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Epilepsy is often considered to be a progressive neurological disease, and the nature of this progression remains unclear. Understanding the overall and common metabolic changes of epileptic seizures can provide novel clues for their control and prevention. Herein, a chronic kindling animal model was established to obtain generalized tonic-clonic seizures via the repeated injections of pentylenetetrazole (PTZ) at subconvulsive dose. Dynamic metabolomic changes in plasma and urine from PTZ-kindled rats at the different kindling phases were explored using NMR-based metabolomics, in combination with behavioral assessment, brain neurotransmitter measurement, electroencephalography and histopathology. The increased levels of glucose, lactate, glutamate, creatine and creatinine, together with the decreased levels of pyruvate, citrate and succinate, ketone bodies, asparagine, alanine, leucine, valine and isoleucine in plasma and/or urine were involved in the development and progression of seizures. These altered metabolites reflected the pathophysiological processes including the compromised energy metabolism, the disturbed amino acid metabolism, the peripheral inflammation and changes in gut microbiota functions. NMR-based metabolomics could provide brain disease information by the dynamic plasma and urinary metabolic changes during chronic epileptic seizures, yielding classification of seizure stages and profound insights into controlling epilepsy via targeting deficient energy metabolism.

Immobilized protein has advanced many areas like drug discovery. While this field evolved rapidly over the three decades, the immobilization platform for G-protein-coupled receptor (GPCR) remains unpromising due to its instability under the relatively harsh conditions of current methodologies. Taking beta2-adrenoceptor (β2-AR) as an example, we presented here a general strategy for immobilization of GPCRs by combing his-tag trap system, conformation-specific aptamer, and target binding induced DNA hybridization. Morphology characterization by diverse assays confirmed a monolayer of β2-AR on the microsphere surface. Radio-ligand binding assay and immuno-transmission electron microscopy displayed desirable ligand- and antibody-binding activities. Owing to the competitive strand displacement during the immobilization, the method proved to be capable of sensitively and directly determining the receptor density on the surface which enormously challenges most of the reported assays. A case study of chromatography using the immobilized receptor as stationary phase exhibited a demonstrable conformation specificity that enables the selective recognition of the receptor agonists or antagonists. This method is possible to turn into a general strategy for immobilization of GPCRs with defined orientation, conformation, function, and density, thus paving a way to precisely realize the receptor-ligand binding interaction and screen the receptor agonist or antagonist with high efficiency.

Siraitia grosvenorii (Swingle) C. Jeffrey (SG) is widely used as a natural sweetener and traditional medicine for respiratory diseases. The anti-respiratory compounds in the plant and their mechanism remain elusive due to the lack of a high-throughput screening method. In this work, immobilization of the muscarinic-3 acetylcholine receptor (M

α-Asaronol from

Immobilized protein has advanced in many areas like drug discovery. While this field evolved rapidly over the last three decades, the immobilization platform for the G-protein-coupled receptor (GPCR) remains unpromising due to its instability under the relatively harsh conditions of current methodologies. Taking beta

Small GTPase or Ras superfamily, including Ras, Rho, Rab, Ran and Arf, are fundamental in regulating a wide range of cellular processes such as growth, differentiation, migration and apoptosis. They share structural and functional similarities for binding guanine nucleotides and hydrolyzing GTP. Dysregulations of Ras proteins are involved in the pathophysiology of multiple human diseases, however there is still a stringent need for effective treatments targeting these proteins. For decades, small GTPases were recognized as 'undruggable' targets due to their complex regulatory mechanisms and lack of deep pockets for ligand binding. NMR has been critical in deciphering the structural and dynamic properties of the switch regions that are underpinning molecular switch functions of small GTPases, which pave the way for developing new effective inhibitors. The recent progress of drug or lead molecule development made for small GTPases profoundly delineated how modern NMR techniques reshape the field of drug discovery. In this review, we will summarize the progress of structural and dynamic studies of small GTPases, the NMR techniques developed for structure-based drug screening and their applications in early-stage drug discovery for small GTPases.

The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drug-receptor interaction analysis and ligand screening. In our example assay using beta2-adrenergic receptor (β2-AR), the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) fusion receptor expressed in Escherichia coli was directly captured onto polyethylene glycol polyacrylamide (PEGA) resin. We observed even distribution and physiological functions of β2-AR on the resin. The immobilized β2-AR as a stationary phase enabled us to rapidly determine the binding of four drugs to β2-AR. By coupling this assay to mass spectrometry, we screened rosmarinic acid as a bioactive compound targeting β2-AR in Fructus Perillae. We concluded that O6-benzylguanine derivative-functionalized supporter is promising for specific immobilization of hAGT-tagged proteins; immobilized receptor chromatography has great potential in screening receptor-binding leads from herbal plants or traditional medicine recipes.

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe heart disease when compared with healthy subjects. Given the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on an exploratory multi-centre investigation in patients with no urinary tract infections to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients being treated for severe heart disease or those conditions associated with inflammation (WBC ≥ 10 ×10

Human calcium-sensing receptor (CaSR), a member of the G-protein-coupled receptor superfamily (GPCR), has been a therapeutic target for developing new drugs against calciotropic disorders and non-calciotropic diseases. The highly efficient methodologies for pursuing novel ligands/drugs remained a challenge due to the redundant purification processes of membrane protein in some widely-used methods including NMR, X-ray crystallography, Fluorescence Titration Spectroscopy, and Circular Dichroism. Herein, extracellular domain (ECD) of CaSR as its functional fragment was used to develop a rapid chromatographic method, which involved the synthesis of stationary phase material based on the site-specific covalent reaction of Halogenated alkane dehalogenase (Halo)-tagged ECD of CaSR in cell lysate with 6-chlorocaproic acid modified silica beads, the use of the immobilized CaSR column for revealing the interaction of three known agonists with CaSR and further screening ligands from complex matrix like Chinese herb medicine 'Shuangdan'. The immobilized CaSR column was prepared rapidly without the protein purification and retained a good stability and specificity for at least 35 days. It was revealed that one type of binding sites occurred on CaSR with the binding affinity of neomycin gentamicin-C / kanamycin, presumably which related to the number of structural amino groups attached. This method allowed for recognizing specifically novel ligands from 'Shuangdan', demonstrating one type of binding sites on CaSR with the binding affinity of gallic acid caffeic acid paeonol. These results indicated that, the immobilization of a representative extracellular domain of CaSR to silica beads as biomaterial is feasible to develop a new rapid method, which can be successfully applied in screening novel ligands efficiently from complex matrices.

α-Asaronol [or (E)-3′-hydroxyasarone; systematic name: (E)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-ol; C12H16O4] was synthesized towards the development of a potential antiepileptic drug. Following purification by recrystallization, single crystals of α-asaronol were obtained by a liquid interface diffusion method at room temperature. The product was characterized by 1H and 13C NMR, and FT–IR spectroscopic analysis. X-ray crystallography revealed the title crystal to belong to the orthorhombic space group P212121. Preliminary bioassays with mouse neuroblastoma N2a cells demonstrated the neuroprotective activities of the synthesized α-asaronol.

Screening and identification of potential compounds from herbal medicine is a prevailing way to find a lead for the development of innovative drugs. This promotes the development of new methods that are feasible in complex matrices. Here, we described a one-step reversible methodology to immobilize nuclear peroxisome proliferator-activated receptor gamma (PPARγ) onto amino microsphere coated with a DNA strand specifically binding to the receptor. The specific interaction allowed us to achieve the immobilization of PPARγ by mixing the DNA modified microspheres with E. coli lysates expressing the receptor. Characterization of the immobilized receptor was carried out by morphology and binding specificity analysis. Feasibility of immobilized PPARγ in the drug-receptor interaction analysis was performed by an injection amount-dependent method. Besides, immobilized PPARγ was also applied in screening modulators of the receptor from Coptidis Rhizoma extract. The binding of the screened compounds to PPARγ was examined by time-resolved fluorescence resonance energy transfer assay. The results showed that immobilized PPARγ was stable for thirty days with a high-specificity of ligand recognition at the subtype receptor level. Berberine and palmatine were the bioactive compounds of Coptidis Rhizoma specifically binding to PPARγ. The two compounds exhibited half maximal inhibitory concentrations of 4.11 and 2.98 μM during their binding to the receptor. We concluded that the current method is possible to become a common strategy for the immobilization of nuclear receptors, and the immobilized receptor is a high throughput method for recognizing and separating the receptor modulators from complex matrices including herbal medicine.

Drug discovery based on natural products like medicinal herbs remains challenging due to the technique limitations for rapidly screening and validating leads. To address the challenges, we employ the immobilized β

The kinetic study in drug-receptor interaction analysis is of great significance for the efficacy and safety of a drug. In previous work, we established a one-step method to immobilize the endothelin receptor A (ETA) on the surface of macroporous silica gel through a covalent interaction between the epidermal growth factor tyrosine kinase (EGFR) at the C terminal of ETA and the covalent inhibitor ibrutinib. The affinity stationary phase was used for lead screening and investigating the thermodynamic binding of ligands and ETA. Herein, we intend to introduce the ETA column in the kinetic study of the receptor and three specific ligands (bosentan, macitentan, and ambrisentan) as well as three leads (ferulic acid, berberine, and palmatine) from herbal medicines. Three classical methods in affinity chromatography, nonlinear chromatography, peak decay, and peak profiling, were performed to determine the dissociation rate constants of the ligands to ETA. A comparison of the three methods was made and the binding parameters were discussed. The three methods gave the same order of the dissociation constants between the six ligands and ETA. The results indicated that the immobilized ETA can be used for not only thermodynamic studies but also kinetic investigations, which provides a reliable and fast evaluation for drug-receptor interaction analysis.

Bornyl caffeate was initially discovered as a bioactive compound in medicinal plants. Despite the promising pharmacological activities including anti-tumor and antibacterial activities, the pharmacokinetics of the compound remain open. This work developed a high performance liquid chromatography-tandem mass spectrometric method for the determination of bornyl caffeate and caffeic acid (major metabolite and a main unit of bornyl caffeate) in vivo. Successful application of the method included identification of its metabolites and investigation on the drug pharmacokinetics. A total of 30 compounds were identified as the metabolites of bornyl caffeate in rats. We attributed these metabolites to phase I metabolic routes of reduction, oxidation, hydrolysis and phase II metabolic reactions of glucuronidation, sulfation, O-methylation and glycine. Glucuronidation, sulfation, O-methylation and reduction were the main metabolic pathways of bornyl caffeate. The method presented a linear range of 1–4000 ng mL−1. The pharmacokinetic profile of bornyl caffeate was found to be a three compartment open model, while caffeic acid fitted to a two compartment open model when it was administered alone or served as the main metabolite of bornyl caffeate. The time to peak concentration (Tmax) and the maximum plasma concentration (Cmax) of bornyl caffeate were 0.53 h and 409.33 ng mL−1. Compared with original caffeic acid, the compound displayed an increased half-life of elimination (T1/2β), area under the concentration time curve from 0 to t (AUC0–t) and area under the concentration time curve from 0 to ∞ (AUC0–∞), a decreased half-life of absorption (T1/2α) and an identical Cmax. Taking together, we concluded that bornyl caffeate is able to rapidly initiate therapeutic effect and last for a relatively long time in rats; metabolic pathways of O-methylation and reduction is key to interpret the mechanism and toxicity of bornyl caffeate.

Objective To understand the in vivo metabolic fate of 1,2,3,4,6-Penta-O-galloyl-β- d -glucose (PGG) naturally existed in many medicinal herbs and food plants such as Rhus chinensis, Paeonia suffruticosa, Paeonia lactiflora and Mango. Methods The metabolites of PGG in rat biofluids were characterized using high performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS). Results Ten metabolites in urine, five metabolites in feces and two metabolites in plasma, were observed when the rats were administrated with a single intravenous injection of PGG (20 mg/kg). Conclusion PGG is firstly metabolized to gallic acid, then gallic acid undergoes sulfation, glucuronidation and methylation by rat liver. The determination of metabolites and the proposed metabolic pathway of PGG in vivo will be benefit to gain deeper insights into its pharmacological activities.

Drug discovery based on natural products like medicinal herbs remains challenging due to the technique limitations for rapidly screening and validating leads. To address the challenges, we employ the immobilized β2- adrenergic recepotor (β2-AR), an identified target of asthma, as the stationary phase in chromatographic column to screen compounds extracted from Stemonae Radix, Playtycodonis Radix, and Glycyrrhizae Radix et Rhizoma. To analyze binding properties of the extracted compounds to the immobilized receptors, we measured their retention behavior in the receptor chromatography and compared with six clinical asthma drugs. We identified tuberostemonine, platycodin D, and glycyrrhizic acid as the potential leads against asthma by our β2-AR chromatography coupled with mass spectrum (MS). The association constants of the three compounds to β2-AR were 2.85 × 10−5, 2.55 × 10−4, and 4.07 × 10−6 M with the dissociation rate constants of 6.91 ± 0.35, 11.88 ± 0.60, and 9.49 ± 0.64 min−1, respectively. Tuberostemonine, a pentacyclic Stemona alkaloids, presented the most optimum values of binding efficiency index (BEI) and surface efficiency index (SEI) as close to the diagonal of SEI–BEI optimization plane when it is compared with platycodin D, glycyrrhizic and the six clinical drugs. Our results suggest that tuberostemonine is a promising natural product to be developed for treating asthma because it exhibits better drug-like binding properties to β2-AR than the clinical drugs. As such, we demonstrate a chromatographic strategy to identify bioactive natural products based on the β2-AR immobilization, which can be widely adopted to screen natural products from mixture of herbal extracts.

Inspired by the traditional Chinese herbal pair of Polygala tenuifolia-Acori Tatarinowii for treating epilepsy, 33 novel substituted cinnamic α-asaronol esters and analogues were designed by Combination of Traditional Chinese Medicine Molecular Chemistry (CTCMMC) strategy, synthesized and tested systematically not only for anticonvulsant activity in three mouse models but also for LDH inhibitory activity. Thereinto, 68–70 and 75 displayed excellent and broad spectra of anticonvulsant activities with modest ability in preventing neuropathic pain, as well as low neurotoxicity. The protective indices of these four compounds compared favorably with stiripentol, lacosamide, carbamazepine and valproic acid. 68–70 exhibited good LDH1 and LDH5 inhibitory activities with noncompetitive inhibition type, and were more potent than stiripentol. Notably, 70, as a representative agent, was also shown as a moderately positive allosteric modulator at human α1β2γ2 GABAA receptors (EC50 46.3 ± 7.3 μM). Thus, 68–70 were promising candidates for developing into anti-epileptic drugs, especially for treatment of refractory epilepsies such as Dravet syndrome.

The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drug-receptor interaction analysis and ligand screening. In our example assay using beta

Objective To determine the distribution of metabolites in the root barks of different tree peony cultivars for quality assessment. Methods Seven tree peony phenotypic cultivars with different colors were systematically analyzed using NMR-based metabolomics. Results A total of 16 metabolites from their methanol extracts were simultaneously identified and quantified, including one primary metabolite (sucrose) and 15 secondary ones (acetophenones, phenolics, monoterpene glycosides, flavonoids, and unsaturated fatty acids). The quantitative data indicated that sucrose (90-180 mg/g) and acetophenones (15-100 mg/g), and non-phenolics, monoterpene glycosides, flavonoids, and unsaturated fatty acids (2-15 mg/g) were the major metabolites in these tree peony cultivars. The significantly increasing levels of paeonoside with bioactivity were observed in “Xiangyu”, “Wujinyaohui”, “Roufurong”, “Yaohuang”, “Zhaofen”, “Doulű”, and “Yingrihong” in order. Opposite trends in the levels of paeonoside and paeonol were observed in “Xiangyu” and “Yingrihong”, suggesting that the changes of the secondary metabolites in plants were influenced by primary metabolites, such as sucrose/glucose, and the different physiological processes occurred in different tree peony cultivars. Conclusion “Yingrihong” with red flower has the highest medicine quality whereas “Xiangyu” with white flower has the worst one based on the content of paeonoside.

Shaoyao-Gancao decoction (SGD), a traditional Chinese herbal medicine, was originally described in the Treatise on Cold Pathogenic Diseases and is composed of Paeoniae Radix alba and Glycyrrhiza Radix et rhizoma. It has been widely used in treating spasmolysis and asthma. In the present study, the therapeutic effects of SGD were assessed, and the detailed mechanisms of SGD for asthma treatment were investigated. We established a rat asthma model and analyzed the changes in the metabonomic profiles of plasma and urine after SGD administration. The metabonomic variations were characterized in control, model, and SGD treatment groups through a combination of nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analysis. Seventy-four metabolites were detected in rat plasma and urine and analyzed using the orthogonal projection to latent structures discriminate analysis (OPLS-DA). The results showed that some physiological changes and also metabolic disorders were induced in model rats. SGD treatment reduced the injury and maintained metabolic balance, possibly by the regulation of energy metabolism, amino acid metabolism, and metabolism of other metabolites. These findings provide new insights into the pathways associated with the pathogenesis of asthma and the effects of SGD on the endogenous metabolism in rats.

Tanshinol borneol ester (DBZ) is a novel experimental compound that consists of two chemical structural units from danshensu and borneol. It exhibits efficacious anti-ischemic and anti-atherosclerosis activities in rats. A fecal metabolomics based on Liquid Chromatography-Mass Spectrometry combined with clinical histopathology and blood lipid estimation was employed to assess the efficacy and the metabolic changes caused by administration of DBZ in atherosclerotic rats. There were the typical pathological features of atherosclerosis and significantly increased levels of TC, TG and LDL-C in the atherosclerotic rat group. Nevertheless, atherosclerotic rats administered both DBZ (at a dose of 40 mg kg(-1)) and simvastatin (at a dose of 20 mg kg(-1)) showed good therapeutic effects. The results of the metabolomics studies showed that 55 differential metabolites such as sebacic acid, enterodiol, nonanedioic acid, dodecanedioic acid, cholic acid, 13(S)-HPODE, deoxycholic acid, some phosphatidylglycerol and phosphatidic acids were found, indicating that abnormal metabolism occurred in the pathways of fatty acid oxidation, linoleic acid metabolism, bile acid biosynthesis and glycerophospholipid metabolism in atherosclerotic rats. Compared to those in the model group, the contents of 41 differential metabolites showed a tendency to recover to a healthy level after DBZ administration. Metabolomics studies suggested that DBZ exhibited good treatment efficacy against atherosclerosis by adjusting disturbed metabolic pathways related to atherosclerosis. This study could provide an experimental basis for DBZ's application to act as a candidate drug with anti-atherosclerosis activity.

Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-Q-TOF-MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid-methanol solution (v:v, 5:95), chromatographic separation by an Agilent Eclipse XDB-C18 column (150 × 4.6 mm i.d., 5 µm) and online identification of the metabolites by Q-TOF-MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including 4 phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O-methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. This article is protected by copyright. All rights reserved.

It is extremely important and necessary to develop a highly sensitive and excellent selective fluorescence probe for detection of ClO− in living systems because the excess production of ClO− was related to a series of diseases. Herein, a novel turn-on colorimetric fluorescent probe ADT-MAM based on naphthalenone scaffold was successfully designed and synthesized for highly selective and sensitive detection of ClO− with fast response time, a large Stokes shift, low limit of detection and wide pH range in applications. Meanwhile, probe ADT-MAM allowed colorimetric detection of ClO− by naked eyes. Fluorescence titration experiments showed that the probe has an excellent linear relationship (R2 = 0.9949) and a low detection limit of 0.15 μM. HOMO-LUMO energy based on density functional theory (DFT) has been calculated in order to provide a better understanding of optical property for ADT-MAM and DTC. Moreover, the probe could be applied to sense exogenous imaging of ClO− in HeLa cells and A549 cells with negligible cytotoxicity, cell membrane permeability and good biocompatibility. The success of subcellular imaging suggested that such a novel probe ADT-MAM could be applied to further reveal essential biological functions and pathological roles of ClO− in extensive biological systems.

International audience; A novel naked-eye colorimetric and fluorescent turn-on pH sensor based on naphthalenone scaffold was rationally designed and facilely synthesized. The probe exhibited strong alkaline pH-dependent (pKa=9.92) behavior, high selectivity, sensitivity, high quantum efficiency (Ф= 0.64) and rapid respond to pH fluctuations (within 1 min) in the range of 9.0-14.0. In addition, probe DDTM displayed a notably large Stokes shift of 198 nm. The response mechanism of the fluorescent probes was proposed to be caused by OH-induced structure changes from the nonfluorescent DDTM form to the highly emissive addition product (more than 150-fold fluorescence enhancement) along with ICT changes. Live cell imaging data revealed that probe DDTM could selectively monitor pH changes with low cytotoxicity and cell membrane permeability. Therefore, this probe could be acted as an effective intracellular pH imaging sensor under extreme alkaline condition in the biomedical and biological fields.

An approach is established for the specific immobilization of GPCRs from cell lysates that circumvents labor intensive purification procedures and minimize loss of activity., Protein immobilization techniques play an important role in the development of assays for disease diagnosis and drug discovery. However, many of these approaches are not applicable to transmembrane proteins. G protein-coupled receptors (GPCRs) are the largest protein superfamily encoded by the human genome and are targeted by a quarter of all prescription drugs. GPCRs are highly dynamic and sensitive to changes in the ambient environment, and current immobilization methodologies are not suitable for GPCRs. We used haloalkane dehalogenase (Halo) as an immobilization tag fused to the β2-adrenoceptor (β2-AR), angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors. The engineered Halo-tag covalently binds to a specific substrate chloroalkane through Asp 106 in the catalytic pocket. The Halo-tagged GPCRs were expressed in Escherichia coli at a suitable yield. Accordingly, we loaded cell lysate containing Halo-tagged GPCRs onto a macroporous silica gel coated with chloroalkane. Morphological characterization indicated a homogeneous monolayer of immobilized Halo-tagged GPCRs on the silica gel surface. The immobilized receptors proved to be surrounded by specific bound phospholipids including PG C18:1/C18:1. We observed a radio-ligand binding ability and ligand-induced conformational changes in the immobilized GPCRs, suggesting the preservation of bioactivity. This method is a one-step approach for the specific immobilization of GPCRs from cell lysates and validates that immobilized receptors retain canonical ligand binding capacity. Our immobilization strategy circumvents labor-intensive purification procedures and minimizes loss of activity. The immobilized receptors can be applied to high-throughput drug and interaction partner screening for GPCRs.

Phenylpropanoids comprise a broad spectrum of biologically active natural products. As part of our ongoing research on antiepileptic active compounds from traditional Chinese herb, Acorus calamus var. angustatus Besser, three undescribed phenylpropanoids and twenty-two known ones were isolated. All the undescribed structures were determined by a combination of 1D and 2D NMR, HRMS. In addition, γ-asaronol was identified as racemates and its absolute configuration were determined by the modified Mosher's method and ECD spectral data. Furthermore, some selected isolated compounds were evaluated for their cell viability and neuroprotective activities in H2O2-induced SH-SY5Y cells. α-Asaronol, β-asaronol, 3-(2,4,5-trimethoxyphenyl)propan-1-ol and 1,2,4-trimethoxy-5-(3-methoxypropyl)benzene exerted potential protective activity from neuronal oxidative stress in all test concentrations ranging from 0.01 to 100 μM, in which the neuroprotective activity of β-asaronol was the best.

Introduction The distribution of metabolites in the different root parts of Cortex Moutan (the root bark of Paeonia suffruticosa Andrews) is not well understood, therefore, scientific evidence is not available for quality assessment of Cortex Moutan. Objective To reveal metabolomic variations in Cortex Moutan in order to gain deeper insights to enable quality control. Methods Metabolomic variations in the different root parts of Cortex Moutan were characterised using high-performance liquid chromatography combined with mass spectrometry (HPLC–MS) and multivariate data analysis. The discriminating metabolites in different root parts were evaluated by the one-way analysis of variance and a fold change parameter. Results The metabolite profiles of Cortex Moutan were largely dominated by five primary and 41 secondary metabolites . Higher levels of malic acid, gallic acid and mudanoside-B were mainly observed in the second lateral roots, whereas dihydroxyacetophenone, benzoyloxypaeoniflorin, suffruticoside-A, kaempferol dihexoside, mudanpioside E and mudanpioside J accumulated in the first lateral and axial roots. The highest contents of paeonol, galloyloxypaeoniflorin and procyanidin B were detected in the axial roots. Accordingly, metabolite compositions of Cortex Moutan were found to vary among different root parts. Conclusion The axial roots have higher quality than the lateral roots in Cortex Moutan due to the accumulation of bioactive secondary metabolites associated with plant physiology. These findings provided important scientific evidence for grading Cortex Moutan on the general market. Copyright © 2014 John Wiley & Sons, Ltd.

Metabolomic analysis is an important molecular phenotyping method for understanding plant ecotypic variations. Here, we systematically characterized the metabolomic variations associated with five Chinese cultivars of tree peony (Paeonia suffruticosa Andrews) using high performance liquid chromatography-mass spectrometry (HPLC-MS) and multivariate data analysis. Our results indicated that the metabolite profile of the root bark in tree peony was largely dominated by 5 primary metabolites and 41 secondary ones including 7 phenolics, 7 flavonoids, 16 monoterpene glycosides and 11 acetophenones. The distribution of these secondary metabolites varied in the different tree peony cultivars. Some secondary metabolites, such as galloyl glucoses, procyanidins, mudanpiosides and acetophenones, will become the novel and potential chemotaxonomic markers to differentiate tree peony cultivars when the conventional classification methods are not practicable. These results demonstrated that HPLC-MS based metabolomics was an effective tool for the classification of phenotypic cultivars and provided novel and potential chemotaxonomic characteristics of tree peony.

Weak self-interaction plays an important role in interpreting the biomechanisms and modes of drug action. The structure-dependent self-association of five phenolic acids with various bioactivities, including danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA), was investigated by 1H NMR. These phenolic acids have similar condensed structures, with a CA moiety and varying numbers of DSS moieties. The strengths of the self-association constants are in the order DSS

A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high-performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 104/m. The number of the binding site involving the interaction between caffeic acid and HSA was 69 nm. The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug–protein interaction. The proposed model also has the advantages of ligand saving and rapid operation. Copyright © 2014 John Wiley & Sons, Ltd.

Affinity chromatography is the most widespread and widely accepted methodology for exploring drug-protein and protein-protein interactions. Despite the successful application of frontal analysis and zonal elution in affinity chromatography, research into the creation of new mathematical tools for data processing is encouraged due to these two methods' drawbacks of long analysis times and high ligand consumption. In this work, we created a novel mathematical model using the relationship between the molar amount of an injected solute and its capacity factor. We validated the method by analyzing the binding of drugs to human serum albumin (HSA) and β2-adrenoceptor (β2-AR). The association constants of omeprazole, propranolol and promethazine binding to HSA were determined to be (4.10±0.24)×10(4), (2.30±0.12)×10(4) and (1.24±0.14)×10(4)M(-1), respectively. These constants agreed with previously reported literature results of 4.60×10(4), 2.30×10(4) and 1.40×10(4)M(-1). Salbutamol, norepinephrine, isoprenaline, bamethane and methoxyphenamine were found to bind to β2-AR with association constants of (1.11±0.06)×10(3), (0.95±0.03)×10(3), (1.66±0.12)×10(3), (0.47±0.04)×10(3) and (0.43±0.02)×10(3)M(-1), respectively, which positively correlated to the negative logarithm of the dissociation constants obtained via radio-ligand binding assays. The proposed model is relatively fast and conserves ligand, and it has the potential to serve as an alternative method for rapidly revealing drug-protein and protein-protein interactions.

A method based on reaction with a diazonium salt was developed to immobilise oriented His-tagged protein onto silica gel. The binding efficiency of the phenylamine-group-coated gel was determined to be 65 %, providing a binding capacity of His-tagged protein up to the gram level. Using His-tagged β2-adrenoceptor (β2-AR) as a probe, we developed a new mathematical model to elucidate the interactions between the receptor and five ligands (methoxyphenamine, terbutaline, salbutamol, tulobuterol and fenoterol). These drugs proved to only have one type of binding site on the immobilised β2-AR, yielding higher association constants and numbers of binding sites than random attachment assays. The association constants determined by the new model positively correlated to the values from a radioligand binding method, with a regression equation of y = 1.75x - 7.18 and a correlation coefficient of 0.9807. The oriented method resulted in a high binding capacity and quantitative immobilisation of the His-tagged protein. The proposed model can be used to determine the interactions between the ligands and the immobilised protein with the advantages of drug and time saving.

A pilot-scale process has been developed for green and scalable synthesis of (±)-β-(3,4-dihydroxyphenyl) lactic acid ((±)-DSS) and their two important derivatives, namely, (±)-IDHP [(±)-isopropyl 2-hydroxy-3-(3,4-dihydroxyphenyl)propanoate] and (±)-DBZ [(±)-bornyl 2-hydroxy-3-(3,4-dihydroxyphenyl)propanoate]. Subsequent hydrogenation has been carried out by employing Raney Ni as catalyst. The improved process results in higher yields of 47.5% for (±)-DBZ and 49.2% for (±)-IDHP compared to the initial process with a yield of 12% for (±)-DBZ and 18% for (±)-IDHP in our original medicinal chemistry route. Furthermore, kilograms of optical DBZ [(−)-S-DBZ and (+)-R-DBZ, >99% ee] and IDHP [(−)-S-IDHP and (+)-R-IDHP, >99% ee] have been produced by chiral high-performance liquid chromatography in good yield (>84%).

A simple, sensitive and rapid method for the analysis of jasminoidin in rabbit plasma by liquid chromatography coupled to tandem mass spectrometry was developed. Detection was by positive ion electrospray ionization in multiple reactions monitoring mode. The method included a chromatographic run of 5.0 min using a C18 analytical column and the calibration curve was linear over the concentration range of 0.5–2,000 ng mL−1 with a correlation coefficient R of 0.998 or better. The intra- and inter-day precision ranged from 3.4 to 5.6% and 4.3 to 8.2%. The intra- and inter-day assay accuracy was between −7.4 and 8.6%. The method was successfully applied for the pharmacokinetic study on jasminoidin in rabbit after a single dose oral administration of Gardenia jasminoides Ellis (Gardenia) and Gardenia coupling Coptis chinensis Franch (Coptidis) extracts.

Metabonomic analysis is an important molecular phenotyping method for understanding plant ecotypic variations and gene functions. Here, we systematically characterized the metabonomic variations associated with three Salvia miltiorrhiza Bunge (SMB) cultivars using the combined NMR and LC-DAD-MS detections in conjunction with multivariate data analysis. Our results indicated that NMR methods were effective to quantitatively detect the abundant plant metabolites including both the primary and secondary metabolites whereas the LC-DAD-MS methods were excellent for selectively detecting the secondary metabolites. We found that the SMB metabonome was dominated by 28 primary metabolites including sugars, amino acids, and carboxylic acids and 4 polyphenolic secondary metabolites, among which N-acetylglutamate, asparate, fumurate, and yunnaneic acid D were reported for the first time in this plant. We also found that three SMB cultivars growing at the same location had significant metabonomic differences in terms of metabolisms of carbohydrates, amino acids, and choline, TCA cycle, and the shikimate-mediated secondary metabolisms. We further found that the same SMB cultivar growing at different locations differed in their metabonome. These results provided important information on the ecotypic dependence of SMB metabonome on the growing environment and demonstrated that the combination of NMR and LC-MS methods was effective for plant metabonomic phenotype analysis.

Plant metabonomic analysis is essential for understanding plant systems responses to osmotic stresses. To understand the comprehensive metabolic responses of Salvia miltiorrhiza Bunge (SMB) to continuous and exhaustive water depletion, we characterized the SMB metabonomic variations induced by three different drying processes using the combined NMR and LC-DAD-MS method. NMR results showed that SMB extracts were dominated by 29 primary metabolites such as sugars, carboxylic acids and amino acids, which were comprehensively reported for the first time, and 8 secondary metabolites including polyphenolic acids and diterpenoids. LC-DAD-MS methods detected 44 secondary metabolites, among which 5 polyphenolic acids together with genipin, umbelliferone and tormentic acid were found for the first time in this plant. We found that aqueous methanol was efficient in extracting both primary metabolites and polyphenolic acids, whereas chloroform-methanol was effective in selectively extracting diterpenoids. We further found that air- and sun-drying markedly affected both primary and secondary metabolisms of SMB by enhancing tanshinone and glutamate-mediated proline biosynthesis and altering carbohydrate and amino acid metabolisms. The shikimate-mediated biosynthesis of polyphenolic acids was promoted by air-drying but suppressed by sun-drying. These findings fill the gap of our understandings to the metabolic responses of S. miltiorrhiza Bunge to water depletion and demonstrated effectiveness of the combined NMR and LC-DAD-MS methods in plant metabonomic analysis.

Metabolite identification is a key step for metabonomics study. A fully automated hyphenation of HPLC-diode-array detector (DAD) mass spectrometry (MS) solid phase extraction (SPE)-NMR spectroscopy (HPLC-DAD-MS-SPE-NMR) is one of the most efficient methods to determine the structure of a given unknown metabolite in a complex mixture (metabonome) and hence represents one of the most important analytical techniques for the further development of metabonomics. In this review, some recent applications of this technique in identifying novel and trace metabolites in plant extracts and drug metabolism have been discussed. Modification of this hyphenated technique, enabling multiple trappings of strong polar metabolites for biofluids, needs further development. Copyright (C) 2009 John Wiley & Sons, Ltd.

Humans have evolved intimate symbiotic relationships with a consortium of gut microbes (microbiome) and individual variations in the microbiome influence host health, may be implicated in disease etiology, and affect drug metabolism, toxicity, and efficacy. However, the molecular basis of these microbe–host interactions and the roles of individual bacterial species are obscure. We now demonstrate a“transgenomic” approach to link gut microbiome and metabolic phenotype (metabotype) variation. We have used a combination of spectroscopic, microbiomic, and multivariate statistical tools to analyze fecal and urinary samples from seven Chinese individuals (sampled twice) and to model the microbial–host metabolic connectivities. At the species level, we found structural differences in the Chinese family gut microbiomes and those reported for American volunteers, which is consistent with population microbial cometabolic differences reported in epidemiological studies. We also introduce the concept of functional metagenomics, defined as “the characterization of key functional members of the microbiome that most influence host metabolism and hence health.” For example, Faecalibacterium prausnitzii population variation is associated with modulation of eight urinary metabolites of diverse structure, indicating that this species is a highly functionally active member of the microbiome, influencing numerous host pathways. Other species were identified showing different and varied metabolic interactions. Our approach for understanding the dynamic basis of host–microbiome symbiosis provides a foundation for the development of functional metagenomics as a probe of systemic effects of drugs and diet that are of relevance to personal and public health care solutions.

The distribution of metabolites in the different root parts of Cortex Moutan (the root bark of Paeonia suffruticosa Andrews) is not well understood, therefore, scientific evidence is not available for quality assessment of Cortex Moutan.To reveal metabolomic variations in Cortex Moutan in order to gain deeper insights to enable quality control.Metabolomic variations in the different root parts of Cortex Moutan were characterised using high-performance liquid chromatography combined with mass spectrometry (HPLC-MS) and multivariate data analysis. The discriminating metabolites in different root parts were evaluated by the one-way analysis of variance and a fold change parameter.The metabolite profiles of Cortex Moutan were largely dominated by five primary and 41 secondary metabolites . Higher levels of malic acid, gallic acid and mudanoside-B were mainly observed in the second lateral roots, whereas dihydroxyacetophenone, benzoyloxypaeoniflorin, suffruticoside-A, kaempferol dihexoside, mudanpioside E and mudanpioside J accumulated in the first lateral and axial roots. The highest contents of paeonol, galloyloxypaeoniflorin and procyanidin B were detected in the axial roots. Accordingly, metabolite compositions of Cortex Moutan were found to vary among different root parts.The axial roots have higher quality than the lateral roots in Cortex Moutan due to the accumulation of bioactive secondary metabolites associated with plant physiology. These findings provided important scientific evidence for grading Cortex Moutan on the general market.

Weak self-interaction plays an important role in interpreting the biomechanisms and modes of drug action. The structure-dependent self-association of five phenolic acids with various bioactivities, including danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA), was investigated by (1)H NMR. These phenolic acids have similar condensed structures, with a CA moiety and varying numbers of DSS moieties. The strengths of the self-association constants are in the order DSS CA RA LA SA, which corresponds to the increasing molecular size of these phenolic acids and roughly corresponds to the increasing number of DSS moieties. The binding site for the self-aggregation of these phenolic acids has been identified to be on the CA moiety, rather than on the DSS moiety, as a result of CA's stronger aromatic π-π interactions, which cause larger chemical shift variations. The thermodynamic parameters for the self-association of these phenolic acids show that the self-association is spontaneous and enthalpically favorable at room temperature in all cases. It was inferred that π-π interactions and intermolecular hydrogen bonding stabilize the stacking structures of the phenolic acids. Knowledge of self-association processes will enable us to quantitatively assess the possible effects of self-aggregation on the interaction between drug and protein.

A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high-performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 10(4) /m. The number of the binding site involving the interaction between caffeic acid and HSA was 69 nm. The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug-protein interaction. The proposed model also has the advantages of ligand saving and rapid operation.

Gallic acid (GA) and its metabolites are polyphenolic compounds present in daily diets and herbal medicines. To understand the GA effects on the endogenous metabolism of mammals, we systematically analyzed the metabonomic responses of rat plasma, liver, urine, and feces to a single GA dosage of 120 and 600 mg/kg, which were below the no-obvious-adverse-effect-level of 1 g/kg for rats. Clinical chemistry and histopathological assessments were conducted to provide complementary information. Our results showed that GA intake induced significant metabonomic changes in multiple rat biological matrices. Such changes were more outstanding in liver than in the other matrices and clearly showed dose- and time-dependence. The results suggested GA-induced promotion of oxidative stress as the major effect. High-dose GA caused significant metabolic changes involving glycogenolysis, glycolysis, TCA cycle, and metabolism of amino acids, purines, and pyrimidines, together with gut microbiota functions. Low-dose GA only caused some urinary metabonomic changes and to a much less degree. The GA-induced liver metabonomic changes were not completely recoverable within a week, although such recovery completed in plasma, urine, and feces within 80 h. These findings provided new essential information on the effects of dietary polyphenols and demonstrated the great potential of this nutrimetabonomics approach.

A novel high performance liquid chromatography-electrochemical detector (HPLC-ECD) analytical system was developed in this study by integratedly utilizing ionic liquid (IL) of 1-butyl-3-methylimidazolium bromide and an additive of gold nanoparticles. The resulted pilot study was first performed to assess the effects of 1-butyl-3-methylimidazolium bromide and gold nanoparticles on the chromatographic characteristics of five phenolic acids in Xuebijing injection, including danshensu (DSS), protocatechuic acid (PA), protocatechuic aldehyde (PAH), hydroxy safflower yellow A (HSYA) and ferulic acid (FA). It was notable to observe that retainability of the phenolic acids were markly lowered by IL addition. Compared with the cases without IL addition, the retention times of DSS, PA, PAH, HSYA and FA have decreased 2.851, 1.532, 1.53, 0.818 and 0.552 min, respectively when 0.6% IL in the mobile phase. In addition, the corresponding theoretical plate numbers and peak areas for these compounds were significantly increased. Area response for DSS, PA, PAH, HSYA and FA were enhanced by 772%, 628%, 584%, 703% and 600%, respectively. It was observed that nano-gold catalysis power enabled peak areas of DSS, PAH, FA and PA to enhance 5.7, 6.2, 8.5 and 66.5 times relative to the case with addition of IL. Altogether, the optimized HPLC-ECD system was successfully applied to the pharmacokinetics study of Xuebijing injection with underlying applicability to in vivo and in vitro analysis of a variety of natural product from Chinese medicine plants, TCM formulae and associated patent TCM preparation.

Diabetes mellitus is a complex polygenic disease caused by gene-environment interactions with multiple complications, and metabonomic analysis is crucial for pathogenesis, early diagnosis, and timely interventions. Here, we comprehensively analyzed the dynamic metabolic changes in rat urine and plasma, which were induced by the well-known diabetogenic chemical streptozotocin (STZ), using (1)H NMR spectroscopy in conjunction with multivariate data analysis. The results showed that a single intraperitoneal injection of STZ with a moderate dosage (55 mg/kg) induced significant urinary metabonomic changes within 24 h. These changes showed time-dependence and heterogeneity among the treated animals with an animal recovered within 11 days. STZ-induced metabonomic alterations were related to suppression of glycolysis and TCA cycle, promotion of gluconeogenesis and oxidation of amino acids, alterations in metabolisms of basic amino acids associated with diabetic complications, and disruption of lipid metabolism and gut microbiota functions. With diffusion-edited NMR spectral data, we further observed the STZ-induced significant elevation of monounsaturated fatty acids and total unsaturated fatty acids together with reductions in PUFA-to-MUFA ratio in the blood plasma. These findings provided details of the time-dependent metabonomic changes in the progressive development of the STZ-induced diabetes mellitus and showed the possibility of detecting the biochemical changes in the early stage of type 1 diabetic genesis.

In the title compound, C10H12O5, the dihedral angle between the benzene ring is almost coplanar with the attached C(O)—O—C group [dihedral angle = 0.32 (15)°]. In the crystal, two intermolecular O—H...O hydrogen bonds make R44(26) ring mofits.

Identification of new compounds especially those with new skeletons from plant kingdom has long been a vital aspect for understanding phytochemistry, plant metabolisms and discovering new bioactive compounds. In this study, we identified and isolated three novel polyphenolic compounds, origanine A-C, from a well-researched plant Origanum vulgare L. using the hyphenated LC-DAD-SPE-NMR/MS methods. Based on the combined information from UV-visible, accurate mass and 2D NMR spectra together with computational calculations, we found that these compounds all had a novel skeleton of cyclohexenetetracarboxylic acids attached with some well-known bioactive moieties including 3,4-dihydroxyphenyl, 4-(β-d-glucopyranosyloxy)benzyl alcohol (gastrodin), and 3-(3,4-dihydroxyphenyl)lactic acid (danshensu) residues. These findings provided crucial information to fill the gaps in our knowledge in terms of the plant secondary metabolism. This study also indicated the necessity for further research in plant secondary metabolism for even well-studied plants and demonstrated the powerfulness of the hyphenated LC-DAD-SPE-NMR/MS methods for comprehensive analysis of plant metabolites in particular for discovering new natural compounds.