An Integrated Approach to Characterize Intestinal Metabolites of Four Phenylethanoid Glycosides and Intestinal Microbe-Mediated Antioxidant Activity Evaluation In Vitro Using UHPLC-Q-Exactive High-Resolution Mass Spectrometry and a 1,1-Diphenyl-2-picrylhydrazyl-Based Assay.

Intestinal bacteria have a significant role in metabolism and the pharmacologic actions of traditional Chinese medicine active ingredients. Phenylethanoid glycosides (PhGs), as typical phenolic natural products, possess wide bioactivities, but low oral bioavailability. The aim of this work was to elucidate the metabolic mechanism underlying PhGs in the intestinal tract and screen for more active metabolites. In this study, a rapid and reliable method using an effective post-acquisition approach based on advanced ultra-high-performance liquid chromatography (UHPLC) coupled with hybrid Quadrupole-Orbitrap high resolution mass spectrometry (Q-Exactive-HRMS) provided full MS and HCD MS2 data. Thermo Scientific™ Compound Discoverer™ software with a Fragment Ion Search (FISh) function in one single workflow was developed to investigate the intestinal microbial metabolism of four typical PhGs. Furthermore, antioxidant activity evaluation of PhGs and their related metabolites was simultaneously carried out in combination with a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay to understand how intestinal microbiota transformations modulate biological activity and explore structure-activity relationships (SARs). As a result, 26 metabolites of poliumoside, 42 metabolites of echinacoside, 42 metabolites of tubuloside, and 46 metabolites of 2'-acetylacteoside were identified. Degradation, reduction, hydroxylation, acetylation, hydration, methylation, and sulfate conjugation were the major metabolic pathways of PhGs. Furthermore, the degraded metabolites with better bioavailability had potent antioxidant activity that could be attributed to the phenolic hydroxyl groups. These findings may enhance our understanding of the metabolism, pharmacologic actions, and real active forms of PhGs.

Identification of the Chemical Constituents of an Anti-Arthritic Chinese Medicine Wen Luo Yin by Liquid Chromatography Coupled with Mass Spectrometry.

Wen Luo Yin (WLY), a well-known traditional Chinese medicine formulation, has been used as a complementary therapy for the treatment of rheumatoid arthritis in clinical settings. However, the chemical constituents of WLY remain unclear. In this study, a high-performance liquid chromatography coupled with tandem mass spectrometry method was established to separate and comprehensively identify the chemical constituents of WLY. The analytes were eluted with a mobile phase of acetonitrile and 0.1% aqueous acetic acid. Mass detection was performed in both positive and negative ion mode. The MS/MS fragmentation pathways were proposed for the identification of the components. A total of 42 compounds including sesquiterpenes, alkaloids, biflavonoids, polyacetylenes, phenylpropanoids and acetylenic phenols were identified unambiguously or tentatively according to their retention times and mass behavior with those of authentic standards or literature data. The identification and structural elucidation of chemical constituents may provide important information for quality control and pharmacological research of WLY.

Shengjiang Xiexin Decoction Alters Pharmacokinetics of Irinotecan by Regulating Metabolic Enzymes and Transporters: A Multi-Target Therapy for Alleviating the Gastrointestinal Toxicity.

Shengjiang Xiexin decoction (SXD), a classic traditional Chinese medical formula chronicled in Shang Han Lun, is used in modern clinical practice to decrease gastrointestinal toxicity induced by the chemotherapeutic drug irinotecan (CPT-11). In this study, the effect of SXD on the pharmacokinetics of CPT-11 and its active metabolites (SN-38 and SN-38G), and the underlying mechanisms were further examined. An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous quantification of CPT-11, SN-38, and SN-38G in the plasma, bile, liver, intestine, and intestinal contents of control and SXD-pre-treated rats after intravenous administration of CPT-11. SXD pretreatment increased the area under the curve (AUC) and the initial plasma concentration (C0) of CPT-11 but decreased the plasma clearance (CL). The AUC and the maximum plasma concentration (Cmax) of SN-38 decreased, whereas the Cmax of SN-38G increased. Compared with that of the control group, the biliary excretion of CPT-11, SN-38, and SN-38G was inhibited. The CPT-11, SN-38, and SN-38G concentrations in the liver, intestine, and intestinal contents were different between the two groups. Furthermore, the hepatic expression of multidrug resistance-associated protein-2 (Mrp-2), P-glycoprotein (P-gp), and carboxylesterase 2 (CES2) was significantly down-regulated by SXD, while the hepatic and jejunal uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) expression was elevated. The hydrolysis of CPT-11 to SN-38 by CES and the glucuronidation of SN-38 to SN-38G by UGT were affected by liver and jejunum S9 fractions from rats pre-treated with SXD. Therefore, this study demonstrated for the first time that SXD could alter the pharmacokinetics of CPT-11 and its metabolites to alleviate CPT-11-induced diarrhea. And the underlying mechanism of drug interaction between CPT-11 and SXD involves decreasing hepatic Mrp-2 and P-gp expression and altering the activities of CES and UGT.

Comparison of the Chemical Profiles and Antioxidant Activities of Different Parts of Cultivated Cistanche deserticola Using Ultra Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry and a 1,1-Diphenyl-2-picrylhydrazyl-Based Assay.

In this study, a sensitive ultra-performance liquid chromatography-photodiode array coupled to quadruple time-of-flight mass (UPLC-PDA-Q/TOF-MS) method and a 1,1-diphenyl-2- picrylhydrazyl (DPPH)-based assay were used to determine the chemical constituents and screen the antioxidant activity profiles of the methanol extracts of different parts of cultivated Cistanche deserticola (C. deserticola). First, qualitative and quantitative chemical composition analyses of the different parts of cultivated C. deserticola were conducted. Obvious differences were observed between the chemical profiles and content distribution of phenylethanoid glycosides (PhGs) from the different cultivated C. deserticola parts. The average contents of the six PhGs parts varied from 4.91 to 72.56 mg/g DW (milligrams of extract per gram of plant dry weight) in the six different parts of Cistanche deserticola, displaying a significant decreasing trend from the bottom to the top of cultivated C. deserticola and the highest content in the stems. From the bottom to the top of the plant, the echinacoside and cistanoside A content decreased and the 2 ' -acetylacteoside content increased. Second, an offline DPPH assay revealed that the total scavenging activities of all parts within the range of 20-500 µ g/mL increased in a concentration-dependent manner and that good antioxidant activities were found in all plant parts, particularly in the stems, which could be related to their higher PhG content. Additionally, a DPPH-UPLC-PDA method was successfully applied to rapidly screen the antioxidant profiles and antioxidant components of the different cultivated C. deserticola parts. According to the antioxidant profiles before and after the DPPH reaction, there were wide variations in the antioxidant activities of different cultivated C. deserticola parts. Moreover, the antioxidant profiles revealed the presence of major free radical scavengers identified as PhGs using UPLC-Q/TOF-MS. Finally, the established DPPH-UPLC-PDA method was reagent saving, rapid and feasible for correlating the chemical profile of traditional chinese medicines (TCMs) with their bioactivities without isolation and purification and may be used for multicomponent analysis of active substances in other foods and herbs. Therefore, to better harness C. deserticola resources, using this method to evaluate cultivated C. deserticola, a promising herb material with obvious antioxidant activity, is crucial.

Identification of Metabolites of the Cardioprotective Alkaloid Dehydrocorydaline in Rat Plasma and Bile by Liquid Chromatography Coupled with Triple Quadrupole Linear Ion Trap Mass Spectrometry.

Dehydrocorydaline (DHC), a quaternary alkaloid from Corydalis yanhusuo, has been demonstrated to be the active constituent in the treatment of coronary heart disease. In this study, a high-performance liquid chromatography-electrospray ionization-triple quadrupole linear ion trap mass spectrometry (HPLC-ESI-QTRAP MS) technique was used to identify DHC metabolites in plasma and bile after oral administration of DHC to rats. A total of 18 metabolites (M1 to M18) were identified and characterized by LC-MS/MS in the positive ion mode. These 18 metabolites were all present in rat bile, while only 9 were detected in plasma. O-demethylation, hydroxylation, di-hydroxylation, glucuronidation of O-demethyl DHC, sulfation of O-demethyl DHC and di-hydroxylation of dehydro-DHC were the major metabolic pathways of DHC. This is the first time that these metabolites of DHC have been identified in rat plasma and bile, which provides useful information for further analysis of the biotransformation of DHC and other quaternary protoberberine-type alkaloids.

Fingerprint-efficacy study of the quaternary alkaloids in Corydalis yanhusuo.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis yanhusuo is a well-known Chinese herbal medicine that is commonly applied as an analgesic agent in clinic using for thousands of years. Resent research showed that the quaternary ammonium alkaloids from Corydalis yanhusuo have a significant effect on myocardial ischemia. However, the corresponding anti-myocardial ischemia components that represent the efficacy of the quaternary ammonium alkaloids have not been elucidated. AIM OF THE STUDY: Explore the anti-myocardial ischemia components of Corydalis yanhusuo and develop a method of quality control for Corydalis yanhusuo. Chemical fingerprints of quaternary ammonium alkaloids extracted from Corydalis yanhusuo samples from 37 different sources were identified using UPLC-Q-TOF MS. The protective effects of the 37 samples with respect to H9C2 cell hypoxia-reoxygenation were detected by MTT assays. The fingerprint-efficacy relationship between the chemical fingerprints and cardioprotection afforded by Corydalis yanhusuo was investigated using three chemometric methods. RESULTS: Because of their inherent differences in chemical compositions, the protective effects to H9C2 cell hypoxia-reoxygenation were different. The results of three chemometric methods showed that the source of the Corydalis yanhusuo has an important influence on both the chemical fingerprint and efficacy. In particular, dehydrocorybulbine, 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and palmatine appear to be the main effective components for quality control of this TCM. CONCLUSION: This work provides a general model of combination of UPLC and cardioprotection efficiency to study the fingerprint-efficacy relationship of Corydalis yanhusuo which can offer some references for detecting principal components of Corydalis yanhusuo on cardioprotection efficiency. Fingerprint-efficacy studies also provide a powerful method of quality control in Corydalis yanhusuo and other TCMs.