Fabrication of a surface molecularly imprinted polymer membrane based on a single template and its application in the separation and extraction of phenytoin, phenobarbital and lamotrigine.

An innovative molecularly imprinted polymer membrane (MIPM) was prepared with polyvinylidene difluoride (PVDF) as the support, phenytoin (PHT) as the single template, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross-linking reagent, azobisisobutyronitrile as the initiator, and acetonitrile-dimethylformamide (1 : 1.5, v/v) as the porogen. These materials were characterized via scanning electron microscopy, Fourier transform infrared spectroscopy, Brunauer-Emmett-Teller measurements and X-ray photoelectron spectroscopy. Their adsorption performances were evaluated through a series of experiments including isothermal adsorption, kinetic adsorption, selective adsorption, adsorption-desorption, reusability, and preparation reproducibility. Additionally, the application was explored by investigating the extraction recovery of MIPMs towards PHT, phenobarbital (PHB) and lamotrigine (LTG) in different matrices including methanol, normal saline (NS), phosphate buffer solution (PBS) and plasma. The results showed that MIPMs with rough and porous surfaces were successfully constructed, which offered good preparation reproducibility, reusability and selectivity. The adsorption capacities of MIPMs towards PHT, PHB and LTG were 2.312, 2.485 and 2.303 mg g-1, respectively, while their corresponding imprinting factors were 8.538, 12.122 and 4.562, respectively. The adsorption equilibrium of MIPMs was achieved within 20 min at room temperature without stirring or ultrasonication. The extraction recoveries of MIPMs for PHT, PHB or LTG in methanol, NS and PBS were more than 80% with an RSD% value of less than 3.64. In the case of plasma, the extraction recovery of MIPMs for PHT and PHB was more than 80% with an RSD% value of less than 2.41, while that of MIPMs for LTG was more than 65% with an RSD% value of less than 0.99. All the results indicated that the preparation method for MIPMs was simple, stable, and reliable, and the prepared MIPMs possessed excellent properties to meet the extraction application of PHT, PHB and LTG in different matrices.

Mulberry (Morus alba L.) leaf water extract attenuates type 2 diabetes mellitus by regulating gut microbiota dysbiosis, lipopolysaccharide elevation and endocannabinoid system disorder.

ETHNOPHARMACOLOGICAL RELEVANCE: Mulberry (Morus alba L.) leaf is a well-known herbal medicine and has been used to treat diabetes in China for thousands of years. Our previous studies have proven mulberry leaf water extract (MLWE) could improve type 2 diabetes mellitus (T2D). However, it is still unclear whether MLWE could mitigate T2D by regulating gut microbiota dysbiosis and thereof improve intestinal permeability and metabolic dysfunction through modulation of lipopolysaccharide (LPS) and endocannabinoid system (eCBs). AIM OF STUDY: This study aims to explore the potential mechanism of MLWE on the regulation of metabolic function disorder of T2D mice from the aspects of gut microbiota, LPS and eCBs. MATERIALS AND METHODS: Gut microbiota was analyzed by high-throughput 16S rRNA gene sequencing. LPS, N-arachidonoylethanolamine (AEA) and 2-ararchidonylglycerol (2-AG) contents in blood were determined by kits or liquid phase chromatography coupled with triple quadrupole tandem mass spectrometry, respectively. The receptors, enzymes or tight junction protein related to eCBs or gut barrier were detected by RT-PCR or Western blot, respectively. RESULTS: MLWE reduced the serum levels of AEA, 2-AG and LPS, decreased the expressions of N-acylphophatidylethanolamine phospholipase D, diacylglycerol lipase-α and cyclooxygenase 2, and increased the expressions of fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA), alpha/beta hydrolases domain 6/12 in the liver and ileum and occludin, monoacylglycerol lipase and cannabinoid receptor 1 in the ileum of T2D mice. Furthermore, MLWE could change the abundances of the genera including Acetatifactor, Anaerovorax, Bilophila, Colidextribacter, Dubosiella, Gastranaerophilales, Lachnospiraceae_NK4A136_group, Oscillibacter and Rikenella related to LPS, AEA and/or 2-AG. Moreover, obvious improvement of MLWE treatment on serum AEA level, ileum occludin expression, and liver FAAH and NAAA expression could be observed in germ-free-mimic T2D mice. CONCLUSION: MLWE could ameliorate intestinal permeability, inflammation, and glucose and lipid metabolism imbalance of T2D by regulating gut microbiota, LPS and eCBs.

Mulberry leaf water extract alleviates type 2 diabetes in mice via modulating gut microbiota-host co-metabolism of branched-chain amino acid.

Elevations in circling branched-chain amino acids (BCAAs) levels associated with insulin resistance and type 2 diabetes mellitus (T2DM). Morus alba L. water extracts (MLE) show hypoglycemic function, but the precise mechanism remains obscure. This study is designed to investigate the association of the antidiabetes effect of MLE with the BCAAs co-metabolism modulated by host and gut microbiota. Tissue-specific expressions of BCAA-catabolizing enzymes were detected by RT-PCR and western blot, respectively. The components of the intestinal microflora were analyzed by high-throughput 16S rRNA gene sequencing. The results showed that MLE administration improved blood glucose and insulin level, decreased inflammatory cytokines expression, and lowered serum and feces BCAAs levels. Furthermore, MLE reversed the abundance changes of the bacterial genera correlated with serum and feces BCAAs, such as Anaerovorax, Bilophila, Blautia, Colidextribacter, Dubosiella, Intestinimonas, Lachnoclostridium, Lachnospiraceae_NK4A136, Oscillibacter, and Roseburia. Functionality prediction indicated that MLE potentially inhibited bacterial BCAAs biosynthesis, and promoted the tissue-specific expression of BCAAs catabolic enzyme. More importantly, MLE had obvious impacts on BCAA catabolism in germ-free-mimic T2DM mice. Those results indicated that MLE improving T2DM-related biochemical abnormalities is associated with not only gut microbiota modification but also the tissue-specific expression of BCAAs catabolic enzyme.

Identification of geographic origins of Morus alba Linn. through surfaced enhanced Raman spectrometry and machine learning algorithms.

The leaves of Morus alba Linn., which is also known as white mulberry, have been commonly used in many of traditional systems of medicine for centuries. In traditional Chinese medicine (TCM), mulberry leaf is mainly used for anti-diabetic purpose due to its enrichment in bioactive compounds such as alkaloids, flavonoids and polysaccharides. However, these components are variable due to the different habitats of the mulberry plant. Therefore, geographic origin is an important feature because it is closely associated with bioactive ingredient composition that further influences medicinal qualities and effects. As a low-cost and non-invasive method, surface enhanced Raman spectrometry (SERS) is able to generate the overall fingerprints of chemical compounds in medicinal plants, which holds the potential for the rapid identification of their geographic origins. In this study, we collected mulberry leaves from five representative provinces in China, namely, Anhui, Guangdong, Hebei, Henan and Jiangsu. SERS spectrometry was applied to characterize the fingerprints of both ethanol and water extracts of mulberry leaves, respectively. Through the combination of SERS spectra and machine learning algorithms, mulberry leaves were well discriminated with high accuracies in terms of their geographic origins, among which the deep learning algorithm convolutional neural network (CNN) showed the best performance. Taken together, our study established a novel method for predicting the geographic origins of mulberry leaves through the combination of SERS spectra with machine learning algorithms, which strengthened the application potential of the method in the quality evaluation, control and assurance of mulberry leaves.

Effects of UGT1A, CYP2C9/19 and ABAT polymorphisms on plasma concentration of valproic acid in Chinese epilepsy patients.

Aim: To evaluate the association between genetic polymorphisms and plasma concentration-to-dose ratio of valproic acid (CDRV) in Chinese epileptic patients. Methods: A total of 46 epileptic patients treated with valproic acid therapy were enrolled. 18 SNPs in nine genes related to valproic acid were directly sequenced with Sanger methods. Results: Patients carrying UGT1A6 heterozygous genotypes had significantly lower CDRV than those carrying the wild-type genotypes. In contrast, patients with the homozygote genotypes of CYP2C9 and ABAT had higher CDRV than those with the wild-type genotypes and patients with the heterozygous genotypes of CYP2C19 had higher CDRV. Conclusion: Detection of genetic polymorphism in these genes might facilitate an appropriate dose of valproic acid for epileptic patients. Further studies with larger cohorts are necessary to underpin these observations.

A simple and easy non-derivatization gas chromatography-mass spectrometry method for simultaneous quantification of valproic acid, gabapentin, pregabalin, and vigabatrin in human plasma.

Immunoassays are currently not available in commercial kits for the quantification of valproic acid, vigabatrin, pregabalin, and gabapentin, which also cannot suffer the limitations of interferences of substances with similar structures. Chromatography is a good alternative to immunoassay. In this study, a simple and robust non-derivatization gas chromatography-mass spectrometry method for simultaneous determination of the above four drugs in human plasma was developed and validated for therapeutic drug monitoring purposes. This method employed benzoic acid as the internal standard with hydrochloric acid for plasma acidification and ACN for precipitate protein. The supernatant was directly injected into gas chromatography-mass spectrometry for analysis. Good linearity was obtained with linear correlation coefficients of the four analytes of 0.9988-0.9996. Extraction recoveries of valproic acid, vigabatrin, pregabalin, and gabapentin were respectively in the ranges of 91.3%-94.5%, 90.0%-90.9%, 90.0%-92.1%, and 88.0%-92.2% with the relative standard deviation values less than 12.6%. Intra- and inter-batch precision and accuracy, and stability assays were all acceptable. Taken together, the novel method developed in this study provided easy plasma pretreatment, good extraction yield, and high chromatographic resolution, which has been successfully validated through the quantification of valproic acid in the plasma of 46 patients with epilepsy.

Synthesis of Molecularly Imprinted Polymers Based on a New Monomer "2-(4-Vinylphenyl) Quinoline-4-Carboxylic Acid" for the Selective Solid-Phase Extraction of Lamotrigine.

A new molecularly imprinted polymers (MIPs) have been prepared for the high selective extraction of lamotrigine (LTG), a widely used antiepileptic drug, in human serum. The MIPs were polymerized by bulk polymerization using our synthesized compound, 2-(4-vinylphenyl) quinolin-4-carboxylic acid, as functional monomer, which achieved better adsorption specificity than universal MIPs. Then, the molecularly imprinted solid phase extraction (MISPE) based on this material was coupled with high-performance liquid chromatography (HPLC) for the detection of LTG in human serum. The results of method validation showed that the developed method presented a good precision and accuracy, and the linearity was in the range of 1.50-40.00 mg/mL with the limit of quantitation (LOQ) at 0.20 mg/mL. The recovery ranged from 80.8% to 83.8% with RSD ranges from 5.5% to 11.1%. The validated method was successfully used to determine the concentration of LTG in human simulate serum samples.

Development of magnetic molecularly imprinted polymers with double templates for the rapid and selective determination of carbamazepine and lamotrigine in serum.

A dual-template magnetic molecularly imprinted polymer (Dt-MMIP) with a specific recognition capability for carbamazepine (CBZ) and lamotrigine (LTG) was synthesized using methacrylic acid as a functional monomer, and ethylene glycol dimethylmethacrylate as a cross-linking agent. A magnetic non-molecularly imprinted polymer without templates (MNIP) was also prepared using the same procedure. The prepared polymers were characterized using scanning electron microscopy, Fourier-transform infrared spectroscopy and adsorption experiments. Results indicated that both Dt-MMIPs and MNIPs were microspherical nanoparticles, and the surface of the Dt-MMIP was rougher than that of the MNIP. In addition, the prepared Dt-MMIPs possessed a higher adsorption capacity and better selectivity for CBZ and LTG than the MNIPs. The maximum static adsorption capacities of Dt-MMIP for CBZ and LTG were 249.5 and 647.9 µg g-1, respectively, whereas those of MNIP were 75.8 and 379.8 µg g-1, respectively. The obtained Dt-MMIPs were applied as a magnetic solid-phase extraction sorbent for the rapid and selective extraction of CBZ and LTG in rat serum samples, and determination was performed by high-performance liquid chromatography with UV detection (HPLC-UV). The developed method of dispersive SPE based on Dt-MMIPs coupled to HPLC-UV has good rapidity and selectivity, and application prospects in serum.

Development and evaluation of a simple and easy high-performance liquid chromatography-ultraviolet system simultaneously suitable for determination of 24 anti-epileptic drugs in plasma.

We aim to establish a simple and easy high-performance liquid chromatography system coupled with an ultraviolet detector suitable for simultaneous determination of 24 antiepileptic drugs in human plasma. Optimized chromatographic separation was performed on a ZORBAX Eclipse Plus-C18 (4.6 × 150 mm2 , 3.5 µm) column with acetonitrile and 5 mM potassium dihydrogen phosphate water solution as mobile phase. Note that, 24 antiepileptic drugs were divided into three groups and eluted with different gradient procedures, respectively. The column temperature was maintained at 35°C and the detection wavelength was set at 210 nm. Plasma was processed with ethyl acetate or acetonitrile. The calibration curves of 24 antiepileptic drugs demonstrated good linearity within the test range (r > 0.996). The intra- and inter-batch precision and accuracy were all less than 15%, while extraction recoveries were in the range of 74.57-90.89% with the relative standard deviation values less than 15%. The validated methods have been successfully applied to determination of some antiepileptic drugs in rat or patient plasma. Those results indicated that the developed methods were simple and easy, and could be suitable for the determination of 24 antiepileptic drugs in plasma just by changing the gradient elution procedures of mobile phase.

Lipid metabolism disorders and lipid mediator changes of mice in response to long-term exposure to high-fat and high sucrose diets and ameliorative effects of mulberry leaves.

Mulberry leaves exhibit anti-lipogenic and lipid-lowering effects. However, the lipid biomarkers and underlying mechanisms for the improvement of the action of mulberry leaves on obesity and lipid metabolism disorders have not been sufficiently investigated yet. Herein, biochemical analysis combined with metabolomics targeting serum lipid mediators (oxylipins) were used to explore the efficacy and underlying mechanisms of mulberry leaf water extract (MLWE) in high-fat and high-sucrose diet (HFHSD)-fed mice. Our results showed that MLWE supplementation not only decreased body weight gain, serum total triglycerides, low-density lipoprotein cholesterol, alanine transaminase and aspartate transaminase levels, but also increased the serum level of high-density lipoprotein cholesterol. In addition, MLWE supplementation also ameliorated hepatic steatosis and lipid accumulation. These beneficial effects were associated with down-regulating genes involved in oxidative stress, inflammation, and lipogenesis such as acetyl-CoA carboxylase and fatty acid synthase, and up-regulating genes related to lipolysis that encoded peroxisome proliferator-activated receptor α, adiponectin (ADPN), adiponectin receptor (AdipoR) 1, AdipoR2, adenosine monophosphate-activated protein kinase (AMPK) and hormone-sensitive lipase. Moreover, a total of 54 serum lipid mediators were differentially changed in HFHSD-fed mice, among which 11 lipid mediators from n-3 polyunsaturated fatty acids (PUFAs) were apparently reversed by MLWE. These findings indicated that the ADPN/AMPK pathway, anti-inflammation, anti-oxidation, and n-3 PUFA metabolism played important roles in anti-obesity and improvement of lipid metabolism disorders modulated by MLWE supplementation.

Analysis of plasma free amino acids in diabetic rat and the intervention of Ginkgo biloba leaves extract using hydrophilic interaction liquid chromatography coupled with tandem mass-spectrometry.

Amino acids (AAs) are important metabolites that are related with diabetes. However, their roles in the initiation and development of diabetes mellitus (DM), especially in the treatment of Ginkgo biloba leaves extract (GBE) have not been fully explored. Thus, we investigated the roles that AAs played in the progression and GBE supplementation of DM rat induced by streptozotocin. The rats were randomly divided into a normal control group treated with drug-free solution, a normal control group treated with GBE, a DM group treated with drug-free solution, and DM group treated with GBE; and maintained on this protocol for 9 weeks. Rat plasma was collected from the sixth week to the ninth week and then analyzed with the optimized hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method. A total of 17 AAs with differential levels were monitored to indicate dysfunction of AAs metabolism to confirm the occurrence and development of DM. Treatment with GBE partially reversed the changes seen in seven AAs including leucine, isoleucine, tyrosine, glutamic acid, asparagines, lysine and alanine in DM rats, indicating that GBE could prevent the occurrence and development of DM by acting on AAs metabolism. The improvement of those AAs metabolism disorders may play a considerable role in the treatment of GBE on the occurrence and development of DM. Those findings potentially promote the understanding of the pathogenic progression of DM and reveal the therapeutic mechanism of GBE against DM.

Chemical Antioxidant Quality Markers of Chrysanthemum morifolium Using a Spectrum-Effect Approach.

Traditionally, the quality evaluation of Chrysanthemum morifolium (CM) cv. (Juhua) attributes its habitats and processing methods, however, this strategy of neglecting bioactive ingredients usually results in deviation of quality evaluation. This study aims to explore the quality marker (Q-marker) based on spectrum-effect relationship and quality control strategy of CMs. The chromatographic fingerprint of 30 flower head samples of CMs from five different habitats including Hang-baiju, Gongju, Huaiju, Taiju and Boju were constructed by high performance liquid chromatography and analyzed through chemometrics methods such as similarity analysis (SA), cluster analysis (CA) and principal component analysis (PCA). The common peaks were quantified by external standard method and relative correction factor method. The in-vitro radical scavenging capacity assays of DPPH·, ·OH and ABTS were carried out. The Q-marker was explored by the correlation analysis between the contents of common peaks and in-vitro radical scavenging capacity, and then used to evaluate the quality of 30 flower head samples of CMs. A total of eight common peaks were appointed in 30 flower head samples of CMs, and their similarities ranged from 0.640 to 0.956. CA results showed that 30 flower head samples of CMs could be divided into five categories with reference to the Euclidean distance of 5. PCA results showed that common peaks played a major role in differential contribution of CMs. The quantification of common peaks hinted that their contents possessed significant variation whether for different accessions or the same accessions of CMs. The correlation analysis showed that chlorogenic acid, 3,5-O-dicaffeoylquinic acid, unknown peak 1, 4,5-O-dicaffeoylquinic acid and kaempferol-3-O-rutinoside could be used as the Q-markers for the quality evaluation of 30 flower head samples of commercially available CMs. The analysis strategy that combines chromatographic fingerprint analysis, multiple ingredients quantification, in-vitro chemical anti-oxidant activity evaluation and spectrum-effect relationship analysis clarified the therapeutic material basis and discovered the Q-markers, which possibly offers a more comprehensive quality assessment of CMs.

Morus alba L. water extract changes gut microbiota and fecal metabolome in mice induced by high-fat and high-sucrose diet plus low-dose streptozotocin.

Gut microbiota plays a key role in the pathophysiology of type 2 diabetes mellitus (T2D). Mulberry leaf has a hypoglycemic effect, but the potential mechanism is not fully understood. This study aimed to explore the influences and potential mechanisms of mulberry leaf water extract (MLWE) intervention on mice with T2D induced through a high-fat and high-sucrose diet combined with streptozotocin by the combination of fecal metabolomics and gut microbiota analysis. Results showed that MLWE could decrease fasting blood glucose and body weight while ameliorating lipid profiles, insulin resistance, liver inflammation, and the accumulation of lipid droplets in T2D mice. MLWE could reverse the abundances of the phyla Actinobacteria and Bacteroidetes and the ratio of Firmicutes/Bacteroidetes, and increase the abundances of the phyla Cyanobacteria and Epsilonbacteraeota in the feces of T2D mice. The abundances of genera Alloprevotella, Parabacteroides, Muribaculaceae, and Romboutsia in the feces of T2D mice could be reversed, while Oscillatoriales_cyanobacterium and Gastranaerophilales could be reinforced by MLWE supplementation. The levels of nine metabolites in the feces of T2D mice were improved, among which glycine, Phe-Pro, urocanic acid, phylloquinone, and lactate were correlated with Romboutsia and Gastranaerophilales. Taken together, we conclude that MLWE can effectively alleviate T2D by mediating the host-microbial metabolic axis.

Amelioration of lipid accumulations and metabolism disorders in differentiation and development of 3T3-L1 adipocytes through mulberry leaf water extract.

BACKGROUND: Obesity is a worldwide problem that resulted from the excessive fat accumulation in adipose tissue, leading to the impairment of individual health. Mulberry leaf is an important traditional Chinese medicine and has been used to alleviate obesity for a long term. However, its underlying molecular mechanisms have not been fully elucidated yet. PURPOSE: In this study, we aimed to investigate the inhibition effects of mulberry leaf water extract (MLWE) on lipid accumulation during the process of differentiation of 3T3-L1 preadipocytes and development of mature adipocytes through the combination of molecular biology assays and metabolomic analysis. METHODS: The quality consistency and main chemical ingredients of MLWE were analyzed by high performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), respectively. Oil red O staining was used to mirror lipid accumulation. Lipogenesis-, lipolysis- and inflammation-related genes were evaluated by real-time PCR and western blot, respectively. Untargeted metabolomics were performed by LC-MS/MS. RESULTS: Prepared method and quality of MLWE were stable and reliable. A total of 34 compounds were identified and 14 of them were undoubtedly confirmed. MLWE supplementation could dose-dependently inhibit the aggregation of lipid droplets, and the expressions of sterol regulatory element-binding protein (SREBP)-1c, peroxisome proliferator-activated receptor (PPAR) γ, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), tumor necrosis factor (TNF)-α and interleukin (IL)-6, and increase the expressions of adenosine monophosphate-activated protein kinase (AMPK), hormone-sensitive lipase (HSL) and IL-10 in the differentiation of preadipocytes. Furthermore, MLWE treatment could dose-dependently decrease the level of triglycerides and the expressions of ACC, FAS, TNF-α, and IL-6, and up-regulate the level of glycerol and the expressions of PPARα, adiponectin (ADPN), adiponectin receptor (AdipoR) 1, AdipoR2, AMPK, HSL, and IL-10 in the development of mature adipocytes. Untargeted metabolomics showed that a total of 5 and 18 differential metabolites were reversed by MLWE intervention in the differentiation of preadipocytes and the development of mature adipocytes, respectively, which involved in the biosynthesis of unsaturated fatty acids, arachidonic acid metabolism and glycerophospholipids metabolism. CONCLUSION: Taken together, this study firstly verified that MLWE could effectively alleviate lipid accumulation and inflammation by regulating ADPN/AMPK-mediated signaling pathways and relevant metabolic disturbances including biosynthesis of unsaturated fatty acids, arachidonic acid metabolism and glycerophospholipids metabolism.

MS-based metabolite analysis of two licorice chalcones in mice plasma, bile, feces, and urine after oral administration.

Isoliquiritigenin (ILG) and isoliquiritin (ILQ), two kinds of major flavonoids in licorice, are biological active substances with antioxidant, anti-inflammatory, and tumor-suppressive effects. However, their in vivo metabolites, possible material basis of this two licorice chalcones for the treatment of diseases, have not been studied completely. To determine the metabolism of ILG and ILQ, after oral administration of 100 mg/kg/day of these compounds for consecutive 8 days, the metabolites of these two licorice chalcones in mice plasma, urine, feces, and bile were determined using liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry in this study. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism law, and reference standards-matching. As a result, a total of 25 and 29 metabolites of ILG and ILQ were identified, respectively. Seven main metabolic pathways, oxidation and reduction, deglycosylation and glycosylation, dehydroxylation and hydroxylation, demethoxylation and methoxylation, acetylation, glucuronidation, and sulfation, were summarized to tentatively explain how the metabolites were biologically transformed. These results provide the important information on the metabolism of ILG and ILQ, which may be helpful for the further research of their pharmacological mechanism.

Metabolic profiling of mice plasma, bile, urine and feces after oral administration of two licorice flavonones.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is an ancient food and medicinal plant. Liquiritigenin and liquiritin, two kinds of major flavonoes in licorice, are effective substances used as antioxidant, anti-inflammatory and tumor-suppressive food, cosmetics or medicines. However, their in vivo metabolites have not been fully explored. AIM OF STUDY: To clarify the metabolism of liquiritigenin and liquiritin in mice. MATERIALS AND METHODS: In this study, we developed a liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry approach to determine the metabolites in mice plasma, bile, urine and feces after oral administration of liquiritigenin or liquiritin. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism laws or reference standard matching. RESULTS: A total of 26 and 24 metabolites of liquiritigenin or liquiritin were respectively identified. The products related with apigenin, luteolin or quercetin were the major metabolites of liquiritigenin or liquiritin in mice. Seven main metabolic pathways including (de)hydrogenation, (de)hydroxylation, (de)glycosylation, (de)methoxylation, acetylation, glucuronidation and sulfation were summarized to tentatively explain their biotransformation. CONCLUSION: This study not only can provide the evidence for in vivo metabolites and pharmacokinetic mechanism of liquiritigenin and liquiritin, but also may lay the foundation for further development and utilization of liquiritigenin, liquiritin and then licorice.

Metabolomics Study of Metabolic Changes in Renal Cells in Response to High-Glucose Exposure Based on Liquid or Gas Chromatography Coupled With Mass Spectrometry.

Diabetic nephropathy (DN) is one of the most serious microvascular complications and the leading causes of death in diabetes mellitus (DM). To find biomarkers for prognosing the occurrence and development of DN has significant clinical value for its prevention, diagnosis, and treatment. In this study, a non-targeted cell metabolomics-based ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry and gas chromatography coupled with mass spectrometry was developed and performed the dynamic metabolic profiles of rat renal cells including renal tubular epithelial cells (NRK-52E) and glomerular mesangial cells (HBZY-1) in response to high glucose at time points of 12 h, 24 h, 36 h, and 48 h. Some potential biomarkers were then verified using clinical plasma samples collected from 55 healthy volunteers, 103 DM patients, and 57 DN patients. Statistical methods, such as principal component analysis and partial least squares to latent structure-discriminant analysis were recruited for data analyses. As a result, palmitic acid and linoleic acid (all-cis-9,12) were the potential indicators for the occurrence and development of DN, and valine, leucine, and isoleucine could be used as the prospective biomarkers for DM. In addition, rise and fall of leucine and isoleucine levels in plasma could be used for prognosing DN in DM patients. Through this study, we established a novel non-targeted cell dynamic metabolomics platform and identified potential biomarkers that may be applied for the diagnosis and prognosis of DM and DN.

[Study on blood enrichment mechanism of steamed notoginseng based on metabolomics method].

In this paper,ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOFMS) technique was used to study the effects of steamed notoginseng on endogenous markers in plasma of rats with hemolytic anemia induced by N-acetyl phenyl hydrazine( APH). The aim was to find out the potential biomarkers and possible blood enriching mechanism of steamed notoginseng on hemolytic anemia rats. In the experiment,steamed notoginseng medicine pair( steamed notoginseng-ginseng)and compound medicines( Sanqi Yangxue Capsules) were used respectively to intervene in APH-induced hemolytic anemia model rats.Then blood routine indexes such as red blood cells( RBC),hemoglobin( Hb) and related organ indexes were determined. As compared with the blank group,the RBC and Hb levels in the model group were substantially decreased( P< 0. 01),while the liver and spleen organ indexes were increased( P< 0. 05). The results of blood routine and organ index demonstrated that the blood deficiency model was successfully established. Steamed notoginseng can significantly increase the RBC level of rats( P<0. 01),and the related indicators of each drug group had a trend of returning to normal levels,verifying the blood enriching effect of steamed notoginseng. The UPLC-Q-TOF-MS technique,principal component analysis( PCA) and partial least squares-discrimination analysis( PLS-DA) were used to analyze the metabolic profiles between the normal group and the model group. Twenty-six potential biomarkers for hemolytic anemia were screened in plasma. Nine metabolites such as retinol,L-valine,and arachidonic acid were down-regulated in the blood deficiency rats,and 17 metabolites such as protoporphyrin Ⅸ and niacinamide were up-regulated. The metabolic level of biomarkers could be changed to a normal state after rats were given with steamed notoginseng,drug pairs,and compound prescriptions. It can be speculated that steamed notoginseng may play a role of blood tonifying by improving biosynthesis of valine,leucine and isoleucine,as well as metabolic pathways such as retinol metabolism and arachidonic acid metabolism.

Segmented scan modes and polarity-based LC-MS for pharmacokinetic interaction study between Fufang Danshen Dripping Pill and Clopidogrel Bisulfate Tablet.

Fufang Danshen Dripping Pill (FDDP) and Clopidogrel Bisulfate Tablet (CBT) are usually combined for treatment of coronary artery diseases in clinical. To investigate the pharmacokinetic interaction between FDDP and CBT after oral administration of FDDP, CBT and their combination in rats, a novel LC-MS method with segmented scan modes (multiple reaction monitoring and selected ion monitoring) and polarity (positive and negative ionization) was developed. Clopidogrel and the main active ingredients of FDDP, with different chemical and ionization properties, were simultaneously quantified in plasma in a single run. The method was validated in terms of specificity, linearity, precision, accuracy, recovery, matrix effect and stability. As a result, co-administration of FDDP and CBT significantly altered the pharmacokinetic parameters of danshensu, ginsenoside Rb1, dihydrotanshinone I, tanshinone I and tanshinone IIA of FDDP, as well as clopidogrel. Mechanism studies suggested that induction of liver cytochrome P450 isozymes CYP2C11 and CYP3A1 by co-administration, as well as inhibition of carboxyl esterase 1, was partly responsible for FDDP-CBT pharmacokinetic interactions. The developed LC-MS method could be used to simultaneously quantify different types of in vivo analytes in a single run, and the results could be used for clinical medication guidance of FDDP and CBT.