LC-MS/MS profiling of colon oxysterols and cholesterol precursors in mouse model of ulcerative colitis.

Oxysterols and cholesterol precursors are being increasingly investigated in humans and laboratory animals as markers for various diseases in addition to their important functions. However, the quantitative analysis of these bioactive molecules is obstructed by high structural similarity, poor ionization efficiency and low abundance. The current assay methods are still cumbersome to be of practical use, and their applicability in different bio-samples needs to be evaluated and optimized as necessary. In the present work, chromatographic separation conditions were carefully studied to achieve baseline separation of difficult-to-isolate compound pairs. On the other hand, an efficient sample purification method was established for colon tissue samples with good recoveries of sterols, demonstrating negligible autoxidation of cholesterol into oxysterols. The developed UPLC-APCI-MS/MS method was thoroughly validated and applied to measure oxysterols and cholesterol precursors in colon tissue of dextran sulfate sodium (DSS)-induced mouse colitis models, and it is expected to be successfully applied to the quantitative determination of such components in other tissue samples.

Discovery of a novel class of reversible monoacylglycerol lipase inhibitors for potential treatment of depression.

Biological studies on the endocannabinoid system (ECS) have suggested that monoacylglycerol lipase (MAGL), an essential enzyme responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), is a novel target for developing antidepressants. A decrease of 2-AG levels in the hippocampus of the brain has been observed in depressive-like models induced by chronic stress. Herein, employing a structure-based approach, we designed and synthesized a new class of (piperazine-1-carbonyl) quinolin-2(1H)-one derivatives as potent, reversible and selective MAGL inhibitors. And detailed structure-activity relationships (SAR) studies were discussed. Compound 27 (IC50 = 10.3 nM) exhibited high bioavailability (92.7%) and 2-AG elevation effect in vivo. Additionally, compound 27 exerted rapid antidepressant effects caused by chronic restraint stress (CRS) and didn't show signs of addictive properties in the conditioned place preference (CPP) assays. Our study is the first to report that reversible MAGL inhibitors can treat chronic stress-induced depression effectively, which may provide a new potential therapeutic strategy for the discovery of an original class of safe, rapid antidepressant drugs.

Effects of Tibetan medicine Longdan zhike tablet on chronic obstructive pulmonary disease through MAPK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Longdan zhike tablet (LDZK) is a Tibetan medicine formula commonly used in the highland region of Tibet, China, to ameliorate respiratory diseases, such as acute bronchitis and asthma. In Chinese traditional medicine, some herbal formulas with anti-inflammatory properties targeting the respiratory system are clinically adopted as supplementary therapies for chronic obstructive pulmonary disease (COPD). However, the specific anti-COPD effects of LDZK remain to be evaluated. AIM OF THE STUDY: The aim of this study is to identify the principal bioactive compounds in LDZK, and elucidate the effects and mechanisms of the LDZK on COPD. METHODS: High-resolution mass spectrometry was utilized for a comprehensive characterization of the chemical composition of LDZK. The therapeutic effects of LDZK were assessed on the LPS-papain-induced COPD mouse model, and LPS-induced activation model of A549 cells. The safety of LDZK was evaluated by orally administering a single dose of 30 g/kg to rats and monitoring physiological and biochemical indicators after a 14-day period. Network pharmacology and Western blot analysis were employed for mechanism prediction of LDZK. RESULTS: A comprehensive analysis identified a total of 45 compounds as the major constituents of LDZK. Oral administration of LDZK resulted in notable ameliorative effects in respiratory function, accompanied by reduced inflammatory cell counts and cytokine levels in the lungs of COPD mice. Acute toxicity tests demonstrated a favorable safety profile at a dose equivalent to 292 times the clinically prescribed dose. In vitro studies revealed that LDZK exhibited protective effects on A549 cells by mitigating LPS-induced cellular damage, reducing the release of NO, and downregulating the expression of iNOS, COX2, IL-1ß, IL-6, and TNF-α. Network pharmacology and Western blot analysis indicated that LDZK primarily modulated the MAPK signaling pathway and inhibited the phosphorylation of p38/ERK/JNK. CONCLUSIONS: LDZK exerts significant therapeutic effects on COPD through the regulation of the MAPK pathway, suggesting its potential as a promising adjunctive therapy for the treatment of chronic inflammation in COPD.

Health risk assessment for human mercury exposure from Cinnabaris-containing Baizi Yangxin Pills in healthy volunteers Po administration.

BACKGROUND: Cinnabaris (α-HgS), a mineral traditional Chinese material medica, has been used in combination with other herbs manifesting some definite therapeutic effects for thousands of years. But the currently reported mercury poisoning incidents raised the doubts about the safety of Cinnabaris-containing traditional Chinese medicines (TCMs). Baizi Yangxin Pills (BZYXP) is a Cinnabaris-containing TCM widely used in clinical practice. This study evaluated the health risk of mercury exposure from BZYXP in healthy volunteers based on the total mercury and mercury species analysis of blood and urine after single and multiple doses of BZYXP. METHODS: Blood pharmacokinetics and urinary excretion studies of mercury were compared between single (9 g, once daily) and multiple doses (9 g, twice daily, continued for 7 days) of BZYXP. The whole blood and urine samples were collected at the specific points or periods after the administration of BZYXP. The total mercury and mercury species in blood and urine samples were determined by cold vapor-atomic fluorescence spectrometry (CV-AFS) and HPLC-CV-AFS, respectively. RESULTS: The mercury was excreted slowly and accumulated obviously after continuous exposure of BZYXP. Moreover, the well-known neurotoxin methylmercury (MeHg) was detected in blood samples after 7 days' administration of BZYXP. In the urine samples, only Hg(II) was detected. Therefore, long-term use of BZYXP will cause mercury poisoning due to mercury's high accumulative properties and MeHg formation. CONCLUSION: Cinnabaris-containing TCMs such as BZYXP should be restricted to cases in which alternatives are available, and the blood mercury species profile should be monitored during the long-term clinical medication.

Hepatic Stellate Cell-Targeting Micelle Nanomedicine for Early Diagnosis and Treatment of Liver Fibrosis.

Diagnosing and treating liver fibrosis is a challenging yet crucial endeavor due to its complex pathogenesis and risk of deteriorating into cirrhosis, liver failure, and even hepatic cancer. Herein, a silica cross-linked micelles (SCLMs) based nano-system is developed for both diagnosing and treating liver fibrosis. The SCLMs are first modified with peptide CTCE9908 (CT-SCLMs) and can actively target CXCR4, which is overexpressed in activated hepatic stellate cells (HSCs). To enable diagnosis, an ONOO--responded near-infrared fluorescent probe NOF2 is loaded into the CT-SCLMs. This nano-system can target the aHSCs and diagnose the liver fibrosis particularly in CCl4-induced liver damage, by monitoring the reactive nitrogen species. Furthermore, a step is taken toward treatment by co-encapsulating two anti-fibrosis drugs, silibinin and sorafenib, within the CT-SCLMs. This combined approach results in a significant alleviation of liver injury. Symptoms associated with liver fibrosis, such as deposition of collagen, expression of hydroxyproline, and raised serological indicators show notable improvement. In summary, the CXCR4-targeted nano-system can serve as a promising theragnostic system of early warning and diagnosis for liver fibrosis, offering hope against progression of this serious liver condition.

Hybrids of selective COX-2 inhibitors and active derivatives of edaravone as COX-2 selective NSAIDs with free radical scavenging activity: Design, synthesis and biological activities.

Novel hybrids of selective COX-2 inhibitors (coxibs) and active derivatives of free radical scavenger edaravone were designed to overcome the risk of cardiovascular events and stroke increased by NSAIDs (nonsteroidal anti-inflammatory drugs) in this study. All the hybrids were assayed for the COX-2 inhibitory and DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging activities in vitro. Finally, we found a series of hybrids with good inhibitory activity and selectivity of COX-2 and excellent free radical scavenging activity in vitro. The most promising compound 6a (WYZ90) exhibited very potent COX-2 inhibitory activity (COX-2, IC50 = 75 nM), weak COX-1 inhibitory activity (COX-1, IC50 = 5734 nM), better free radical scavenging activity (DPPH, IC50 = 19.9 µM) than edaravone, moderate drug-likeness and ADME properties in silico, acceptable pharmacokinetic properties (T1/2 = 4.16 h, 10 mg/kg, o.p.) and oral bioavailability (F% = 36.03 %) in mice. In addition, compound WYZ90 showed similar analgesic activity to the selective COX-2 inhibitor celecoxib in acetic acid-induced mice and better antioxidant activity in Fe2+-induced lipid peroxidation in mouse liver tissue homogenate than edaravone. In conclusion, this study provided a novel class of coxibs containing edaravone moiety as COX-2 selective NSAIDs with free radical scavenging activity and the candidate compound WYZ90 showed not only similar selective COX-2 inhibitory and analgesic activity to celecoxib but also better free radical scavenging and antioxidant activity than edaravone.

Identification of degradation products of brivaracetam using liquid chromatography quadrupole time-of-flight tandem mass spectrometry: Degradation pathway elucidation.

RATIONALE: Pyrrolidone-based drugs find widespread use in treating conditions such as epilepsy and Alzheimer's disease, and in various other medical applications. Brivaracetam, the latest generation of pyrrolidone drugs, has exhibited significant promise owing to chemical structure modifications. Its affinity to the SV2A receptor is double that of the previous-generation drug, levetiracetam. Consequently, brivaracetam holds substantial potential for diverse applications. As a novel drug not yet included in the pharmacopeias of developed nations, comprehensive analysis and research are necessary to guarantee its safe utilization in clinical settings. METHODS: A liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC/QTOFMS) method has been developed to effectively separate, identify and characterize both the degradation products and process-related substances of brivaracetam. Stress testing of the sample was carried out following the guidelines outlined in ICH Q1A(R2). The structures of these impurities were identified through positive electrospray ionization QTOF high-resolution MS and NMR spectroscopy. Additionally, the formation mechanism of each degradation product is thoroughly discussed. RESULTS: Under the analytical conditions outlined in this paper, brivaracetam and its degradation products were effectively separated. Thirteen degradation products were detected and characterized, shedding light on their origins and degradation pathways. Among these, three degradation products align with previously reported impurities, and two unreported degradation products were synthesized and confirmed through NMR spectroscopy. The stress testing results revealed the instability of brivaracetam under acidic, alkaline, oxidative and thermal stress conditions, while it exhibited relative stability under photolytic stress conditions. CONCLUSION: The study developed an analytical method for brivaracetam that enabled the effective detection and separation of brivaracetam and its 13 degradation products. This method addresses a gap in both current domestic and foreign drug standards. The structures of all the major degradation products were characterized by high-resolution LC/QTOFMS, which is essential for quality control during the drug production process, stability evaluation and the establishment of proper storage conditions.

X-ray crystallography study and optimization of novel benzothiophene analogs as potent selective estrogen receptor covalent antagonists (SERCAs) with improved potency and safety profiles.

Endocrine therapy (ET) is a well-validated strategy for estrogen receptor α positive (ERα + ) breast cancer therapy. Despite the clinical success of current standard of care (SoC), endocrine-resistance inevitably emerges and remains a significant medical challenge. Herein, we describe the structural optimization and evaluation of a new series of selective estrogen receptor covalent antagonists (SERCAs) based on benzothiophene scaffold. Among them, compounds 15b and 39d were identified as two highly potent covalent antagonists, which exhibits superior antiproliferation activity than positive controls against MCF-7 cells and shows high selectivity over ERα negative (ERα-) cells. More importantly, their mode of covalent engagement at Cys530 residue was accurately illustrated by a cocrystal structure of 15b-bound ERαY537S (PDB ID: 7WNV) and intact mass spectrometry, respectively. Further in vivo studies demonstrated potent antitumor activity in MCF-7 xenograft mouse model and an improved safety profile. Collectively, these compounds could be promising candidates for future development of the next generation SERCAs for endocrine-resistant ERα + breast cancer.

DNA Repair-Responsive Engineered Whole Cell Microbial Sensors for Sensitive and High-Throughput Screening of Genotoxic Impurities.

Genotoxic impurities (GTIs) occurred in drugs, and food and environment pose a threat to human health. Accurate and sensitive evaluation of GTIs is of significance. Ames assay is the existing gold standard method. However, the pathogenic bacteria model lacks metabolic enzymes and requires mass GTIs, leading to insufficient safety, accuracy, and sensitivity. Whole-cell microbial sensors (WCMSs) can use normal strains to simulate the metabolic environment, achieving safe, sensitive, and high-throughput detection and evaluation for GTIs. Here, based on whether GTIs causing DNA alkylation required metabolic enzymes or not, two DNA repair-responsive engineered WCMS systems were constructed including Escherichia coli-WCMS and yeast-WCMS. A DNA repair-responsive promoter as a sensing element was coupled with an enhanced green fluorescent protein as a reporter to construct plasmids for introduction into WCMS. The ada promoter was screened out in the E. coli-WCMS, while the MAG1 promoter was selected for the yeast-WCMS. Different E. coli and yeast strains were modified by gene knockout and mutation to eliminate the interference and enhance the GTI retention in cells and further improved the sensitivity. Finally, GTI consumption of WCMS for the evaluation of methyl methanesulfonate (MMS) and nitrosamines was decreased to 0.46-8.53 µg and 0.068 ng-2.65 µg, respectively, decreasing 2-3 orders of magnitude compared to traditional methods. This study provided a novel approach to measure GTIs with different DNA damage pathways at a molecular level and facilitated the high-throughput screening and sensitive evaluation of GTIs.

Chemical index components and quality control of Traditional Chinese Medicine: "Never change a winning team"? -A case study of volatile oil from Bupleuri radix.

Chemical index components, especially those defined as quality control (QC) markers through spectrum-effect relationship approach, are commonly suggested and adopted as indicator for quality control of Traditional Chinese Medicines (TCMs). However, are chemical index components and quality control of TCMs "never change a winning team"? In this study, under the ponderation of the applicability of QC markers strategy, spectrum-effect relationship and OPLS-DA between GC×GC-MS fingerprint and inhibitory effect on the expression of extracellular secretory TNF-α of volatile oil from Bupleuri radix (BVO) was studied with the purpose of discovery of QC markers and establish a bioactive compounds-based QC method. 290 compounds of BVO were identified by GC×GC-MS. Besides, BVO had significant inhibitory effects on the expression of extracellular secretory TNF-α in a dose-dependent manner. The potency of different batches of BVOs could be distinguished with this bioassay-based method, which has been validated in terms of intermediate precision, repeatability, linearity, range and credibility tests. The QC markers of BVO were investigated by Spearman's correlation test and OPLS-DA. It is regrettable that there were no ideal QC markers of BVO could be found. In conclusion, quality control method relayed on chemical QC markers is not feasible for TCMs with complex composition but lack of ingredients that dominate in content, just like BVO. Alternatively, a bioassay-based method established in our study is suitable for quality control of BVO.

A chemical derivatization-based pseudotargeted LC-MS/MS method for high coverage determination of dipeptides.

Dipeptides (DPs) have attracted more and more attention in many research fields due to their important biological functions and promising roles as disease biomarkers. However, the determination of DPs in biological samples is very challenging owing to the limited availability of commercial standards, high structure diversity, distinct physical and chemical characteristics, wide concentration range, and the extensive existence of isomers. In this study, a pseudotargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with chemical derivatization for the simultaneous analysis of 400 DPs and their constructing amino acids (AAs) in biospecimens is established. Dansyl chloride (Dns-Cl) chemical derivatization was introduced to provide characteristic MS fragments for annotation and improve the chromatographic separation of DP isomers. A retention time (RT) prediction model was constructed using 83 standards (63 DPs and 20 AAs) based on their quantitative structural retention relationship (QSRR) after the Dns-Cl labeling, which largely facilitated the annotation of the DPs without standards. Finally, we applied this method to investigate the profile change of DPs in a cisplatin-induced acute kidney injury (AKI) rat model. The established workflow provides a platform to profile DPs and expand our understanding of these little-studied metabolites.

Therapeutic effects of Zhuling Jianpi capsule on experimental ulcerative colitis and characterization of its chemical constituents and metabolomics using UHPLC-Q-TOF-MS.

Zhuling Jianpi Capsule (Zhuling) is a traditional Chinese medicinal formula used to treat symptoms such as abdominal pain, bloating and diarrhea associated with inflammatory bowel disease (IBD). However, the protective effects of Zhuling on experimental ulcerative colitis (UC) and the effective substance responsible for its efficacy have rarely been reported. In this study, we evaluated the therapeutic effects of orally administrated Zhuling on DSS-induced UC in mice. The chemical constituents and metabolomics of Zhuling were qualitatively analyzed by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The results showed that Zhuling treatment markedly alleviated DSS-induced clinical symptoms, restrained the secretion of pro-inflammatory cytokines, and improved intestinal epithelial barrier function. Furthermore, a total of 167 compounds have been identified or characterized, and 120 prototype components were detected in the urine, plasma, bile and feces of mice. Among them, altogether 26 representative prototypes were associated with 139 metabolites via the corresponding biotransformation pathways, and both of them mainly contained flavonoids, alkaloids, organic acids, monoterpenes, phenylpropanoids, triterpenoids, sesquiterpenoids and anthraquinones. Finally, 12 potent compounds mainly containing flavonoids, terpenoids and phenylpropanoids were screened out as potential quality control index components and might be the main substances that exert a pharmacological effect. Our data indicated that Zhuling administration prominently alleviates DSS-induced colitis in mice. Additionally, the chemical and metabolic profiling provided helpful information on the potential pharmacodynamic substances of Zhuling, which can be further investigated in the future.

Structural optimization of tetrahydroisoquinoline-hydroxamate hybrids as potent dual ERα degraders and HDAC inhibitors.

Both estrogen receptor α (ERα) and histone deacetylases (HDACs) are valid therapeutic targets for anticancer drug development. Combination therapies using diverse ERα antagonists or degraders and HDAC inhibitors have been proven effective in endocrine-resistant ER + breast cancers based on the crosstalk between ERα and HDAC pathway. In this study, we reported the optimization of a series of methoxyphenyl- or pyridinyl- substituted tetrahydroisoquinoline-hydroxamates, which were optimized from 31, a dual ERα degrader/HDAC inhibitor previously reported by our group. Most of the synthesized compounds displayed potent ERα degradation efficacy and antiproliferative activity. Among them, A04 demonstrated the best anti-proliferation activity (MCF-7 IC50 = 1.96 µM) and HDAC6 inhibitory activity (HDAC6 IC50 = 25.96 nM), which is slightly more potent than the lead compound 31 (MCF-7 IC50 = 4.38 µM, HDAC6 IC50 = 63.03 nM). In addition, compound A04 exerted ERα-independent HDAC6-inhibiting effect without agonistic activity in endometrial cells. These results demonstrated that A04 is a novel and promising dual ERα degrader/HDAC inhibitor worthy of further development.

Harnessing nanobiotechnology for cerebral ischemic stroke management.

Cerebral ischemic stroke remains one of the most serious neurological disorders that pose threats to human health, causing a large amount of long-term disability or even death throughout the world. Based on its physiologic and pathological features, there are limited available therapeutic options for effective ischemic stroke management. Encouragingly, a rapid advancement of nanobiotechnology is bringing new insights into exploring more alternative strategies against cerebral ischemic stroke, which can cleverly overcome the limitations related to conventional treatment methods. Therefore, this review focuses on the recent achievements of nanobiotechnology for ischemic stroke management, which emphasizes diverse targeted delivery strategies using various nanoplatforms including liposomes, micelles, polymeric nanoparticles, nanogels, inorganic nanomaterials, and cell-derived nano-vectors based on the pathophysiological features of ischemic stroke. Moreover, different therapeutic approaches against ischemic stroke such as neuroprotection, anti-inflammation, thrombolysis, increased blood-brain barrier penetration and reactive oxygen species scavenging are highlighted. Meanwhile, this review discusses how these versatile nanoplatforms were designed to assist in the treatment of ischemic stroke. Based on this, challenges, opportunities, and future perspectives using nanobiotechnology through rational design for effective ischemic stroke management are revealed.

Discovery of selective covalent cathepsin K inhibitors containing novel 4-cyanopyrimidine warhead based on quantum chemical calculations and binding mode analysis.

Cathepsin K (Cat K), mainly expressed by osteoclasts, plays an important role in bone resorption. Covalent Cat K inhibitors will show great potential in the future treatment of osteoporosis. It has been reported that the selectivity of covalent cathepsin K inhibitors was related to the drug's safety. The type of warhead has a crucial influence on the enzyme bioactivity and selectivity of covalent inhibitors. In order to develop novel covalent inhibitors with the selective new warhead, quantum chemical calculations were performed to estimate the reactivity of the nitrile warheads. Moreover, binding mode analysis between ligands and high homology Cat K, S and B revealed differences in non-covalent interactions. Novel covalent Cat K inhibitors containing 4-cyanopyrimidine warhead (11) were determined for the first time. Among them, compound 34 significantly inhibited Cat K (IC50 = 61.9 nM) with excellent selectivity compared to Cat S (>810-fold) and Cat B (>1620-fold), respectively. Binding mode analysis of Cat K-34 complex provided the basis for further optimization. Compound 34 could be a valuable lead compound for further research on safe and effective Cat K inhibitors.

Discovery of potential active ingredients of Er-Zhi-Wan, a famous traditional Chinese formulation, in model rat serum for treating osteoporosis with kidney-yin deficiency by UPLC-Q/TOF-MS and molecular docking.

Er-Zhi-Wan (EZW), a classical traditional Chinese formulation, has attracted more and more attention. This study was carried out to analyze the constituents of EZW absorbed into blood and find out the potential active ingredients for treating osteoporosis (OP) with kidney-yin deficiency (KYD). The rat model of OP with KYD was achieved by ovariectomies and using the mixture of thyroxine and reserpine. Then ultra-high performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer (UPLC-Q/TOF-MS) combined with statistical analysis was used to analyze the constituents of EZW absorbed into blood and differential components between the normal and OP with KYD rats. Finally, the components identified in OP with KYD rats were docked with targets of OP with KYD found in online databases. The results of molecular docking were adopted to find the potential active ingredients and further verified in vitro experiment. A total of 21 prototype compounds and 69 metabolites were identified in serum. Among them, 63 components in model rats and 50 components in normal rats were summarized, respectively. Most of the identified metabolites in serum of model rats were produced by hydrolysis, oxidation or glucuronidation, while in serum of normal rats were produced by hydrolysis, oxidation and methylation. According to the results of molecular docking, specnuezhenide, salidroside, tyrosol, echinacoside and verbascoside could be classified as potential active ingredients. The activity of salidroside and a metabolite was verified by pharmacodynamics analysis. In summary, UPLC-Q/TOF-MS system was combined with molecular docking to search the potential active ingredients from model rats of OP with KYD, which provided a new idea for the research on the pharmacodynamic material basis of other traditional medicine. Moreover, the result of this study lays the foundation for further study regarding the mechanism of EZW in treating OP with KYD.

Design and Synthesis of Novel Celastrol Derivatives as Potential Anticancer Agents against Gastric Cancer Cells.

Gastric cancer (GC) is a common malignant disease worldwide, and finding novel agents and strategies for the treatment of GC are of urgent need. Celastrol (CEL) is a well-known natural product with antineoplastic activity. In this study, pyrazole analogues were introduced at the C-29 position of CEL. A total of 24 new derivatives were designed, synthesized, and evaluated for their mechanism and antitumor activity in vitro and in vivo. Among them, compound 21 exhibited the best activity against BGC-823 cells (IC50 = 0.21 ± 0.01 µM). Further biological studies showed that 21 significantly raised the reactive oxygen species (ROS) levels to activate the apoptotic pathway, causing mitochondrial dysfunction in BGC-823 cells. In addition, 21 also arrested cells in the G2/M phase to induce tumor cell apoptosis. In a nude mouse tumor xenograft model, 21 exhibited a better tumor inhibition rate (89.85%) than CEL (inhibition rate 76.52%). Taken together, the present study has provided an anticancer lead compound candidate, 21, and has revealed that increased ROS generation may be an effective strategy in the treatment of GC.

Metabolic analysis of rhubarb extract by rat intestinal bacteria using liquid chromatography-tandem mass spectrometry.

Through investigation of the metabolism of rhubarb extract by rat intestinal bacteria, a total of 14 components in rhubarb extract were found to be biotransformed. These components included aloe-emodin-O-glucosides, emodin-O-glucosides, chrysophanol-O-glucosides, physcion-O-glucosides and the corresponding aglycones. Rhein also could be biotransformed by rat intestinal bacteria. Twelve major metabolites were detected in the incubation sample. Under ESI tandem mass conditions, the sequential fragmentation patterns of [M H](-) ions were similar to those of free anthraquinones, thus allowing the rapid identification of the metabolites formed in incubation samples. The results suggested that the proposed hydrolysis of glycoside group followed by hydrogenation in quinoid moiety and/or further acetylation was the major biotransformation pathway for these anthraquinone glycosides by rat intestinal bacteria.

In vivo metabolism study of rhubarb decoction in rat using high-performance liquid chromatography with UV photodiode-array and mass-spectrometric detection: a strategy for systematic analysis of metabolites from traditional Chinese medicines in biological samples.

High-performance liquid chromatography with diode-array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) was used for separation and identification of metabolites in rat urine, bile and plasma after oral administration of rhubarb decoction. Based on the proposed strategy, 91 of the 113 potential metabolites were tentatively identified or characterized. Besides anthraquinones metabolites, gallic acid, (-)-epicatechin and (+)-catechin metabolites were also detected and characterized in these biological samples. Our results indicated that glucuronidation and sulfation were the main metabolic pathways of anthraquinones, while methylation, glucuronidation and sulfation were the main metabolic pathways of gallic acid, (-)-epicatechin and (+)-catechin. Phase I reactions (e.g., hydroxylation and reduction) played a relatively minor role compared to phase II reactions in metabolism of phenolic compounds of rhubarb decoction. The identification and structure elucidation of these metabolites provided essential data for further pharmacological and clinical studies of rhubarb and related preparations. Moreover, the results of the present investigations clearly indicated the relevance and usefulness of the combination of chromatographic, spectrophotometric, and mass-spectrometric analysis to detect and identify metabolites.

HPLC/DAD comparison of sixteen bioactive components between Da-Cheng-Qi decoction and its parent herbal medicines.

Differences in the contents of sixteen bioactive components (three tannins, five anthraquinones, six flavonoids and two neolignans) between Da-Cheng-Qi decoction (DCQD) and its three constitutional herbal medicines (Radix et Rhizoma Rhei, Cortex Magnoliae officinalis, and Fructus Aurantii Immaturus) were compared using validated HPLC/DAD methods. The results indicated that there existed some kinds of interactions between these constitutional natural medicines during the DCQD preparation procedure, which could either enhance or depress the extractive rates of bioactive components.