Identification of metabolites of rupestonic acid in rat urine by liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Rupestonic acid, a potential anti‐influenza agent, is an important and characteristic compound in Artemisia rupestris L., a well‐known traditional Uighur medicine for the treatment of colds. In the present study, high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was used to detect and identify the metabolites in rat urine after oral administration of rupestonic acid. A total of 10 metabolites were identified or partially characterized. The structure elucidations of the metabolites were performed by comparing the changes in accurate molecular masses and fragment ions with those of the parent compound. The results showed that the main metabolites of rupestonic acid in rat urine were formed by oxidation, hydrogenation and glucuronidation. A metabolism pathway was proposed for the first time based on the characterized structures. This metabolism study can provide essential information for drug discovery, design and clinical application of rupestonic acid. Copyright © 2014 John Wiley & Sons, Ltd.     

Chemical Profiles and Pharmacological Properties with in Silico Studies on Elatostema papillosum Wedd

The current study attempted, for the first time, to qualitatively and quantitatively determine the phytochemical components of Elatostema papillosum methanol extract and their biological activities. The present study represents an effort to correlate our previously reported biological activities with a computational study, including molecular docking, and ADME/T (absorption, distribution, metabolism, and excretion/toxicity) analyses, to identify the phytochemicals that are potentially responsible for the antioxidant, antidepressant, anxiolytic, analgesic, and anti-inflammatory activities of this plant. In the gas chromatography-mass spectroscopy analysis, a total of 24 compounds were identified, seven of which were documented as being bioactive based on their binding affinities. These seven were subjected to molecular docking studies that were correlated with the pharmacological outcomes. Additionally, the ADME/T properties of these compounds were evaluated to determine their drug-like properties and toxicity levels. The seven selected, isolated compounds displayed favorable binding affinities to potassium channels, human serotonin receptor, cyclooxygenase-1 (COX-1), COX-2, nuclear factor (NF)-κB, and human peroxiredoxin 5 receptor proteins. Phytol acetate, and terpene compounds identified in E. papillosum displayed strong predictive binding affinities towards the human serotonin receptor. Furthermore, 3-trifluoroacetoxypentadecane showed a significant binding affinity for the KcsA potassium channel. Eicosanal showed the highest predicted binding affinity towards the human peroxiredoxin 5 receptor. All of these findings support the observed in vivo antidepressant and anxiolytic effects and the in vitro antioxidant effects observed for this extract. The identified compounds from E. papillosum showed the lowest binding affinities towards COX-1, COX-2, and NF-κB receptors, which indicated the inconsequential impacts of this extract against the activities of these three proteins. Overall, E. papillosum appears to be bioactive and could represent a potential source for the development of alternative medicines; however, further analytical experiments remain necessary.     

Metabonomics study of the effects of traditional Chinese medicine formula Ermiaowan on hyperuricemic rats

To explore the global mechanism of Ermiaowan on hyperuricemia regulation, the holistic function of Ermiaowan for hyperuricemia in rats was firstly assessed by the urinary metabonomics method which was based on ultra‐high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry. The urinary targeted metabonomics approach combined with the serum biochemical analysis and histological assay was conducted to verify the research result. As a result, the significant differences in metabolic profiles were observed from Ermiaowan‐treated group, model group, and healthy control group by using multivariate statistical approaches. Twenty therapeutic related metabolites were identified in response to the therapeutic effects of Ermiaowan, which were mainly associated with purine metabolism, pyrimidine metabolism, tryptophan metabolism, tricarboxylic acid cycle, and tyrosine metabolism. In addition, the urinary targeted metabonomics study showed that Ermiaowan can better restore the disturbed pathways than Phellodendri cortex and Atractylodis rhizome. The biochemical assay and histopathological assay confirmed that Ermiaowan could significantly reduce uric acid and fibrosis areas of kidney. These results provided new insights into the mechanism of Ermiaowan on hyperuricemia.     

Comparative in vivo constituents and pharmacokinetic study in rats after oral administration of ultrafine granular powder and traditional decoction slices of Chinese Salvia

Salvia miltiorrhiza, one of the most well‐known herbal medicines, is commonly used for the treatment of coronary heart diseases in China. Besides traditional decoction slices (TDS), another relatively new product of S. miltiorrhiza, ultrafine granular powder (UGP; D90 < 45 μm), is also increasingly being used. In this paper, a UHPLC‐LTQ‐Orbitrap MS technique was developed for a metabolite profile study after oral administration of UGP and TDS of S. miltiorrhiza. The results showed that the number of in vivo absorbed compounds from UGP was much greater than that from TDS, and different types of products from S. miltiorrhiza will have different metabolic processes in vivo. Furthermore, a UHPLC‐Q‐Trap MS/MS method for simultaneously determining four tanshinones (tanshinone IIA, dihydrotanshinone I, tanshinone I and cryptotanshinone) was established and applied to assess the pharmacokinetics of the two types of products. All of the analytes displayed significant higher area under the concentration–time curve and peak concentration after oral administration of UGP than after TDS, indicating that ultrafine powder product could improve the bioavailability and absorption of cryptotanshinon,tanshinone II A,dihydrotanshinonE I and tanshinone I in vivo. The present study provides scientific information for further exploration of the pharmacology of these two types of S. miltiorrhiza and offers a reference for clinical administration of S. miltiorrhiza.     

Simultaneous quantitation of 23 bioactive compounds in Tanreqing capsule by high‐performance liquid chromatography electrospray ionization tandem mass spectrometry

Tanreqing capsule (TRQC) is a formulation frequently used in traditional Chinese medicine to treat pyrexia, cough, expectoration and pharyngalgia. Since the pharmacological action of traditional Chinese medicines is closely related to their complex and diverse constituents, understanding the exact composition of TRQC is important to elucidate its clinical effectiveness and mechanism of action as well as to establish quality control methods and resolve safety issues. Herein, we employed high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry for the simultaneous quantitation of 23 bioactive compounds in five batches of TRQC; the analytes could be categorized into five types: organic acids (seven compounds), flavonoids (10 compounds), iridoids (two compounds), phenylethanoid glycosides (two compounds) and bile acids (two compounds). The calibration curves for all analytes showed good linearity (r > 0.9953), and the inter‐ and intra‐day precisions did not exceed 4.94 and 4.97%, respectively. The recoveries varied from 90.47% to 109.80%; the corresponding relative standard deviations (RSDs) did not exceed 4.94%; and the repeatability (RSD < 4.72%) and stability (RSD < 4.88%) were also within acceptable limits. Thus, this study can be viewed as a fundamental reference for setting comprehensive TRQC quality standards.     

A biochemometrics strategy combining quantitative determination,bioactivity evaluation and relationship analysis for identification of analgesic alkaloids of raw and vinegar‐processed Corydalis turtschaninovii

A biochemometrics strategy combining quantitative determination, bioactivity evaluation, and relationship analysis was proposed for identification of analgesic components of herbs. First, a robust liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of nine major alkaloids in crude and vinegar‐processed Corydalis turtschaninovii. Nine alkaloids were separated on a BEH C₁₈ column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid and then detected by multiple reactions monitoring in the positive ion mode. Nitidine chloride was employed as the internal standard. The method displayed good linearity and the precisions of intra‐day and inter‐day were all within 3.0%. The recovery rates of each alkaloid ranged from 97.1 to 102.9%. The method was successfully applied for quantitative analysis of nine alkaloids in ten batches of crude and vinegar‐processed Corydalis turtschaninovii. Second, the analgesic effects of crude and vinegar‐processed Corydalis turtschaninovii were evaluated in mice. Third, principle component analysis, canonical correlation analysis, and partial least squares regression were used to analysis the relationship between the contents of nine major alkaloids and the analgesic effect of different crude and vinegar‐processed samples. Tetrahydropalmatine, coptisine, and dehydrocorydaline have a close positive correlation with the analgesic effect.     

Comprehensive two‐dimensional PC‐3 prostate cancer cell membrane chromatography for screening anti‐tumor components from Radix Sophorae flavescentis

Radix Sophorae flavescentis is generally used for the treatment of different stages of prostate cancer in China. It has ideal effects when combined with surgical treatment and chemotherapy. However, its active components are still ambiguous. We devised a comprehensive two‐dimensional PC‐3 prostate cancer cell membrane chromatography system for screening anti‐prostate cancer components in Radix Sophorae flavescentis . Gefitinib and dexamethasone were chosen as positive and negative drugs respectively for validation and optimization the selectivity and suitability of the comprehensive two‐dimensional chromatographic system. Five compounds, sophocarpine, matrine, oxymatrine, oxysophocarpine, and xanthohumol were found to have significant retention behaviors on the PC‐3 cell membrane chromatography and were unambiguously identified by time‐of‐flight mass spectrometry. Cell proliferation and apoptosis assays confirmed that all five compounds had anti‐prostate cancer effects. Matrine and xanthohumol had good inhibitory effects, with half maximal inhibitory concentration values of 0.893 and 0.137 mg/mL, respectively. Our comprehensive two‐dimensional PC‐3 prostate cancer cell membrane chromatographic system promotes the efficient recognition and rapid analysis of drug candidates, and it will be practical for the discovery of prostate cancer drugs from complex traditional Chinese medicines.     

Identification and analysis of chemical constituents and rat serum metabolites in Suan‐Zao‐Ren granule using ultra high performance liquid chromatography quadrupole time‐of‐flight mass spectrometry combined with multiple data processing approaches

Suan‐Zao‐Ren granule is widely used to treat insomnia in China. However, because of the complexity and diversity of the chemical compositions in traditional Chinese medicine formula, the comprehensive analysis of constituents in vitro and in vivo is rather difficult. In our study, an ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry and the PeakView® software, which uses multiple data processing approaches including product ion filter, neutral loss filter, and mass defect filter, method was developed to characterize the ingredients and rat serum metabolites in Suan‐Zao‐Ren granule. A total of 101 constituents were detected in vitro. Under the same analysis conditions, 68 constituents were characterized in rat serum, including 35 prototype components and 33 metabolites. The metabolic pathways of main components were also illustrated. Among them, the metabolic pathways of timosaponin AI were firstly revealed. The bioactive compounds mainly underwent the phase I metabolic pathways including hydroxylation, oxidation, hydrolysis, and phase II metabolic pathways including sulfate conjugation, glucuronide conjugation, cysteine conjugation, acetycysteine conjugation, and glutathione conjugation. In conclusion, our results showed that this analysis approach was extremely useful for the in‐depth pharmacological research of Suan‐Zao‐Ren granule and provided a chemical basis for its rational.     

Quality assessment of crude and processed Arecae semen based on colorimeter and HPLC combined with chemometrics methods

Raw Arecae Semen, the seed of Areca catechu L., as well as Arecae Semen Tostum and Arecae semen carbonisata are traditionally processed by stir‐baking for subsequent use in a variety of clinical applications. These three Arecae semen types, important Chinese herbal drugs, have been used in China and other Asian countries for thousands of years. In this study, the sensory technologies of a colorimeter and sensitive validated high‐performance liquid chromatography with diode array detection were employed to discriminate raw Arecae semen and its processed drugs. The color parameters of the samples were determined by a colorimeter instrument CR‐410. Moreover, the fingerprints of the four alkaloids of arecaidine, guvacine, arecoline and guvacoline were surveyed by high‐performance liquid chromatography. Subsequently, Student's t test, the analysis of variance, fingerprint similarity analysis, hierarchical cluster analysis, principal component analysis, factor analysis and Pearson's correlation test were performed for final data analysis. The results obtained demonstrated a significant color change characteristic for components in raw Arecae semen and its processed drugs. Crude and processed Arecae semen could be determined based on colorimetry and high‐performance liquid chromatography with a diode array detector coupled with chemometrics methods for a comprehensive quality evaluation.     

Rapid separation and simultaneous quantitative determination of 13 constituents in Psoraleae Fructus by a single marker using high‐performance liquid chromatography with diode array detection

Psoraleae Fructus is one of the most popular traditional Chinese medicines. Coumarins, flavonoids, and meroterpenes are the main contributors to the biological activity of Psoraleae Fructus. In this study, a new method for the quality control of Psoraleae Fructus was developed, through the quantitative analysis of multicomponents by single marker with diode array detector. Thirteen components, including psoralenoside, isopsoralenoside, psoralen, isopsoralen, psoralidin, neobavaisoflavone, bavachin, corylin, isobavachalcone, corylifol A, bavachinin, bavachalcone, and bakuchiol were rapidly separated and identified within 12 min by the newly developed method. The feasibility and reliability of this method were corroborated. The method was also compared to the external standard method and detection by corona charged aerosol detector. The results of percent difference (%) and cos (θ) have shown that there were no significant differences observed between the quantitative analysis of multicomponents by single marker and external standard method analyses; psoralen and isopsoralen were undetectable with the corona charged aerosol detector due to their but the sensitivity for all the compounds except bakuchiol detected by corona charged aerosol detector are higher than those obtained by diode array detector. In addition, the newly method developed was applied to the quality evaluation of Chinese patent medicines containing Psoraleae Fructus.