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The metabolites of salvianolic acid A and salvianolic acid B in rats were analyzed and compared by ultra-high-perfor-mance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS). After the rats were administrated by gavage, plasma at different time points and urine within 24 hours were collected to be treated by solid phase extraction(SPE), then they were gradient eluted by Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) and 0.1% formic acid solution(A)-acetonitrile(B) mobile phase system, and finally all biological samples of rats were analyzed under negative ion scanning mode. By obtaining the accurate relative molecular mass and multi-level mass spectrometry information of metabolites, combined with the characteristic cleavage law of the reference standard and literature reports, a total of 30 metabolites, including salvianolic acid A and B, were identified. Among them, there were 24 metabolites derived from salvianolic acid A, with the main metabolic pathways including ester bond cleavage, dehydroxylation, decarboxylation, hydrogenation, methylation, hydroxylation, sulfonation, glucuronidation, and their multiple reactions. There were 15 metabolites of salvianolic acid B, and the main biotransformation pathways were five-membered ring cracking, ester bond cleavage, decarboxylation, dehydroxylation, hydrogenation, methylation, sulfonation, glucuronidation, and their compound reactions. In this study, the cross-metabolic profile of salvianolic acid A and B was elucidated completely, which would provide reference for further studies on the basis of pharmacodynamic substances and the exploration of pharmacological mechanism.
Subject(s)
Animals , Rats , Benzofurans , Caffeic Acids , Chromatography, High Pressure Liquid , Lactates , Mass Spectrometry , TechnologyABSTRACT
A method of ultra-high performance liquid chromatography coupled with quadrupole/electrostatic field Obitrap high-resolution mass spectrometry(UHPLC-Q-Exactive MS) was established to comprehensively identify the metabolites of carnosic acid in rats. After oral gavage of carnosic acid CMC-Na suspension in rats, urine, plasma and feces samples were collected and pretreated by solid phase extraction(SPE). Acquity UPLC BEH C_(18 )column(2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid solution(A)-acetonitrile(B) as the mobile phase for the gradient elution. Biological samples were analyzed by quadrupole/electrostatic field Obitrap high-resolution mass spectrometry in positive and negative ion mode. Based on the accurate molecular mass, fragment ion information, and related literature reports, a total of 28 compounds(including carnosic acid) were finally identified in rat samples. As a result, the main metabolic pathways of carnosic acid in rats are oxidation, hydroxylation, methylation, glucuronide conjugation, sulfate conjugation, S-cysteine conjugation, glutathione conjugation, demethylation, decarbonylation and their composite reactions. The study showed that the metabolism of carnosic acid in rats could be efficiently and comprehensively clarified by using UHPLC-Q-Exactive MS, providing a reference for clarifying the material basis and metabolic mechanism of carnosic acid.
Subject(s)
Animals , Rats , Abietanes , Chromatography, High Pressure Liquid , Mass Spectrometry , Solid Phase ExtractionABSTRACT
Aim To study the material basis of hepatotoxicity induced by the ripe fruit of Terminalia chebula Retz. var. tomentella Kurt using high content screening. Methods Shikimic acid, benzoic acid, gallic acid, and 1,2,3,4,6-o-pentagalactosyl glucose were applied to HepG2 cells, respectively, and the cells were stained with fluorescent stains such as Hoechst 33342. The imagewas scannedand the collected datawere input into the Assay Template. Finally, the dose-response curves of cell numbers, DNA content, GSH reduction level, ROS content, MMP and other indicators were obtained for different monomers at different concentrations, thereby the hepatotoxicity of the monomers was determined. Results Aspirin and shikimic acid showed negative results. Ticlopidine, benzoic acid, 1,2,3,4,6-o-pentagalloglucose, gallic acid caused a significant decrease in cell number and increase in ROS content. There was a risk of liver-toxicity. Conclusions Gallic acid, benzoic acid, 1,2,3,4,6-o-pentagalactosylglucose have the risk of hepatotoxicity, and the risk of hepatotoxicity caused by gallic acid is the largest. Basically, gallic acid is safer when administered at concentrations below 50 mg·L-1
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AIM:To explore the effects of neuraminidase 3 (NEU3) on the viability, invasion and apoptosis of human prostate cancer DU145 cells and the molecular mechanism.METHODS:The human prostate cancer DU145 cells were divided into blank control group and treatment group.The cells in treatment group were treated with either neuraminidase inhibitor DANA, or NEU3 small interfering RNA (siRNA) to knock down the expression of NEU3.The cell viability was measured by CCK-8 assay.The cell invasion ability was detected by Transwell assay.The effects of the treatments on the mRNA level of Bcl-2 were detected by q PCR.The effects of the treatments on the protein levels of matrix metalloproteinase 2 (MMP2) and apoptotic inhibitory protein Bcl-2 were determined by Western blot.Apoptosis of the cells was analyzed by flow cytometry.RESULTS:The protein level of NEU3 and the apoptotic rate in DANA group were not significantly different from those in blank control group.The viability of DANA-treated DU145 cells was increased, and the invasion ability, MMP2 protein level, and Bcl-2 mRNA and protein levels were all decreased in these cells, compared with blank control group.On the other hand, the levels of NEU3 protein and Bcl-2 mRNA and protein in NEU3 siRNA group were significantly decreased compared with blank control group, while the viability and apoptotic rate of the cells with NEU3 siRNA transfection were increased (P<0.05).However, the protein expression of MMP2 and the invasion ability of the cells were not significantly changed after NEU3 siRNA treatment.CONCLUSION:The inhibition of NEU3 in enzyme activity and expression decreases the viability, and enhances the apoptosis of human prostate cancer DU145 cells.However, it has no obvious effect on the invasion ability of DU145 cells.
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Background Epidemiologic studies found that the incidence of myopia is higher in Hong Kong and Taiwan regions of China than that of the mainland.So whether the general reading words with traditional Chinese characters and simplified characters is associated with myopia deserves attention.Objective This study was to test the accommodative responses and the regression levels of nearwork-induced transient myopia (NITM) induced by traditional complex Chinese characters and modern simplified ones in the same size,and to explore the differences and inherent relationship of the accommodative regulations based on the structures of the two types of Chinese character in causing reading triggered myopia.Methods Twenty two volunteers aged 24-29 years were included in this study with informed consent.The corrected vision of both eyes from each subject was ≥ 1.0 with a mean spherical equivalence of (-1.86±2.34)D.Accommodative response was tested with 4 different reading texts using the rapid sequence visual presenting model with the GRAND SEIKO-WV5500 infrared autorefractor,and this procedure was performed after full correction of refractive error.An initial test of looking at a certain distance was performed (as baseline),and then the subjects read intensively at the targets for 10 minutes at 33 cm to calculate the accommodative responses.After a 10 second pause,the ocular refractive status was obtained exactly at 15 seconds,20 seconds.The one-way ANOVA method was used to determine the effects of the different font types and sizes on the adjustive responses and the causation of NITM.Results Accommodative response induced by simplified and traditional Chinese characters showed an accommodative lag of (1.11 ±0.38),(0.95 ±0.43),(1.18 ±0.33) and (1.06±0.28) D,showing a significant difference among the 9 pt and 12 pt simplified and traditional Chinese characters (F =1.62,P =0.19),and significantly different accommodative lag values between 12 pt simplified characters and 9 pt traditional characters was found (t =5.56,P =0.02).NITM induced by the four different targets were (-0.45 ±0.45),(-0.47 ±0.46),(0.45 ±0.82) and (-0.46±0.78) D in the 4 types of characters,without a significant difference among them (F=0.01,P =0.99).Conclusions Near-distance reading causes accommodative lag regardless of the type of reading texts.The target demonstrated stimuli spatial frequency and font size play an impact on accommodative responses.NITM appears when one reads simplified or traditional Chinese for 10 minutes.The accommodative lag and NITM trend might be responsible for the onset or regression of myopia,yet it is not supportive for the hypothesis that reading traditional Chinese causes more strain since there is no difference between the two.