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.

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.

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.

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.

Constituents of Da-Cheng-Qi decoction and its parent herbal medicines determined by LC-MS/MS.

Da-Cheng-Qi decoction (DCQD) is a purgative prescription used in China and East Asia. To profile the constituents of this complex traditional Chinese medicine (TCM), a high-performance liquid chromatographic, electrospray ionization, tandem mass spectrometric (HPLC-ESI/MS/MS) analytical method was developed. After separation on a reversed-phase C18 analytical column using gradient elution, samples were analyzed by ESI-MS/MS in negative mode. As a result, a total of 37 compounds were detected, of which two tannins, three anthraquinones, two sennosides, five flavonoids and two lignans were unambiguously identified by comparison with standard compounds, and sixteen compounds were either tentatively identified or deduced according to their MS/MS data. The fragmentation pathways of many of the observed compounds, such as the tannins and lignans are reported for the first time. In addition, the identity of each peak in DCQD was explored by comparison with those of its three constituent herbs. The results indicated that tannins, anthraquinones and sennosides in DCQD originated from Radix et Rhizoma Rhei, flavonoids from Fructus Aurantii Immaturus, and lignans from Cortex Magnoliae officinalis. The present study provides an example of chemical constitution profiling in complex TCM systems using LC/MS/MS.

Pharmacokinetic comparison in rats of six bioactive compounds between Da-Cheng-Qi decoction and its parent herbal medicines.

Da-Cheng-Qi decoction (DCQD) is a purgative compound prescription used in China and East Asia. In this paper, pharmacokinetic differences of six major active components (rhein, emodin, aloe-emodin, magnolol, naringenin and hesperetin) between DCQD and its three constitutional herbal medicines, i.e., Radix et Rhizoma Rhei, Cortex Magnoliae officinalis and Fructus Aurantii Immaturus were investigated in rats after oral administration. Plasma samples were analyzed for the quantification of the six active components using validated LC-MS/MS methods. Unpaired Student's t-test was used for statistical comparison. Significant differences (p < 0.05) in the main pharmacokinetic parameters for rhein, emodin, aloeemodin, magnolol, naringenin and hesperetin were found between DCQD and the decoction of its constitutional single herbal medicines, which demonstrated the presence of drug-drug interactions between these constitutional raw materials of DCQD occurring either in the procedure of decoction or during ADME process.

Chemical fingerprinting of Andrographis paniculata (Burm. f.) Nees by HPLC and hierarchical clustering analysis.

The aim is to develop a simple and specific method for the extraction and chemical fingerprinting of Andrographis paniculata (Burm. f.) Nees and to apply the method to this drug from different regions. High-performance liquid chromatographic (HPLC) with gradient elution is used for developing the fingerprints, and liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) technique is employed to identify the component of the fingerprints. Nine peaks are selected as common peaks, and six compounds are elucidated by MS data. Twenty-three samples of A. paniculata from different regions of China are collected and detected by HPLC fingerprinting. Comparisons of the chromatograms show that there are obvious differences in the content of each component contained between the habitat samples in China. The results of hierarchical cluster analysis show that these samples can be clustered reasonably into three groups, and the growth of A. paniculata and their internal quality are related to their habitat. The HPLC fingerprint developed allows simple identification of A. paniculata from many natural drugs.

Structural elucidation of in vitro metabolites of emodin by liquid chromatography-tandem mass spectrometry.

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was employed to investigate the in vitro metabolism of emodin. Emodin was incubated with rat liver microsomes in the presence of a NADPH-generating system, followed by extraction with ethyl acetate. After separation on a reversed-phase C18 analytical column with a linear gradient elution of methanol and 0.1% formic acid in water, negative electrospray ionization tandem mass spectrometry experiments were performed. As a result, the parent drug and its six metabolites were detected from rat liver microsomal incubations. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. Besides three mono-hydroxylated metabolites (omega-hydroxyemodin, 2-hydroxyemodin, 4-hydroxyemodin), three other metabolites were identified, which were emodic acid, 3-carbomethoxy-6-methoxy-1,8-dihydroxyanthraquinone and physcion, respectively.

Rapid simultaneous quantification of five active constituents in rat plasma by high-performance liquid chromatography/tandem mass spectrometry after oral administration of Da-Cheng-Qi decoction.

A rapid, sensitive and specific liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) method has been developed and validated for simultaneous determination of five active constituents (including magnolol, honokiol, rhein, emodin and aloe-emodin) from Da-Cheng-Qi decoction (DCQD) in rat plasma. After the addition of gliquidone as the internal standard (IS), plasma samples were prepared by one-step protein precipitation using methanol and separated by HPLC on a short reversed phase C(18) column packed with smaller particles (100 mm x 3.0 mm, 3.5 microm) using a mobile phase of methanol-0.1% formic acid aqueous solution (70:30, v/v). Analytes were determined in a triple-quadrupole mass spectrometer in the selected reaction-monitoring (SRM) mode using electrospray source with negative mode. The method was proved to be rapid, sensitive, specific, accurate and reproducible and has been successfully applied to the determination of the five compounds in rat plasma after oral administration of low dose DCQD for pharmacokinetic study.