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Comprehensive characterization of metabolites and metabolic profiles in plasma has considerable sig-nificance in determining the efficacy and safety of traditional Chinese medicine(TCM)in vivo.However,this process is usually hindered by the insufficient characteristic fragments of metabolites,ubiquitous matrix interference,and complicated screening and identification procedures for metabolites.In this study,an effective strategy was established to systematically characterize the metabolites,deduce the metabolic pathways,and describe the metabolic profiles of bufadienolides isolated from Venenum Bufonis in vivo.The strategy was divided into five steps.First,the blank and test plasma samples were injected into an ultra-high performance liquid chromatography/linear trap quadrupole-orbitrap-mass spectrometry(MS)system in the full scan mode continuously five times to screen for valid matrix compounds and metabolites.Second,an extension-mass defect filter model was established to obtain the targeted precursor ions of the list of bufadienolide metabolites,which reduced approximately 39％of the interfering ions.Third,an acquisition model was developed and used to trigger more tandem MS(MS/MS)fragments of precursor ions based on the targeted ion list.The acquisition mode enhanced the acquisition capability by approximately four times than that of the regular data-dependent acquisition mode.Fourth,the acquired data were imported into Compound Discoverer software for identification of metabolites with metabolic network prediction.The main in vivo metabolic pathways of bufadienolides were elucidated.A total of 147 metabolites were characterized,and the main biotransformation reactions of bufadienolides were hydroxylation,dihydroxylation,and isomerization.Finally,the main prototype bufadienolides in plasma at different time points were determined using LC-MS/MS,and the metabolic profiles were clearly identified.This strategy could be widely used to elucidate the metabolic profiles of TCM preparations or Chinese patent medicines in vivo and provide critical data for rational drug use.
ABSTRACT
Objective To evaluate the clinical effect of venenum bufonis combined with antibiotics in the treatment of bacterial liver abscess .Methods 60 patients with bacterial liver abscess were enrolled ,and all patients took percutaneous transhepatic puncture guided by ultrasound .They were randomly divided into two groups according to the digital table ,30 cases in each group .The control group received antibiotics treatment ,and the treatment group received venenum bufonis plus antibiotics intravenous drip ,once daily ,one course of treatment lasted seven days .The body temperature , blood routine , procalcitonin and other project changes of the two groups were detected .Results Compared with the control group,the effective rate of the treatment group was higher (96.7％ vs.80.0％,χ2 =4.043,P=0.044).In the treatment group,the time of body temperature returning to normal [(7.00 ±1.67)d vs. (9.00 ±1.41)d],leukocyte recovery time [(7.83 ±2.32) d vs.(9.82 ±1.94) d],procalcitonin recovery time [(7.00 ±1.67)d vs.(9.00 ±1.41)d],symptom disappearance time [(5.17 ±1.72)d vs.(7.50 ±1.87)d],disap-pearance time of abscess[(12.00 ±3.41)d vs.(16.00 ±2.37)d]were shorter than those in the control group(t=-2.601,-2.890,-2.236,-2.248,-2.362,P=0.026,0.016,0.049,0.044,0.041).Conclusion Venenum bufonis combined with antibiotics can significantly increase the curative rate and accelerate infection control , there-fore,it is worthy of popularizing in clinical practice .
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Objective To investigate the function of aurora kinase (AURK) in liver cancer and the mechanism of cinobufagin and bufalin-induced liver cancer HepG2 cells growth inhibition by down-regulating AURK family. Methods Kaplan-Meier survival method analyzed the relationship between mRNA expression levels of AURKA and AURKB and survival periods. The viability and cell cycle of HepG2 cells were detected by MTT method and flow cytometry. Western blotting analyzed the expression levels of spindle-associated protein AURKA, AURKB, TPX2, SMC2, TOP2A, and cyclin-dependent kinase CDK1. Results Kaplan-Meier survival analysis presented a significantly negative correlation between mRNA expression levels of AURKA and AURKB and survival periods. Cinobufagin and bufalin inhibited the growth of HepG2 cells in a time- and dose-dependent manner, and induced the cell cycle G2/M phase arrest. They all down-regulated the expression of AURKA, AURKB, TPX2, SMC2, TOP2A, and CDK1 (P < 0.05). Conclusion There is a significantly negative correlation between mRNA expression levels of AURKA and AURKB and survival periods. Cinobufagin and bufalin could induce HepG2 cells growth inhibition and cell cycle arrest by down-regulating the expression of AURKA and AURKB and other mitosis-regulating proteins.
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Ulta performance liqiuid chromatography-triple quadrupole tandem mass spectrometry ( UPLC-MS/MS) was used to monitor the relative levels of bufadienolides in toad venom in normal and bensulfuron-polluted groups. Methanol extract of toad venom was separated by UPLC ( ODS-C18 ) using a gradient elution of water contains 0. 1% formic acid and acetonitrile. Mass spectrometry was used in an ESI source operated in positive ion and MRM mode. The parameters in the source were set as follows: capillary voltage 3. 0 kV; sampling cone voltage 30 V; and desolvation temperature 500℃. In this method, external calibrations of 6 standards were typically constructed (R2=0. 9953-0. 9992). The LOD was 0. 42-4. 86 ng/mL. Intra- and inter-day precision was 3. 8%-6. 8% and 4. 0%-8. 8%, respectively. The recovery of standard was evaluated by spiking the standard compound into toad venom. Their average recoveries were 96. 9%-109. 6%, and RSDs were 2. 0%-8. 1%. This method was further employed into monitoring the levels of 36 bufadienolides. The levels of more than 20 bufadienolides were greatly different after bensulfuron pollution, suggesting that the bensulfuron pollution could change the chemical expression pattern of bufadienolides in toad venom.
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AIM: To isolate three kinds of bufadienolides composition from Venenum bufonis in order to investigate their antitumor activity in vitro.METHODS: Alcohol extraction and isolation by Silica gel colume were employed in this study.Their antitumor activities in vitro were evaluated by MTT colorimetric method.RESULTS: With such methods,three kinds of bufadienolides,including Bufalin,cinobufagin,and resibufogenin with purity above 90% were prepared.The in-vitro results showed that three kinds of bufadienolides had prominent inhibition effect on human lung cancer A549 cells,human breast cancer MDA-MB-435 cells in the range of 0.001 ?g/mL ～ 100 ?g/mL.CONCLUSION: Isolation by Silica gel colume can be used to separate three kinds of bufadienolides from Venenum bufonis and its constituents has prominent anti-cancer effects.
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AIM: To establish HPLC fingerprint of Venenum Bufonis in an attempt to become a standard of quality control. METHODS: The HPLC method was set up using Alltima C_(18)(250 mm?4.6 mm,5 ?m) column with mobile phase of acetonitrile-0.5% potassium dihydrogen phosphate solution of water;UV detection wavelength at 296 nm and column temperature at 30 ℃ with the flow rate of 1.0 mL/min;20 ?L of the injection volume. RESULTS: In this chromatogram condition,10 peaks were identified as the characteristic fingerprints of Venenum Bufonis.All samples showed the content differences among the samples.The retention times for resibufogenin、cinobufagin、bufalin、bufotalin and cinobufotalin in Venenum Bufonis were consistent with each other.The fingerprint showed good similarity up to 93% in samples from different habitats. CONCLUSION: The method is exact、simple and accurate,and can be used for the identification and quality control of Venenum Bufonis.