difference in chemical constituents using different processing methods, the two-dimensional (2D) ¹H-¹³C heteronuclear single quantum correlation (¹H-¹³C HSQC)-based metabolomics approach was applied to extensively characterize the difference in the chemical components in the extracts of SR processed using different processing methods. In total, 20 compounds were identified as potential chemical markers that changed significantly with different steaming durations. Seven compounds can be used as potential chemical markers to differentiate processing by sweating, hot-air drying, and steaming for 4 h. These findings could elucidate the change of chemical constituents of the processed SR and provide a guide for the processing. In addition, our protocol may represent a general approach to characterizing chemical compounds of traditional Chinese medicine (TCM) and therefore might be considered as a promising approach to exploring the scientific basis of traditional processing of TCM.     

Understanding the traditional aspect of Chinese medicine in order to achieve meaningful quality control of Chinese materia medica

Although sophisticated and technologically advanced, current quality control methods for Chinese medicines (syn. Chinese materia medica or CMM) lack comprehensiveness and practicability. They are more suited for analyzing single-chemical drugs or specific, known chemical components that have already been isolated. While these methods can fully satisfy the modern scientific requirements for identity, purity and quality in the assessment of chemical drugs, they are not suitable for handling the complex chemical nature of traditional CMM whose multifunctional components along with their inherent holistic activities are frequently unknown and thus are not adequately analyzed by these methods. In order to assess properly and meaningfully the identity and quality of complex CMM (also known as Chinese herbs and Chinese herbal medicines), additional measures that can retain the traditional aspect of CMM need to be included. This requires a basic understanding of traditional Chinese medicine (TCM).     

Static headspace‐multicapillary column with gas chromatography coupled to ion mobility spectrometry as a simple approach for the discrimination of crude and processed traditional Chinese medicines

The processing procedure can alter the nature and chemical transformation of traditional Chinese medicine to accommodate different clinical dispensing and preparation requirements. In this study, static headspace‐multicapillary column with gas chromatography coupled to ion mobility spectrometry was developed for the rapid and sensitive discrimination of crude and processed traditional Chinese medicine. Using Radix Paeoniae Alba as a traditional Chinese medicine model, the combined power of this approach was illustrated by classifying the crude and processed Radix Paeoniae Alba samples into two main categories. The contents of the main components in Radix Paeoniae Alba varied significantly. The established method could promote the use of ion mobility spectrometry in intrinsic quality control and differentiation of herbal medicines from other processed products or preparations.     

Chromatographic fingerprinting and metabolomics for quality control of TCM

Chromatographic fingerprinting technique of traditional Chinese medicine (TCM) has proved to be a comprehensive strategy for assessing the intact quality of herbal medicine. In general, one could use the chromatographic techniques to obtain a relatively complete picture of herbal medicines, which are in common called chromatographic fingerprints of herbal medicines to represent the so-called phytoequivalence. Based on this, the features of chromatographic fingerprints of herbal medicines have been discussed in some detail. The technique based on chromatographic fingerprinting is essentially a kind of high-throughput and integral tools to explore the complexity of herbal medicines. In order to further control the comprehensive quality of TCMs, some new strategies are proposed to trace the chemical changes of chromatographic fingerprints both in product processing and/or after their administration by modern chromatographic techniques and chemometrics. Combined with metabolomics, it seems possible for one to reveal the working mechanism of TCMs and to further control their intrinsic quality. Finally, the intensive study of chromatographic fingerprinting coupled with multivariate analysis tools developed in bioinformatics and chemometrics are emphasized in order to achieve the aim to reveal the working mechanisms of TCMs and to further control and strengthen TCMs' intrinsic quality in a comprehensive manner.     

Multiple chromatographic fingerprinting and its application to the quality control of herbal medicines

Recently, chromatographic fingerprinting has become one of the most powerful approaches to quality control of herbal medicines. However, the performance of reported chromatographic fingerprinting constructed by single chromatogram sometimes turns out to be inadequate for complex herbal medicines, such as multi-herb botanical drug products. In this study, multiple chromatographic fingerprinting, which consists of more than one chromatographic fingerprint and represents the whole characteristics of chemical constitutions of the complex medicine, is proposed as a potential strategy in this complicated case. As a typical example, a binary chromatographic fingerprinting of “Danshen Dropping Pill” (DSDP), the best-sold traditional Chinese medicine in China, was developed. First, two HPLC fingerprints that, respectively, represent chemical characteristics of depsides and saponins of DSDP were developed, which were used to construct binary chromatographic fingerprints of DSDP. Moreover, the authentication and validation of the binary fingerprints were performed. Then, a data-level information fusion method was employed to capture the chemical information encoded in two chromatographic fingerprints. Based on the fusion results, the lot-to-lot consistency and frauds can be determined either using similarity measure or by chemometrics approach. The application of binary chromatographic fingerprinting to consistency assessment and frauds detection of DSDP clearly demonstrated that the proposed method was a powerful approach to quality control of complex herbal medicines.     

A Rapid and Sensitive Assay for Determining the Main Components in Processed Fructus Corni by UPLC�CQ-TOF-MS

Fructus Corni is the dried sarcocarp of Cornus officinalis Sieb. et Zucc., and has been widely used as an important traditional Chinese medicine, which is used to nourish liver and kidney conditions and to astringe semen as well as to solidify collapse. Precise structural identification of the main compounds in processed Fructus Corni has been established using ultra performance liquid chromatography (UPLC) combined with quadrupole-time-of-flight-mass spectrometry (Q-TOF-MS), and BEH C₁₈ column for direct analysis of total water extracts. Mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in negative mode. The MS data for candidates, containing accurate mass for both precursors and their fragment ions, were acquired selectively. Based on the MS data, the mass spectrometric fingerprint (MSFP) for candidates, consisting of chemical formula and dissociation pattern, was determined. By this method, eight iridoid glycosides and seven other compounds were identified or tentatively identified. Therefore, MS combined with selective enrichment provided a powerful means for analyzing main compounds in processed Fructus Corni. LC?CQ-TOF-MS based chemical profiling is a rapid and powerful approach for holistic quality evaluation of processed Fructus Corni, and should also be useful for the global quality investigation of decoctions derived from other herbal medicines.     

Rapid identification and comparative analysis of the chemical constituents and metabolites of Phellodendri amurensis cortex and Zhibai dihuang pill by ultra‐performance liquid chromatography with quadrupole TOF‐MS

Zhibai dihuang pill is a famous traditional Chinese medicine formula containing the herbal medicine Phellodendri amurensis cortex. In this work, an integrative pattern recognition approach including principal component analysis and orthogonal partial least squared discriminant analysis based on ultra‐performance liquid chromatography coupled to MS was successfully applied for the rapid discovery of natural compounds from herbal medicines. In a 24 min analysis, 93 compounds were identified or tentatively characterized from Zhibai dihuang pill based on their fragmentation behaviors, 23 of which were from Phellodendri amurensis cortex. A total of 26 metabolites in plasma were identified from Phellodendri amurensis cortex in Zhibai dihuang pill formula, among them, 12 prototypes and 14 metabolites through metabolic pathways of demethylation, methylation, hydrolysis, sulfate conjugation, and glucuronide conjugation, which were seven metabolites more than that of the single drug, suggesting the importance of the compatibility of traditional Chinese medicine. The present study provided important structural information on the metabolism of Zhibai dihuang pill. Furthermore, the results of this work have demonstrated the feasibility of ultra‐performance liquid chromatography coupled to MS for the rapid and reliable characterization of metabolites from herbal medicines. Based on these results, this method could be a novel approach for identifying potentially bioactive components in other traditional Chinese medicines.     

Identification of chemical ingredients of peanut stems and leaves extracts using UPLC‐QTOF‐MS coupled with novel informatics UNIFI platform

Peanut stems and leaves have been used traditionally as both herbal medicines and special food in Asia. In this study, the main functional compounds of peanut stems and leaves extracts were identified using UPLC separation coupled to high resolution mass spectrometry (QTOF‐MS), and a traditional medicine library. Three different extraction solvents (ethyl acetate, petroleum ether and n‐butanol) were evaluated to prepare the extracts of peanut stems and leaves. A total of 283 chemical compounds were identified in peanut stems and leaves extracts, of which 207 compounds are tentatively new identifications in Genus Arachis. The integration of data acquisition and processing with the traditional medicine library provides a simple, efficient process to effectively facilitate the identification of chemical ingredients in complex natural product extracts. The integrated workflow for separation, detection and identification of functional compounds in natural products using UPLC/QTOF‐MS greatly improves productivity for development of traditional herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification of chemicals and their metabolites from PHY906, a Chinese medicine formulation,in the plasma of a patient treated with irinotecan and PHY906 using liquid chromatography/tandem mass spectrometry (LC/MS/MS)

Traditional Chinese Medicine (TCM) is increasingly being used in combination with Western medicine. In general, TCM is comprised of multiple components in sharp contrast to Western medicine, where a single active chemical is used. Presently, there are no well-established standards for most of the chemical compounds of TCM and their respective metabolites. Moreover, there are no formal analytical methods for the identification of these chemicals, especially in trace amounts. The ability to measure the pharmacokinetic behaviors of chemicals and their metabolites from these herbal formulations are critical in understanding of the action of TCM. This paper describes the use of LC/MS/MS along with enzyme treatments and n-octanol/water partition coefficient, to investigate the chemical components of PHY906 and their metabolites in the plasma of a patient with metastatic colorectal cancer (mCRC) treated with irinotecan and PHY906. The chemicals from an aqueous extract of PHY906 and the plasma from a patient was prepared and separated on an Agilent ZORBAX-SB C₁₈ column, and eluted with acetonitrile/0.05% (v/v) formic acid. From the PHY906 aqueous extract, a total of 57 compounds and 27 metabolites were identified and tentatively assigned structures based on their identified mass spectrometry, enzyme digestion and n-octanol/water partition coefficient. In contrast, analysis of patient plasma identified only 33 chemicals and new metabolites. These findings demonstrated that LC/MS/MS was and effective and reliable method for studying the parent chemicals of the Chinese herbal medicine PHY906 and their metabolites in a patient with metastatic colorectal cancer.     

Software Assisted Multi-Tiered Mass Spectrometry Identification of Compounds in Traditional Chinese Medicine: Dalbergia odorifera as an Example

The complexity of metabolites in traditional Chinese medicine (TCM) hinders the comprehensive profiling and accurate identification of metabolites. In this study, an approach that integrates enhanced column separation, mass spectrometry post-processing and result verification was proposed and applied in the identification of flavonoids in Dalbergia odorifera. Firstly, column chromatography fractionation, followed by liquid chromatography–tandem mass spectrometry was used for systematic separation and detection. Secondly, a three-level data post-processing method was applied to the identification of flavonoids. Finally, fragmentation rules were used to verify the flavonoid compounds. As a result, a total of 197 flavonoids were characterized in D. odorifera, among which seven compounds were unambiguously identified in level 1, 80 compounds were tentatively identified by MS-DIAL and Compound Discoverer in level 2a, 95 compounds were annotated by Compound discoverer and Peogenesis QI in level 2b, and 15 compounds were exclusively annotated by using SIRIUS software in level 3. This study provides an approach for the rapid and efficient identification of the majority of components in herbal medicines.     

A multi-evaluating strategy for raw and processed Veratrum nigrum L.: Fingerprinting combined with quantitative analysis based on multivariate chemometric methods

Veratrum nigrum L. (VN) is a well-known herbal medicine and rich in chemical components with multiple pharmacological activities including antihypertensive, anticancer, and antifungal effects. In the current experiment, the quality of VN from different habitats was evaluated based on combinative method of fingerprint, multi-component quantification and chemical pattern recognition. Fifteen batches of VN were collected, and intrinsic chemical composition were identified using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, which is a method for analyzing the similarity between samples, coupled with fingerprint of traditional Chinese medicine. The fingerprint similarity model show that 22 common peaks were selected covering 15 batches of and the similarity > 0.963. The total of 22 joint components were tentatively identified by comparison with standard substances or literature. A ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry method for simultaneous determination of 8 compounds was established to evaluate the contents of raw and processed Veratrum nigrum L. Multivariate analysis was then applied to compare different batches of herbs based on ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry data. All raw and processed samples were classified by partial least squares discriminant analysis based on the 8 analyzed compounds. The findings suggested that veratramine and polydatin with a variable importance for the project (VIP) > 1 were identified as significant constituents, the presence of which can be used to differentiate between raw and processed Veratrum nigrum L. samples. These results indicate that processing methods show important effects on the composition of Veratrum nigrum L..     

Editorial: Current Issues on Future Researches and Applications of Natural Product Medicines

The use of natural product medicine has emerged from traditional to modern therapy in order to increase the quality of health worldwide. To prove pharmacological effects of medicinal plants and to further develop the rational use of herbal medicines, scientific approaches are essential. The development of sciences and technologies have highly supported the research on natural product medicines in all aspects. Recent findings from research in natural product medicines were a major focus in the International Seminar on Natural Product Medicines 2012 which was held in Bandung and organized by School of Pharmacy, Bandung Institute of Technology (ITB) and Indonesian Society on Natural Product Researchers (PERHIPBA). Furthermore, there was an interesting sharing of experiences and knowledge on how to develop and practice some well-known traditional medicines such as Traditional Chinese Medicine (TCM), ayurveda and jamu     

Identifying the chemical markers in raw and wine-processed Scutellaria baicalensis by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry coupled with multiple statistical strategies

Herb processing is a typical pharmaceutical preparation process for traditional Chinese medicine. After processing, its clinical applications and pharmacological effects vary greatly, which is most commonly attributed to the changing chemical properties between raw herb and processed products. In this work, a total of 53 chemical compounds were detected, among which 17 compounds were identified as discriminatory chemicals between raw and wine-processed Scutellaria baicalensis, and 10 components were identified as chemical markers with a cumulative content contribution of 88.75%. In addition, this work revealed that the best wine-processed time was 18 min by investigating the changes of chemical markers in S. baicalensis during processing. This work demonstrated that ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry coupled with multiple statistical strategies is an effective approach for screening and identifying discriminatory chemical markers in complex traditional Chinese medicine.     

Global detection and identification of components from crude and processed traditional Chinese medicine by liquid chromatography connected with hybrid ion trap and time‐of‐flight‐mass spectrometry

We herein present a chemical profiling method to efficiently process the information acquired by ultra fast liquid chromatography (UFLC)‐electrospray ionization source in combination with hybrid ion trap and high‐resolution time‐of‐flight mass spectrometry (UFLC‐(ESI)‐IT‐TOF/MS), facilitating the structural determination of serial components contained in crude or processed traditional Chinese medicine (TCM). Under the optimized UFLC and IT‐TOF‐MSⁿ conditions, over 39 compounds were separated and detected in crude or processed Fructus corni within 25 min. The components were identified by comparing the mass spectra and retention time with reference compounds, or tentatively assigned by elucidating low‐energy collision‐induced dissociation (CID) fragment ions and matching empirical molecular formula with that of the published compounds. Several factors in the processing procedure were examined. The experimental results demonstrate that the chemical reactions that occurred in the processing procedure can be used to elucidate the processed mechanism of F. corni, which is regularly affected by the processing conditions. This study provides a novel approach and methodology to identify the complicated components from various complex mixtures such as crude TCM, processed TCM, and biological samples. It can be used as a valid analytical method for further understanding the processing mechanism of TCM, along with the intrinsic quality control of TCM and its processed product.     

Identification of the chemical constituents of Chinese medicine Yi‐Xin‐Shu capsule by molecular feature orientated precursor ion selection and tandem mass spectrometry structure elucidation

The incomplete identification of the chemical components of traditional Chinese medicinal formula has been one of the bottlenecks in the modernization of traditional Chinese medicine. Tandem mass spectrometry has been widely used for the identification of chemical substances. Current automatic tandem mass spectrometry acquisition, where precursor ions were selected according to their signal intensity, encounters a drawback in chemical substances identification when samples contain many overlapping signals. Compounds in minor or trace amounts could not be identified because most tandem mass spectrometry information was lost. Herein, a molecular feature orientated precursor ion selection and tandem mass spectrometry structure elucidation method for complex Chinese medicine chemical constituent analysis was developed. The precursor ions were selected according to their two‐dimensional characteristics of retention times and mass‐to‐charge ratio ranges from herbal compounds, so that all precursor ions from herbal compounds were included and more minor chemical constituents in Chinese medicine were identified. Compared to the conventional automatic tandem mass spectrometry setups, the approach is novel and can overcome the drawback for chemical substances identification. As an example, 276 compounds from the Chinese Medicine of Yi‐Xin‐Shu capsule were identified.     

Recent Research Progress on Natural Stilbenes in Dendrobium Species

Dendrobium is the second biggest genus in the Orchidaceae family, and many of them have been utilized as a traditional Chinese medicine (TCM) for thousands of years in China. In the last few decades, constituents with great chemical diversity were isolated from Dendrobium, and a wide range of biological activities were detected, either for crude extracts or for pure compounds. Stilbene compound is one of the primary active constituents in the genus Dendrobium. At present, 267 stilbene compounds with clarified molecular structures have been extracted and isolated from 52 species of Dendrobium, including 124 phenanthrenes and 143 bibenzyls. At the same time, activity studies have indicated that 157 compounds have pharmaceutical activity. Among them, most of the compounds showed antitumor activity, followed by antioxidant, anti-inflammatory and anti-α-glucosidase inhibitory activities. Additionally, 54 compounds have multiple pharmacological activities, such as confusarin (14), 2,4,7-trihydroxy-9,10-dihydro-phenanthrene (43), moscatilin (148), gigantol (150) and batatasin III (151). This review summarizes current knowledge about the chemical composition of stilbene, bioactivities and pharmacologic effects in 52 species of Dendrobium. We also expect to provide a reference for further research, development and utilization of stilbene constituents in the Dendrobium genus.     

Screening and Identification of Many of the Compounds Present in Rauvolfia verticillata by Use of High-Pressure LC and Quadrupole TOF MS

A simple approach is described for LC–Q-TOFMS screening and identification of many of the compounds present in Rauvolfia verticillata. Combined isolation and identification of the complex chemical composition was conducted when investigating traditional Chinese medicine. Twenty-two components of Rauvolfia verticillata were identified by comparing retention times and molecular weights with those of available standards and with reference data. This study provided a rapid, sensitive, economical, and systematic method for identification and evaluation of the quality of complex traditional Chinese medicines.     

Rapid quality assessment of Radix Aconiti Preparata using direct analysis in real time mass spectrometry

This study presents a novel and rapid method to identify chemical markers for the quality control of Radix Aconiti Preparata, a world widely used traditional herbal medicine. In the method, the samples with a fast extraction procedure were analyzed using direct analysis in real time mass spectrometry (DART MS) combined with multivariate data analysis. At present, the quality assessment approach of Radix Aconiti Preparata was based on the two processing methods recorded in Chinese Pharmacopoeia for the purpose of reducing the toxicity of Radix Aconiti and ensuring its clinical therapeutic efficacy. In order to ensure the safety and effectivity in clinical use, the processing degree of Radix Aconiti should be well controlled and assessed. In the paper, hierarchical cluster analysis and principal component analysis were performed to evaluate the DART MS data of Radix Aconiti Preparata samples in different processing times. The results showed that the well processed Radix Aconiti Preparata, unqualified processed and the raw Radix Aconiti could be clustered reasonably corresponding to their constituents. The loading plot shows that the main chemical markers having the most influence on the discrimination amongst the qualified and unqualified samples were mainly some monoester diterpenoid aconitines and diester diterpenoid aconitines, i.e. benzoylmesaconine, hypaconitine, mesaconitine, neoline, benzoylhypaconine, benzoylaconine, fuziline, aconitine and 10-OH-mesaconitine. The established DART MS approach in combination with multivariate data analysis provides a very flexible and reliable method for quality assessment of toxic herbal medicine.     

Quality assessment of raw and processed Arctium lappa L. through multicomponent quantification,chromatographic fingerprint,and related chemometric analysis

In traditional Chinese medicine, raw and processed herbs are used to treat different diseases. Suitable quality assessment methods are crucial for the discrimination between raw and processed herbs. The dried fruit of Arctium lappa L. and their processed products are widely used in traditional Chinese medicine, yet their therapeutic effects are different. In this study, a novel strategy using high‐performance liquid chromatography and diode array detection coupled with multivariate statistical analysis to rapidly explore raw and processed Arctium lappa L. was proposed and validated. Four main components in a total of 30 batches of raw and processed Fructus Arctii samples were analyzed, and ten characteristic peaks were identified in the fingerprint common pattern. Furthermore, similarity evaluation, principal component analysis, and hierachical cluster analysis were applied to demonstrate the distinction. The results suggested that the relative amounts of the chemical components of raw and processed Fructus Arctii samples are different. This new method has been successfully applied to detect the raw and processed Fructus Arctii in marketed herbal medicinal products.