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ABSTRACT: A novel poly-N-acetylhexosamine (polyhexNAc) about 6kDa average molecular weight (MW) was isolated from the low-MW fraction of exopolysaccharide produced by liquid fermentation of a medicinal fungus Cordyceps sinensis Cs-HK1. The composition and linkage of sugar residues were determined by mass spectrometry and methylation analysis, and the anomeric configuration and chain linkage were confirmed by NMR. From the analytical results, the molecular structure was elucidated as a [-4-β-d-ManNAc-(1→3)-β-d-GalNAc-(1→] disaccharide repeating unit in the main chain with a Gal branch occurring randomly at the 3-position of ManNAc. This polyhexNAc showed notable antioxidant activities with a Trolox equivalent antioxidant capacity of 330μmol Trolox/g, a ferric reducing ability of plasma of 45.7μmol Fe(II)/g, and significant cytoprotective effect against H2O2-induced PC12 cell injury. This is the first report on the structure and bioactivity of an extracellular amino-polysaccharide from the Cordyceps species.
Carbohydrate polymers. 04/2013; 94(1):332-8.
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ABSTRACT: : This chapter reviews the various biotic and abiotic elicitors applied to hairy root cultures and their stimulating effects on the accumulation of secondary metabolites. Elicitors generally refer to the agents that stimulate the defense responses of plants. As a major response of plants to biotic and abiotic stress, the accumulation of secondary metabolites in plant tissue cultures can be stimulated by the elicitors. Among the many elicitors applied to hairy root cultures as well as plant cell suspension cultures, the most common and effective elicitors are fungal cell extracts, polysaccharides from fungal and plant cells, and heavy metal salts. With the crude fungal cell extracts, it is essential to observe the preparation conditions carefully for achieving reproducible effects. In addition to the chemical agents, UV-radiation, hyperosmotic stress and temperature shift have been shown effective for some plant species/metabolites. Elicitor type, dose, and treatment schedule are major factors determining the effects on the secondary metabolite production. In addition to the accumulation of products in roots, elicitor treatments often stimulate the release of intracellular products. Although elicitation is mainly effective to increase specific product yield on per unit mass of roots, the incorporation of nutrient feeding strategies can be applied to enhance the volumetric product yield. The integration of in situ product recovery from the roots/liquid medium is another synergistic strategy with the elicitor treatment to improve the process.
Advances in biochemical engineering/biotechnology 03/2013; · 1.64 Impact Factor
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ABSTRACT: This work is to examine the molecular weight (MW)-anticoagulation activity relationship of sulfated cellulose derivatives
(cellulose sulfates, CS). The initial CS with a degree of substitution (1.59) was prepared by homogeneous sulfation of microcrystalline
cellulose in an ionic liquid [C4mim]Cl. It was then hydrolyzed in a dilute acidic solution and separated into four MW fractions of 59, 23, 10×103g/mol and below 2.7×103g/mol. The anticoagulation activities based on in vitro assays of coagulation time and coagulation factors in human plasma
showed a positive correlation with the MW of CS, while the activity assay of clotting time in rats exhibited a negative correlation
with the MW 10–59×103g/mol, and the fraction with a MW <2.7×103g/mol exhibited a more moderate and durable activity. The results indicate that MW is a major factor on the anticoagulant
properties of CS derivatives and higher MW range is favorable for in vitro, and lower MW suitable for in vivo applications.
KeywordsSulfated cellulose derivatives-Acidic degradation-Anticoagulation-Molecular weight-Ionic liquid
Cellulose 04/2012; 17(5):953-961. · 3.60 Impact Factor
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ABSTRACT: Beauvericin (BEA) is a proven and potent antibiotic compound useful for bio-control and a potential antifungal and anticancer
agent for human. This study was to evaluate and optimize the nutrient medium for BEA production in mycelial liquid culture
of a high BEA-producing fungus Fusarium redolens Dzf2 isolated from a medicinal plant. Among various organic and inorganic carbon and nitrogen sources, glucose and peptone
were found the most favorable for the F. redolens Dzf2 mycelial growth and BEA production. Through a Plackett-Burman screening test on a basal medium, glucose, peptone, and
medium pH were identified as the significant factors for mycelial growth and BEA production. These factors were optimized
through central composite design of experiments and response surface methodology, as 49.0 g/L glucose, 13.0 g/L peptone and
pH 6.6, yielding 198 mg/L BEA (versus 156 mg/L in the basal medium). The BEA yield was further increased to 234 mg/L by feeding
10 g/L glucose to the culture during exponential phase. The results show that F. redolens Dzf2 mycelial fermentation is a feasible and promising process for production of BEA.
Keywords
Fusarium redolens
-mycelial culture-liquid medium-beauvericin-statistical optimization
Biotechnology and Bioprocess Engineering 04/2012; 15(3):460-466. · 1.28 Impact Factor
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ABSTRACT: EPS-1 was an exopolysaccharide produced by the medicinal fungus Cordyceps sinensis (Cs-HK1). In the present study, EPS-1 was sulfated with chlorosulfonic acid (CSA)-pyridine (Pyr) at different volume ratios, yielding four sulfated derivatives, SEPS-1A, B, C and D, with different degrees of substitution (DS: 0.25-1.38) and molecular weights (17.1-4.1 kDa). The sulfation of EPS-1 occurred most frequently at the C-6 hydroxyl groups due to their higher reactivity. In aqueous solution, the native EPS-1 formed random coils or aggregated networks, but the sulfated derivatives formed single helices. The antioxidant activities of the sulfated EPS-1 derivatives for scavenging hydroxyl radicals (•OH) and 2,2-azinobis-3-ehtylbenzothiazolin-6-sulfonic acid radicals (ABTS•+) were significantly increased with increasing DS and decreasing molecular weight (MW). Sulfation has thus been shown to be an effective and favorable strategy for improving the physico-chemical properties and bioactivities of fungal polysaccharides.
Molecules 01/2012; 18(1):167-77. · 2.39 Impact Factor
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ABSTRACT: This study investigated the effects of surfactant additives and medium pH on mycelial morphology and exopolysaccharide (EPS) production in liquid culture of a valuable medicinal fungus Cordyceps sinensis Cs-HK1. In the medium containing 20 g l⁻¹ glucose and 6 g l⁻¹ peptone as the sole nitrogen source, the Cs-HK1 fungal mycelia formed smooth and spherical pellets about 1.8-mm mean diameter. The mycelial pellets became less uniform at pH (4.0-5.0) lower than the optimum (pH 6.0) or turned to filamentous form at higher pH (8-9). Surfactants added to the medium inhibited pellet formation, resulting in smaller and looser pellets. Tween 80 exhibited a remarkable promoting effect on EPS production, increasing the EPS yield more than twofold at 1.5% (w/v), which was most probably attributed to the stimulation of EPS biosynthesis and release from the fungal cells by Tween 80.
Journal of Industrial Microbiology 12/2011; 39(4):623-8. · 1.80 Impact Factor
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ABSTRACT: This study examines the roles of anion channels and ATP binding cassette (ABC) protein transporters in mediating elicitor-induced ATP release in Salvia miltiorrhiza hairy root cultures. The elicitor-induced ATP release was effectively blocked by two putative membrane anion channel blockers, niflumic acid and Zn(2+), but not by a specific Cl(-) channel blocker, phenylanthranilic acid. The elicitor-induced ATP release was also significantly suppressed by two ABC inhibitors, glibenclamide and ethacrynic acid. Notable ATP release from the hairy roots was also induced by verapamil (2mM), an ABC activator in animal cells. The verapamil-induced ATP release was effectively blocked by niflumic acid, but only slightly inhibited by the ABC inhibitors. Another notable effect of verapamil was the induction of exocytosis, the secretion of vesicle-like particles to the root surface. The verapamil-induced exocytosis was not inhibited by nifulumic acid and YE did not induce the exocytosis. Overall, the results suggest a significant role of anion channels, a possible involvement of ABC proteins and no significant involvement of exocytosis in mediating the ATP efflux in hairy root cells.
Journal of plant physiology 01/2011; 168(2):128-32. · 2.50 Impact Factor
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ABSTRACT: Beauvericin (BEA) is a cyclic hexadepsipeptide mycotoxin with notable phytotoxic and insecticidal activities. Fusarium redolens Dzf2 is a highly BEA-producing fungus isolated from a medicinal plant. The aim of the current study was to develop a simple and valid kinetic model for F. redolens Dzf2 mycelial growth and the optimal fed-batch operation for efficient BEA production. A modified Monod model with substrate (glucose) and product (BEA) inhibition was constructed based on the culture characteristics of F. redolens Dzf2 mycelia in a liquid medium. Model parameters were derived by simulation of the experimental data from batch culture. The model fitted closely with the experimental data over 20-50 g l(-1) glucose concentration range in batch fermentation. The kinetic model together with the stoichiometric relationships for biomass, substrate and product was applied to predict the optimal feeding scheme for fed-batch fermentation, leading to 54% higher BEA yield (299 mg l(-1)) than in the batch culture (194 mg l(-1)). The modified Monod model incorporating substrate and product inhibition was proven adequate for describing the growth kinetics of F. redolens Dzf2 mycelial culture at suitable but not excessive initial glucose levels in batch and fed-batch cultures.
Journal of Industrial Microbiology 11/2010; 38(9):1187-92. · 1.80 Impact Factor
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ABSTRACT: 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.
Applied Microbiology and Biotechnology 09/2010; 88(2):437-49. · 3.42 Impact Factor
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ABSTRACT: This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Four classes of elicitors were tested, heavy metal ions (Co2+, Ag+, Cd2+), polysaccharides (yeast extract and chitosan), plant response-signaling compounds (salicylic acid and methyl jasmonate), and hyperosmotic stress (with sorbitol). Of these, Ag (silver nitrate), Cd (cadmium chloride), and polysaccharide from yeast extract (YE) were most effective to stimulate the tanshinone production, increasing the total tanshinone content of cell by more than ten-fold (2.3 mg g(-1) versus 0.2 mg g(-1) in control). The stimulating effect was concentration-dependent, most significant at 25 microM of Ag and Cd and 100 mg l(-1) (carbohydrate content) of YE. Of the three tanshinones detected, cryptotanshinone was stimulated most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold. Meanwhile, most of the elicitors suppressed cell growth, decreasing the biomass yield by about 50% (5.1-5.5 g l(-1) versus 8.9 g l(-1) in control). The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity. The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.
Applied Microbiology and Biotechnology 03/2010; 87(1):137-44. · 3.42 Impact Factor
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ABSTRACT: High-power ultrasound (20 kHz) was applied to modify the physicochemical properties of a high-molecular weight (MW) exopolysaccharide (EPS) from mycelial culture of a medicinal fungus. At 35 W/cm(2) or higher ultrasound power, the apparent and intrinsic viscosities of EPS solution dropped by nearly 85% within 10 min, and the water solubility was increased by more than fourfold. The ultrasonic treatment led to a notable reduction of the maximum MW and a more uniform MW distribution, but no significant change in the primary structure of the EPS molecules. In contrast, the intrinsic viscosity of EPS was reduced by only 20% in 1.0M sulfuric acid at 50 degrees C for 9h. Ultrasound was proven an effective and favorable means for improving the solution properties of high-MW bioactive polysaccharides in mild conditions.
Bioresource technology 02/2010; 101(14):5517-22. · 4.25 Impact Factor
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ABSTRACT: Four isaindigotone derivatives (5a,b and 6a,b) designed as telomeric G-quadruplex ligands have been synthesized and characterized. The unfused aromatic rings in these compounds allow a flexible and adaptive conformation in G-quadruplex recognition. The interaction of human telomeric G-quadruplex DNA with these designed ligands was explored by means of FRET melting, fluorescence titration, CD spectroscopy, continuous variation, and molecular modeling studies. Our results showed that the adaptive scaffold might not only allow the ligands to well occupy the G-quartet but also perfectly bind to the grooves of the G-quadruplex. The synergetic effect of the multiple binding modes might be responsible for the improved binding ability and high selectivity of these ligands toward G-quadruplex over duplex DNA. Long-term exposure of HL60 and CA46 cancer cells to compound 5a showed a remarkable decrease in population growth, cellular senescence phenotype, and shortening of the telomere length, which is consistent with the behavior of an effective telomeric G-quadruplex ligand and telomerase inhibitor.
Journal of Medicinal Chemistry 05/2009; 52(9):2825-35. · 4.80 Impact Factor
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ABSTRACT: Homogeneous sulfation of bagasse cellulose (BC) with chlorosulfonic acid-dimethylformamide was accomplished in an ionic liquid 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl). The BCS products from the sulfation had degrees of substitution (DS) in the range of 0.52-2.95 and a simultaneous substitution pattern at C-6, C-2 and C-3 positions. The sulfated BCS attained significant anticoagulation activity, causing a dose-dependent prolongation of coagulation time and inhibition of FIIa and FXa activities in human plasma. The anticoagulation activity of BCS showed a positive correlation with DS, and some of the activity indexes exceeded those of heparin.
Bioresource technology 03/2009; 100(4):1687-90. · 4.25 Impact Factor
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ABSTRACT: G-quadruplex structures are promising targets for design of cancer drugs with high selectivity and low toxicity. This review provides an update of the progress made over the last few years in the design of selective G-quadruplex ligands, and a comprehensive summary of the major design strategies and structural characteristics.
Mini Reviews in Medicinal Chemistry 11/2008; 8(11):1163-78. · 2.53 Impact Factor
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ABSTRACT: An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).
Biotechnology and Bioengineering 06/2008; 101(5):996-1004. · 3.95 Impact Factor
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ABSTRACT: A series of novel oxoisoaporphine alkaloid derivatives, 9-aminoalkanamido-1-azabenzanthrone (general formula Ar-NHCO(CH(2))(n)NR(2), Ar=1-azabenzanthrone, n=1, 2 or 3), had been synthesized. Compared with 1-azabenzanthrone, the derivatives had significantly higher DNA binding affinity with calf thymus DNA, and higher potent cytotoxicity against different tumor cell lines. The cytotoxicity and the structure-activity relationship of the prepared compounds were studied. The derivatives with two methylene groups (n=2), and piperidine or ethanolamine functional group in the side chain exhibited highest DNA binding affinity and cytotoxicity.
European Journal of Medicinal Chemistry 06/2008; 43(5):973-80. · 3.35 Impact Factor
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ABSTRACT: The application of a polysaccharide elicitor from yeast extract, YE, to Salvia miltiorrhiza hairy root cultures induced transient release of ATP from the roots to the medium, leading to a dose-dependent increase in the extracellular ATP (eATP) level. The eATP level rose to a peak (about 6.5 nM with 100 mg l(-1) YE) at about 10 h after YE treatment, but dropped to the control level 6 h later. The elicitor-induced ATP release was dependent on membrane Ca2+ influx, and abolished by the Ca2+ chelator EGTA or the channel blocker La3+. The YE-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors. On the other hand, the application of exogenous ATP at 10-100 microM to the cultures also induced rapid and dose-dependent increases in H2O2 production and medium pH, both of which were effectively blocked by RB and EGTA. The non-hydrolyzable ATP analog ATPgammaS was as effective as ATP, but the hydrolyzed derivatives ADP or AMP were not so effective in inducing the pH and H2O2 increases. Our results suggest that ATP release is an early event and that eATP plays a signaling role in the elicitation of plant cell responses; Ca2+ is required for activation of the elicitor-induced ATP release and the eATP signal transduction. This is the first report on ATP release induced by a fungal elicitor and its involvement in the elicitor-induced responses in plant cells.
Plant and Cell Physiology 05/2008; 49(4):617-24. · 4.70 Impact Factor
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ABSTRACT: Hyperosmotic stress (OS, created with 50 g/L sorbitol) and a yeast elicitor (YE, polysaccharide fraction of yeast extract) applied to Salvia miltiorrhiza hairy root cultures had a synergistic effect on the diterpenoid tanshinone production. With a single OS+YE treatment and nutrient feeding, the total tanshinone content of roots was increased by sevenfold (from 0.2 to 1.6 mg/g dry weight (dw)) and the volumetric yield by 13-fold (from 1.95 to 27.4 mg/L) compared to the batch control culture. With repeated feeding of OS and nutrient medium in an extended fed-batch culture process (i.e., 10 mL fresh medium with 50 g/L sorbitol 25 mg/L YE, every 5 days from day 21 to day 60), the total tanshinone content of roots was increased to 18.1 mg/g dw (or 1.8 wt.%) and the volumetric tanshinone yield to 145 mg/L, which were about 100-fold and 70-fold of those, respectively, in the batch control. Another interesting finding was the presence of root fragments (fine particles) with extremely high tanshinone content in the OS+YE treated cultures. It was also possible to reuse the sorbitol medium for the hairy root growth and tanshinone production to reduce the medium expenses.
Applied Microbiology and Biotechnology 04/2008; 78(3):441-8. · 3.42 Impact Factor
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ABSTRACT: Salvia miltiorrhiza Bunge (Chinese sage; Lamiaceae) is a valuable Chinese herbal plant, and its rhizome, known as Danshen in Chinese because of its characteristic red pigment, is the part of the plant used in herbal medicine. The red pigment in S. miltiorrhiza roots is mainly composed of numerous diterpenoid tanshinones, as the major bioactive ingredients of the herb. In plants, diterpenes are synthesized through the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway in the plastids, and DXR [DXP (1-deoxy-D-xylulose 5-phosphate) reductoisomerase] is an enzyme catalysing the first step of the MEP pathway. In the present study, a full-length cDNA encoding DXR (GenBank Nucleotide Sequence Database accession no. DQ991431) was cloned from the hairy roots of S. miltiorrhiza in culture. The enzyme activity of DXR protein was verified by complementation of an Escherichia coli mutant deficient in dxr. The transcription level of the dxr gene in the hairy roots was up-regulated after exposure to hyperosmotic stress and a yeast elicitor in parallel with increased tanshinone accumulation in the hairy roots. This is the first report, to our knowledge, of elicitor-induced dxr transcription and its correlation with the accumulation of diterpenoid tanshinones in S. miltiorrhiza roots.
Biotechnology and Applied Biochemistry 03/2008; 52(Pt 1):89-95. · 1.53 Impact Factor
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ABSTRACT: Extracellular ATP (eATP) is a novel signalling agent, and nitric oxide (NO) is a well-established signal molecule with diverse functions in plant growth and development. This study characterizes NO production induced by exogenous ATP and examines its relationship with other important signalling agents, Ca(2+) and H(2)O(2) in Salvia miltiorrhiza hairy root culture. Exogenous ATP was applied at 10-500 microM to the hairy root cultures and stimulated NO production was detectable within 30 min. The NO level increased with ATP dose from 10-100 microM but decreased from 100-200 muM or higher. The ATP-induced NO production was mimicked by a non-hydrolysable ATP analogue ATPgammaS, but only weakly by ADP, AMP or adenosine. The ATP-induced NO production was blocked by Ca(2+) antagonists, but not affected by a protein kinase inhibitor. ATP also induced H(2)O(2) production, which was dependent on both Ca(2+) and protein kinases, and also on NO biosynthesis. On the other hand, ATP induced a rapid increase in the intracellular Ca(2+) level, which was dependent on NO but not H(2)O(2). The results suggest that NO is implicated in ATP-induced responses and signal transduction in plant cells, and ATP signalling is closely related to Ca(2+) and ROS signalling.
Journal of Experimental Botany 02/2008; 59(14):4007-16. · 5.36 Impact Factor
