Zhaoguang Zheng

Development of self-microemulsifying drug delivery system for oral bioavailability enhancement of berberine hydrochloride

The purpose of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) to improve the oral bioavailability of Berberine hydrochloride (BBH), an important bioactive compound from Chinese Medicines with poor water solubility. Pseudoternary phase diagrams were constructed using oil, surfactant and co-surfactant types to identify the efficient self-microemulsification region. SMEDDS was characterized by morphological observation, droplet size, zeta-potential determination, stability, in vitro release and in vivo bioavailability study. The optimal formulation with the best self-microemulsifying and solubilization ability consisted of 40% (w/w) of ethyl linoleate and oleic acid (2:1), 35% (w/w) Tween-80 and 25% (w/w) glycerol. The SMEDDS of BBH could exhibit good stability. In vitro release test showed a complete release of BBH from SMEDDS was in 5 h. In vivo results indicated that the peak plasma concentration (Cmax) and the area under the curve (AUC0→12 h) of SMEDDS of BBH were higher than the commercial tablet by 163.4% and 154.2%, respectively. The relative bioavailability of SMEDDS of BBH was enhanced about 2.42-fold compared with the commercial tablet in rats. The study confirmed that the SMEDDS formulation could be used as a possible alternative to traditional oral formulations of BBH to improve its bioavailability.

Inhibitory effects of two major isoflavonoids in Radix Astragali on high glucose-induced mesangial cells proliferation and AGEs-induced endothelial cells apoptosis.

Radix Astragali, the dried roots of Astragalus membranaceus var. mongholicus, is well known to have a protective effect on diabetic nephropathy. However, the effects of isoflavonoids in Radix Astragali on glomerular cells, which play a key role in the development of diabetic vascular complications, remain largely unknown. Thus, the purpose of this study was to investigate in vitro the effect of calycosin and calycosin-7-O-β-D-glucoside, two major isoflavonoids in Radix Astragali, on high glucose-induced rat mesangial cells proliferation and AGEs-induced human glomerular endothelial cell apoptosis. The results indicated that both calycosin and calycosin-7-O-β-D-glucoside (10-100 µM) could inhibit high glucose-induced mesangial cell early proliferation. Additionally, AGEs-mediated cell apoptosis was also attenuated by treatment of glomerular endothelial cells with either calycosin or calycosin-7-O-β-D-glucoside (1-100 µM). Therefore, the results obtained in this study suggest that both calycosin and calycosin-7-O-β-D-glucoside have a significant therapeutic potential to modulate the development and/or progression of diabetic nephropathy.

Phytoestrogen calycosin‐7‐O‐β‐D‐glucopyranoside ameliorates advanced glycation end products‐induced HUVEC damage

Vasculopathy including endothelial cell (EC) apoptosis and inflammation contributes to the high incidence of stroke and myocardial infarction in diabetic patients. The aim of the present study was to investigate the effect of calycosin‐7‐O‐β‐D‐glucopyranoside (CG), a phytoestrogen, on advanced glycation end products (AGEs)‐induced HUVEC damage. We observed that CG can significantly ameliorate AGEs‐induced HUVEC oxidative stress and apoptosis. The ratio of SOD/MDA was significantly increased to the normal level by CG pretreatment. CG preincubation dramatically increased anti‐apoptotic Bcl‐2 while decreased pro‐apoptotic Bax and Bad expressions as detected by immunocytochemistry. Moreover, CG ameliorated macrophage migration and adhesion to HUVEC; the monocyte chemotactic protein‐1 and interleukin‐6 levels in the culture supernatant were dramatically reduced by CG as determined by ELISA; the expressions of inflammatory proteins including ICAM‐1, TGF‐β1, and RAGE in both protein and mRNA levels were significantly reduced to the normal level by CG pretreatment as determined by immunocytochemistry and real‐time RT‐PCR. The intracellular investigation suggests that CG can reverse AGEs‐activated ERK1/2 and NF‐κB phosphorylation, in which estrogen receptors were involved in. Our results strongly indicate that CG can modulate EC dysfunction by ameliorating AGEs‐induced cell apoptosis and inflammation. J. Cell. Biochem. 112: 2953–2965, 2011.

Macrophage biospecific extraction and HPLC–ESI-MSn analysis for screening immunological active components in Smilacis Glabrae Rhizoma

A cell-permeable membrane, as typified by Transwell insert Permeable Supports, permit accurate repeatable invasion assays, has been developed as a tool for screening immunological active components in Smilacis Glabrae Rhizoma (SGR). In this research, components in the water extract of SGR (ESGR) might conjugate with the receptors or other targets on macrophages which invaded Transwell inserts, and then the eluate which contained components biospecific binding to macrophages was identified by HPLC-ESI-MS(n) analysis. Six compounds, which could interact with macrophages, were detected and identified. Among these compounds, taxifolin (2) and astilbin (4) were identified by comparing with the chromatography of standards, while the four others including 5-O-caffeoylshikimic acid (1), neoastilbin (3), neoisoastilbin (5) and isoastilbin (6), were elucidated by their structure clearage characterizations of tandem mass spectrometry. Then compound 1 was isolated and purified from SGR, along with 2 and 4, was applied to the macrophage migration and adhesion assay in HUVEC (Human Umbilical Vein Endothelial Cells) -macrophages co-incultured Transwell system for immunological activity assessment. The results showed that compounds 1, 2 and 4 with concentration of 5μM (H), 500nM (M) and 50nM (L) could remarkably inhibit the macrophage migration and adhesion (Vs AGEs (Advanced Glycation End Produces) group, 1-L, 2-H and 4-L groups: p<0.05; other groups: p<0.01). Moreover, 1 and 4 showed satisfactory dose-effect relationship. In conclusion, the application of macrophage biospecific extraction coupled with HPLC-ESI-MS(n) analysis is a rapid, simple and reliable method for screening immunological active components from Traditional Chinese Medicine.

Kuwanon G Preserves LPS-Induced Disruption of Gut Epithelial Barrier In Vitro

Defects in the gut epithelial barrier have now been recognized to be responsible for diabetic endotoxemia. In everyday life, Mulberry leaf tea is widely used in Asian nations due to its proposed benefits to health and control of diabetes. Evidence indicates the potential role of Kuwanon G (KWG), a component from Morus alba L., on blocking the gut epithelial barrier. In lipopolysaccharides (LPS)-damaged Caco-2 cells, it was found that KWG increased the viability of cells in a concentration-dependent manner. KWG administration significantly elevated the anti-oxidant abilities via increasing ratio of superoxidase dismutase (SOD)/malondialdehyde (MDA) and decreasing reactive oxygen species (ROS) within the cells. During KWG incubation, pro-inflammatory cytokines including interleukin (IL)-1β and tumor necrosis factor (TNF)-α were significantly reduced, tight junction proteins including zonula occludens (ZO)-1, intercellular adhesion molecule (ICAM)-1 and Occludin were dramatically increased as detected by immunofluorescence assay, trans-epithelial electrical resistance was significantly increased and the transmission of albumin-fluorescein isothiocyanate (FITC) across the barrier was decreased. In conclusion, the present study demonstrated that KWG could ameliorate LPS-induced disruption of the gut epithelial barrier by increasing cell viability and tight junction between cells, and decreasing pro-inflammatory cytokines and oxidative damage.

Calycosin Rebalances Advanced Glycation End Products-Induced Glucose Uptake Dysfunction of Hepatocyte In Vitro.

Diabetes mellitus (DM) often accompanies liver dysfunction. Astragali Radix is a traditional Chinese herbal medicine that is widely administrated to ameliorate the symptoms of diabetes as well as liver dysfunction, but its acting mechanism is still not yet fully recognized. Advanced glycation end products (AGEs) play a key role in promoting diabetic organ dysfunction. Both hyperglycemia and AGEs can induce insulin resistance, hepatocyte damage and liver dysfunction. We designed this study to explore the effects of the phytoestrogen Calycosin, a major active component of Astragali Radix, on AGEs-induced glucose uptake dysfunction in the hepatocyte cell line and relevant mechanisms. MTT and BrdU methods were applied to evaluate cell viability. 2-NBDG was used to observe glucose uptake by a live cell imaging system. Immunofluorescence method was carried out to investigate GLUT1, GLUT4, and RAGE protein expressions on cell membrane. cAMP content was determined by an EIA method. We found Calycosin concentration-dependently ameliorated AGEs-induced hepatocyte viability damage. AGEs dramatically reduced basal glucose uptake in hepatocytes, and this reduction could be reversed by Calycosin administration. By immunofluorescence detection, we observed that Calycosin could inhibit AGEs-induced GLUT1 expression down-regulation via estrogen receptor (ER). Furthermore, Calycosin decreased AGEs-promoted RAGE and cAMP elevation in hepatocytes. These findings strongly suggest that Calycosin can ameliorate AGEs-promoted glucose uptake dysfunction in hepatocytes; the protection of cell viability and ER-RAGE and GLUT1 pathways play a significant role in this modulation.