Application of UPLC-QTOF-MS non-targeted metabonomics in mechanism study of property differences of ginseng and American ginseng / 中国中药杂志

This study adopted ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)-based untargeted metabolomic approaches for exploring the changes in endogenous metabolites of rat serum related to property differences between ginseng and American ginseng. Then the action mechanisms of them with warm and cool properties and the effects of processing on their property changes were investigated. Based on principal component analysis(PCA), the differences in metabolite profiles between ginseng, red ginseng, American ginseng, and red American ginseng were compared. After that, 16 potential differential endogenous biomarkers were identified by orthogonal partial least squares discriminant analysis(OPLS-DA) and online database searching. And the related metabolic pathways were systematically analyzed. By comparing content variations of these 16 potential differential endogenous biomarkers, we have found that 10 potential differential biomarkers were responsible for the warm property of ginseng and red ginseng, and 9 were related to the cool property of American ginseng and red American ginseng. As demonstrated by in-depth analysis of related metabolic pathways of differential biomarkers, ginseng and American ginseng mainly played a role in regulating the energy metabolism of amino acid, glycolysis, and fatty acids, during which they exhibited differences in property. The comparison of content variations of these differential endogenous between groups revealed that the energy metabolism of red ginseng group was stronger than that of ginseng group, consistent with the traditional processing theory that the warming and tonifying effects of ginseng could be enhanced after processing. The property of red American ginseng was similar to that of American ginseng, both cool in property, but American ginseng was cooler than red American ginseng. It can be seen that non-targeted metabolomic approaches can be utilized to study mechanisms underlying property differences of Chinese medicines and the effects of processing on their property changes.

Clinical Value of Serum Amyloid A and Misfolded Transthyretin for Relapsed/Refractory Diffuse Large B-Cell Lymphoma Patients / 中国实验血液学杂志

OBJECTIVE@#To evaluate the clinical value of serum amyloid A (SAA1/2) and misfolded transthyretin (TTR) for relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) patients.@*METHODS@#30 R/R DLBCL patients were enrolled as observation group, 20 remission/stabilization DLBCL and 10 chronic lymphadenitis patients were enrolled as control group. SELDI technique, Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electro-phoresis, the shotgun-LTQ-MS method, and bioinformatics technique were used to detected and analyzed SAA and TTR in R/R DLBCL patients. SPSS 21.0 software was used to analyze the relationship between the high expression of SAA, misfolded TTR in serum and the clinicopathological features, survival time of R/R DLBCL. patients Chi-square test was used to analyze clinical count data, Kaplan-Meier curve was used for survival analysis, and Log-Rank test was used to compare single-factor survival differences.@*RESULTS@#The high expression of SAA and TTR (SAA@*CONCLUSION@#Both SAA and misfolded TTR are poor prognosis factors of R/R DLBCL patients.

Berberine Inhibits Mechanical Stretch Stress induced MAPK Phosphorylation，Proliferation and Migration of Vascular Smooth Muscle Cells / 中山大学学报(医学科学版)

@#【Objective】To observe whether berberine can inhibit vascular smooth muscle cells（VSMC）proliferation induced by mechanical strength stress and to investigate the role of MAPK pathway in it.【Methods】The cultured VSMC were divided into 4 groups：negative control group（NC group），stretch stress group（SS group），berberine pretreated and stretch stress stimulation group（BBR+SS group），and berberine group. In NC group，phosphate buffer saline was used as a negative control；in SS group，stretch stress was given to VSMC；in BBR+SS group，VSMC were pretreated with berberine for 1 hour and then exposed to stretch stress；in BBR group，VSMC were treated only with berberine for 1 hour and cultured in serum- free DMEM afterwards. We collected VSMC in each group ，detected and analyzed their MAPK phosphorylation，proliferation and migration by using Western blotting，immunofluorescence and wound-healing assay respectively. 【Results】 Compare with NC group，stretch stress markedly induced VSMC proliferation and migration ，which could be inhibited significantly by berberine. Stretch stress obviously increased phosphorylation of MAPK （ERK，JNK，p38），which could be inhibited by berberine in a concentration dependent manner. 【Conclusion】 Berberine inhibits hypertension-induced proliferation and migration of VSMC through MAPK pathway. The results revealed the new use and mechanism of berberine，and provided important data for further study on the prevention and treatment of vascular remodeling caused by abnormal increase of mechanical stress in hypertension.

Candidate genes involved in the biosynthesis of lignan in Schisandra chinensis fruit based on transcriptome and metabolomes analysis / 中国天然药物

Schisandra chinensis Turcz. (Baill.) is a plant species with fruits that have been well known in Far Eastern medicine for a long time. It has traditionally been used as a stimulating and fortifying agent in cases of physical exhaustion and to inhibit fatigue. The major bioactive compounds found in S. chinensis are lignans with a dibenzocyclooctadiene skeleton, but little is known about their biosynthesis in plants. S. chinensis is the ideal medicinal plant for studying the biosynthesis of lignans, especially the dibenzocyclooctadiene skeleton. Genomic information for this important herbal plant is unavailable. To better understand the lignan biosynthesis pathway, we generated transcriptome sequences from the fruit during ripening and performed de novo sequence assembly, yielding 136 843 unique transcripts with N50 of 1778 bp. Putative functions could be assigned to 41 824 transcripts (51.57%) based on BLAST searches against annotation databases including GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, 22 candidate cytochrome P450 genes and 15 candidate dirigent proteins genes that were most likely involved in the lignan biosynthesis pathway were discovered based on transcriptome sequencing of S. chinensis. The genomic data obtained from S. chinensis, especially the identification of putative genes involved in the lignan biosynthesis pathway, will facilitate our understanding of lignan biosynthesis at the molecular level. The lignan metabolite profiles were analyzed by metabolomes, the accumulation patterns of 30 metabolites involved in the lignan pathway were studied. Co-expression network of lignan contents and transcriptional changes showed 355 strong correlations (correlation coefficient, R > 0.9) between 21 compounds and 153 transcripts. Furthermore, the comprehensive analysis and characterization of the genes involved in lignan pathways and the metabolite profiles of lignans are expected to provide better insight regarding the diversity of the chemical composition, synthetic characteristics, and regulatory mechanisms of this medical herb.

Chemical Constituents of Ginseng Radix et Rhizoma with Different Growth Years and Different Origins Based on LC-MS / 中国实验方剂学杂志

Objective::To study the components of Ginseng Radix et Rhizoma of different origins and growth years. Method::Rapid liquid chromatography-time of flight mass spectrometry (RRLC-Q-TOF-MS) was applied to detect the raw data of Ginseng Radix et Rhizoma.After peak extraction, alignment, and normalization, the multivariate statistical analysis was made for the resulted dataset to find out the different compounds.The compounds were identified by using accurate molecular weight and tandem mass spectra, and the standard references were used to further confirm the identification.The changing trends of these components in different ginseng samples were observed. Result::The ginseng samples of different growth years and different origins were divided into different groups in the score plot of PLS-DA, and the variables with the variable importance in projection (VIP) value of more than 1 were considered to contribute more to the separations, then the t-test was applied to determine whether potential biomarkers were statistically significant (P<0.05) between the two groups.The contents of eleven compounds, including ginsenoside Rb1, Rh4, and Rk2, were significantly different between ginseng samples aged 3 and 5 years, and the contents of these compounds increased as the rise of the ginseng growth years.Ginsenosides Rg1, Rf, Rh1, Rb1, and other six compounds were significantly different in ginseng samples from Jilin and Heilongjiang province. Conclusion::LC-MS is a rapid and accurate method for the analysis of ginseng samples, and could help to find out the different components among samples.

High fat diet-induced elevation of blood glucose impairs the microvascular function of testis in mice / 基础医学与临床

Objective To observe the effects of high fat dietinduced elevation of blood glucose on the microvascular function of testis and male reproduction in C57BL/6 mice. Methods A total of 40 male C57BL/6 mice were randomly divided into control group and high fat diet (HFD) group (n =20). The mice in HFD group were fed with high fat diet for 20 weeks. Blood glucose and body weight were measured weekly. The permeability of bloodtestis barrier was evaluated by intraperitoneal injection of Evans blue. The blood flow of testicular microcircu-lation and the frequency and amplitude of microvascular vasomotion were detected by laser Doppler blood flow ima-ging system. The morphology of testicular tissue was observed by HE staining. The expressions of platelet- endothelial cell adhesion molecule-1 (PECAM-1/CD31) in testicular microvascular endothelial cells and prolifera-ting cell nuclear antigen (PCNA) in spermatogenic cells were detected by immunohistochemistry. The apoptosis of spermatogenic cells was observed by TUNEL staining. Results The body weight and blood glucose of HFD group were significantly higher than those in control group (P＜0.01). Evans blue staining showed that the integrity of blood-testis barrier of HFD group was damaged, and increased permeability was observed in seminiferous tubules. In HFD group, the mean blood flow of testis and the frequency and amplitude of microvascular vasomotion were sig-nificantly lower than those in control group (P＜0.01). The number of spermatogenic epithelial cells and the thickness of seminiferous epithelium decreased. The expressions of CD31 in microvascular endothelial cells and PCNA in spermatogenic cells were significantly lower in HFD group than those in control group (P＜0.01). The apoptosis level of spermatogenic cells was higher than that in the control group (P <0.01). Conclusions Increased blood glucose level induced by high fat diet in mice can impair the testicular microvasculature and damage the integrity of blood-testis barrier and injure the structure of seminiferous epithelium in mice.

A Comparative Pharmacokinetic Study of Ginsenoside Re after Oral Administration in Normal and Ultraviolet B Irradiation-induced Damage Rats / 分析化学

A methodology of quantitative analysis on ginsenoside Re (G-Re) in rat plasma by ultra performance liquid chromatography-triple quadrupole mass spectrometry was developed for comparing the pharmacokinetic profiles between normal rats and Ultraviolet B (UVB) irradiation-induced damage rats after oral administration. The sample separation was carried out on an Ascentis?Express C18column (5.0 mm× 3.0 mm,2.7 μm) with 0.1% formic acid in water and acetonitrile as the mobile phase under gradient elution. MS analysis was operated in multiple-reaction monitoring (MRM) mode using electrospray ionization (ESI) with negative ion mode,and the ions for quantification were m/z 991.54/945.53/475.60. The limit of detection (LOD,S/N=3), limit of quantification (LOQ, S/N=10) were 4.0 ng/mL and 13.5 ng/mL, respectively. G-Re was in good linearity between 15 ng/mL and 20000 ng/mL(r=0.999),the intra-day and inter-day precisions, recovery, matrix effect and stability could meet the pharmacokinetic analysis requirement. The results indicated that the metabolic process of G-Re conformed to a two-compartment pharmacokinetic model after single oral administration in the normal and model groups. The t1/2αwere(0.21± 0.04) h and (0. 69 ± 0. 07) h, respectively; t1/2βwere (17. 08 ± 0. 53) and (21. 40 ± 16. 77) h, respectively;AUC(0-t)were (321.91±2.27) μg/(L·h) and (474.99±194.96) μg/(L·h), respectively;AUC(0-∞)were (332. 44 ± 1. 66) μg/(L·h) and (518. 64 ± 231. 39) μg/(L·h), respectively; the pharmacokinetic parameters were significantly different between normal and UVB irradiated rats (p<0.05), except for t1/2α. This UHPLC-QQQ-MS method showed excellent separation, accuracy, high sensitivity, specificity and good repeatability,and it was suitable for the pharmacokinetic study of G-Re in vivo.

Simultaneous Determination of Six Neurotransmitters in Rat Plasma, Adrenal Gland and Hypothalamus with Liquid Chromatography-Tandem Mass Spectrometry / 分析化学

A rapid Liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of six neurotransmitters in rat plasma,adrenal gland and hypothalamus,including γ-aminobutyric acid (GABA),serotonin(5-HT), tyrosine(L-Tyr), dopamine(DA), norepinephrine(NE) and epinephrine(E). This method was used for the neurotransmitters detection in rat adrenal, blood and hypothalamus. The samples were pre-column derivatized with dansyl chloride,and 5-HTP and CA were used as internal standards. A Thermo C18column (50 mm×3 mm,2.7 μm) was used for sample separation with 0.1% aqueous formic acid solution as phase A and methanol as phase B at a flow rate of 0.2 mL/min, and the injection volume was set at 2 μL. The detection was carried out with multi-reaction monitoring(MRM) in electron spray ionization (ESI) positive mode. The linear ranges for detection of GABA,5-HT, L-Tyr, DA, NE and E were 0.26-620.80 μmol/L,0.03-11.20 μmol/L,1.20-88.00 μmol/L,0.03-41.02 μmol/L, 0.01-47.20 μmol/L, 0. 01-90. 24 μmol/L, respectively, with recoveries varying from 91. 16% to 116.20%. This method can detect the neurotransmitters rapidly and accurately, providing a platform for the determination of neurotransmitters in rat plasma, adrenal gland and hypothalamus in pharmacological experiments.

Progress of the regulation effect of ginsenosides on HPA axis / 药学学报

Ginseng is a typical adaptogen which has resistance to various stresses. This effect is related to the hypothalamic-pituitary-adrenal (HPA) axis. As the main active ingredients, saponin has the similar structure to steroids. The regulation characteristics of ginseng saponin on the HPA axis are narrated from the aspects of total saponin and saponin monomers in this paper after the introduction of adaptation definition and HPA axis regulation mechanisms. Pharmacological effects of ginseng saponin and the regulation effect of HPA axis are summarized finally.

Research on construction of sheep lung adenomas virus pEGFP-C1/exJSRV-env and induction of malignant transformation in NIH3T3 / 病毒学报

This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.

Urinary metabonomics of stomach cancer assessed by rapid resolution liquid chromatography/time-of-fight mass spectrometry / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Stomach cancer is among the most commonly occurring malignancies worldwide. It would be beneficial to develop a urine-based assay whereby patients with undiagnosed stomach cancer could be screened and their cancer detected in the earliest stages.</p><p><b>METHODS</b>A urinary metabonomics method based on ultra-performance liquid chromatography combined with quadruple time-of-flight mass spectrometry was used to analyze urine samples from patients with stomach cancer and healthy controls.</p><p><b>RESULTS</b>Statistical analysis revealed a clear separation of patients and healthy controls using the aforementioned methodology. Some significantly changed metabolites were identified.</p><p><b>CONCLUSIONS</b>Use of the metabonomics method in patients with stomach cancer could effectively detect distinct changes in urinary metabolites and had the capacity to detect cancer; therefore, it may be a valuable tool in earlier diagnosis. Furthermore, the detection and identification of altered metabolites in the current study may help elucidate possible mechanisms involved in stomach cancer.</p>

Change of the content of chemical constituents and anti-oxidative action of the decoction of radix ginseng combined with Flos Lonicerae, radix Polygoni Multiflori and Radix Astragali / 药学学报

Ginsenosides in the decoction of Radix Ginseng, Radix Ginseng with Flos Lonicerae, Radix Polygoni Multiflori or Radix Astragali have been investigated by high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometric method (ESI-MS). Change of the content of ginsenosides was nonlinear in diverse combinative proportion of Radix Ginseng with Flos Lonicerae, while the stripping of ginsenosides was promoted by a small amount of Radix Polygoni Multiflori. In the combinative decoction of Radix Ginseng with Radix Astragali, ginsenosides contents were increased compared to single decoction of Radix Ginseng. Besides, ferric reducing antioxidant power (FRAP) method was developed for determination of the total antioxidative activity of n-butanol and water-soluble extracts from the decoction. The experimental results showed that antioxidative activity was better in the combinative decoction than that in single decoction, and the FRAP values of n-butanol extract were also greater compared with that of water extract.

Studies on change of chemical composition in coptis-scute herb couple by using HPCE and HPLC / 药学学报

Quantitative analysis on different proportions of couple of Coptis and Scute. The proportions of Coptis and Scute couple were 6:1, 6:4, 6:7, 6:10, 6:13, separately. The contents of alkaloids in Coptis and decoction of Coptis and Scute were determined by HPCE and the contents of flavones in Scute and decoction of Coptis and Scute were determined by HPLC, separately. Precipitates were generated after the couple of Coptis and Scute, which results the decrease of contents of effective ingredients.

Lignans from different processed products of Schisandra chinensis fruits / 药学学报

To study the lignan components of different processed products of Schisandra chinensis fruits, the study was conducted with electrospray ionization mass spectrometry (ESI-MS) and high performance liquid chromatography (HPLC). There was no new lignan components detected from the products obtained with different processed methods, the difference was in the content of lignan components. The method is accurate and effective, which can be used to evaluate the quality of different processed products of Schisandra chinensis fruits.

Interaction between anticancer drugs and DNA studied by using electrospray ionization mass spectrometry / 药学学报

To elucidate further sequence selectivity and nature of the binding of anticancer drugs to DNA, the interaction between anticancer drugs, which are minor groove ligands (distamycin A, DM and netropsin, NP) and intercalator (mitoxantrone, MT), and DNA were studied by electrospray ionization mass spectrometry. The 2 : 1 specific complex of DM and AT-rich DNA were observed principally, while only 1 : 1 specific complex of NP and AT-rich DNA were observed. MT specifically binds to GC-rich DNA. In addition, DM binds to DNA containing 5 A/T bases minor groove almost in a 2 : 1 mode and does not bind to DNA containing 3 A/T bases minor groove. NP binds most strongly to DNA containing 4 A/T bases minor groove. The 1 : 1 specific complex of MT and 6-mer DNA was also observed. The result of competitive binding experiment shows that DM binds more strongly to AT-rich DNA than NP does. These results provide bases for investigating the mechanism of interaction between the drugs and DNA and for improving the structure of target drug.

Analysis of aconite alkaloids in the combination of radix aconiti lateralis preparata with different herbs by ESI-MS spectrometry / 药学学报

The method was established study the influence of different herbal combination with Radix Aconiti in the traditional medical formulae on content of the aconite alkaloids, for elucidating the scientific basis of reducing the toxicity of aconite in traditional Chinese medical formulation. The samples for ESI-MS study were prepared by decocting a mixture of Radix Aconiti Lateralis Preparata ( RALP) with Radix Glycyrrhizae Preparata (RGP) , Radix Paeoniae Alba ( RPA) , Rhizoma Zingiberis (RZ) or Radix Et Rhizoma Rhei ( RERR) , separately, and extracting the residue of the above mentioned mixtures after decocting. The diester-diterpenoid alkaloids (DDAs) was lower in the herb couples of RALP-RGP, RALP-RPA, RALP-RZ and RALP-RERR, and lipo-alkaloids was increased in the herb couples of RALP-RGP, RALP-RPA and RALP-RZ. The reason of reducing toxic effect principle is that the components of RGP, RPA and RZ have ester-exchange reactions with DDAs in RALP to produce lipo-alkaloids of low toxicity in the decocting process of the herb couples. The combination of RALP-RERR can reduce the content of DDAs in decoction and residue due to the formation of water insoluble alkaloid compound.

Effect of iipopclysaccharide on ENaC expression in the lung of rats with acute lung injured / 中华急诊医学杂志

Objective To study the effect of lipopolysaccharide(LPS)on the endo-pulmonary natrium channel(ENaC)expression in the lung of rats with acute lung injured.Method Sixteen rats were randomly divided into normal control group and LPS-group.Rats of normal control group and LPS-group were killed at 6 hours after intravenous injection of normal saline(8 ml/kg)or LPS(8 mg/kg).The extent of lung injury was assessed by arterial blood gas analysis and histological examination.At the same time,?-ENaC protein and???- ENaC mRNA expression in the lung tissue were analyzed by immunohistochemistry and RT-PCR.Results PaO_2 in LPS-group was noticeably lower than in normal control group(P

Study on Aconitum diterpenoid alkaloids from flowers of Aconitum kusnezoffi and its decoction by ESI-MS / 药学学报

<p><b>AIM</b>To study the change of chemical compound after decocting the flowers of Aconitum kusnzoffii Reichb (FAK) by analyzing the alkaloids in FAK and the decoction of FAK qualitatively.</p><p><b>METHODS</b>The alkaloid extracts of FAK and alkaloid mixtures in the decoction of FAK were directly analyzed by electrospray ionization trap tandem mass spectrometry via by pump.</p><p><b>RESULTS</b>Three novel aconitum alkaloids were found in FAK. In addition, it is found that both diester diterpenoid alkaloids and triester diterpenoid alkaloids were hydrolyzed in the decoction of FAK. The hydrolysis products of the diester-alkaloids are benzoylaconines and aconines; the hydrolysis products of the triester-alkaloids are 3-acetyl-aconines.</p><p><b>CONCLUSION</b>ESI-MS" method is suitable for the analysis of Aconitum alkaloids in the decoction of FAK as well as other Aconitum plants, which offers the advantages of convenience, speed, sensitivity and specificity.</p>

Study on the combination principles about aconite roots by electrospray ionization mass spectrometry / 药学学报

<p><b>AIM</b>To explore the combination principle about aconite roots from the chemical point of view.</p><p><b>METHODS</b>Analyzing and comparing the nordeterpenoid alkaloids in the crude aconite roots, the decoction of crude aconite roots and aconite roots with some other Chinese traditional herbs by electrospray ionization trap tandem mass spectrometry. Detecting the pH value of the decoction of crude aconite roots and of some other Chinese traditional herbs; studying the influence of acidity to the hydrolysis of standard sample of mesaconitine.</p><p><b>RESULTS</b>Most of diester-diterpenoid alkaloids in the decoction of aconite roots and the decoction of aconite roots with liquorice roots are hydrolyzed, and the hydrolysis of alkaloids are inhibited in the decoction of aconite roots with Pinellia tuber or Schisandra. The acidity of the decoction of aconite roots or liquorice is lower than the decoction of Pinellia tuber or Schisandra; standard sample of mesaconitine can not be hydrolyzed in acid water.</p><p><b>CONCLUSION</b>Acidity is a key factor to influence the hydrolysis of diester-diterpenoid alkaloids, and the hydrolysis of alkaloids are inhibited in high acidity, which lead to the high toxicity of the decoction containing aconite roots.</p>