Cerium-Promoted Ginsenosides Accumulation by Regulating Endogenous Methyl Jasmonate Biosynthesis in Hairy Roots of Panax ginseng.

Among rare earth elements, cerium has the unique ability of regulating the growth of plant cells and the biosynthesis of metabolites at different stages of plant development. The signal pathways of Ce3+-mediated ginsenosides biosynthesis in ginseng hairy roots were investigated. At a low concentration, Ce3+ improved the elongation and biomass of hairy roots. The Ce3+-induced accumulation of ginsenosides showed a high correlation with the reactive oxygen species (ROS), as well as the biosynthesis of endogenous methyl jasmonate (MeJA) and ginsenoside key enzyme genes (PgSS, PgSE and PgDDS). At a Ce3+ concentration of 20 mg L-1, the total ginsenoside content was 1.7-fold, and the total ginsenosides yield was 2.7-fold that of the control. Malondialdehyde (MDA) content and the ROS production rate were significantly higher than those of the control. The activity of superoxide dismutase (SOD) was significantly activated within the Ce3+ concentration range of 10 to 30 mg L-1. The activity of catalase (CAT) and peroxidase (POD) strengthened with the increasing concentration of Ce3+ in the range of 20-40 mg L-1. The Ce3+ exposure induced transient production of superoxide anion (O2•-) and hydrogen peroxide (H2O2). Together with the increase in the intracellular MeJA level and enzyme activity for lipoxygenase (LOX), there was an increase in the gene expression level of MeJA biosynthesis including PgLOX, PgAOS and PgJMT. Our results also revealed that Ce3+ did not directly influence PgSS, PgSE and PgDDS activity. We speculated that Ce3+-induced ROS production could enhance the accumulation of ginsenosides in ginseng hairy roots via the direct stimulation of enzyme genes for MeJA biosynthesis. This study demonstrates a potential approach for understanding and improving ginsenoside biosynthesis that is regulated by Ce3+-mediated signal transduction.

Highly Selective Production of Compound K from Ginsenoside Rd by Hydrolyzing Glucose at C-3 Glycoside Using ß-Glucosidase of Bifidobacterium breve ATCC 15700.

To investigate a novel ß-glucosidase from Bifidobacterium breve ATCC 15700 (BbBgl) to produce compound K (CK) via ginsenoside F2 by highly selective and efficient hydrolysis of the C-3 glycoside from ginsenoside Rd, the BbBgl gene was cloned and expressed in E. coli BL21. The recombinant BbBgl was purified by Ni-NTA magnetic beads to obtain an enzyme with specific activity of 37 U/mg protein using pNP-Glc as substrate. The enzyme activity was optimized at pH 5.0, 35°C, 2 or 6 U/ml, and its activity was enhanced by Mn2+ significantly. Under the optimal conditions, the half-life of the BbBgl is 180 h, much longer than the characterized ß-glycosidases, and the Km and Vmax values are 2.7 mM and 39.8 µmol/mg/min for ginsenoside Rd. Moreover, the enzyme exhibits strong tolerance against high substrate concentration (up to 40 g/l ginsenoside Rd) with a molar biotransformation rate of 96% within 12 h. The good enzymatic properties and gram-scale conversion capacity of BbBgl provide an attractive method for large-scale production of rare ginsenoside CK using a single enzyme or a combination of enzymes.

Increase of rutin antioxidant activity by generating Maillard reaction products with lysine.

Rutin exists in medicinal herbs, fruits, vegetables, and a number of plant-derived sources. Dietary sources containing rutin are considered beneficial because of their potential protective roles in multiple diseases related to oxidative stresses. In the present study, the change and antioxidation activity of rutin in Maillard reaction with lysine through a heating process were investigated. There is release of glucose and rhamnose that interact with lysine to give Maillard reaction products (MRPs), while rutin is converted to less-polar quercetin and a small quantity of isoquercitrin. Because of their high cell-membrane permeability, the rutin-lysine MRPs increase the free radical-scavenging activity in HepG2 cells, showing cellular antioxidant activity against Cu(2+)-induced oxidative stress higher than that of rutin. Furthermore, the MRPs significantly increased the Cu/Zn SOD (superoxide dismutase) activity and Cu/Zn SOD gene expression of HepG2 cells, consequently enhancing antioxidation activity.

Synthesis and receptor binding assay of indolin-2-one derivatives as dopamine D4 receptor ligands.

Five indolin-2-one derivatives bearing piperazinylbutyl side chains attached to the amide nitrogen were synthesized from 2-indolinone. 1-(4-Bromobutyl)-indolin-2-one was reacted with 1-piperazinecarboxaldehyde to form 1-(4-(4-formyl-1-piperazinyl)butyl)indolin-2-one (2). In the presence of H2SO4, the aldehyde moiety was removed from 1-(4-(4-formyl-1-piperazinyl)butyl)indolin-2-one and then 1-(4-(1-piperazinyl)butyl)indolin-2-one (3) was obtained, this compound was reacted with benzaldehyde derivatives to give the target compounds 4 a-e by N-alkylation reaction. The structures of the intermediates and the target compounds were characterized by 1H NMR, ESI-MS spectra and elemental analyses. In vitro receptor binding assays at D2, D3, D4 receptor subtypes of the target compounds were performed and the five compounds showed selectivity towards D2-like receptors. Among them, 1-(4-(4-(4-hydroxybenzy)-1-piperazinyl)butyl) indolin-2-one (4c) exhibited a remarkable affinity and selectivity to D4 receptor with K(i) value of 0.5 nM. The results indicated that 1-(4-(4-(4-hydroxybenzy)-1-piperazinyl)butyl)indolin-2-one might be a potential dopamine D4 receptor ligand.

Enhancement of ginsenoside Rg(1) in Panax ginseng hairy root by overexpressing the α-L-rhamnosidase gene from Bifidobacterium breve.

OBJECTIVES: To improve the production of ginsenoside Rg1 in Panax ginseng. RESULTS: The α-L-rhamnosidase gene from Bifidobacterium breve (BbRha) was overexpressed into hairy root culture system using Agrobacterium rhizogenes A4. Ginsenoside Rg1 in hairy roots was obtained following transformation via overexpressed gene representing 2.2-fold higher than those of control lines. Several overexpression transgenic hairy root lines were obtained exhibiting markedly increased levels of the corresponding α-L-rhamnosidase enzymatic activity relative to control. Ginsenoside Rg1 levels in the transgenic lines were higher (2.2-fold) than those of control after following 30 days culturing, while ginsenoside Re contents in tested transgenic lines were found to be lower. The transgenic hairy roots harboring α-L-rhamnosidase gene improved the accumulation of ginsenoside Rg1 up to 3.6 mg g(-1) dry weight. CONCLUSION: BbRha gene selectively enhances the production of ginsenoside Rg1 in P. ginseng hairy roots.

Biotransformation of rutin to isoquercitrin using recombinant α-L-rhamnosidase from Bifidobacterium breve.

OBJECTIVES: To biotransform rutin into isoquercitrin. RESULTS: A α-L-rhamnosidase from Bifidobacterium breve was produced by using Escherichia coli BL21 for biotransformation of rutin to isoquercitrin. The enzyme was purified by Ni(2+)-NTA chromatography to yield a soluble protein with a specific activity of 56 U protein mg(-1). The maximum enzyme activities were at pH 6.5, 55 °C, 20 mM rutin, and 1.2 U enzyme ml(-1). Under optimal conditions, the half-life of the enzyme was 96 h. The K m and V max values were 2.2 mM, 56.4 µmol mg(-1) min(-1) and 2.1 mM, 57.5 µmol mg(-1) min(-1) using pNP-Rha and rutin as substrates, respectively. The kinetic behavior indicated that the recombinant α-L-rhamnosidase has good catalytic performance for producing isoquercitrin. 20 mM rutin was biotransformed into 18.25 and 19.87 mM isoquercitrin after 60 and 240 min. CONCLUSION: The specific biotransformation of rutin to isoquercitrin using recombinant α-L-rhamnosidase from B. breve is a feasible method for use in industrial processes.

[Cloning of human adiponectin gene by PCR-driven overlap extension and expression in Pichia pastoris].

Gene of human adiponectin (ADPN) was cloned by PCR-driven overlap extension. The ADPN gene was linked into pGEM-T vector. After the sequence was determined, the ADPN gene was subcloned into expression vector pPIC3.5K to yield the recombinant expression vector pPIC3.5K-ADPN. The recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation, then the recombinant strain was identified by PCR and Southern blotting. After induction by methanol, ADPN was expressed in GS115, then the protein was identified by Western blotting. The results showed that the ADPN was expressed successfully. The optimum conditions of expression were 30 degrees C and 1% methanol inducing 48 h.