ABSTRACT
Objective:To study on the material basis of Sanguisorbae Radix by column chromatography and liquid chromatography-ion trap-time-of-flight mass spectrometry (LCMS-IT-TOF), and analyze the distribution of different components in Sanguisorbae Radix water extract on D101 macroporous resin and polyamide resin. Method:Sanguisorbae Radix water extract was separated by D101 macroporous resin and polyamide resin, and LCMS-IT-TOF was used for detection, chromatography separation was achieved on an ACQUITY UPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm) with the mobile phase consisted of water (A) and acetonitrile (B) for gradient elution (0-10 min, 5%-20%B; 10-18 min, 20%-35%B; 18-23 min, 35%-50%B; 23-28 min, 50%-90%B; 28-30 min, 90%B; 30-33 min, 90%-5%B; 33-35 min, 5%B), the flow rate was 0.3 mL·min-1, the column temperature was 30 ℃. Data acquisition was carried out in electrospray ionization (ESI) under the positive and negative ion modes, the scanning range was m/z 100-1 200. According to mass spectrometry data such as accurate molecular mass and fragment information, combined with literature, different chemical components in loading effluents and ethanol eluents of Sanguisorbae Radix water extract were identified. A heat map of the distribution of components in each fraction was drawn by extracting mass spectrum peak intensity data of each sample. The elution rules of various components were compared visually. Result:The enrichment and separation of D101 macroporous resin and polyamide resin were obvious. Tannins in Sanguisorbae Radix water extract was mainly concentrated in loading effluent of macroporous resin and its water eluent, triterpenoids were mainly distributed in the 90% ethanol eluent of macroporous resin. In the above effluents and eluents, a total of 63 compounds (including isomers) were identified. Among them, 6 compounds, ellagic acid-4-pyranoarabinoside or its isomer, 6-O-galloylnorbergerin, 3-O-galloylnorbergerin, (6-acetyloxy-5,7-dihydroxy-8-methoxy-4-oxochromen-2-yl) acetate, ethyl 2-methyl-5,6-bis (sulfooxy) benzofuran-3- carboxylate were first discovered in Sanguisorbae Radix. Conclusion:The method can quickly and accurately identify the distribution of components in aqueous extract of Sanguisorbae Radix after column chromatography, providing experimental basis for exploring the pharmacodynamic components and mechanism of Sanguisorbae Radix.
ABSTRACT
The aim of this study is to establish the in vitro methods for the study of induction and inhibition on CYP450 by drugs, and to validate the analytical method and incubation system. A method for the simultaneous determination of eight metabolites of seven subtypes of CYP450 enzymes probe substrates in human liver microsomes (HLM) was established and validated. The incubation system was optimized to confirm the incubation time and protein concentration of HLM, the enzyme activity of seven subtypes of CYP450 enzymes in HLM was determined, and the inhibition effects on each CYP450s were checked by positive controls. The method for the simultaneous determination of three metabolites of subtypes of CYP450 enzymes was established and validated in human primary cultured hepatocytes (HPCH) using the incubation medium. The enzyme activity of three subtypes of CYP450 enzymes in HPCH was determined, and the total RNA was extracted from HPCH after incubation. The expression of CYP450 enzymes were measured by Taqman fluorescence probe method. The induction effects on each CYP450s were examined using the positive controls. The established methods for the determination of metabolites of probe substrates were fully validated, and the results were conformed to the requirements of bioanalytical method validation. The induction and inhibition effects on each CYP450s were checked by positive controls. The established in vitro methods for the study of drug induction and inhibition on CYP450 were simple and reliable, which could be used in the investigation of enzyme induction or inhibition properties of new drug candidates and to evaluation the metabolic interactions of concomitant medication in clinical.
ABSTRACT
AIM@#To synthesize the baicalein amino acid derivatives and evaluate their cytotoxicity activities in vitro.@*METHODS@#Amino acids were subjected to methylation and aminoacylation reaction, then reacted with formaldehyde and baicalein to synthesize baicalein-8 benzyl amino acid derivatives. Through carboxyl group protection and aminoacylation of amino acid and benzyl protection of baicalein, derivatives of baicalein-6-O-amino acid esters were obtained. All of the target compounds were identified by IR, MS and (1)H NMR.@*RESULTS@#Thirteen novel derivatives were synthesized and characterized. Their cytotoxic activities were assessed by the MTT method on the inhibition of HepG2 cells in vitro.@*CONCLUSION@#Compounds 4c, 4d, 7a and 7b showed a significant increase in cytotoxicity compared with baicalein.