Micelle to trapping solution stacking in micellar electrokinetic chromatography

An analytical strategy micelle to trapping solution stacking (MSS) was developed in acidic buffer in micellar electrokinetic chromatography (MEKC). The stacking mechanism is based on the transport, release, capturing of molecules bound to micelle carriers that are made to collapse into trapping solution (TS) to serve as the medium to contain and stacking the analytes. Tetrandrine and fangchinoline were selected as model mixture using sodium dodecyl sulfate (SDS) micelles as carrier to demonstrate this stacking method. The experiments by MSS-MEKC were carried out and further compared with those by normal MEKC. The results reveal that 113–123-fold improvements in the detection sensitivity was obtained for the analytes, and separation and determination of tetrandrine and fangchinoline in Stephaniae tetrandrae S. Moore and Fengtongan capsules were finished under optimum conditions using the sample matrix containing 8.0 mM SDS and TS containing 50 mM H₃PO₄–55% (v/v) ethanol.     

Tetrandrine Suppresses Human Brain Glioblastoma GBM 8401/luc2 Cell-Xenografted Subcutaneous Tumors in Nude Mice In Vivo

Tetrandrine (TET), a bisbenzylisoquinoline (BBI) alkaloid, is isolated from the plant Stephania tetrandra S. Moore and has a wide range of biological activity, including anticancer properties in vitro and in vivo. At first, we established a luciferase-expressing stable clone that was named GBM 8401/luc2 cells. Herein, the primary results indicated that TET reduced the total cell viability and induced cell apoptosis in GBM 8401/luc2 human glioblastoma cells. However, there is no available information showing that TET suppresses glioblastoma cells in vivo. Thus, we investigated the effects and mechanisms of TET on a GBM 8401/luc2 cell-generated tumor in vivo. After the tumor volume reached 100–120 mm³ in subcutaneously xenografted nude mice, all of the mice were randomly divided into three groups: Group I was treated with phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 25 mg/kg of TET, and Group III with 50 mg/kg of TET. All mice were given the oral treatment of PBS or TET by gavage for 21 days, and the body weight and tumor volumes were recorded every 5 days. After treatment, individual tumors, kidneys, livers, and spleens were isolated from each group. The results showed that TET did not affect the body weights, but it significantly decreased the tumor volumes. The TET treatment at 50 mg/kg had a two-fold decrease in tumor volumes than that at 25 mg/kg when compared to the control. TET decreased the total photon flux, and treatment with TET at 50 mg/kg had a lower total photon flux than that at 25 mg/kg, as measured by a Xenogen IVIS imaging system. Moreover, the higher TET treatment had lower tumor volumes and weights than those of the lower dose. The apoptosis-associated protein expression in the tumor section was examined by immunohistochemical analysis, and the results showed that TET treatment reduced the levels of c-FLIP, MCL-1, and XIAP but increased the signals of cleaved-caspase-3, -8, and -9. Furthermore, the hematoxylin and eosin (H & E) staining of kidney, liver, and spleen tissues showed no significant difference between the TET-treated and control groups. Overall, these observations demonstrated that TET suppressed subcutaneous tumor growth in a nude-mice model via the induction of cell apoptosis.     

Ultra high performance liquid chromatography with tandem mass spectrometry method for the determination of tetrandrine and fangchinoline in rat plasma after oral administration of Fangji Huangqi Tang and Stephania tetrandra S. Moore extracts

A rapid, sensitive, and reliable analytical method based on ultra high performance liquid chromatography with tandem mass spectrometry has been developed for the simultaneous determination of fangchinoline and tetrandrine in rat plasma. Plasma samples were pretreated by protein precipitation with acetonitrile. The analysis was performed on a C₁₈ reversed‐phase ultra high performance liquid chromatography column (2.1 mm × 100 mm, 3.5 μm, Agilent), and the flow rate was set at 0.4 mL/min. Under the experimental conditions, extraction recoveries from plasma were 68.1–72.8% for fangchinoline and 69.2–76.5% for tetrandrine. The plasma concentrations of tetrandrine and fangchinoline in rats at assigned time points were all successfully detected through exactly validated method. Good linearities were obtained in the range of 0.92–184 ng/mL for tetrandrine and in the range of 0.83–166 ng/mL for fangchinoline. The intra‐ and interday precisions for both analytes were within 11.1% and the accuracies were within the range of –10.7 to 11.3%. The developed method was then successfully applied to investigate the pharmacokinetic properties of these two alkaloids after oral administration of Stephania tetrandra S. Moore extracts and Fangji Huangqi Tang. The comparative results provided a meaningful basis for a better understanding of the practical value of the compatibility theory of traditional Chinese medicine in the clinic.     

Syntheses and Anti-cancer Activities of Derivatives of Tetrandrine and Fangchinoline

A series of derivatives of tetrandrine and fangchinoline was designed and synthesized to find more active anti-cancer compounds. Their anti-cancer activities were tested against human hcpatocellular carcinoma BEL-7402 and PLC/PRF/5 cells, human lung adenocarcinoma A549 cells as well as human leukaemia K562 cells, and the structure-activity relationship（SAR） was also studied. All the compounds except BI exhibited superior inhibitory ac- tivities against PLC/PRF/5 cells with half maximal inhibitory conccntration（ICs0） values of less than 15 μmol/L, and compounds A2, A4, B2 and B4 showed IC50 values of less than 9 pmol/L. Compounds A2, A6, B2 and B4 showed potent anti-cancer activities against all the tested cells with 1C5o values of less than 10 pmol/L. The results show that terandrine and fangchinoline derivatives are potential suppressors to human cancer.     

Online concentration by field-amplified sample injection in acidic buffer for analysis of fangchinoline and tetrandrine in herbal medicine by flow injection-micellar electrokinetic capillary chromatography

A novel, rapid, and continuous online concentration approach based on field-amplified sample injection for the analysis of fangchinoline and tetrandrine was developed in this paper by combination of flow injection-MEKC. The BGE used was a solution composed of 75 mM H3PO4-triethylamine-2.5% v/v polyoxyethylene sorbitan monolaurate-20% v/v methanol buffer (pH* 5.0). The analytes prepared in 50% v/v aqueous ethanol were used as the test analytes. Sample was injected electrokinetically between plugs of water. When the cations reached the boundary between the water plug and BGE, they slowed down and became concentrated. Thereafter, MEKC was initiated for the separation. This results in 6.8-8.9-fold improvement in concentration sensitivity relative to conventional CE methods. The separation could be achieved within 10 min and sample throughput rate can reach up to 50/h. The repeatability (defined as RSD) was 4.8, 4.4% with peak height evaluation and 3.6, 0.94% with peak area evaluation for TET and FAN, respectively.     

Complexation of tetrandrine with calcium ion probed by various spectroscopic methods and molecular modeling

The formation of the complex between tetrandrine and the calcium ion, in solution, was studied using FTIR, UV-Vis, ¹H NMR, ¹³C NMR and electrospray mass spectroscopy spectroscopic methods and molecular modeling. The calcium salts used were: Ca(ClO₄)₂·4H₂O and Ca(Picrate)₂ in the solvents: acetonitrile (CH₃CN), deuterated acetonitrile (CD₃CN) and tetrahydrofurane (THF). The determined complex stability constant was: 20277±67 dm³ mol⁻¹ and corresponding free energy ΔG₀=−5.820±0.002 kcal mol⁻¹. The molecular simulation of the complex formation with the MM3 Augmented force field integrated in CAChe provided useful data about its energy. Combining the experimental results and molecular modeling we propose a model for the structure of tetrandrine-Ca complex with an eight coordinated geometry.     

Application of nonaqueous capillary electrophoresis for quantitative analysis of quinolizidine alkaloids in Chinese herbs

An easy, rapid method for simultaneous determination of tetrandrine (TET), fangchinoline (FAN), sinomenine (SIN) and tetrahydropalmatine (TEP) in Chinese herbs was developed by nonaqueous capillary electrophoresis without pretreatment for the first time. Optimum separation was achieved with a fused-silica capillary column ( i.d.) and a running buffer containing 50 mM ammonium acetate, 1.0% acetic acid and 20% acetonitrile in methanol medium. The applied voltage was 20.0 kV. The analytes were detected by UV at 214 nm. The effects of concentration of ammonium acetate, acetic acid and organic modifier on electrophoretic behavior of the analytes were studied. Regression equations revealed linear relationships (correlation coefficients: 0.9991-0.9999) between the peak area of each analyte and the concentration. The levels of analytes in Stephania tetrandra S. Moore, Rhizoma corydalis and Sinomenium acutum Rehd. et. Wils were easily determined with recoveries ranging from 96 to 107%.     

微量量热法研究大肠杆菌对粉防己碱和粉防己总生物碱的耐药性

微量量热法研究大肠杆菌对粉防己碱和粉防己总生物碱的耐药性;粉防己碱;粉防己总生物碱;耐药性;大肠杆菌;微量量热法     

Ionic liquid‐based ultrasound‐assisted extraction of fangchinoline and tetrandrine from Stephaniae tetrandrae

An ionic liquid‐based ultrasound‐assisted extraction method has been developed for the effective extraction of fangchinoline and tetrandrine from Stephaniae tetrandrae. The effects of some ultrasound‐assisted extraction parameters including the concentration of [BMIM][BF₄], pH, ultrasonic power and time were investigated to optimize the ultrasound‐assisted extraction conditions. Compared to the regular ultrasound‐assisted extraction and traditional refluent extraction, the proposed [BMIM][BF₄]‐based ultrasound‐assisted extraction offered shorter extraction times (from 6 h to 40 min) and remarkable higher efficiencies (approximately 30% improved), which supported the suitability of the proposed approach. In addition, the proposed approach was confirmed by the good correlation coefficient (R²), recovery and reproducibility (RSD, n = 5), which were in the range of 0.9992–0.9995, 85.5–101.1%, and 1.87–4.33%, respectively.     

毛细管电泳高频电导法测定粉防己药材中的生物碱

建立了千金藤属植物粉防己中生物碱测定的毛细管电泳高频电导法,以融硅毛细管(150μm×60cm)为分离柱,1mmol LHAc 2mmol LNH4Ac 10mmol Lβ CD 2mmol LSDS缓冲液为电泳介质,分离电压14.0kV,用高频电导法检测,粉防己药材中粉防己碱和去甲粉防己碱可实现定量分离和检测。探讨了缓冲液的种类、浓度、添加剂、分离电压和进样量对分离和检测的影响;在优化条件下的线性范围分别为:粉防己碱12 5～900μg mL,去甲粉防己碱25 0～900μg mL;检出限分别为:粉防己碱6 25μg mL,去甲粉防己碱12.5μg mL;样品加标回收率分别为92.7%～98.9%和95.9%～101%。