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ObjectiveTo evaluate the success of Qi-deficiency model of Balb/C-nu mice established by swimming exhaustion test from the view of biomarkers and metabolic pathways by metabonomics. MethodBalb/C-nu mice were randomly divided into the normal group and Qi-deficiency group， Qi-deficiency model was established by swimming with 5% body weight metal fixed at the tail for 15 consecutive days until exhaustion （nose tip immersion time>5 s）. The urine metabonomics was analyzed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF/MS）， and the mobile phase was acetonitrile （A）-0.1% formic acid aqueous solution （B） for gradient elution （0-1 min， 5%-8%A； 1-4 min， 8%-8.5%A； 4-5 min， 8.5%-12%A； 5-10 min， 12%-40%A； 10-12 min， 40%-100%A； 12-15 min， 100%A）， the flow rate was 0.3 mL·min-1， the injection volume was 10 μL， electrospray ionization （ESI） in positive and negative ion modes was used in MS analysis， the MS data were acquired in full-scan mode from m/z 50-1 000. Principal component analysis （PCA）， orthogonal partial least squares-discriminant analysis （OPLS-DA）， human metabolome database （HMDB）， high collision energy ion fragments collected by MSE and tandem MS （MS/MS） ion information were used to identify potential biomarkers. Kyoto Encyclopedia of Genes and Genomes （KEGG） database and MetaboAnalyst 5.0 were used to analyze the corresponding metabolic pathways and pathway enrichment of biomarkers. ResultEndogenous substances in urine of mice in normal group and Qi-deficiency group were obviously separated， and there were 24 biomarkers with significant difference. The metabolic pathways involved in these biomarkers were tricarboxylic acid cycle， glycolysis metabolism， amino acid metabolism， fatty acid metabolism， pyrimidine metabolism， steroid hormone biosynthesis and tryptophan metabolism. Among them， the metabolic pathways related to energy were tricarboxylic acid cycle， glycolysis metabolism， amino acid metabolism， fatty acid metabolism， pyrimidine metabolism and steroid hormone biosynthesis. ConclusionThrough the investigation of urine metabonomics， combined with the physical signs， the Qi-deficiency model established by swimming exhaustion test in Balb/C-nu mice is successfully evaluated， and it is also verified that there is a close correlation between Qi-deficiency and energy metabolism.
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ObjectiveTo evaluate the success of Qi-deficiency model of Balb/C-nu mice established by swimming exhaustion test from the view of biomarkers and metabolic pathways by metabonomics. MethodBalb/C-nu mice were randomly divided into the normal group and Qi-deficiency group， Qi-deficiency model was established by swimming with 5% body weight metal fixed at the tail for 15 consecutive days until exhaustion （nose tip immersion time>5 s）. The urine metabonomics was analyzed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF/MS）， and the mobile phase was acetonitrile （A）-0.1% formic acid aqueous solution （B） for gradient elution （0-1 min， 5%-8%A； 1-4 min， 8%-8.5%A； 4-5 min， 8.5%-12%A； 5-10 min， 12%-40%A； 10-12 min， 40%-100%A； 12-15 min， 100%A）， the flow rate was 0.3 mL·min-1， the injection volume was 10 μL， electrospray ionization （ESI） in positive and negative ion modes was used in MS analysis， the MS data were acquired in full-scan mode from m/z 50-1 000. Principal component analysis （PCA）， orthogonal partial least squares-discriminant analysis （OPLS-DA）， human metabolome database （HMDB）， high collision energy ion fragments collected by MSE and tandem MS （MS/MS） ion information were used to identify potential biomarkers. Kyoto Encyclopedia of Genes and Genomes （KEGG） database and MetaboAnalyst 5.0 were used to analyze the corresponding metabolic pathways and pathway enrichment of biomarkers. ResultEndogenous substances in urine of mice in normal group and Qi-deficiency group were obviously separated， and there were 24 biomarkers with significant difference. The metabolic pathways involved in these biomarkers were tricarboxylic acid cycle， glycolysis metabolism， amino acid metabolism， fatty acid metabolism， pyrimidine metabolism， steroid hormone biosynthesis and tryptophan metabolism. Among them， the metabolic pathways related to energy were tricarboxylic acid cycle， glycolysis metabolism， amino acid metabolism， fatty acid metabolism， pyrimidine metabolism and steroid hormone biosynthesis. ConclusionThrough the investigation of urine metabonomics， combined with the physical signs， the Qi-deficiency model established by swimming exhaustion test in Balb/C-nu mice is successfully evaluated， and it is also verified that there is a close correlation between Qi-deficiency and energy metabolism.
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Enrichment of trace bioactive constituents and metabolites from complex biological samples is chal-lenging. This study presented a one-pot synthesis of magnetic polydopamine nanoparticles (Fe3O4@-SiO2@PDA NPs) with multiple recognition sites for the magnetic dispersive solid-phase extraction (MDSPE) of ginsenosides from rat plasma treated with white ginseng. The extracted ginsenosides were characterized by combining an ultra-high-performance liquid chromatography coupled to a high-resolution mass spectrometry with supplemental UNIFI libraries. Response surface methodology was statistically used to optimize the extraction procedure of the ginsenosides. The reusability of Fe3O4@-SiO2@PDA NPs was also examined and the results showed that the recovery rate exceeded 80％ after recycling 6 times. Furthermore, the proposed method showed greater enrichment efficiency and could rapidly determine and characterize 23 ginsenoside prototypes and metabolites from plasma. In com-parison, conventional methanol method can only detect 8 ginsenosides from the same plasma samples. The proposed approach can provide methodological reference for the trace determination and charac-terization of different bioactive ingredients and metabolites of traditional Chinese medicines and food.
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An untargeted urinary metabonomics method based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry ( UPLC-Q-TOF-MS ) has been established to investigate the mechanism of Schisandra chinensis in treating diabetes and its complications. The urinary biomarkers related to the therapeutic effects of Schisandra chinensis on the diabetes rats were analyzed. In urine, 28 kinds of endogenous metabolites were identified as potential biomarkers, including 13 endogenous metabolites in positive ion mode, 15 endogenous metabolites in negative ion mode, and hippuric acid detected both in positive and negative ion modes. The results revealed that Schisandra chinensis mainly affected the pathways of pentose and glucuronate interconversions, riboflavin metabolism, pantothenate and CoA biosynthesis, arginine and proline metabolism, intestinal bacteria metabolism, ascorbate and aldarate metabolism and tryptophan metabolism in diabetic rats. Combined with biological analysis of these pathways, the therapeutic mechanism of Schisandra chinensis on diabetes and its complications was verified. Based on the biological function of each pathway, the effect of Schisandra chinensis on diabetic nephropathy is stronger. Moreover, it also has the effects of protecting liver, decreasing fat and antioxidant activity.
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Phenylketonuria (PKU) is a newborn inherited metabolic disorder caused by the genetic deficiency of hepatic enzyme phenylalanine hydroxylase (PAH) which thus in metabolic disorder of phenylalanine. In this study, ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was used to analyze the accurate amount of coenzyme Q10 (CoQ10) and the relative amount of CoQ9 in newborn dried blood spot (DBS) collected from 5 PKU newborns (3 and 30 days after birth, respectively) and 20 healthy newborns. The content of CoQ10 was (122.1±24.9 ng/mL) and (59.0±12.0 ng/mL) in DBS from healthy newborns and PKU newborns, respectively. The relative contents of cholesterol and glucose in the DBS were determined by gas chromatography-mass spectrometry (GC-MS). In comparison with healthy newborn group, the levels of CoQ10, CoQ9, cholesterol and glucose were all significantly decreased in PKU newborns. The increased content of Phe and the decreased content of CoQ10 showed significant inverse correlation in the DBS from PKU. This study provides references for diet therapy of PKU newborns.
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The brain pharmacokinetics of hyperoside and 1 , 5-dicaffeoylquinic acid and the effects of acanthopanax senticosus leaves on neurotransmitters in cerebral ischemic rat brain were investigated. In the study, acute incompleteness cerebral ischemia model was developed by ligating the bilateral common carotid arteries of rats. The differences of brain pharmacokinetics of hyperoside and 1,5-dicaffeoylquinic acid between control and cerebral ischemic rats were determined by online microdialysis coupled with MS/MS method. At the same time, the contents of glutamic acid (Glu), aspartic acid (Asp), γ-aminobutyric acid (GABA), serotonin (5-HT) , dopamine ( DA) , and acetylcholine( Ach) in rat hippocampus among different groups were determined simultaneously in MRM mode. ACE 5 C18-AR column was used for separation. Results showed that the online analytical method had excellent linearity (R2>0. 99). The accuracy and precision could meet the analysis requirement. Besides, the hyperoside and 1,5-dicaffeoylquinic acid penetrated the blood-brain barrier successfully and metabolized quickly, but the absorption reduced and elimination became faster in cerebral ischemic rats; the contents of Glu, Asp, GABA and DA in brain issue of cerebral ischemic rats decreased significantly ( p<0 . 01 ) and the level of Ach increased remarkably ( p<0 . 05 ) after a preventive medication with the extract of acanthopanax senticosus leaves for one week.
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A serumal metabonomics method based on UPLC/Q-TOF-MS was established to investigate the mechanism of Schisandra chinensis to treating diabetic nephropathy. The diabetic model was established by feeding with high-fat and high-sucrose chow and streptozotocin intraperitoneal injection. After intragastric administration for 12 weeks, the content of protein and creatinine in rat urine was detected. The results showed that Schisandra chinensis could reduce the content of protein in urine of diabetic rat ( p<0 . 05 ) and ameliorate the condition of diabetic nephropathy. The serum metabolic profiling was analysed by using UPLC/Q-TOF-MS and partial least squares-discriminated analysis ( PLS-DA) was used for data analysis. The score of PLS-DA showed that there was significant difference among the metabolic profile of control group, model group and Schisandra chinensis group ( WWZ ) . Potential biomarkers of Schisandra chinensis ameliorating diabetic nephropathy were selected by orthogonal partial least squares ( OPLS)-DA model. According to the results of OPLS-DA, the MS/MS data of each compound which provided greater contribution to separation of each group were searched from the HMDB databases. Seven endogenous metabolites were identified as potential biomarkers such as xanthurenic acid, palmitic amide, oleamide, uric acid, 5-hydroxyhexanoic acid, p-cresol sulfate, and p-cresol glucuronide. The results revealed that Schisandra chinensis mainly affected the pathways of tryptophan metabolism, gut microbiota metabolism, purine metabolism and fatty acid metabolism to treating type 2 diabetes mellitus. The gut microbiota metabolism and purine metabolism was an important pathway on diabetic nephropathy.
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Using a UPLC-MS/MS (MRM) and cocktail probe substrates method, the metabolic fingerprint of the compatibility of Radix Aconite (RA) and Radix Paeoniae Alba (RPA) and its effect on CYP450 enzymes were investigated. These main CYP isoforms include CYP 1A2, CYP 2C, CYP 2E1, CYP 2D and CYP 3A. Compared with the inhibition effect of RA decoctions on CYP450 isoforms, their co-decoctions of RA and RPA with different proportions can decrease RA' inhibition on CYP3A, CYP2D, CYP2C and CYP1A2, but can not reduce RA' effect on CYP2E1. The metabolic fingerprints of RA decoction and co-decoctions with different proportions of RPA in CYP450 of rat liver were analyzed by UPLC-MS. Compared with the metabolic fingerprints of RA decoction, the intensity of diester-diterpenoid aconitum alkaloids decreased significantly, while the intensity of monoester-diterpenoid alkaloids significantly increased in the metabolic fingerprints of co-decoctions of RA and RPA. The results suggest that RA coadministration with RPA increased the degradation of toxic alkaloid and show the effect of toxicity reducing and efficacy enhancing.
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To evaluate the effects of ginseng aqueous extract on learning and memory abilities and neurochemicals in diabetic encephalopathy rat brain. Diabetes mellitus model was established. Cognitive abilities were evaluated with Morris water maze test by the indexes of escape latency time ( ELT) , times of passing through the target area and the percent of swimming track in goal quadrant. We applied online MD-LC-MS/MS method to determine eight neurochemicals in rat hippocampus among different groups in MRM mode. Venusil C18 column was used for separation. Treatment with ginseng aqueous extract significantly improved the cognitive abilities of diabetic rats (p0. 99). The accuracy and precision could meet the analysis requirement. Comparing with the model group, the concentrations of taurine and acetylcholine were significantly increased ( p<0 . 01 ) after treated with ginseng aqueous extract, but the glutamic acid, aspartic acid,γ-aminobutyric acid, serine, dopamine and serotonine decreased dramatically. Ginseng aqueous extract can efficiently regulate the levels of eight neurochemials in diabetic rat brain to normal.
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High-speed counter-current chromatography (HSCCC) was used to high performance separate and prepare lignans from Schisandrae chinensis fructus. The solvent system is composed of n-hexane-ethyl acetate-methanol-water (9 : 1 : 5 : 5) and n-hexane-ethyl acetate-methanol-water (9 : 1 : 9 : 5), speed is at 900 r.min-1, and flow rate is at 2.0 mL.min-1. Five fractions from Schisandrae chinensis fructus extract were separated and prepared with one HSCCC process. They were identified as schisandrin, gomisin J, schisandrol B, schisantherin A and deoxyschizandrin by electrospray ionization-multiple tandem mass spectrometry (ESI-MSn), respectively. Their contents were obtained in 98.74%, 94.32%, 99.53%, 94.23% and 98.68% by ultra high performance liquid chromatography (UPLC), separately. The rapid and simple method can be applied for the preparation of lignans from Schisandrae chinensis fructus.
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Theultraperformanceliquidchromatographycoupledwithmassspectrometry(UPLC-MS)was used to investigate the metabolic difference of the decoction of Radix Aconiti Preparata ( RAP ) and its co-decoctions with Radix Paeoniae Alba ( RAP-RPA ) or Radix Stephaniae Tetrandrae ( RAP-RST ) in rat intestinal bacteria. The principal component analysis ( PCA) of the relative contents of Aconitum alkaloids after metabolism was performed by SIMCA-P software. The score plots of PCA could successfully distinguish the three groups of RAP, RAP-RPA and RAP-RST. The result indicated that the differences of biotransformation among the groups of PAP, RAP-RPA and RAP-RST were significant. With the loading plot and independent-samples T test, seven relevant markers with the significant differences were found in the group of RAP-RPA, six relevant markers were obtained in the group of RAP-RST. The relative content of four markers in RAP-RPA was higher than that in RAP, and one marker in RAP-RST was higher than that in RAP. The relative contents of other markers were all lower than that in RAP. These markers may be the effective substance for explaining the different effects of Radix Aconiti Preparata before and after combination with Radix Paeoniae Alba and Radix Stephaniae Tetrandrae.
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Effects of six kinds of Chinese herb extracts, including Folium Crataegi extract, Herba Epimedii extract, Folium Acanthopanacis Senticosi extract, Trifolium pratense L. extract, Folium Ginkgo extract and Radix Puerariae extract, on the activities of CYP450 isozymes (CYP1A2, CYP2C, CYP2E1, CYP2D, CYP3A) in rat hepatic microsomals were studied by using a UPLC-MS/MS (MRM) and cocktail probe substrates method. The results showed that effects of six kinds of Chinese herb extracts on each CYP450 isozyme activity were inhibitory. The IC50 of Folium Crataegi extract for the inhibition of rat microsomal CYP2D activity was only for 4.04 microg x mL(-1), which showed the highest inhibition; Trifolium pratense L. extract had strong inhibitory action to CYP2D, the IC50 value was 5.73 microg x mL(-1); Folium Crataegi extract also had strong inhibitory action on CYP2E1, the IC50 value was 10.91 microg x mL(-1). Furthermore, the IC50 of Folium Ginkgo extract for the inhibition of rat microsomal CYP3A, 2D, 2E1 activities were 45.12, 35.45 and 22.41 microg x mL(-1), respectively, and the IC50 of Folium Acanthopanacis Senticosi extract on the inhibition of rat microsomal CYP2E1 activity was 32.89 microg x mL(-1). In addition, mechanism of inhibition experimental results showed that the inhibiting abilities of Folium Crataegi extract and Radix Puerariae extract on each CYP450 isozyme increased with the increasing of the preincubation time, therefore, the inhibitory effects were a mechanism-based inhibition.
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Mesaconitine was incubated with rat liver microsomes in vitro. The metabolites of mesaconitine in rat liver microsomes were identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with high resolution power. A typical reaction mixture of 100 mol L-1 Tris-HCI buffer (pH 7.4) containing 0.5 gL-1 microsomal protein and 50 micro molL-1 mesaconitine was prepared. The above reaction mixture was divided into six groups, and the volume of each group was 200 micro L. The incubation mixture was pre-incubated at 37 degrees C for 2 min and the reactions were initiated by adding NADPH generating system. After 90 min incubation at 37 degrees C, 200 micro L of acetonitrile was added to each group to stop the reaction. The metabolites of mesaconitine were investigated by UPLC-MS/MS method. Mesaconitine and 6 metabolites M1-M6 were found in the incubation system. The structures were characterized according to the data from MS/MS spectra and literatures. The metabolic reactions of mesaconitine in rat liver microsomes included the demethylation, deacetylation, dehydrogenation and hydroxylation. The major metabolic pathways of mesaconitine in rat liver microsomes were determined by UPLC-MS/MS on multiple reaction monitoring (MRM) mode combined with specific inhibitors of cytochrome P450 (CYP) isoforms, including alpha-naphthoflavone (CYP1A2), quinine (CYP2D), diethyldithiocarbamate (CYP2E1), ketoconazole (CYP3A) and sulfaphenazole (CYP2C), separately. Mesaconitine was mainly metabolized by CYP3A. CYP2C and CYP2D were also more important CYP isoforms for the metabolism reactions of mesaconitine, but CYP1A2 and CYP2E1 haven't any contribution to MA metabolism in rat liver microsomes.
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<p><b>OBJECTIVE</b>To establish a quick method for evaluation of the antioxidant activities based on the correlation analysis of lignans content and antioxidant activities of Schisandra chinensis fruits.</p><p><b>METHOD</b>The content of five lignans components in 37 batches of S. chinensis fruits from different regions of Jilin province were measured by HPLC. Simultaneously, the antioxidant activities of the above samples were detected, such as lipid peroxidation inhibition activity in liver (LPIL), kidney (LPIK) and brain (LPIB) and the clearance rate of DPPH (CRD). Bivariate correlation analysis and stepwise regression analysis were carried out by the software of SPSS for windows 11.5.</p><p><b>RESULT</b>The results of bivariate correlation analysis showed that deoxyschizandrin was negative correlation (P<0.01) to the activity of LPIL, LPIB, CRD. Schisandrin was positive correlation (P<0.01) to the activity of LPIL, LPIB, CRD. Schisandrol B was also positive correlation (P<0.05 or P<0.01) to the above four kinds of antioxidant activity. The results of stepwise regression analysis were mostly consistent with the bivariate correlation analysis results. For the other 10 batches of samples, the simulated antioxidant activities according to the regression equation calculated was consistent with the measured activities.</p><p><b>CONCLUSION</b>By using the bivariate correlation analysis and linear stepwise regression analysis, the bioactive components related to the antioxidant activity of S. chinensis fruits were found. Meanwhile, the antioxidant activity of samples will be inferred according to the content of Schisandra lignans.</p>
Subject(s)
Antioxidants , Chemistry , Fruit , Chemistry , Lignans , Chemistry , Schisandra , ChemistryABSTRACT
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.
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Three compounds of metalloporphyrins were studied using electrospray ionization mass spectrometry.The bonding power between substitutional phenyl and porphyrin cycle and the coordinate conditions of metalloporphyrins with imidazole were disscused. The experimental result indicated that the bonding power between substitutional phenyl and porphyrin cycle in metalloporphyrins became weak from Mn,Fe to Co.The complexes abundances formed by metallophorphyrin with imidazole were stronger with the increase of the ligand concentration.At the same ligand concentration,the abundance of the complexes was intensified gradually and the stability of the ligands was become stronger from Mn,Fe to Co.
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Objective To study how and why the lipo-alkaloids changed in decocting the flowers of Aconitum kusnezoffii (FAK). [WT5HZ]Methods The alkaloids in ethanol extract of FAK, FAK decoction, and the residues of decocted FAK were analyzed and compared by electrospray ionization tandem mass spectrometry (ESI-MSn). Results No lipo-alkaloids were detected in FAK decoction, however, triester-lipo-alkaloids become the dominant components in the residues of FAK decoction. Conclusion Besides hydrolysis reactions, ester-exchange reactions happen for diester-aconitines and triester-aconitines in the decocting of FAK. It is the first time to report that C_8-acetyl is displaced by long chain fatty acyl for triester-aconitines in the ester-exchange reactions.
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Objective To study the solubility of lipo-alkaloids from prepared aconite root(Radix Aconiti Lateralis Preparata) and identify the sources of lipo-alkaloids in Sini Decoction.Methods The electrospray ionization mass spectrometry was employed to analyze the alkaloids in crude aconite roots,Sini Decoction,the gruffs of concocted aconite roots and the decoction of prepared aconite root and pinellia tuber(Rhizoma Pinellae).Results Lipo-alkaloids were observed in Sini Decoction,however,no lipo-(alkaloids) were found in the decoction of aconite roots with pinellia tuber.Lipo-alkaloids were the dominant components in the gruffs of concocted aconite roots.Conclusion Lipo-alkaloids are barely soluble in boiling water.The alkaloids existed in the Sini Decoction stem from the ester-exchange reactions of diester-diterpenoid alkaloids and fatty acid in the boiling water.
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Object To study the chemical constituents of the extracts S 1 and S 7 in GINSENG SINI TANG, which has the effect of antihemorrhagic shock Methods The constituents of S 1 and S 7 were isolated and purified by silica gel column chromatographic methods and analyzed by ESI/MS n, MALDI TOF/MS Results The 12 compounds were identified as ginsenosides Ra 1, Ra 2, Rb 1, Rb 2, Rb 3, Rc, Rd, Re, Rg 1, Rg 2, Rg 3, Rf from constituent S 7 in GINSENG SINI TANG The six compounds of diterpenoid alkaloid were identified as 14 benzoylhypaconine 8 linoleate (HAL), 14 benzoyldeoxyaconine 8 oleate (HAO), 14 benzoylhypaconine 8 palmitate (HAP), benzoylmesaconitine (BM), benzoylaconitine (BA), benzoylhypaconitine (BH) from constituent S 1 in GINSENG SINI TANG Conclusion All these compounds were obtained from GINSENG SINI TANG and identified for the first time