Changyuan Yu

Urinary metabonomics study of anti-depressive effect of Chaihu-Shu-Gan-San on an experimental model of depression induced by chronic variable stress in rats

Chaihu-Shu-Gan-San (CSGS), a traditional Chinese medicine (TCM) formula, has been effectively used for the treatment of depression in clinic. However, studies of its anti-depressive mechanism are challenging, accounted for the complex pathophysiology of depression, and complexity of CSGS with multiple constituents acting on different metabolic pathways. The variations of endogenous metabolites in rat model of depression after administration of CSGS may offer deeper insights into the anti-depressive effect and mechanism of CSGS. In this study, metabonomics based on ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to profile the metabolic fingerprints of urine obtained from chronic variable stress (CVS)-induced depression model in rats with and without CSGS treatment. Through partial least squares-discriminate analysis, it was observed that metabolic perturbations induced by chronic variable stress were restored in a time-dependent pattern after treatment with CSGS. Metabolites with significant changes induced by CVS, including 3-O-methyldopa (1), pantothenic acid (2), kynurenic acid (3), xanthurenic acid (4), 2,8-dihydroxyquinoline glucuronide (5), 5-hydroxy-6-methoxyindole glucurnoide (8), l-phenylalanyl-l-hydroxyproline (9), indole-3-carboxylic acid (10), proline (11), and the unidentified metabolites (6, 2.11min_m/z 217.0940; 7, 2.11min_m/z 144.0799), were characterized as potential biomarkers involved in the pathogenesis of depression. The derivations of all those biomarkers can be regulated by CSGS treatment except indole-3-carboxylic acid (10), which suggested that the therapeutic effect of CSGS on depression may involve in regulating the dysfunctions of energy metabolism, tryptophan metabolism, bone loss and liver detoxification. This study indicated that the rapid and noninvasive urinary metabonomics approach may be a powerful tool to study the efficacy and mechanism of complex TCM prescriptions.

UPLC-Q/TOF MS standardized Chinese formula Xin-Ke-Shu for the treatment of atherosclerosis in a rabbit model

Xin-Ke-Shu (XKS), a patent traditional Chinese medicine (TCM) preparation, has been commonly used for the treatment of coronary heart disease in China. In order to understand its mechanism of action, a metabonomic approach based on ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) was utilized to profile the plasma metabolic fingerprints of atherosclerosis (AS) rabbits with and without XKS treatment. The metabolic profile of model group clearly separated from normal, and that of XKS group was closer to the control group. Metabolites with significant changes during atherosclerosis were characterized as potential biomarkers related to the development of atherosclerosis by using orthogonal partial least-squares-discriminate analysis (OPLS-DA). Twenty potential biomarkers, including l-acetylcarnitine (1), propionylcarnitine (2), unknown (3), phytosphingosine (4), glycoursodeoxycholic acid (5), LPC(14:0) (6), sphinganine (7), LPC(20:5) (8), LPC(16:1) (9), LPC(18:2) (10), LPC(18:3) (11), LPC(22:5) (12), LPC(16:0) (13), LPC(18:1) (14), LPC(22:4) (15), LPC(17:0) (16), LPC(20:2) (17), elaidic carnitine (18), LPC(18:0) (19) and LPC(20:1) (20), were identified by their accurate mass and MS(E) spectra. The derivations of those biomarkers can be regulated by administration of XKS, which suggested that the intervention effect of XKS against AS may involve in regulating the lipid perturbation including fatty acid β-oxidation pathway, sphingolipid metabolism, glycerophospholipid metabolism and bile acid biosynthesis. This study indicated that the UPLC-Q/TOF MS-based metabonomics not only gave a systematic view of the pathomechanism of AS, but also provided a powerful tool to study the efficacy and mechanism of complex TCM prescriptions.

Norcyperone, a Novel Skeleton Norsesquiterpene from Cyperus rotundus L.

A novel norsesquiterpene, named norcyperone (1), and three known compounds: (-)-clovane-2,9-diol (2), rosenonolactone (3), and 5α,8α-epidioxy-(20S,22E,24R)-ergosta-6,22-dien-3β-ol (4) were isolated from the rhizomes of Cyperus rotundus L. The structure of 1 was elucidated as 8,11,11-trimethylbicyclo[5.3.1]undecane-5α, 8α-epoxy-3-one on the basis of extensive spectroscopic analyses, including 1D- and 2D-NMR, MS experiments, and single-crystal X-ray diffraction. This is the first report of a 8,11,11-trimethyl- bicyclo[5.3.1]undecane-3-one type norsesquiterpene with a tetrahydrofuran ring at C-5 and C-8.

Comparative Pharmacokinetics of Naringin in Rat after Oral Administration of Chaihu-Shu-Gan-San Aqueous Extract and Naringin Alone

Chaihu-Shu-Gan-San (CSGS), a traditional Chinese medicine (TCM) formula containing seven herbal medicines, has been used in the clinical treatment of gastritis, peptic ulcer, irritable bowel syndrome and depression in China. In order to explore the interaction between naringin and other constituents in CSGS, the pharmacokinetic difference of naringin in rats after oral administration of CSGS aqueous extract and naringin alone was investigated. The pharmacokinetic parameters of naringin in rats were achieved by quantification of its aglycone, naringenin by LC-MS/MS method. The double peaks phenomenon was observed in both serum profiles of rats after orally administered CSGS aqueous extract and naringin alone. However, the T1/2β was significantly decreased in rats given CSGS aqueous extract compared with naringin alone, and the mean residence time (MRT) and the area under the serum concentration–time curve (AUC0-τ) were higher than those of naringin, which indicated that naringin in CSGS had higher bioavailability, longer term efficacy and somewhat faster metabolism and excretion than those of naringin. The results suggested that certain ingredients co-exist in CSGS could influence pharmacokinetic behavior of naringin. This also provides a reference for human studies.

Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay

Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.

Living cell intracellular temperature imaging with biocompatible dye-conjugated carbon dots

Within living cells, the biological functions of subcellular organelles are highly dependent on the distribution of local temperature. In this regard, sensitive and accurate measurement of cellular temperature with subcellular resolution is fundamentally and practically significant. In this work, we demonstrated a fluorescence-based self-calibration method for the quantitative analysis of living cell temperature. A ratiometric temperature sensor was designed by conjugating a temperature sensitive dye, Rhodamine B (RhB), with biocompatible carbon dots (CDs) via a covalent bond. The as-prepared CDs-RhB probe showed excellent ratiometric temperature sensing capability in solution under a single wavelength excitation and displayed a linear temperature sensing range from 5 to 50 °C, which matches the requirement well for the physiologically relevant temperature assay. Furthermore, this probe was applied to map intracellular temperature inside living cells. The fast and accurate temperature response of the probe makes it a promising tool for monitoring the intracellular temperature change, which will promote a better understanding of the temperature relevant biological processes inside living cells.

Fimbriatols A-J, Highly Oxidized ent-Kaurane Diterpenoids from Traditional Chinese Plant Flickingeria fimbriata (B1.) Hawkes.

Fimbriatols A–J (1–10), ten new ent-kaurane diterpenoids possessing differently highly oxidized sites, were isolated from Flickingeria fimbriata (B1.) Hawkes. The structures of these new compounds were determined by HRESI-MS, NMR, CD spectra and X-ray diffraction analysis. Compound 1 displayed moderately inhibitory ratio (48.5%) compared with the positive compound NSC-87877 (81.6%) at the concentration of 0.022 μg/mL. Compounds 7–10 possess 3, 4-seco-ent-kaurane skeleton containing a disaccharide moiety with an unusual linkage at C-2′ to C-1′′ instead of the common linkage at C-6′ to C-1′′, and this is the first report in 600 more ent-kauranes found in nature, which might be originated from ent-kaurane diterpenoids through post-modified reactions of Baeyer-Villiger oxygenation and glycosylation.

Simultaneous HPLC Determination of Costunolide and Dehydrocostuslactonein Xin-ke-shu Preparations

The objective of this study is to establish a quantitative method for simultaneous determination of costunolide and dehydrocostuslactone in Xin-ke-shu preparations. The HPLC quantitative analysis was established on a Hypersil BDS column (100� 4.6 mm, 2.4 μm, Thermo) with UV detection at 225 nm. The mobile phase was water and methanol (30:70, v/v), and used at a flow rate at 0.5 mL·min - 1 . The established method showed a good linearity (R 2 > 0.9999) over the investigated concentration ranges (0.07-2.80 μg), good inter-day and intra-day precisions (less than 3%) and good recoveries (from 99.82% to 99.98%) for both target compounds. The method was found to be suitable for simultaneous determination of costunolide and dehydrocostuslactone in Xin-ke-shu preparations and can be used as a comprehensive approach to the previous reported quality control of Xin-ke-shu preparations.

A dynamic sandwich assay on magnetic beads for selective detection of single-nucleotide mutations at room temperature

Single-nucleotide mutation (SNM) has proven to be associated with a variety of human diseases. Development of reliable methods for the detection of SNM is crucial for molecular diagnosis and personalized medicine. The sandwich assays are widely used tools for detecting nucleic acid biomarkers due to their low cost and rapid signaling. However, the poor hybridization specificity of signal probe at room temperature hampers the discrimination of mutant and wild type. Here, we demonstrate a dynamic sandwich assay on magnetic beads for SNM detection based on the transient binding between signal probe and target. By taking the advantage of mismatch sensitive thermodynamics of transient DNA binding, the dynamic sandwich assay exhibits high discrimination factor for mutant with a broad range of salt concentration at room temperature. The beads used in this assay serve as a tool for separation, and might be helpful to enhance SNM selectivity. Flexible design of signal probe and facile magnetic separation allow multiple-mode downstream analysis including colorimetric detection and isothermal amplification. With this method, BRAF mutations in the genomic DNA extracted from cancer cell lines were tested, allowing sensitive detection of SNM at very low abundances (0.1-0.5% mutant/wild type).

Metabolic Profile Changes of CCl4-Liver Fibrosis and Inhibitory Effects of Jiaqi Ganxian Granule

Jiaqi Ganxian Granule (JGG) is a famous traditional Chinese medicine, which has been long used in clinical practice for treating liver fibrosis. However, the mechanism underlying its anti-hepatic fibrosis is still not clear. In this study, an Ultra-Performance Liquid Chromatography-Time-Of-Flight Mass Spectrometry (UPLC-TOF-MS)-based metabolomics strategy was used to profile the metabolic characteristic of serum obtained from a carbon tetrachloride (CCl4)-induced hepatic fibrosis model in Sprague-Dawley (SD) rats with JGG treatment. Through Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA), it was shown that metabolic perturbations induced by CCl4 were inhibited after treatment of JGG, for 17 different metabolites related to CCl4. Among these compounds, the change tendency of eight potential drug targets was restored after the intervention with JGG. The current study indicates that JGG has a significant anti-fibrosis effect on CCl4-induced liver fibrosis in rats, which might be by regulating the dysfunction of sphingolipid metabolism, glycerophospholipid metabolism, N-acylethanolamine biosynthesis, fat digestion and absorption, while glycerophospholipid metabolism played vital roles in the inhibitory effects of JGG on hepatic fibrosis according to Metabolic Pathway Analysis (MetPA). Our findings indicated that the metabolomics approach may provide a useful tool for exploring potential biomarkers involved in hepatic fibrosis and elucidate the mechanisms underlying the action of therapies used in traditional Chinese medicine.