Xiaoping Zhao

Astragaloside IV Stimulates Angiogenesis and Increases Hypoxia-Inducible Factor-1α Accumulation via Phosphatidylinositol 3-Kinase/Akt Pathway

Astragaloside IV is the major active constituent of Astragalus membranaceus, which has been widely used for the treatment of cardiovascular diseases in China. The aim of this study was to determine the angiogenic effect of astragaloside IV and its underlying mechanism. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay, Western blotting, real-time polymerase chain reaction, and immunofluorescence to detect the effect of astragaloside IV on proliferation of human umbilical vein endothelial cells (HUVECs), the phospho-Akt protein level, hypoxia-inducible factor-1α (HIF-1α) accumulation, vascular endothelial growth factor mRNA expression, and applied cell migration, tube formation, and chick chorioallantoic membrane assays to study the angiogenic effect of astragaloside IV. Results indicate that astragaloside IV promoted cell proliferation and stimulated HIF-1α accumulation during hypoxia. Mechanism studies revealed that astragaloside IV did not affect the degradation of HIF-1α protein or the level of HIF-1α mRNA. In contrast, astragaloside IV apparently activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which regulates HIF-1α protein synthesis. Moreover, astragaloside IV also stimulated cell migration, increased tube formation, and promoted angiogenesis in the chick chorioallantoic membrane assay. All angiogenic effects of astragaloside IV were reversed by the PI3K inhibitor. Taken together, our data collectively reveal that astragaloside IV is a novel regulator of HIF-1α and angiogenesis through the PI3K/Akt pathway in HUVECs that are exposed to hypoxia.

Development of fluorescence imaging-based assay for screening cardioprotective compounds from medicinal plants

Medicinal plants have been widely recognized as a renewable resource for the discovery of novel leads and drug. In this study, an approach for screening and identification compounds with cardioprotective activity from medicinal plant extracts by cellular-fluorescence imaging technique was developed. It is a cell-based assay for measuring mitochondrial membrane potential changes in H9c2 cardiac muscle cells exposed to H(2)O(2) by using a fluorescence automatic microscopy screening platform. Rhodamine 123 was used as the fluorescent dye to indicate the change of mitochondrial membrane potential. The sensitivity and linear range of the proposed approach were evaluated and validated using vitamin C, an antioxidative compound. The method was applied to screen active components with potent cardioprotective effects from a traditional Chinese formula. The potential cardioprotective components were identified by liquid chromatography coupled with mass spectrometry (LC/MS). Moreover, the utility of the proposed approach was further validated by three compounds (salvianolic acid B, protocatechuic aldehyde, and tanshinone II A) identified from the formula which showed cardioprotective effects in a dose-dependent manner. These applications suggested that the proposed rapid and sensitive screening approach offers an efficient way to discover active components or compounds from medicinal plants.

Proteomic study on usnic-acid-induced hepatotoxicity in rats.

Usnic acid, a lichen metabolite, is used as a dietary supplement for weight loss. However, clinical studies have shown that usnic acid causes hepatotoxicity. The present study aims to investigate the mechanism of usnic acid hepatotoxicity in vivo. Two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to analyze the expression profiles of differentially regulated and expressed proteins in rat liver after usnic acid administration. The results reveal the differential expression of 10 proteins in usnic-acid-treated rats compared to the normal controls. These proteins are associated with oxidative stress, lipid metabolism, and several other molecular pathways. The endoplasmic reticulum and mitochondria may be the primary targets of usnic-acid-induced hepatotoxicity.

Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures

Lipase is the key enzyme for catalyzing triglyceride hydrolysis in vivo, and lipase inhibitors have been used in the management of obesity. We present the first report on the use of lipase-adsorbed halloysite nanotubes as an efficient medium for the selective enrichment of lipase inhibitors from natural products. A simple and rapid approach was proposed to fabricate lipase-adsorbed nanotubes through electrostatic interaction. Results showed that more than 85% lipase was adsorbed into nanotubes in 90 min, and approximately 80% of the catalytic activity was maintained compared with free lipase. The specificity and reproducibility of the proposed approach were validated by screening a known lipase inhibitor (i.e., orlistat) from a mixture that contains active and inactive compounds. Moreover, we applied this approach with high performance liquid chromatography-mass spectrometry technique to screen lipase inhibitors from the Magnoliae cortex extract, a medicinal plant used for treating obesity. Two novel biphenyl-type natural lipase inhibitors magnotriol A and magnaldehyde B were identified, and their IC50 values were determined as 213.03 and 96.96 μM, respectively. The ligand-enzyme interactions of magnaldehyde B were further investigated by molecular docking. Our findings proved that enzyme-adsorbed nanotube could be used as a feasible and selective affinity medium for the rapid screening of enzyme inhibitors from complex mixtures.

A Bioactive Chemical Markers Based Strategy for Quality Assessment of Botanical Drugs: Xuesaitong Injection as a Case Study

Current chemical markers based quality assessment methods largely fail to reflect intrinsic chemical complexity and multiple mechanisms of action of botanical drugs (BD). The development of novel quality markers is greatly needed. Here we propose bioactive chemical markers (BCM), defined as a group of chemo-markers that exhibit similar pharmacological activities comparable to the whole BD, which can therefore be used to effectively assess the quality of BD. As a proof-of-concept, a BCM-based strategy was developed and applied to Xuesaitong Injection (XST) for assessing the efficacy and consistency of different batches. Firstly, systemic characterization of chemical profile of XST revealed a total number of 97 compounds. Secondly, notoginsenoside R1, ginsenoside Rg1, Re, Rb1 and Rd were identified as BCM of XST on treating cardiovascular and cerebrovascular diseases according to Adjusted Efficacy Score following an in vivo validation. Analytical method for quantification of BCM was then developed to ensure the efficacy of XST. Finally, chemical fingerprinting was developed and used to evaluate the batch-to-batch consistency. Our present case study on XST demonstrates that BCM-based strategy offers a rational approach for quality assessment of BD and provides a workflow for chemistry, manufacturing, and controls (CMC) study of BD required by regulatory authority.

Enrichment and Purification of the Bioactive Flavonoids from Flower of Abelmoschus manihot (L.) Medic Using Macroporous Resins

Flower of Abelmoschus manihot (FAM) is clinically effective to treat chronic kidney disease (CKD) with a relatively high dosage. To improve the efficacy and the compliance of patients, macroporous resins were adopted to enrich and purify flavonoids from FAM, which are thought to be the major renal protective constituents in FAM. After screening six different kinds of macroporous resins, HPD-100 was selected for its great adsorption and desorption capacity. Then, orthogonal design tests were used to optimize parameters in the processes of impurity removal and flavonoids of FAM desorption on column chromatogram. Moreover, process scale-up was performed, and purification effects maintained after amplification. After purification, the content of seven main flavonoids in the product increased from 8.29% to 51.43%. Protective and anti-inflammatory effects of crude extract and the flavonoid component of FAM after purification were investigated on the adriamycin-damaged HK-2 cells and lipopolysaccharide-stimulated Raw 264.7 cells models. Both bioactivities were improved greatly after purification for these two cell models. Therefore, the purification process had enriched the main bioactive constituents with potential alleviating kidney injury activities. The flavonoid component of FAM is worthy of being developed as an improved remedy for CKD with better patients’ compliance.