An integrated approach to uncover quality marker underlying the effects of Alisma orientale on lipid metabolism, using chemical analysis and network pharmacology.

BACKGROUND: Quality control of traditional Chinese medicines is currently a great concern, due to the correlation between the quality control indicators and clinic effect is often questionable. According to the "multi-components and multi-targets" property of TCMs, a new special quality and bioactivity evaluation system is urgently needed. PURPOSE: Present study adopted an integrated approach to provide new insights relating to uncover quality marker underlying the effects of Alisma orientale (AO) on lipid metabolism. METHODS: In this paper, guided by the concept of the quality marker (Q-marker), an integrated strategies "effect-compound-target-fingerprint" was established to discovery and screen the potential quality marker of AO based on network pharmacology and chemical analysis. Firstly, a bioactivity evaluation was performed to screen the main active fractions. Then the chemical compositions were rapidly identified by chemical analysis. Next, networks were constructed to illuminate the interactions between these component and their targets for lipid metabolism, and the potential Q-marker of AO was initially screened. Finally, the activity of the Q-markers was validated in vitro. RESULTS: 50% ethanol extract fraction was found to have the strongest lipid-lowering activity. Then, the network pharmacology was used to clarify the unique relationship between the Q-markers and their integral pharmacological action. CONCLUSION: Combined with the results obtained, five active ingredients in the 50% ethanol extract fraction were given special considerations to be representative Q-markers: Alisol A, Alisol B, Alisol A 23-acetate, Alisol B 23-acetate and Alisol A 24-acetate, respectively. The chromatographic fingerprints based Q-marker was establishment. The integrated Q-marker screen may offer an alternative quality assessment of herbal medicines.

The single-probe: a miniaturized multifunctional device for single cell mass spectrometry analysis.

We have developed a new mass spectrometry (MS) technology, the Single-probe MS, capable of real-time, in situ metabolomic analysis of individual living cells. The Single-probe is a miniaturized multifunctional sampling and ionization device that is directly coupled to the mass spectrometer. With a sampling tip smaller than individual eukaryotic cells (<10 µm), the Single-probe can be inserted into single cells to sample the intracellular compounds for real-time MS analysis. We have used the Single-probe to detect several cellular metabolites and the anticancer small molecules paclitaxel, doxorubicin, and OSW-1 in individual cervical cancer cells (HeLa). Single cell mass spectrometry (SCMS) is an emerging scientific technology that could reshape the analytical science of many research disciplines, and the Single-probe MS technology is a novel method for SCMS that, through its accessible fabrication protocols, can be broadly applied to different research areas.

A sensitive spectrofluorometric method for determination of ergosta-4,6,8(14),22-tetraen-3-one in rat plasma, feces, and urine for application to pharmacokinetic studies using cerium(III) as a probe.

Ergosta-4,6,8(14),22-tetraen-3-one (ergone) isolated from Polyporus umbellatus possesses a variety of pharmacological activities in vivo and in vitro, including cytotoxic, diuretic, and immunosuppressive effect. The interaction of cerium ions (Ce(3+)) with ergone was studied by fluorescence and absorption spectroscopy. Spectra data revealed that Ce(3+) ions exhibited emission maxima around 350 nm when the excitation wavelength was fixed at 255 or 290 nm, and the fluorescence of Ce(3+) ions was quenched by the addition of ergone, indicating that a Ce(3+)-ergone complex was formed. According to the modified Benesi-Hildebrand equation, the binding constant of interaction of Ce(3+) ions with ergone was obtained at room temperature. Based on this, a sensitive spectrofluorometric method using Ce(3+) ions as a probe was applied for the identification and quantification of ergone in rat plasma, feces, and urine. The linear ranges of the calibration curves were 1.31 to 4.50 µM for plasma, 1.12-9.87 µM for feces, and 1.28-3.42 µM for urine, and the ergone recoveries were found to be 97.1 ± 0.9%, 98.2 ± 0.7% and 96.5 ± 1.4% for plasma, feces, and urine, respectively. The intraday and inter-day relative standard deviations were less than 9.7%. The proposed spectrofluorometric method is simple and rapid for the quantitative determination of ergone in rat plasma, feces, and urine, and it is affordable for most laboratories because it has few requirements and uses low cost, easy to operate equipment.

An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.

The level of 7-dehydrocholesterol (7-DHC) is elevated in tissues and fluids of Smith-Lemli-Opitz syndrome (SLOS) patients due to defective 7-DHC reductase. Although over a dozen oxysterols have been identified from 7-DHC free radical oxidation in solution, oxysterol profiles in SLOS cells and tissues have never been studied. We report here the identification and complete characterization of a novel oxysterol, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), as a biomarker for 7-DHC oxidation in fibroblasts from SLOS patients and brain tissue from a SLOS mouse model. Deuterated (d7)-standards of 7-DHC and DHCEO were synthesized from d7-cholesterol. The presence of DHCEO in SLOS samples was supported by chemical derivatization in the presence of d7-DHCEO standard followed by HPLC-MS or GC-MS analysis. Quantification of cholesterol, 7-DHC, and DHCEO was carried out by isotope dilution MS with the d7-standards. The level of DHCEO was high and correlated well with the level of 7-DHC in all samples examined (R = 0.9851). Based on our in vitro studies in two different cell lines, the mechanism of formation of DHCEO that involves 5α,6α-epoxycholest-7-en-3ß-ol, a primary free radical oxidation product of 7-DHC, and 7-cholesten-3ß,5α,6ß-triol is proposed. In a preliminary test, a pyrimidinol antioxidant was found to effectively suppress the formation of DHCEO in SLOS fibroblasts.

Increased atherosclerosis and vascular inflammation in APP transgenic mice with apolipoprotein E deficiency.

OBJECTIVE: Atherosclerosis is associated with Alzheimer's disease (AD) in humans, but the nature of this link is still elusive. Aim of this study was to investigate aortic atherosclerosis development in a mouse model with central nervous system (CNS) restricted beta-amyloid precursor protein (APP) overexpression. METHODS AND RESULTS: APP23 mice, overexpressing the Swedish mutated human APP selectively in the brain, were crossed with mice lacking apolipoprotein E (ApoE KO). Nine weeks old mice were fed a western type diet for eight weeks, then atherosclerotic lesions, aortic wall and cortical tissues gene expression and beta-amyloid (Abeta) deposition were evaluated. Compared with ApoE KO, APP23/ApoE KO mice developed larger aortic atherosclerotic lesions and showed significantly increased expression of MCP-1, IL-6, ICAM-1 and MTPase 6, a marker of oxidative stress in the vascular wall. Of note brain limited APP synthesis was associated with an increased microglia and brain endothelial cells activation, in spite of the absence of beta-amyloid deposits in the brain or alteration in the levels of oxidized metabolites of cholesterol such as 4-cholesten-3-one. CONCLUSION: Our study suggests that the vascular pro-inflammatory effects of CNS-localised APP overexpression lead to atherogenesis before parenchymal Abeta deposition and neuronal dysfunction.

[Studies on chemical components of Lobelia chinensis].

OBJECTIVE: To study on the chemical constituents of Lobelia chinensis. METHOD: The coloumn chromatographic techniques were applied to isolate constituents, and their structures were elucidated by means of spectral data analysis. RESULT: Sixteen compounds were isolated and identified as daucosterol (1), diosmetin (2), apigenin (3), chrysoeriol (4), loteolin (5), hesperidin (6), loteolin-7-O-beta-D-glucoside (7), apigenin-7-O-beta-D-glucoside (8), linarin (9), diosmin(10), 5,7-dimethoxy-8- hydroxycoumarin (11), palmitinic acid (12), lacceroic acid (13), stearic acid (14), beta-sitosterol (15), daucosterol (16). CONCLUSION: All of these compouds were obtained from L. chinensis for the first time.

[Studies on the marker compounds for standardization of traditional Chinese medicine "polyporus sclerotium ([symbol: see text])"].

Natural products have been used for healthcare and pharmacotherapy. Because difficulties in quality control affect their production, processing, and marketing, it is necessary to establish adequate marker compounds for their effective application. Ergosta-4,6,8(14),22-tetraen-3-one(I) was studied in the screening of the marker compounds for the standardization of Polyporus Sclerotium ([symbol: see text]), which has the advantage of easy qualitative and quantitative analysis because of its fluorescence. Its applicability in the standardization of Polyporus Sclerotium is discussed based on comparative studies of 30 crude samples of Polyporus Sclerotium and some other fungi herbs using TLC and HPLC analysis with I it as the marker compound, as well as its chemical synthesis.

[Analysis of steroids in crude drugs by capillary gas chromatography].

The steroids, cholest-4-ene-one and cholestane-3, 6-dion, were determined in 8 species of crude drugs of Haima and Hailong by capillary gas chromatography.