Guan-Hua Du

Comparison of Two Different Astragali Radix by a 1H NMR-Based Metabolomic Approach

Astragali Radix (AR) is a commonly used herbal drug in traditional chinese medicine and is widely used for the treatment of diabetes, cardiovascular diseases, nephropathy, and neuropathy. The main source of AR in China is the dried root of Astragalus membranaceus var. mongholicus (Bge.) Hsiao, and both cultivated and wild ARs are used clinically. A systematic comparison of cultivated AR (GS-AR) and wild AR (SX-AR) should be performed to ensure the clinical efficacy and safety. In this study, the chemical composition of the two different ARs, which were collected in the Shanxi (wild) and Gansu (cultivated) provinces, were compared by NMR-based metabolic fingerprint coupled with multivariate analysis. The SX-AR- and GS-AR-induced metabolic changes in the endogenous metabolites in mice were also compared. The results showed that SX-AR and GS-AR differed significantly not only in the primary metabolites but also in the secondary metabolites. However, alterations among the endogenous metabolites in the serum, lung, liver, and spleen were relatively small. This study provided a novel and valuable method for the evaluation of the consistency and diversity of herbal drugs, and further studies should be conducted on the difference in polysaccharides as well as the biological effects between the two kinds of AR.

(1)H NMR based metabolomic profiling revealed doxorubicin-induced systematic alterations in a rat model.

Doxorubicin (DOX) is used as a chemotherapy drug with severe carditoxicity. In this study, an integrated echocardiography along with pathological examination and (1)H NMR analysis of multiple biological matrices (urine, serum, heart, and kidney) was employed to systemically assess the toxicity of DOX. Echocardiographic results showed that impaired left ventricular contractility and degenerative pathology lesions in DOX group, which were in consistent with pathology. The endogenous metabolites in the urine, serum, heart and kidney was identified by comparison with the data from the literature and databases. Multivariate analysis, including PCA and OPLS, revealed 8 metabolites in urine, including succinate, 2-ketoglutarate, citrate, hippurate, methylamine, benzoate, allantion, and acetate were the potential changed biomarkers. In serum, perturbed metabolites include elevation of leucine, β-glucose, O-acetyl-glycoprotein, creatine, lysine, glycerin, dimethylglycine, trimethylamine-N-oxide, myo-inositol, and N-acetyl-glycoprotein, together with level decreases of acetone, lipid, lactate, glutamate, phosphocholine, acetoacetate and pyruvate. For heart, DOX exposure caused decline of lipid, lactate, leucine, alanine, glutamate, choline, xanthine, glycerin, carnitine, and fumarate, together with elevation of glutamine, creatine, inosine, taurine and malate. Metabolic changes of kidney were mainly involved in the accumulation of α-glucose, lactate, phosphocholine, betaine, threonine, choline, taurine, glycine, urea, hypoxanthine, glutamate, and nicotinamide, coupled with reduction of asparagine, valine, methionine, tyrosine, lysine, alanine, leucine, ornithine, creatine, lipid, and acetate. In addition, alterations of urinary metabolites exhibited a time-dependent manner. Complementary evidences by multiple matrices revealed disturbed pathways concerning energy metabolism, fatty acids oxidation, amino acids and purine metabolism, choline metabolism, and gut microbiota-related metabolism. In addition, the change of endogenous metabolites in rats urine, serum, heart and kidney were correlated with the echocardiography parameters. This integrative study should help to develop a systematic understanding of cardiomyopathy-related diseases and their metabolic events.

Chemical and biological comparison of raw and vinegar-baked Radix Bupleuri.

ETHNOPHARMACOLOGICAL RELEVANCE Radix Bupleuri (RB) is a commonly used herbal drug in Traditional Chinese Medicine (TCM), and it can be baked with vinegar to afford vinegar-baked Radix Bupleuri (VBRB), which is used in TCM for liver diseases treatment. In this study, the chemical compositions and biological effects between raw and two processed RBs by different vinegars were systematically compared. MATERIALS AND METHODS The chemical compositions of raw and two processed RBs were analyzed by (1)H NMR spectroscopy coupled with multivariate analysis. Two different extraction procedures were used, including direct extraction and liquid-liquid partition. Then HPLC was applied to determine the changes of saikosaponin contents. In addition, their liver protective effects against CCl4 induced liver injury were also investigated, and the biochemical parameters and histopathology were measured after treatment of mice with raw RB and two processed RBs (5 g/kg/day) for 14 days. RESULTS Multivariate analysis showed clear differences between the raw and the two processed RBs, and the vinegar-baking process induced elevated contents of ssb1, ssb2, acetic acid, malic acid, citric acid, 5-HMF, and ligustrazine, as well as the decreased contents of ssa, ssd, sucrose, glycine, succinic acid etc. In addition, both raw and processed RBs showed liver protective effects against CCl4 induced liver injury, and the vinegar-baked RBs showed better effects than that of raw RB. CONCLUSIONS The raw and vinegar-baked RBs differed not only in the chemical compositions but also in the pharmacological effects. And two processed RBs also showed chemical differences, suggesting that the type of vinegar had an important effect on vinegar baking. In order to ensure the therapeutic effect and safety of TCM, the effect of different vinegars on processing of herbal drugs should be further studied.

UPLC/Q-TOF MS-Based Metabolomics and qRT-PCR in Enzyme Gene Screening with Key Role in Triterpenoid Saponin Biosynthesis of Polygala tenuifolia

Background The dried root of Polygala tenuifolia, named Radix Polygalae, is a well-known traditional Chinese medicine. Triterpenoid saponins are some of the most important components of Radix Polygalae extracts and are widely studied because of their valuable pharmacological properties. However, the relationship between gene expression and triterpenoid saponin biosynthesis in P. tenuifolia is unclear. Methodology/Findings In this study, ultra-performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS)-based metabolomic analysis was performed to identify and quantify the different chemical constituents of the roots, stems, leaves, and seeds of P. tenuifolia. A total of 22 marker compounds (VIP>1) were explored, and significant differences in all 7 triterpenoid saponins among the different tissues were found. We also observed an efficient reference gene GAPDH for different tissues in this plant and determined the expression level of some genes in the triterpenoid saponin biosynthetic pathway. Results showed that MVA pathway has more important functions in the triterpenoid saponin biosynthesis of P. tenuifolia. The expression levels of squalene synthase (SQS), squalene monooxygenase (SQE), and beta-amyrin synthase (β-AS) were highly correlated with the peak area intensity of triterpenoid saponins compared with data from UPLC/Q-TOF MS-based metabolomic analysis. Conclusions/Significance This finding suggested that a combination of UPLC/Q-TOF MS-based metabolomics and gene expression analysis can effectively elucidate the mechanism of triterpenoid saponin biosynthesis and can provide useful information on gene discovery. These findings can serve as a reference for using the overexpression of genes encoding for SQS, SQE, and/or β-AS to increase the triterpenoid saponin production of P. tenuifolia.

NMR-based metabonomics and correlation analysis reveal potential biomarkers associated with chronic atrophic gastritis.

HighlightsA first report about the metabolic characteristics and potential biomarkers by associated with CAG.A goal of discriminating specific biomarkers of CAG progression.1 H NMR‐based metabonomics with correlation analysis was employed.3 plasma and 2 urine biomarkers were obtained.New avenues for discriminating biomarkers and elucidating the underlying mechanisms of CAG. ABSTRACT Chronic atrophic gastritis (CAG) is one of the most important pre‐cancerous states with a high prevalence. Exploring of the underlying mechanism and potential biomarkers is of significant importance for CAG. In the present work, 1H NMR‐based metabonomics with correlative analysis was performed to analyze the metabolic features of CAG. 19 plasma metabolites and 18 urine metabolites were enrolled to construct the circulatory and excretory metabolome of CAG, which was in response to alterations of energy metabolism, inflammation, immune dysfunction, as well as oxidative stress. 7 plasma biomarkers and 7 urine biomarkers were screened to elucidate the pathogenesis of CAG based on the further correlation analysis with biochemical indexes. Finally, 3 plasma biomarkers (arginine, succinate and 3‐hydroxybutyrate) and 2 urine biomarkers (&agr;‐ketoglutarate and valine) highlighted the potential to indicate risks of CAG in virtue of correlation with pepsin activity and ROC analysis. Here, our results paved a way for elucidating the underlying mechanisms in the development of CAG, and provided new avenues for the diagnosis of CAG and presented potential drug targets for treatment of CAG.

NMR-based metabonomic and quantitative real-time PCR in the profiling of metabolic changes in carbon tetrachloride-induced rat liver injury

Carbon tetrachloride (CCl4) is commonly used as a model toxicant to induce chronic and acute liver injuries. In this study, metabolite profiling and gene expression analysis of liver tissues were performed by nuclear magnetic resonance and quantitative real-time polymerase chain reaction to understand the responses of acute liver injury system in rats to CCl4. Acute liver injury was successfully induced by CCl4 as revealed by histopathological results and significant increase in alanine aminotransferase and serum aspartate aminotransferase. We found that CCl4 caused a significant increase in lactate, succinate, citrate, dimethylgycine, choline and taurine. CCl4 also caused a decrease in some of the amino acids such as leucine/isoleucine, glutamine/glutathione and betaine. Gene function analysis revealed that 10 relevant enzyme genes exhibited changes in expressions in the acute liver injury model. In conclusion, the metabolic pathways, including tricarboxylic acid cycle, antioxidant defense systems, fatty acid β-oxidation, glycolysis and choline and mevalonate metabolisms were impaired in CCl4-treated rat livers. These findings provided an overview of the biochemical consequences of CCl4 exposure and comprehensive insights into the metabolic aspects of CCl4-induced hepatotoxicity in rats. These findings may also provide reference of the mechanisms of acute liver injury that could be used to study the changes in functional genes and metabolites.

1H NMR based metabolic profiling of the processing effect on Rehmanniae Radix

The roots of Rehmannia glutinosa Libosch, Rehmanniae Radix, also known as Dihuang in China, are an important herbal drug in Traditional Chinese Medicine (TCM). According to the theory of TCM, the raw and processed Rehmanniae Radix are used quite differently. In this study, the chemical change of Rehmanniae Radix induced by processing is investigated by 1H NMR spectroscopy based fingerprinting coupled with multivariate data analysis. The results indicated that big chemical change had occurred after the processing of Rehmanniae Radix. Due to the high concentration of sugars, liquid–liquid partition approach was used to eliminate the interfering effect from sugars, in which more metabolites could be detected, and phenethylalcohol glycosides were concentrated in the EtOAc fractions, while the iridoid glycoside were enriched in the n-BuOH part. In addition, various statistical methods were used to find the differential metabolites between the groups. The results obtained in this study suggest that more than one extraction and statistical method should be used for comprehensive metabolic profiling of medical plants, which contained various metabolites in different concentrations.

Analysis of Polygala tenuifolia Transcriptome and Description of Secondary Metabolite Biosynthetic Pathways by Illumina Sequencing

Radix polygalae, the dried roots of Polygala tenuifolia and P. sibirica, is one of the most well-known traditional Chinese medicinal plants. Radix polygalae contains various saponins, xanthones, and oligosaccharide esters and these compounds are responsible for several pharmacological properties. To provide basic breeding information, enhance molecular biological analysis, and determine secondary metabolite biosynthetic pathways of P. tenuifolia, we applied Illumina sequencing technology and de novo assembly. We also applied this technique to gain an overview of P. tenuifolia transcriptome from samples with different years. Using Illumina sequencing, approximately 67.2% of unique sequences were annotated by basic local alignment search tool similarity searches against public sequence databases. We classified the annotated unigenes by using Nr, Nt, GO, COG, and KEGG databases compared with NCBI. We also obtained many candidates CYP450s and UGTs by the analysis of genes in the secondary metabolite biosynthetic pathways, including putative terpenoid backbone and phenylpropanoid biosynthesis pathway. With this transcriptome sequencing, future genetic and genomics studies related to the molecular mechanisms associated with the chemical composition of P. tenuifolia may be improved. Genes involved in the enrichment of secondary metabolite biosynthesis-related pathways could enhance the potential applications of P. tenuifolia in pharmaceutical industries.

NMR based metabolomic approach revealed cyclophosphamide-induced systematic alterations in a rat model

Cyclophosphamide (CY) is the most commonly used antineoplastic drug, but various side effects are often observed. In this work, a 1H NMR based metabolomic approach combined with biochemical assay and histopathological inspection were employed to study the toxic effects of CY on rats. The endogenous metabolites in liver, kidney, and heart were identified. Multivariate analysis revealed that the 18 metabolites in the liver, 14 metabolites in the kidney, and 6 metabolites in the heart were significantly altered after CY treatment. Metabolic pathway analysis showed that the metabolic alterations were related to amino acids metabolism, energy metabolism, choline metabolism, nucleotide metabolism and oxidative stress-related metabolism. This integrative study should be useful for the clinical safety evaluation of CY, and shows the power of a 1H NMR-based metabolomic approach to evaluate the toxicity of drugs systematically.

Identification of Cultured and Natural Astragalus Root Based on Monosaccharide Mapping

As the main substances responsible for immunomodulatory activity, saccharides can be used as quality indicators for Astragalus root (RA). Saccharide content is commonly determined by ultraviolet spectroscopy, which lacks species specificity and has not been applied in the Chinese Pharmacopoeia. Monosaccharide mapping based on trifluoroacetic acid (TFA) hydrolysis can be used for quantitative analysis of saccharide compositions. In addition, species specificity can be evaluated by analysis of the mapping characteristics. In this study, monosaccharide mapping of soluble saccharides in the cytoplasm and polysaccharides in the cell wall of 24 batches of RA samples with different growth patterns were obtained based on TFA hydrolysis followed by gas chromatography-mass spectrometry. Results indicated that the mapping and the molar ratios of saccharide compositions of the cultured and natural RA samples were different for both cytoplasm and cell wall. For example, the molar ratio of mannose and arabinose was more than 3.5:1 in cytoplasm in cultured RA, whereas the ratio was less than 3.5:1 in natural RA. This research not only lays a foundation for screening indicators for RA, but also provided new ways of evaluating the quality of Chinese medicinal materials in which saccharides are the main bioactive substances.