Screening potential mitochondria-targeting compounds from traditional Chinese medicines using a mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry method

Mitochondria regulate numerous crucial cell processes, including energy production, apoptotic cell death, oxidative stress, calcium homeostasis and lipid metabolism. Here, we applied an efficient mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry（LC/MS） method,also known as screening method for mitochondria-targeted bioactive constituents（SM-MBC）. This method allowed searching natural mitochondria-targeting compounds from traditional Chinese medicines（TCMs）, including Puerariae Radix（PR） and Chuanxiong Radix（CR）. A total of 23 active compounds were successfully discovered from the two TCMs extracts. Among these 23 hit compounds, 17 were identified by LC/MS, 12 of which were novel mitochondria-targeting compounds. Among these, 6 active compounds were analyzed in vitro for pharmacological tests and found able to affect mitochondrial functions. We also investigated the effects of the hit compounds on Hep G2 cell proliferation and on loss of cardiomyocyte viability induced by hypoxia/reoxygenation injury. The results obtained are useful for in-depth understanding of mechanisms underlying TCMs therapeutic effects at mitochondria level and for developing novel potential drugs using TCMs as lead compounds. Finally, we showed that SM-MBC was an efficient protocol for the rapid screening of mitochondria-targeting constituents from complex samples such as PR and CR extracts.

Determination of α-glucosidase inhibitors from Scutellaria baicalensis using liquid chromatography with quadrupole time of flight tandem mass spectrometry coupled with centrifugal ultrafiltration

The present study aimed at identifying potential lead compounds for diabetes mellitus drug discovery. We developed a novel method involving centrifugal ultrafiltration separation subsequent liquid chromatography with quadrupole time of flight tandem mass spectrometry （LC-Q/TOF-MS/MS） determination to screen a-glucosidase inhibitors in complex Scutellaria baicalensis Georgi （SBG） extract. By adding a second filter to the screening process, the level of non-specific binding of Compounds 1, 3, 10 and 11 was significantly decreased, and the level of non-specific binding of Compounds 5 and 15 also was reduced. As a result, five flavonoids identified as baicalein, baicalein, wogonin, chrysin, and oroxylin A, were rapidly found to interact with α-glucosidase and possess potent anti-a-glueosidase aetivity in vitro. Specific binding of ligands to a-glucosidase was demonstrated though the proposed method and the ligands could be ranked in order of affinity for α-glucosidase, which were corresponded to the order of inhibitory activity in vitro. In conclusion, our results indicated that the developed method is a rapid and effective screening method for rat intestinal α-glucosidase inhibitors from complex herbal medicines such as SBG.

Determination of free ceftriaxone concentration and its application in predicting lung tissue concentration

The purpose of this study was to establish a method for determining the free concentration of ceftriaxone based on hollow fiber centrifugal ultrafiltration(HFCF-UF)technology in combination with high-performance liquid chromatography(HPLC)for free pharmacokinetic studies and the prediction of ceftriaxone concentrations in lung tissue.This method only required centrifugation for a short time,and the filtrate could be injected directly for HPLC analysis without further treatment.The specificity,linearity,precision and stability of this method were validated for quantification of free ceftriaxone.Under the optimized conditions,the absolute recoveries were more than 92.5%.The intraday and interday precision RSDs were less than 3.6%.Additionally,nonspecific adsorption(NSB)between the analyte and the ultrafiltration membrane was considered.This method was successfully applied to the analysis of the free ceftriaxone concentration in rat plasma and lung tissue.The free ceftriaxone concentration of lung tissue could be predicted by using the linear formula Cfl=Cfp(0.342 x–0.0129)(x:time).This method also provides a reliable alternative for accurate monitoring of the free ceftriaxone concentration in therapeutic drug monitoring(TDM).