Metabonomics Study of TCM Formula: Qutan Huayu Tongmai Granule as an Effective Treatment for Atherosclerosis in Mini-Pigs

To investigate the effects of QuTan HuaYu TongMai granule (QHTG) on the perturbed metabolism of atherosclerosis mini-pig model, a serum metabonomics method based on Liquid chromatography/mass spectrometry was developed. Principal component analysis and partial least squares-discriminate analysis models were established for the metabonomics analysis. The effect of high dose QHTG group is equivalent with those of positive drugs group in PCA score plots. Some significantly changed metabolites, including lyso-PCs, metabolites of vitamin D₃ and fatty acids were found to be reasonable in explaining the anti-atherosclerosis mechanism of QHTG. Metabonomic approach is helpful to further understand the pathophysiology of atherosclerosis in mini-pigs and the therapeutic mechanism of QHTG. Our work also indicated that the LC–MS based metabonomics method is a potential tool for performing intervention and mechanism research of traditional Chinese medicine (TCM).

     

Metabonomics study of the effects of traditional Chinese medicine formula Ermiaowan on hyperuricemic rats

To explore the global mechanism of Ermiaowan on hyperuricemia regulation, the holistic function of Ermiaowan for hyperuricemia in rats was firstly assessed by the urinary metabonomics method which was based on ultra‐high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry. The urinary targeted metabonomics approach combined with the serum biochemical analysis and histological assay was conducted to verify the research result. As a result, the significant differences in metabolic profiles were observed from Ermiaowan‐treated group, model group, and healthy control group by using multivariate statistical approaches. Twenty therapeutic related metabolites were identified in response to the therapeutic effects of Ermiaowan, which were mainly associated with purine metabolism, pyrimidine metabolism, tryptophan metabolism, tricarboxylic acid cycle, and tyrosine metabolism. In addition, the urinary targeted metabonomics study showed that Ermiaowan can better restore the disturbed pathways than Phellodendri cortex and Atractylodis rhizome. The biochemical assay and histopathological assay confirmed that Ermiaowan could significantly reduce uric acid and fibrosis areas of kidney. These results provided new insights into the mechanism of Ermiaowan on hyperuricemia.     

Urinary metabonomics study of Wu-tou-tang and its co-decoction with Pinelliae Rhizoma in adjuvant-induced arthritis rats

Wu-tou-tang was applied to treat rheumatoid arthritis,rheumatic arthritis,and joint pain for thousands of years.Aconiti Radix Cocta is the primary component of Wu-tou-tang,and Aconiti Radix and Pinelliae Rhizoma is one of the famous combination taboos in traditional Chinese medicine.A urinary metabonomics method based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) had been established to quantify the changes of the endogenous metabolites in the urine of adjuvant-induced arthritis rats treated by Wutou-tang and Wu-tou-tang and Pinelliae Rhizoma(WP) co-decoctions.There was a clear separation between Wu-tou-tang and WP co-decoction treated groups in the PCA model.16 potential biomarkers had been identified which correlated with how Pinelliae Rhizoma influenced the therapeutic effect made by Wu-tou-tang.Results showed that it could reduce the therapeutic effects or make some side effects when Pinelliae Rhizoma is added in Wu-tou-tang.This is the first time to use metabonomics to investigate the reason of combination taboo.     

基于主成分分析的骨碎补药材乙醇和环己烷提取物的高效液相色谱指纹图谱及指标成分的定量分析

建立了骨碎补药材乙醇和环己烷提取物的高效液相色谱(HPLC)指纹图谱,并利用主成分分析法(PCA)对指纹图谱进行统计分析,以各主要色谱峰的保留时间和峰面积为变量得到score图和loading图。在score图和loading图中,骨碎补的正品和非正品可明显区分,且揭示出对此区分贡献最大的4个潜在指标成分,其中已知成分为柚皮苷、新北美圣草苷和E-4-O-β-D-葡萄糖酰咖啡酸。同时测定了这3种成分在19批正品和非正品骨碎补药材中的含量,其中10批骨碎补药材正品中3种成分的含量为: 柚皮苷6.36～10.1 mg/g,新北美圣草苷5.14～9.21 mg/g,E-4-O-β-D-葡萄糖酰咖啡酸1.87～3.19 mg/g。该方法更全面地反映了药材的化学成分信息,并能从定性和定量两方面控制骨碎补药材的内在质量。     

Metabonomics study on the effect of Siwu Decoction for blood deficiency syndrome in rats using UPLC–Q/TOF–MS analysis

Siwu decoction (SWD), a traditional Chinese medicinal formula with over 1000 years of clinical history, is widely used for gynecological disease, especially blood deficiency syndrome, which is similar to anemia in modern medicine. In view of metabonomics being useful approach to investigate the potential mechanisms of action from the point of view of systems biology, in this study an ultra‐performance liquid chromatography–quadrupole‐time of flight mass spectrometry method was employed for a holistic evaluation of SWD on a blood‐deficiency rat model induced by N‐acetylphenylhydrazine and cyclophosphamide via plasma metabonomics study. Routine blood examination results showed that SWD could significantly improve the declining hemogram indices. Meanwhile, the plasma metabonomics profiles in different groups were analyzed and differentiating metabolites were primarily visualized through chemometric analysis. Seven biomarkers were identified in plasma samples of blood‐deficiency rat model compared with the normal group. Five main metabolism pathways were suggested using the Kyoto Encyclopedia of Genes and Genomes Pathway Analysis and Pathway Activity Profiling algorithm analysis. This indicated that SWD played a therapeu role in blood deficiency by regulating the aberrant endogenous metabolites. To sum up, this study provides clear evidence that a metabonomics study could serve as a useful tool to elucidate the systematic therapeutic profiles and mechanisms for blood deficiency syndrome of Chinese herbal medicines.     

中药质量控制的研究策略和色谱技术

综述了色谱技术作为现代高效分离分析技术在中药药效物质基础研究和中药质量控制中的重要作用。提出中药药效物质组学研究策略,以实现中药药效物质基础与作用机理研究的一体化,进而从深层次揭示和全面控制中药的内在质量。论述结果表明,现代色谱及其联用技术是中药质量研究的重要技术平台之一。     

Strategy and Chromatographic Technology of Quality Control for Traditional Chinese Medicines

The application of chromatography, a modern technology, for highly efficient separation and analysis in the research of therapeutic basis matter and quality control of traditional Chinese medicines (TCM), is summarized. A new strategy with metabonomics is put forth to achieve integral study on the therapeutic basis matter and the action mechanism of TCM and to reveal and control the comprehensive quality of TCM. The result shows that modern chromatography and hyphenated techniques are one of the important technique platforms in the quality control of TCM.     

Metabolomics study of the effect of Danggui Buxue Tang on rats with chronic fatigue syndrome

Danggui Buxue Tang (DBT), a traditional Chinese medicine (TCM) formula for ‘invigorating qi and enriching blood’, has been reported to produce a good effect on chronic fatigue syndrome (CFS). However, the related mechanism remains largely undetermined. This study devised a metabolomics approach with GC–MS combined with pattern recognition to estimate the extent to which DBT alleviated CFS induced by food restriction and force swimming in rats. After 4 weeks of treatment, the endurance capability of rats was significantly better, and the motionless time was significantly shorter in the DBT group than in the CFS model group. Moreover, the activities of superoxide dismutase and glutathione peroxidase increased, whereas the levels of malondialdehyde, interleukin 6, and tumor necrosis factor alpha decreased in the DBT treatment group. Fifteen significantly changed metabolites were observed in the serum of rats with CFS, which was reversed markedly by DBT treatment. Metabolic pathway analysis showed that DBT could possibly alleviate CFS in rats by regulating the biosynthesis of phenylalanine, tyrosine, and tryptophan and the metabolism of glycine, serine, threonine, glycerolipid, glyoxylate, dicarboxylate, and tyrosine. It was observed that the metabolism of glycine, serine, and threonine was most closely related to the improvement in CFS by DBT treatment. This study showed that DBT could improve CFS effectively, and metabolomics was a powerful means to gain insights into the TCM formulas against CFS.     

色谱-质谱联用技术在中药代谢组学研究中的应用

代谢组学是研究生物体被扰动后其代谢产物种类、数量及变化规律的科学,研究理念与中医药理论的整体、动态观念非常一致,目前很多工作已将代谢组学应用于中药药效物质基础、作用机制、复方及配伍规律等研究中,有望推动中医药现代化进程。色谱-质谱联用技术是代谢组学的主要分析技术平台,该文综述了近3年来色谱-质谱联用技术在中药代谢组学研究中的应用,重点介绍不同分离技术的特点及最新进展,并讨论了其存在的问题。     

Metabonomics Study of the Hematopoietic Effect of Medicinal Wine Maoji Jiu on a Blood Deficiency Rat Model by Ultra-High-Performance Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry and a Pattern Recognition Approach

Maoji Jiu (MJ) is a kind of medicinal wine that has been widely used by Chinese people for many years to nourish and promote blood circulation. The purpose of this study was to investigate the hematopoietic effect of MJ on the metabolism of blood deficient rats and to explore the underlying hematopoietic regulation mechanisms. Blood deficiency model rats were induced by subcutaneous injection of N-acetylphenylhydrazine (APH) and intraperitoneal injection of cyclophosphamide (CTX). The plasma metabolic fingerprints of blood deficiency model rats with and without MJ treatment were obtained by using metabonomics based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC–QTOF/MS). Orthogonal partial least squares-discriminant analysis (OPLS–DA) was used to evaluate the hematopoietic effect of MJ and identify potential biomarkers in the plasma of blood deficiency model rats. The levels of white blood cells (WBC), red blood cells (RBC) and hemoglobin (HGB) and the activity of antioxidant capacity showed a recovery trend to the control group after MJ treatment, while the dose of 10 mL/kg showed the best effect. In this study, thirteen potential biomarkers were identified, which were mainly related to seven metabolic pathways, including linoleic acid metabolism, d-glutamine and d-glutamate metabolism, alanine, aspartate and glutamate metabolism, tryptophan metabolism, pyrimidine metabolism, porphyrin and chlorophyll metabolism and arginine biosynthesis. Metabolomics was applied frequently to reflect the physiological and metabolic state of organisms comprehensively, indicating that the rapid plasma metabonomics may be a potentially powerful tool to reveal the efficacy and enriching blood mechanism of MJ.     

Metabonomics Study of Heart Homogenates from Myocardial Infarction Rats Using Liquid Chromatography/Time of Flight Mass Spectrometry

In this study, a metabonomics analysis of heart homogenates from myocardial ischemic rats was performed by LC–TOF–MS. Hydrophilic interaction chromatography (HILIC) was used to separate the endogenous metabolites in heart homogenates. Partial least squares to latent structure-discriminant analysis (PLS-DA) was used for data analysis. Good separations were observed between the normal and model groups and 15 potential biomarkers were identified. The major disturbed metabolic pathways were purine metabolism, pyrimidine metabolism, urea cycle, and energy metabolism. The results demonstrated that a metabonomics approach based on HILIC-MS was useful for studying metabolic mechanism on target tissue of the myocardial infarction rat.

     

Quality control and discrimination of pericarpium citri reticulatae and pericarpium citri reticulatae viride based on high-performance liquid chromatographic fingerprints and multivariate statistical analysis

High-performance liquid chromatographic (HPLC) fingerprints of Pericarpium Citri Reticulatae (PCR) and Pericarpium Citri Reticulatae Viride (PCRV) were firstly measured for deliberately collected 39 authentic samples and 21 commercial samples. Both correlation coefficients of similarity for chromatograms and absolute peak areas of characteristic compounds were calculated for quantitative expression of the HPLC fingerprints. After principal component analysis (PCA) successfully distinguished the ‘mixed peels’ samples from authentic samples, partial least squares-linear discrimination analysis (PLS-LDA) was then effectively applied to class separation between authentic PCR and PCRV. Furthermore, the unequivocally determined compounds, hesperidin, nobiletin and tangeretin, were screened out by loadings plots of PCA and PLS-LDA. The results indicated that they could be used as chemical markers for discrimination among different groups of samples. The proposed method shows an efficient strategy for quality control of PCR and PCRV, which cannot only distinguish the ‘mixed peels’ but also discriminate authentic PCR and PCRV. This method has potential perspective for quality control of traditional Chinese medicine (TCM).     

The absolute configuration of streptonigrin

A theoretical study of the molecular circular dichroism (CD) of the antitumor antibiotic natural product streptonigrin is described, aiming at the secure assignment of its absolute configuration by comparison of the CD spectra predicted for M and P, with the experimental one. The stereostructure of streptonigrin was previously investigated by two other groups, yet leading to two different attributions. Although streptonigrin possesses two biaryl axes, only the ‘southern’ one is configurationally stable and thus responsible for the chiroptical properties, since the ‘northern’ AB-ring system of the molecule is kept in plane with ring C by hydrogen bonding. All computational methods applied within this work to simulate the CD spectrum—semiempirical approaches and time-dependent density functional theory (TDDFT)—consistently attribute the M-configuration to streptonigrin.     

Metabolic profiling study on potential toxicity and immunotoxicity‐biomarker discovery in rats treated with cyclophosphamide using HPLC‐ESI‐IT‐TOF‐MS

Despite the recent advances in understanding toxicity mechanism of cyclophosphamide (CTX), the development of biomarkers is still essential. CTX‐induced immunotoxicity in rats by a metabonomics approach was investigated using high‐performance liquid chromatography coupled with ion trap time‐of‐flight mass spectrometry (HPLC‐ESI‐IT‐TOF‐MS). The rats were orally administered CTX (30 mg/kg/day) for five consecutive days, and on the fifth day samples of urine, thymus and spleen were collected and analyzed. A significant difference in metabolic profiling was observed between the CTX‐treated group and the control group by partial least squares‐discriminant analysis (PLS‐DA), which indicated that metabolic disturbances of immunotoxicity in CTX‐treated rats had occurred. One potential biomarker in spleen, three in urine and three in thymus were identified. It is suggested that the CTX‐toxicity mechanism may involve the modulation of tryptophan metabolism, phospholipid metabolism and energy metabolism. This research can help to elucidate the CTX‐influenced pathways at a low dose and can further help to indicate the patients' pathological status at earlier stages of toxicological progression after drug administration. Copyright © 2014 John Wiley & Sons, Ltd.     

Pre-analytical method for NMR-based grape metabolic fingerprinting and chemometrics

Although metabolomics aims at profiling all the metabolites in organisms, data quality is quite dependent on the pre-analytical methods employed. In order to evaluate current methods, different pre-analytical methods were compared and used for the metabolic profiling of grapevine as a model plant. Five grape cultivars from Portugal in combination with chemometrics were analyzed in this study. A common extraction method with deuterated water and methanol was found effective in the case of amino acids, organic acids, and sugars. For secondary metabolites like phenolics, solid phase extraction with C-18 cartridges showed good results. Principal component analysis, in combination with NMR spectroscopy, was applied and showed clear distinction among the cultivars. Primary metabolites such as choline, sucrose, and leucine were found discriminating for ‘Alvarinho’, while elevated levels of alanine, valine, and acetate were found in ‘Arinto’ (white varieties). Among the red cultivars, higher signals for citrate and GABA in ‘Touriga Nacional’, succinate and fumarate in ‘Aragonês’, and malate, ascorbate, fructose and glucose in ‘Trincadeira’, were observed. Based on the phenolic profile, ‘Arinto’ was found with higher levels of phenolics as compared to ‘Alvarinho’. ‘Trincadeira’ showed lowest phenolics content while higher levels of flavonoids and phenylpropanoids were found in ‘Aragonês’ and ‘Touriga Nacional’, respectively. It is shown that the metabolite composition of the extract is highly affected by the extraction procedure and this consideration has to be taken in account for metabolomics studies.     

Microchip device for rapid screening and fingerprint identification of phenolic pollutants

A new microchip protocol has been developed for rapid measurements of the ‘total’ content of phenolic compounds, as well as for a detailed fingerprint identification of the ‘individual’ ones. The protocol involves the use of a microchip flow-injection analysis for fast screening and early detection of phenols and switching to the separation (fingerprint) mode once such compounds are detected. This is readily accomplished by exchanging the run buffers in the separation channel. While operating with an acidic run buffer (pH 5) offers high speed flow-injection measurements of the ‘total’ phenolic content, on chip switching to a basic buffer (pH 8) leads to ionization of the phenolic compounds and to their effective separation and detection. Under optimum conditions, assay rates of about 120 and 18 samples/h can be realized for the ‘total’ and ‘individual’ measurements, respectively. The effect of the buffer pH, switching (washing) time, applied voltages and other relevant variables, is described. The concept is illustrated in connection to amperometric detection and is attractive for a wide range of environmental-monitoring applications.     

Fully automated immunoassay for detection of prostate-specific antigen using nano-magnetic beads and micro-polystyrene bead composites, 'Beads on Beads'

Magnetic beads have served as a conventional bioassay platform in biotechnology. In this study, a fully automated immunoassay was performed using novel nano- and microbead-composites constructed by assembling nano-magnetic beads onto polystyrene microbeads, designated ‘Beads on Beads’. Nano-sized bacterial magnetic particles (BacMPs) displaying the immunoglobulin G (IgG)-binding domain of protein A (ZZ domain) were used for the construction of ‘Beads on Beads’ via the interaction of biotin-streptavidin. The efficient assembly of ‘Beads on Beads’ was performed by gradual addition of biotin-labeled BacMPs onto streptavidin-coated polystyrene microbeads. Approximately 2000 BacMPs were uniformly assembled on a single microbead without aggregation. The constructed ‘Beads on Beads’ were magnetized and separated from the suspension by using an automated magnetic separation system with a higher efficiency than BacMPs alone. Furthermore, fully automated detection of prostate-specific antigens was performed with the detection limit of 1.48 ng mL⁻¹. From this preliminary assay, it can be seen that ‘Beads on Beads’ could be a powerful tool in the development of high-throughput, fully automated multiplexed bioassays.     

非靶向尿液代谢组学方法研究五味子治疗精尿病并发症作用机制

采用超高效液相色谱-串联四级杆飞行时间质谱联用技术(Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,UPLC/Q-TOF-MS),结合主成分分析技术(Principal component analysis,PCA),进行了五味子治疗糖尿病并发症大鼠尿液代谢组学研究,对五味子治疗糖尿病并发症大鼠后尿液生物标志物变化进行了考察.采用UPLC/Q-TOF-MS方法分析了健康组、糖尿病并发症模型组和五味子给药组的大鼠尿液,采用偏最小二乘法-判别分析(Partial least squares discriminant analysis,PLS-DA)和正交偏最小二乘法-判别分析(Orthogonal partial least squares discriminant analysis,OPLS-DA)载荷图筛选,通过分析对各组分离贡献较大(VIP>1,P°0.05)化合物的串联质谱数据,再经Human Metabolome Data-base(HMDB)等数据库检索,进行质谱信息匹配,尿液中共鉴定出28种潜在生物标记物,其中正谱13种,负谱15种.这些生物标记物主要影响戊糖和葡糖醛酸相互转化通路、核黄素代谢、泛酸和CoA合成、精氨酸和脯氨酸代谢、肠内菌代谢、嘌吟代谢、Vc代谢胆酸合成、色氨酸代谢等通路,五味子从能量代谢、氨基酸代谢、肠内菌代谢、脂类代谢等各个角度发挥治疗糖尿病并发症的作用.从各个通路的相关生物功能分析,五味子治疗糖尿病肾病的作用较强,此外还具有降脂、抗氧化的功效.     

Synthetic aspects of the oxidative amidation of phenols

The oxidative amidation of phenols effects the conversion of appropriately substituted phenols into 4-amidodienones (‘para-oxidative amidation’) or 2-amidodienones (‘ortho-oxidative amidation’) by the action of hypervalent iodine reagents. The reagent, (diacetoxyiodo)benzene (‘DIB’) is especially effective in these transformations. This paper focuses on techniques for the desymmetrization of the dienoes thus obtained, leading to the stereocontrolled creation of N-substituted spiro carbons. The methodology creates new opportunities in alkaloid synthesis, as apparent from a number of examples.