Kunming Qin

Screening and identification of multiple constituents and their metabolites of Fangji Huangqi Tang in rats by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry basing on coupling data processing techniques

Fangji Huangqi Tang (FHT) is a classical formula widely used in Chinese clinical application. In this paper, a novel and advanced strategy has been developed for the multiple constituent identification of FHT in rats, which was basing on an ultra-high performance liquid chromatography equipped with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS) method combined with dynamic background subtract (DBS) data acquisition and enhance peak list (EPL) data processing techniques. Firstly, a total of 58 potential bioactive compounds including alkaloids, flavonoids, saponins, saccharides and terpenoids were detected from FHT. Their chemical structures were identified by comparing the retention time and mass spectrometry data, as well as retrieving the reference literatures. Based on the same instrumental conditions, 33 compounds were found in rat serum after oral administration of FHT. After a considerate comparison with the former chemical identification results of FHT, 33 compounds were found, which turned out to be 8 original compounds of FHT as well as 25 metabolites, including 20 phase I and 5 phase II metabolites. The results indicated that the metabolic reactions included hydroxylation, hydrogenation, demethylation, tarine conjugation and acetylation. This study firstly reported the metabolism description of fangchinoline and tetrandrine in vivo, which could be very useful for further pharmacological and clinical studies of FHT. Meanwhile, it provided a practical strategy for rapid screening and identifying of multiple constituents and their metabolites of complex traditional Chinese medicine in biological matrix.

Profiling and analysis of multiple compounds in rhubarb decoction after processing by wine steaming using UHPLC–Q‐TOF‐MS coupled with multiple statistical strategies

Rhubarb is one of the most popular traditional Chinese medicines and has been used for thousands of years in many Asian countries. Prepared rhubarb is obtained by steaming raw rhubarb with glutinous rice wine until it turned black in appearance both inside and outside. After processing, the therapeutic effects of prepared rhubarb change a lot. To find out the exact compound changes of the chemical profile in a decoction of rhubarb after processing and to clarify the material basis of the changed therapeutic effects, an ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry method coupled with automated data analysis software and statistical strategy was developed. As a result, 63 peaks in raw rhubarb and 54 peaks in prepared rhubarb were detected, and a total of 45 chemical compounds were identified. The analysis data were subjected to a principle component analysis and a t-test. Based on the results, 16 peaks were found to be the main contributors to the significant difference (p < 0.05) between raw and prepared rhubarb. Compared with raw rhubarb, the content of 15 components in prepared rhubarb was lower, while only rhein (1,8-dihydroxy-3-carboxy anthraquinone) showed a higher intensity.

Investigation on the spectrum-effect relationships of Da-Huang-Fu-Zi-Tang in rats by UHPLC-ESI-Q-TOF-MS method

ETHNOPHARMACOLOGICAL RELEVANCE Da-Huang-Fu-Zi-Tang (DHFZT) is a crucial TCM formula commonly used for the treatment of acute pancreatitis in Chinese clinical application. Our previous work found that DHFZT could act against pancreatic injury in rats with severe acute pancreatitis (SAP). The goal of this paper was to study the underlying correlations between the chemical spectra and the protective effect of DHFZT on pancreatic acinar cell to reveal the real bioactive compounds in DHFZT. MATERIALS AND METHODS The fingerprint chromatograms of rat serum after oral administration of DHFZT were established by UHPLC-ESI-Q-TOF-MS technique. At the same time, the model of anti-acute pancreatitis on cells was established by adding 10(-7) mol/L cerulein to AR42J cell line, and the protective effects of the serum on pancreatic acinar cell from injury was evaluated by detecting the efficacy of amylase. Then, the spectrum-effect relationships between UHPLC fingerprints and anti-acute pancreatitis activities were evaluated using canonical correlation analysis (CCA) statistical method. The chromatogram separation was performed on a C18 reversed phase UHPLC column (2.1 mm × 100 mm, 3.5 μm, Agilent), the column temperature was set at 35°C. The mobile phase consisted of 0.1% formic acid and acetonitrile with gradient elution. The serum samples were analyzed both in negative and positive ion mode. The mother and productive ions were scanned within the mass range of m/z 100-1200 and 50-1200, respectively. A thorough analysis of a great deal of information of the constituents in the rat serum was undertaken. The structure identification of the detected compounds was achieved by using high resolution MS values as well as the MS/MS fragments. RESULTS Eighteen peaks in rat serum after oral administration of DHFZT were detected within only 30 min recorded chromatograms. The structure of the 18 compounds were then given out, of which 10 were the original form of compounds absorbed from DHFZT, 8 were the metabolites of the compounds existed in rat serum. According to the CCA results, talatisamine, rhein glucoside, rhein isomer methylation, hypaconine, hydroxyl-chrysophanol, emodin glucuronide conjugation, and chrysophanol glucuronide conjugation were finally found to be the main anti-acute pancreatitis components in DHFZT. CONCLUSIONS The model presented in this paper successfully discovered the spectrum-effect relationships of DHFZT, which showed a representative way to discover the primary active ingredients from the complicated herbal drugs.

Profiling and characterization of volatile components from non-fumigated and sulfur-fumigated Flos Lonicerae Japonicae using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry coupled with chemical group separation.

Flos Lonicerae Japonicae (FLJ) is a popular herb used for many centuries in Traditional Chinese Medicine as a treatment of fever and inflammation. Non-fumigated processing of FLJ has been the traditional approach used in post-harvest preparation of the commodity for commercial use. However, in recent years, natural drying processing of FLJ has been replaced by sulfur-fumigation for efficiency and pest control. Sulfur-fumigation can induce changes in the volatile compounds of the herb, altering its medicinal properties. A comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF/MS) method was established for the resolution and determination of volatile components in non-fumigated and sulfur-fumigated FLJ. In this paper, analysis of the volatile oils in non-fumigated and sulfur-fumigated (including lab-prepared sulfur- fumigated and industrial sulfur-fumigated) FLJ was performed using GC×GC-TOF/MS. Seventy-three representative volatile components were identified, including furans, alkalies, acids, aldehydes, ketones, alcohols, terpenes, esters, and others, as the main components of FLJ volatile oils. The proposed method was successfully applied for rapid and accurate quality evaluation of FLJ and its related medicinal materials and preparations.

Comparative pharmacokinetics studies of benzoylhypaconine, benzoylmesaconine, benzoylaconine and hypaconitine in rats by LC-MS method after administration of Radix Aconiti Lateralis Praeparata extract and Dahuang Fuzi Decoction

A rapid and sensitive high-performance liquid chromatography-mass spectrometric (HPLC-MS) method was developed and validated for simultaneous determination of benzoylhypaconine (BHA), benzoylmesaconine (BMA), benzoylaconine (BAC) and hypaconitine (HA) in rat plasma for the first time. The analytes were separated on a Kromasil C₁₈ column with a total running time of 11 min. The validation data demonstrated a sound feasibility for the newly developed method and it was then applied to the pharmacokinetic study of these analytes in rats. Pharmacokinetic behaviors of BHA, BMA, BAC and HA in rats were studied after oral administration of Radix Aconiti Lateralis Praeparata extract (FZ) and Dahuang Fuzi Decoction (DFD). The main parameters for the two groups of subjects were compared, and significant differences between Radix Aconiti Lateralis Praeparata extract group and Dahuang Fuzi Decoction group in calculated parameters, such as the area under the plasma concentration-time from zero to the last quantifiable time-point (AUC(₀-t)), the area under the plasma concentration-time curve from zero to infinity (AUC(₀-∞)), peak plasma concentration (C(max)), half-life of elimination (T₁/₂), mean retention time (MRT(₀-t)), plasma clearance (CL), volume of distribution (V(d)) and time to reach Cmax (T(max)), were found. After oral administration of DFD, the AUC(₀-t), AUC(₀-∞) and C(max) of BHA, BMA, BAC and HA decreased remarkably (p < 0.05) compared with those of the FZ extract group. Vd and CL values of BHA, BMA, BAC and HA increased, two of which showed significant difference (p < 0.05). T₁/₂ and MRT(₀-t) values of BHA, BMA and BAC in the DFD group were significantly delayed compared with those of FZ extract group. Only the T(max) of HA, the toxic ingredient in FZ, delayed significantly in DFD group compared with the value of FZ group. All these pharmacokinetic parameters were statistically compared, and the rationality of the combination for DFD was clearly demonstrated.

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.

Study on chemical fingerprinting of crude and processed Atractylodes macrocephala from different locations in Zhejiang province by reversed-phase high-performance liquid chromatography coupled with hierarchical cluster analysis

Background: In China, Atractylodes macrocephala is mainly distributed in Zhejiang province. It is, therefore, desirable to determine a reliable and accurate methodology to differentiate the samples collected from Zhejiang province. Although some studies on the fingerprints of Atractylodes macrocephala using HPLC have been published, none of them compared the processed product of Atractylodes macrocephala from different areas of Zhejiang province. Materials and Methods: Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with hierarchical cluster analysis was employed in the fingerprint analysis of Atractylodes macrocephala from Zhejiang province, China. The LC assay was performed on a reversed-phase C18 column with linear gradient elution using water and acetonitrile. The LC data showed considerable variation of chemical constituents among Atractylodes macrocephala populations. Results: 21 and 22 characteristic peaks in the 14 grants of Atractylodes macrocephala and its processed product were determined in samples from different habitats of Zhejiang province, respectively. Their chromatographic patterns were generally consistent although their contents of chemical compositions were greatly different. The results of hierarchical cluster analysis showed that the samples could be divided into four groups; it was able to select excellent resources from the groups. Conclusion: This was the first report of hierarchical cluster analysis of crude and processed Atractylodes macrocephala according to their chemical fingerprints and could be applied to the intrinsic quality control of crude and processed Atractylodes macrocephala. The processing technique of Atractylodes macrocephala through the pilot-scale experiment was first studied and is simple and suitable for prepared yin pian of Atractylodes macrocephala industrial manufacture.

Multi-component analysis in sun-dried and sulfur-fumigated Angelicae Sinensis Radix by single marker quantitation and chemometric discrimination

Background: A new method has been developed for the simultaneous determination of ferulic acid, senkyunolide A, and Z-ligustilide in Angelicae Sinensis Radix before and after sulfur-fumigation using quantitative analysis of multi-components by a single marker (QAMS). Materials and Methods: The feasibility and accuracy of QAMS were checked by the external standard method, and various high-performance liquid chromatographic instruments and chromatographic conditions were investigated to verify its applicability. Using ferulic acid as the internal reference substance, and the contents of senkyunolide A and Z-ligustilide were calculated according to relative correction factors by high-performance liquid chromatography. Meanwhile, the influence of sulfur-fumigation on these chemical components in Angelicae Sinensis Radix were evaluated and discriminated by chromatographic fingerprint and chemometrics. Results: There was no significant difference observed between the QAMS method and the external standard method. Furthermore, sulfur-fumigation reduced the contents of ferulic acid, senkyunolide A, and Z-ligustilide in Angelicae Sinensis Radix by some degree, and the sun-drying and sulfur-fumigation processing could be easily discriminated by chromatographic fingerprint and chemometrics. Conclusion: QAMS is a convenient and accurate approach to analyzing multi-component when reference substances are unavailable, simultaneously, chemometrics is an effective way to discriminate sun-dried and sulfur-fumigated Angelicae Sinensis Radix.

Screening and analysis of the multiple absorbed bioactive components and metabolites of Baihe Zhimu Tang by the metabolic fingerprinting technique and liquid chromatography/diode array detection-electrospray ionization-mass spectrometry

Background: Baihe Zhimu Tang (BZT) is a widely used traditional Chinese medicinal formula in treating various diseases; however, its active components have remained unknown. Materials and Methods: Based on the metabolic fingerprinting technique and liquid chromatography/diode array detection-electrospray ionization-mass spectrometry (LC/DAD-ESI-MS), a method for rapid screening and analysis of the multiple absorbed bioactive components and metabolites of an oral solution of Baihe Zhimu Tang (BZT) in rabbit plasma, urine and feces after oral administration of BZT was developed. Results: The results obtained from a comprehensive comparative analysis of the fingerprints of the BZT and its metabolic fingerprints in rabbit biological samples indicated that 19 components in the BZT were absorbed into the rabbits body. Both of them were tentatively identified from their MS and UV spectra and retention behaviors by comparing the results with the reported literature. In addition, only six components were found in the metabolic fingerprints, which suggested that they might be metabolites of some components in the BZT. Conclusion: The findings demonstrated that the proposed method could be used to rapidly and simultaneously analyze and screen the multiple absorbed bioactive constituents and metabolites in a formula of traditional Chinese medicines (TCMs) by comparing and contrasting the chromatographic fingerprints with its metabolic fingerprints. This is very important not only for the pharmaceutical discovery process and the quality control of crude drugs, but also for explaining the curative mechanism of TCMs.

Identification and differentiation of major components in three different “Sheng-ma” crude drug species by UPLC/Q-TOF-MS

Cimicifugae Rhizoma (Sheng ma) is a Ranunculaceae herb belonging to a composite family and well known in China. has been widely used in traditional Chinese medicine. The Pharmacopoeia of the People׳s Republic of China contains three varieties (Cimicifuga dahurica (Turcz.), Cimicifuga foetida L. and Cimicifuga heracleifolia Kom.) which have been used clinically as “Sheng-ma”. However, the chemical constituents of three components of “Sheng-ma” have never been documented. In this study, a rapid method for the analysis of the main components of “Sheng-ma” was developed using ultra-high performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The present study reveals the major common and distinct chemical constituents of C. dahurica, C. foetida and C. heracleifolia and also reports principal component and statistical analyses of these results. The components were identified by comparing the retention time, accurate mass, mass spectrometric fragmentation characteristic ions and matching empirical molecular formula with that of the published compounds. A total of 32 common components and 8 markers for different “Sheng-ma” components were identified. These findings provide an important basis for the further study and clinical utilities of the three “Sheng-ma” varieties.