Jian Wen Wang

Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.

Glaucocalyxin A induces apoptosis in human leukemia HL-60 cells through mitochondria-mediated death pathway

Glaucocalyxin A (GLA) is a biologically active ent-kauranoid diterpenoid isolated from Rabdosia japonica var. glaucocalyx, a traditional Chinese medicinal herb, which has been shown to inhibit tumor cell proliferation. However, the mechanism underlying GLA-induced cytotoxicity remains unclear. In this study, we focused on the effect of GLA induction on apoptosis, the mitochondria-mediated death pathway and the accumulation of reactive oxygen species (ROS) in human leukemia cells (HL-60). GLA could induce a dose-dependent apoptosis in HL-60 cells as characterized by cell morphology, DNA fragmentation, activation of caspase-3, -9 and an increased expression ratio of Bax/Bcl-2. The mitochondrial membrane potential (Δψ(m)) loss and cytochrome c release from mitochondria to cytosol were observed during the induction. Moreover, GLA caused a time- and dose-dependent elevation of intracellular ROS level in HL-60 cells, and N-acetyl-l-cysteine (NAC, a well-known antioxidant) could block GLA-induced ROS generation and apoptosis. These data suggest that GLA induces apoptosis in HL-60 cells through ROS-dependent mitochondrial dysfunction pathway.

Nitric oxide elicitation for secondary metabolite production in cultured plant cells

Nitric oxide (NO) is an important signal molecule in stress responses. Accumulation of secondary metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. NO has been reported to play important roles in elicitor-induced secondary metabolite production in tissue and cell cultures of medicinal plants. Better understanding of NO role in the biosynthesis of such metabolites is very important for optimizing the commercial production of those pharmaceutically significant secondary metabolites. This paper summarizes progress made on several aspects of NO signal leading to the production of plant secondary metabolites, including various abiotic and biotic elicitors that induce NO production, elicitor-triggered NO generation cascades, the impact of NO on growth development and programmed cell death in medicinal plants, and NO-mediated regulation of the biosynthetic pathways of such metabolites. Cross-talks among NO signaling and reactive oxygen species, salicylic acid, and jasmonic acid are discussed. Some perspectives on the application of NO donors for induction of the secondary metabolite accumulation in plant cultures are also presented.

Glaucocalyxin A and B-induced Cell Death is Related to GSH Perturbation in Human Leukemia HL-60 Cells

Glaucocalyxin (Gla) A-C are major ent-kauranoid diterpenoids isolated from Rabdosia japonica var. glaucocalyx, a plant used in Chinese traditional medicine as an antitumor and anti-inflammatory agent. The present investigation was carried out to observe whether cellular reduced glutathione (GSH) plays important roles in Gla -induced cytotoxicity. Among major ent-kauranoid diterpenoids isolated, Gla A and B dose-dependently decreased the growth of HL-60 cells with an IC50 of approximately 6.15 and 5.86 µM at 24 h, respectively. Both Gla A and B could induce apoptosis, G2/M-phase cycle arrest, DNA damage and the accumulation of reactive oxygen species (ROS) in HL-60 cells. Moreover, Gla A, B caused rapid decrease of the intracellular GSH content, while inhibition of cellular GSH synthesis by buthionine sulfoximine (BSO) augmented the induced cytotoxicity and apoptosis in HL-60 cells. On the other hand, the administration of GSH or GSH precursor N-acetyl-cysteine (NAC) could rescue Gla A, B-depleted cellular GSH, and abrogate the induced cytotoxicity, G2/M-phase cycle arrest, DNA damage and ROS accumulation in HL-60 cells. Furthermore, Gla A, B decreased the activity of the GSH-related enzymes including glutathione reductase (GR) and glutathione peroxidase (GPX). These data suggest that the intracellular GSH redox system plays important roles in regulating the Gla A, B-induced cytotoxicity on HL-60 cells.

The microbial transglutaminase immobilization on carboxylated poly(N-isopropylacrylamide) for thermo-responsivity

Microbial transglutaminase (mTG) is widely utilized in the PEGylation of pharmaceutical proteins. mTG immobilization can be achieved via covalent bonding on solid supports. However, the catalytic efficiency of mTG immobilized on solid supports was significantly reduced by mass transfer limitation. To overcome this limitation, mTG was covalently immobilized on the thermo-responsive carboxylated poly(N-isopropylacrylamide) (pNIPAM). The pNIPAM-mTG conjugate exhibited reversibly solubility in aqueous solution with a low critical solution temperature (LCST) at 39°C, i.e., it was insoluble above 39°C and soluble below 39°C. The pH dependence of the pNIPAM-mTG conjugate was similar with that of the native mTG. Upon conjugation to pNIPAM, the optimal temperature of mTG shifted down from 50-55°C to 40-45°C, and the thermal stability of the conjugate was elevated. The easy separation of the pNIPAM-mTG conjugate with its substrate and the catalytic efficiency of the pNIPAM-mTG conjugate were demonstrated by employing the pNIPAM-mTG conjugate to cross-link bovine serum albumin (BSA) and catalyze PEGylation of therapeutic protein, cytochrome c (Cyt C), respectively. The thermo-responsive mTG is suitable to modify proteins in food processing and biomedical engineering.

Antifungal Properties of Ag-SiO2 Core-Shell Nanoparticles against Phytopathogenic Fungi

Silver nanoparticles have been extensively used as the anti-microbial agent in medicine. The present study addressed their antifungal potential against phytopathogenic fungi. Ag-SiO2 core–shell nanoparticles (Ag-SiO2 NPs) with an average size of 118.2 ± 4.6 nm were successfully prepared. The released Ag+ slowly through the outer porous SiO2 coating layer from the silver cores was detected in potato dextrose medium, reaching a peak value of 0.016 ppm around day 4. Ag-SiO2 NPs have displayed excellent antifungal effects against tested phytopathogenic fungi even at a low concentration of 0.5 ppm. The antifungal activity of these Ag-SiO2 NPs is not just the effect of dissolved Ag+ released from the nanoparticles. Our results suggested that the induced reactive oxygen species (ROS) by Ag-SiO2 NPs were involved in the effect of growth inhibition on tested fungi.