Meiqing Feng

Purification Method Improvement and Characterization of a Novel Ginsenoside-Hydrolyzing β-Glucosidase from Paecilomyces Bainier sp. 229

The purification method for a novel ginsenoside-hydrolyzing β-glucosidase from Paecilomyces Bainier sp. 229 was successfully simplified by the application of microcrystalline cellulose (MCC) as a novel chromatographic matrix. Only two chromatographic steps, Q-Sepharose FF and MCC column in sequence, were required to purify the enzyme to apparent homogeneity. The purified enzyme, with a native molecular weight estimated to be 305 KDa, was composed of three identical subunits of approximately 102 KDa each. The optimal enzyme activity was observed at pH 3.5 at 55 °C. It was stable within pH 3–7 and at temperatures lower than 50 °C. The optimal substrate for the enzyme was p-nitrophenyl-β-D-glucoside, followed by ginsenoside Rd, gentiobiose, and ginsenoside Rb1. It converted ginsenoside Rb1 to ginsenoside Rg3 specifically and efficiently. The hydrolyzing pathway of ginsenoside Rb1 by the enzyme was Rb1→Rd→Rg3. The specific activities against ginsenoside Rb1 and Rd were 56.7 μmol/min/mg and 129.4 μmol/min/mg respectively.

Biotransformation of ginsenoside Rb1 to ginsenoside Rd by highly substrate-tolerant Paecilomyces bainier 229-7

Paecilomyces bainier 229-7 was obtained after UV irradiation for 8 min in the presence of 0.4% LiCl and selection on potato dextrose media containing 30 mg/mL saponin from Panax notoginseng leaves (SPNL). The mutant produces ginsenoside Rd from ginsenoside Rb1 with a bioconversion rate as high as 94.9% under optimized culture conditions in shake flasks when supplied with 20 mg/mL of SPNL. Scale-up in 10-L fermenter resulted in an 89% bioconversion rate. Ginsenoside Rd was purified from the culture medium by a macroporous resin with a chromatographic purity of 92.6%. These results suggest that P. bainier 229-7 could be useful for the preparation of ginsenoside Rd in the pharmaceutical industry.

In vitro and in vivo anti-hepatitis B virus activities of a plant extract from Geranium carolinianum L.

Natural products provide a large reservoir of potentially active agents with anti-hepatitis B virus (HBV) activity. We examined the effect of the polyphenolic extract from Geranium carolinianum L. (PPGC) on HBV replication both in vitro and in vivo. In the human HBV-transfected liver cell line HepG(2) 2.2.15, PPGC effectively suppressed the secretion of the HBV antigens in a dose-dependent manner with IC(50) values of 46.85 microg/ml for HBsAg and 65.60 microg/ml for HBeAg at day 9. Consistent with the HBV antigen reduction, PPGC (100 microg/ml) also reduced HBV DNA level by 35.9%. In the duck hepatitis B virus (DHBV) infected ducks, after PPGC was dosed intragastricly (i.g.) once a day for 10 days, the plasma DHBV DNA level was reduced, with an ED(50) value of 47.54 mg/kg. In addition, Southern blot analysis confirmed the in vivo anti-HBV effect of PPGC in ducks and PPGC also reduced the plasma and the liver DHBV DNA level in a dose-dependent manner. Furthermore, significant improvement of the liver was observed after PPGC treatment, as evaluated by the histopathological analysis.

Studies on the preparation, crystal structure and bioactivity of ginsenoside compound K

Microbial transformation of Panax notoginseng saponins (PNS) using Aspergillus niger afforded, as the main metabolite, ginsenoside compound K (20-O-β-glucopyranosyl-20(S)-protopanaxadiol). Its structure was determined spectroscopically and by X-ray analysis, and this is the first time the crystal structure of ginsenoside has been reported. In comparison with ginsenoside Rb1, the pro-drug for this metabolite, compound K exhibits potent cytotoxic activity against tumor cell lines. The mean concentrations of compound K needed to inhibit the proliferation of cells by 50% (IC50) were 12.7, 11.4, 8.5 and 9.7 μM for mouse high-metastatic melanoma (B16-BL6), human hepatoma (HepG2), human myeloid leukemia (K562) and human high-metastatic lung carcinoma (95-D) cell lines, respectively. The data show that ginsenoside compound K is a good antitumor drug candidate.

Recombinant high-density lipoprotein complex as a targeting system of nosiheptide to liver cells

Nosiheptide is a lipophilic peptide of significant anti-hepatitis B virus (anti-HBV) activity in cell culture, but has poor distribution to liver in vivo. In this study, recombinant high-density lipoprotein (rHDL) complexes of nosiheptide were constructed to target this anti-HBV agent to hepatocytes. The optimized rHDL–nosiheptide complex had a high drug-loading efficiency (>80%) and a diameter smaller than 30 nm. The concentration of nosiheptide in an optimized rHDL–nosiheptide complex to achieve 50% virus inhibition (IC50) in HepG2 2.2.15 cells was 0.63 μg/ml, which was 40 times lower than the IC50 of nosiheptide in control liposome (2.5 μg/ml) and 200 times lower than the IC50 of the free nosiheptide (12.5 μg/ml). The complex targeted most of the administered nosiheptide to the liver within 30 min after i.v. injection to male Wistar rats. Together, this report provides early evidence that it is feasible to develop efficient, HDL-based drug delivery systems against HBV, utilizing apolipoprotein A-I as the targeting moiety.

Diosgenin induces ROS-dependent autophagy and cytotoxicity via mTOR signaling pathway in chronic myeloid leukemia cells

BACKGROUND Diosgenin, a steroidal saponin isolated from legumes and yams, has been confirmed to possess potent anticancer effect on multifarious tumors including chronic myeloid leukemia (CML). PURPOSE We aimed to further determine the anti-cancer activity of diosgenin and its mechanisms in CML cells. METHODS The cell vitality was detected by MTT assay. Autophagic flux and reactive oxygen species (ROS) production were analyzed by laser scanning confocal microscope. Apoptosis was observed by flow cytometry. All proteins expression was examined by western blotting. RESULTS Autophagy induction was demonstrated by examination of autophagic flux including autophagosomes accumulation, autophagosome-lysosome fusion and degradation of autophagosomes. Moreover, blocking autophagy with inhibitor chloroquine (CQ) and 3-methyladenine (3-MA), enhanced diosgenin-induced apoptosis, indicating the protective effect of autophagy in diosgenin-treated CML cells. Further study suggested that diosgenin-induced autophagy and cytotoxicity were accompanied by reactive oxygen species (ROS) generation and mammalian target of rapamycin (mTOR) signaling pathway inhibition. N-acetyl-L-cysteine (NAC) administration, a scavenger agent of ROS, could down-regulate diosgenin-induced autophagy via reversion of mTOR pathway inhibition. CONCLUSION These results indicate that diosgenin obviously generates ROS and this oxidative pressure not only produces cytotoxic effect on CML cells but also induces autophagy. Whats more, autophagy functions as a cytoprotective mechanism to overcome cytotoxicity of diosgenin in tumor cells and inhibition of autophagy can enhance the anti-CML activity of diosgenin.

Delivery of hydrophilic drug doxorubicin hydrochloride-targeted liver using apoAI as carrier

Abstract High-density lipoprotein (HDL) particles can deliver cholesterol from peripheral tissues to the liver through apolipoprotein A1 (Apo A1), which specifically binds to the scavenger receptor class B type 1 (SR-B1) receptor on the surface of hepatocytes. Therefore, ApoA1 can be potentially used to target drugs to the liver. In this study, we successfully loaded doxorubicin hydrochloride (Dox or Dox-HCl), which is a hydrophilic drug used in a wide variety of clinical applications, into the core of reconstituted HDL (rHDL prepared by apoAI and egg phospholipids) to form a doxorubicin-HDL complex (rHDL-Dox). The MTT assays showed that rHDL-Dox particles also had higher cytotoxicity against several cells lines compared to free drug or Dox encapsulated into liposomes. A cellular uptake assay demonstrated that rHDL-Dox had higher absorption in SR-BI receptor positive liver cells. Importantly, in vivo experiments showed that rHDL-Dox can reduce tumor growth more effectively than liposomes. In addition, an in vitro hemolysis assay showed that rHDL-Dox caused only limited hemolysis in the case of high doses. Taken together, our findings indicate that rHDL is a safe and effective drug delivery system for targeting liver.

Baicalin inhibits autophagy induced by influenza A virus H3N2.

Baicalin, a natural product isolated from Scutellariaradix, has been reported to have significant in vivo and in vitro anti-influenza virus activity, but the underlying mechanism remains poorly understood. In this study, we found that baicalin inhibited autophagy induced by influenza virus A3/Beijing/30/95 (H3N2) in both A549 and Ana-1 cells. The results showed that H3N2 induced autophagy by suppressing mTOR signaling pathway, which however could be significantly inhibited by baicalin. Baicalin could suppress the expression of Atg5-Atg12 complex and LC3-II, and attenuate autophagy induced by starvation. Thus, the inhibition of autophagy induced by virus may account for the antiviral activities of baicalin against H3N2. Autophagy may be a potential marker in developing novel anti-influenza drugs.

Autophagy Plays a Critical Role in ChLym-1-Induced Cytotoxicity of Non-Hodgkin’s Lymphoma Cells

Autophagy is a critical mechanism in both cancer therapy resistance and tumor suppression. Monoclonal antibodies have been documented to kill tumor cells via apoptosis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin’s Lymphoma (NHL) cells. Interestingly, inhibition of autophagy by pharmacological inhibitors (3-methyladenine and NH4Cl) or genetic approaches (siRNA targeting Atg5) suppresses chLym-1-induced growth inhibition, apoptosis, ADCC and CDC in Raji cells, while induction of autophagy could accelerate cytotoxic effects of chLym-1 on Raji cells. Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1. Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2). These results elucidate the critical role of autophagy in cytotoxicity of chLym-1 antibody and suggest a potential therapeutic strategy of NHL therapy by monoclonal antibody chLym-1 in combination with autophagy inducer.

Antioxidant effect of apolipoprotein A-I on high-fat diet-induced non-alcoholic fatty liver disease in rabbits

Non-alcoholic fatty liver disease (NAFLD) is an increasingly recognized condition that encompasses a spectrum of liver abnormalities. It has been suggested that oxidative stress and lipid peroxidation are key pathophysiological mechanisms in NAFLD. Although an antioxidant effect of apolipoprotein A-I (apoA-I) has been reported, its influence on NAFLD has not been reported. The aim of this study was to determine whether apoA-I could improve the biochemical and histological abnormalities associated with high-fat diet-induced NAFLD through its antioxidant actions in rabbits. Liver damage was evaluated by hepatic coefficient, hepatic lipid assay, liver apparent abnormalities as well as hematoxylin-eosin staining of liver sections. Lipid peroxidation was assessed by measuring malondialdehyde (MDA) level in liver. Oxidative stress was assessed by measuring superoxide dismutase (SOD), glutathione peroxidase (GPx), and inducible nitric oxide synthase (iNOS) activities in serum and liver. Also, the mRNA expressions levels of SOD, GPx, and catalase (CAT) were determined by real-time quantitative polymerase chain reaction method. The results showed that apoA-I (20 or 40 mg/kg/w) was effective in reducing hepatic steatosis, inflammation, hepatic coefficient, and liver total cholesterol, triglyceride, low-density lipoprotein-cholesterol, and MDA levels in high-fat diet rabbits. In addition, apoA-I increased SOD and GPx activities while reducing iNOS activity in serum and liver. Moreover, apoA-I significantly increased the mRNA expression levels of SOD, GPx, and CAT in liver. This study showed that apoA-I exerted protective effects against fatty liver disease in rabbits induced by a high-fat diet, possibly through its antioxidant actions.