Construction of a prognostic model for autophagy in Wilm's tumor.

BACKGROUND: Wilm's tumor (WT) is one of the most common childhood urological tumors, ranking second in the incidence of pediatric abdominal tumors. The development of WT is associated with various factors, and the correlation with autophagy is currently unclear. PURPOSE: To develop a new prognostic model of autophagy-related genes (ATG) for WT. METHODS: Using the Therapeutically applicable research to generate effective treatments (TARGET) database to screen for differentially expressed ATGs in WT and normal tissues. ATGs were screened for prognostic relevance to WT using one-way and multifactorial Cox regression analyses and prognostic models were constructed. The risk score was calculated according to the model, and the predictive ability of the constructed model was analyzed using the ROC (receiver operating characteristic) curve to verify the significance of the model for the prognosis of WT. RESULTS: Sixty-eight differentially expressed ATGs were identified by univariate Cox regression analysis, and two critical prognostic ATGs (CXCR4 and ERBB2) were identified by multivariate Cox regression analysis. Patients were divided into high-risk and low-risk groups according to the differential expression of these two ATGs. Kaplan-Meier (KM) curves showed a significant difference in survival time between the two groups. The critical prognostic ATGs were combined with race, age, and stage in a multifactorial regression analysis, and the final prognostic model was produced as a line graph. CONCLUSION: The prognostic model of autophagy-related genes composed of the CXCR4 gene and ERBB2 gene has a specific predictive value for the prognosis of WT, and the present study provides a clear basis for future research on biomarkers and therapeutic targets.

Discrepancy Study of the Chemical Constituents of Panax Ginseng from Different Growth Environments with UPLC-MS-Based Metabolomics Strategy.

Panax ginseng (P. ginseng), the dried root and rhizome of P. ginseng C. A. Meyer, is widely used in many fields as dietary supplements and medicine. To characterize the chemical constituents in P. ginseng cultivated in different growth environments, a UPLC-TOF-MS method was established for qualitative analysis. Four hundred and eight ginsenosides, including 81 new compounds, were characterized in P. ginseng from different regions. Among the detected compounds, 361 ginsenosides were recognized in P. ginseng cultivated in the region of Monsoon Climate of Medium Latitudes, possessing the largest amount of ginsenosides in all samples. Furthermore, 41 ginsenosides in 12 batches of P. ginsengs were quantified with a UPLC-MRM-MS method, and P. ginsengs from different regions were distinguished via chemometric analysis. This study showed that the different environments have a greater influence on P. ginseng, which laid a foundation for further quality control of the herb.

Characterization of Ginsenosides from the Root of Panax ginseng by Integrating Untargeted Metabolites Using UPLC-Triple TOF-MS.

To compare the chemical distinctions of Panax ginseng Meyer in different growth environments and explore the effects of growth-environment factors on P. ginseng growth, an ultra-performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS) was used to characterize the ginsenosides obtained by ultrasonic extraction from P. ginseng grown in different growing environments. Sixty-three ginsenosides were used as reference standards for accurate qualitative analysis. Cluster analysis was used to analyze the differences in main components and clarified the influence of growth environment factors on P. ginseng compounds. A total of 312 ginsenosides were identified in four types of P. ginseng, among which 75 were potential new ginsenosides. The number of ginsenosides in L15 was the highest, and the number of ginsenosides in the other three groups was similar, but it was a great difference in specie of ginsenosides. The study confirmed that different growing environments had a great influence on the constituents of P. ginseng, and provided a new breakthrough for the further study of the potential compounds in P. ginseng.

A comprehensive study on the relieving effect of Lilium brownii on the intestinal flora and metabolic disorder in p-chlorphenylalanine induced insomnia rats.

CONTEXT: The bulb of Lilium brownii F. E. Brown (Liliaceae) (LB) is a common Chinese medicine to relieve insomnia. OBJECTIVE: To investigate the molecular mechanism of LB relieving insomnia. MATERIALS AND METHODS: Insomnia model was induced by intraperitoneally injection p-chlorophenylalanine (PCPA) in Wistar rats. Rats were divided into three groups: Control, PCPA (400 mg/kg, i.p. 2 days), LB (598.64 mg/kg, oral 7 days). The levels of 5-hydroxytryptamine (5-HT), norepinephrine (NE), melatonin (MT), and the expression of GABAA, 5-HT1A and MT receptors, as well as pathological changes in hypothalamus, were evaluated. 16S rDNA sequencing and UPLC-MS/MS were used to reveal the change of the intestinal flora and metabolic profile. RESULTS: The adverse changes in the abundance and diversity of intestinal flora and faecal metabolic phenotype altered by PCPA in rats were reversed after LB treatment, accompanied by the up-regulated levels of 5-HT as 8.14 ng/mL, MT as 16.16 pg/mL, 5-HT1A R and GABAA R, down-regulated level of NE as 0.47 ng/mL, and the improvement of pathological phenomena of cells in the hypothalamus. And the arachidonic acid metabolism and tryptophan metabolism pathway most significantly altered by PCPA were markedly regulated by LB. Besides, it was also found that LB reduced the levels of kynurenic acid related to psychiatric disorders and trimethylamine-N-oxide associated with cardiovascular disease. CONCLUSION: The mechanism of LB relieving insomnia involves regulating flora and metabolites to resemble the control group. As a medicinal and edible herb, LB could be considered for development as a health-care food to relieve increasing insomniacs in the future.

Anti-pandemic influenza A (H1N1) virus potential of Xilingjiedu capsule in vitro.

This study is aimed to investigate the effect of Xilingjiedu capsule (XLC), one of a preparation of traditional Chinese medicine, on influenza A (H1N1) virus as well as its preliminary mechanism. The median cell mortality (TC50) to A549 cells and half effective inhibition concentration (IC50) of influenza A (H1N1) virus of XLC were determined by MTT assay. Reed-Muench method was used to calculated the 50% tissue culture infective dose (TCID50) of H1N1 virus to A549 cells. In mechanism research, the mRNA expression levels of MyD88, TLR4, TLR7 and TRAF6 and the protein expression level of MyD88 were detected by using RT-PCR and Western blot, respectively. The results suggested that XLC showed good anti influenza A (H1N1) virus activity. The antiviral mechanism of XLC was related to the Toll-like signaling pathway. It could drown regulate the mRNA expression level of MyD88 and TLR4 and the protein level of MyD88. This research provides reference for the application of XLC in anti influenza virus.

Poly-pharmacokinetic strategy represented the synergy effects of bioactive compounds in a traditional Chinese medicine formula, Si Shen Wan and its separated recipes to normal and colitis rats.

Si Shen Wan is a classic traditional Chinese medicine formula, which has been used to treat chronic colitis for thousands of years. Many research and experience show that Si Shen Wan was developed by the combination of two sets of "Herb Pairs," Er Shen Wan and Fructus Schisandrae Chinensis Powder. This research aimed to revealing the effective substances, guide the clinical treatment, and represent the synergy effects from the view of pharmacokinetics. An ultra high performance liquid chromatography with tandem mass spectrometry method was established and validated for simultaneous quantification of 26 main bioactive compounds in normal and colitis rat plasma after oral administration of Si Shen Wan and its "Herb Pairs" extract. The method validation results illustrated that the experimental method was reliable and reproducible for quantitative determination of the biological samples. The pharmacokinetic behaviors in different groups were compared and discussed comprehensively, which indicated that the treatment of Si Shen Wan has a superiority in synthetic action of the "Herb Pairs" for the higher peak concentrations and bioavailability of some mainly components. Furthermore, the synergy effect was still existing backed up again for the longer eliminate time and a better bioavailability in colitis groups. The pharmacokinetics research of multiple components in Si Shen Wan and its "Herb Pairs" supplied a significant basis for better understanding the metabolic mechanism of these formulas in both normal and pathological state.

Metabolic map of the antiviral drug podophyllotoxin provides insights into hepatotoxicity.

Podophyllotoxin (POD) is a natural compound with antiviral and anticancer activities. The purpose of the present study was to determine the metabolic map of POD in vitro and in vivo.Mouse and human liver microsomes were employed to identify POD metabolites in vitro and recombinant drug-metabolizing enzymes were used to identify the mono-oxygenase enzymes involved in POD metabolism. All in vitro incubation mixtures and bile samples from mice treated with POD were analysed with ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry.A total of 38metabolites, including six phase-I metabolites and 32 phase-II metabolites, of POD were identified from bile and faeces samples after oral administration, and their structures were elucidated through interpreting MS/MS fragmentation patterns.Nine metabolites, including two phase-I metabolites, five glucuronide conjugates, and two GSH conjugates were detected in both human and mouse liver microsome incubation systems and the generation of all metabolites were NADPH-dependent. The main phase-I enzymes involved in metabolism of POD in vitro include CYP2C9, CYP2C19, CYP3A4, and CYP3A5.POD administration to mice caused hepatic and intestinal toxicity, and the cellular damage was exacerbated when 1-aminobenzotriazole, a broad-spectrum inhibitor of CYPs, was administered with POD, indicating that POD, but not its metabolites, induced hepatic and intestinal toxicities.This study elucidated the metabolic map and provides important reference basis for the safety evaluation and rational for the clinical application of POD.

Systematic analysis of the metabolites of Roukou Wuwei pills by ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry after oral administration to rats.

Roukou Wuwei pill is one of the most commonly used Mongolian medicinal prescriptions and historically used for the treatment of depression. This research aimed to illustrate the metabolic characteristic of Roukou Wuwei pills in vivo. To address this objective, an ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry method based metabonomics approach was used to detect and analyze the metabolites of Roukou Wuwei pills. The chromatographic separation was completed on an Agilent SB-C18 column (1.8 µm, 2.1 × 50 mm) with a gradient elution system (acetonitrile and 0.1% formic acid-water). Electrospray ionization was operated in a full-scan mode at m/z 100-1000. The data were collected in positive and negative ion modes. The Masslynx 4.1 and SIMCA-P 13.0 software were used to analyze the mass spectrometry data and select the potential metabolites by using an orthogonal partial least-squares discriminant analysis, which was applied to investigate the differences between the blank and drug groups in biosamples of rats. Finally, totally 87 metabolites were detected based on their tandem mass spectrometry data. Among them, 69 metabolites are potential new compounds.

Simultaneous detection and quantification of 57 compounds in Spatholobi Caulis applying ultra-fast liquid chromatography with tandem mass spectrometry.

A method of ultra-fast liquid chromatography in series with tandem mass spectrometry for the rapid and sensitive detection of 57 compounds in Spatholobi Caulis (the vine stem of Spatholobus suberectus Dunn) within 35 min was established. This assay can simultaneously determine a variety of compounds without matrix interference in multiple reaction monitoring mode including evaluating the quality of different batches of Spatholobi Caulis from several areas and further identifying the characteristic compounds efficiently. After comprehensive validation, this method can be used to determinate samples rapidly, precisely, accurately, repeatably, and sensitivity. There were significant content differences in 12 batches of Spatholobi Caulis, which were further classified and systematically differentiated applying multivariate statistical analysis. Furthermore, orthogonal partial least squares discrimination analysis results indicated that (-)-gallocatechin (10), (-)-epiafzelechin (20), 4,7,2'-trihydroxy-4'-methoxyisoflavanol (51), and biochanin A (53) characterize compounds to discernment internal quality of Spatholobi Caulis, and recommended as quality control indicators. Hence, presented work provides a method for further study on pharmaceutic preparation, metabolism, as well as for the design, production optimization process, and clinical application.

MFG-E8 regulates inflammation and apoptosis in tendon healing, and promotes tendon repair: A histological and biochemical evaluation.

Several studies have identified potential roles for MFG-E8 in promoting tissue repair. However, the effects of MFG-E8 on tendon repair have not yet been investigated. Therefore, we explored the role of MFG-E8 on tendon repair using a rat model of patellar tendon injury. The patellar tendons of Sprague Dawley rats (n = 24/group) received window defects and, after modeling, three groups were randomly assigned: (a) recombinant MFG-E8 (rMFG-E8) group, implantation with MFG-E8 and fibrin glue (400 ng in 10 µl); (b) fibrin group, implantation with fibrin only; and (c) control group, without any treatment. Histopathology, immunohistochemistry, and gene expression analyses were performed at 2 and 4 weeks after healing. Administration of rMFG-E8 in injury sites significantly improved tendon healing histologically at 4 weeks after injury. In addition, numbers of M1 macrophages and M1-stimulator genes, including IFNG, Il-1B, and Il-6, were reduced in the repair sites at 2 weeks by rMFG-E8 administration. In parallel, rMFG-E8 significantly increased the number of M2 macrophages and expression of anti-inflammatory IL-10 and IL-4 at 2 weeks after injury. Treatment with rMFG-E8 markedly decreased tendon cell apoptosis. Moreover, rMFG-E8 significantly enhanced the expression of genes related to tendon matrix formation at 2 weeks after injury, including Col1a1and tenascin-C. We conclude that MFG-E8 could regulate inflammatory responses and apoptotic cell accumulation in tendon repair, and promote the healing process of injured tendon tissue. Thus, exogenous application of MFG-E8 might have therapeutic potential for repair of tendon injuries.

Anti-Inflammatory Activity of Some Characteristic Constituents from the Vine Stems of Spatholobus suberectus.

The dried vine stems of Spatholobus suberectus are commonly used in traditional Chinese medicine for treating gynecological and cardiovascular diseases. In this study, five new compounds named spasuberol A (2), homovanillyl-4-oxo-nonanoate (5), spasuberol C (6), spasuberoside A (14), and spasuberoside B (15), together with ten known compounds (1, 3, 4, 7-13), were isolated from the dried vine stems of S. suberectus. Their chemical structures were analyzed using spectroscopic assays. This is the first study interpreting the detailed structural information of 4. The anti-inflammatory activity of these compounds was evaluated by reducing nitric oxide overproduction in RAW264.7 macrophages stimulated by lipopolysaccharide. Compounds 1 and 8-10 showed strong inhibitory activity with half maximal inhibitory concentration (IC50) values of 5.69, 16.34, 16.87, and 6.78 µM, respectively, exhibiting higher activity than the positive drug l-N6-(1-iminoethyl)-lysine (l-NIL) with an IC50 value of 19.08 µM. The IC50 values of inhibitory activity of compounds 2 and 4-6 were 46.26, 40.05, 45.87, and 28.29 µM respectively, which were lower than l-NIL, but better than that of positive drug indomethacin with an IC50 value of 55.44 µM. Quantitative real-time polymerase chain reaction analysis revealed that assayed compounds with good anti-inflammatory activity, such as 1, 6, 9, and 10 at different concentrations, can reduce the messenger RNA (mRNA) expression of some pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2). The anti-inflammatory activity and the possible mechanism of the compounds mentioned in this paper were studied preliminarily.

Metabolic Profiling of the Novel Hypoxia-Inducible Factor 2α Inhibitor PT2385 In Vivo and In Vitro.

PT2385 is a first-in-class, selective small-molecule inhibitor of hypoxia-inducible factor-2α (HIF-2α) developed for the treatment of advanced clear cell renal cell carcinoma. Preclinical results demonstrated that PT2385 has potent antitumor efficacy in mouse xenograft models of kidney cancer. It also has activity toward metabolic disease in a mouse model. However, no metabolism data are currently publically available. It is of great importance to characterize the metabolism of PT2385 and identify its effect on systemic homeostasis in mice. High-resolution mass spectrometry-based metabolomics was performed to profile the biotransformation of PT2385 and PT2385-induced changes in endogenous metabolites. Liver microsomes and recombinant drug-metabolizing enzymes were used to determine the mechanism of PT2385 metabolism. Real-time polymerase chain reaction analysis was employed to investigate the reason for the PT2385-induced bile acid dysregulation. A total of 12 metabolites of PT2385 was characterized, generated from hydroxylation (M1, M2), dihydroxylation and desaturation (M3, M4), oxidative-defluorination (M7), glucuronidation (M8), N-acetylcysteine conjugation (M9), and secondary methylation (M5, M6) and glucuronidation (M10, M11, and M12). CYP2C19 was the major contributor to the formation of M1, M2, and M7, UGT2B17 to M8, and UGT1A1/3 to M10-M12. The bile acid metabolites taurocholic acid and tauro-ß-muricholic acid were elevated in serum and liver of mice after PT2385 treatment. Gene expression analysis further revealed that intestinal HIF-2α inhibition by PT2385 treatment upregulated the hepatic expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis. This study provides metabolic data and an important reference basis for the safety evaluation and rational clinical application of PT2385.

Structure-Activity Relationships of the Main Bioactive Constituents of Euodia rutaecarpa on Aryl Hydrocarbon Receptor Activation and Associated Bile Acid Homeostasis.

Rutaecarpine (RUT), evodiamine (EOD), and dehydroevodiamine (DHED) are the three main bioactive indoloquinazoline alkaloids isolated from Euodia rutaecarpa, a widely prescribed traditional Chinese medicine. Here, the structure-activity relationships of these analogs for aryl hydrocarbon receptor (AHR) activation were explored by use of Ahr-deficient (Ahr-/-) mice, primary hepatocyte cultures, luciferase reporter gene assays, in silico ligand-docking studies, and metabolomics. In vitro, both mRNA analysis of AHR target genes in mouse primary hepatocytes and luciferase reporter assays in hepatocarcinoma cell lines demonstrated that RUT, EOD, and DHED significantly activated AHR, with an efficacy order of RUT > DHED > EOD. Ligand-docking analysis predicted that the methyl substitute at the N-14 atom was a key factor affecting AHR activation. In vivo, EOD was poorly orally absorbed and failed to activate AHR, whereas RUT and DHED markedly upregulated expression of the hepatic AHR gene battery in wild-type mice, but not in Ahr-/- mice. Furthermore, RUT, EOD, and DHED were not hepatotoxic at the doses used; however, RUT and DHED disrupted bile acid homeostasis in an AHR-dependent manner. These findings revealed that the methyl group at the N-14 atom of these analogs and their pharmacokinetic behaviors were the main determinants for AHR activation, and suggest that attention should be given to monitoring bile acid metabolism in the clinical use of E. rutaecarpa.

SIRT1 activator isolated from artificial gastric juice incubate of total saponins in stems and leaves of Panax ginseng.

Panax ginseng as a traditional Chinese medicine has been extensively used for the treatment of many diseases, especially in prolonging life and anti-tumor. Dammarane-type triterpenoids from P. ginseng have diverse beneficial effects and their chemical structures can be modified in the gastrointestinal tract after oral administration. In this paper, the dammarane-type triterpenoids were isolated from artificial gastric juice incubate of total saponins in the stems and leaves of P. ginseng through column chromatographic methods and their chemical structures were determined based on spectral data. Two new dammarane-type triterpenoids named ginsenotransmetins B (1) and C (2), along with twenty-nine known compounds (3-31), were obtained. All 31 compounds isolated were investigated for their activities of SIRT1 using SIRT1 fluorometric drug discovery assay kit. Among them, compounds 11, 17, 18, 20, 23, 24, 28, and 29, which were found to be potential as SIRT1 activators, exhibited significant stimulation of SIRT1 activity. The results showed that these compounds may be considered to be a useful medicinal resource for prolonging life and anti-tumor. In addition, the results were helpful to explain the longevity effect of ginseng from the new field of view.

Pharmacokinetics Studies of 12 Alkaloids in Rat Plasma after Oral Administration of Zuojin and Fan-Zuojin Formulas.

Zuojin formula (ZJ) is a traditional Chinese medicine (TCM) prescription consisted of Coptidis Rhizoma (CR) and Euodiae Fructus (EF), and has been used to treat gastrointestinal (GI) disease for more than 700 years. Fan-Zuojin formula (FZJ) is a related TCM prescription also consisted of CR and EF with the opposite proportion. In recent years, ZJ was getting more attention for its antitumor potential, but the indeterminate pharmacokinetic (PK) behavior restricted its clinical applications, and the PK differences between ZJ and FZJ were also largely unknown. Consequently it is necessary to carry out a full-scale PK study to demonstrate the physiological disposition of ZJ, as well as the comparative PK study between ZJ and FZJ to illustrate the compatibility dose effects. Therefore a liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method was established and validated for the determinations of coptisine, epiberberine, palmatine, berberine, 8-oxocoptisine, 8-oxoepiberberine, noroxyhydrastinine, corydaldine, dehydroevodiamine, evodiamine, wuchuyuamide-I, and evocarpine in rat plasma. PK characteristics of 12 alkaloids after oral administration of ZJ and FZJ were compared, and the result was analyzed and discussed with the help of an in silico study. Then an integrated PK study was carried out with the AUC-based weighting method and the total drug concentration method. The established method has been successfully applied to reveal the PK profiles of the 12 alkaloids in rat plasma after oral administration of ZJ and FZJ. The results showed that: (1) double peaks were observed in the plasma concentration-time (C-T) curves of the alkaloids after ZJ administration; but the C-T curves approximately matched the two-compartment model after FZJ administration; (2) There were wide variations in the absorption levels of these alkaloids; and even for a certain alkaloid, the dose modified systemic exposure levels and elimination rate also varied significantly after administration of ZJ and FZJ extracts. The results could be interpreted as follows: firstly, inhibition effect on GI motility caused by the high content CR alkaloids (especially berberine) in ZJ could delay the Tmax, and increase the absorption and systemic exposure levels of the other alkaloids, and also lead to the double peak phenomenon of these alkaloids. However, for quaternary protoberberine alkaloids (QPA), double peaks were primarily caused by the different Ka value in two intestinal absorption sites. Secondly, absorption was the major obstacle to the systemic exposure level of the alkaloids from CR and EF. In silico and PK studies suggested that the absorption of these alkaloids, except QPAs, mainly depended on their solubility rather than permeability. Thirdly, EF could promote the absorption and accelerate the elimination of QPAs, and had a greater influence on the former than the latter. At last the integrated PK analysis suggested that berberine and dehydroevodiamine could be regarded as the representative components to reflect the PK behaviors of CR and EF alkaloids after administration of ZJ and FZJ. In conclusion, the absorption, elimination and systemic exposure level of these alkaloids were mainly influenced by the proportion of EF and CR, the pharmacological effect on GI motility, and the physicochemical property of these alkaloids. These findings would be helpful for a better understanding of the activities and clinical applications of ZJ, FZJ and other related TCM prescriptions.

Anti-Inflammatory Phenolic Acid Esters from the Roots and Rhizomes of Notopterygium incisium and Their Permeability in the Human Caco-2 Monolayer Cell Model.

A new ferulic acid ester named 4-methyl-3-trans-hexenylferulate (1), together with eight known phenolic acid esters (2-9), was isolated from the methanolic extract of the roots and rhizomes of Notopterygium incisium. Their structures were elucidated by extensive spectroscopic techniques, including 2D NMR spectroscopy and mass spectrometry. 4-Methoxyphenethyl ferulate (8) NMR data is reported here for the first time. The uptake and transepithelial transport of the isolated compounds 1-9 were investigated in the human intestinal Caco-2 cell monolayer model. Compounds 2 and 6 were assigned for the well-absorbed compounds, compound 8 was assigned for the moderately absorbed compound, and compounds 1, 3, 4, 5, 7, and 9 were assigned for the poorly absorbed compounds. Moreover, all of the isolated compounds were assayed for the inhibitory effects against nitric oxide (NO) production in the lipopolysaccharide-activated RAW264.7 macrophages model and L-N6-(1-iminoethyl)-lysine (L-NIL) was used as a positive control. Compounds 1, 5, 8, and 9 exhibited potent inhibitory activity on NO production with the half maximal inhibitory concentration (IC50) values of 1.01, 4.63, 2.47, and 2.73 µM, respectively, which were more effective than L-NIL with IC50 values of 9.37 µM. These findings not only enriched the types of anti-inflammatory compounds in N. incisum but also provided some useful information for predicting their oral bioavailability and their suitability as drug leads or promising anti-inflammatory agents.

[Chemical constituents from lipophilic parts in roots of Angelica dahurica cv.Yubaizhi].

The chemical constituents from lipophilic parts in the roots of Angelica dahurica cv. Yubaizhi were studied in this paper. The compounds were separated and purified by repeated column chromatographic methods on silica gel and HPLC, and the chemical structures of compounds were determined by spectral data analyses. Thirty-three compounds were obtained and identified as isoimperatorin (1), imperatorin (2), stigmasterol (3), isooxypeucedanin (4), pabulenol (5), psoralen (6), bergapten (7), isodemethylfuropinarine (8), phellopterin (9), osthenol (10), alloimperatorin (11), xanthotoxin (12), xanthotoxol (13), isopimpinellin (14), alloisoimperatorin (15), ß-sitosterol (16), oxyalloimperatorin (17), pabularinone (18), 5-hydroxy-8-methoxypsoralen (19), columbianetin (20), heracol (21), isogosferol (22), 2″R-neobyakangelicol (23), byakangelicin ethoxide (24), byakangelicin (25), oxypeucedanin hydrate (26), uracil (27), umbelliferone (28), bergaptol (29), demethylfuropinarine (30), isobyakangelicol (31), oxypeucedanin ethanolate (32), heraclenol (33). Among them, compounds 8, 10, 17, 21, and 30 were obtained from the roots of title plant for the first time.

Tissue distribution study of columbianadin and its active metabolite columbianetin in rats.

Columbianadin, one of the main bioactive constituents of the roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan, has been found to possess obvious pharmacological effects in previous studies. In this study, a valid and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method was established and validated for the determination of columbianadin (CBN) and its active metabolite columbianetin (CBT) in rat tissue samples. Sample separation was performed on an RP-HPLC column using a mobile phase of MeOH-H2 O (75:25, v/v) at a flow rate of 1.0 mL/min. The UV absorbance of the samples was measured at the wavelength 325 nm. The calibration curves for CBN were linear over the ranges of 0.5-20 µg/g for brain, testes and muscle, 1.0-10.0 µg/g for stomach and intestine, and 0.2-20.0 µg/g for heart, liver, spleen, lung and kidney. The calibration curves for CBT were linear over the ranges of 0.5-25 µg/g for stomach and intestine, and 0.1-10.0 µg/g for heart, liver, spleen, lung and kidney. The analysis method was successfully applied to a tissue distribution study of CBN and CBT after intravenous administration of CBN to rats. The results of this study indicated that CBN could be detected in all of the selected tissues after i.v. administration. CBN was distributed to rat tissues rapidly and could be metabolized to CBT in most detected tissues. Of the detected tissues, heart had the highest uptake of CBN, which suggested that heart might be one of the main target tissues of CBN. Concentrations of CBT were obviously higher in the digestive system than in other assayed tissues. The information provided by this research is very useful for gaining knowledge of the capacities of CBN and CBT to access different tissues.

Liquid Chromatography with Tandem Mass Spectrometry: A Sensitive Method for the Determination of Dehydrodiisoeugenol in Rat Cerebral Nuclei.

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is developed for the quantification of dehydrodiisoeugenol (DDIE) in rat cerebral nuclei after single intravenous administration. DDIE and daidzein (internal standard) were separated on a Diamonsil™ ODS C18 column with methanol-water containing 0.1% formic acid (81:19, v/v) as a mobile phase. Detection of DDIE was performed on a positive electrospray ionization source using a triple quadrupole mass spectrometer. DDIE and daidzein were monitored at m/z 327.2→188.0 and m/z 255.0→199.2, respectively, in multiple reaction monitoring mode. This method enabled quantification of DDIE in various brain areas, including, cortex, hippocampus, striatum, hypothalamus, cerebellum and brainstem, with high specificity, precision, accuracy, and recovery. The data herein demonstrate that our new LC-MS/MS method is highly sensitive and suitable for monitoring cerebral nuclei distribution of DDIE.

High-Performance Liquid Chromatography with Diode Array Detector and Electrospray Ionization Ion Trap Time-of-Flight Tandem Mass Spectrometry to Evaluate Ginseng Roots and Rhizomes from Different Regions.

Ginseng, Panax ginseng C. A. Meyer, is an industrial crop in China and Korea. The functional components in ginseng roots and rhizomes are characteristic ginsenosides. This work developed a new high-performance liquid chromatography coupled with electrospray ionization ion trap time-of-flight multistage mass spectrometry (LC-ESI-IT-TOF-MS(n)) method to identify the triterpenoids. Sixty compounds (1-60) including 58 triterpenoids were identified from the ginseng cultivated in China. Substances 1, 2, 7, 15-20, 35, 39, 45-47, 49, 55-57, 59, and 60 were identified for the first time. To evaluate the quality of ginseng cultivated in Northeast China, this paper developed a practical liquid chromatography-diode array detection (LC-DAD) method to simultaneously quantify 14 interesting ginsenosides in ginseng collected from 66 different producing areas for the first time. The results showed the quality of ginseng roots and rhizomes from different sources was different due to growing environment, cultivation technology, and so on. The developed LC-ESI-IT-TOF-MS(n) method can be used to identify many more ginsenosides and the LC-DAD method can be used not only to assess the quality of ginseng, but also to optimize the cultivation conditions for the production of ginsenosides.