Study the interactions between multiple flavonoids and bovine serum albumin by the developed equilibrium dialysis.

The therapeutic function of traditional Chinese medicine (TCM) is based on the combination effect of multiple active ingredients. However, the current pharmacological studies mainly focus on the protein binding of the single component from TCM, which is difficult to explain the overall therapeutic mechanism. Thus in this work the equilibrium dialysis method combined with high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to study the interactions between multi-components and protein. Firstly, the binding constants of seven different structural types of flavonoids with bovine serum albumin (BSA) were determined. The results showed that the binding affinity of flavones and flavonols with BSA was stronger than that of dihydroflavonoids, and the substitution of glycosides would reduce the binding affinity with BSA. The results of competitive displacement experiment showed that there existed competitive interactions among the four flavonoids (rutin, luteolin, hesperetin and kaempferol). The binding constants of flavonoids to BSA were significantly changed under the condition of multi-components coexistence. Especially, the binding constant of hesperetin to BSA increased from 9.46 × 104 L/mol to 1.49 × 106 L/mol under the coexistence of rutin. The results of fluorescence spectroscopy showed that the reason for competitive binding was that the four flavonoids were mainly bound to the IIA region of BSA. Finally, the method was successfully applied to study the binding of multiple components in Radix Scutellariae (RS) extract with BSA. Five flavonoids in RS extract were identified by UPLC-MS/MS, they had different degrees of binding to BSA, among which oroxylin A had the strongest binding degree. In conclusion, the equilibrium dialysis was reliable and sufficiently accurate for study of the interaction between multi-components or TCM extract and protein, which can provided a theoretical basis for the scientific explanation of the overall treatment mechanism of TCM.

[Methods and techniques of capillary electrophoresis for drug screening].

Capillary electrophoresis (CE) shows enormous potential for application in new drug research and development. Because of the aqueous medium employed as the running buffer in CE, drug screening can be carried out in an environment similar to that in physiological testing media. Drug screening methods based on CE are different from other instrumental measurements in vitro. CE can not only sustain the biological activity of the screened molecules and ligands, but also help evaluate the interactions between the receptors and the ligands. Based on these interactions, some important pharmacological parameters related to drug screening, such as the association constant Kb, bonding rate constant Kon, and dissociation rate constant Koff, can be determined by CE. Thus, CE is an effective tool for simulating and predicting the entire interaction process between receptors and drugs in vivo. In this review, the history of CE for drug screening is revisited. The theories, common methods for drug screening by CE, and some application examples and related technologies are reviewed. The methods of drug screening by means of affinity CE and kinetic CE are introduced. Some selected studies on different ligands at the molecular and cellular level are reported, along with examples several types of drugs. Techniques based on a combination of CE with mass spectrometry and chemiluminescence are reviewed, with focus on the screening of candidate drugs and active compounds from traditional Chinese medicine. The application prospect of drug screening by CE combined with a DNA-encoded compound library is introduced. This paper discusses the core of the fraction collection step in CE and emphasizes the significance of combining CE with systematic evolution of ligands by exponential enrichment. In conclusion, various optional methods for CE drug screening would pave the way for new concepts related to drug screening and evaluation in the future.

Curcumin-loaded galactosylated BSA nanoparticles as targeted drug delivery carriers inhibit hepatocellular carcinoma cell proliferation and migration.

BACKGROUND: The main objective of this study was to develop novel BSA nanoparticles (BSA NPs) for improving the bioavailability of curcumin as an anticancer drug, and those BSA NPs were galactosylated for forming the curcumin-loaded galactosylated BSA nanoparticles (Gal-BSA-Cur NPs), thus enhancing their ability to target asialoglycoprotein receptor (ASGPR) overexpressed on hepatocellular carcinoma (HCC) cells. MATERIALS AND METHODS: Gal-BSA-Cur NPs were prepared by the desolvation method and showed a spherical shape and well distribution with the average particle size of 116.24 nm. RESULTS: In vitro drug release assay exhibited that Gal-BSA-Cur NPs had higher release rates and improved the curcumin solubility. Cell uptake studies confirmed that Gal-BSA-Cur NPs could selectively recognize receptors on the surface of HCC (HepG2) cells and improve internalization ability of drug compared with BSA NPs-loaded curcumin (BSA-Cur NPs), which might be due to high affinity to galactose. Further, the effects of Gal-BSA-Cur NPs were evaluated by cytotoxicity assay, crystal violet assay, cell apoptosis assay, and wound healing assay, respectively, which revealed that Gal-BSA-Cur NPs could inhibit HepG2 cells proliferation, induce cell apoptosis, and inhibit cell migration. CONCLUSION: Immunofluorescence staining has proved that the effects of Gal-BSA-Cur NPs related to the suppression of the nuclear factor κB-p65 (NF-κB-p65) expression in HepG2 cell nucleus. Therefore, these results indicate that novel Gal-BSA-Cur NPs are potential candidates for targeted curcumin delivery to HCC cells.

[Study on double fingerprints of isatidis radix micropowder].

OBJECTIVE: To establish the double HPLC fingerprints of water-soluble composition and amino acids precolumn derivative reagent of 13 batches of Isatidis Radix micropower. METHOD: The gradient elution was adopted with Hypersil BDS C18 column (4.6 mm x 250 mm, 5 microm). Water-soluble ingredients were detected with acetonitrile-0.1% phosphoric acid solution as mobile phase, flow rate 0.5 mL x min(-1), column temperature 20 degrees C, and the injection volume 10 microL. Amino acid ingredient were derived by PITC, and then were detected with mobile phase of 0.1 mol x L(-1) sodium acetate buffer solution (pH 6.5) - acetonitrile, flow rate 1.0 mL x min(-1), column temperature 30 degrees C, and the injection volume 5 microL. Both of the absorption wavelengths were 254 nm. Pharmacopoeia Commission "Chinese chromatographic fingerprint evaluation system (version 2.0)" was adopted to analyse, fingerprints of Isatidis Radix micropower were established, at the same time 4 main ingredients were recognized by the SPSS cluster analysis. RESULT: Common mode of Fingerprint to water-soluble and amino acids ingredient of Isatidis Radix micropower was established, then adenosine, epigoitrin and 15 amino acids were identified as characteristic peaks. Cluster analysis showed that different kinds of the herbal Isatidis Radix micropower from different areas were different levels in the main ingredients. CONCLUSION: Double fingerprints of Isatidis Radix micropower is established. Each peak is optimally separated in chromatogram, which provides a scientific basis for quality control of Isatidis Radix micropower.