Mingmei Zhou

Metabolite Profiling of Panax notoginseng Using UPLC–ESI-MS

The metabolite profiling of different parts of Panax notoginseng was carried out using rapid ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESI-MS) and multivariate statistical analysis. Principal component analysis (PCA) of the UPLC-ESI-MS data showed a clear separation of compositions among the flower buds, roots and rhizomes of P. notoginseng. The saponins accounting for such variations were identified through the corresponding loadings weights and were further verified by accurate mass, tandem mass and retention times of available standard saponins using UPLC quadrupole time-of-flight mass spectrometer (UPLC-QtofMS). Finally, the influential factors of different metabolic phenotypes of P. notoginseng was elucidated. The currently proposed UPLC-ESI-MS/MS analytical method coupled with multivariate statistical analysis can be further utilized to evaluate chemical components obtained from different parts of the plant and/or the plant of different geographical locations, thereby classifying the medicinal plant resources and potentially elucidating the mechanism of inherent phytochemical diversity.

Metabonomics Approach to Understanding Acute and Chronic Stress in Rat Models

The effects of acute and chronic stress on the production of systemic metabolites were investigated in male Sprague-Dawley (SD) rats. Metabolites excreted in urine were analyzed using GC/MS in conjunction with multivariate and univariate statistical techniques. SD rats were subjected to two kinds of acute stress and chronic unpredictable mild stress, respectively. Metabolic analysis demonstrated that urinary expression of a number of metabolites including glutamate, glutamine, homovanillate, proline, succinate, citrate, and tyrosine altered in the acute stress model in the same way as in the chronic model, while pimelate and hippurate changed in the opposite trend. The results suggested that the stress induced metabolic perturbations were reversible and nonspecific. Metabolic response to chronic combined stress revealed biochemical clues to depression-like symptoms validated by behavior and physiologic results. This study provides a noninvasive and dynamic analytical strategy for the characterization of endogenous metabolic perturbations induced by external stress.

Traditional Chinese medicine: balancing the gut ecosystem

Gut microflora has become a topic of interest in life sciences in the context of global systems biology, in which human biological system is viewed as ‘superorganisms’ involving an internal ecosystem of diverse microbiome. We conceive that multi‐pathway modulations of the human gut microbial system exerted by traditional Chinese medicines (TCMs) to restore the balance of the gut ecology may account for a large portion of their effectiveness in host during treatment. Such a concept is evidenced by series of studies which have revealed an interactive relationship between gut microflora and TCM, involving the two important aspects: gut microflora‐dependent drug metabolism in TCM and gut microflora‐targeted modulation of physiological conditions, both of which highlight the significance of gut microflora involvement in the future TCM investigation. Copyright

A rapid ultra‐performance liquid chromatography–electrospray Ionisation mass spectrometric method for the analysis of saponins in the adventitious roots of Panax notoginseng

INTRODUCTION Saponins are bioactive compounds employed in the prevention and treatment of cardiovascular and cerebrovascular diseases. The adventitious roots of Panax notoginseng may offer an alternative source of saponins. Identification and determination of saponins in the crude extract is challenging owing to their similar structures and the lack of standards. OBJECTIVE To develop a rapid, sensitive and accurate method based on solid-phase extraction followed by ultra-performance liquid chromatography-electrospray ionisation mass spectrometry (UPLC-ESI-MS) for the identification and quantification of saponins in P. notoginseng. METHODOLOGY Following extraction using Waters Oasis(TM) HLB cartridges, the analytes were subjected to a UPLC system with a Waters Acquity BEH C(18) chromatographic column and a binary mobile phase system consisting of 0.05% formic acid in water and acetonitrile under gradient elution conditions, with final detection by ESI-MS in the positive ion mode. RESULTS The UPLC-ESI-MS method gave limits of detection and quantification within the range 0.015-0.382 and 0.052-1.124 microg/mL, respectively, for 15 studied saponins. The instrumentation/injection precision (RSD) was 4.5% for a low concentration and 3.2% for an intermediate concentration sample. The intra- and inter-day repeatability was less than 2.65% (RSD). The method described was validated using spiked samples with different amounts of saponin standards. CONCLUSION This UPLC-ESI-MS assay provides a suitable quality control method for the tentative identification and determination of major biological active constituents in adventitious and native roots of P. notoginseng.

Metabonomics approach to assessing the modulatory effects of St John's Wort, ginsenosides, and clomipramine in experimental depression

The protective effects of St Johns Wort extract (SJ), ginsenosides (GS), and clomipramine (CPM) on chronic unpredictable mild stress (CUMS)-induced depression in rats were investigated by using a combination of behavioral assessments and metabonomics. Metabonomic analyses were performed using gas chromatography/mass spectrometry in conjunction with multivariate and univariate statistical analyses. During and at the end point of the chronic stress experiment, food consumption, body weight, adrenal gland, thymus and spleen indices, behavior scores, sucrose consumption, and stress hormone levels were measured. Changes in these parameters reflected characteristic phenotypes of depression in rats. Metabonomic analysis of serum, urine, and brain tissue revealed that CPM and SJ mainly attenuated the alteration of monoamine neurotransmitter metabolites, while GS affected both excitatory/inhibitory amino acids and monoamine neurotransmitter metabolites. GS also attenuated the stress-induced alterations in cerebrum and peripheral metabolites to a greater extent than CPM and SJ. These results provide important mechanistic insights into the protective effects of GS against CUMS-induced depression and metabolic dysfunction.

Multiparametric analysis of amino acids and organic acids in rat brain tissues using GC/MS

Using design of experiment (DOE) theory coupled with multivariate statistical analysis, we have developed a simple and reliable GC/MS-based analytical assay for simultaneous analysis of amino acids and organic acids in rat brain tissue samples. The process of water extraction (pH 10.0) was extensively evaluated using brain tissue samples and a set of 21 reference standards. Acceptable calibration curves were obtained over a wide concentration range, 0.2-35.0 microg/mL for standards and 15.0-2.4 mL/g (tissue) for brain tissue samples. The precision was mostly better than 10% for both the mixed standards and the brain tissue samples. The brain tissue samples exhibited good stability within 48 h with RSD generally less than 15%. Furthermore, the developed analytical method was successfully applied in distinguishing the subtle variation among different parts of the brain tissues, such as cerebral cortex, hippocampus, and thalamus.

GC/MS-based urinary metabolomics reveals systematic differences in metabolism and ethanol response between Sprague–Dawley and Wistar rats

Metabolic differences of experimental animals contribute to pharmacological variations. Sprague–Dawley (SD) and Wistar rats are commonly used experimental rats with similar genetic background, and considered interchangeable in practical researches. In this study, we present the urinary metabolomics results, based on gas chromatography coupled to mass spectrometry (GC/MS), which reveal the systematic metabolic differences between SD and Wistar rats under different perturbations such as fasting, feeding, and consecutive acute ethanol interventions. The different metabotypes between the two strains of rats involve a number of metabolic pathways and symbiotic gut microflora. SD rats exhibited higher individualized metabolic variations in the fasting and feeding states, and a stronger ability to recover from an altered metabolic profile with less hepatic injury from the consecutive ethanol exposure, as compared to Wistar rats. In summary, the GC/MS-based urinary metabolomics studies demonstrated an intrinsic metabolic difference between SD and Wistar rats, which warrants consideration in experimental design using these animal strains.

Urinary metabolite variation is associated with pathological progression of the post-hepatitis B cirrhosis patients.

Cirrhosis is a common and terminal outcome of many chronic liver conditions. A urinary metabonomic study using gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOFMS) was carried out to elucidate the pathophysiological basis of posthepatitis B cirrhosis in 63 posthepatitis B cirrhosis patients and 31 health controls. Urinary metabolic profile and corresponding differential metabolites associated with Child-Pugh (CP) grading of liver function were characterized, in addition to the blood routine, liver, and renal function tests. Multivariate statistical tools including principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) were employed in the metabolite analysis along with a univariate statistical method, Wilcoxon-Mann-Whitney test. The alterations of differential metabolites contributing to the intergroup variation between healthy controls and cirrhotic patients, and among cirrhosis of CP grade A, B and C were also investigated. Six metabolites, α-hydroxyhippurate, tyrosine-betaxanthin, 3-hydroxyisovalerate, canavaninosuccinate, estrone, and glycoursodeoxycholate, were significantly altered among cirrhotic patients with CP A, B, and C, reflecting abnormal metabolism of amino acid, bile acids, hormones, and intestinal microbial metabolism. The results show that dynamic alteration of urinary metabolome, characterized by the changes of a panel of the differential metabolite markers, is indicative of an exacerbated liver function, highlighting their diagnostic and prognostic potential for the liver cirrhosis development.

The Brain Metabolome of Male Rats across the Lifespan

Comprehensive and accurate characterization of brain metabolome is fundamental to brain science, but has been hindered by technical limitations. We profiled the brain metabolome in male Wistar rats at different ages (day 1 to week 111) using high-sensitivity and high-resolution mass spectrometry. Totally 380 metabolites were identified and 232 of them were quantitated. Compared with anatomical regions, age had a greater effect on variations in the brain metabolome. Lipids, fatty acids and amino acids accounted for the largest proportions of the brain metabolome, and their concentrations varied across the lifespan. The levels of polyunsaturated fatty acids were higher in infancy (week 1 to week 3) compared with later ages, and the ratio of omega-6 to omega-3 fatty acids increased in the aged brain (week 56 to week 111). Importantly, a panel of 20 bile acids were quantitatively measured, most of which have not previously been documented in the brain metabolome. This study extends the breadth of the mammalian brain metabolome as well as our knowledge of functional brain development, both of which are critically important to move the brain science forward.

Metabolic Profiling Reveals the Protective Effect of Diammonium Glycyrrhizinate on Acute Hepatic Injury Induced by Carbon Tetrachloride

Diammonium glycyrrhizinate (DG), a constitutent of Glycyrrhiza uralensis, has a protective effect on hepatic injury, hepatisis and cirrhosis. To date, the mechanism has been poorly understood, especially at the metabolic level. A metabolomic profiling study was performed to characterize the carbon tetrachloride (CCl4) induced global metabolic alteration and the protective effects of DG in Sprague-Dawley rats. Urinary and hepatic tissue metabolic profiling revealed that CCl4 perturbed the amino acid metabolism (alanine, glycine, leucine), tricarboxylic acid cycle (citrate), lipid metabolism (unsaturated fatty acids) and gut microbiota related metabolites. Our results also indicated that DG was able to attenuate CCl4 perturbed metabolic pathways and ameliorated biochemical markers of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and Total cholesterol (TCHO). This global metabolomic approach also revealed full metabolic recovery takes longer than apparent and conventional histological and biochemical markers.